Drosophila Histone Locus Body assembly and function involves … · 2020. 3. 16. · 66 are constitutively localized in the HLB (Duronio and Marzluff, 2017). The HLB provides a 67

1

1 Drosophila Histone Locus Body assembly and function involves 2

multiple interactions 3 4 5

Running title: Drosophila HLB assembly and function 6 7 8 9 10

Kaitlin P. Koreski1,, Leila E. Rieder2, Lyndsey M. McLain3, 11 William F. Marzluff,1,3,4-6* and Robert J. Duronio1,3,4,6,7* 12

13 14

15

16

Affiliations 17

1 Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 18

27599, USA. 19

2 Department of Biology, Emory University, Atlanta, GA 30322, USA 20

3 Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA. 21

4 Integrative Program for Biological and Genome Sciences, University of North Carolina, Chapel 22

Hill, NC 27599, USA. 23

5 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 24

27599, USA. 25

6 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, 26

USA. 27

7 Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA. 28

* Correspondence: [email protected], [email protected] 29

.CC-BY-NC-ND 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.994483doi: bioRxiv preprint

https://doi.org/10.1101/2020.03.16.994483

http://creativecommons.org/licenses/by-nc-nd/4.0/

2

Abstract 30

The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and 31

concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome 32

has a single locus comprised of ~100 copies of a tandemly arrayed repeat unit containing one 33

copy of each of the 5 RD histone genes. To determine sequence elements required for D. 34

melanogaster HLB formation and histone gene expression, we used transgenic gene arrays 35

containing 12 copies of the histone repeat unit that functionally complement loss of the ~200 36

endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were 37

replaced with H2a-H2b promoters does not form an HLB or express high levels of RD histone 38

mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is 39

active in HLB assembly and RD histone gene expression in the absence of the endogenous RD 40

histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. 41

The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array 42

contains all known factors present in the wild type HLB including CLAMP, which normally 43

binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein 44

and/or protein-DNA interactions contribute to HLB formation, and that the large number of 45

endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic 46

arrays, thereby preventing HLB formation and gene expression. 47

48



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Introduction 49

An important organizing principle in cells is the use of membraneless compartments to 50

spatially and temporally regulate diverse biological processes (Mitrea and Kriwacki, 2016). 51

Numerous membraneless compartments have been identified in both the nucleus (e.g. nucleoli, 52

Cajal bodies, histone locus bodies) and the cytoplasm (e.g. P-bodies, stress granules, germ 53

granules) and are collectively referred to as biomolecular condensates (Banani et al., 2017). 54

There is increasing evidence suggesting that membraneless compartments are formed through 55

liquid-liquid phase separation or condensation (Alberti et al., 2019). This occurs when proteins 56

and/or nucleic acids in the nucleoplasm or cytoplasm coalesce or demix into a condensed phase 57

that often resembles liquid droplets. Large nuclear condensates that are visible under light 58

microscopy are most often referred to as nuclear bodies (NBs), and represent an important 59

organizing feature of the nucleus. 60

The histone locus body (HLB) is a conserved NB that assembles at replication-dependent 61

(RD) histone genes and concentrates factors required for RD histone mRNA biogenesis (Duronio 62

and Marzluff, 2017). RD histone mRNAs are the only eukaryotic mRNAs that are not 63

polyadenylated (Marzluff and Koreski, 2017). The unique stem loop at the 3’end of RD histone 64

mRNAs results from a processing reaction requiring a specialized suite of factors, some of which 65

are constitutively localized in the HLB (Duronio and Marzluff, 2017). The HLB provides a 66

powerful system to study how NBs form and function because it contains a well characterized set 67

of factors involved in producing a unique class of cell-cycle regulated mRNAs. We previously 68

demonstrated that concentrating factors (e.g. FLASH and U7 snRNP) in the Drosophila 69

melanogaster HLB is critical for efficient histone pre-mRNA processing (Wagner et al., 2007; 70



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Tatomer et al., 2016b). However, a full understanding of how the HLB participates in histone 71

mRNA biosynthesis requires knowledge of HLB assembly at the molecular level. 72

Prior studies of NBs have provided several important assembly concepts that are 73

applicable to the HLB. Many NB components have an intrinsic ability to self-associate, an 74

observation leading to two different models of NB assembly: (1) interactions among NB 75

components occur stochastically, wherein individual factors can be recruited to the body in any 76

order, or (2) components assemble in an ordered or hierarchical pathway, wherein the 77

recruitment of components is predicated on prior recruitment of others (Dundr and Misteli, 2010; 78

Rajendra et al., 2010). The HLB appears to employ a hybrid version of these two possibilities. 79

For example, genetic loss of function experiments suggest a partially ordered assembly pathway 80

of the Drosophila HLB with some components being required for subsequent recruitment of 81

others (White et al., 2011; Terzo et al., 2015; Tatomer et al., 2016a). The scaffolding protein 82

Mxc (Multi sex combs), the Drosophila ortholog of human NPAT (Nuclear Protein, Ataxia-83

Telangiectasia Locus), and FLASH (FLICE-Associated Huge protein) likely form the core of the 84

HLB and are required for subsequent recruitment of other factors (White et al., 2011). Tethering 85

experiments indicate that ectopic HLB formation also may be induced by several different HLB 86

components, supporting a stochastic model of assembly (Shevtsov and Dundr, 2011). 87

The initiation event in self-organizing NB assembly is the key step in the process and is 88

not well understood. A prevalent model postulates a “seeding” event that initiates the nucleation 89

of critical components that form a platform for further recruitment of other components (Dundr, 90

2011; Shevtsov and Dundr, 2011; Altmeyer et al., 2015; Falahati et al., 2016; Stanek and Fox, 91

2017; Gomes and Shorter, 2019). In some instances, RNA is thought to help seed NB assembly, 92

and NBs such as the nucleolus and ectopic paraspeckles can form at sites of specific transcription 93



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(Matera et al., 2009; Mao et al., 2011; Falahati et al., 2016). Blocking transcription prevents 94

complete HLB assembly in both zebrafish and flies (Salzler et al., 2013; Heyn et al., 2017; Hur, 95

2019). However, the HLB is present at RD histone genes even in G1 when the genes are not 96

active, raising the possibility that histone genes themselves participate in seeding HLB assembly 97

(Zhao et al., 1998; Liu et al., 2006). 98

In Drosophila, the RD histone genes are present at a single locus with ~100 copies of a 99

tandemly arrayed 5kb repeat unit, each of which contains one copy of the divergently transcribed 100

H2a-H2b and H3-H4 gene pairs as well as the gene for linker histone H1 (Lifton et al., 1978; 101

McKay et al., 2015; Bongartz and Schloissnig, 2019). Using transgenes containing a wild type or 102

mutant derivative of a single histone repeat, we previously demonstrated that the bidirectional 103

H3-H4 promoter stimulated HLB assembly and transcription of the single histone repeat in 104

salivary glands (Salzler et al., 2013). We subsequently showed that the conserved GAGA repeat 105

elements present in the H3-H4 promoter region are targeted by the zinc-finger transcription 106

factor CLAMP (Chromatin-Linked Adaptor for MSL Proteins), and that this interaction 107

promotes HLB assembly (Rieder et al., 2017). Thus, the H3-H4 promoter region might act to 108

seed HLB assembly. 109

In this work, we leveraged transgenic histone gene arrays to test whether the H3-H4 110

promoter region is necessary for in vivo function of the RD histone locus. We found that 111

replacement of H3-H4 promoters with H2a-H2b promoters results in an attenuated transgenic 112

histone gene array that does not function in the presence of the intact endogenous RD histone 113

locus, but surprisingly provides full in vivo function, including normal HLB assembly and 114

histone gene expression, when the endogenous RD histone locus is absent. These results suggest 115



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that multiple elements in the histone genes and core HLB proteins are involved in HLB 116

assembly. 117

118 Results 119

To study histone gene regulation and the role of histone post-translational modifications 120

in chromatin we previously developed an experimental system in which BAC-based, transgenic 121

histone gene arrays containing 12 copies of the 5kb histone repeat unit assemble HLBs and 122

functionally complement homozygous loss of the ~100 gene copies at the endogenous RD 123

histone locus, HisC (McKay et al., 2015). To study histone gene expression, we created a 124

12xHWT (Histone Wild Type) transgenic construct containing a synonymous polymorphism in the 125

H2a gene (i.e. mutation of a XhoI site) that allows us to distinguish transgenic H2a gene 126

expression from endogenous H2a gene expression (McKay et al., 2015). Here, we extended this 127

design and created a “Designer Wild Type” (DWT) 5kb histone repeat unit that has all five 128

histone genes marked in a similar manner (Fig. 1A, Fig. S1A). We also introduced restriction 129

enzyme sites around the mRNA 3’ end processing signals, the stem loop and histone downstream 130

element (Marzluff and Koreski, 2017), to allow us to readily manipulate those sequences (Fig. 131

S1). We used this DWT repeat to create a 12xDWT construct, and found that a transgenic version 132

of this array behaved identically to the original 12xHWT array. It rescued the lethality caused by 133

homozygous deletion of HisC, resulting in viable, fertile adult flies. In addition, we can maintain 134

a stock of flies lacking the endogenous RD histone locus and containing a homozygous 12xDWT 135

transgene. 136

137

The H3-H4 promoter stimulates HLB formation 138



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To test whether the H3-H4 promoter region is necessary for histone locus function in vivo, we 139

engineered a derivative of the 12xDWT array in which all H3-H4 promoters were replaced with 140

H2a-H2b promoters. In this “Promoter Replacement” (12xPR) construct, we replaced the entire 141

298nt sequence between the initiation codons of the divergently transcribed H3 and H4 genes 142

with the 226nt sequence between the initiation codons of the divergently transcribed H2a and 143

H2b genes, leaving the H1 and H2a and H2b genes intact (Fig. 1A, Fig. S1B). One difference 144

between the two promoters is that the H2a-H2b region lacks the CLAMP-binding GAGA repeats 145

present in the H3-H4 promoter (Rieder et al., 2017). Otherwise it is an intact, native bidirectional 146

histone gene promoter. Consequently, the 12xPR construct should retain the ability to initiate 147

transcription from all RD histone genes. The 72nt size difference between the promoters 148

provides a way to unambiguously distinguish between the 12xPR and 12xDWT array genotypes 149

using PCR primers in the H3 and H4 coding regions in flies heterozygous (Fig. 1B) or 150

homozygous (Fig. 1C) for the HisC deletion. 151

We first assessed whether the 12xPR and 12xDWT transgenes could support HLB formation 152

in the presence of the endogenous histone genes in polytene chromosome spreads from 3rd instar 153

larval salivary glands. In these polyploid cells, the genome is amplified greater than 1000-fold 154

and sister chromatids line up in register, resulting in large chromosomes providing high-155

resolution cytology. We visualized the HLB by immunofluorescence using antibodies that 156

recognize one of several HLB components. These components include Multi sex combs (Mxc), 157

the Drosophila ortholog of human NPAT, which is necessary for HLB assembly and histone 158

gene expression (White et al., 2011; Terzo et al., 2015); histone pre-mRNA processing factors 159

FLASH (Yang et al., 2009) and Lsm11, a component of the U7 snRNP (Godfrey et al., 2009; 160

Burch et al., 2011); and Muscle wasted (Mute), an ortholog of human YARP (YY1AP-related 161



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protein 1) that is a putative repressor of histone gene expression (Bulchand et al., 2010). In these 162

preparations, we also visualized both the endogenous histone locus and the ectopic, transgenic 163

histone genes by FISH using a probe derived from the entire histone repeat unit. We observed 164

HLB formation at the control 12xDWT transgenic locus but not at the 12xPR transgenic locus (Fig. 165

1D). These data are consistent with our previous results that sequences within the H3-H4 166

promoter are necessary for HLB assembly in the presence of the endogenous histone genes 167

(Salzler et al., 2013). 168

Both single copy transgenic histone gene repeats that fail to form an HLB (Salzler et al., 169

2013) and Mxc mutants that don’t form an HLB (Terzo et al., 2015) result in reduced histone 170

mRNA levels, suggesting that the HLB is necessary for efficient histone mRNA biosynthesis. 171

We therefore tested whether the 12xPR transgene could support histone gene expression in the 172

absence of HLB assembly. To determine expression from the ectopic 12xDWT or 12xPR genes in 173

the presence of the endogenous RD histone genes, we randomly prime cDNA from total RNA 174

and then amplify a fragment of each coding region containing the restriction enzyme site that is 175

changed in the DWT construct. By digesting the PCR fragment with the appropriate restriction 176

enzyme and resolving the fragments by gel electrophoresis we can determine the relative level of 177

expression from the transgenic and endogenous RD histone genes. For example, RT-PCR of the 178

H2a gene results in the same size amplification product from both the transgenic and endogenous 179

genes, but only the product from the endogenous histone genes is sensitive to digestion with 180

XhoI (Fig. 1E). When assayed in the presence of the endogenous genes, we found that H2a was 181

expressed from the 12xDWT at a higher level than from the 12xPR transgene, even though identical 182

promoters drove H2a in each transgenic array (Fig. 1E, Fig. 2A, lanes 2 and 4; Fig. 2B, lanes 2 183

and 3). These data are consistent with our previous observations using transgenes with a single 184



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histone gene repeat unit (Salzler et al., 2013; Terzo et al., 2015). Together these results 185

demonstrate that the H3-H4 promoter is required both for HLB formation and histone gene 186

expression in the presence of the endogenous RD histone genes. 187

188

A histone gene array lacking the H3-H4 promoter forms HLBs and is expressed in the 189

absence of the endogenous genes 190

To assess the biological activity of the 12xPR transgene, we determined the developmental 191

outcome of having a 12xPR transgene as the only zygotic source of histone mRNA. Due to large 192

stores of maternal histone protein and mRNAs, embryos homozygous for a deletion that removes 193

the endogenous RD histone gene array develop normally through S phase of cell cycle 14, but 194

require zygotic RD histone gene expression for progression through S phase of cycle 15 and 195

beyond (Gunesdogan et al., 2010, 2014). Consequently, embryos lacking RD histone genes 196

arrest at nuclear cycle 15 and do not hatch. As noted above, this embryonic lethality is rescued 197

by a single 12xDWT transgene, which supports development of histone deletion progeny into 198

viable, fertile, adult flies. Surprisingly, we found that embryos lacking endogenous histone genes 199

and containing the 12xPR transgene hatched and developed into nearly the expected number of 200

fertile adults without any overt developmental delays, similar to embryos lacking endogenous 201

histone genes and rescued by the 12xDWT transgene. These data suggest that the 12xPR transgene 202

provides normal amounts of histone gene function when the endogenous RD histone genes are 203

absent. 204

We interrogated this unexpected result more thoroughly by analyzing 1) histone gene 205

expression, 2) HLB formation, and 3) histone pre-mRNA processing in both 5-7 hour old 206

embryos and 3rd instar larvae lacking endogenous histone genes and containing either the 12xDWT 207



