The barrier element is located in intergenic sequences 3′ to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.Regulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate  Major histocompatibility complex class I (MHC I) molecules provide immune surveillance against intracellular pathogens We previously reported the generation of transgenic mice carrying MHC class I gene, PD1. The PD1 transgene is a 9 Kb genomic DNA fragment that contains 4 Kb of swine MHC class I coding sequences, 1 Kb of sequences 5′ to the transcription start site (TSS) and 4 Kb downstream of the polyadenylation site. In mice, the PD1 transgene displays patterns of expression and cytokine responses that parallel those of the endogenous classical MHC class I gene in vivo and ex vivo molecular analyses of the promoter of MHC class I gene have identified a series of regulatory DNA sequence elements 5′ to the coding sequences that serve to establish proper patterns of gene expression Detailed Despite the detailed understanding of the role of promoter DNA sequence elements and transcription factors in establishing tissue-specific levels of MHC class I expression and hormone/cytokine-mediated responses, little is known about either the role of downstream sequence elements or of chromatin structure in the regulation of this gene family. Recently, we reported the surprising finding that chromatin structure does not actively regulate transcription of the class I gene: nucleosomal occupancy and positioning are indistinguishable in tissues that differ in class I expression levels by an order of magnitude and do not vary upon either induction or inhibition of transcription. Rather, the chromatin organization functions to keep the core promoter poised and accessible for transcription, allowing rapid activation of the gene without chromatin remodeling in vivo. Rather, stable PD1 expression and transcription-permissive chromatin organization depend on the presence of a 3′ barrier element downstream of the PD1 gene. Distinct barrier element sequences are necessary to prevent silencing and to maintain an open chromatin organization across the MHC class I gene both in transgenic mice and in stably transfected cells. In addition, sequence elements within the introns contribute to the proper regulation of tissue specific expression.In the present study, we have examined the role of sequences downstream of the promoter in regulating MHC class I gene expression. We demonstrate that the promoter alone is not sufficient for proper regulation of PD1 expression in vivo expression, transgenic mice were generated from a construct containing the 1 Kb PD1 promoter segment ligated to a human CD2 reporter in vivo.To determine whether the PD1 promoter contains all the regulatory elements necessary for normal in vivo interferon treatment . Thus, all of the regulatory elements necessary for appropriate MHC class I gene expression in vivo reside within the 5.3 Kb PD1/Bam DNA segment.To begin to map the minimal 3′ sequence requirements for stable MHC class I expression, we first truncated the 9 Kb genomic fragment used to generate the PD1 transgenic mouse, removing 3.7 Kb of distal intergenic sequences 3′ to the PD1 gene, leaving 730 bp immediately downstream of the polyadenylation site. In this construct (PD1/Bam), the 1 Kb regulatory region upstream of the TSS, the entire coding region and the poly A addition site remained intact  of the Pin vivo. A transgenic construct further deleted of all introns except intron 7 (PD1cDNA/Bam), was also expressed in vivo (To further map the necessary downstream elements and to investigate their role(s) in regulating expression, we generated transgenic mice from a series of constructs in which introns were successively removed. A transgenic construct deleted of introns 3–6  PD1cDNA and patt in vivo , as asse in vivo .per se does not depend on the introns, introns 1 and/or 2 contain sequences necessary for normal tissue-specific patterns of PD1 expression in vivo. Importantly, the reproducibility of PD1cDNAint1-2/Bam expression and proper tissue distribution among multiple independent transgenic lines indicates that this short 4.0 Kb construct contains the minimal PD1 regulatory unit.Taken together, these data demonstrate that while PD1 promoter activity in vivo expression of the PD1 gene does not depend on introns 1–6 suggested that the required downstream sequences are located within the 3′ intergenic region, which is present in all of the constructs that are expressed in vivo. To test this possibility, we next asked whether the 3′ terminus of the PD1/Bam clone could restore expression to the silent PD1-CD2 transgene  mice expressed surface CD2 on PBL, the patterns of expression of the CD2 transgene on lymphocyte populations and in the tissues differed markedly from the endogenous MHC class I and PD1 transgene patterns  did not significantly augment class I promoter activity relative to PD1-CD2 in transient transfections of either murine L cells or human HeLa cells (The 3′ regulatory element(s) necessary for La cells .These findings indicate the presence of a barrier element in the 3′ intergenic segment of the PD1 gene.The minimal 3′ sequences necessary for barrier activity were determined by 3′ truncation of PD1 cDNAint1-2/Bam intergenic sequence to the poly A addition site (PD1 cDNAint1-2/PolyA) . Most ofin vivo expression of PD1 and thus provide barrier function.This variegation in the expression of the PD1cDNAint1-2/PolyA construct among the independent founder lines contrasts sharply with transgenic mice derived with the PD1cDNAint1-2/Bam, all eight of which expressed with normal tissue-specific patterns. Therefore, sequences within the 3′ intergenic region are required for normal Similar variegated expression was observed when the PD1 cDNAint1-2/PolyA, which does not retain barrier activity, was extended by 79 bp to a downstream Sac site , the PD1cDNAint1-2/polyA construct is stably expressed, both in the presence and absence of the selective medium (data not shown).Taken together, these studies functionally identified a complex boundary element within the 3′ downstream sequence of the MHC class I gene between the poly A addition site and the distal Bam site. Full boundary function depends on the presence of both the 79 bp segment downstream of the polyA site and the DNA segment between the Sac I site and the distal Bam H1 site see . NeitherBarrier elements are required to maintain nucleosome organization and histone modifications associated with an open chromatin structure Surprisingly, loss of expression of the PD1cDNAint1-2/Sac construct does not correlate with nucleosomal density, which was almost uniformly low across the gene and indistinguishable from the expressing PD1cDNAint1-2/Bam clone . These fTo examine the relationship between barrier function and histone modifications, we assessed chromatin modifications associated with active chromatin across the gene in the same clones that were analyzed for nucleosomal density. Specifically, we assayed by ChIP analysis for the presence of histone H3K9/K14 acetylation and H3K4 dimethylation. As shown in Interestingly, we did not observe increases in modifications associated with stable heterochromatin, namely DNA CpG methylation  or histoHistone modifying enzymes have been reported to be associated with barrier elements, thereby actively regulating chromatin modifications Among the transcription factors that have been identified within boundary complexes are Upstream Factors 1 and 2 (USF1 and USF2), which recruit histone modifying enzymes to the beta-globin barrier element The enhancer-blocking factor, CTCF, interacts with both β-globin The presence of histone variant H2A.Z in chromatin has been associated with barrier function In summary, the above studies have demonstrated the presence of a complex barrier activity within the 3′ intergenic region of the MHC class I, PD1, gene that is required for stable expression of the gene and for maintaining an open chromatin structure.in vivo depends on both intronic and 3′ intergenic sequences. Element(s) within the introns are necessary for normal, tissue-specific levels of expression. A novel barrier element located in the 3′ intergenic region is necessary for sustained in vivo PD1 promoter activity and thus is an important component of MHC class I immune function.The MHC class I genes, whose products play a critical role in immune homeostasis, are primarily regulated transcriptionally. Past studies on understanding the relationship between transcriptional regulation and immune function have largely focused on characterizing the regulatory elements and transcription factors that govern their transcription. The core promoter and 5′ extended promoter region of the MHC class I gene, PD1, contain elements that mediate regulation of both constitutive, tissue-specific levels of MHC class I expression and of immune activated expression Although transcribed at very different levels in different tissues, the PD1 promoter is constitutively in an open chromatin conformation regardless of transcriptional activity, enabling the cells to respond rapidly to infection by intracellular pathogens by increasing MHC class I transcription rates without chromatin remodeling The separation of chromatin modifying activities and sustained expression found in the mammalian MHC class I, PD1, barrier element is similar to that observed in the chicken β-globin 5′ compound boundary element that separates the gene locus from an upstream 16 Kb heterochromatin domain Although alone it is not sufficient to sustain transcription, active chromatin structure contributes to MHC class I barrier function. The binding of CBP and PCAF to the 3′ intergenic region of PD1 suggests that the mechanism of the MHC class I barrier function involves active recruitment of histone modifying enzymes. Indeed, histone modifying enzymes have been implicated in mediating the activities of barriers associated with other genes in vivo or in in vitro gel shift assays (data not shown), despite the presence of a consensus E box binding sites within the 3′ intergenic barrier region. The transcription factor(s) that recruits CBP and PCAF to the PD1 barrier remains to be identified. In initial gel shift studies with HeLa nuclear extract, a complex dependent on sequences within the 79 bp segment between the polyA addition site and the Sac I site was observed (data not shown). However, the sequence was not homologous to any known canonical binding sites and it was not possible to identify the cognate factors. The HMG box containing protein, HBP1, has been shown to bind to an element with barrier function within the locus control region of the human CD2 gene TTCATTCATTCA, does not. Moreover, DNA sequence homology searches did not identify any additional known binding sites within the entire 3′ intergenic region.Since neither CBP nor PCAF bind DNA directly, they must be recruited to the barrier element by one or more transcription factors. USF1/2 has been shown to play a critical role in mediating barrier activity of the chicken β globin gene, recruiting histone modifying enzymes The histone variant, H2A.Z, interacts with both promoters and barrier elements in yeast Interestingly, loss of PD1 expression did not depend on the acquisition of repressive chromatin marks. Modifications associated with silenced genes, H3K9 dimethylation and promoter CpG methylation, did not appear in the absence of the barrier element. Consistent with these findings, several studies have shown that transcriptional silencing by heterochromatin propagation is a gradual process, composed of stepwise formation of