Developmental Cell, Volume 28

Supplemental Information

Polycomb Potentiates Meis2 Activation

in Midbrain by Mediating Interaction

of the Promoter with a Tissue-Specific Enhancer

Takashi Kondo, Kyoichi Isono, Kaori Kondo, Takaho A. Endo, Shigeyoshi Itohara, Miguel

Vidal, and Haruhiko Koseki

Inventory for Supplemental Information

There are 3 supplemental figures (Figure S1-3), 1 supplemental table (Table S1).

-Figure S1 related to Figure 1. (A) ChIP-seq data at promoter and RBS of Meis2 gene, (B)

section planes employed in Figure 1, (C) distribution of distances between promoter/RBS, (D)

statistical analysis on the FISH experiments in Figures 1D, 1E and 1F.

-Figure S2 related to Figure 2. (A) genomic map of tested fragments for transgenic analysis, (B)

summary results for transgenic study, (C) 3C results between promoter and enhancer candidates,

(D) ChIP-seq data by H3K4me1 and H3K27ac antibodies at the region surrounding Meis2 locus,

(E) statistical analysis on the FISH experiments in Figure 2C and 2D, and (F) Immuno-FISH for

PRT/MBE/RING1B.

-Figure S3 related to Figure 3. (A) summary for the tripartite interaction between

promomter/MBE/RBS in Figures 3B and 3C, (B) summary for association between RING1B

and each FISH probe at different stage embryos Figures 3D and 3E, and (C) summary of the

RING1B binding sites surrounding genes and several examples of gene loci bound by RING1B.

-Table S1 related to Figure 4. (A) summarizes promoter/MBE/RBS association in Figure 4B, and

(B) summarizes promoter/MBE/RBS association in Figure 4C.

-Supplemental Figure Legends

-Supplemental Experimental Procedures

-Supplemental References

Kondo T. Figure S1

section planes

LM

MB

LGEMGE

CM

A

C

1050Log10(P-value)

PRT/RBS

MB

LGE

MGE

CM

LM

average distance (μm)

0.290

0.344

0.143

0.126

0.137

SD

0.136

0.169

0.143

0.121

0.0956

n

297

209

195

246

226

wt

wt

MB LGE MGE CM LM

MB

LGE

MGE

CM

LM

average distance (μm)

0.293

0.313

0.287

0.253

0.274

SD

0.124

0.126

0.107

0.0998

0.136

n

264

209

223

214

234

R1

Ring1 mut

MB LGE MGE CM LM

D

0 10

250

Distance (µm)

Num

ber o

f pai

red

foci

Cluster 2(50%)

Cluster 1(44%)

Cluster 3(6%)

0.16µm

71.2% 28.8%68.7% 31.3% 72.8% 27.2%MGE LM CM

0 0.75 0 0.75 0 0.75 0 0.750

100

Distance (μm)

Num

bers

LGE12.0% 88.0%

MB14.8% 85.2%

0 0.75

FB

1 2 3 4 5

B

RBS promoter

115920000

Meis2

ES

chr2:115660000 115710000 115870000

FB

MB

LM

2810405F15Rik

4.20 x 10-19.76 x 10-151.19 x 10-147.59 x 10-146.55 x 10-145.87 x 10-1

0

500

P value

TissueExpression

FB+

MB+

LM-

ES-

Rea

ds /

M re

ads

TSS

ES

TSSRING1B

RING1B

RING1B

RING1B

H3K27me3

Human chromosome: chr15 37,110,000-37,710,000 (hg19)

a b c d e f g

37,618k 37,318k37,418k37,518k 37,218k 37,118k

Meis2 (37,183,222-37.392,341)

mouse

chicken

fugu

100 %

50 %

75 %

100 %

50 %

75 %

100 %

50 %

75 %

1 kb

EcoRI EcoRIEcoRIEcoRI EcoRIEcoRIEcoRIEcoRI

promoter sequence c sequence vsequence d

v

mouse corresponding sequences

1

0.8

0.6

0.4

0.2

p-c p-d p-vassociating region

rela

tive

valu

e fo

r 3C

forebrain

midbrain

limb bud

A

RP11-597G23 (37,381,873-37,589,340)RP11-991D13 (37,381,873-37,589,340)

sequence a sequence b

sequence c sequence d sequence e sequence f sequence g

WISH RP11-597G23 RP11-991D13

B

C

E

1050Log10(P-value)

MB

LGE

MGE

CM

LM

average distance (μm)