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or 12xPR transgene. In both genotypes, all histone mRNA in either 5-7 hr embryos (Fig. 2A, 208

lanes 3 and 5) or 3rd instar larvae (Fig. 2B, lanes 4 and 5) was derived only from the ectopic 209

histone gene array, indicating that the maternal histone mRNA stores had been degraded 210

normally. We observed robust histone gene expression from the 12xPR transgene when the 211

endogenous histone genes are absent, in stark contrast with the low level of expression from the 212

12xPR transgene when endogenous histone genes are present (Fig. 2A, lane 4; Fig. 2B lane 3). 213

High levels of histone gene expression are strongly correlated with the ability to form an 214

HLB (Salzler et al., 2013; Terzo et al., 2015; Rieder et al., 2017). Given that the 12xPR can 215

support histone gene expression in the absence of the endogenous genes, we assayed for HLB 216

formation in polytene chromosome spreads from 3rd instar larval salivary glands and in embryos. 217

We detected robust HLB formation at the 12xPR transgenic locus with antibodies against FLASH 218

and Lsm11 (Fig. 2C), or Mxc and Mute (Fig. 2D), similar to that observed at the 12xDWT locus. 219

Thus, the 12xPR transgene, which lacks H3-H4 promoter sequences, can support HLB formation 220

and histone gene expression in the absence of endogenous RD histone genes. 221

222

Histone mRNA from the 12xPR transgenic locus is properly processed 223

An important function of the HLB is concentrating factors to promote efficient histone pre-224

mRNA processing (Tatomer et al., 2016b). Therefore, we reasoned it was possible that the 225

attenuated 12xPR gene array might affect other aspects of histone mRNA biosynthesis, including 226

pre-mRNA processing. All Drosophila RD histone genes contain cryptic polyadenylation signals 227

downstream of the normal processing sites that are only used when the histone pre-mRNA 228

processing reaction is compromised, resulting in the production and accumulation of poly(A)+ 229

histone mRNA (Lanzotti et al., 2002; Godfrey et al., 2009; Tatomer et al., 2016b). We examined 230



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pre-mRNA processing efficiency in flies rescued by the 12xPR transgene. Our PCR based assays 231

to detect histone mRNA expression cannot differentiate between properly processed and 232

misprocessed histone mRNAs. We therefore used northern blotting with a probe against the H3 233

coding region to determine whether histone pre-mRNA was efficiently processed. In contrast to a 234

FLASH mutant, which expresses large amounts of poly(A)+ histone mRNA, we did not detect 235

poly(A)+ histone mRNA from early embryos (Fig. 2E) or from whole 3rd instar larvae (Fig. 2F) 236

in HisC deletion animals rescued by the 12xPR transgene. Note also that the levels of histone 237

mRNA from the 12xPR transgenes are similar to wild type levels. These results indicate that the 238

HLB formed on the 12xPR array in the absence of endogenous histone genes supports efficient 239

histone pre-mRNA processing. 240

241

HLB assembly at the 12xPR transgenic locus occurs at the normal time during embryogenesis 242

The above data suggest that the 12xPR represents an attenuated histone gene array that 243

provides normal biological function when not in competition with the wild type endogenous 244

histone genes. To further explore this model, we determined if an HLB assembles on the 12xPR 245

array in the early embryo at the same time that it assembles on the 12xDWT array. The HLB 246

begins assembling in syncytial blastoderm embryos just prior to the onset of zygotic histone 247

transcription (White et al., 2007; White et al., 2011). We previously reported that a “proto-HLB” 248

consisting of Mxc and FLASH forms in nuclear cycle 10, followed by the onset of zygotic 249

histone gene expression and further recruitment of additional HLB components (Mute and 250

Lsm11) in cycle 11 (White et al., 2011; Salzler et al., 2013). To determine if the HLB forms at 251

the 12xPR transgenic locus with normal timing, we stained syncytial blastoderm embryos lacking 252

endogenous histone genes and rescued by a 12xPR transgene with antibodies against Mxc (Fig. 253



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2G). In these experiments, HLB assembly during the syncytial blastoderm cycles was 254

indistinguishable from that of histone deletion embryos rescued by the control 12xDWT transgene. 255

Thus, in the absence of the endogenous genes, an HLB assembles on the 12xPR array at the same 256

time in early development as it does on a wild type array. 257

258

CLAMP is present in the 12xPR HLB in the absence of the endogenous RD histone genes 259

The H3-H4 promoter is highly conserved among Drosophilids and contains conserved GAGA 260

repeats (Salzler et al., 2013; Rieder et al., 2017), which we previously showed are essential for 261

HLB formation and expression of RD histone genes in the presence of the endogenous histone 262

locus (Rieder et al., 2017). Although in vitro these repeats can bind both the zinc-finger GA-263

repeat binding protein CLAMP, and the major Drosophila GAGA repeat binding protein GAF 264

(GAGA factor; trithorax-like) (Gilmour et al., 1989) only CLAMP is bound to the histone locus 265

in wild-type animals (Rieder et al., 2017). The H2a-H2b promoter and the rest of the histone 266

repeat unit do not contain any GAGA repeats longer than 4 nts, and CLAMP preferentially binds 267

to longer GAGA repeats (Kuzu et al., 2016). We asked whether CLAMP is recruited to the 12xPR 268

transgene in salivary gland polytene chromosomes. As with all other HLB components we 269

tested, CLAMP is not recruited to the 12xPR transgene or a 12x histone gene array in which the 270

GAGA sequences are replaced with lacO binding sites (GAM, Fig. 3A) when the endogenous RD 271

histone genes are present (Rieder et al., 2017). Surprisingly, we found that in the absence of 272

endogenous RD histone genes, CLAMP (Fig. 3C), but not GAF (Fig. 3E), is recruited to the 273

12xPR transgenic locus with similar intensity to CLAMP recruitment to the 12xDWT transgenic 274

locus (Fig. 3B, D). Furthermore, in this genotype the 12x GAM array supports H2a gene 275

expression in the absence of the endogenous genes (Fig. 3F). These data indicate that CLAMP 276



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can be recruited to a histone gene array lacking GAGA repeats when the preferred GAGA 277

binding sites within the H3-H4 promoter at the endogenous RD histone locus are absent. The H3 278

and H4 genes are transcribed, indicating that CLAMP is not an essential DNA-binding 279

transcription factor for the H3 and H4 genes, but likely serves as a factor that alters chromatin 280

structure (Rieder et al, 2017). 281

We carried out ChIP-qPCR experiments on embryos containing only the 12xDWT array or 282

the 12xPR array to determine whether CLAMP is interacting with either the H2a-H2b or H3-H4 283

genes. In agreement with ChIP-seq results on the endogenous genes, which showed that CLAMP 284

is localized precisely to the H3-H4 promoter (Rieder et al., 2017), CLAMP was bound to the H3-285

H4 promoter but not to the H2a-H2b genes or promoter on the 12xDWT array (Fig. 3G). In 286

contrast, CLAMP was not bound to either gene pair on the 12xPR array (Fig. 3G), despite our 287

observation that CLAMP is present in the HLB at the 12xPR array (Fig. 3C). These data suggest 288

that CLAMP can be recruited to RD histone genes by protein-protein interactions independently 289

of its binding to DNA. 290

291

A wild type array can activate the 12xPR array when present in trans at the homologous locus 292

The results above demonstrate that the 12xPR transgenic locus does not form an HLB in 293

the presence of the endogenous histone genes unlike the wild type 12xDWT transgene, which 294

does. We tested whether juxtaposing a wild type histone gene array near the 12xPR locus would 295

nucleate a functional HLB and activate expression from the 12xPR transgene. Our BAC based 296

transgenes are inserted into the genome via site-specific recombination at the same chromosomal 297

location. We created flies in which the 12xPR was placed at position VK33 on chromosome 3 in 298

trans to the 12xHWT transgene used in our initial studies (McKay et al., 2015), and analyzed these 299



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transgenes in the presence of the endogenous RD histone genes on chromosome 2. We examined 300

HLB formation at the ectopic VK33 location by staining whole mount salivary glands with 301

antibodies against Mxc and FLASH. Using this approach, we detect only a single, large HLB on 302

the endogenous RD histone genes in wild type nuclei (Fig. 4A-1, 4B). When 12xPR was the only 303

transgene present in addition to the endogenous RD histone genes, none of the nuclei contained 304

an ectopic HLB and we only detected the endogenous HLB (Fig. 4A-2, 4B), consistent with the 305

results of staining spread salivary gland polytene chromosomes (Fig. 1D). In contrast, we 306

observed formation of a second small HLB in addition to the single large endogenous HLB in 307

100% of the 12xHWT/12xPR nuclei (Fig. 4A-5, 4B), as expected since this HLB also forms when 308

only the 12xHWT array is present at this site. 309

We measured histone gene expression from the 12xPR transgene in these genotypes. In 310

the 12xHWT array the histone H2a gene, and not the other histone genes, is marked with a 311

restriction site change (McKay et al., 2015), whereas the 12xPR array has all five histone genes 312

marked with a restriction site change. Therefore, to specifically detect histone gene expression 313

from the 12xPR transgene, we digested embryonic H3 RT-PCR products with SacI, whose 314

recognition site is missing from the 12xPR H3 gene but is present in both the endogenous and 315

12xHWT H3 genes (Fig. 4C). Strikingly, the 12xPR transgene supports stronger H3 gene 316

expression in the presence compared to the absence of the endogenous histone genes when 317

present in trans with the 12xHWT transgene (Fig. 4C, lanes 1 and 2). These data demonstrate that 318

the wild type histone sequences in the 12xHWT were able to activate the 12xPR transgenic locus in 319

the presence of the endogenous histone genes, likely by nucleating ectopic HLB formation that 320

encompasses the paired homologous chromosomes. This result is similar to transvection, a 321



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phenomenon in which transcription of a gene can be activated by an enhancer located in trans 322

through pairing of homologous chromosomes (Duncan, 2002; Fukaya and Levine, 2017). 323

324

Transgenes with a single copy of the wild type repeat partially activate a 12x array in trans 325

Since 12 copies of a wild type histone repeat at the same integration site stimulated 326

expression from the 12xPR transgene, we tested whether a single copy could do the same using 327

two different approaches; one where the single copy is present in trans and the other where it is 328

present in cis. For the first approach, we inserted a 1x HWT transgene at the VK33 integration 329

site. In these strains we detected the HLB in 90% of the salivary gland nuclei in the presence of 330

the endogenous genes (Fig. 4B), similar to what we observed previously (Salzler et al., 2013). 331

When this 1x HWT transgene was placed in trans with the 12xPR array, we detected an ectopic 332

HLB in 94% of the nuclei examined (Fig. 4A-6, 4B). We tested whether there was any activation 333

of expression of the 12xPR array using our RT-PCR assay. There was a modest activation of the 334

expression 12xPR array, but much weaker than when the 12xHWT was in trans with the 12xPR as 335

measured by the ratio of the bands (Fig. 4D, lanes 1 and 3). 336

We next tested if including a single wild type repeat in the 12xPR array could stimulate 337

HLB formation and histone mRNA expression from that array in the presence of the endogenous 338

genes. We created a 12x array (12xPR-1) containing one wild type histone repeat unit in the center 339

of 11 PR repeat units (Fig. 4E). Like 12xPR, the 12xPR-1 transgene rescued a deletion of the 340

endogenous histone genes, resulting in viable and fertile adults and indicating that it is likely 341

fully active when present as the only source of RD histone genes. We then examined HLB 342

formation in intact salivary glands from animals containing both the 12xPR-1 transgene and the 343

endogenous RD histone genes. In this genotype, we detected HLB formation at the ectopic 344



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12xPR-1 transgenic locus in 59% of the nuclei (Fig. 4A-4, 4B), compared to 0% of nuclei from the 345

12xPR. We also analyzed expression from this array in the presence of the endogenous RD 346

histone genes and observed only modest activation of histone mRNA expression (Fig. 4D, lane 347

2) and less than the level of expression when the 1X WT was in trans to the 12X array (Fig. 4D, 348

lane 3). Thus, a single histone repeat unit can stimulate HLB formation, but activates expression 349

of neighboring genes better when placed in trans (i.e. on the other homolog) rather than in cis 350

(i.e. in the same 12x array). 351

We next tested a transgene containing only the H3-H4 promoter with no coding or 3’ end 352

sequences, which we previously demonstrated could form an HLB in salivary glands when 353

inserted into a ectopic site (Salzler et al., 2013). Ectopic HLBs were detected in 70% of the 354

salivary glands when only the H3-H4p promoter transgene was present and 69% when it was 355

placed in trans the 12xPR array (Fig. 4A-3, 4A-7, 4B). We assayed the expression of the H3 gene 356

from the 12xPR when the H3-H4p transgene was in trans to the 12xPR array. We detected low 357

level expression, similar to the expression detected when the 1xWT transgene was present 358

opposite the 12xPR array (Fig. 4D, lane 4). Collectively, these data demonstrate that the presence 359

of an HLB nucleating sequence in trans to 12xPR can induce formation of an ectopic HLB and 360

histone gene expression in the presence of the endogenous histone genes. Further, these data 361

emphasize that the H3-H4 promoter is a critical element in promoting HLB formation, consistent 362

with our previous observations (Salzler et al., 2013; Rieder et al., 2017). 363

364

Discussion 365

In this study, we used our histone gene replacement platform to analyze the cis acting 366

elements within the Drosophila histone repeat unit that are necessary for HLB formation and 367



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histone gene expression. Previously we showed using a single, transgenic histone gene repeat 368

unit that the promoter region of the divergently transcribed H3-H4 gene pair is capable of 369

stimulating HLB formation (Salzler et al., 2013). We subsequently further mapped this 370

functionality using a 12x gene array to conserved GAGA repeats in this region that are targeted 371

by the CLAMP protein (Rieder et al., 2017). Here, we present evidence that although a 12xPR 372

histone gene array devoid of the H3-H4 promoter and lacking CLAMP binding elements cannot 373

assemble an HLB in the presence of the ~100 RD histone gene copies at the endogenous locus 374

(HisC), it surprisingly can rescue homozygous deletion of HisC and fully support the entire 375

Drosophila life cycle. In the HisC deletion background, the 12xPR array assembles an HLB and 376

expresses the same amount of properly processed histone mRNAs as the endogenous genes or as 377

a 12xDWT wild type array. Below we discuss the implications of these observations on HLB 378

assembly and organization. 379

380

The RD histone locus stimulates HLB formation in Drosophila 381

Biomolecular condensates form via a seeding event that promotes a high concentration of 382

factors at a discrete location, leading to recruitment of additional factors that ultimately results in 383

a structure that can be observed by light microscopy (Gomes and Shorter, 2019). A number of 384

putative seeding events for biomolecular condensates have been described (Dellaire et al., 2006; 385

Dundr, 2011; Mao et al., 2011; Shevtsov and Dundr, 2011; White et al., 2011), but in many 386

cases the precise mechanism of seeding is not known. Nucleic acids, particularly RNA, have 387

been proposed to seed different nuclear bodies. Both the nucleolus and the HLB are associated 388

with specific genomic loci (Mao et al., 2011; Shevtsov and Dundr, 2011; Salzler et al., 2013), 389

and it is likely that the DNA (or chromatin) and/or nascent RNA at the locus participates in the 390



