High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency. Here we report that hd-siRNAs induce mouse Eri-1 (meri-1) expression through the recruitment of Sp1, Ets-1, and STAT3 to the meri-1 promoter and the formation of an Sp1-Ets-1-STAT3 complex. In addition, hd-siRNAs also abolish the 3′ untranslated region (UTR) mediated posttranscriptional repression of meri-1. Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.The  RNA interference (RNAi) is widely accepted as a precise and effective method for specific gene knockdown in eukaryotes. It has been extensively studied as a powerful tool for experimental and therapeutic purpose. However, several unexpected effects of RNAi compromise the efficiency and spectrum of its applications. These effects include immune activation, unintended gene inhibition/activation, and potential endogenous microRNAs interference C. elegans, several negative regulators of RNAi, including eri-1, eri-3, eri-5 and eri-6/7, have been identified through genetic screening eri-1 gene (meri-1) was upregulated by high-dose exogenous non-silencing siRNA but not by low-dose siRNA meri-1 gene could rescue the effectiveness of RNAi meri-1 played an important role in this negative regulation. However, the exact molecular mechanism underlying the regulation is poorly understood.Even RNAi delivered with high efficiency is restricted in its activity. Several negative regulatory mechanisms of RNAi have been described Sp1 is a general transcription factor that regulates the expression of numerous genes. Both the DNA binding ability and the transactivational activity of Sp1 are influenced by posttranslational modifications such as phosphorylation, glycosylation, and acetylation Ets-1 belongs to the Ets family that regulates gene expression in a variety of tissues and cell types STAT3 mediates a variety of biological events including inflammation, cellular transformation, survival, angiogenesis, and metastasis of cancer through collaboration with other transcription factors, including Sp1 meri-1 expression through interfering with the miRNA-mediated regulation in our previous study. In the miRNA pathway, after forming the miRNA-induced silencing complexes (miRISCs) on target mRNA 3′-UTR, the mRNA and miRISCs containing Argonaute proteins accumulate in discrete cytoplasmic foci known as P-bodies or GW bodies In animals, when small double-stranded RNAs possess near-perfect complementarity to target sequences positioned in either coding or 3′-UTR regions of mRNAs, they can direct endoribonucleolytic cleavage of mRNA by Argonaute 2 (Ago2) through an RNAi-like mechanism induced by siRNAs meri-1 gene and its interaction with Sp1/Ets-1/STAT3. In response to the hd-siRNAs treatment, Sp1 is acetylated and binds to the meri-1 promoter where it forms a complex with transcription factors Ets-1 and STAT3 to promote meri-1 transcription. In addition, hd-siRNAs also abolish the 3′-UTR mediated posttranscriptional repression of meri-1 expression. Together, these results provide key mechanisms to illustrate how hd-siRNAs induce enhanced meri-1 expression, which is a critical determinant in reducing RNAi efficiency.In the present study, we analyzed the promoter region of the meri-1 transcription in mouse liver in vitro meri-1 transcription in response to hd-siRNA treatment, we measured meri-1 mRNA level by qPCR in CHO cells. As shown in meri-1 transcription was significantly upregulated in CHO cells treated with >0.4 µg/24-well plate of Nc siRNA. This meri-1 transcription peaked at 12 h after Nc siRNA transfection and gradually decreased to its basal level after 36 h -GFP reporter construct into the cells alone. Four hours later, the medium was removed and the cells were washed three times with fresh medium and treated with 1 µg/24-well plate of Nc siRNA_1. Without the transfection procedure, this direct addition of siRNA was unable to induce GFP reporter expression  upon siRNA treatment was observed for the 1.8 kb promoter (Peri-1(−1654)), the 500 bp (Peri-1(−386)), and the 200 bp fragments (Peri-1(−87)) ) : panel 7http://mapper.chip.org/mapper/mapper-main) CCGCCC) at −87 to −76, a potential NRF-1 