EUKARYOTIC CELL, Jan. 2010, p. 136–147 Vol. 9, No. 11535-9778/10/$12.00 doi:10.1128/EC.00281-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Active VSG Expression Sites in Trypanosoma bruceiAre Depleted of Nucleosomes�†

Tara M. Stanne and Gloria Rudenko*The Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford OX1 3SY, United Kingdom

Received 29 September 2009/Accepted 5 November 2009

African trypanosomes regulate transcription differently from other eukaryotes. Most of the trypanosomegenome is constitutively transcribed by RNA polymerase II (Pol II) as large polycistronic transcription unitswhile the genes encoding the major surface proteins are transcribed by RNA polymerase I (Pol I). Inbloodstream form Trypanosoma brucei, the gene encoding the variant surface glycoprotein (VSG) coat isexpressed in a monoallelic fashion from one of about 15 VSG bloodstream form expression sites (BESs). Littleis known about the chromatin structure of the trypanosome genome, and the chromatin state of active versussilent VSG BESs remains controversial. Here, we determined histone H3 occupancy within the genome of T.brucei, focusing on active versus silent VSG BESs in the bloodstream form. We found that histone H3 was mostenriched in the nontranscribed 50-bp and 177-bp repeats and relatively depleted in Pol I, II, and III tran-scription units, with particular depletion over promoter regions. Using two isogenic T. brucei lines containingmarker genes in different VSG BESs, we determined that histone H3 is 11- to 40-fold depleted from active VSGBESs compared with silent VSG BESs. Quantitative PCR analysis of fractionated micrococcal nuclease-digested chromatin revealed that the active VSG BES is depleted of nucleosomes. Therefore, in contrast toearlier views, nucleosome positioning appears to be involved in the monoalleleic control of VSG BESs in T.brucei. This may provide a level of epigenetic regulation enabling bloodstream form trypanosomes to efficientlypass on the transcriptional state of active and silent BESs to daughter cells.

In eukaryotes nuclear DNA is packaged into linear arrays ofnucleosomes, which provides one of the main determinants ofaccessibility to DNA binding proteins (37, 60, 68, 82). Nucleo-somes consist of �146 bp of DNA wrapped around a histoneoctamer composed of two copies each of histones H2A, H2B,H3, and H4 (44). The ability to change how DNA is packagedwithin nucleosomes allows variation in the accessibility of dif-ferent DNA binding sites and permits fine modulation of pro-moter activity (33). Recently, there have been major advancesin our understanding of how chromatin structure impacts theregulation of RNA polymerase II (Pol II) transcription units.New developments (72) have enabled the determination of theglobal organization of nucleosomes in organisms includingbudding yeast, Drosophila, and humans (3, 35, 36, 48, 71, 83).These studies have shown evidence for nucleosome depletionat transcriptionally active regulatory regions, with the level ofnucleosome occupancy inversely proportional to the rate oftranscription initiation at the promoter (3, 36, 83). However,nucleosome remodeling at promoters does not always appearto be simply a consequence of transcriptional activity but isalso thought to mechanistically regulate transcription throughmodulating the access of trans-acting factors (74).

Our understanding of the role that chromatin structure playsin the regulation of RNA Pol I transcription has relativelylagged behind that of Pol II (reviewed in references 6, 21, and

49). This is in part due to the repetitive nature of the nearlyidentical ribosomal DNA (rDNA) transcription units, which inSaccharomyces cerevisiae (and presumably most eukaryotes)are the only transcription units transcribed by Pol I (54). Ap-proximately half of the rDNA repeats are transcriptionallyactive at any time (11–13). Silencing of the inactive rDNA unitsis mediated by the nucleolar remodeling complex (NoRC)(69), which silences the inactive rRNA genes by changing nu-cleosome positioning (38, 77). Despite the clear role that chro-matin remodeling plays in transcriptional regulation of therRNA, the precise nature of the chromatin present in activeversus silent rRNA transcription units remains unclear (49).Studies using psoralen cross-linking (11) or more recent anal-yses combining this with chromatin endogenous cleavage(ChEC) (50) have suggested that transcriptionally activerDNA units are essentially devoid of nucleosomes. However,other recent studies have argued that active rDNA has a nu-cleosomal structure (30).

In African trypanosomes the majority of the trypanosomegenome is constitutively transcribed as extensive Pol II tran-scription units. Very few regulatory regions have been defined,and only a few promoters have been well characterized. Try-panosomes appear to largely lack gene regulation at the levelof transcription and, instead, rely on posttranscriptional con-trol acting at the level of RNA processing and stability (10).Due to this very different way of regulating expression of theirgenes, it is unclear whether nucleosome positioning plays arole in trypanosomes similar to that of other eukaryotes.

Trypanosomes are further unconventional in that they usePol I to transcribe not only the multicopy rDNA but also thegenes encoding their major surface proteins: variant surfaceglycoprotein (VSG) in the bloodstream form or procyclin in

* Corresponding author. Mailing address: The Peter MedawarBuilding for Pathogen Research, University of Oxford, South ParksRoad, Oxford OX1 3SY, United Kingdom. Phone: 44 1865 281548.Fax: 44 1865 281894. E-mail: gloria.rudenko@medawar.ox.ac.uk.

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 13 November 2009.

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the procyclic (insect mid-gut stage) form (22, 32, 64). Thisunique ability to use an unorthodox RNA polymerase to tran-scribe some of their protein-coding genes is presumably madepossible by trans-splicing, which adds a capped 39-nucleotidespliced leader (SL) RNA to the 5� end of each mRNA, therebymaking an uncapped Pol I-derived transcript available fortranslation (34, 39, 42, 47). In order to provide the largeamount of SL RNA necessary for trans-splicing, the trypano-some has up to 200 SL RNA transcription units which arehighly transcribed by Pol II. The SL RNA promoter is the onlyPol II promoter in trypanosomes which has been well charac-terized (14, 23, 70).

In bloodstream form T. brucei antigenic variation of a VSGcoat is used to escape host antibodies. This is accomplishedthrough monoallelic exclusion of VSG expression, wherebywithin a single cell only one of about 15 telomeric VSG bloodstream form expression sites (BESs) is transcribed at a time byPol I (7, 9, 58). The nature of the chromatin structure of VSGBESs has been a controversial issue. Earlier studies have ar-gued that there are no detectable differences in nucleosomalorganization between active and silent VSG BESs, althoughactive VSG BESs are sensitive to digestion by endonucleasesincluding single-strand-specific endonucleases (20, 51, 57).Studies using exogenous T7 RNA polymerase as a probe forchromatin accessibility also did not find evidence that activeVSG BESs in bloodstream form T. brucei were more accessiblefor transcription than inactive ones (52). Recently, however,the role of chromatin in the downregulation of silent BESs isbeing reevaluated as chromatin remodeling proteins have beenshown to be important for VSG BES control (15). First, thechromatin remodeling protein T. brucei ISWI (TbISWI) hasbeen shown to be important for VSG BES silencing in bothbloodstream and insect forms of T. brucei (28). In addition,downregulation of the histone modification protein DOT1Baffects the kinetics of VSG BES switching, arguing that histonemodification plays a role in monoallelic transcription (16).