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seeding event. The activation of zygotic transcription of rRNA leads to the precise 391

spatiotemporal formation of the nucleolus in Drosophila embryos (Falahati et al., 2016). In the 392

absence of rDNA, Drosophila nucleolar components still form high concentration assemblies, 393

but these are smaller, more numerous, and do not form at the same time in the early embryo as 394

the wild type nucleolus. 395

Drosophila HLB components also stochastically assemble smaller and more unstable foci 396

in embryos lacking the RD histone locus (White et al., 2007; Salzler et al., 2013; Hur, 2019), 397

suggesting that HLBs and the nucleolus form similar seeding events. Indeed, the dynamics of 398

HLB assembly in single early Drosophila embryos display properties consistent with liquid-399

liquid phase transition seeded by HisC (Hur, 2019). Blocking transcription in the early embryo 400

prevents normal HLB growth (Hur, 2019), and a defective H3-H4 promoter (with mutated 401

TATA boxes) does not support HLB formation in the context of a single copy histone gene 402

repeat in salivary glands (Salzler et al., 2013). These data suggest that active transcription is 403

essential for forming a complete HLB. 404

It is important to note that HLBs assemble and persist in non-proliferating Drosophila 405

tissues that do not express histone mRNAs (Liu et al., 2006; White et al., 2007), and are also 406

present in G0/G1 mammalian cells (Ma et al., 2000; Zhao et al., 2000). Histone gene expression 407

is activated as a result of phosphorylation of Mxc/NPAT by Cyclin E/Cdk2, resulting in changes 408

in the HLB that promote histone gene transcription and pre-mRNA processing (Wei et al., 2003; 409

Ye et al., 2003; White et al., 2011). We propose that in early embryonic development the histone 410

locus DNA and/or chromatin seeds HLB assembly in Drosophila, with the H3-H4 promoter 411

region being particularly important. We further propose that subsequent transcriptional activation 412

of histone genes then drives HLB growth and maturation. 413



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Formation of an HLB on a transgenic RD histone gene array requires that this array 414

compete effectively with the endogenous HisC locus for recruitment of HLB components. This is 415

the situation with 12xHWT and 12XDWT arrays, which form HLBs in the presence of HisC. These 416

results also indicate that there are no other elements within HisC that are necessary for HLB 417

formation. Because the 12xPR array does not form an HLB in the presence of HisC, but it does so 418

in the absence of HisC, we hypothesize that the endogenous RD histone gene array sequesters 419

critical HLB components, likely including Mxc and CLAMP, thereby preventing HLB assembly 420

at the transgenic locus. By removing the H3-H4 promoter from the transgene, we removed an 421

element that provided additional interactions with HLB components, notably CLAMP, 422

weakening the overall ability of the locus to stably nucleate an HLB. 423

424

HLB assembly involves multiple interactions among components 425

Interactions among multivalent proteins, or multivalent protein-nucleic acid interactions, 426

are driving forces in the assembly of biomolecular condensates (Shin and Brangwynne, 2017). 427

Mxc is likely the critical factor that together with histone genes seeds Drosophila HLB formation 428

and activates histone gene expression. Mxc is a large (~1800 aa) protein that oligomerizes in 429

vivo and likely provides a scaffold for multivalent interactions (Terzo et al., 2015). A C-terminal 430

truncation mutant of Mxc that fails to recruit histone pre-mRNA processing factors still forms an 431

HLB and activates histone gene expression at sufficient levels to complete development, 432

underscoring the multivalent nature of Mxc (White et al., 2011; Landais et al., 2014; Tatomer et 433

al., 2016b). 434

Surprisingly, the HLB that assembles on the 12xPR array in the absence of HisC contains 435

CLAMP, even though we have removed all of the known CLAMP binding sites from the histone 436



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repeat. Although CLAMP may bind another sequence in the 12xPR array, no other favorable 437

GAGA repeats are present, and we were unable to detect CLAMP bound to any of the histone 438

promoters by ChIP-qPCR experiments. More likely, CLAMP may interact with other HLB 439

components, possibly Mxc or the Mxc-FLASH complex, providing multivalent contacts between 440

CLAMP and other HLB components. Deleting the GAGA sequences from the H3-H4 promoter 441

did not affect transcription of the H3 or H4 genes in the absence of HisC, suggesting that 442

CLAMP’s major function is to promote HLB assembly and not to act as a DNA binding 443

transcription factor. Supporting this interpretation is the observation that another, more abundant 444

transcription factor that binds to GAGA repeats, GAF, is not found at the HLB unless CLAMP is 445

absent (Rieder et al., 2017), consistent with CLAMP’s critical interactions with both the GAGA 446

repeats and HLB factors in seeding the HLB. 447

Because the 12xPR array is capable of assembling a completely functional HLB in the 448

absence of HisC, the H3-H4 promoter is not absolutely essential for HLB formation. One 449

possibility is that there are multiple pathways for assembling functional HLBs. Previous work 450

suggests that not all seeding events are equivalent in their ability to assemble biomolecular 451

condensates. In artificial systems, changes in scaffold stoichiometry, which can stem from 452

changes in valence, alter the recruitment of components (Banani et al., 2016). Further, 453

mathematical modeling has revealed that scaffolds can nucleate distinct complexes when at 454

different concentrations, and that this can qualitatively alter the transcriptional output (Yang and 455

Hlavacek, 2011). Additionally, P-bodies can form in multiple ways through different protein-456

protein or protein-nucleic acid interactions, with different interactions predominating under 457

different conditions (Rao and Parker, 2017). Therefore, different nucleators of the HLB (i.e. the 458

H3-H4 promoter or other sequences in the locus) may result in similar but not identical 459



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outcomes. Collectively our results suggest that HLB formation results from the contribution of 460

many molecular interactions, and the loss of any single one may be overcome by other 461

multivalent interactions within the body. 462

463

Methods 464

Culture condition and fly strains 465

Original fly strains and crosses were used as in McKay et al. 2015. Stocks were maintained on 466

standard corn medium. Viability studies were performed as in (Penke et al., 2016). 467

Transgenic histone gene array construction 468

Construction of the 5kb histone repeat designed for this study was performed using the HiFi 469

DNA Assembly system from New England Biolabs. PCR amplification of fragments from 470

existing histone repeats, in addition to in vitro synthesized gblocks (IDT) containing the desired 471

nucleotide changes were employed for the building blocks of the reaction. Manufactures protocol 472

was followed with slight modification to the incubation time of the reaction. Engineered 5kb 473

histone repeats were then arrayed to 12 copies in pMultiBac as in (McKay et al., 2015). All 474

histone gene arrays were inserted into the VK33 attP site on chromosome 3L (65B2) 475

(Venken et al., 2006). 476

Northern Analysis 477

Northern analysis was performed using a 7 M 6% urea acrylamide gel to resolve histone 478

mRNAs. ~1ug of RNA from embryos or larvae was used with a radio-labeled probe to the 479

coding region of H3 as in (Lanzotti et al., 2002). 480

Histone Expression Analysis 481



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Total RNA was prepared in Trizol and cDNA synthesized with random hexamers using 482

Superscript II (Invitrogen), according to the manufacturer’s instructions. RT-RCR was 483

performed using gene-specific primers to H2a (McKay et al. 2015) and H3. Each reaction was 484

performed at least 3 times with similar results. PCR products were digested using XhoI (H2a) or 485

SacI (H3). Digested amplicons were run on an 8% acrylamide gel or a 1.5% agarose gel. 486

Immunofluorescence 487

We used primary antibodies at the following concentrations: rabbit anti-CLAMP (1:1000), 488

guinea pig anti-Mxc (1:2000), guinea pig anti-Mute (1:5000), rabbit anti-FLASH (1:2000), rabbit 489

anti-Lsm10 (1:1000), mouse anti-MPM-2 (1:100; Millipore), rabbit anti-GAF (1:1000), mouse 490

anti-LacI (1:1000; Millipore). We used Alexa fluor secondary antibodies (Thermo Fisher 491

Scientific) at a concentration of 1:1000. In situ probes were detected using 15 µg/mL 492

streptavidin-DyLight-488 (Vector Laboratories). Salivary gland dissections and squashes were 493

performed as in (Tatomer et al., 2016b). Images were acquired with using a Zeiss Lsm710 with 494

ZEN DUO software. Images were analyzed using ImageJ. Ectopic HLBs in embryos were 495

quantified as (Salzler et al., 2013). 496

Fluorescent in situ hybridization 497

FISH probes were made from a PCR product that spanned the entire wild-type histone repeat 498

(primers [AAAGGAGGTTGGTAGGCAGC] and [ACGCTAGCGCTTTATCTGCA]). 499

Biotinylated FISH probes were made with nick translation by incubating 1 µg of purified PCR 500

product for 2 h at 15°C in a total of 50 µL containing 1×DNAPolI buffer (Fisher Optizyme); 501

0.05mM each of dCTP, dATP, and dGTP; 0.05 mM biotin-11-dUTP (Thermo Scientific); 10 502

mM 2-mercaptoethanol; 0.004 U of DNaseI (Fisher Optizyme); and 10 U of DNAPol I (Fisher 503

Optizyme). The reaction was purified on a PCR purification column (Thermo Scientific) and 504



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diluted in hybridization buffer (2xSSC, 10%dextran sulfate, 50%formamide, 0.8 mg/mL salmon 505

sperm DNA) to a final volume of 220 µL. FISH probes were diluted in hybridization mixture and 506

added to slides containing salivary gland polytene chromosome spreads before heating. We 507

added a coverslip, sealed it with rubber cement, and heated the slide for 2 min on a 91°C heat 508

block. Slides were placed in a humid box and incubated at 37°C overnight. Immunostaining was 509

then performed by incubating the slides in primary antibody overnight at 4°C in a humid box. 510

Chromatin Immunoprecipitation 511

ChIP was performed according to (Blythe and Wieschaus, 2015) with the following particulars. 512

2-4 hours old embryos (~200 embryos for each of three biological replicates) from HisC/HisC; 513

12xDWT or 12xPR strains were collected, fixed and stored at −80°C until further processing. 514

Immunoprecipitation of chromatin preparations was performed using rabbit anti-CLAMP 515

antibody (Rieder et al., 2017) and a polyclonal rabbit IgG (Millipore-Sigma, 12-370). qPCR was 516

performed as described in (Urban et al., 2017). Three biological replicates and two technical 517

replicates were used for each genotype. We normalized CLAMP IP target abundance against IgG 518

IP target abundance. We analyzed data using a Student's t-test, comparing target abundance 519

between the DWT and PR lines. 520

521

Acknowledgements 522

We thank Erica Larschan’s lab at Brown University for assistance with ChIP experiments. This 523

work was supported by NIH grant R01GM058921 to R.J.D and W.F.M and F32GM109663, 524

K99HD092625, and R00HD092625 to L.E.R. 525

526

References 527



https://doi.org/10.1101/2020.03.16.994483


24

Alberti, S., Gladfelter, A., and Mittag, T. (2019). Considerations and Challenges in Studying 528 Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 176, 419-434. 529

Altmeyer, M., Neelsen, K.J., Teloni, F., Pozdnyakova, I., Pellegrino, S., Grofte, M., Rask, M.B., 530 Streicher, W., Jungmichel, S., Nielsen, M.L., and Lukas, J. (2015). Liquid demixing of 531 intrinsically disordered proteins is seeded by poly(ADP-ribose). Nat Commun 6, 8088. 532

Banani, S.F., Lee, H.O., Hyman, A.A., and Rosen, M.K. (2017). Biomolecular condensates: 533 organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18, 285-298. 534

Banani, S.F., Rice, A.M., Peeples, W.B., Lin, Y., Jain, S., Parker, R., and Rosen, M.K. (2016). 535 Compositional Control of Phase-Separated Cellular Bodies. Cell 166, 1-13. 536

Blythe, S.A., and Wieschaus, E.F. (2015). Zygotic genome activation triggers the DNA 537 replication checkpoint at the midblastula transition. Cell 160, 1169-1181. 538

Bongartz, P., and Schloissnig, S. (2019). Deep repeat resolution-the assembly of the Drosophila 539 Histone Complex. Nucleic Acids Res 47, e18. 540

Bulchand, S., Menon, S.D., George, S.E., and Chia, W. (2010). Muscle wasted: a novel 541 component of the Drosophila histone locus body required for muscle integrity. J Cell Sci 123, 542 2697-2707. 543

Burch, B.D., Godfrey, A.C., Gasdaska, P.Y., Salzler, H.R., Duronio, R.J., Marzluff, W.F., and 544 Dominski, Z. (2011). Interaction between FLASH and Lsm11 is essential for histone pre-mRNA 545 processing in vivo in Drosophila. RNA 17, 1132-1147. 546

Dellaire, G., Eskiw, C.H., Dehghani, H., Ching, R.W., and Bazett-Jones, D.P. (2006). Mitotic 547 accumulations of PML protein contribute to the re-establishment of PML nuclear bodies in G1. J 548 Cell Sci 119, 1034-1042. 549

Duncan, I.W. (2002). Transvection effects in Drosophila. Annu Rev Genet 36, 521-556. 550

Dundr, M. (2011). Seed and grow: a two-step model for nuclear body biogenesis. J Cell Biol 551 193, 605-606. 552

Dundr, M., and Misteli, T. (2010). Biogenesis of nuclear bodies. Cold Spring Harb Perspect Biol 553 2, a000711. 554

Duronio, R.J., and Marzluff, W.F. (2017). Coordinating cell cycle-regulated histone gene 555 expression through assembly and function of the Histone Locus Body. RNA Biol 14, 726-738. 556

Falahati, H., Pelham-Webb, B., Blythe, S., and Wieschaus, E. (2016). Nucleation by rRNA 557 Dictates the Precision of Nucleolus Assembly. Curr Biol 26, 277-285. 558

Fukaya, T., and Levine, M. (2017). Transvection. Curr Biol 27, R1047-R1049. 559



https://doi.org/10.1101/2020.03.16.994483


25

Gilmour, D.S., Thomas, G.H., and Elgin, S.C. (1989). Drosophila nuclear proteins bind to 560 regions of alternating C and T residues in gene promoters. Science 245, 1487-1490. 561

Godfrey, A.C., White, A.E., Tatomer, D.C., Marzluff, W.F., and Duronio, R.J. (2009). The 562 Drosophila U7 snRNP proteins Lsm10 and Lsm11 are required for histone pre-mRNA 563 processing and play an essential role in development. RNA 15, 1661-1672. 564

Gomes, E., and Shorter, J. (2019). The molecular language of membraneless organelles. J Biol 565 Chem 294, 7115-7127. 566

Gunesdogan, U., Jackle, H., and Herzig, A. (2010). A genetic system to assess in vivo the 567 functions of histones and histone modifications in higher eukaryotes. EMBO Rep 11, 772-776. 568

Gunesdogan, U., Jackle, H., and Herzig, A. (2014). Histone supply regulates S phase timing and 569 cell cycle progression. Elife 3, e02443. 570

Heyn, P., Salmonowicz, H., Rodenfels, J., and Neugebauer, K.M. (2017). Activation of 571 transcription enforces the formation of distinct nuclear bodies in zebrafish embryos. RNA Biol 572 14, 752-760. 573