site (TGCGCCTGCGC) at −75 to −65, a potential Ahr-ARNT site (GCGTG) at −63 to −59, and a potential Ets-1 site (CCGGAAGTA) at −42 to −34 -SEAP reporter plasmid. SEAP assays revealed that the knockdown of Sp1 and Ets-1 significantly decreased meri-1 promoter activity  lacking the transactivation domain of Sp1 meri-1 promoter activity.To confirm the roles of Sp1 and Ets-1 in the hd-siRNA-stimulated meri-1 promoter, competition EMSAs were carried out. The EMSA results  were performed using CHO cell extracts  inhibitor TSA K703A, was constructed. It carries a mutation at lysine residue 703 of Sp1 that resides in the DNA binding domain and is the only known acetylated residue of Sp1 K703A suppressed the transcriptional activity of meri-1 promoter , STAT3D and STAT3ΔN (162–770). STAT3β retains the tyrosine residue at position 705 critical for dimerization but lacks a serine residue at position 727 which is a phosphorylation site meri-1 promoter activity , and control plasmid, pSEAP2-control (0.17 µg/24-well plate), were transfected into CHO cells. As shown in meri-1. This indicates that the 3′-UTR of meri-1 mediates the gene repression. There were no differences in the quantity of SEAP mRNA from constructs with or without the meri-1 3′-UTR as assessed by qPCR  by DAPA. Results showed that the recruitment of Sp1 to the meri-1 promoter was attenuated when the lysine-703 was substituted by alanine. Meanwhile, the overexpression of mutant Sp1 (K703A) significantly repressed reporter gene expression in CHO cells. This evidence indicates that the acetylation is critical for meri-1 expression induced by hd-siRNAs. Based on the results presented in the current studies, potential roles of acetylation in regulating Sp1 DNA binding activity may be proposed. One possibility is that the acetylation is a prerequisite for Sp1 binding to the meri-1 promoter, which may modify its biological activity including altering protein-protein interactions, DNA recognition, or/and protein stability. Acetylation might induce a conformational change in the DBD of Sp1 resulting in an increase in its affinity for DNA binding or selecting the partner proteins interacting with Sp1 which may further enhance the transcriptional competence of Sp1 and the stability of transcriptional complex. The second possibility is that the acetylation is just a concomitant consequence of Sp1 recruiting HAT to the meri-1 promoter to activate the gene expression. It has been reported that the HAT region of p300 interacts with the DBD of Sp1 to stimulate Sp1 DNA binding activity by physical interaction rather than acetylation, despite Sp1 being acetylated by the p300 acetyltransferase region. Once Sp1 is acetylated and binds to DNA, the affinity for p300 and Sp1 was reduced et al. reported data that p300 interacted with mutant Sp1 (K703A) more than with wild-type Sp1 It is now widely recognized that acetylation is a crucial posttranslational modification for regulation of Sp1 activity. Many findings indicated that acetylation may increase Sp1-mediated transcription meri-1 promoter activity. This finding is distinct from the classical concept that STAT3 should bind to its own consensus DNA element. Similar to this finding, STAT3 also participated in the regulation of DINE  without phosphorylation and DNA binding trans-acting factors could give them functional versatility and ensure the cells' proper response to redundant stimuli by integrating multiple signals. Trans-acting factors activated by different pathways construct a gene-specific architecture of the transcription complex to mediate the selective control of transcriptional activity. The present study revealed that there was a function synergy of different trans-acting factors in the complex. In addition to STAT3 functioning as a bridge, Sp1 might function as a recruiter or scaffold for other transcription factors in the complex. Consistently, some studies have revealed that STAT3 or c-Jun can activate the promoters without their binding sites, through the interaction with Sp1 meri-1 promoter activity, the acetylation of Sp1 was enhanced by Nc siRNA treatment, which suggested that the overall