heterochromatin, leading to the existence of intermediate state heterochromatin, in which silencing is partial and histone modifications are a mixture of active and inactive chromatin In conclusion, the PD1 barrier element, like other functionally defined barrier elements, serves to sustain gene expression and to maintain histone modifications and low nucleosomal density associated with active transcription PD1/Bam extends from the Hind III site approximately 1 Kb 5′ of the PD1 promoter through the entire coding sequence (CDS),and 730 bp beyond the poly A addition sites to a Bam HI site in the 3′ intergenic region cloned in pBR322 . PD1cDNATransgenic mice were generated as described previously 6 HeLa cells were transfected with 10 µg of DNA using Lipofectamine  according to manufacturer's protocol. M12 cells were transfected as described before For transient transfection assays, 1×106 cells were co-transfected with 0.5 µg of the indicated clone DNA and 10 µg plasmid containing the thymidine kinase gene (TK) using Lipofectamine. The cells were maintained in DMEM for 48 hours and then the medium was replaced with fresh DMEM containing 1% hypoxanthine-aminopterine-thymidine (HAT). Isolated clones were analyzed for PD1 expression by FACS.For stably transfected Ltk(−) clones, 1×10FACS was carried out as described before RNA was prepared using Trizol (Invitrogen). Northern blotting and real-time RT-PCR for PD1 analyses were done as described before 8 Ltk(−) cells stably co-transfected with TK plasmid and different PD1 constructs according to the protocol published by O'Neill and Turner Nuclei were prepared from 1×10Native chromatin immunoprecipitation (N-ChIP) was performed to analyze histones modifications according to the protocol published by O'Neill and Turner 6 Ltk(−) cells stably transfected with PD1cDNAint1-2/5-1 construct were crosslinked as detailed in the published protocol, followed by addition of 0.125 M glycine for five minutes at room temperature to stop the crosslinking reaction. Cells were washed twice with ice-cold PBS in the presence of protease inhibitors and then resuspended in SDS lysis buffer. The cells lysates were then sonicated in the presence of glass beads to give fragments size ranging from 1–0.3 Kb. ChIP proceeded according to the published protocol, using the following antibodies: anti CTCF , anti USF1 , anti USF2 , and anti CBP , followed by reversal of crosslinking by over night incubation with NaCl at 65°C. A no antibody control was included as well.Cross-linked ChIP (X-ChIP) was used to test for binding factor according to protocol published by Upstate and using Upstate ChIP assay kit  with a few modifications. Briefly, 1×10DNA immunoprecipitated by N-ChIP or X-ChIP reactions was analyzed by real time PCR using the following primers:Promoter primers:5′ TACATATGAAACACTCCTGCTACCTTCC; antisense: 5′ CCAGTAAAGGTTGTATTCCATGA(-1000): sense: 5′ TGTGCGGGGCT TTTACATTTC; antisense: 5′ CACTGGAGGTTTATGTCTGCTTCTG(-600): sense: 5′ CGCAACCTGTGTGGGAC; antisense: 5′ GGGTGGGTGGAGAGTTT(-100): sense: Coding region:5′ GAACAAGGCCGCTGCG; antisense: 5′ CAGCAGA GTCGCACCTTCintron 1: sense: 5′ CAGACCCTGCTCAGCCC 3′; antisense: 5′ G GGTGGGTGGAGAGTTTExon 5; sense: 3′ end:5′ TCTGTGTTCCTATGAGCATCC; antisense: 5′GAACAC AGGTCAGGGTGAG3′UTR(1): sense: 5′CTGACATCTCCATCCTTACT; antisense: 5′ CCTCATGGCACCAATTAGAA3′UTR(2): sense: 5′ TGCCATGAGGAGTTGAGG GGATAATAAA; antisense: 5′ GGATTCTGGAAGGTTCTCA3′UTR(3): sense: 5′ ATTCAGGAGCTTGGTTAGCAA; antisense: 5′ GCCAAATACA AACACAGAATAAA3′distal(1): sense: 5′ CCTCCTTCCAGAGATGTTTA; antisense: 5′ ACCTCCATGTGCCGTGAGT3′distal(2): sense: 5′ GGGTCTAATCAGAGCTACAG; antisense: 5′ AGCATTGCTGTGTGTTGTGG3′distal(3): sense: Quantitative real-time PCR was performed using ABI7900 with SYBR green real time PCR kit (Applied Biosystems). Results were calculated as percentage bound/input DNA as compared to no antibody control.Genomic DNA was prepared from Ltk(−) clones stably transfected with PD1cDNAint1-2/Bam, PD1/Sac and PD1/polyA. The DNA was digested with HpaII (CpG methylation sensitive) or Msp I (CpG methylation insensitive). After digestion, the DNA was precipitated and a promoter region spanning two HpaII/Msp I restriction sites was amplified in a real time qPCR reaction using (−100) primers (see above). Results were calculated as percentage PCR product of non-digested DNA amplified with the same primers.Figure S1Tissue expression pattern of PD1cDNAint1-2/Bam transgenic mice parallels that of PD1/Bam transgenic mice. RNA was isolated from the indicated tissues and assessed by qPCR for the levels of PD1 specific RNA using a primer that spans exons 2–3. RNA levels were quantitated relative to an 18S rRNA internal control.(0.01 MB PDF)Click here for additional data file.Figure S2Loss of PD1 expression in the absence of 3′ intergenic sequences is not associated with promoter CpG methylation. DNA was isolated from L cell clones stably transfected with PD1cDNAint1-2/Bam, PD1cDNAint1-2/Sac and PD1cDNAint1-2/polyA and maintained in the absence of HAT medium. Although all three clones retain the respective transgenes, only the PD1cDNAint1-2/Bam line expresses PD1. CpG methylation at the promoter in each of the clones was determined as described in Supplementary Experimental Procedures. Results show real time PCR amplification of PD1 promoter region after digestion with Hpa II or Msp I, relative to non-digested DNA. Results are representative of three independent clones from each construct.(0.06 MB TIF)Click here for additional data file.Figure S33′ intergenic sequences do not affect nucleosomal organization of expressed PD1 genes in the presence of selective pressure. Nucleosomal occupancy was determined across the PD1 gene in L cell clones stably transfected with PD1cDNAint1-2/Bam, PD1cDNAint1-2/Sac and PD1cDNAint1-2/polyA and maintained in the presence of HAT medium. All three clones expressed the PD1 gene to comparable levels.(1.27 MB TIF)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file.Table S2(0.03 MB DOC)Click here for additional data file.Table S3(0.03 MB DOC)Click here for additional data file.


Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5′ splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/. Trypanosoma brucei add a leader sequence to their mRNAs through a reaction called trans-splicing. Until now the splice sites for most mRNAs were unknown in T. brucei. Using high throughput sequencing we have developed a method to identify the splice sites and at the same time measure the abundance of the corresponding mRNAs. Analyzing three different life cycle stages of the parasite we identified the vast majority of splice sites in the organism and, to our great surprise, uncovered more than 2500 alternative splicing events, many of which appeared to be specific for one of the life cycle stages. Alternative splicing is a result of the addition of the leader sequence to different positions on the mRNA, leading to mixed mRNA populations that can encode for proteins with varying properties. One of the most obvious changes caused by alternative splicing is the gain or loss of targeting signals, leading to differential localization of the corresponding proteins. Based on our findings we hypothesize that alternative splicing is a major mechanism to regulate gene expression in T. brucei and could contribute to protein diversity in the parasite.Some organisms like the human and animal parasite  Trypanosoma brucei is a unicellular eukaryotic parasite with a digenetic life cycle alternating between the tsetse fly and a variety of mammalian hosts. Besides its importance as a human and veterinary pathogen it has been key to the discovery and understanding of general biological principles such as RNA editing, antigenic variation, GPI anchoring and trans-splicing T. brucei revealed a compact structure containing about 9000 predicted genes, including 900 pseudogenes and 1700 genes specific to T. bruceitrans-splicing of a 39 nt leader sequence to the 5′ end and polyadenylation of the 3′ end Trypanosoma cruzi, where approximately half of the genome is regulated at the level of transcript abundance T. brucei genome for such elements it is crucial to delineate the 5′ UTR of each expressed gene. In the past, bioinformatics approaches have been used to predict 5′ splice sites in T. brucei, but few of these have been confirmed experimentally trans-splicing of a spliced leader has been identified not only in protozoa but also in cnidarians, nematodes, flatworms and ascidians, SLT could potentially be applied to a wide variety of organisms Long slender and short stumpy bloodstream form trypanosomes are the proliferative and quiescent life cycle stages, respectively, in the mammalian host, while the procyclic form is a proliferative form in the midgut of the insect host. In order to map the 5′splice sites and quantify abundance of the corresponding transcripts in these three life cycle stages, poly(A)-RNA was purified and first strand cDNA was synthesized using random hexamers . In ordeT. brucei, the Antat 1.1 variant surface glycoprotein  or the telomeric expression sites . Depending on the library, 77% to 79% of all genes had at least one tag associated with their 5′ UTR or annotated coding sequence. The median number of tags per gene ranged from 12 in the long slender bloodstream form to 24 in the procyclic form  indicated large variations in transcript abundance between adjacent genes. Of 270 monocistronic mRNA transcription units that have been annotated, 76 had major splice sites less than 2kb upstream of the ATG and eight were differentially expressed.It has recently been shown that procyclin-associated genes (PAGs) are located preferentially at a subset of strand-switch regions, or between polycistrons that are transcribed by different polymerases, and that a region within PAG1 could silence transcription by Pol I −5; In order to evaluate if the expression profiles were consistent with previously published data we first analyzed 20 well-studied genes and found good agreement between the direction and magnitude of change in transcript abundance between life cycle stages . These iet al.  and mapped the sequence tags to the active VSG expression site . The mosT. brucei genome with a median of two splice sites per gene  at the N-terminus as determined by MITOPROT . Thus thtrans-splicing. This was tested on T. brucei, where we obtained 85% coverage of all genes with fewer than 1 million sequence tags. A major improvement over traditional microarray technology is the possibility of sequencing directly from the spliced leader, which allows selective analysis of the transcriptome of intracellular parasites like the amastigote forms of Trypanosoma cruzi or forms that are closely associated with their host and extremely difficult to purify like the epimastigote form of T. brucei in tsetse salivary glands.We have developed a cost effective method to analyze whole genome expression and splicing profiles from organisms employing leader T. brucei. Each sequence tag covered at least 24 nucleotides 3′ of the spliced leader/5′ UTR junction. Even though the analyzed strains (Antat 1.1 and Mitat 1.2) were not identical to the genome strain (TREU 927) mapping of the 24mer sequence tags onto the TREU 927 genome was very successful, given that the majority (98%) could be aligned with high statistical significance using a combination of mismatch and sequence quality scores (see Using the SLT approach we sequenced more than 1 million splice site tags from poly(A)-mRNA from each of three life cycle stages of ores see . In the In general, our data are in good agreement with the current genome annotation, and strongly