Hur, W., Tarzia, M., Deneke, V. E., Terzo, E. A., Duronio, R. J., Di Talia, S. (2019). CDK-574 regulated phase separation seeded by histone genes ensures precise growth and function of 575 Histone Locus Bodies. BioRXiv https://doi.org/10.1101/789933. 576

Kuzu, G., Kaye, E.G., Chery, J., Siggers, T., Yang, L., Dobson, J.R., Boor, S., Bliss, J., Liu, W., 577 Jogl, G., Rohs, R., Singh, N.D., Bulyk, M.L., Tolstorukov, M.Y., and Larschan, E. (2016). 578 Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the 579 X-Chromosome to Promote Drosophila Dosage Compensation. PLoS Genet 12, e1006120. 580

Landais, S., D'Alterio, C., and Jones, D.L. (2014). Persistent replicative stress alters polycomb 581 phenotypes and tissue homeostasis in Drosophila melanogaster. Cell Rep 7, 859-870. 582

Lanzotti, D.J., Kaygun, H., Yang, X., Duronio, R.J., and Marzluff, W.F. (2002). Developmental 583 control of histone mRNA and dSLBP synthesis during Drosophila embryogenesis and the role of 584 dSLBP in histone mRNA 3' end processing in vivo. Mol Cell Biol 22, 2267-2282. 585

Lifton, R.P., Goldberg, M.L., Karp, R.W., and Hogness, D.S. (1978). The organization of the 586 histone genes in Drosophila melanogaster: functional and evolutionary implications. Cold Spring 587 Harb Symp Quant Biol 42, 1047-1051. 588

Liu, J.L., Murphy, C., Buszczak, M., Clatterbuck, S., Goodman, R., and Gall, J.G. (2006). The 589 Drosophila melanogaster Cajal body. J Cell Biol 172, 875-884. 590

Ma, T., Van Tine, B.A., Wei, Y., Garrett, M.D., Nelson, D., Adams, P.D., Wang, J., Qin, J., 591 Chow, L.T., and Harper, J.W. (2000). Cell cycle-regulated phosphorylation of p220(NPAT) by 592 cyclin E/Cdk2 in cajal bodies promotes histone gene transcription. Genes Dev 14, 2298-2313. 593



https://doi.org/10.1101/2020.03.16.994483


26

Mao, Y.S., Sunwoo, H., Zhang, B., and Spector, D.L. (2011). Direct visualization of the co-594 transcriptional assembly of a nuclear body by noncoding RNAs. Nat Cell Biol 13, 95-101. 595

Marzluff, W.F., and Koreski, K.P. (2017). Birth and Death of Histone mRNAs. Trends Genet 33, 596 745-759. 597

Matera, A.G., Izaguire-Sierra, M., Praveen, K., and Rajendra, T.K. (2009). Nuclear bodies: 598 random aggregates of sticky proteins or crucibles of macromolecular assembly? Dev Cell 17, 599 639-647. 600

McKay, D.J., Klusza, S., Penke, T.J., Meers, M.P., Curry, K.P., McDaniel, S.L., Malek, P.Y., 601 Cooper, S.W., Tatomer, D.C., Lieb, J.D., Strahl, B.D., Duronio, R.J., and Matera, A.G. (2015). 602 Interrogating the function of metazoan histones using engineered gene clusters. Dev Cell 32, 603 373-386. 604

Mitrea, D.M., and Kriwacki, R.W. (2016). Phase separation in biology; functional organization 605 of a higher order. Cell Commun Signal 14, 1. 606

Penke, T.J., McKay, D.J., Strahl, B.D., Matera, A.G., and Duronio, R.J. (2016). Direct 607 interrogation of the role of H3K9 in metazoan heterochromatin function. Genes Dev 30, 1866-608 1880. 609

Rajendra, T.K., Praveen, K., and Matera, A.G. (2010). Genetic analysis of nuclear bodies: from 610 nondeterministic chaos to deterministic order. Cold Spring Harb Symp Quant Biol 75, 365-374. 611

Rao, B.S., and Parker, R. (2017). Numerous interactions act redundantly to assemble a tunable 612 size of P bodies in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 114, E9569-E9578. 613

Rieder, L.E., Koreski, K.P., Boltz, K.A., Kuzu, G., Urban, J.A., Bowman, S.K., Zeidman, A., 614 Jordan, W.T., 3rd, Tolstorukov, M.Y., Marzluff, W.F., Duronio, R.J., and Larschan, E.N. (2017). 615 Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP. 616 Genes Dev 31, 1494-1508. 617

Salzler, H.R., Tatomer, D.C., Malek, P.Y., McDaniel, S.L., Orlando, A.N., Marzluff, W.F., and 618 Duronio, R.J. (2013). A sequence in the Drosophila H3-H4 Promoter triggers histone locus body 619 assembly and biosynthesis of replication-coupled histone mRNAs. Dev Cell 24, 623-634. 620

Shevtsov, S.P., and Dundr, M. (2011). Nucleation of nuclear bodies by RNA. Nat Cell Biol 13, 621 167-173. 622

Shin, Y., and Brangwynne, C.P. (2017). Liquid phase condensation in cell physiology and 623 disease. Science 357, 1253. 624

Stanek, D., and Fox, A.H. (2017). Nuclear bodies: news insights into structure and function. Curr 625 Opin Cell Biol 46, 94-101. 626



https://doi.org/10.1101/2020.03.16.994483


27

Tatomer, D.C., Terzo, E., Curry, K.P., Salzler, H., Sabath, I., Zapotoczny, G., McKay, D.J., 627 Dominski, Z., Marzluff, W.F., and Duronio, R.J. (2016a). Concentrating pre-mRNA processing 628 factors in the histone locus body facilitates efficient histone mRNA biogenesis. J Cell Biol. 629

Tatomer, D.C., Terzo, E., Curry, K.P., Salzler, H., Sabath, I., Zapotoczny, G., McKay, D.J., 630 Dominski, Z., Marzluff, W.F., and Duronio, R.J. (2016b). Concentrating pre-mRNA processing 631 factors in the histone locus body facilitates efficient histone mRNA biogenesis. J Cell Biol 213, 632 557-570. 633

Terzo, E.A., Lyons, S.M., Poulton, J.S., Temple, B.R., Marzluff, W.F., and Duronio, R.J. (2015). 634 Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of 635 the Drosophila histone locus body. Mol Biol Cell 26, 1559-1574. 636

Urban, J.A., Doherty, C.A., Jordan, W.T., 3rd, Bliss, J.E., Feng, J., Soruco, M.M., Rieder, L.E., 637 Tsiarli, M.A., and Larschan, E.N. (2017). The essential Drosophila CLAMP protein 638 differentially regulates non-coding roX RNAs in male and females. Chromosome Res 25, 101-639 113. 640

Venken, K.J., He, Y., Hoskins, R.A., and Bellen, H.J. (2006). P[acman]: a BAC transgenic 641 platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314, 1747-642 1751. 643

Wagner, E.J., Burch, B.D., Godfrey, A.C., Salzler, H.R., Duronio, R.J., and Marzluff, W.F. 644 (2007). A genome-wide RNA interference screen reveals that variant histones are necessary for 645 replication-dependent histone pre-mRNA processing. Mol Cell 28, 692-699. 646

Wei, Y., Jin, J., and Harper, J.W. (2003). The Cyclin E/Cdk2 Substrate and Cajal Body 647 Component p220(NPAT) Activates Histone Transcription through a Novel LisH-Like Domain. 648 Mol Cell Biol 23, 3669-3680. 649

White, A.E., Burch, B.D., Yang, X.C., Gasdaska, P.Y., Dominski, Z., Marzluff, W.F., and 650 Duronio, R.J. (2011). Drosophila histone locus bodies form by hierarchical recruitment of 651 components. J Cell Biol 193, 677-694. 652

White, A.E., Leslie, M.E., Calvi, B.R., Marzluff, W.F., and Duronio, R.J. (2007). Developmental 653 and cell cycle regulation of the Drosophila histone locus body. Mol Biol Cell 18, 2491-2502. 654

Yang, J., and Hlavacek, W.S. (2011). Scaffold-mediated nucleation of protein signaling 655 complexes: elementary principles. Math Biosci 232, 164-173. 656

Yang, X.C., Burch, B.D., Yan, Y., Marzluff, W.F., and Dominski, Z. (2009). FLASH, a 657 proapoptotic protein involved in activation of caspase-8, is essential for 3' end processing of 658 histone pre-mRNAs. Mol Cell 36, 267-278. 659

Ye, X., Wei, Y., Nalepa, G., and Harper, J.W. (2003). The cyclin E/Cdk2 substrate p220(NPAT) 660 is required for S-phase entry, histone gene expression, and Cajal body maintenance in human 661 somatic cells. Mol Cell Biol 23, 8586-8600. 662



https://doi.org/10.1101/2020.03.16.994483


28

Zhao, J., Dynlacht, B., Imai, T., Hori, T., and Harlow, E. (1998). Expression of NPAT, a novel 663 substrate of cyclin E-CDK2, promotes S- phase entry. Genes Dev 12, 456-461. 664

Zhao, J., Kennedy, B.K., Lawrence, B.D., Barbie, D.A., Matera, A.G., Fletcher, J.A., and 665 Harlow, E. (2000). NPAT links cyclin E-Cdk2 to the regulation of replication-dependent histone 666 gene transcription. Genes Dev 14, 2283-2297. 667 668

669



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Figure Legends 670

671

Figure 1. The H3-H4 bidirectional promoter is required for ectopic HLB formation in the 672

presence of the endogenous RD histone genes. 673

A) Schematic of the endogenous Drosophila RD histone gene repeat, which is found in a tandem 674

array of ~100 copies at HisC, and the BAC-based DWT and PR synthetic histone repeats, which 675

arrayed to 12 copies and inserted at VK33 on chromosome 3. The colors indicate that each 676

transgenic histone gene was engineered to be distinguishable molecularly from the endogenous 677

histone genes (see Fig. S1). In the PR construct the H2a-H2b promoters (green) replaced the H3-678

H4 promoters (blue). B, C) Primers that anneal to H3 and H4 genes (A) were used to amplify the 679

promoter region from genomic DNA extracted from whole flies heterozygous (B) or embryos 680

homozygous (C) for a deletion of the HisC locus and containing either the 12xDWT or 12xPR 681

transgene. The amplicon from a wild type locus is larger (637nts) than the PR locus (565nts). D) 682

Polytene chromosome squashes from 3rd instar larval salivary glands containing both HisC and 683

either the 12xDWT or 12xPR transgenes hybridized with a probe to the histone locus and stained 684

for HLB components Lsm11 and Mxc. Note that absence of an HLB at 12xPR (arrows) when 685

HisC is present (right panels). Scale bar 10 microns. E) Assay for specific detection of 686

endogenous versus transgenic RD histone gene expression. Total RNA was prepared from 3rd 687

instar larvae containing the endogenous genes and one copy of the 12xDWT or 12xPR transgene. 688

RT-PCR with H2a primers followed by digestion with Xho I cleaves cDNA from the endogenous 689

genes but not the transgenes, which was visualized using an 8% polyacrylamide gel. 690

691

692



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30

Figure 2. The PR transgene is expressed and forms an HLB in the absence of the 693

endogenous RD histone genes. 694

A, B) RNA was isolated from 5-7 hr embryos (A) or 3rd instar larvae (B) containing the 12xDWT 695

or 12xPR transgene in the presence or absence of HisC, with wild-type embryos as a control (lane 696

1). RT-PCR products using H2a primers were digested with Xho I and visualized using a 1.5% 697

agarose gel (which does not resolve the two restriction fragments from the endogenous genes as 698

in Fig. 1E). C, D) Polytene chromosomes from HisC deletion homozygous 3rd instar larval 699

salivary glands rescued by either the 12xDWT or 12xPR transgene stained with antibodies against 700

HLB components Lsm11 and FLASH (C) or Mxc and Mute (D). E, F) Total RNA from 0-3h or 701

3-5h embryos (D) or 3rd instar larvae (E) from wild-type (yw) or HisC deletion homozygotes 702

containing either the 12xDWT or 12xPR transgene analyzed by Northern blotting using a 703

radiolabeled H3 coding region probe. * = normally processed H3 mRNA. A FLASH mutant that 704

cannot recruit the processing machinery to the HLB (Tatomer et al., 2016b) was used as a 705

positive control for production of poly(A)+ histone mRNA. G) Syncytial, embryos from HisC 706

deletion homozygous stocks rescued by 12xDWT or 12xPR transgenes and stained with Mxc to 707

monitor HLB formation in development. 3 consecutive syncytial nuclear cycles (top to bottom) 708

were analyzed. 709

710



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31

Figure 3. CLAMP but not GAF is recruited to HLBs that form in the absence of GA repeats 711 712 A) Schematic of the WT, PR, and GA mutant (GAM) synthetic histone repeats. For GAM, GA 713

sequences in the H3-H4 promoter were mutated to lacO sites or scrambled (Rieder et al., 2017). 714

B-E) Polytene chromosome squashes from 3rd instar larval salivary glands lacking endogenous 715

histone genes and rescued by a either a 12xDWT transgene (B,D) or a 12xPR transgene (C,E) were 716

stained with antibodies against FLASH and CLAMP (B,C) or FLASH and GAF (D,E). Scale bar 717

10 microns. F) RT-PCR analysis with H2a primers performed on cDNA from 5-7 hr embryos of 718

indicated genotypes and visualized with ethidium bromide on a 0.8% agarose gel. G) 2-4 hr old 719

HisC homozygous deletion embryos rescued by a 12xDWT or 12xPR transgene were analyzed by 720

ChIP-PCR using primers recognizing the indicated promoters. 721

722

Figure 4 Activation of PR transgenes by wild type histone gene arrays 723

A-1 to A-8) Intact salivary gland nuclei from 3rd instar larvae of the indicated genotypes, all of 724

which contained HisC and stained for the HLB components Mxc and FLASH. Ectopic HLBs are 725

indicated by arrows and dashed boxes, and were present in all genotypes except the 12xPR. Scale 726

bar 10 microns. B) Quantification of the percentage of ectopic HLBs in the salivary gland nuclei 727

in panel B. C) Schematic of the assay used to distinguish 12xPR from 12xHWT cDNA. RT-PCR 728

amplification of H3 mRNA from 5-7 hr old embryos of the indicated genotypes, followed by 729

digestion with SacI and visualized on an 8% polyacrylamide gel. D) RT-PCR amplification of 730

H2a mRNA from larvae of indicated genotypes, followed by digestion with XhoI and 731

visualization on an agarose gel. E) Top: Schematic of the BAC-based PR-1 histone array 732

containing 11 PR and 1 DWT repeat unit. Bottom: Analysis of BAC constructs containing WT 733

and PR repeats using PCR as in panel 1B and C. 734



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32

Supplemental Figure 1. Annotated maps of engineered histone gene repeat unit used to 735

make transgenic 12x replication dependent histone gene arrays. A) Designer Wild Type. B) 736

Promoter Replacement. Restriction enzyme sites that were either added or deleted are shown 737

below each line of sequence. The maps were made using Snapgene. 738



https://doi.org/10.1101/2020.03.16.994483


merge

FISH

Lsm11

DWT

MxcMxc

ectopicEndo ectopicEndo

PR

Genotype Gel.