The way in which African trypanosomes regulate tran-scription is unique compared with most eukaryotes, andlittle is known about the chromatin structure of the trypano-some genome. Here, we investigated nucleosome distribu-tion within the genome of both bloodstream and insectforms of T. brucei. In general, we find that nucleosomes areenriched on the nontranscribed repeat arrays and are de-pleted from the promoter regions of a variety of differenttranscription units (including examples transcribed by Pol I,II, and III) in both bloodstream and insect forms of T.brucei. We used two isogenic bloodstream form T. bruceilines containing marker genes in silent and active VSG BESsto investigate whether differences in chromatin structureexist between VSG BESs when they are either transcription-ally active or silent. Strikingly, we find that the active VSGBES in bloodstream form T. brucei is particularly depletedof nucleosomes compared with all other regions analyzedand shows a more open chromatin structure than silent VSGBESs. This difference may provide a level of epigeneticcontrol whereby bloodstream form trypanosomes are able toefficiently pass on the transcriptional states of active andsilent BESs to daughter cells.

MATERIALS AND METHODS

Trypanosome strains and culturing. Bloodstream form and procyclic-form T.brucei subsp. brucei strain 427 was used for all experiments. Wild-type procycliccells were grown at 27°C in SDM-79 medium containing 10% fetal bovine serum(8). Bloodstream form trypanosomes were grown at 37°C in HMI-9 mediumcontaining 10% fetal bovine serum and 10% SerumPlus (SAFC Biosciences)(26). Two isogenic bloodstream form T. brucei cell lines containing a hygromycinresistance gene downstream of the promoter of the VSG221 BES and a neomycinresistance gene downstream of the promoter of the VSGVO2 BES were used (T.brucei HNI). In T. brucei HNI (221�) the VSG221 BES was active, and theVSGVO2 BES was silent, while in T. brucei HNI (VO2�) the VSGVO2 BES wasactive, and the VSG221 BES was silent (66).

ChIP. Chromatin immunoprecipitation (ChIP) was performed essentially asdescribed previously (43). In brief, cultures of T. brucei were fixed in 1% form-aldehyde for 20 min at room temperature. The reaction was stopped by theaddition of glycine to a final concentration of 125 mM. Chromatin was sonicatedto an average DNA size of about 200 to 500 bp using a BioRuptor (WolfLaboratories) and clarified by centrifugation at 15,000 � g for 5 min at 4°C. Thesonicated extract was precleared using protein A-Sepharose CL-4B beads (GEHealthcare) and incubated for 16 h either with or without the relevant anti-histone antibody. Chromatin from 7 � 107 cell equivalents was used per immu-noprecipitation (IP). Antibodies used for the ChIP experiments include anti-histone H3 (ab1791; AbCam) at a concentration of 2 �g per IP, anti-T. bruceihistone H4 (kind gift of the George Cross laboratory) at a concentration of 10 �gper IP (75), and anti-histone H2A (07-146; Upstate) at a final dilution of 1:58.The protein-DNA complexes were incubated with protein A-Sepharose CL-4Bbeads for 2 h and eluted from the beads after a wash in 1% SDS–0.1 M NaHCO3

preheated to 65°C. The cross-links were reversed by adding NaCl to a finalconcentration of 325 mM and incubated at 65°C overnight. Following RNase Aand proteinase K treatment, DNA was purified using a QIAquick PCR purifi-cation kit (Qiagen). Each ChIP experiment was performed two or three times, asindicated in the figure legends, and the results were analyzed by hybridization ofslot blots or quantitative PCR (qPCR).

Slot blot analysis. ChIP material was analyzed on slot blots using radiolabeledprobes. Twenty microliters of input chromatin (10% of total) and immunopre-cipitated DNA was diluted in TE (Tris-EDTA) buffer (pH 8.0) and denatured byheating to 65°C for 30 min in a final concentration of 0.3 M NaOH. Thedenatured DNA were neutralized by the addition of ammonium acetate, pH 7.0,to a final concentration of 1.0 M and immediately transferred to Hybond XLmembrane (GE Healthcare) using a Schleicher and Schuell Minifold II slot blotapparatus. Membranes were hybridized with a probe for the 50-bp repeatsdirectly flanking VSG BESs (84) or the 177-bp repeats comprising the bulk of theT. brucei minichromosomes (81). The radioactive probes were labeled with[�-32P]dCTP using Ready To-Go DNA Labeling Beads and Illustra ProbeQuantG-50 Micro Columns according to the manufacturer’s instructions (GE Health-care). Membranes were hybridized with radiolabeled probes at 42°C overnightand washed to an end stringency of 0.3� SSC (1� SSC is 0.15 M NaCl plus 0.015M sodium citrate). Blots were exposed to a PhosphorImager screen (Bio-Rad),and the percentage of DNA immunoprecipitated was determined using Quan-tityOne software (Bio-Rad).

Quantitative PCR. The ChIP material obtained was analyzed using qPCR.Input chromatin (10% of total) and immunoprecipitated material were analyzedusing the primers listed in Table S1 in the supplemental material. All qPCRsamples were amplified in triplicate in a 20-�l total reaction volume containing19 �l of qPCR reaction mix (LightCycler480 SYBR green Master Mix; Roche),0.5 �M (each) forward and reverse primers, and 1 �l of the DNA template.Reaction mixtures were amplified using a LightCycler480 Real-Time PCR Sys-tem (Roche) according to the manufacturer’s instructions. All PCR primers andconditions were optimized to produce a single amplicon of the correct size.