support both the number and positions of putative transcription start sites identified by binding of specific histones The large variation in transcript abundance that we detected within transcription units was expected and is in good agreement with previous studies, strengthening the notion that steady state RNA levels in trypanosomes are regulated mainly post-transcriptionally at the level of RNA processing and/or RNA stability. When we analyzed the monocistronic transcription units we found >70% unlikely to be expressed in the life cycle stages analyzed because we could not detect any splice sites within 2 kb upstream of the start codon. Of the remaining 76 transcripts, only 8 showed differential expression.T. brucei using genomic arrays or single probe arrays have indicated a rather static transcriptome with relatively few changes between the bloodstream and procyclic forms. Estimates ranged from 2–6% of the genome being regulated at the transcript abundance level et al. using eight oligonucleotides per gene on a Nimblegen microarray found that up to 700 transcripts or (8%) change expression between the two life cycle stages T. cruzi is regulated at the level of transcript abundance Previous microarray studies of et al. published a study describing the expression profile in bloodstream and procyclic forms of T. brucei using RNAseq 5 to 106). However, there are also a number of features that do not correlate so well, most striking of which is the difference in expression profile. We identified almost 40% of the genes as being regulated significantly while Siegel et al. found only about 10%. There are several reasons that could explain the differences observed: (i) strain differences and the number of life stages analyzed, (ii) growth conditions for bloodstream forms (in vitro versus in vivo), (iii) cDNA preparation, with RNAseq being more likely to capture precursors and breakdown intermediates (e.g. intron sequences), and (iv) scaling of the data (tags/million versus constant median count/gene). The major advantage of SLT when compared to conventional RNAseq is the cost effective mapping of 5′ splice sites in splice leader bearing organisms, especially intracellular parasites where the purification of RNA is very difficult.During the revision of this manuscript Siegel According to our study, VSG transcripts account for 7–11% of spliced mRNAs in bloodstream forms trypanosomes, which is in excellent agreement with the value obtained from hybridization data T. brucei genome (T. cruzi (LYT1) and for alternative cis-splicing in other systems T. cruziThe data presented here describes the splice sites for 85% of the annotated genes in the i genome . Splicedi genome . The leai genome . Interesbona fide start codon is within the open reading frame. A second set of transcripts (>600) indicated the possibility of 5′ extensions to the currently annotated open reading frames, which would effect changes in the N-terminus of the corresponding protein. The evaluation of these N-terminal extensions is much more difficult and will require additional experiments. However, by screening the recently published proteomics dataset from Panigrahi and co-workers, we were able to identify 11 candidates where peptides corresponding to a region upstream of the annotated start codon are expressed in the procyclic form trypanosomes More than 500 transcripts with splice sites exclusively 3′ of the annotated start site were identified by SLT. While we cannot exclude that a very small fraction is still spliced upstream of the annotated AUG, we consider it much more likely that the T. brucei used different splice sites on an artificial construct, but it remained unclear if, and how frequently, this might occur in the T. brucei transcriptome Most surprising was that a large number of transcripts showed differential abundance of alternative splice variants in the three life stages analyzed. A previous report indicated that T. brucei brucei AnTat 1.1 and MITat 1.2 (221) were used in this study. Late procyclic forms of AnTat 1.1 were cultivated in SDM79 supplemented with 10% FBS. Bloodstream forms were grown in mice. For bloodstream forms of AnTat 1.1, mice were immunosuppressed with 260mg/kg cyclophosphoamide (Sigma) 24 hours prior to intraperitoneal injection of 106 parasites. Short stumpy parasites were harvested 5 days post-infection at a density of 2–4×108/ml blood. 75–80% of the cells showed a short stumpy phenotype as determined by light microscopy of blood smears after methanol fixation. Long slender forms were harvested from untreated mice at day three post-infection at a density of <5×107 cells/ml blood. MITat 1.2 bloodstream forms were grown in mice as described above for long slender bloodstream forms of AnTat 1.1. Parasites were purified from whole blood using DE-52 anion exchange resin equilibrated to pH 8 with a bicine glucose buffer. After purification cells were centrifuged and resuspended in TriPure RNA isolation reagent .RNA was extracted using TriPure  according to the manufacturer.5′- AATGATACGGCGACCACCGAGATCTACAGTTTCTGTACTATATTG -3′), 2 units Taq polymerase  and 4.5 µl H2O by incubation for 5 minutes at 50°C and then 5 minutes at 72°C. The cDNA was purified on a Qiagen MinElute column  and eluted in 10ul TE buffer. Adapter ligation to the purified dsDNA was done using , 10.0 µl H2O, 1.0 µl Fasteris customized bar-coded paired-end Illumina adapter, 600 units T4 DNA Ligase  for 1 hour at room temperature. The ligation mix was purified from unligated linker with streptavidin beads (Dynabeads). After incubation at room temperature for 15 minutes beads were separated on a magnetic stand, and washed twice and then resuspended in 20 µl 10 mM Tris buffer. 5 µl was used for PCR amplification with primers (5′- AATGATACGGCGACCACCGA -3′/5′- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAAC -3′) following the standard Illumina mRNA-SEQ library amplification protocol. Fragments in the range 120–160 bp were separated on a 2% agarose-TBE gel and subsequently purified on Qiagen Gel Extraction MinElute columns . Quality control for insert size was performed by TOPO cloning and subsequent ABI sequencing. Sequencing on the Illumina Genome Analyzer was carried out using the following sequencing primer (5′- ACCGAGATCTACAGTTTCTGTACTATATTG -3′).Poly(A) mRNA was purified from 7.5 µg total RNA using Dynabeads oligo-(dT) beads according to the manufacturer . First strand cDNA was synthesized from poly(A) RNA using random hexamers and Superscript II reverse transcriptase  in a final volume of 20 µl for 1 hour at 42°C. Half of the first strand mix was used for second strand synthesis . Second strand synthesis was done using 2 µl 10× Thermopol buffer (New England BioLabs), 1 µl dNTPs 10 mM, 1 µl 2nd strand primer 10 µM , one from procyclic form mRNA (Antat1.1), 2×2 RNAi libraries (uninduced and induced), where each pair could be considered a biological replicate. Lastly we prepared and sequenced one conventional RNAseq library from procyclic mRNA (Antat1.1).T. brucei TREU927 using maq (http://maq.sourceforge.net) with n = 3 and an effective first read length of 24. Single mapping reads were separated from multi mapping reads by an alignment quality threshold of 30. Tags ending in the same position were joined together, and their counts were added  and then visualized using Gbrowse bona fide (CDS) start codon. Sequences surrounding the splice sites were extracted using bioperl and visualized as sequence logos using WebLogo −5. Scatterplots of the differential gene expression levels of all libraries were produced using R −5). Significant differences were termed differential trans-splicing events. Expression levels were pooled over T. brucei specific KEGG pathways and visualized as heatmaps after log2 transform and hierarchical average linkage clustering of euclidian distances using MeV .Base calling was performed using the Genome Analyzer Pipeline (Illumina). Linker sequences were removed while separating reads according to identified barcodes. Only sequence reads of inserts with a length of at least 24 nucleotides were retained. A pipeline was set up using languages with open source interpreters  to automate the following analysis. The reads were mapped to the genome sequence of et al. with the mapped splice sites substituted for the previous small mapping EST set Anti-sense splice sites were detected using bioperl. In order to simulate gene coverage a subset of reads was drawn randomly without replacement from one library and the mapping and analysis pipeline executed for each subset. This was repeated five times for each subset size. Saturation curves were drawn for several parameters, with error bars given as confidence intervals assuming normal distribution. Comparison to splice model predictions were made according to Benz CGCTATTATTAGAACAGTTTCTGTAC-3′ (Tm 55°C), and reverse primers 5′- GTTGCATCCGGTGTTCTTTT -3′ (Tb11.02.2700) and 5′-AGCAGTCATCAATTCTTCCT-3′ (Tb927.6.4240) were used. The reaction was performed in 25µl, , 0.2mM dNTP and 30ng/ul cDNA) for 30 cycles. RT qPCR was done essentially as described previously. cDNA for RT-qPCR was prepared as described above. The primers were designed such they would amplify regions of 80 to 150 nucleotides using the online software tool for real time PCR from Genescript  in the presence of 1mM dNTPs, 360 ng random hexamers, 80 U RNasIn  and 60 U AMV reverse transcriptase . Subsequently 1µl of this reaction was used for PCR. For PCR the splice leader primer nescript . Real-tihttp://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE22571 or through our website http://splicer.unibe.ch/ or in future also from TriTrypDB (http://tritrypdb.org/tritrypdb/).The sequence data can be accesed through GEO .Figure S1Spliced leader trapping approach. Schematic description of (A) library preparation, sequencing and (B) bioinformatics analysis.(0.62 MB PDF)Click here for additional data file.Figure S2et al. (2008). Transcripts more abundant in procyclic forms than in the bloodstream forms correlated with a coefficient of 0.77 or 0.93 to the SLT approach depending on the statistics (fspma or limma) used in the study by Koumandou and coworkers Microarray correlation. Correlation between the SLT approach and a recent microarray study by Koumandou (0.66 MB PDF)Click here for additional data file.Figure S3et al. (2008). Transcripts more abundant in bloodstream forms than the procyclic forms correlated with a coefficient of 0.43 or 0.23 to the SLT approach depending on the statistics (fspma or limma) used in the study by Koumandou and coworkers Microarray correlation. Correlation between the SLT approach and a recent microarray study by Koumandou (0.38 MB PDF)Click here for additional data file.Figure S42 changes for KEGG pathway genes for three life cycle stages of T. brucei. The dendrogram was obtained using hierarchical average linkage clustering of euclidian distances. LS, long slender bloodstream form; SS: short stumpy bloodstream form; PC, procyclic form.Expression profile of KEEG pathways. Heatmap of log(0.18 MB PDF)Click here for additional data file.Figure S52 fold.Expression profile glycolysis pathway. Regulation of the glycolytic pathway as annotated in KEGG. The differences in expression between long slender bloodstream and short stumpy (maroon), short stumpy and procyclic (light green) and long slender bloodstream from and procyclic form (dark green) are shown in log(0.09 MB PDF)Click here for additional data file.Figure S62 fold.Expression profile oxidative phosphorylation pathway. The regulation of the citric acid cycle pathway as annotated in KEGG. The differences in expression between long slender bloodstream and short stumpy (maroon), short stumpy and procyclic (light green) and long slender bloodstream form and procyclic form (dark green) are shown in log(0.26 MB PDF)Click here for additional data file.Figure S72 fold changes of expression are shown for the SLT (blue) and RT qPCR. A positive value indicates higher steady state level RNA abundance for the corresponding gene in procyclic cells.Correlation RT qPCR and SLT. Comparison of expression levels between SLT and RT qPCR from procyclic over long slender bloodstream form cells. Log(0.17 MB PDF)Click here for additional data file.Figure S8Comparison RNAi libraries uninduced induced. Comparison of SLT and Northern hybridization. (A and B) Relative levels of RNA transcripts detected by either SLT or Northern blot analysis using RNAi cell lines Alba1 and Alba 3/4. −Tet/+Tet refers to RNA isolated from uninduced and induced cell lines after 7 days. Quantitation of Northern blots was performed with a Phosphoimager using the 18S rRNA as a loading control. RNA levels from uninduced cells were set at 100%. (C and D) Scatter plots of mRNA expression levels (log10 tags per million) between two libraries. Each dot represents one gene. (C) RNAi experiment Alba1_N non-induced vs. Alba1_I induced. (D) RNAi experiment Alba34_N non-induced vs. Alba34_I induced. (E) Overall correlation of steady state RNA levels of the entire genome from uninduced and induced cell lines.