DWT PR

H3-H4p - 637ntsH2a-H2bp- 565 nts

+/+ | HisCΔ / +

H3-H4p - 637ntsH2a-H2bp- 565 nts

+/+ | HisCΔ /HisCΔ

XhoI

CTCGAG

Endogenous H2aXhoI

CTGGAA

Transgenic H2a

Trans H2a: 391nts

Endo H2a: 200+191

Endogenous Transgene

+ +DWT

H2a PCR Product digested with Xho I

FISH

Lsm11

merge

+PR

D

A

B C

E

H1DWT

PR

H3 H4 H2a H2b

H1H3 H4 H2a H2b

H1WT H3 H4 H2a H2b

DWT PR

x 100

x 12

x 12

Mxc

Koreski, et al., Figure 1



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PR

Lsm11

FLASH

merge

FLASH

Lsm11

merge

DWT

A

B

E

C

F

DTransgene Endogenous

DWT DWT PR PR+ + +

1 2 3 4 5

Transgene Endogenous + + +

DWT PR DWT PR

1 2 3 4 5

Mxc Mxc/DNA

PR DWT

Trans H2a

Endo H2a

Trans H2a

Endo H2a

DWTpoly(A

) +

0-3hr 3-5hr

yw DWT PR yw PR FLASH mutant

H3

DWT*

yw DWT PR FLASH mutant

H3

poly(A) +

*

merge

Mute

Mxc

PR

G

merge

Mute

Mxc

DWT

Mxc Mxc/DNA

Koreski et al., Figure 2



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HWT

A

FLASH

CLAMP

mergeΔHisC; PR

FLASH

CLAMP

mergeΔHisC; DWT

ΔHisC; DWT merge

GAF

FLASHFLASH

GAF

merge

G

ΔHisC; PR

F

B C

D E

GAM

DWTPR PRGAM DWTGAM

+

Trans H2a

Endo H2a

ΔΔ ΔTransgene

Endogenous + +

++ ++ + + +

0

20

40

60

80

100

120

140

H3H4 H2AH2B H1Fold

Enr

ichm

ent o

f CLA

MP

IP o

ver I

gG IP

PR DWT

H1DWT H3 H4 H2a H2b

PR H1H3 H4 H2a H2b

H1H3 H4 H2a H2b

Koreski, et al., Figure 3



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DWT copies

PR copies

DWTPR

1

1

1

7

1 1

3 11

yw

PR-1

B

mergeMxcFLASH mergeMxcFLASH

212xPR

1yw

412XPR-1

61xHWT

12XPR

512xHWT

12XPR

3H3H4p

7H3H4p

12XPR

812xPR-1

12XPR

D

Transgene #1

Endogenous DWT

HWT

PR PR

+ +PR PR

HWT

Sac I

Endogenous andtransgenic HWT H3

Sac I

TransgenicDWT and PR H3

1 2 3 4 5

Transgene #2

Transgene #1

Endogenous PR PR-1

+ +

PR

H3H4PTransgene #2

+ +

1xHWT

PR E

1 2 3 4

A

C

PR5 DWT1 PR6

tg

endo

Koreski et al., Figure 4



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Page 1

BspDIClaI

SalI AccI

5

3

80

H1-H3 intergenic region

G T C G A C T A A T G C A T A T G T G G C G A G G A A A T C G A T T G A T T T C A G A A C A A A T T A T T T T A A A A T A T G C A T G A A A A C A C A T T A A T

C A G C T G A T T A C G T A T A C A C C G C T C C T T T A G C T A A C T A A A G T C T T G T T T A A T A A A A T T T T A T A C G T A C T T T T G T G T A A T T A

160


A A C A A G C A A A C A C A T T A A T A A T T T A A G A A A A T A T T A T T T A T T A T A T T A A T A T T A T G T T A T T T A A G A A A G T A T C T G T A T T T

T T G T T C G T T T G T G T A A T T A T T A A A T T C T T T T A T A A T A A A T A A T A T A A T T A T A A T A C A A T A A A T T C T T T C A T A G A C A T A A A

PvuI

240


T T A A C G A T C G A A A A T T A T T T C T G A A T G C T G C T T T A A A G C A A A T T T T T C T G T A G T T C A A T G T G A A C T T A A A T C A A G T A A T A

A A T T G C T A G C T T T T A A T A A A G A C T T A C G A C G A A A T T T C G T T T A A A A A G A C A T C A A G T T A C A C T T G A A T T T A G T T C A T T A T

PacI

320


A A G T A T C T T A A T T A A T A A T A G A C G C T T C T T T T A G A A G C C C T T C A A G G G G A T G A A C G T T T C A A T T T T A A T A A A C A T T A C G A

T T C A T A G A A T T A A T T A T T A T C T G C G A A G A A A A T C T T C G G G A A G T T C C C C T A C T T G C A A A G T T A A A A T T A T T T G T A A T G C T

400


A T T A G T G A A A T A T T T G C A A T G A T T C T T A T T T T A T T A G A T G T T T T T T T A T A A A T T G G T C C A G T T A A A A A T T T G A A T A T A A A

T A A T C A C T T T A T A A A C G T T A C T A A G A A T A A A A T A A T C T A C A A A A A A A T A T T T A A C C A G G T C A A T T T T T A A A C T T A T A T T T

480


A A T T C A A T C A A C A T T T G A A A G T C T C A A A A C C C A T A T T A C A T C C T T T T A A A A A T G G A A A G T G A C G A A A A A A T T A T T T A A A A

T T A A G T T A G T T G T A A A C T T T C A G A G T T T T G G G T A T A A T G T A G G A A A A T T T T T A C C T T T C A C T G C T T T T T T A A T A A A T T T T

560


G T G T A G A A C T A T T A A A A C C T T A T T T T T A T T A A T G A T T T A A A A T A C T A A A A A A T T A A A A A A A G T T T A C A C T T C A A G C A A A C

C A C A T C T T G A T A A T T T T G G A A T A A A A A T A A T T A C T A A A T T T T A T G A T T T T T T A A T T T T T T T C A A A T G T G A A G T T C G T T T G

BsaBIPshAI

640

H1-H3 intergenic region H1 core promoter

T T C G A C A T A G T A A A T G A C T G A T G T C A G T A G C A T T G T T A A A G T G C T C T C C T C C T C G A T T C T C A T C A G A G C A A A G G A G G T T G

A A G C T G T A T C A T T T A C T G A C T A C A G T C A T C G T A A C A A T T T C A C G A G A G G A G G A G C T A A G A G T A G T C T C G T T T C C T C C A A C

BssHII

720

H1 transcription startH1 5'UTR

H1 transl. start

H1 coding region

G T A G G C A G C G C G C G A G C C A T T T T T A A C A G A A A A A A A G T G T T C T C A G T G A A A A A A A G A T G T C T G A T T C T G C A G T T G C A A C G

C A T C C G T C G C G C G C T C G G T A A A A A T T G T C T T T T T T T C A C A A G A G T C A C T T T T T T T C T A C A G A C T A A G A C G T C A A C G T T G C

800

H1 coding region

T C C G C T T C C C C A G T G G C T G C C C C A C C A G C G A C A G T T G A G A A G A A A G T G G T C C A A A A A A A G G C A T C T G G A T C T G C T G G C A C

A G G C G A A G G G G T C A C C G A C G G G G T G G T C G C T G T C A A C T C T T C T T T C A C C A G G T T T T T T T C C G T A G A C C T A G A C G A C C G T G

A Koreski et al., Figure S1.CC-BY-NC-ND 4.0 International licenseavailable under a

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.994483doi: bioRxiv preprint

https://doi.org/10.1101/2020.03.16.994483


Page 2

AflII

880

H1 coding region

Add AflII

A A A G G C A A A G A A A G C C T C T G C G A C G C C G T C A C A T C C G C C A A C T C A G C A A A T G G T G G A C G C T T C C A T T A A A A A T C T T A A G G

T T T C C G T T T C T T T C G G A G A C G C T G C G G C A G T G T A G G C G G T T G A G T C G T T T A C C A C C T G C G A A G G T A A T T T T T A G A A T T C C

960

H1 coding region

A A C G T G G C G G T T C A T C A C T T C T G G C A A T C A A A A A A T A T A T C A C T G C C A C T T A T A A A T G C G A C G C C C A A A A G T T A G C G C C A

T T G C A C C G C C A A G T A G T G A A G A C C G T T A G T T T T T T A T A T A G T G A C G G T G A A T A T T T A C G C T G C G G G T T T T C A A T C G C G G T

1040

H1 coding region

H1 Tag- Remove ScaI

T T C A T C A A G A A G T A T T T A A A A T C G G C C G T G G T C A A T G G A A A G C T T A T T C A A A C T A A G G G A A A G G G T G C A T C T G G A T C T T T

A A G T A G T T C T T C A T A A A T T T T A G C C G G C A C C A G T T A C C T T T C G A A T A A G T T T G A T T C C C T T T C C C A C G T A G A C C T A G A A A

1120

H1 coding region

Removed BamHI

C A A A C T G T C G G C C T C T G C C A A G A A G G A A A A A G A T C C G A A G G C A A A G T C G A A G G T T T T G T C T G C T G A G A A A A A A G T T C A A A

G T T T G A C A G C C G G A G A C G G T T C T T C C T T T T T C T A G G C T T C C G T T T C A G C T T C C A A A A C A G A C G A C T C T T T T T T C A A G T T T

1200

H1 coding region

G C A A G A A G G T A G C C T C T A A G A A G A T T G G T G T C T C C T C C A A A A A A A C T G C C G T T G G G G C T G C T G A C A A A A A G C C C A A A G C T

C G T T C T T C C A T C G G A G A T T C T T C T A A C C A C A G A G G A G G T T T T T T T G A C G G C A A C C C C G A C G A C T G T T T T T C G G G T T T C G A

1280

H1 coding region

A A G A A G G C T G T G G C T A C C A A A A A G A C T G C C G A A A A T A A G A A A A C T G A G A A G G C A A A A G C C A A G G A T G C C A A G A A A A C T G G

T T C T T C C G A C A C C G A T G G T T T T T C T G A C G G C T T T T A T T C T T T T G A C T C T T C C G T T T T C G G T T C C T A C G G T T C T T T T G A C C

1360

H1 coding region

A A T C A T A A A G T C G A A G C C C G C C G C A A C A A A G G C G A A A G T G A C T G C A G C G A A G C C A A A G G C T G T A A T A G C G A A A G C G T C A A

T T A G T A T T T C A G C T T C G G G C G G C G T T G T T T C C G C T T T C A C T G A C G T C G C T T C G G T T T C C G A C A T T A T C G C T T T C G C A G T T

1440

H1 coding region

A G G C A A A G C C A G C G G T G T C T G C A A A A C C C A A A A A G A C G G T G A A G A A A G C A T C G G T T T C T G C T A C C G C C A A G A A G C C G A A A

T C C G T T T C G G T C G C C A C A G A C G T T T T G G G T T T T T C T G C C A C T T C T T T C G T A G C C A A A G A C G A T G G C G G T T C T T C G G C T T T

HpaI

1520

H1 coding region

H1 transl. stopAdded HpaI

H1 3'UTR [3']

H1 3'UTR [5']

G C G A A G A C T A C G G C T G C C A A A A A G T A A G T T A A C A T T G T G A A A A A G T G C A G T A T T T G G T A C A T G T T C G C A A T T A A A A T T T T

C G C T T C T G A T G C C G A C G G T T T T T C A T T C A A T T G T A A C A C T T T T T C A C G T C A T A A A C C A T G T A C A A G C G T T A A T T T T A A A A



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PmeI

1600

H1 3'UTR [3'] H1 HDE

Removed BglIIH1 stem loop

A G A T T T A T G T T T T A T A G A C C T G A A A T T T G T T T A A A C A A G T C C T T T T C A G G G C T A C A A C G T T C C G T T G C A A G A G A A A A A A A

T C T A A A T A C A A A A T A T C T G G A C T T T A A A C A A A T T T G T T C A G G A A A A G T C C C G A T G T T G C A A G G C A A C G T T C T C T T T T T T T

BspHIZraI AatII

1680

H1 HDEAdded AatII

C T T T T A T G A C G T C T T T C T T C C A C T T A T T T A T T A G C T G A C G T T C G C A G C A A C A A T A A A A C G T T T C A T G T C A T G A A T T A C A T

G A A A A T A C T G C A G A A A G A A G G T G A A T A A A T A A T C G A C T G C A A G C G T C G T T G T T A T T T T G C A A A G T A C A G T A C T T A A T G T A

1760

T G C A T G T T G G T C G C A T T C A G T T T T C G T T C C C G A T T T T T T T G T A T T T A T T T G A A C A T T A C C C A A T T A C C C A T A T T G C G G G T

A C G T A C A A C C A G C G T A A G T C A A A A G C A A G G G C T A A A A A A A C A T A A A T A A A C T T G T A A T G G G T T A A T G G G T A T A A C G C C C A

BstBI

1840

Add BstBIH2b HDE

H2b 3'UTR

A A A T A A G T T T T A T T T G T A A A T T C A T A T T C G A T G A T T G G T G G T T C G A A T T G A A A A A T G C A T T T C T T T G G T A T A A C A C A T T G

T T T A T T C A A A A T A A A C A T T T A A G T A T A A G C T A C T A A C C A C C A A G C T T A A C T T T T T A C G T A A A G A A A C C A T A T T G T G T A A C

BglII

1920

H2b 3'UTRAdd BglII

H2b transl. stop

H2b coding sequence

H2b stem loop

T G G C C C T G A A A A G G G C C G T T T T G G A T T A T T G T C C G C A T T C G C A G G A G A A A A A G A T C T T T A T T T A G A G C T G G T G T A C T T G G

A C C G G G A C T T T T C C C G G C A A A A C C T A A T A A C A G G C G T A A G C G T C C T C T T T T T C T A G A A A T A A A T C T C G A C C A C A T G A A C C

SphI

2000

H2b coding sequence

T G A C A G C C T T G G T T C C C T C A C T G A C A G C A T G C T T G G C C A A C T C T C C A G G C A A A A G C A G G C G A A C A G C C G T T T G G A T C T C C

A C T G T C G G A A C C A A G G G A G T G A C T G T C G T A C G A A C C G G T T G A G A G G T C C G T T T T C G T C C G C T T G T C G G C A A A C C T A G A G G

NruI

2080

H2b coding sequence

Added NruI

C G A C T G G T G A T G G T C G A G C G C T T G T T G T A G T G A G C T A G T C G C G A C G C T T C G G C A G C A A T T C G C T C G A A A A T A T C A T T T A C

G C T G A C C A C T A C C A G C T C G C G A A C A A C A T C A C T C G A T C A G C G C T G C G A A G C C G T C G T T A A G C G A G C T T T T A T A G T A A A T G

BpuEIBspMIBfuAI

2160

H2b coding sequence

A A A G C T G T T C A T T A T G C T C A T C G C C T T C G A C G A A A T T C C G G T G T C A G G A T G G A C C T G C T T G A G A A C C T T G T A A A T G T A G A