Micrococcal nuclease digestion of T. brucei chromatin. Digitonin was used topermeabilize bloodstream form T. brucei prior to micrococcal nuclease (MNase)treatment, as previously described (43, 51). In brief, 3 � 108 T. brucei HNI(221�) or T. brucei HNI (VO2�) cells were incubated in permeabilization buffer(100 mM KCl, 10 mM Tris [pH 8.0], 25 mM EDTA, 1 mM dithiothreitol [DTT])containing digitonin at a final concentration of 40 �M for 5 min at roomtemperature. Cells were pelleted at 1,200 � g for 5 min at 4°C, and unincorpo-rated digitonin was removed by washing the cells in isotonic buffer (100 mM KCl,10 mM Tris [pH 8.0], 10 mM CaCl2, 5% glycerol, 1 mM DTT, 1 mM phenyl-methylsulfonyl fluoride [PMSF], and 1 �g/ml each of pepstatin A, leupeptin, andaprotinin). Cells were then centrifuged at 1,200 � g for 5 min at 4°C andresuspended in MNase buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mMMgCl2, 0.25 M sucrose, 3 mM CaCl2, 100 �M PMSF) to a concentration of 5 �

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105 cells �l�1. Six units of MNase (Worthington Biochemicals) was added to thecell suspension (two units per 1 � 108 cells) and incubated for 5 min at 37°C. Thereaction was stopped by the addition of EDTA and EGTA to a final concentra-tion of 10 mM. Nuclei were pelleted at 10,000 � g for 10 min at 4°C, and thesupernatant was discarded. Chromatin was solubilized by resuspending the nu-clear pellet in 200 �l of RSB buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3mM MgCl2, 5 mM EDTA, 5 mM EGTA, 0.05% NP-40, 200 mM NaCl). Celldebris was removed by centrifuging at 10,000 � g for 10 min at 4°C. A 15-�lsample of the supernatant was removed as an input control, and the remainingsupernatant was loaded immediately on a preprepared 5 to 30% sucrose gradi-ent, as described below.

Sucrose density gradient fractionation of multinucleosomes. MNase-digestedDNA was separated into mono-, di-, tri-, and tetranucleosome fractions using 5to 30% sucrose gradients, essentially as described previously (24). Discontinuoussucrose gradients were prepared containing 2 ml each of 5, 10, 15, 20, 25, and30% sucrose solution in gradient buffer (10 mM Tris-HCl [pH 7.5], 0.25 mMEDTA, 100 mM NaCl, 0.1 mM PMSF, and 0.5 �g/ml each of pepstatin A,leupeptin, and aprotinin) 24 h prior to use and stored at 4°C. The supernatantfrom the MNase treatment was carefully loaded on the 5 to 30% sucrose gradientand centrifuged at 40,000 rpm (Beckman SW41 Ti rotor) for 16 h at 4°C.Twenty-four 500-�l fractions were isolated. Proteins were removed by treatingfractions with proteinase K for 30 min at 37°C, followed by phenol-chloroformand chloroform extractions and ethanol precipitation. Aliquots from each of theMNase-digested DNA fractions were analyzed on a 2% agarose gel for thepresence of mono- and multinucleosomes. Fractions containing di-/trinucleo-somes (fractions 18 to 21) and tri-/tetranucleosomes (fractions 22 and 23) werepooled for analysis by qPCR using the primer pairs indicated in the figures,sequences of which can be found in Table S1 in the supplemental material. Thedata were plotted as the fold difference between T. brucei HNI (221�) and T.brucei HNI (VO2�). Error bars in the figures reflect standard deviations of theaverage signal obtained from three independent experiments.

RESULTS

Histone H3 is enriched in nontranscribed repeat sequencescompared with regions highly transcribed by Pol II or Pol III.Most of the trypanosome genome is transcribed as extensivepolycistronic transcription units extending for many tens ofkilobases (10, 75), with relatively few regions remaining non-transcribed. Notable exceptions which are kept transcription-ally silent include the 50-bp and 177-bp repeat arrays. The50-bp repeats form the upstream flanks of all known VSGBESs and typically extend for 40 to 50 kb (66, 73, 84). The177-bp repeat arrays comprise the bulk of the transcriptionallysilent T. brucei minichromosomes (81). This highly abundantclass of about 100 small chromosomes (each ranging between50 to 150 kb) makes up about 10% of the T. brucei nucleargenome (81).

To determine the distribution of histone H3 over differenttypes of genomic regions in T. brucei, we first looked at histoneH3 occupancy over the nontranscribed 50-bp and 177-bp re-peat arrays. Histones are extremely well conserved within eu-karyotes, and an antibody against the C terminus of humanhistone H3 specifically recognizes T. brucei histone H3 (46).Chromatin immunoprecipitation (ChIP) using formaldehydecross-linked T. brucei chromatin was performed with the anti-histone H3 antibody or no antibody as a negative control. Inputchromatin (10% of total) and immunoprecipitated materialwere analyzed by slot blot using radiolabeled probes for eitherthe 50-bp repeats or the 177-bp repeats (Fig. 1A). The signalon the blots was quantitated, and values obtained from theno-antibody control were subtracted from the values obtainedusing the anti-histone H3 antibody. In both bloodstream formand procyclic-form T. brucei, about 8% of the input DNA wasimmunoprecipitated with anti-histone H3 antibody, showing

that histone H3 is very abundant in these nontranscribed re-gions (Fig. 1D).

We next looked at the distribution of histone H3 on activelytranscribed regions of the T. brucei genome. As with the ma-jority of genes in T. brucei, the genes encoding the RNA poly-merase I large subunit (Pol I) and �-tubulin are present inlarge polycistronic transcription units constitutively transcribedby Pol II in both bloodstream form and procyclic form T.brucei. Quantitative PCR (qPCR) was used to determine therelative abundance of these two genes in the material immu-noprecipitated with the anti-histone H3 antibody. The primersused for the qPCR experiments are shown in Table S1 in thesupplemental material. In both life cycle stages, about 5 to 6%of the input DNA was immunoprecipitated (Fig. 1D), indicat-ing that these Pol II-transcribed genes contain less histone H3and are in a more open chromatin conformation than the silentrepeats.

In trypanosomes, maturation of the precursor mRNAs re-quires the addition of a 39-nucleotide spliced leader (SL) RNAonto the 5� end of every mRNA (39). In order to provide thelarge amount of SL RNA necessary for trans-splicing, the try-panosome has up to 200 SL RNA transcription units which aretranscribed by RNA Pol II (19, 70). The repetitive SL genesare highly transcribed regions in the T. brucei genome, and arethe only Pol II-transcribed genes with a well-characterizedpromoter (14, 23, 70). Here, we used ChIP followed by qPCRto determine the distribution of histone H3 in the genomicregion containing the SL RNA transcription unit repeats. Fig-ure 1B depicts a portion of a genomic region containing 10 to11 tandem SL RNA transcription units, as well as a retrotrans-posable element called the SL-associated conserved sequence(SLACS), which has integrated exclusively in the SL RNAgenes (1, 55). Using ChIP-qPCR, we found that the SLACSelements contain amounts of histone H3 similar to those of thegenes encoding the Pol I large subunit or �-tubulin, which arealso transcribed in both life cycle stages by Pol II (Fig. 1D).However, approximately 3-fold less histone H3 was found onthe SL gene promoters, and about 2-fold less histone H3 waspresent on the SL RNA genes themselves or the SL RNAspacer region (Fig. 1D). This indicates that the SL RNA tran-scription units are more transcriptionally open than those ar-eas containing SLACSs.