0.118

0.301

0.258

0.269

0.259

SD

0.0898

0.118

0.0962

0.0963

0.0965

n

307

243

236

219

212

wt

MB

LGE

MGE

CM

LM

average distance (μm)

0.272

0.255

0.257

0.246

0.255

SD

0.0933

0.118

0.102

0.0984

0.105

n

392

266

222

217

209

R1

wt Ring1 mutPRT/MBE

MB LGE MGE CM LMMB LGE MGE CM LM

D

F

chr2; 115,500,000 116,000,000

H3K4me1

H3K27ac

C15orf41 Meis2

mid

brai

n

MBE

MB

2 μm

LMCMMGELGE

PRT MBERING1B

H3K4me1

H3K27ac

fore

brai

n

Kondo T. Figure S2

22%

55%

20%2%

RING1B binding sites

UpstreamPromoter (-4kb,TSS)Promoter (TSS,+4kb)Gene bodyDownstream

Meis1

chr11:18760000 18939000

Foxd1

chr13:99104000 99147000

Onecut3

chr10:79937000 80001000

Tbr1

chr2:61622000 61673000

10kb

CpG island

GSM656523

Pax2

chr19:44811000 44931000

Kondo T. Figure S3

B

6.5 dpc

16-20s

22-26s

colocalization

stage

RING1BPRT

82.0 (128)

82.8 (99)

22.2 (135)

RING1BMBE

28.7 (108)

79.8 (99)

22.2 (135)

RING1BRBS

87.5 (128)

79.0 (119)

78.6 (117)

association

stage

6.5 dpc epi

16-20 somite

22-26 somite

P/M/R

25.4

61.5

24.8

P/R (w/o M)

55.6

12.8

6.0

P/M (w/o R)

5.9

19.8

56.0

no proximity

13.1

5.9

13.2

totalnumber

205

187

234

A

C

P/M

P/R

P/M

P/R

average distance (μm)

0.148

0.160

0.240

0.272

SD

0.0919

0.136

0.109

0.159

n

173

173

190

190

cont.

mut.

A

Baverage distance (μm)

0.118

0.290

0.272

0.293

0.146

0.268

SD

0.0898

0.136

0.0933

0.124

0.0898

0.133

n

307

297

392

264

219

219

cont

rol

P/M

P/R

P/M

P/R

P/M

P/R

8.5

dpc

9.5

dpc

Kondo T. Table S1

SUPPLEMENTAL FIGURE LEGENDS

Figure S1, related to Figure 1. Section planes of 11 dpc embryos used for FISH analyses

and statistical analysis for promoter/RBS FISH in wild-type and Ring1 mutant embryos

at 11.5 dpc.

(A) RING1B distribution around Meis2 RBS region (mm9/Chr. 2:115660-115710 kb) and

promoter region (mm9/Chr. 2:115870-115920 kb) in ES cells (ES: GEM656523), forebrain,

midbrain and limb mesenchyme (FB, MB, and LM: GSE48464) revealed by ChIP-seq

analysis. Genomic structure/location of Meis2 and 2810405F15Rik genes is depicted below

the ChIP-seq data. (Right panel) Sequences mapped around Meis2 TSS (from -4kb to +4kb,

shaded rectangle) were collected for each tissue (forebrain (FB), midbrain (MB), limb bud

(LB) and ES cell) and statistically evaluated. P-values indicated in above graphs were

obtained using Wilcoxon test for normalized mapped read counts with 100 bp bins in these

regions and colored in blue (not significant) or red (significant, P < 10-2). (B) Representative

section planes used for FISH analyses of forebrain (FB), midbrain (MB) and limb

mesenchyme (LM) in Figures 1, 2 and 4 are indicated on a Meis2 in situ hybridization image

of an 11.5 dpc embryo. Immuno-histological image of a representative section plane through

the forebrain is shown in the right panel. Positions of lateral ganglionic eminence (LGE),

medial ganglionic eminence (MGE) and cephalic mesoderm (CM) are indicated by open

boxes. Note that MEIS2 is expressed only in LGE. (C) Determination of a threshold

promoter/RBS distance to distinguish paired and unpaired foci. We classified the distribution

of paired foci into three clusters. Details are described in Supplemental Experimental

Procedures. In the lower panels, the composition of cluster 1 and that of cluster 2 plus 3 in

midbrain (MB), LGE, MGE, limb mesenchyme (LM) and cephalic mesoderm (CM) are

shown. (D) Chi-square test of the distribution of promoter/RBS distances. In the top tables,

average distances, standard deviations (SD) and numbers of pairs tested in midbrain (MB),

LGE, MGE, limb mesenchyme (LM) and cephalic mesoderm (CM) in wild type (left) and

Ring1 mutant (right) are summarized. At the bottom, the results of chi-square test among

tissues are summarized by matrices. An example matrix is shown at the left. The chi-square

test results between sample 1 and 4 are indicated by a grey box in this matrix (pointed by

arrows). In the wild type, the average promoter/RBS distances in midbrain and LGE are

significantly different from those of MGE, limb mesenchyme and cephalic mesoderm, but are

not different from each other in the Ring1 mutants.