(0.27 MB PDF)Click here for additional data file.Figure S910 tags per million) between SLT and RNAseq from poly(A) procyclic T. brucei mRNA.Scatter plot expression profile comparison SLT and RNA seq. Scatter plot of mRNA expression levels (log(0.09 MB PDF)Click here for additional data file.Figure S10Alternative splice variant A. Example of splicing type A, no downstream AUG is found in the reading frame of the annotated gene. This also represents an example of a differentially spliced transcript, where the major splice site changes between long slender (LS), short stumpy (SS) and procyclic form (PC). RT qPCR amplicon shows the region that was used for RT qPCR. Ratio BS/PC (SLT) indicates the ratio of SLT tags, for the downstream tags the upstream tags are added in order to be comparable to the qPCR results (i.e. 1 downstream+9 upstream).(0.07 MB PDF)Click here for additional data file.Figure S11Aternative splice variant B. Example of splicing type B with a downstream AUG (M) in the reading frame of the annotated gene, but use of that AUG would lead to loss of the signal peptide predicted by SingalP. This also represents an example of a differentially spliced transcript where the major splice site changes between the long slender (LS), short stumpy (SS) and the procyclic form (PC).(0.08 MB PDF)Click here for additional data file.Figure S12Alternative splice variant C. Example of splicing type C with several uORFs between the two alternative splice sites. uORFs are in color according to the reading frame. The minimum length for an uORF was set to six amino acids. Long slender (LS), short stumpy (SS) and the procyclic form (PC).(0.08 MB PDF)Click here for additional data file.Figure S13Alternative splice variant D. Example of splicing type D with an overlapping open reading frame of 384 bases (ORF2). Long slender (LS), short stumpy (SS) and procyclic form (PC).(0.09 MB PDF)Click here for additional data file.Table S1Correlation of SLT expression profile(0.04 MB PDF)Click here for additional data file.Table S2Robustly regulated transcripts(0.08 MB PDF)Click here for additional data file.Table S3KEGG pathways and their expression levels in three life cycle stage(0.08 MB PDF)Click here for additional data file.Table S4Correlation of expression profile between SLT and qPCR for 10 selected genes(0.03 MB PDF)Click here for additional data file.Table S5Proteins with N-terminal extensions(0.04 MB PDF)Click here for additional data file.Table S6Comparison of expression levels from differential splice sites using RT qPCR and SLT(0.04 MB PDF)Click here for additional data file.Table S7Alternative splicing and dual localization of tRNA Synthetases(0.07 MB PDF)Click here for additional data file.Table S8Oligonucleotide sequences(0.04 MB PDF)Click here for additional data file.


Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N–bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In  Although all cells of a developing organism have the same DNA, they express different genes and transmit these gene expression patterns to daughter cells through multiple rounds of cell division. This cellular memory for gene expression states is maintained by two groups of proteins: Polycomb-group proteins (PcG), which establish and maintain stable gene silencing, and trithorax group proteins (trxG), which counteract silencing and enable gene activation. It is unknown how this balance works and how exactly these proteins are recruited to their target sequences. By mapping the genome-wide distribution of PcG and trxG factors and proteins known to recruit them to chromatin, we found that putative PcG recruiters are not only colocalized at PcG binding sites, but also bind to many other genomic regions that are actually the binding sites of the Trithorax complex. We identified new DNA sequences important for the recruitment of both PcG and trxG proteins and showed that the differential binding of the recruiters PHO and PHOL may discriminate between active and inactive regions. Finally, we found that the two fragments of the Trithorax protein have different chromosomal distributions, suggesting that they may have distinct nuclear functions. Comparison of the genome-wide distribution of PcG, trxG, and sequence-specific DNA binding proteins allowed the identification of key signals leading to Polycomb or Trithorax recruitment. Drosophila, PcG and trxG proteins are recruited to chromatin by regulatory DNA elements called PcG and trxG response elements . These elements were shown to drive epigenetic inheritance of silent and active chromatin states throughout development [Scm-related gene containing four MBT domains). Several trxG complexes have been identified: TAC1 (Trithorax Acetylation Complex) with the histone methyltransferse Trithorax (TRX), NURF, SWI/SNF, ASH1, and ASH2  and trithorax group (trxG) proteins are conserved chromatin factors that maintain, respectively, the memory of inactive or active states of homeotic genes throughout development. They also regulate many other target genes (reviewed in ) and miselopment ,4. Biochelopment ). PRC2-telopment . PRC1 coelopment . The Phoews, see ,8). Inteews, see ,10. Howeews, see . These ppho/phol double mutants despite lack of detectable PHO and PHOL proteins [pho/phol double-mutant wing discs [PcG and trxG complexes (except PhoRC) do not bind their target DNA in a sequence-specific manner in vitro, but are recruited to PRE/TRE sequences in vivo. A simple pathway for PcG protein recruitment based on stepwise recruitment of PRC2 proteins by PhoRC, followed by PRC1 recruitment by the H3K27me3 mark deposited by PRC2 has been suggested . Howeverproteins . Howeverng discs , suggestng discs ). MutatiDrosophila embryos of two PRC1 components and their associated histone mark H3K27me3, the N- and the C-terminal part of the TRX protein and their associated histone mark H3K4me3 as well as four sequence-specific DNA binding proteins known to be involved in recruitment of Polycomb proteins. Our results show the complementarity between PcG and trxG protein binding in the genome and suggest that multiple DNA binding proteins participate in setting up this PcG and trxG protein distribution.Recently, the distribution of several core components of PcG members and their associated histone modifications has been analyzed in fly as well as mammalian cells –22. Yet,Drosophila melanogaster embryos coupled with genome-wide high-density tiling arrays, we mapped the distribution of the PRC1 components: PC and PH, the N- and the C-terminal part of the Trithorax protein , and the histone H3K27me3 and H3K4me3 marks. We also determined the genome-wide binding profile of GAF, PHO, PHOL and DSP1, four DNA binding proteins thought to be involved in PcG recruitment. Reproducibility of biological replicates is shown in p-values for enrichment separated by small intervening subregions that were enriched although their p-values were not significant  in 4–12-h-old cant see A. The secant see . Surpriscant see B. Bindincant see , but thecant see C where T studies ,22 and f studies . In addi studies ,25), whoRecent analysis of H3K4me3 and H3K27me3 in mouse and human cells revealed the coexistence of these two marks in a large fraction of the H3K27me3 regions –29. ThesWe sought a comprehensive characterization of the joint distribution of PcG and trxG factors and associated marks. Many of the data tracks are highly correlated among themselves  and S6, ph, the hh, and the en genes were previously identified as PREs, suggesting that in general, most of the PH clusters are indeed PREs. The H3K27me3 supercluster also included three clusters with lower levels of PC and a general lack of PH and cofactors. We labeled them as “Strong,” “Medium,” and “Weak” PC clusters.As shown in Similarly, the H3K4me3-marked supercluster was subdivided by the algorithm into four clusters. These clusters reflect clear organization around annotated TSSs, as identified by their TSS enrichment statistics B and binPC and H3K27me3 were bound in large regions, often greater than 5 kb, with the largest ones spanning several hundred kilobases see  and S4A.Nearly all PH peaks were specific to PC and H3K27me3 regions  of them did not overlap any bound regions in S2 cells. These data are corroborated by the analysis of the distribution of the PC protein which, similar to H3K27me3, forms large domains. In general, H3K27me3 differences between embryos and S2 cells paralleled differences in PC binding. The same was observed in a comparison between ChIP on chip binding of PC from embryos and the PC DamID profile obtained previously in Kc cells . InteresTo gain more insight into PRC1 recruitment to chromatin, we examined the distribution of PcG recruitment factors at PH sites that are also bound by PC (PRC1 sites). The combination of different PcG recruitment factors at the PRC1 sites as compared to the genome is listed in The K4me3-recruiter cluster (including strong GAF and DSP1 and medium to weak H3K4me3 levels) is located in a position just upstream to the TSS. The K4me3-TSS cluster  is usually following it and is almost exclusively observed over the 2 kb around the TSS. Finally, the K4me3 cluster (high H3K4me3 levels without TF occupancy) is enriched 3′ to the TSS. This organization suggests that binding of GAF and DSP1 can promote the activation of a TSS upon binding of TRX-N and the PHO/PHOL factors. Therefore, PH target promoters are strongly bound by PHO and TRX-C and depleted of PHOL and TRX-N B, whereaWe further analyzed active promoters and PREs/TREs by analyzing TRX binding. The human TRX homolog MLL1 is cleaved by Taspase1, generating an N-terminal and a C-terminal fragment, which can heterodimerize in vitro ,10. Low In the case of PHO, PHOL, and GAF, sequence-specific DNA binding in vitro has been shown previously ,33. By aIn order to determine whether other sequence features may characterize PREs specifically, we further developed the unsupervised spatial clustering methodology  to allowTwo motifs (GAGA and the CA repeat motif) are marking clearly the PH sites and the K4me3-recruiter clusters. Three additional motifs are strongly marking the K4me3-TSS cluster and clearly discriminating it from the spatially coupled K4me3-recruiter