T T T C G A C A A G T A A T A C G A G T A G C G G A A G C T G C T T T A A G G C C A C A G T C C T A C C T G G A C G A A C T C T T G G A A C A T T T A C A T C T

2240

H2b coding sequence

T G G C A T A G C T C T C C T T C C T T T T G C G C T T C T T T T T C T T G T C G G T C T T G G T G A T G T T C T T C T G A G C C T T G C C A G C C T T C T T G

A C C G T A T C G A G A G G A A G G A A A A C G C G A A G A A A A A G A A C A G C C A G A A C C A C T A C A A G A A G A C T C G G A A C G G T C G G A A G A A C



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Page 4

PspOMISpeI

2320

H2b coding sequence

H2b transl. start

H2b 5' UTR

H2B transcription start site

G C T G C C T T T C C A C T A G T T T T C G G A G G C A T T G T T C A C G T T A C T T A T A T T T T C A C A A A C A C A A T T C A C T T A T T G T A A T G T G G

C G A C G G A A A G G T G A T C A A A A G C C T C C G T A A C A A G T G C A A T G A A T A T A A A A G T G T T T G T G T T A A G T G A A T A A C A T T A C A C C

FspIApaIBanII

2400

H2b TATAAA box

G C C C G A A C G C G T T C A C A T T T A T A C T T T T T T T C G A G C A G T C A A T T C A G G T C T A A G T C C C C C A C C C C T A A C T G A A T G C G C A G

C G G G C T T G C G C A A G T G T A A A T A T G A A A A A A A G C T C G T C A G T T A A G T C C A G A T T C A G G G G G T G G G G A T T G A C T T A C G C G T C

2480

H2A TATAAA box H2a transcription start siteH2a 5'UTR

G C A A A C G G A A A A G T A T A A A T A T T T C G C T G T C G G G G T T A G G C G A G C A T T C G T G T T C C G T G T G T A A A G T G A A C T A A G T G A A A

C G T T T G C C T T T T C A T A T T T A T A A A G C G A C A G C C C C A A T C C G C T C G T A A G C A C A A G G C A C A C A T T T C A C T T G A T T C A C T T T

XbaI*BmgBI

2560

H2a 5'UTR

H2a transl. start

H2A coding region

Add XbaI

T A A A C G C A A A G C A A A A T G T C T G G A C G T G G A A A A G G T G G C A A A G T G A A G G G A A A G G C A A A G T C T A G A T C A A A C C G T G C C G G

A T T T G C G T T T C G T T T T A C A G A C C T G C A C C T T T T C C A C C G T T T C A C T T C C C T T T C C G T T T C A G A T C T A G T T T G G C A C G G C C

2640

H2A coding region

Removed BspEI

T C T T C A A T T C C C T G T G G G C C G T A T T C A C C G T T T G C T T C G G A A G G G A A A C T A C G C A G A G C G T G T T G G T G C A G G C G C T C C A G

A G A A G T T A A G G G A C A C C C G G C A T A A G T G G C A A A C G A A G C C T T C C C T T T G A T G C G T C T C G C A C A A C C A C G T C C G C G A G G T C

BssS IBbvCI

2720

H2A coding region

T T T A C C T A G C T G C C G T A A T G G A A T A T C T G G C C G C T G A G G T T C T G G A A T T G G C T G G C A A T G C T G C T C G T G A C A A C A A G A A G

A A A T G G A T C G A C G G C A T T A C C T T A T A G A C C G G C G A C T C C A A G A C C T T A A C C G A C C G T T A C G A C G A G C A C T G T T G T T C T T C

2800

H2A coding region

A C T A G A A T T A T T C C G C G T C A T C T G C A A C T G G C C A T C C G C A A C G A C G A G G A G T T A A A C A A G C T G C T C T C C G G C G T C A C A A T

T G A T C T T A A T A A G G C G C A G T A G A C G T T G A C C G G T A G G C G T T G C T G C T C C T C A A T T T G T T C G A C G A G A G G C C G C A G T G T T A

StuIBsu36I Acc65I

KpnI

2880

H2A coding region

H2a transl. stop Add Acc65I to remove 3'UTRH2a 3'UTR

T G C A C A A G G T G G C G T G T T G C C T A A T A T A C A G G C T G T T C T G T T G C C C A A G A A G A C C G A G A A G A A G G C C T A A G G T A C C A C G T

A C G T G T T C C A C C G C A C A A C G G A T T A T A T G T C C G A C A A G A C A A C G G G T T C T T C T G G C T C T T C T T C C G G A T T C C A T G G T G C A

BlpI

2960

H2a 3'UTRH2a HDE

H2a stem loop

T T C A A A G G C T A A G C T A A A A A C C T A C A T G T A C A T A A A A T C G T C A A T C A A A C C G T C C T T T T C A G G A C G A C C A A A T T A T T A C C

A A G T T T C C G A T T C G A T T T T T G G A T G T A C A T G T A T T T T A G C A G T T A G T T T G G C A G G A A A A G T C C T G C T G G T T T A A T A A T G G



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BspEI

3040

H2a HDEAdd BspEI

A A A G A A T T G A A A A A T T T T T T T C C G G A A G C T T G G C A A T T T C T T G T A A T T A G T A A A T C A T A A A G A A T T A T T A A C G T A A A G A T

T T T C T T A A C T T T T T A A A A A A A G G C C T T C G A A C C G T T A A A G A A C A T T A A T C A T T T A G T A T T T C T T A A T A A T T G C A T T T C T A

3120

G G T A A T G T A G T A A G G G T T T T C T A C T A T A T G C G G T A T A A A C T A T A A T T T G C T T C T T T A A A C A A T C G C A C A C C A C G A T G T G A

C C A T T A C A T C A T T C C C A A A A G A T G A T A T A C G C C A T A T T T G A T A T T A A A C G A A G A A A T T T G T T A G C G T G T G G T G C T A C A C T

3200

T G C T G T A C A T G C G G T G T C T G A A A C C A T T T G T A C A G T C T G T A C A A A T C C A T G T T A G A A A T A C A C A T T C T A T T T G A A A G A G T

A C G A C A T G T A C G C C A C A G A C T T T G G T A A A C A T G T C A G A C A T G T T T A G G T A C A A T C T T T A T G T G T A A G A T A A A C T T T C T C A

AgeI

3280

AgeI Added H4 HDE

A C G A A C G A C A G A C A T T T A T T T T T A G T T T A A C A T A T T T T T T G G G A G T C C C G A C C A A T A A A A T T A A A T A C T T T A C C G G T T T G

T G C T T G C T G T C T G T A A A T A A A A A T C A A A T T G T A T A A A A A A C C C T C A G G G C T G G T T A T T T T A A T T T A T G A A A T G G C C A A A C

3360

H4 HDE H4 3'UTR

H4 stem loop

A A A A T C T T C C T C C T T T T A A A A A C T G A A T G G T G G T C C T G A A A A G G A C C G A T T G C T T A A T A G G G G T A C A C A G G A T G T A C A C T

T T T T A G A A G G A G G A A A A T T T T T G A C T T A C C A C C A G G A C T T T T C C T G G C T A A C G A A T T A T C C C C A T G T G T C C T A C A T G T G A

BclI*

3440

H4 3'UTR Add BclIH4 transl. stop [3'] [3']

H4 coding region

H4 Tag Removed NcoI [*]

T T T G A T C A T T A A C C G C C A A A T C C G T A G A G G G T G C G G C C T T G C C T C T T C A G A G C G T A C A C A A C A T C C A T G G C T G T A A C T G T

A A A C T A G T A A T T G G C G G T T T A G G C A T C T C C C A C G C C G G A A C G G A G A A G T C T C G C A T G T G T T G T A G G T A C C G A C A T T G A C A

3520

H4 coding region

C T T C C T C T T G G C G T G T T C C G T G T A G G T C A C G G C A T C A C G A A T T A C G T T C T C C A A G A A A A C C T T C A G A A C G C C A C G C G T T T

G A A G G A G A A C C G C A C A A G G C A C A T C C A G T G C C G T A G T G C T T A A T G C A A G A G G T T C T T T T G G A A G T C T T G C G G T G C G C A A A

NgoMIV NaeI

3600

H4 coding region

Added NaeI

C C T C G T A T A T G A G T C C A G A T A T G C G C T T C A C A C C G C C T C G A C G G G C C A A A C G G C G G A T A G C C G G C T T C G T G A T A C C T T G G

G G A G C A T A T A C T C A G G T C T A T A C G C G A A G T G T G G C G G A G C T G C C C G G T T T G C C G C C T A T C G G C C G A A G C A C T A T G G A A C C

3680

H4 coding regionH4 [*]

H4 transl. start [3']

A T G T T A T C A C G C A G C A C T T T G C G A T G A C G C T T G G C G C C A C C C T T T C C C A A G C C T T T G C C T C C T T T A C C A C G A C C A G T C A T

T A C A A T A G T G C G T C G T G A A A C G C T A C T G C G A A C C G C G G T G G G A A A G G G T T C G G A A A C G G A G G A A A T G G T G C T G G T C A G T A

3760

H4 5' UTRH4 transcription start site

T T T T C A C T G T T C T A T A C T A T T A T A C A C G C A C A G C A C G A A A G T C A C T A A A G A A C T A A T T T C A A C G T T T C T G T G T G C C C C T A

A A A A G T G A C A A G A T A T G A T A A T A T G T G C G T G T C G T G C T T T C A G T G A T T T C T T G A T T A A A G T T G C A A A G A C A C A C G G G G A T



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3840

H4 TATAAA box short GA repeat

T T T A T A G G T A A A A C G A C A A A A A C C C G A G A G A G T A C G A A C G A T A T G T T C G T T C G C T T T T C G C T C G T C A A A T G A A A T G G C C T

A A A T A T C C A T T T T G C T G T T T T T G G G C T C T C T C A T G C T T G C T A T A C A A G C A A G C G A A A A G C G A G C A G T T T A C T T T A C C G G A

3920

long GA repeatH3 TATAA box

C T G T T T C T C T C T C T C T C T C T C T C T C T C T C T T T C A C C C T C C A C G A T T G C T A T A T A A G T A G G T A G C A A A T G C T C T G A T C G T T

G A C A A A G A G A G A G A G A G A G A G A G A G A G A G A A A G T G G G A G G T G C T A A C G A T A T A T T C A T C C A T C G T T T A C G A G A C T A G C A A

XcmI

4000

H4 transcription start site

H3 5'UTR


H3 coding region

T A T T G T G T T T T C A A A C G T G A A G T A G T G A A C G T G A A C T T T A G T G A A A C C C A A A T C G G A G A T G G C T C G T A C C A A G C A A A C T G

A T A A C A C A A A A G T T T G C A C T T C A T C A C T T G C A C T T G A A A T C A C T T T G G G T T T A G C C T C T A C C G A G C A T G G T T C G T T T G A C

BsrBISacII

4080

H3 coding region

Added SacII

C T C G C A A A T C G A C T G G T G G A A A G G C G C C G C G G A A A C A A C T G G C T A C T A A G G C C G C T C G C A A G A G T G C T C C A G C C A C C G G A

G A G C G T T T A G C T G A C C A C C T T T C C G C G G C G C C T T T G T T G A C C G A T G A T T C C G G C G A G C G T T C T C A C G A G G T C G G T G G C C T

4160

H3 coding region

G G T G T G A A G A A G C C C C A C C G C T A T C G C C C T G G A A C C G T G G C C T T G C G T G A A A T T C G T C G C T A C C A A A A G A G C A C C G A G C T

C C A C A C T T C T T C G G G G T G G C G A T A G C G G G A C C T T G G C A C C G G A A C G C A C T T T A A G C A G C G A T G G T T T T C T C G T G G C T C G A

PflMI

4240

H3 coding region

H3 Tag Remove SacI

T C T A A T C C G C A A G C T G C C T T T C C A G C G T C T G G T G C G T G A A A T C G C T C A G G A C T T T A A G A C G G A C T T G C G A T T C C A G A G T T

A G A T T A G G C G T T C G A C G G A A A G G T C G C A G A C C A C G C A C T T T A G C G A G T C C T G A A A T T C T G C C T G A A C G C T A A G G T C T C A A

BsaISexAI*

4320

H3 coding region

H3 Tag Remove SacI Remove BstBI

C G G C G G T T A T G G C T C T G C A G G A A G C T A G C G A A G C C T A C C T G G T T G G T C T C T T T G A A G A T A C C A A C T T G T G T G C C A T T C A T

G C C G C C A A T A C C G A G A C G T C C T T C G A T C G C T T C G G A T G G A C C A A C C A G A G A A A C T T C T A T G G T T G A A C A C A C G G T A A G T A

BsiWI

4400

H3 coding region

H3 transl. stopBsiWI Added H3 3'UTR

G C C A A G C G T G T C A C C A T A A T G C C C A A A G A C A T C C A G T T A G C G C G A C G C A T T C G C G G C G A G C G T G C T T A A C G T A C G G C T G A

C G G T T C G C A C A G T G G T A T T A C G G G T T T C T G T A G G T C A A T C G C G C T G C G T A A G C G C C G C T C G C A C G A A T T G C A T G C C G A C T

4480

H3 3'UTR H3 HDE

H3 stem loop

C A C G G C A T T A A C T T G C A G A T A A A G C G C T A G C G T A C T C T A T A A T C G G T C C T T T T C A G G A C C A C A A A C C A G A T T C A A T G A G A

G T G C C G T A A T T G A A C G T C T A T T T C G C G A T C G C A T G A G A T A T T A G C C A G G A A A A G T C C T G G T G T T T G G T C T A A G T T A C T C T



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4560

H3 HDENcoI added


T A A A A T T T T C T G T T C C A T G G G C C G A C T A T T T A T A A C T T T A A A A A A A A A A T A A G A A C A A A A T T C A T A T T C T A T T A T T T A T G

A T T T T A A A A G A C A A G G T A C C C G G C T G A T A A A T A T T G A A A T T T T T T T T T T A T T C T T G T T T T A A G T A T A A G A T A A T A A A T A C

4640


G C G C A A A C G G T A C T G G G T C T T A A A T C A T A T G T A A A A A T A C T A A T T C T G C C A G A G A A G G A A T A A A A A T A A T C T T A T T T T A A

C G C G T T T G C C A T G A C C C A G A A T T T A G T A T A C A T T T T T A T G A T T A A G A C G G T C T C T T C C T T A T T T T T A T T A G A A T A A A A T T

4720


T T G T C A G C T C A A C A T T T A T T A A A T T A A A G A A G A G G T T A A T A C A A A A A T A T A T A T T T T T A T T T G T T C T T T G T G C G A A C A T C

A A C A G T C G A G T T G T A A A T A A T T T A A T T T C T T C T C C A A T T A T G T T T T T A T A T A T A A A A A T A A A C A A G A A A C A C G C T T G T A G

4800


C T T T A A A G C A G T G A A A G T G T C G T G C G G G G C A A G G G A C T C T G A A C C T T A A A C A T C T A A A A A A A A A A T C T G A A T T C T G T G T A