Figure S2, related to Figure 2. Transgenic analyses to identify Meis2 enhancers and

statistical analysis for promoter/MBE FISH in wild-type and Ring1 mutant embryos at

11.5 dpc.

(A) Genomic sequence similarity of Meis2 and its surrounding regions from mouse, chicken

and fugu compared to the human counterpart (hg19/Chr. 15: 37,110,000-37,710,000) as

revealed by the VISTA tool (http://genome.lbl.gov/vista/index.shtml). Genomic regions

corresponding to human BAC clones used in this study (RP11-597G23 and RP11-991D13)

are shown by bold lines below the homology plot panel. Thirteen transgenic mice harboring

the RP11-597G23 reporter were obtained, but none of them showed midbrain fluorescence.

On the other hand, the RP11-991D13 reporter gave 4 midbrain positive embryos out of 26

transgenic animals. Boxes indicate evolutionary highly conserved DNA sequences outside of

the coding regions. Mouse genomic sequences corresponding to the regions indicated by

boxes as “a” to “g” were tested for their transcriptional regulatory activity by conventional

transgenic assays. Positions corresponding to mouse genomic sequences used for 3C

(chromosomal conformation capture) assays are also shown. Evolutionarily highly conserved

regions within respective EcoRI fragments are indicated by open boxes. The mouse genomic

sequence corresponding to the region indicated by a dotted box as “v” is another midbrain

enhancer candidate from an enhancer browser site (http://enhancer.lbl.gov/) corresponding to

hs181 and was used as a negative control for the 3C assay. (B) Identification of the sequence

“d” as a midbrain-specific enhancer. Whole mount in situ image for Meis2 is also shown

(WISH). (C) Association of the sequence “d” with the promoter specifically in midbrain. (D)

Accumulation of histone H3K4me1 and H3K27ac in 11.5 dpc midbrain at the MBE (dotted

red box) revealed by ChIP-seq analyses. (E) Average distances, SD, numbers of pairs and

matrices of Chi-square test of the distribution of promoter/MBE distances tested in midbrain,

LGE, MGE, limb mesenchyme and cephalic mesoderm in wild type (left) and Ring1 mutant

(right) are summarized. (F) Immuno-FISH analyses of the topology of promoter, MBE and

RING1B bodies in midbrain (MB), LGE, MGE, cephalic mesoderm (CM) and limb

mesenchyme (LM).

Figure S3, related to Figure 3. Summary of colocalization of promoter/MBE/RBS FISH

and RING1B protein and analysis of the position of RING1B in the mouse genome.

(A) Supplement for Figures 3B and 3C. A summary of promoter/MBE/RBS interactions in

6.5 dpc epiblasts and 16-20 and 22-26 somites stage midbrain. (B) Supplement for Figures

3D and E. A summary of the association of promoter, MBE or RBS with RING1B bodies in

6.5 dpc epiblasts and 16-20 and 22-26 somites stage midbrain. (C) Distribution, relative to

the gene body, of the most prominent RING1B-binding sites in the mouse genome based on

ChIP-seq data from ES cells (GEM656523). Several genes harboring additional and more

prominent RING1B binding sites outside of their promoters are shown below the graph.

Tables S1, related to Figure 4. Summary table corresponding to Figure 4B and 4C,

respectively.

(A) Summary table shows averages, standard deviations and numbers of paired foci within

Figure 4B. (B) Summary table shows averages, standard deviations and numbers of paired

foci within Figure 4C.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Animals

Transgenic constructs were made with PCR-isolated mouse genomic fragments linked with

the Hsp68-LacZ reporter plasmid (Sasaki and Hogan, 1996). PCR fragments were obtained

by Phusion DNA Polymerase (Thermo Fischer Scientific) and their positions within the

mouse genome (mm9) are listed below. Whole mount in situ hybridization was done using an

established protocol. The Meis2 probe corresponding to the 3’ UTR of Meis2 mRNA was

isolated and cloned by PCR.