cluster sites. Two of them are motifs bound by the Myc, Max, and Mad/Mnt proteins  and inclImportantly, we also discovered motifs that discriminate between K4me3-recruiters and PH sites. The CAACAACAA motif is enriched around K4me3-recruiters, but not in and around PH sites see also . On the In addition to these motifs, the consensus sites for PHO/PHOL, DSP1, and GAF are more strongly enriched at the 300-bp core regions around the maximal binding peak of PH than around the other genomic regions bound by the factors without PH –S8. ThusOf particular interest is the distribution of the PHO motif around PH sites and the K4me3-TSS clusters. Unlike the GAGA (or CACA) motif, the frequency of motifs with sequence similarity to consensus PHO motifs is high, but these motifs are not well localized at PH sites. High predicted PHO affinities  and toy (twin of eyeless) have very low expression in haltere/third leg discs and are highly expressed in eye discs  as compared to eye discs. Both so and toy TSS showed higher levels of PH binding in haltere/third leg discs, where these genes are silenced (OFF), as compared to eye imaginal discs (ON). At the Ubx TSS and the bx PRE, levels of PHOL were significantly higher in haltere/third leg discs (ON) as compared to eye discs (OFF). With regards to PHO, stronger binding was observed at the PREs in eye discs (OFF state), whereas at so and toy stronger binding was observed in haltere/third discs (OFF state) compared to eye discs (ON). In summary, a significant decrease in the levels of PH in tissue where target genes are active correlates with a decrease in the PHO/PHOL ratio. On the other hand, increased PH levels at genes that are OFF in a certain tissue correlates well with an increased PHO/PHOL ratio.PHO and PHOL share sequence homology, were shown to bind the same DNA motif in vitro, and have been proposed to play redundant roles in PcG-mediated silencing (reviewed in ). Notablal discs  and is rye discs A. For Ubd region B. PH, PHd region  and S16.1pho homozygous , and gene activation is even stronger for the Antp gene (between 10- and 30-fold) .To further examine the function of the PHO/PHOL ratio in Polycomb-dependent gene silencing, we performed quantitative reverse-transcriptase PCR (RT-PCR) on eye, haltere/third leg and wing imaginal discs from wild-type and e of PHO ) third i30-fold) A. These 1pho mutation in haltere/third leg discs where the Ubx gene is transcribed and in wing discs where Antp is active. We detected a consistent, yet slight, decrease of their transcripts   B. These Chc and Rp49, which are bound by PHO in wild-type embryos. Again, Chc expression decreased 1.6 times in both eye and haltere/third leg discs and Rp49 1.3 times in eye discs from 1pho mutant larvae (phol81A null mutant background (unpublished data), pointing to a redundant role of PHOL in gene activationBecause we found a high colocalization of PHO and PHOL with TRX-N at many gene promoters not related to PcG-mediated silencing, we performed quantitative RT-PCR to check the expression of two constitutively transcribed genes such as t larvae C. In conThese results, together with the recent work of Beisel et al. , indicatDrosophila embryos revealed several important features. First, similar to the PcG distribution in Drosophila cell lines, PcG proteins strongly colocalize and form large domains containing multiple binding sites. Second, the N-terminal and C-terminal fragments of TRX show different binding affinities to repressed and active chromatin. The N-terminal fragment of TRX has low affinity to PcG binding sites but is strongly bound to thousands of active promoter regions that are trimethylated on H3K4, whereas the C-terminal fragment of TRX only showed high binding affinity to PcG binding sites. Third, the majority of PcG recruiter binding sites are associated with H3K4me3 and TRX-N foci and not with PH binding. The binding ratio between the PHO protein and its homolog PHOL is a major predictive feature of PcG versus TRX recruitment. Finally, supervised and unsupervised sequence analysis methods led to the identification of sequence motifs that discriminate between most of the PcG and TRX binding sites, but these motifs are likely to be working jointly, and none of them seems to drive recruitment by itself.The genome-wide mapping of PcG factors, TRX, their associated histone marks, and potential PcG recruiter proteins in Drosophila. These elements are not defined by a conserved sequence, but include several conserved motifs, which are recognized by known DNA binding proteins like GAGA factor (GAF), Pipsqueak (PSQ), Pleiohomeotic and Pleiohomeotic-(like) (PHO and PHOL), dorsal switch protein (DSP1), Zeste, Grainyhead (GH), and SP1/KLF. Our genomic profiles provide a comprehensive view on the potential role of these factors in the establishment of PcG domains.To date, PREs have been only characterized in Fab-7  PRE/TRE [The presence of PHO at all PREs indicates that PHO is a crucial determinant of PcG-mediated silencing, consistent with earlier analysis on one particular PRE ,33,42–46 PRE/TRE . Therefo PRE/TRE ,49 to re PRE/TRE ,50,51. TIn addition to an increased density of motifs for GAF, PHO, and PHOL, unsupervised spatial cluster analysis identified specific motifs that distinguish the PH sites from the K4me3 cluster. Although the identity of the factors binding to these motifs is unknown, this suggests that the DNA sequence of PREs contains much of the information needed to recruit PcG proteins and to define silent or active chromatin states. With this distinction, it may be possible to develop an algorithm to faithfully predict the genomic location of PREs. Earlier attempts to predict PREs in the fly genome have made progress toward this goal, but they are still far from reaching the required sensitivity and specificity ,22,24,25Our ChIP on chip data showed that PHO binding comes in two distinct flavors. In one class of target sites, PHO binding coincides with PH sites within PC domains, whereas outside these domains, it is largely colocalized with PHOL, TRX-N, and H3K4me3 . PHOL biUbx TSS, the bx PRE, and the bxd PRE (the same primers were used in our study) by comparing haltere/third leg imaginal discs (ON state) with wing imaginal discs (OFF state). They found a 50% reduction of PH binding levels at the bx PRE, a minor decrease at bxd, and no change in the Ubx TSS. Our ChIP experiments demonstrated a 50% decrease in PH levels at bx PRE and at the Ubx TSS and a minor decrease at bxd PRE when comparing haltere/third leg imaginal discs to eye imaginal discs. We also observed a slight decrease in the levels of PHO in haltere/third leg disc (ON state) as compared to eye imaginal discs (OFF state) at the bx and bxd PRE, whereas Papp and Muller [Ubx, whereas all cells silence this gene in eye imaginal discs.Papp and Muller  analyzedd Muller  did not 1pho mutant eye discs, the absence of PHO causes derepression of the homeotic genes Ubx and Antp. However, the expression levels in 1pho mutants are still much weaker compared to tissues where these genes are normally expressed. This low degree of activation could be explained by compensatory binding of PHOL to the PHO sites in order to maintain PcG-mediated silencing, even if the PHOL-dependent rescue function is incomplete as 1pho mutants die as pharate adults. PHO and PHOL have indeed been described as redundant in their role in PcG-mediated silencing since they bind to the same DNA sequence motif in vitro. However, out of the 1,757 places wherein both PHO and PHOL were significantly bound, only 807 shared the same local maxima (46%). Another 559 (32%) peaks were within 250 bp of each other. This suggests that, in vivo, these two proteins prefer slightly different sequences, with PHO more strongly attracted to PREs, whereas PHOL binds better to promoters. Moreover, PHO interacts directly with PC and PH [In C and PH , as wellC and PH . StrongeDrosophila polytene chromosomes. They then showed that PcG and TRX proteins bind to a PRE mutually exclusively in salivary gland chromosomes [Here, we report for the first time, to our knowledge, the genome-wide distribution of TRX. This protein has been proposed to counteract PcG-mediated silencing . Petruk omosomes . In contomosomes ,41 foundomosomes  and one omosomes ,41. Notaomosomes , but theOur genome-wide mapping studies using the same antibody against the N-terminal fragment (TRX-N) as used by Petruk et al. , showed Drosophila cell lines, H3K27me3 also forms large domains in Drosophila embryos. These large PcG domains could provide the basis of a robust epigenetic memory to maintain gene expression states during mitosis. As previously suggested [Similar to mapping studies in uggested , stably Drosophila embryos correlate well with profiles from Drosophila cell lines, it has recently been shown that PcG protein binding profiles are partially remodeled during development [Although genome-wide PcG profiles in elopment ,30. Compelopment  showed telopment ,56, simiAll antibodies used in this study are listed in w1118 line of Drosophila melanogaster. The complete experimental details of the ChIP experiments are available in p-value < 0.0001) for all profiles are shown in ChIP assays were performed on 4–12-h-old embryos of the Oregon-R Spatial clustering was performed by training a Hidden Markov Model (HMM) to fit the available genomic profiles using a small set of clusters. The HMM represents both the relations between clusters and the joint profile distribution emitted from each cluster. We developed a hierarchical version of the algorithm so that the two layers of genomic organization in the data can be characterized  for 12 min . Sheared chromatin had an average length of 500 to 1,000 bp. Antibodies used for IP  were diluted 1:100 (PH and PHO) or 1:20 (PHOL). Enrichment of specific DNA fragments was analyzed by real-time PCR, using Roche Light Cycler equipment and accessories as described in Comet et al. [robo3 gene. Primer sequences are listed in ChIP assays of imaginal discs were performed as described for embryos with the following modifications: third instar larval eye discs and haltere/third leg discs were dissected in SS M3 insect medium and kept on ice during dissection. A hundred discs were used per immunoprecipitation (IP). Discs were pelleted by centrifugation at 4.000 t et al. . Enrichm1pho homozygous larvae were collected from a stock ey-GAL4/ey-GAL4; pho1/GS15194 kindly provided by R. Paro's lab [1/pho1pho mutant larvae were dissected in PBS, and 40 eye or haltere/third leg discs were taken for RNA isolation using TRIzol reagent (Invitrogen). RT-PCR was performed using Superscript III First Strand Synthesis Kit from Invitrogen following the manufacturer's instructions. Reverse transcription was primed using hexamer primers. Quantitative polymerase chain reaction (qPCR) analysis was done as described for ChIP experiments. The copy number for each investigated gene was normalized to the copy number of the 18S RNA gene. Primer sequences are listed in ro's lab . Wild-tyExperiment, first part : E-MEXP-1708.Additionally, all employed microarray designs have their own accessions: PhysicalArrayDesign name: 2005-08-08_Henikoff_Dros_ChIP_1, ArrayExpress accession: A-MEXP-1251; PhysicalArrayDesign name: 2005-08-08_Henikoff_Dros_ChIP_2, ArrayExpress accession: A-MEXP-1252; PhysicalArrayDesign name: 2005-08-08_Henikoff_Dros_ChIP_3, ArrayExpress accession: A-MEXP-1253; PhysicalArrayDesign name: 2007-03-13_Henikoff_Dros_ChIP_1, ArrayExpress accession: A-MEXP-1254; PhysicalArrayDesign name: 2007-03-13_Henikoff_Dros_ChIP_2, ArrayExpress accession: A-MEXP-1255; PhysicalArrayDesign name: 2007-03-13_Henikoff_Dros_ChIP_3, ArrayExpress accession: A-MEXP-1256; and PhysicalArrayDesign name: Cavalli_Dmel_1_tiling, ArrayExpress accession: A-MEXP-1257.Antp: FBgn0000095; ato: FBgn0010433; cad: FBgn0000251; Chc: FBgn0000319; Dll: FBgn0000157; dsx: FBgn0000504; grn: FBgn0001138; hb: FBgn0001180; robo3: FBgn0041097; Rp49: FBgn0002626; so: FBgn0003460; toy: FBgn0019650; and ubx: FBgn0003944.Gene accession numbers: Figure S12 ratio of replicate 1 versus replicate 2 for each profile. Probes having a statistically significant log2 ratio  are highlighted in red. The significant probes show good correlation between biological replicates.Plots showing correlation between normalized log(482 KB PPT)Click here for additional data file.Figure S22 ratio  are highlighted in red.In each pair of rows, the upper panel shows correlation plots between replicate 1 versus replicate 2 mock (green) signal intensities, whereas the lower panel shows correlation plots between replicate 1 versus replicate 2–specific IP (dark red) signal intensities for each chromatin profile. Probes having a statistically significant log(1.35 MB PPT)Click here for additional data file.Figure S3p-value < 1E−04. Note that in all the profiles except H3K4me3, the majority of bound regions were of length less than 2,000 bp.Only profiles that showed localized binding (and PC) were analyzed. All the bound regions were with (968 KB PPT)Click here for additional data file.Figure S4(A) Size distribution of H3K27me3 domains.