acetylation status of the meri-1 promoter, which is maintained through a balance of HAT and HDAC activities, is pro-acetylation. It has been reported that the NH2-terminal acetylation of STAT3 is necessary for IL-6-dependent gene transcription and the decrease of HDAC1 reduced the nuclear export of STAT3, leading to nuclear accumulation of acetyl STAT3 and enhanced target gene transcription meri-1 promoter was impeded by hd-siRNA treatment (data not shown) and that NH2-terminal deletion of STAT3 suppressed the meri-1 promoter activity. Together, we conclude that Sp1 regulates meri-1 promoter activity not only by its transcriptional activity, but also regulating the steady state level of HATs and HDACs to control the reversible acetylation of the non-histone protein STAT3. Consequently, the subcellular distribution and nucleocytoplasmic shuttling of STAT3 dictated the duration and degree of gene activation in response to hd-siRNA.In this study, we found that the transactivational activity of STAT3 was independent of the phosphorylation and DNA binding activity in the hd-siRNA-stimulated meri-1 promoter. This occurs in conjunction with release of HDACs from the promoter. Then, STAT3 is recruited to the meri-1 promoter and bridges Sp1 with the co-activator Ets-1 to form the transcriptional complex and elevate the level of gene transcription.In conclusion, as shown in meri-1 gene is regulated by a 3′-UTR-mediated posttranscriptional repression. The expression of the reporter gene was repressed when the 3′-UTR of meri-1 was downstream of the reporter gene. Moreover, the increase of reporter gene expression was not proportional with the dose of reporter gene transfected. When the amount of reporter vector was lower than 0.4 µg per 24-well plate, the expression of reporter gene was almost completely repressed. A significantly dosage-responsive increase emerged when the amount of reporter vector transfected was over 0.4 µg per 24-well plate. This threshold of the repression effect indicates that the amount of the components exerting repression is limited. Considering that miRNA and siRNA pathways share common components meri-1 3′-UTR-mediated expression repression could be abolished by hd-siRNA in a sequence-independent manner. However, although the abolishment of the repression occurred rapidly after siRNA treatment and the effect of hd-siRNAs is sequence-independent, it couldn't exclude the possibility that hd-siRNAs decrease the expression of miRNAs which regulate the expression of meri-1. More details of the mechanism under hd-siRNA abolishing the 3′-UTR- mediated repression of meri-1 wait to be elucidated in further studies.We have begun to realize the important role of the 3′-UTR of mRNA, with miRNA activity playing a major role in gene regulation et al.RNAi is a powerful research tool, and the clinical potential of RNAi as therapeutics has been demonstrated. Some studies have demonstrated clearly that the efficacy of RNAi is based on multiple factors. Some key mechanistic aspects need to be investigated before RNAi becomes a real therapeutic modality. The amount of siRNA used is a crucial factor, as the use of non-optimal siRNA minimizes the knockdown effect meri-1 promoter fragments of various lengths were generated by PCR amplification from the genomic DNA of C57BL/6 mice, using primer P+115 (5′-cGAATTCGCCGCCTCTCGCGGGCCGCGAGAC-3′) as the antisense primer and the following primers as the sense primers, including P−1654 (5′-cgAGATCTCGGGATCCATTAATGGGCTGGTG-3′), P−386 (5′-cgAGATCTATTAATGGAATCTAGACCCTAGGAACGC-3′), P−87 (5′-cgAGATCTATTAATGGACCCGCCCACTGCGCCTGC-3′), P−76 (5′-cgAGATCTCTGCGCCTGCGCGGCGTGACAAC-3′), P−67 (5′-cgAGATCTCGCGGCGTGACAACGCCACAC-3′), P−47 (5′-cgAGATCTCGCTCCCGGAAGTAGGAGGC-3′), P−27 (5′-cgAGATCTATTAATGTCCGACGGAGTTTGTGTGG-3′), P−7 (5′-cgAGATCTATTAATCGGCCGCCGCGGGAACGTG-3′), P+16 (5′-cgAGATCTATTAATCGCGGTAGTTTTTCAAGGGAG-3′), P−84Δ−70–−48 (5′-cgAGATCTCCCGCCCACTGCGCCGCTCCCGGAAGTAGGAGGCG-3′), and P−84Δ−70–−30 (5′-cgAGATCTCCCGCCCACTGCGCGTCCGACGGAGTTTGTGTGGC-3′). Each primer was named according to the first nucleotide relative to the transcription start site (+1) of meri-1 gene. These fragments