Positions of fragments in transgenic analysis (positions are based on mm9:chr.2)

fragment position from to

a 116,026,875 116,028,222

b 115,952,276 115,955106

c 115,843,835 115,846,217

d 115,825,100 115,828,518

e 115,805,413 115,806,189

f 115,722,985 115,724,514

g 115,700,636 115,702,700

ChIP primers

Primers designed for ChIP-qpCR are summarized below.

ChIP primers used in this study (genomic position; mm9:chr.2)

name

(forward)

sequence position

from

to

name

(reverse)

sequence position

from

to

promoter 1f ttgactgaggtgtgcatggc 115,895,419 115,895,438 promoter 1r caacagcgaggtgtccacac 115,895,252 115,895,271

RBS 1f agggtgtgattatatgtgctc 115,681,932 115,681,952 RBS 1r tcaacaacgattccactcgg 115,681,822 115,681,841

MBE 1f cttagcgtgggatttatggc 115,827,231 115,827,250 MBE 1r agtggagagtttatgctccc 115,827,111 115,827,130

Co5 1f cagtactctgtgaggtagcc 115,968,325 115,968,344 Co5 1r gaggaaagacactgggatcg 115,968,153 115,968,172

Co3 1f tctgagctgtgagatgtctg 115,658,666 115,658,685 Co3 1r gtgagccacatatcaacagg 115,658,539 115,658,558

CoI 1f gtggtctatgcacaatcctg 115,809,788 115,809,07 CoI 1r tcagcaggctttgggtaatg 115,809,688 115,809,707

3D-FISH and Immuno-FISH analyses

FISH technology has been described previously (Chambeyron et al., 2005; Tanemura et al.,

2005; Isono et al., 2013). Mouse embryonic samples were fixed with 4% paraformaldehyde

and embedded in paraffin. Detailed information about the fosmids used as FISH probes is

summarized below. Probes were labeled with Alexa Fluor® 488-5-dUTP (Life

Technologies-Molecular Probe C11397), Alexa Fluor® 647-OBFA-dCTP (Life

Technologies-Molecular Probe C21559) and Cy3-dCTP (GE Healthcare PA53021) by

nick-translation (Roche 976776). The histological protocols have been described previously

(Tanemura et al., 2005). The thickness of the sections was 5 µm. After deparaffinization, all

sections were soaked and permeabilized in the HistoVT One solution (Nakalai Tesque,

06380-05) in a glass vessel, which was then incubated in a 100°C boiling water bath for 20

minutes. Immediately after the treatment, sections were washed with water to remove the

detergent prior to performing the FISH or Immuno-FISH processes. In the case of

Immuno-FISH, immuno-staining was carried out prior to the FISH process. The primary

antibody was a mouse monoclonal RING1B (Atsuta et al., 2001) or mouse monoclonal

MEIS2 (Abcam ab55647). Secondary antibodies were anti mouse IgGs coupled with Alexa

Fluor from Life Technologies (355; 10035, 488; A-11001, 555; A21422, 647; A-21235).

After the secondary antibody reaction, sections were postfixed with 4% PFA for 15 minutes,

and then were processed to the cooled 70% ethanol incubation step of the FISH reaction

(Isono et al., 2013).

Fosmids used for FISH analysis

probe name fosmid location (mm9:chr.2)

promoter WI1-703C19 115,882,765-115,924,771

MBE WI1-1321D12 115,795,659-115,836,845

RBS WI1-275J3 115,659,499-115,695,402

The confocal images were captured with an inverted microscope IX71 with oil immersion

objectives (Olympus UPlanSApo 100x NA 1.40 and PlanApo N 60x NA 1.42) as described

previously (Isono et al., 2013). Images with 65 nm pixels in X-Y and 300 nm steps in Z were

deconvoluted one iteration with AutoDeblur®/AutoVisualize® software (AutoQuant Imaging,

Inc.). Deconvoluted images along the Z-axis were analyzed with Volocity software

(Improvision) (Isono et al., 2013).

We determined a threshold promoter/RBS distance to distinguish paired and unpaired foci as

following procedure. Distribution of distances between promoter/RBS FISH signals in

midbrain, LGE, MGE, limb mesenchyme and cephalic mesoderm are summed (Figure S1C;

top panel). The summed distribution was fitted to mixed Gaussian distributions using the EM

algorithm. The numbers of distributions were estimated with Bayesian information criteria

(BIC) so that we applied three normal distributions (cluster 1, 2, and 3). We classified three

clusters of FISH probes using these distributions and determined 0.16µm, which represents

the boundary of cluster 1 and 2, as the threshold to distinguish paired and unpaired signals.