G A A A T T T C G T C A C T T T C A C A G C A C G C C C C G T T C C C T G A G A C T T G G A A T T T G T A G A T T T T T T T T T T A G A C T T A A G A C A C A T

4880


A G A C A G T T T G A A A T T A A T G A A A T T A C A T T G A T G A C G G C A A T A T T T A T A A A A T A A C A G A A A A T A A T A A A A T A A A A C T A G C T

T C T G T C A A A C T T T A A T T A C T T T A A T G T A A C T A C T G C C G T T A T A A A T A T T T T A T T G T C T T T T A T T A T T T T A T T T T G A T C G A

4960


Removed HpaI Removed BsiWI

A T T T T A T A T T T T T T C C A T G T G C T A A C T G A A G A A T G T G T T A T T A T T G A A G A G G T C G T A T G G G A C A A T T G A C A C T G T C C C T T

T A A A A T A T A A A A A A G G T A C A C G A T T G A C T T C T T A C A C A A T A A T A A C T T C T C C A G C A T A C C C T G T T A A C T G T G A C A G G G A A

5040


C A A A C G T C T G T A A A A A A T A A A A C C T A T G T A A A A T T C A G C A C G G A A A T T G G C T A A T T T T G T T G C G G A A T G T A A T A T A T A T T

G T T T G C A G A C A T T T T T T A T T T T G G A T A C A T T T T A A G T C G T G C C T T T A A C C G A T T A A A A C A A C G C C T T A C A T T A T A T A T A A

PaeR7IXhoI

5120


A C A T A A T A A A G G A T A A T A C A A A A A T T G T T T C T T T T T A T T T T T T A T T T G A T T T A T T T A T T T G A C T A C A T A G A C G G C T C G A G

T G T A T T A T T T C C T A T T A T G T T T T T A A C A A A G A A A A A T A A A A A A T A A A C T A A A T A A A T A A A C T G A T G T A T C T G C C G A G C T C

NotI

3

5

5140

A A G G G C G A A T T C G C G G C C G C

T T C C C G C T T A A G C G C C G G C G



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SalI AccI

5

3


G T C G A C T A A T G C A T A T G T G G C G A G G A A A T C G A T T G A T T T C A G A A C A A A T T A T T T T A A A A T A T G C A T G A A A A C A C A T T A A T

C A G C T G A T T A C G T A T A C A C C G C T C C T T T A G C T A A C T A A A G T C T T G T T T A A T A A A A T T T T A T A C G T A C T T T T G T G T A A T T A


A A C A A G C A A A C A C A T T A A T A A T T T A A G A A A A T A T T A T T T A T T A T A T T A A T A T T A T G T T A T T T A A G A A A G T A T C T G T A T T T

T T G T T C G T T T G T G T A A T T A T T A A A T T C T T T T A T A A T A A A T A A T A T A A T T A T A A T A C A A T A A A T T C T T T C A T A G A C A T A A A

PvuI


T T A A C G A T C G A A A A T T A T T T C T G A A T G C T G C T T T A A A G C A A A T T T T T C T G T A G T T C A A T G T G A A C T T A A A T C A A G T A A T A

A A T T G C T A G C T T T T A A T A A A G A C T T A C G A C G A A A T T T C G T T T A A A A A G A C A T C A A G T T A C A C T T G A A T T T A G T T C A T T A T

PacI


A A G T A T C T T A A T T A A T A A T A G A C G C T T C T T T T A G A A G C C C T T C A A G G G G A T G A A C G T T T C A A T T T T A A T A A A C A T T A C G A

T T C A T A G A A T T A A T T A T T A T C T G C G A A G A A A A T C T T C G G G A A G T T C C C C T A C T T G C A A A G T T A A A A T T A T T T G T A A T G C T


A T T A G T G A A A T A T T T G C A A T G A T T C T T A T T T T A T T A G A T G T T T T T T T A T A A A T T G G T C C A G T T A A A A A T T T G A A T A T A A A

T A A T C A C T T T A T A A A C G T T A C T A A G A A T A A A A T A A T C T A C A A A A A A A T A T T T A A C C A G G T C A A T T T T T A A A C T T A T A T T T


A A T T C A A T C A A C A T T T G A A A G T C T C A A A A C C C A T A T T A C A T C C T T T T A A A A A T G G A A A G T G A C G A A A A A A T T A T T T A A A A

T T A A G T T A G T T G T A A A C T T T C A G A G T T T T G G G T A T A A T G T A G G A A A A T T T T T A C C T T T C A C T G C T T T T T T A A T A A A T T T T


G T G T A G A A C T A T T A A A A C C T T A T T T T T A T T A A T G A T T T A A A A T A C T A A A A A A T T A A A A A A A G T T T A C A C T T C A A G C A A A C

C A C A T C T T G A T A A T T T T G G A A T A A A A A T A A T T A C T A A A T T T T A T G A T T T T T T A A T T T T T T T C A A A T G T G A A G T T C G T T T G

BsaBIPshAI

H1-H3 intergenic region H1 core promoter

T T C G A C A T A G T A A A T G A C T G A T G T C A G T A G C A T T G T T A A A G T G C T C T C C T C C T C G A T T C T C A T C A G A G C A A A G G A G G T T G

A A G C T G T A T C A T T T A C T G A C T A C A G T C A T C G T A A C A A T T T C A C G A G A G G A G G A G C T A A G A G T A G T C T C G T T T C C T C C A A C

BssHII

H1 transcription startH1 5'UTR

H1 transl. start

H1 coding region

G T A G G C A G C G C G C G A G C C A T T T T T A A C A G A A A A A A A G T G T T C T C A G T G A A A A A A A G A T G T C T G A T T C T G C A G T T G C A A C G

C A T C C G T C G C G C G C T C G G T A A A A A T T G T C T T T T T T T C A C A A G A G T C A C T T T T T T T C T A C A G A C T A A G A C G T C A A C G T T G C

H1 coding region

T C C G C T T C C C C A G T G G C T G C C C C A C C A G C G A C A G T T G A G A A G A A A G T G G T C C A A A A A A A G G C A T C T G G A T C T G C T G G C A C

A G G C G A A G G G G T C A C C G A C G G G G T G G T C G C T G T C A A C T C T T C T T T C A C C A G G T T T T T T T C C G T A G A C C T A G A C G A C C G T G

B Koreski et al., Figure S1

80

160

240

320

400

480

560

640

720

800



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AflII

H1 coding region

Add AflII

A A A G G C A A A G A A A G C C T C T G C G A C G C C G T C A C A T C C G C C A A C T C A G C A A A T G G T G G A C G C T T C C A T T A A A A A T C T T A A G G

T T T C C G T T T C T T T C G G A G A C G C T G C G G C A G T G T A G G C G G T T G A G T C G T T T A C C A C C T G C G A A G G T A A T T T T T A G A A T T C C

H1 coding region

A A C G T G G C G G T T C A T C A C T T C T G G C A A T C A A A A A A T A T A T C A C T G C C A C T T A T A A A T G C G A C G C C C A A A A G T T A G C G C C A

T T G C A C C G C C A A G T A G T G A A G A C C G T T A G T T T T T T A T A T A G T G A C G G T G A A T A T T T A C G C T G C G G G T T T T C A A T C G C G G T

H1 coding region

H1 Tag- Remove ScaI

T T C A T C A A G A A G T A T T T A A A A T C G G C C G T G G T C A A T G G A A A G C T T A T T C A A A C T A A G G G A A A G G G T G C A T C T G G A T C T T T

A A G T A G T T C T T C A T A A A T T T T A G C C G G C A C C A G T T A C C T T T C G A A T A A G T T T G A T T C C C T T T C C C A C G T A G A C C T A G A A A

H1 coding region

Removed BamHI

C A A A C T G T C G G C C T C T G C C A A G A A G G A A A A A G A T C C G A A G G C A A A G T C G A A G G T T T T G T C T G C T G A G A A A A A A G T T C A A A

G T T T G A C A G C C G G A G A C G G T T C T T C C T T T T T C T A G G C T T C C G T T T C A G C T T C C A A A A C A G A C G A C T C T T T T T T C A A G T T T

H1 coding region

G C A A G A A G G T A G C C T C T A A G A A G A T T G G T G T C T C C T C C A A A A A A A C T G C C G T T G G G G C T G C T G A C A A A A A G C C C A A A G C T

C G T T C T T C C A T C G G A G A T T C T T C T A A C C A C A G A G G A G G T T T T T T T G A C G G C A A C C C C G A C G A C T G T T T T T C G G G T T T C G A

H1 coding region

A A G A A G G C T G T G G C T A C C A A A A A G A C T G C C G A A A A T A A G A A A A C T G A G A A G G C A A A A G C C A A G G A T G C C A A G A A A A C T G G

T T C T T C C G A C A C C G A T G G T T T T T C T G A C G G C T T T T A T T C T T T T G A C T C T T C C G T T T T C G G T T C C T A C G G T T C T T T T G A C C

H1 coding region

A A T C A T A A A G T C G A A G C C C G C C G C A A C A A A G G C G A A A G T G A C T G C A G C G A A G C C A A A G G C T G T A A T A G C G A A A G C G T C A A

T T A G T A T T T C A G C T T C G G G C G G C G T T G T T T C C G C T T T C A C T G A C G T C G C T T C G G T T T C C G A C A T T A T C G C T T T C G C A G T T

H1 coding region

A G G C A A A G C C A G C G G T G T C T G C A A A A C C C A A A A A G A C G G T G A A G A A A G C A T C G G T T T C T G C T A C C G C C A A G A A G C C G A A A

T C C G T T T C G G T C G C C A C A G A C G T T T T G G G T T T T T C T G C C A C T T C T T T C G T A G C C A A A G A C G A T G G C G G T T C T T C G G C T T T

HpaI

H1 coding region

H1 transl. stopAdded HpaI

H1 3'UTR [3']

H1 3'UTR [5']

G C G A A G A C T A C G G C T G C C A A A A A G T A A G T T A A C A T T G T G A A A A A G T G C A G T A T T T G G T A C A T G T T C G C A A T T A A A A T T T T

C G C T T C T G A T G C C G A C G G T T T T T C A T T C A A T T G T A A C A C T T T T T C A C G T C A T A A A C C A T G T A C A A G C G T T A A T T T T A A A A

880

960

1040

1120

1200

1280

1360

1440

1520



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PmeI

1600

H1 3'UTR [3'] H1 HDE

Removed BglIIH1 stem loop

A G A T T T A T G T T T T A T A G A C C T G A A A T T T G T T T A A A C A A G T C C T T T T C A G G G C T A C A A C G T T C C G T T G C A A G A G A A A A A A A

T C T A A A T A C A A A A T A T C T G G A C T T T A A A C A A A T T T G T T C A G G A A A A G T C C C G A T G T T G C A A G G C A A C G T T C T C T T T T T T T

BspHIZraI AatII

1680

H1 HDEAdded AatII

C T T T T A T G A C G T C T T T C T T C C A C T T A T T T A T T A G C T G A C G T T C G C A G C A A C A A T A A A A C G T T T C A T G T C A T G A A T T A C A T

G A A A A T A C T G C A G A A A G A A G G T G A A T A A A T A A T C G A C T G C A A G C G T C G T T G T T A T T T T G C A A A G T A C A G T A C T T A A T G T A

1760

T G C A T G T T G G T C G C A T T C A G T T T T C G T T C C C G A T T T T T T T G T A T T T A T T T G A A C A T T A C C C A A T T A C C C A T A T T G C G G G T

A C G T A C A A C C A G C G T A A G T C A A A A G C A A G G G C T A A A A A A A C A T A A A T A A A C T T G T A A T G G G T T A A T G G G T A T A A C G C C C A

BstBI

1840

Add BstBIH2b HDE

H2b 3'UTR

A A A T A A G T T T T A T T T G T A A A T T C A T A T T C G A T G A T T G G T G G T T C G A A T T G A A A A A T G C A T T T C T T T G G T A T A A C A C A T T G

T T T A T T C A A A A T A A A C A T T T A A G T A T A A G C T A C T A A C C A C C A A G C T T A A C T T T T T A C G T A A A G A A A C C A T A T T G T G T A A C

BglII

1920

H2b 3'UTRAdd BglII

H2b transl. stop

H2b coding sequence

H2b stem loop

T G G C C C T G A A A A G G G C C G T T T T G G A T T A T T G T C C G C A T T C G C A G G A G A A A A A G A T C T T T A T T T A G A G C T G G T G T A C T T G G

A C C G G G A C T T T T C C C G G C A A A A C C T A A T A A C A G G C G T A A G C G T C C T C T T T T T C T A G A A A T A A A T C T C G A C C A C A T G A A C C

SphI

2000

H2b coding sequence

T G A C A G C C T T G G T T C C C T C A C T G A C A G C A T G C T T G G C C A A C T C T C C A G G C A A A A G C A G G C G A A C A G C C G T T T G G A T C T C C

A C T G T C G G A A C C A A G G G A G T G A C T G T C G T A C G A A C C G G T T G A G A G G T C C G T T T T C G T C C G C T T G T C G G C A A A C C T A G A G G

NruI

2080

H2b coding sequence

Added NruI

C G A C T G G T G A T G G T C G A G C G C T T G T T G T A G T G A G C T A G T C G C G A C G C T T C G G C A G C A A T T C G C T C G A A A A T A T C A T T T A C

G C T G A C C A C T A C C A G C T C G C G A A C A A C A T C A C T C G A T C A G C G C T G C G A A G C C G T C G T T A A G C G A G C T T T T A T A G T A A A T G

BpuEIBspMIBfuAI

2160

H2b coding sequence

A A A G C T G T T C A T T A T G C T C A T C G C C T T C G A C G A A A T T C C G G T G T C A G G A T G G A C C T G C T T G A G A A C C T T G T A A A T G T A G A

T T T C G A C A A G T A A T A C G A G T A G C G G A A G C T G C T T T A A G G C C A C A G T C C T A C C T G G A C G A A C T C T T G G A A C A T T T A C A T C T

2240

H2b coding sequence

T G G C A T A G C T C T C C T T C C T T T T G C G C T T C T T T T T C T T G T C G G T C T T G G T G A T G T T C T T C T G A G C C T T G C C A G C C T T C T T G

A C C G T A T C G A G A G G A A G G A A A A C G C G A A G A A A A A G A A C A G C C A G A A C C A C T A C A A G A A G A C T C G G A A C G G T C G G A A G A A C



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SpeI

2320

H2b coding sequence

H2b transl. start

H2b 5' UTR

H2B transcription start site

G C T G C C T T T C C A C T A G T T T T C G G A G G C A T T G T T C A C G T T A C T T A T A T T T T C A C A A A C A C A A T T C A C T T A T T G T A A T G T G G

C G A C G G A A A G G T G A T C A A A A G C C T C C G T A A C A A G T G C A A T G A A T A T A A A A G T G T T T G T G T T A A G T G A A T A A C A T T A C A C C

2400

H2b TATAAA box

G C C C G A A C G C G T T C A C A T T T A T A C T T T T T T T C G A G C A G T C A A T T C A G G T C T A A G T C C C C C A C C C C T A A C T G A A T G C G C A G