were cloned into the pEGFP-N2 vector  between Ase I and EcoR I sites to replace the CMV promoter upstream of the GFP reporter gene. Similarly, these fragments were also cloned into the pSEAP2-control vector  between Bgl II and EcoR I sites to replace the SV40 early promoter upstream of the SEAP reporter gene. A consensus binding site mutant corresponding to Ets-1  was constructed using the QuikChange site-directed mutagenesis kit . The 3866 bp meri-1 3′-UTR was cloned from the genomic DNA of C57BL/6 mice and inserted into the pSEAP2-control vector downstream of SEAP reporter gene to produce the pSEAP-meri-1_3′-UTR vector.The The cDNA fragments coding for Sp1, Ets-1, STAT3, dominant-negative mutant of Sp1 (Sp1DN) (amino acids 592–758), dominant-negative mutant of Ets-1 (Ets-1DN) (amino acids 306–441), STAT3ΔN (amino acids 162–770), and STAT3β were PCR amplified from their cDNAs and subcloned into the pCMV-HA vector . STAT3D and STAT3 (Y705F) mutants were generated by using the QuikChange II site-directed mutagenesis kit. The Sp1 expression vector pCMV-HA-mSp1 (K703A) carrying the Sp1 mutant (K703A) was similarly generated. All these constructs were verified by sequencing. Sp1, mSp1 (K703A) and STAT3 were also subcloned into pCMV-Tag2  to generate the N-terminal FLAG tagged protein.5′-GGAACAGAGUGGCAACAGUdTdT-3′ and 5′-ACUGUUGCCACUCUGUUCCdTdT-3′5′-GGACAAGCCUGUCAUUCCUdTdT-3′ and 5′-AGGAAUGACAGGCUUGUCCdTdT-3′5′-CTTCAGACCCGCCAACAAAdTdT-3′ and 5′- TTTGTTGGCGGGTCTGAAGdTdT -3′Both enzymatically synthesized siRNAs and chemically synthesized siRNAs were used in this study. The enzymatically synthesized siRNAs for Sp1, Ets-1, and STAT3 were prepared as previously described 5′-UUCUCCGAACGUGUCACGUdTdT-3′, anti-sense: 5′-ACGUGACACGUUCGGAGAAdTdT-3′) was also used as Nc siRNA_2 5′-CGTACGCGGAATACTTCGATT-3′, anti-sense: 5′- TCGAAGTATTCCGCGTACGTT-3′), was used as a negative control in experiments where indicated.The enzymatically synthesized nonsilencing control siRNA (esiHBVP) was derived from the Hepatitis B Virus P protein and prepared as previously described 2. The cells were transfected at 70% confluence using Lipofectamine 2000 .CHO-K1  cells and HEK293  cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum , 100 µg/ml streptomycin, and 100 IU/ml penicillin, at 37°C and 5% COhttp://fokker.wi.mit.edu/primer3/) meri-1 was normalized to β-actin. Threshold cycle numbers (Ct) were determined with SDS 2.2 software and transformed using by the ΔCt comparative method. After PCR amplification, a melting curve of each amplicon was determined to verify its accuracy.Total RNA was isolated from CHO cells transfected with or without Nc siRNAs using a Qiagen RNA isolation kit. First-strand cDNA was synthesized from 1 µg of total RNA in a final volume 10 µl using ReverTra Ace® qRCP RT kit . The qPCR was performed using an ABI Prism 7900HT  and data were analyzed with SDS 2.2 software . All primers were designed using Primer3  at 488 nm.2, 10 mM L-homoarginine, and 1 M diethanolamine, at pH 9.8). The reaction was incubated at 37°C for 15 min and the absorbance was measured at 405 nm using a microplate reader. Parallel experiments using a SEAP constitutive expression construct, pSEAP2-control, were performed to monitor and normalize transfection efficiency.Alkaline phosphatase expression was detected as following. CHO cells were transfected with the pPeri-1-SEAP series of reporter constructs together with either the Nc siRNAs or the 21-bp DNA control. Twenty-four hours later, the SEAP  activity of the culture media was measured by performing a colorimetric assay as recommended CHO cells were transfected with Nc siRNAs. Twelve hours after transfection, the nuclear extracts of the treated cells were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime).2 and 1% NP-40. The reactions were then incubated for 15 min at room temperature. The oligonucleotides used for EMSAs are listed in Twenty femtomoles of 5′ biotinylated double-stranded oligonucleotides (Invitrogen) were added to 2 µg of the nuclear extracts in a final volume of 10 µl that contained 0.5 µg of poly(dI-dC), 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.5 