BAC recombineering and production of BAC transgenic mice

Each BAC was modified by insertion of eGFP in-frame from the Meis2 initiation codon by

BAC recombineering (GENE BRIDGES). The deleted sequence of the delta MBE BAC

corresponds to 37,329,745-37,330,178 of human chromosome 15 (hg19) whose mouse

corresponding sequence is located at 115826623-115827057 of chromosome 2 (mm9) and

has 97.7 % sequence identity. Embryos were isolated at 11.5 dpc. BAC insertions were tested

with 4 pairs of PCR primers listed below.

BAC typing primer (human sequence positions are based on hg19:chr.15)

forward sequence position from to reverse sequence position from to

EGFP-140 f gttcatctgcaccaccggc Gfp Gfp EGFP-424 r ttgtgccccaggatgttgc Gfp Gfp

G-Bf agcttgccgtaggtggcatc Gfp Gfp G-B r cttcgttgtctagactgggc 37,391,824 37,391,843

hMeis2B1f aggttgctgaggttcagagc 37,321,049 37,321,068 hMeis2B1r ctagaggagcttctaggatc 37,320,701 37,320,720

hMeis2B2f ttgtcaccaccttagcgtcc 37,224,146 37,224,165 hMeis2B2r gccctgtgacattgttgcac 37,223,770 37,223,789

3C (chromosomal conformation capture)

The 3C technique was performed as described previously (Splinter et al., 2004) with slight

modifications. 106 to 5 x 106 cells were used for 3C analysis, and chromatin was digested

with 400U of the EcoRI restriction enzyme. Primers used for 3C are summarized below.

Quantification of ligation products was based on standard curves made using the cloned

fusion fragments for each expected product. Relative values for 3C were further normalized

by 3C values obtained from the results using the ERCC3 allele in each experiment.

Primer used for 3C analysis

primer name sequence promoter gtttgtcccacactccagcccttg

sequence C acggattgaagctggtcgctcttc sequence V gaagagaaaggcgactcagtctc Ercc3-1-r gcttgagcatgacgtcactg Ercc3-2-r gctgagatcaaggatgaggc

SUPPLEMENTAL REFERENCE Atsuta, T., Fujimura, S., Moriya, H., Vidal, M., Akasaka, T., and Koseki, H. (2001). Production of monoclonal antibodies against mammalian Ring1B proteins. Hybridoma 20, 43-46. Chambeyron, S., Da Silva, N.R., Lawson, K.A., and Bickmore, W.A. (2005). Nuclear re-organisation of the Hoxb complex during mouse embryonic development. Development 132, 2215-2223. del Mar Lorente, M., Marcos-Gutierrez, C., Perez, C., Schoorlemmer, J., Ramirez, A., Magin, T., and Vidal, M. (2000). Loss-and gain-of- function mutations show a Polycomb group function for Ring1A in mice. Development 127, 5093-5100. De Napoles, M., Merrnoud, J.E., Wakao, R., Tang, Y.A., Endoh, M., Appanah, R., Nesterova, T.B., Silva, J., Otter, A.P., Vidal, M., Koseki, H., and Brockdorff, N. (2004). Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev. Cell 7, 663-676. Isono, K., Endo, T.A., Ku, M., Yamada, D., Suzuki, R., Sharif, J., Ishikura, T., Toyoda, T., Bernstein, B.E., and Koseki, H. (2013). SAM domain polymerization links subnuclear clustering of PRC1 to gene silencing. Dev. Cell 26, 565-577. Kondo, T., and Duboule, D. (1999). Breaking colinearity in the mouse HoxD complex. Cell 97, 407-417. Sasaki, H. and Hogan, B.L.M. (1996). Enhancer analysis of HNF-3b gene: regulatory elements for node/notochord and floor plate are independent and consist of multiple sub-elements. Genes to Cells 1, 59-72. Splinter, E., Grosveld, F., and de Laat, W. (2004). 3C technology: analyzing the spatial

organization of genomic loci in vivo. Methods Enzymol. 375, 493-507. Tanemura, K., Ogura, A., Cheong, C., Gotoh, H., Matsumoto, K., Sato, E., Hayashi, Y., Lee, H.W., and Kondo, T., (2005). Dynamic rearrangement of telomeres during spermatogenesis in mice. Dev. Biol. 281, 196-207.