We next determined the distribution of histone H3 acrossthe 5S rDNA transcription units transcribed by RNA Pol III.5S rRNA is an integral component of the large subunit ofribosomes, and a schematic depicting part of a region of eighttandem 5S rRNA transcription units is shown in Fig. 1C. Thehistone H3 distribution across the 5S rDNA region was slightlylower than that found within the Pol II-transcribed polycis-tronic transcription units and was constant between both T.brucei life cycle stages (Fig. 1D).

Histone H3 is particularly depleted around the promoters ofthe Pol I-transcribed rDNA transcription units. It has beenshown in a variety of eukaryotes that the ribosomal DNAtranscription units are transcribed at a very high rate by Pol I.In T. brucei there are about 11 to 12 rDNA transcription unitsarranged over six to seven chromosomes (4). In order to de-termine the distribution of histone H3 across these highlytranscribed rDNA transcription units, we designed qPCRprimers spanning the length of an rDNA transcription unit as

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well as in the nontranscribed rDNA spacer (80) (Fig. 2A). Weperformed ChIP-qPCR in both bloodstream form and procy-clic-form T. brucei and found that the rDNA promoter regioncontains approximately 3-fold less histone H3 (approximately 1to 2% input precipitated) than the nontranscribed rDNAspacer (5 to 6% of the input was immunoprecipitated) (Fig.2B). This is compatible with an open chromatin structure thatis depleted of nucleosomes immediately around the transcrip-tionally highly active Pol I promoters. Additionally, a gradientof histone H3 distribution was found along the rDNA tran-scription unit, with levels that were lowest immediately down-stream of the promoter and gradually increasing up to thoseobserved in the spacer region (Fig. 2B).

Histone H3 is depleted from the procyclin gene promoters inboth bloodstream form and procyclic form T. brucei. In mosteukaryotes RNA Pol I exclusively transcribes rDNA transcrip-tion units. However, T. brucei is unique in its ability to use PolI to transcribe the major cell surface proteins of both procyclicform and bloodstream form trypanosomes (22, 32, 64). The

surface coat of procyclic form trypanosomes is composed ofmembers of the procyclin family of glycoproteins including theEP and GPEET procyclin variants (62, 63), of which one EPprocyclin transcription unit is depicted in Fig. 3A. Using ChIP-qPCR analysis, we found that histone H3 is more than 3-folddepleted on the procyclin EP promoter in both bloodstreamand procyclic T. brucei (less than 1.5% ChIP material immu-noprecipitated) compared with regions upstream of the EPprocyclin transcription unit (Fig. 3B). The EP procyclin genescontain more histone H3 than the promoter region (about 3%of input immunoprecipitated) (Fig. 3B); however, this is 2-foldless than that observed in genes present in the Pol II-tran-scribed polycistronic arrays, indicating a more open chromatinstructure (Fig. 1D).

Analysis of these two Pol I transcription units (rDNA andprocyclin) is complicated by the fact that the genes within themare present in multiple, nearly identical copies. In a number ofother eukaryotes, it has been shown that about half of therDNA transcription units are transcriptionally active (11). In T.

FIG. 1. Histone H3 is relatively abundant on nontranscribed repeat regions in T. brucei chromatin compared with the SL and 5S RNAtranscription units. (A) Histone H3 distribution analyzed by ChIP performed using an anti-histone H3 antibody (�-H3) or no antibody (No Ab)in bloodstream form (BF) or procyclic form (PF) T. brucei, as described previously (43). Input material (10% of total [10% In]) was compared withimmunoprecipitated samples using slot blot analysis. The slot blots were hybridized with radiolabeled probes for the transcriptionally silent 50-bprepeat regions flanking VSG BESs (Fig. 4A) or the 177-bp repeats, which make up the bulk of the transcriptionally silent minichromosomes. Threeindependent ChIP experiments were performed, and a representative slot blot is shown. (B) Schematic of the genomic region containing the SLRNA transcription units transcribed by RNA Pol II. The SL promoters are indicated with flags, and the SL genes, as well as characteristictransposable elements found within the spliced leader arrays (SLACS) (1) are indicated with black boxes. Regions analyzed by qPCR are indicatedwith lettered brackets. (C) Schematic of the 5S rDNA transcription units, transcribed by RNA Pol III. The 5S rDNA promoters are indicated withflags, the 5S rDNA genes are indicated with black boxes, and regions amplified using qPCR are indicated by lettered brackets. (D) Quantificationof histone H3 distribution in bloodstream form and procyclic-form T. brucei analyzed either by hybridization of slot blots (panel A) using aPhosphorimager for quantitation of the 50-bp or 177-bp simple sequence repeats or qPCR for the Pol II- or Pol III-transcribed transcription units.Regions analyzed include the gene encoding the large subunit of RNA Pol I, �-tubulin (�Tub), regions around the SL RNA (a to d), or 5S rRNAtranscription units (e to g) (panels B and C show the location of the amplified regions). Signals are expressed as a percentage of inputimmunoprecipitated after subtraction of signal from the no-antibody control. The data shown are the average of three independent ChIPexperiments, with the standard deviations indicated with error bars. , pseudogene.

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brucei this is also likely to be the case, although this issue hasnot yet been resolved. Multiple procyclin transcription unitsare also present, and within a single cell multiple units can besimultaneously transcriptionally active (reviewed in reference62). In order to better study the chromatin structure of a singlePol I transcription unit, we investigated the chromatin structure ofVSG BESs since in bloodstream form T. brucei a single VSG BESis transcribed by Pol I in a monoallelic fashion (9, 22).