(B) Distribution of PH peaks in H3K27me3 domains. The majority of domains had at least one PH peak. The largest number of PH peaks (30) was found in Hox cluster . Twenty-two PH peaks were not present within H3K27me3 domains. Here, PH peaks denote those that are present along with PC.(C) Distribution of genes in H3K27me3 domains. The majority of domains have at least one gene. The largest number of genes was present in a domain in chromosome 3L : 18 genes.(339 KB PPT)Click here for additional data file.Figure S52 ratios was done for each pair of profiles. K27 denotes H3K27me3, and K4 denotes H3K4me3.(A) Probe by probe correlation of the log(B) Table indicating percentage of the genome covered by each single protein or histone modification.(138 KB PPT)Click here for additional data file.Figure S6x- and y-axis profiles are shown in orange. Note that almost all the probes bound with PH also are enriched for PC and PHO.Green and red denote the significantly enriched probes, and grey denotes the nonsignificant ones. Common significantly enriched probes of (6.15 MB PPT)Click here for additional data file.Figure S7p-value < 1E−04) are shown in red. Gray bars in the “Modes profiles” indicate posterior probabilities for the association of probes with a cluster.Plots showing ChIP on chip profiles aligned with the different spatial clusters (modes) along part of chromosome 3R. ChIP on chip plots show the ratios (fold change) of specific IP versus mock IP. Significantly enriched fragments ((506 KB PPT)Click here for additional data file.Figure S8The “motif length” parameter is 5–10 bp. The MEME E-value of each motif is shown beside its name.(A) Overrepresented DNA motifs at PH- and PC-bound regions with PHO, DSP1, and GAF.(B) Overrepresented DNA motifs at regions bound with PHO, DSP1, and GAF (absence of PH and PC).(1.25 MB PPT)Click here for additional data file.Figure S9(A) Overrepresented motifs in the complete set of DSP1-bound regions . A 300-bp region around the Lmax was taken out for searching motifs. Zoops model and 5–10-bp motif width parameters were used.(B) Overrepresented motifs in DSP1-bound regions wherein GAF binding is not detected . A total of 41/77 bound regions contained this motif.(341 KB PPT)Click here for additional data file.Figure S10(A) Overrepresented motifs in PHO-bound regions with no detectable PHOL (479), E-value 1.8e−100 (set1).(B) Overrepresented motifs in PHOL-bound regions with no detectable PHO (30), E value 2.2e-003 (set2).(330 KB PPT)Click here for additional data file.Figure S11(A) A 300-bp sequence around the local maxima of intensity of a PH-bound region was analyzed for sequence motifs.(B) A 500-bp region around the local maxima of intensity was analyzed. Zoops model in MEME was used and the E-value of each motif is shown beside its name. Both sequence sets yielded the same overrepresented motifs. Note the absence of PHO motif in the enriched motifs list.(1.42 MB PPT)Click here for additional data file.Figure S12Average intensities for GAF, DSP1, and PHO, but not PHOL, are higher at PH binding sites as compared to non-PH–bound regions.(77 KB PPT)Click here for additional data file.Figure S13Drosophila embryos. Before amplification and hybridization on microarrays, specific enrichments of several regions were quantified by qPCR. Three regions that were known to be bound by PcG proteins  and two control regions (Rp49 and robo3) were analyzed.ChIP assays were performed on 4–12-h-old whole (A) ChIP assays with PC, PH, H3K27me3, H3K4me3, and TRX antibodies. Copy number of the PCR fragments enriched in the ChIP experiments are represented for each region analyzed.(B) ChIP assays with PcG recruitment factors PHO, PHOL, DSP1, and GAF. Copy number of the PCR fragments enriched in the ChIP experiment are represented for each region analyzed.robo3 amplicon is located in the coding region of the gene; hence, enrichment of all examined proteins is low.(C). ChIP assays of PHO and PHOL replotted side by side for better comparison. Note that higher levels of PHO and very low levels of PHOL are seen in PH-bound regions, whereas higher levels of PHOL are seen at the Rp49 promoter. The (86 KB PPT)Click here for additional data file.Figure S14(A) Average intensity ratio of PHO/PHOL in PH-bound sites {PHO OR PHOL AND PH} and non-PH–bound regions {PHO OR PHOL AND NOT PH}.(B) Average intensity of PHO in PH-bound {PHO OR PHOL AND PH} and non-PH–bound {PHO OR PHOL AND NOT PH} regions.(C) Average intensity of PHOL in PH-bound {PHO OR PHOL AND PH} and non-PH–bound {PHO OR PHOL AND NOT PH} regions. In PH and non-PH regions, the first significant peak of PHO/PHOL is looked for. If a significant peak is present for one of them, then the intensity of the other, even if it not significant, is recorded, and a ratio between PHO/PHOL is calculated.(75 KB PPT)Click here for additional data file.Figure S15so and toy in eye and haltere/third leg imaginal discs. The cDNA copy number was quantified using qPCR. Note that both the genes are highly expressed in eye imaginal discs, whereas their expression levels are low (so) or not detectable (toy) in haltere/third leg discs.(A) Expression status of Drosophila embryos of PcG proteins, PHO, PHOL, and H3K27me3 at the Ubx, so (sine oculis), and toy (twin of eyeless) genes.(B) ChIP on chip profile in (178 KB PPT)Click here for additional data file.Figure S16Same experiment as shown in (109 KB PPT)Click here for additional data file.Figure S17A total of 63.79% of our target genes overlapped with Schwartz et al.  (S2 cell(85 KB PPT)Click here for additional data file.Figure S18Each cluster represents a combinatorial pattern among transcription factor (TF) occupancies and histone mark densities (as shown in (285 KB PPT)Click here for additional data file.Figure S19The PcG target genes were functionally categorized using the Gene Ontology (GO) toolbox . The “mo(2.02 MB PPT)Click here for additional data file.Table S1p-value cutoffs.The data are shown for two different (14 KB XLS)Click here for additional data file.Table S2(A) Target genes for PH peaks present in PC- and H3K27me3-bound regions.p-value threshold.(B) Target genes corresponding to PH peaks within PC-bound region but with weak H3K27me3 that does not cross the (23 KB XLS)Click here for additional data file.Table S3(15 KB XLS)Click here for additional data file.Table S4(A) Various combinations of PcG recruitment factors present in the genome. Note that PHO and PHOL do not colocalize at all the regions. However, very few regions are present in the genome where we could see PHOL sites without PHO. At the genome-wide level, DSP1 largely colocalizes with GAF and PHO.(B) Various combinations of PcG recruitment factor binding sites, H3K4me3, and TRX-N in the genome.(C) Various combinations of Zeste-bound regions in the genome with the other PcG recruiters, TRX-N and H3K4me3. P*, PL*, K4*, and Z denote PHO, PHOL, H3K4me3, and Zeste, respectively. The hash mark (#) denotes the number of regions. Zeste ChIP on chip data were taken from (D) Various combinations of TRX-C, TRX-N and H3K4me3 in the genome.(25 KB XLS)Click here for additional data file.Table S5The patser program was used for this analysis. The position-specific probability matrix (PSPM) of the MEME motifs (motif width 5–10 bp) were taken as input for patser. The motifs were counted in PH and non-PH regions bound with recruitment factors (PHO+DSP1+GAF). ‘NC' denotes not calculated. The density of motif in each sequence set was also calculated. The total number of base pairs in each sequence set was calculated after concatenating the entire sequence into a single string. In PRE regions, am1, am3, and bm3 were present in one motif per 168 bp, 66 bp, and 2,464 bp, whereas in non-PRE regions, the same motifs were present at one motif per 356 bp, 112 bp, and 9,047 bp, respectively.(16 KB XLS)Click here for additional data file.Table S6MEME top motif (default “motif width” parameter) sequences used in MEME along with two control sets were taken as input. Control 1 denotes random regions wherein none of our tested proteins/histone modifications showed binding. Control 2 denotes random regions from the genome . The data reveal the specific enrichment of each motif in ChIP on chip bound regions. The MAST program was used for analysis.(15 KB XLS)Click here for additional data file.Table S7The patser program was used for this analysis. The position-specific scoring matrix (PSSM) of the top MEME motif (default “motif width”) was taken as input for patser. The motif was counted in three sets of sequences: Set1: sequences around the Lmax of each ChIP on chip bound region (column 2); Set2: the complete sequence of the bound region (column 3); and Set3: the input sequences taken for MEME (column 4).(15 KB XLS)Click here for additional data file.Table S8t-test was done to look for the difference in distribution of motif frequency between recruitment factor–bound regions with PH and without PH.PHO, DSP1, and GAF motifs had higher frequency in PH-bound regions as compared to other places wherein they were bound without PH. A (14 KB XLS)Click here for additional data file.Table S9A total of 53/344 (15.41%) predicted regions showed PH + PC binding in ChIP on chip; 53/439 (12.07%) of ChIP on chip PH+PC-bound regions were predicted by PREdictor. The predictions of chromosome 3R were marginally more validated in our data as compared to other chromosomes. This could be because the majority of the input PRE regions for PREdictor were taken from chromosome 3R.(14 KB XLS)Click here for additional data file.Table S10p-value < 1E−04 for each factor.All the bound sites had (14 KB XLS)Click here for additional data file.Table S11p-value < 1E-04 for each factor.All the bound sites had (15 KB XLS)Click here for additional data file.Table S12(16 KB XLS)Click here for additional data file.Table S13(15 KB XLS)Click here for additional data file.Table S14(56 KB XLS)Click here for additional data file.Table S15(254 KB XLS)Click here for additional data file.Table S16(43 KB XLS)Click here for additional data file.Table S17(1.04 MB XLS)Click here for additional data file.Text S1(148 KB DOC)Click here for additional data file.