mM DTT, 4% glycerol, 5 mM MgClFor the competition assay, unlabeled double-stranded oligonucleotides were incubated with the nuclear extracts at 4°C for 20 min before adding the labeled probe. For the immunodepletion EMSA, the following procedure was carried out as described previously g, 4°C). The cell extracts, containing 700 µg of protein, were incubated for 16 h with 1 µg of the biotinylated double-stranded oligonucleotides that corresponded to the different regions of the meri-1 promoter, including BPeri-1 ((−88)–(−30)), BPeri-1 ((−99)–(−64)), and BPeri-1((−57)–(−22))  with PMSF and sonication. The cell debris was removed by centrifugation . The annealing sites of these primers are shown in CHO cells were transfected with or without Nc siRNAs. Twelve hours after transfection, ChIP assays were performed using EZ ChIP Kit  and antibodies against Sp1, Ets-1, STAT3 or control IgG. The input control DNA or immunoprecipitated DNA was then subjected to PCR amplification using primers specific to CHO cells were pre-transfected with the FLAG-Sp1 expression plasmid and gene specific siRNAs for twelve hours prior to transfection with Nc siRNA_2. Twelve hours after Nc siRNA_2 transfection, cell lysates were prepared using the Cell Lysis Buffer containing PMSF and TSA  followed by sonication. The cell extracts, containing 1 mg of protein, were incubated with M2-anti-FLAG antibody-agarose beads (Sigma) for 3 h. Protein-antibody-agarose complexes were precipitated, washed, and examined by Western blotting using indicated antibodies.Antibodies to the Sp1 and Ets-1 nuclear proteins were purchased from Santa Cruz Biotechnology . STAT3 antibody was purchased from Cell Signaling Technology . HRP-conjugated FLAG antibody was purchased from Shanghai Westang Bio-tech Co. Ltd. The transfected cell lysate was prepared using lysis buffer containing 50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol and 0.1% bromophenol blue. The proteins were resolved on 10% SDS-PAGE gels and Western blotted using standard protocols. The primary antibodies were diluted at 1∶500 or 1∶2000, and the AP- or HRP-conjugated, goat anti-mouse or goat anti-rabbit secondary antibody was diluted at 1∶2000 before using.t-test. A p value of <0.05 was considered to be statistically significant.Results were expressed as mean ± SD. Statistical significance was evaluated for data from three independent experiments using Student's


Transcription regulation in pluripotent embryonic stem (ES) cells is a complex process that involves multitude of regulatory layers, one of which is post-translational modification of histones. Acetylation of specific lysine residues of histones plays a key role in regulating gene expression.Here we have investigated the genome-wide occurrence of two histone marks, acetylation of histone H3K9 and K14 (H3K9ac and H3K14ac), in mouse embryonic stem (mES) cells. Genome-wide H3K9ac and H3K14ac show very high correlation between each other as well as with other histone marks (such as H3K4me3) suggesting a coordinated regulation of active histone marks. Moreover, the levels of H3K9ac and H3K14ac directly correlate with the CpG content of the promoters attesting the importance of sequences underlying the specifically modified nucleosomes. Our data provide evidence that H3K9ac and H3K14ac are also present over the previously described bivalent promoters, along with H3K4me3 and H3K27me3. Furthermore, like H3K27ac, H3K9ac and H3K14ac can also differentiate active enhancers from inactive ones. Although, H3K9ac and H3K14ac, a hallmark of gene activation exhibit remarkable correlation over active and bivalent promoters as well as distal regulatory elements, a subset of inactive promoters is selectively enriched for H3K14ac.Our study suggests that chromatin modifications, such as H3K9ac and H3K14ac, are part of the active promoter state, are present over bivalent promoters and active enhancers and that the extent of H3K9 and H3K14 acetylation could be driven by cis regulatory elements such as CpG content at promoters. Our study also suggests that a subset of inactive promoters is selectively and specifically enriched