Active VSG expression sites are dramatically depleted of his-tones compared with silent VSG expression sites. VSG BESs areextensive telomeric transcription units typically varying be-

tween 40 to 60 kb and containing a variety of expression site-associated genes (ESAGs) in addition to the telomeric VSGgene (Fig. 4A) (25). Although different members of individualESAG gene families are polymorphic, they remain highly sim-ilar to each other in sequence (25), which greatly complicatesthe analysis of BES transcriptional states. Here, we use two iso-genic bloodstream form T. brucei cell lines containing differentsingle-copy marker genes in both the active and silent VSG BES(Fig. 4B) to investigate the distribution of histone H3 along thelength of VSG BESs as well as between silent and active BESs. T.brucei HNI (221�) has an active VSG221 BES with a hygromycinresistance gene inserted behind the promoter and a neomycinresistance gene inserted behind the promoter of the silent VS-GVO2 BES. In T. brucei HNI (VO2�) the VSG221 BES is silent,and the VSGVO2 BES is active.

Using ChIP-qPCR, we first used these cell lines to investi-gate the distribution of histone H3 along the length of VSGBESs. In T. brucei 427 there are approximately 15 BESs (25),all of which are silent except one. Due to the high degree ofsequence similarity between BESs, most of the primer pairsused for amplifying genes within the BES would be expected torecognize most if not all BESs (BES Fig. 4C) (25). The distri-bution of histone H3 over BESs was found to be particularlylow over the “core” BES promoter (29) (Fig. 4C, qPCR primerpair c) in isogenic T. brucei strains expressing either theVSG221 or VSGVO2 BES (Fig. 4C, red or blue bars, respec-tively). Levels of histone H3 over the BES core promoter wereabout 2-fold lower than in the region upstream of the BESpromoter (primer pair a) and about 3-fold lower than levels inESAG6/7 (primer pair e). The low level of histone H3 presentat the BES core promoter is consistent with data from Figueir-edo and Cross (17) and suggests that the BES promoter regionis in a more open chromatin conformation in multiple BESs.These results are compatible with the observation that there istranscription extending from most if not all silent BES pro-moters in bloodstream form T. brucei, but this transcription

FIG. 2. T. brucei ribosomal DNA transcription units show relative depletion of histone H3 around the promoter regions. (A) Schematicdepicting the region around a T. brucei rDNA transcription unit according to White et al. (80); the rDNA promoter is indicated by a flag, the rDNAgenes are shown as black boxes, and the regions analyzed by qPCR are indicated by lettered brackets. (B) Distribution of histone H3 over therDNA as determined by ChIP using a histone H3 antibody (or no antibody as a negative control) in bloodstream form (BF) or procyclic form (PF)T. brucei. qPCR was used to amplify regions indicated in panel A. Signals are expressed as the percentage of input immunoprecipitated aftersubtraction of signal from the no-antibody control. Results show the average from three independent experiments, with the standard deviationsindicated with error bars.

FIG. 3. Histone H3 is less abundant on procyclin promoters in bothbloodstream form and procyclic form T. brucei. (A) Schematic depictingpart of an EP procyclin locus (PARP B1) (see references 62 and 67). TheEP procyclin promoter is indicated with a flag, the EP procyclin genes areindicated by black boxes, and the genomic regions analyzed using qPCRare indicated by lettered brackets. (B) Histone H3 distribution over theprocyclin locus was investigated using ChIP with an anti-histone H3 an-tibody (or no antibody as a negative control) in bloodstream form (BF)and procyclic form (PF) T. brucei. Quantitative PCR was used to amplifythe procyclin genomic regions indicated in panel A. The data are ex-pressed as the percentage of input immunoprecipitated after subtractionof signal from the no-antibody control. The results shown are the averagesignal from three independent ChIP experiments, with the standard de-viations indicated with error bars.

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attenuates relatively rapidly downstream of the BES promoterin all except for the active BES (2, 65, 79).

We next investigated the distribution of histone H3 betweensilent and active BESs by performing ChIP-qPCR analysis onthe single-copy marker genes present in our two isogenic T.brucei cell lines (Fig. 4C). We found that there is a striking 11-to 40-fold difference in distribution of histone H3 on activeversus silent BESs. Less than 0.5% of input was immunopre-cipitated from the single-copy genes present in the activelytranscribed VSG221 BES (Fig. 4C, primer pairs i to k) andVSGVO2 BES (Fig. 4C, primer pairs l and m) compared toover 4% when the same genes were silent. This indicates thatthe active VSG BES is in a far more open chromatin confor-mation than the silent BESs. As expected, in the isogenic T.

brucei strain where the VSG221 BES has been silenced and theVSGVO2 BES activated, the distribution of histone H3 overthe two BESs is reversed (Fig. 4C).

To determine whether other histones exhibit a similar distribu-tion pattern between silent and active BESs, we performed addi-tional ChIP experiments using antibodies against histone H4 andhistone H2A. qPCR analysis was performed using a subset of theBES primer pairs and �-tubulin as a control. As observed with thehistone H3 ChIP experiments, histone H4 was found to be sig-nificantly depleted (5- to 10-fold) on the active BES comparedwith the silent BES (Fig. 5A) while histone H2A was 2- to 4-folddepleted (Fig. 5B). Taken together, these data strongly suggestthat the active BES in bloodstream form T. brucei is depleted ofnucleosomes.

FIG. 4. Active VSG expression sites (BESs) are depleted of histone H3 compared with silent VSG BESs in bloodstream form T. brucei. (A) Atypical VSG BES (modified from AnTat1.3A) (5, 41) is indicated in the schematic shown, with the promoter indicated by a flag, the expressionsite-associated genes (ESAGs) are indicated with numbered boxes, and characteristic BES repeat arrays are indicated with striped boxes. The areaimmediately around the BES promoter is shown expanded below. The locations of BES regions used for qPCR analysis that are common to allBESs are indicated with lettered brackets. (B) The two isogenic bloodstream form T. brucei 427 cell lines used for investigating the distributionof histone H3 along active versus silent VSG BESs are shown as large red or blue boxes. T. brucei HNI (221�) expresses VSG221 from the activeVSG221 BES, and T. brucei HNI (VO2�) expresses VSGVO2 from the active VSGVO2 BES (66). BESs are shown with the promoters indicatedwith flags. Relevant single copy genes, including hygromycin resistance (Hyg), a VSG pseudogene (), VSG221, neomycin resistance (Neo), andVSGVO2 are indicated. Transcription of the active VSG BES is indicated with arrows. The BES regions analyzed by qPCR are indicated withlettered bars. (C) Histone H3 distribution over active or silent VSG BESs. ChIP analysis was carried out on isogenic bloodstream form T. bruceicell lines containing either an active VSG221 BES [HNI (221�)] or an active VSGVO2 BES [HNI (VO2�)] (panel B). Chromatin from each cellline was immunoprecipitated using an anti-histone H3 antibody or no antibody as a negative control. Quantitative PCR was used to amplifyconserved regions shared by all BESs indicated in panel A or the single-copy marker genes specific for the individual BESs shown in panel B. Aftersubtraction of signal from the no-antibody control, signals are expressed as a percentage of input precipitated. ChIP results from the HNI (221�)cell line are shown with red bars, and results from the HNI (VO2�) cell line are shown with blue bars. The results presented are the average signalobtained from three independent experiments, with the standard deviations indicated with error bars.