A macroarray platform was used to identify binding sites of yeast histone acetyltransferase catalytic subunits and to correlate their positions with acetylation of lysine 14 of histone H3, revealing that Sas3p and Gcn5p are recruited to similar sets of intensely transcribed genes. in vitro and may play a central role in the stability of the nucleosome. This study is focused on the genome-wide binding of Saccharomyces cerevisiae histone acetyltransferases that are specific for Lys14 of histone H3.Specific histone modifications can perform several cellular functions, for example, as signals to recruit trans-acting factors and as modulators of chromatin structure. Acetylation of Lys14 of histone H3 is the main target of many histone acetyltransferases GCN5 deleted strain and the Gcn5p genome occupancy is shown.We have used a variation of the genome-wide location analysis method, based on a macroarray platform, to identify binding sites of yeast histone acetyltransferase catalytic subunits and to correlate their positions with acetylation of Lys14 of histone H3. Our results revealed that the histone acetyltransferases Sas3p and Gcn5p are recruited to a pool of intensely transcribed genes and that there is considerable overlap between the two cohorts of Sas3p and Gcn5p bound gene pools. We also demonstrate a positive correlation between binding sites of both proteins and the acetylation state of Lys14 of histone H3. Finally, a positive correlation between the decrease of H3 Lys14 acetylation in a Our data support a model in which both Gcn5p and Sas3p act as general activators of an overlapping pool of intensely transcribed genes. Since both proteins preferentially acetylate Lys14 of histone H3, our data support the hypothesis that acetylation of this specific residue facilitates the action of the transcriptional apparatus. Most of the protein mass in eukaryotic chromatin is composed of histones, which are crucial for the organization of the highly conserved nucleosomal structure. Histones are subjected to a wide variety of post-translational modifications, most of them reversible ,2, that in vitro have been described to date in Saccharomyces cerevisiae: Gcn5p, Hat1p, Elp3p, Hpa2p, Esa1p, Sas2p, Sas3p, Nut1p and Taf1p. Some of the properties of this set of enzymes are consistent with the histone code hypothesis. For instance, they are able to selectively acetylate particular histone residues. Moreover, most of them have physical and functional connections to transcriptional regulation and act as complexes composed of several subunits, which can modulate their activities. Nevertheless, there are also some features of these enzymes that are hard to reconcile with their proposed role in a combinatorial signaling system, which involves four different histones, each one with several lysine targets. Thus, with the data available to date, if we omit Hat1p [in vitro, for histone H3, and particularly Lys14 (H3K14). Hence, there seems to be something special about this position because most HATs have potential H3K14 acetylation (H3K14ac) activity, exclusively or accompanied by other specificities. More specifically, it has been reported that Gcn5p [in vitro. Nut1p also preferentially acetylates H3, but its specificity is unknown. Finally, the SAS complex, which is composed of the Sas2p acetylase bound to the Sas4 and Sas5 proteins, acetylates free H3 at Lys14 [Several types of enzymatic activities, generically referred to as chromatin regulators , can carit Hat1p  and Esa1it Hat1p ,11, whicat Gcn5p -14, Sas3at Gcn5p , Hpa2p [at Gcn5p , Elp3p [at Gcn5p  and Taf1at Gcn5p  preferenat Lys14 , althougat Lys14 .in vivo are not well known. There are some studies where the acetylation state of bulk H3 obtained from different HAT mutant strains has been analyzed. However, most of these studies used an antibody specific for histone H3 acetylated at both K9 and K14 and, hence, it is not possible to determine if a lower signal in histone H3 isolated from the mutant strains is the consequence of a decrease in the acetylation state of H3K9, H3K14 or both. For instance, the acetylation analysis of a gcn5Δ strain did not definitively show which position was affected [sas3Δ [elp3Δ [hpa2Δ [gcn5Δ strain the results showed that Gcn5p likely acetylates all sites in H3 with the sole exception of H3K14 [in vitro. On the other hand, using chromatin immunoprecipitation of histones and DNA-microarray analysis (ChIP-chip), a positive association between Gcn5p occupancy and transcriptional activity [in vivo acetylation of H3K14 and transcriptional activation. Moreover, it was reported that acetylation of H3K14 peaks at the start sites of active genes and drops substantially across the open reading frames (ORFs) on a genome-wide level [Despite the important role that acetylation of H3K14 could play, the HATs responsible for this modification affected ,21,22. Fd [sas3Δ ,21, elp3of H3K14 . These aactivity ,24 and bde level . These oSaccharomyces cerevisiae HATs that have shown specificity for H3K14. With this aim, we have developed a ChIP-chip method based on the use of macroarrays. Using this technique, we have shown that Sas3p, like Gcn5p, is generally recruited to a similar pool of actively transcribed protein-coding genes. We also have shown that Gcn5p and Sas3p are involved in the acetylation state of H3K14 in vivo. Additionally, we found a relationship between Sas2p and double-strand break repair and ubiquitin-specific protease activity.In this study, we have investigated the genome-wide binding of known Since the development of ChIP-chip technology, this method has been extensively used to analyze the histone modification patterns of chromatin and the binding sites of histone-modifying enzymes in yeast. These studies employ microarrays to hybridize the PCR-amplified, immunoprecipitated DNA. We have developed a method of ChIP-chip  based on the utilization of membrane-based DNA macroarrays. Macroarrays, while less widespread than microarrays, are also used by many laboratories and offer an interesting alternative. Macroarrays can be hybridized many times, up to ten times, without a measurable reduction in their performance , thus grS. cerevisiae. There were several reasons for this selection. First, class III genes in yeast are well known and they have been studied by three independent ChIP-chip studies using different RNA polymerase III subunits [in silico analysis of the positioning of the 275 tRNA genes and found that they are randomly distributed compared to distances between ORFs [The macroarrays used in this study are made with ORF DNA, 93% of which are complete ORFs . ORF macsubunits -30. RNA een ORFs  in the yAmong the 275 tRNA genes, we focused on the 179 tRNA genes that are at less than 400 bp from an ORF because IGRs in yeast showed a distribution with a maximum at lengths from 150 to 800 bp  for a binding ratio ≥1.8 (results not shown). Moreover, when we compared the Sas3p and Gcn5p occupancies related to specific ORFs, a positive correlation was observed . Interestingly, when the threshold of the binding ratio is increased to 2, there are 252 genes bound by Sas2p, 13 of them belonging to categories including double-strand break repair or ubiquitin-specific protease activity  will work poorly for detection of promoter regions and detection of protein binding will weaken as ORF length increases . Howeverin vitro towards histone H3 and, especially, to H3K14. In this study, we have used our methodology to investigate the genome-wide association of known S. cerevisiae HATs that have shown specificity for this residue. Considering that there are at least 12 lysines that could act as possible acetylation sites in the yeast inner histones, H3, H4, H2A and H2B, we think that this preferential specificity for H3K14 may be of particular importance. For instance, this position may have a central role in the stability of the nucleosome. Thus, this specific modification of this H3 residue may directly facilitate the action of the transcriptional apparatus and other chromatin functions. Among the HATs analyzed, genome-wide occupancy of Gcn5p and Sas3p correlates with the transcriptional rate of the genes, whereas in the case of Elp3p, Sas2p, Hpa2p and Nut1p, no significant correlation was observed. These results demonstrate that Sas3p, a protein initially described as implicated in transcriptional silencing, is recruited to intensely transcribed genes, similar to what has been observed for Gcn5p. In this sense, it has recently been proposed that NuA3 is targeted to and/or retained at sites of H3K4me3 through interaction with Yng1, promoting H3K14ac, positively regulating downstream transcription events [in vivo. However, analysis of the genome-wide effect of the loss of GCN5 or SAS3 on the H3K14ac state may be subject to different interpretations. Thus, the negative correlation found between H3K14ac in the gcn5Δ strain and the Gcn5p occupancy using an epitope-tagged strain provides good evidence that Gcn5p acetylates this residue in vivo. On the contrary, the positive correlation found between H3K14ac in the sas3Δ strain and the Sas3p occupancy in the epitope-tagged strain suggests that Sas3p occupancy has a negative influence on the H3K14ac state. This striking result can be interpreted by considering that there is another HAT in vivo with H3K14 specificity that acetylates intensely this position in vivo in the absence of Sas3p. Based on the results shown here, we suggest that this HAT is Gcn5p. In any case, these results show implicitly that, in vivo, different HATs with overlapping specificities compete for chromatin binding sites and that this competition can play an important role in the regulation of the establishment of the histone code signals. Thus, one possible