C G G G C T T G C G C A A G T G T A A A T A T G A A A A A A A G C T C G T C A G T T A A G T C C A G A T T C A G G G G G T G G G G A T T G A C T T A C G C G T C

2480

H2A TATAAA box H2a transcription start siteH2a 5'UTR

G C A A A C G G A A A A G T A T A A A T A T T T C G C T G T C G G G G T T A G G C G A G C A T T C G T G T T C C G T G T G T A A A G T G A A C T A A G T G A A A

C G T T T G C C T T T T C A T A T T T A T A A A G C G A C A G C C C C A A T C C G C T C G T A A G C A C A A G G C A C A C A T T T C A C T T G A T T C A C T T T

XbaI*BmgBI

2560

H2a 5'UTR

H2a transl. start

H2A coding region

Add XbaI

T A A A C G C A A A G C A A A A T G T C T G G A C G T G G A A A A G G T G G C A A A G T G A A G G G A A A G G C A A A G T C T A G A T C A A A C C G T G C C G G

A T T T G C G T T T C G T T T T A C A G A C C T G C A C C T T T T C C A C C G T T T C A C T T C C C T T T C C G T T T C A G A T C T A G T T T G G C A C G G C C

2640

H2A coding region

Removed BspEI

T C T T C A A T T C C C T G T G G G C C G T A T T C A C C G T T T G C T T C G G A A G G G A A A C T A C G C A G A G C G T G T T G G T G C A G G C G C T C C A G

A G A A G T T A A G G G A C A C C C G G C A T A A G T G G C A A A C G A A G C C T T C C C T T T G A T G C G T C T C G C A C A A C C A C G T C C G C G A G G T C

BssS IBbvCI

2720

H2A coding region

T T T A C C T A G C T G C C G T A A T G G A A T A T C T G G C C G C T G A G G T T C T G G A A T T G G C T G G C A A T G C T G C T C G T G A C A A C A A G A A G

A A A T G G A T C G A C G G C A T T A C C T T A T A G A C C G G C G A C T C C A A G A C C T T A A C C G A C C G T T A C G A C G A G C A C T G T T G T T C T T C

2800

H2A coding region

A C T A G A A T T A T T C C G C G T C A T C T G C A A C T G G C C A T C C G C A A C G A C G A G G A G T T A A A C A A G C T G C T C T C C G G C G T C A C A A T

T G A T C T T A A T A A G G C G C A G T A G A C G T T G A C C G G T A G G C G T T G C T G C T C C T C A A T T T G T T C G A C G A G A G G C C G C A G T G T T A

StuIBsu36I Acc65I

KpnI

2880

H2A coding region

H2a transl. stop Add Acc65I to remove 3'UTRH2a 3'UTR

T G C A C A A G G T G G C G T G T T G C C T A A T A T A C A G G C T G T T C T G T T G C C C A A G A A G A C C G A G A A G A A G G C C T A A G G T A C C A C G T

A C G T G T T C C A C C G C A C A A C G G A T T A T A T G T C C G A C A A G A C A A C G G G T T C T T C T G G C T C T T C T T C C G G A T T C C A T G G T G C A

BlpI

2960

H2a 3'UTRH2a HDE

H2a stem loop

T T C A A A G G C T A A G C T A A A A A C C T A C A T G T A C A T A A A A T C G T C A A T C A A A C C G T C C T T T T C A G G A C G A C C A A A T T A T T A C C

A A G T T T C C G A T T C G A T T T T T G G A T G T A C A T G T A T T T T A G C A G T T A G T T T G G C A G G A A A A G T C C T G C T G G T T T A A T A A T G G



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BspEI

3040

H2a HDEAdd BspEI

A A A G A A T T G A A A A A T T T T T T T C C G G A A G C T T G G C A A T T T C T T G T A A T T A G T A A A T C A T A A A G A A T T A T T A A C G T A A A G A T

T T T C T T A A C T T T T T A A A A A A A G G C C T T C G A A C C G T T A A A G A A C A T T A A T C A T T T A G T A T T T C T T A A T A A T T G C A T T T C T A

3120

G G T A A T G T A G T A A G G G T T T T C T A C T A T A T G C G G T A T A A A C T A T A A T T T G C T T C T T T A A A C A A T C G C A C A C C A C G A T G T G A

C C A T T A C A T C A T T C C C A A A A G A T G A T A T A C G C C A T A T T T G A T A T T A A A C G A A G A A A T T T G T T A G C G T G T G G T G C T A C A C T

3200

T G C T G T A C A T G C G G T G T C T G A A A C C A T T T G T A C A G T C T G T A C A A A T C C A T G T T A G A A A T A C A C A T T C T A T T T G A A A G A G T

A C G A C A T G T A C G C C A C A G A C T T T G G T A A A C A T G T C A G A C A T G T T T A G G T A C A A T C T T T A T G T G T A A G A T A A A C T T T C T C A

AgeI

3280

AgeI Added H4 HDE

A C G A A C G A C A G A C A T T T A T T T T T A G T T T A A C A T A T T T T T T G G G A G T C C C G A C C A A T A A A A T T A A A T A C T T T A C C G G T T T G

T G C T T G C T G T C T G T A A A T A A A A A T C A A A T T G T A T A A A A A A C C C T C A G G G C T G G T T A T T T T A A T T T A T G A A A T G G C C A A A C

3360

H4 HDE H4 3'UTR

H4 stem loop

A A A A T C T T C C T C C T T T T A A A A A C T G A A T G G T G G T C C T G A A A A G G A C C G A T T G C T T A A T A G G G G T A C A C A G G A T G T A C A C T

T T T T A G A A G G A G G A A A A T T T T T G A C T T A C C A C C A G G A C T T T T C C T G G C T A A C G A A T T A T C C C C A T G T G T C C T A C A T G T G A

BclI*

3440

H4 3'UTR Add BclIH4 transl. stop [3'] [3']

H4 coding region

T T T G A T C A T T A A C C G C C A A A T C C G T A G A G G G T G C G G C C T T G C C T C T T C A G A G C G T A C A C A A C A T C C A T G G C T G T A A C T G T

A A A C T A G T A A T T G G C G G T T T A G G C A T C T C C C A C G C C G G A A C G G A G A A G T C T C G C A T G T G T T G T A G G T A C C G A C A T T G A C A

3520

H4 coding region

C T T C C T C T T G G C G T G T T C C G T G T A G G T C A C G G C A T C A C G A A T T A C G T T C T C C A A G A A A A C C T T C A G A A C G C C A C G C G T T T

G A A G G A G A A C C G C A C A A G G C A C A T C C A G T G C C G T A G T G C T T A A T G C A A G A G G T T C T T T T G G A A G T C T T G C G G T G C G C A A A

NgoMIV NaeI

3600

H4 coding region

Added NaeI

C C T C G T A T A T G A G T C C A G A T A T G C G C T T C A C A C C G C C T C G A C G G G C C A A A C G G C G G A T A G C C G G C T T C G T G A T A C C T T G G

G G A G C A T A T A C T C A G G T C T A T A C G C G A A G T G T G G C G G A G C T G C C C G G T T T G C C G C C T A T C G G C C G A A G C A C T A T G G A A C C

3680

H4 coding regionH4 [*]


A T G T T A T C A C G C A G C A C T T T G C G A T G A C G C T T G G C G C C A C C C T T T C C C A A G C C T T T G C C T C C T T T A C C A C G A C C A G T C A T

T A C A A T A G T G C G T C G T G A A A C G C T A C T G C G A A C C G C G G T G G G A A A G G G T T C G G A A A C G G A G G A A A T G G T G C T G G T C A G T A

3760

H2b 5' UTRH2B transcription start site H2b TATAAA box

G T T C A C G T T A C T T A T A T T T T C A C A A A C A C A A T T C A C T T A T T G T A A T G T G G G C C C G A A C G C G T T C A C A T T T A T A C T T T T T T

C A A G T G C A A T G A A T A T A A A A G T G T T T G T G T T A A G T G A A T A A C A T T A C A C C C G G G C T T G C G C A A G T G T A A A T A T G A A A A A A



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3840

H2A TATAAA box

T C G A G C A G T C A A T T C A G G T C T A A G T C C C C C A C C C C T A A C T G A A T G C G C A G G C A A A C G G A A A A G T A T A A A T A T T T C G C T G T

A G C T C G T C A G T T A A G T C C A G A T T C A G G G G G T G G G G A T T G A C T T A C G C G T C C G T T T G C C T T T T C A T A T T T A T A A A G C G A C A

3920

H2a transcription start siteH2a 5'UTR


H3 coding region

C G G G G T T A G G C G A G C A T T C G T G T T C C G T G T G T A A A G T G A A C T A A G T G A A A T A A A C G C A A A G C A A A A T G G C T C G T A C C A A G

G C C C C A A T C C G C T C G T A A G C A C A A G G C A C A C A T T T C A C T T G A T T C A C T T T A T T T G C G T T T C G T T T T A C C G A G C A T G G T T C

BsrBISacII

4000

H3 coding region

Added SacII

C A A A C T G C T C G C A A A T C G A C T G G T G G A A A G G C G C C G C G G A A A C A A C T G G C T A C T A A G G C C G C T C G C A A G A G T G C T C C A G C

G T T T G A C G A G C G T T T A G C T G A C C A C C T T T C C G C G G C G C C T T T G T T G A C C G A T G A T T C C G G C G A G C G T T C T C A C G A G G T C G

4080

H3 coding region

C A C C G G A G G T G T G A A G A A G C C C C A C C G C T A T C G C C C T G G A A C C G T G G C C T T G C G T G A A A T T C G T C G C T A C C A A A A G A G C A

G T G G C C T C C A C A C T T C T T C G G G G T G G C G A T A G C G G G A C C T T G G C A C C G G A A C G C A C T T T A A G C A G C G A T G G T T T T C T C G T

PflMI

4160

H3 coding region

C C G A G C T T C T A A T C C G C A A G C T G C C T T T C C A G C G T C T G G T G C G T G A A A T C G C T C A G G A C T T T A A G A C G G A C T T G C G A T T C

G G C T C G A A G A T T A G G C G T T C G A C G G A A A G G T C G C A G A C C A C G C A C T T T A G C G A G T C C T G A A A T T C T G C C T G A A C G C T A A G

BsaISexAI*

4240

H3 coding region

H3 Tag Remove SacI Remove BstBI

C A G A G T T C G G C G G T T A T G G C T C T G C A G G A A G C T A G C G A A G C C T A C C T G G T T G G T C T C T T T G A A G A T A C C A A C T T G T G T G C

G T C T C A A G C C G C C A A T A C C G A G A C G T C C T T C G A T C G C T T C G G A T G G A C C A A C C A G A G A A A C T T C T A T G G T T G A A C A C A C G

BsiWI

4320

H3 coding region

H3 transl. stopBsiWI Added

C A T T C A T G C C A A G C G T G T C A C C A T A A T G C C C A A A G A C A T C C A G T T A G C G C G A C G C A T T C G C G G C G A G C G T G C T T A A C G T A

G T A A G T A C G G T T C G C A C A G T G G T A T T A C G G G T T T C T G T A G G T C A A T C G C G C T G C G T A A G C G C C G C T C G C A C G A A T T G C A T

4400

BsiWI AddedH3 3'UTR

H3 stem loop

C G G C T G A C A C G G C A T T A A C T T G C A G A T A A A G C G C T A G C G T A C T C T A T A A T C G G T C C T T T T C A G G A C C A C A A A C C A G A T T C

G C C G A C T G T G C C G T A A T T G A A C G T C T A T T T C G C G A T C G C A T G A G A T A T T A G C C A G G A A A A G T C C T G G T G T T T G G T C T A A G

4480

H3 HDENcoI added


A A T G A G A T A A A A T T T T C T G T T C C A T G G G C C G A C T A T T T A T A A C T T T A A A A A A A A A A T A A G A A C A A A A T T C A T A T T C T A T T

T T A C T C T A T T T T A A A A G A C A A G G T A C C C G G C T G A T A A A T A T T G A A A T T T T T T T T T T A T T C T T G T T T T A A G T A T A A G A T A A



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4560


A T T T A T G G C G C A A A C G G T A C T G G G T C T T A A A T C A T A T G T A A A A A T A C T A A T T C T G C C A G A G A A G G A A T A A A A A T A A T C T T

T A A A T A C C G C G T T T G C C A T G A C C C A G A A T T T A G T A T A C A T T T T T A T G A T T A A G A C G G T C T C T T C C T T A T T T T T A T T A G A A

4640


A T T T T A A T T G T C A G C T C A A C A T T T A T T A A A T T A A A G A A G A G G T T A A T A C A A A A A T A T A T A T T T T T A T T T G T T C T T T G T G C

T A A A A T T A A C A G T C G A G T T G T A A A T A A T T T A A T T T C T T C T C C A A T T A T G T T T T T A T A T A T A A A A A T A A A C A A G A A A C A C G

4720


G A A C A T C C T T T A A A G C A G T G A A A G T G T C G T G C G G G G C A A G G G A C T C T G A A C C T T A A A C A T C T A A A A A A A A A A T C T G A A T T

C T T G T A G G A A A T T T C G T C A C T T T C A C A G C A C G C C C C G T T C C C T G A G A C T T G G A A T T T G T A G A T T T T T T T T T T A G A C T T A A

4800


C T G T G T A A G A C A G T T T G A A A T T A A T G A A A T T A C A T T G A T G A C G G C A A T A T T T A T A A A A T A A C A G A A A A T A A T A A A A T A A A

G A C A C A T T C T G T C A A A C T T T A A T T A C T T T A A T G T A A C T A C T G C C G T T A T A A A T A T T T T A T T G T C T T T T A T T A T T T T A T T T

4880


Removed HpaI Removed BsiWI

A C T A G C T A T T T T A T A T T T T T T C C A T G T G C T A A C T G A A G A A T G T G T T A T T A T T G A A G A G G T C G T A T G G G A C A A T T G A C A C T

T G A T C G A T A A A A T A T A A A A A A G G T A C A C G A T T G A C T T C T T A C A C A A T A A T A A C T T C T C C A G C A T A C C C T G T T A A C T G T G A

4960


G T C C C T T C A A A C G T C T G T A A A A A A T A A A A C C T A T G T A A A A T T C A G C A C G G A A A T T G G C T A A T T T T G T T G C G G A A T G T A A T

C A G G G A A G T T T G C A G A C A T T T T T T A T T T T G G A T A C A T T T T A A G T C G T G C C T T T A A C C G A T T A A A A C A A C G C C T T A C A T T A

5040


A T A T A T T A C A T A A T A A A G G A T A A T A C A A A A A T T G T T T C T T T T T A T T T T T T A T T T G A T T T A T T T A T T T G A C T A C A T A G A C G

T A T A T A A T G T A T T A T T T C C T A T T A T G T T T T T A A C A A A G A A A A A T A A A A A A T A A A C T A A A T A A A T A A A C T G A T G T A T C T G C

NotIPaeR7IBsoBIXhoIAvaI

BmeT110I

3

5

5067


G C T C G A G A A G G G C G A A T T C G C G G C C G C

C G A G C T C T T C C C G C T T A A G C G C C G G C G



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