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In insect form T. brucei, all BESs are downregulated as VSGis not necessary in this life cycle stage. BES transcriptionalsilencing appears to be more “leaky” in procyclic form than inbloodstream form T. brucei, and significant levels of transcrip-tion extending from most, if not all, BES promoters has beenreported (2, 65, 79). Using a subset of the BES qPCR primerpairs, we next investigated the distribution of histone H3 alongBESs in procyclic-form T. brucei. Less histone H3 was presenton the BES core promoter than the rest of the BES, compat-ible with the transcriptional activity observed in this life cyclestage (Fig. 6).

Active VSG expression sites are depleted of nucleosomes. Toensure that the results obtained from the ChIP experiments

were not an artifact of cross-linking, we investigated BES chro-matin structure using an alternative experimental approach. Inorder to confirm that the relative depletion of histones over theactive BES observed in the ChIP experiments indeed reflectedreduced nucleosome occupancy, we examined the relative sen-sitivity of the active and silent BES to digestion by micrococcalnuclease (MNase). Rather than using the traditional MNaseexperimental procedure requiring large numbers of cells andSouthern blotting, we used an approach which involves frac-tionating MNase-digested chromatin with sucrose gradients,followed by qPCR. This newly developed approach has beenused to establish nucleosome depletion over mammalian pro-moters and provides a more sensitive and quantitative way ofinvestigating chromatin structure than older Southern blotting-based methods (18, 40). Genomic areas which are depleted fornucleosomes are in a more open chromatin structure and aretherefore hypersensitive to digestion by MNase. Using thisreasoning, less qPCR product would be expected to be ampli-fied from oligonucleosomal DNA template in regions of thegenome that are in a relatively more open chromatin state thanin those genomic regions with a more closed chromatin struc-ture.

Chromatin from permeabilized T. brucei HNI (221�) andHNI (VO2�) cell lines was partially digested with MNase toyield oligonucleosomes. This preparation was then separatedon 5 to 30% sucrose gradients, and DNA was isolated from thecollected fractions (Fig. 7A). Fractions enriched in di- andtrinucleosomes were pooled to form nucleosomal fraction I(Fig. 7A, NI), as were fractions enriched in tri- and tetranu-cleosomes (Fig. 7A, NII). These two pools of oligonucleosomalDNA were then subjected to qPCR using a subset of theprimer pairs used in the ChIP experiments (Fig. 7B and C) andplotted as the fold difference in the amount of DNA amplifiedbetween the two isogenic T. brucei lines. For the housekeepinggenes (Pol I, �-tubulin, and �-tubulin) and two regions com-mon to all BESs (BES promoter and ESAG6/7), equivalent

FIG. 5. Histone H4 and histone H2A are relatively depleted fromactive compared with silent VSG BESs. (A) Histone distribution wasdetermined in T. brucei cell lines containing either an active VSG221[HNI (221�)] or an active VSGVO2 [HNI (VO2�)] BES. Diagramsfor these cell lines are shown in Fig. 4B. ChIP experiments wereperformed using an antibody against histone H4 or no antibody as anegative control. Quantitative PCR was used to analyze two regionscommon to all BESs: the BES core promoter (primer c in Fig. 4A) andESAG7/6 (ESAG6 and primer e in Fig. 4A). Additionally, the fivesingle-copy sequences, shown schematically in Fig. 4B, specific foreither the VSG221 or VSGVO2 BES were analyzed: i, hygromycin;j, pseudogene; k, VSG221; l, neomycin; and m, VSGVO2. ChIP datafrom the VSG221-expressing cell line are indicated with red bars, anddata from the VSGVO2-expressing line are shown with blue bars. Asa control, histone H4 presence in the �-tubulin locus (�Tub) was alsoanalyzed. Signals are expressed as a percentage of input immunopre-cipitated, with the results shown being the average of three indepen-dent experiments; the standard deviations are indicated with errorbars. (B) ChIP was performed as described for panel A, except that ananti-histone H2A antibody was used. Signals from T. brucei cell linescontaining an active VSG221 (red bars) or an active VSGVO2 (bluebars) gene are expressed as a percentage of input immunoprecipitatedand are the average signal obtained from two independent experi-ments. The primers used for qPCR analysis are as indicated in panel A.

FIG. 6. Histone H3 distribution over VSG expression sites in pro-cyclic form T. brucei. ChIP was performed in procyclic form T. bruceiusing either an anti-histone H3 antibody or no antibody. QuantitativePCR was used to analyze three regions common to all BESs with theprimers indicated in Fig. 4A: the BES core promoter (ESProm, primerc in Fig. 4A), the area immediately downstream of the BES promoter(ESProm Dn; primer d), and ESAG7/6 (ESAG6; primer e). Addition-ally, the single-copy VSG221 gene (primer k in Fig. 4B) was amplified.The results are expressed as the percentage of input immunoprecipi-tated after the signal obtained from the no-antibody control was sub-tracted. Three independent experiments were performed, and thestandard deviations are indicated with error bars.

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levels of DNA were amplified from the NI and NII fractionsusing both T. brucei HNI (221�) and HNI (VO2�), indicatingthat the chromatin structure within these regions does notdiffer between these two cell lines.