role of Sas3p could be to displace Gcn5p from certain key sites.Most of the HATs present in yeast exhibit HAT activity n events . The obsn events  that GcnS. cerevisiae strains for genome-wide location analysis used in this study are listed in Table The targets of catalytic HAT subunits and the Rpc160 subunit of RNA Pol III from yeast were identified by combining chromatin immunoprecipitation and ORF macroarray hybridization. We designed a specific protocol for ORF macroarrays based on a previously described method originally devised for intergenic microarrays .in vivo by adding formaldehyde to a final concentration of 1%. Cells were incubated for 30 minutes at room temperature with occasional mixing, and cross-linking was quenched by adding glycine to a final concentration of 125 mM. Cells were washed three times with 30 ml of ice-cold phosphate-buffered saline buffer . Pelleted cells were frozen after this step. Cells were thawed on ice and resuspended in 300 μl lysis buffer , 1 mM benzamidine and 1 pill of protease inhibitor cocktail  dissolved in every 25 ml of buffer). The equivalent of 0.2 ml of frozen glass beads  was added to the cellular suspension and cells were lysed at 4°C by 25 minutes of vortexing in a Genie 2 vortex with Turbo mix at maximum power. Lysis buffer (400 μl) was added and the extract was transferred to a new tube. Sonication of DNA was performed in order to obtain DNA fragments between 400 and 3,000 bp, with an average size of 800 bp. Chromatin was sonicated on ice, 3 pulses of 10 s at 38% amplitude in a Vibracell VCX500 . Cell debris was removed by centrifugation at 13,000 rpm at 4°C for 10 minutes. An aliquot of 50 μl of this whole cell extract (WCE) was kept to check the quality and size of the isolated chromatin.Three independent cultures from tagged and control strains were grown. A volume of 40 ml YPD  culture from each one (OD 600 ≈ 0.5-0.8) was set aside, and proteins were cross-linked to their target sites The antibodies used were 2 μg of rat anti-HA 3F10 (Roche), 20 μg of mouse anti-MYC 9E11 (Roche), 15 μl of rabbit α-K14acH3 chip grade  and 20 μg of rabbit α-H3Ct . Rat and mouse antibody complexes were collected using 100 μl of a 50% (v/v) suspension of Protein G Sepharose 4FF  equilibrated in 5 mg/ml bovine serum albumin (BSA) lysis buffer. Rabbit antibody complexes were collected using 50 μl of a suspension of sheep α-rabbit IgG M-280 dynabeads  in a final volume of 120 μl. Suspensions were incubated overnight at 4°C. DNA fragments specifically cross-linked to the epitope-tagged protein were purified by immunoprecipitation with the indicated monoclonal antibodies coupled to Protein G Sepharose for 4 h at 4°C. We included control immunoprecipitations of lysates from wild-type strains with α-HA, α-MYC or α-H3Ct antibodies. Beads were washed twice with lysis buffer, twice with 500 mM NaCl lysis buffer, twice with wash buffer ), once with TE  containing 1 mM PMSF, and finally collected. Two successive elutions were performed with 100 and 150 μl of elution buffer  by incubating for 10 minutes at 65°C each time. The eluted fraction of protein cross-linked to DNA was treated overnight at 65°C to reverse the cross-linking. For WCE, 200 μl of elution buffer were added before reversal of cross-linking (from 1 h to overnight).Proteins were degraded by adding 50 μg of proteinase K and SDS to 2.5% final concentration, and incubation at 37°C for 1 h. DNA was purified by phenol/chloroform/isoamylic alcohol extraction. For WCE, the aqueous phase was directly ethanol precipitated, resuspended in 15 μl of milliQ water, and treated for 30 minutes with 10 μg of RNAse A. The WCE chromatin was visualized by agarose electrophoresis to check the quantity and size of the fragments. For immunoprecipitated DNA samples, the aqueous phase was subsequently purified with Montage PCR columns . The total eluted volume was treated with 10 μg of RNAse A for 30 minutes at 37°C. Ethanol precipitation in the presence of 20 μg of glycogen as a carrier was performed. The immunoprecipitated DNA was blunted by T4 phage DNA polymerase in a reaction volume of 124 μL . The reaction was allowed to proceed for 20 minutes at 12°C. After phenol/chloroform/isoamylic alcohol extraction, DNA was ethanol-precipitated in the presence of 11 μg of glycogen and was ligated in a final volume of 50 μl with annealed linkers oJW102 and oJW103 (1.5 μM of each primer) [2, 0.25 mM dNTPs, 1.25 μM oligonucleotide oJW102). The reaction was started by incubating for 2 minutes at 55°C and paused to add 10 μl of reaction mix . The program was resumed with 5 minutes at 72°C, 2 minutes at 95°C and 33 cycles of 30 s at 95°C, 30 s at 55°C and 2 minutes at 72°C. A 5 μl DNA aliquot of the LM-PCR was analyzed on a 1.2% agarose gel , and the rest was purified with Montage PCR columns. DNA was precipitated overnight and resuspended in 25 μl of milliQ water. The concentration of nucleic acids was determined using a spectrophotometer.Ligation Mediated PCR  was used33P-dCTP, cold dCTP and the rest of dNTPs. The choice of the ratio between hot and cold dCTP is essential to produce long-sized DNA samples. Both Klenow and Taq polymerase were assayed in the presence of different radioactive/cold ratios. The labeling yield was much higher for Taq polymerase at any of the cold dCTP concentrations assayed (results not shown). Increases in the concentration of cold dCTP produced longer sizes of labeled DNA, although the incorporation of 33P-dCTP was reduced. We chose a ratio that produced DNA products with an average size of 600 bp and an acceptable threshold of radioactive labeling. The reaction mixture contained 1.5 μg of amplified DNA in 50 μl . The mix was denatured for 5 minutes at 95°C, annealed 5 minutes at 50°C and was amplified during 35 minutes at 72°C. The reaction product was purified through a ProbeQuant G-50 column (Amersham Biosciences) to remove unincorporated 33P-dCTP and oligonucleotides.The PCR product was used as a template for the radioactive labeling reaction. This reaction consisted of a single amplification step where Taq polymerase was incubated with a mix of S. cerevisiae [6 dpm/ml) for 16-18 h. After hybridization, the membranes were washed once in 2× SSC, 0.1% SDS for 20 minutes, and twice in 0.2× SSC, 0.1% SDS for 30 minutes. They were exposed to an imaging plate  for 48-72 h and read in a PhosphorImager  at 50 μm resolution. After analysis of macroarray filters, they were stripped as described previously [The macroarrays used contained PCR products representing full-length ORFs for 6,049 genes of revisiae  . The signal intensity for each spot was the background subtracted ARM Density (artifact removed median). Only values 1.5 times over the corresponding background were taken as valid measurements. Reproducibility of the replicates was checked using ArrayStat software . We considered data as independent and used a minimum number of two valid replicates in order to calculate the mean and standard deviation values for every gene. Normalization between conditions was done using the global median method. In order to detect the statistically significant, immunoprecipitated genes for condition, a z-test for independent data was applied and, consequently, a z-score for every gene was obtained. A S. cerevisiae, in a ChIP-chip experiment using anti-HA antibodies . The binding ratio is calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies. Additional data files gcn5Δ and sas3Δ strains, respectively. Acetylation at H3K14 was obtained as described in Additional data file The following additional data are available with the online version of this paper. Additional data file S. cerevisiae, in a ChIP-chip experiment using anti-HA antibodies . The binding ratio was calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies.Binding ratio of Rpc-160-HA, a subunit of RNA polymerase III from Click here for fileThe binding ratio was calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies.Click here for fileThe binding ratio was calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies.Click here for fileThe binding ratio was calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies.Click here for fileThe binding ratio was calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies.Click here for fileThe binding ratio was calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies.Click here for fileThe binding ratio was calculated as the ratio of chromatin-immunoprecipitated signals between the epitope-tagged strain and the un-tagged parent, using the same antibodies.Click here for fileAcetylation at H3K14 was obtained by two ChIP-chip experiments. The first was performed by immunoprecipitating WCE with an antibody that recognizes the carboxyl termini of histone H3 (α-H3Ct) and the other one by immunoprecipitating with α-K14acH3. Ratios from the α-K14acH3 ChIP-chip experiment and the control from the α-H3Ct ChIP-chip experiment were calculated and are listed.Click here for fileAcetylation at H3K14 was obtained by two ChIP-chip experiments. The first was performed by immunoprecipitating WCE with an antibody that recognizes the carboxyl termini of histone H3 (α-H3Ct) and the other one by immunoprecipitating with α-K14acH3. Ratios from the α-K14acH3 ChIP-chip experiment and the control from the α-H3Ct ChIP-chip experiment were calculated and are listed.Click here for fileAcetylation at H3K14 was obtained by two ChIP-chip experiments. The first was performed by immunoprecipitating WCE with an antibody that recognizes the carboxyl termini of histone H3 (α-H3Ct) and the other one by immunoprecipitating with α-K14acH3. Ratios from the α-K14acH3 ChIP-chip experiment and the control from the α-H3Ct ChIP-chip experiment were calculated and are listed.Click here for file