Using primers for the five single-copy genes (Fig. 4B), wefound that DNA from silent BESs was about 3-fold moreenriched in the di-/trinucleosomal fraction than DNA from the

active BES (Fig. 7B, NI), indicating that the active BES isindeed more sensitive to digestion by MNase. Comparableresults were obtained using the tri-/tetranucleosomal fraction(Fig. 7C, NII). This result is similar to that observed in theChIP experiments using anti-histone antibodies and confirmsthat the active BES is depleted of nucleosomes. As a control,qPCR analysis was performed on “naked” DNA that was

FIG. 7. The active VSG expression site in T. brucei is relatively depleted of nucleosomes. (A) Chromatin from the T. brucei HNI (221�) or HNI(VO2�) cell lines was partially digested by MNase and fractionated through sucrose gradients to obtain mono- and oligonucleosomes. DNA wasextracted from the input material as well as fractions 9 to 24 (Frac. nr.) and electrophoresed on a 2% agarose gel stained with ethidium bromide.Size markers are indicated on the left, and the locations of mono-, di-, tri-, and tetranucleosomes are indicated on the right. Oligonucleosomalfractions comprised primarily of di- and trinucleosomes were pooled to serve as the template DNA in subsequent qPCR analysis (NI). In addition,fractions containing primarily tri- and tetranucleosomes were pooled (NII). Three independent experiments were performed, and a representativegel is shown. (B) qPCR was used to amplify oligonucleosomal DNA from the NI pool of fractions containing primarily di- and trinucleosomes.These data are plotted as the fold difference in amount of DNA amplified (indicating nucleosome occupancy) between the two isogenic T. bruceilines expressing either VSG221 or VSGVO2. Red bars indicate the values obtained from the VSG221-expressing cells divided by the values obtainedfrom the VSGVO2-expressing cell line (221/VO2). Blue bars represent the reciprocal calculation (VO2/221). Three housekeeping genes weremonitored as a control and include �-tubulin (�tub), �-tubulin (�tub), and the large subunit of RNA Pol I. Two primer pairs common to all BESswere also used and include the BES core promoter (ESPro) and ESAG7/6 (represented in Fig. 4A as primers c and e, respectively). The fivesingle-copy sequences specific for either the VSG221 or VSGVO2 BES were analyzed (shown schematically in Fig. 4B as primers i to m). The resultspresented are the average from three independent experiments, with the standard deviations indicated with error bars. (C) qPCR was used toamplify DNA from the NII pool containing primarily tri- and tetranucleosomes, as described in panel B. Average values are shown from threeindependent experiments, with standard deviations indicated with error bars.

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stripped of nucleosomes and then partially digested withMNase (see Fig. S1A in the supplemental material). As ex-pected, no relative enrichment between the different BESs wasfound, providing further support that nucleosome positioningis responsible for the differences observed between silent andactive expression sites (see Fig. S1B in the supplemental ma-terial).

DISCUSSION

Here, we examined the distribution of nucleosomes through-out the genome of T. brucei using histone H3 as a marker. Wefind that histone H3 is enriched at the extensive nontran-scribed 50-bp and 177-bp simple sequence repeats (8 to 10% ofinput precipitated) in both bloodstream form and procyclic-form T. brucei, compatible with the idea that these nontran-scribed regions are present in a more closed chromatin state.In contrast, histone H3 is relatively depleted in the Pol IItranscription units (�-tubulin or Pol I large-subunit genes with5 to 6% of input precipitated) or Pol III-transcribed 5S rRNAtranscription units (approximately 4% input precipitated),which is compatible with the regions being present in a rela-tively open chromatin state. Histone H3 is particularly de-pleted in the spliced leader (SL) arrays which are highly tran-scribed by Pol II, with only 2% of the input precipitated. Theseresults are in agreement with what has been found in yeast,where histone occupancy has in general been shown to beinversely correlated with rates of transcription (3, 35).

Within the SL RNA genomic loci we observed differences inchromatin structure and found about 3-fold more histone H3present within characteristic SLACS retroposons (about 6%input precipitated) than within the SL transcription unitsthemselves (about 2% input precipitated). This is in agreementwith the observed reduced density of Pol II over the SLACSelements, indicating that transcription through the SLACSopen reading frames (ORFs) is relatively inefficient comparedwith the SL RNA (55). Recently, similar observations havebeen reported for other trypanosomatids. In Trypanosomacruzi relatively few nucleosomes are detected at the SL pro-moter (61), and in Leishmania tarentolae the SL RNA promot-ers and transcribed SL RNA genes are nucleosome free (27).

In the Pol I-transcribed rDNA we find that histone H3occupancy is greatly reduced in the area immediately down-stream of the rDNA promoter (approximately 1% input pre-cipitated) compared with the nontranscribed rDNA spacer (5to 6% input precipitated). Therefore, the promoter regions ofT. brucei transcription units transcribed by all three RNA poly-merases (Pol I, II, and III) appear to be particularly depletedof histone H3. This relative depletion of histone H3 at pro-moter regions is in agreement with previous studies in yeastwhich provide evidence for nucleosome depletion at activeregulatory regions (3, 35, 83). Within the rDNA in T. brucei, wefind evidence for a gradient of histone H3 distribution extend-ing along the length of the rDNA transcription unit, rangingfrom relatively little histone H3 in the neighborhood of therDNA promoter, up to higher levels of histone H3 at the endof the transcription unit and approaching levels found in thenontranscribed spacer. A gradient of histone H3 distributionover the rDNA transcription unit has also been shown in S.cerevisiae (30) although these data appear to show relatively

less histone H3 in the nontranscribed spacer region than wefound using T. brucei.

Procyclin is another Pol I transcription unit in T. bruceiwhich encodes the major surface protein in insect form try-panosomes. We found approximately 3-fold less histone H3 onthe procyclin EP promoter in both bloodstream form and pro-cyclic form T. brucei in comparison with sequences upstream ofthese transcription units. These data are compatible with pre-vious studies which found significant transcriptional activityfrom the procyclin promoter even in bloodstream form T.brucei (56, 78), where the elongation of transcription appearsto be developmentally controlled (78).

However, a complicating factor in the analysis of rDNA PolI transcription units has been the repetitive nature of thenearly identical genes contained within them. This complica-tion applies to the analysis of rDNA as well as procyclin tran-scription in T. brucei. Bloodstream form T. brucei is unique,however, in transcribing a single VSG BES by Pol I using strictmonoallelic exclusion (7, 9, 53). We have been able to inves-tigate the chromatin structure of different single-copy se-quences within VSG BESs using isogenic T. brucei strains withtwo different VSG BESs in either “on” or “off” states. Here, weshow that there is a striking depletion of histone H3 along theentire length of an active VSG BES, with an 11- to 40-folddifference between silent and active sites. Using antibodiesagainst histone H4 and H2A in additional ChIP experiments,we identified a similar trend, albeit less pronounced, with 5- to10-fold and 2- to 4-fold depletion, respectively, compared withthe same BES in its silent state. The fact that VSG BESs aremonoallelically expressed and contain, or can be modified tocontain, single-copy sequences greatly facilitates the analysis ofthe chromatin architecture of these Pol I transcription units.Our results showing the dramatic depletion of histones withinthe active VSG BES are consistent with those from Figueiredoand Cross (17) and are in agreement with studies which arguethat actively transcribed rRNA genes are largely devoid ofhistones (12, 50).

We find more striking differences in the distribution of his-tone H3 than in histone H2A distribution in active versus silentVSG BESs. It is important to note, however, that the anti-histone antibodies used here differ in how efficiently they areable to immunoprecipitate their target. Using the Pol II-tran-scribed �-tubulin genes as a control, the anti-histone H3 anti-body immunoprecipitated �5% of the input (Fig. 1D), andanti-histone H4 immunoprecipitated �2% of the input (Fig.5A) while anti-histone H2A immunoprecipitated only �0.5%of the input (Fig. 5B). The observed variations in abundance ofthese histones could therefore be a consequence of differentantibody affinities. However, in addition these results could beexplained by nucleosome dynamics. Within the nucleosome,DNA is first wrapped around a core histone H3/H4 tetramerbefore two histone H2A/H2B dimers are added (44). Consis-tent with this, H2A/H2B dimers have been shown to be moredynamic in chromatin, and there is a relatively rapid exchangeof histone H2B compared with the histone H3/H4 tetramers,which are more tightly and stably associated in chromatin (31).Our results showing relatively less enrichment of histone H2Ain silent BESs is compatible with a scenario whereby histoneH2A is less stably associated with the nucleosomes present insilent BESs than histone H3.

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Histone H3 was found to be depleted from the multicopyVSG BES promoters in both bloodstream and insect form T.brucei, indicating a more open chromatin conformation. Inbloodstream form T. brucei this depletion is about 2-foldgreater than in the procyclic form T. brucei, with �2% ratherthan 4% of the input immunoprecipitated. These results agreewith the previous observation that a prominent nuclease-hy-persensitive site is present within the core promoter of bothactive and inactive VSG BESs in bloodstream form T. brucei(51). Furthermore, it agrees with the extensive evidence thatthere is significant nonprocessive transcription from multiple“inactive” VSG BESs in both bloodstream and insect form T.brucei (2, 65, 79), which could explain the open chromatinstructure in this region that we observe.

An inherent problem with ChIP experiments is the fact thatformaldehyde cross-linking can potentially inhibit antibody ac-cess to more interior targets. In order to ensure that we werenot observing an artifact related to this, we used an additionalexperimental approach that did not require cross-linking toinvestigate VSG BES chromatin structure. Micrococcal nucle-ase digestion followed by nucleosome fractionation provides apowerful tool to investigate chromatin structure in a quantita-tive fashion using limited material. Using this approach, weconfirm our histone ChIP results and show that the active VSGBESs are in a more open chromatin structure than the silentVSG BESs. This observation that active VSG BESs are highlysensitive to digestion by micrococcal nuclease is in agreementwith results obtained using Southern blot analysis of MNase-digested chromatin (17). These results in some ways contradictthe findings from previous studies, which concluded that nodifference exists in the nucleosome structure between activeand silent VSG BESs.

Why has this difference in nucleosomal structure betweenthe two different VSG BES activation states been missed in thepast? Early studies comparing the nucleosomal structure ofactive versus silent BESs have argued that there is no detect-ably altered nucleosomal organization in active versus silentVSG BESs (20). However, this study did not compare the sameVSG BES sequence (in either an active or silent state) with asingle probe to allow effective comparison. In our study, usingisogenic T. brucei strains, we have been able to compare thesame set of single-copy sequences present within an BES ineither a transcriptionally active or silent state, allowing us todirectly compare differences. A later study concentrated oninvestigating the chromatin structure immediately around theBES core promoter in both an active and silent state in blood-stream form T. brucei and found a prominent DNase I-hyper-sensitive site in both cases (51). Our data are in agreement withthe observation that most, if not all, VSG BES promoters arepresent in an open chromatin state (51). However, using twodifferent methods, we find that the active VSG BES is depletedof nucleosomes along its entire length. An additional studyusing exogenous T7 RNA polymerase to probe for chromatinstructure in T. brucei found that the silent VSG BESs in insectform but not bloodstream form T. brucei were refractory totranscription by T7 RNA polymerase (52). The authors arguedfrom these data that chromatin remodeling plays a role in VSGBES silencing specifically in insect form T. brucei but did notfind evidence for this being the case in bloodstream form T.brucei. However, it is unclear exactly which aspects of eu-

karyotic chromatin can impede transcription by T7 RNApolymerase.

We do not know if the relative depletion of nucleosomes onthe active VSG BES is a direct consequence of transcriptionalactivity or if this open chromatin structure is uncoupled fromtranscription itself. It has been shown in other experimentalsystems that nucleosome loss is not necessarily a consequenceof transcription. For example, after the induction of a heatshock, initial loss of nucleosomes from the Drosophila heatshock locus occurs extremely rapidly and before the first RNApolymerases reach the corresponding region of the gene (59).This initial rapid loss of nucleosomes after heat shock wasshown to be independent of transcription and to occur over alarger region than a single transcription unit (59). In theseexperiments, a second and later wave of nucleosome loss wasdependent on transcription. Although we have found that theactive VSG BES is significantly depleted of nucleosomes, we donot know the order of events and whether nucleosome deple-tion precedes VSG BES activation.

In summary, we show that highly transcribed loci within theT. brucei genome have reduced nucleosomes, with the activeVSG BES showing the lowest nucleosomal occupancy of allregions investigated. The observed depletion of nucleosomeswithin the active VSG BES indicates that chromatin remodel-ing may play a critical role in the monoallelic exclusion neces-sary for VSG BES control in bloodstream T. brucei. The tran-scriptional states of active and repressed BESs are efficientlyinherited in bloodstream form trypanosomes, suggesting thepresence of epigenetic marks. The lack of nucleosomes on theactive BES may be one of the factors involved in marking andpropagating the epigenetic state of the BES from one gener-ation to the next. Future studies will be necessary to allow us todetermine exactly how this chromatin remodeling proceedsduring VSG BES switching.

ACKNOWLEDGMENTS

We are very grateful to Luisa Figueiredo, Nicolai Siegel, and GeorgeCross (Rockefeller University, New York, NY) for providing the T.brucei anti-histone H4 antibody, suggesting the use of the specificanti-histone H2A antibody, and communicating unpublished results onthe chromatin structure of VSG expression sites. We thank Jane Mel-lor and David Clynes (Department of Biochemistry, University ofOxford) for discussions on chromatin structure, for advice on experi-mental procedures, and for generously allowing us to use the Bio-Ruptor sonicator. We thank Mani Narayanan, Megan Lindsay, ManishKushwaha, Viola Denninger, Nadina Vasileva, and Alexander Full-brook for comments on the manuscript.

G.R. is a Wellcome Senior Fellow in the Basic Biomedical Sciences.This research was funded by the Wellcome Trust.

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