for H3K14ac. This observation suggests that histone acetyl transferases (HATs) prime inactive genes by H3K14ac for stimuli dependent activation. In conclusion our study demonstrates a wider role for H3K9ac and H3K14ac in gene regulation than originally thought. Embryonic stem (ES) cells provide an important model system to study developmental regulation and hold significant potential for clinical therapeutics because of their unique capabilities to self re-new and differentiate into multiple lineages (reviewed in ). The chDifferent modifications of chromatin are associated with variable functions. Histone modifications such as trimethylation of H3 lysine 4 (H3K4me3) and hyperacetylation of histone H3 and H4 are known as active marks and are often associated with ongoing transcription . PromoteOne of the most studied modifications of histones is acetylation of specific lysine (K) residues, which generally correlates with gene activation. The level of histone acetylation is regulated by the activity of both histone acetyl transferases (HATs) and histone deacetylases (HDACs), which acetlylate and deacetylate lysine residues of the N terminal histone tails, respectively. Genetic and biochemical studies suggested that HATs have rather specific roles in gene activation, while genome-wide experiments rather suggested that HATs are often recruited simultaneously, and together are acetylating multiple lysine residues at a given loci ,14. ThusThe lack of in-depth studies concerning the role of H3K14ac was due to the absence of reliable and specific antibodies. Antibodies against modified histone tails are central research tools in studying chromatin biology at a genome-wide level. Thus, we have developed a new specific ChIP-grade antibody against the H3K14 acetylation mark. By using the antibody we have developed against H3K14ac and another published commercial antibody against H3K9ac, we made genome-wide location analysis of these two acetylation marks in mouse (m) ES cells, and compared the presence of two marks over various genomic regions. Our study suggests that these two marks are present not only over promoters of actively transcribed genes, but also on the developmentally regulated bivalent promoters, as well as over active enhancers in mES cells. Moreover, the degree of H3K9 and H3K14 acetylation correlates with the CpG content of the promoters and transcription level of the genes. Finally, we observed differential presence of these two acetylation marks over a subset of inactive genes, which is marked by low level of H3K14ac and thus seemed to be prepared for future activation.To understand the role of histone H3 acetylation at positions K9 and K14 in mouse ES cells, we have systematically tested the available antibodies raised against these modified histone tails. We and others have found that the anti-H3K9ac antibody from Abcam (ab4441) is specific for the corresponding modification in various applications including chromatin immunoprecipitation (ChIP) ,21. HoweTo gain insight in the genome-wide acetylation profile of H3K9 and H3K14 residues, ChIP-seq was performed using the commercially available antibody against H3K9ac and the new antibody developed against H3K14ac in this study. Peaks of local enrichment for H3K9ac and H3K14ac were determined after sequence alignment and normalization to input DNA. Further to validate the peaks obtained in ChIP-seqs for H3K9ac (using ab4441 antibody) and H3K14ac (using 13HH3-1A5 antibody), we performed ChIP-qPCR on randomly selected genomic loci enriched for H3K9ac and H3K14ac. All the selected peaks of H3K9 and H3K14 acetylation from the ChIP-seq experiments were validated by ChIP-qPCR ), coding exons, introns and distal intergeneic regions  islands are usually found at the 5’ end of the regulatory regions of genes . CpG islTranscription from enhancers resulting in enhancer RNAs (eRNAs) play important regulatory role in maintenance of gene expression programs -32. EnhaMany promoters of developmentally regulated genes in mES cells are marked by H3K4me3 (active histone mark) as well as polycomb mediated repressive histone mark, H3K27me3 ,7 and arIn order to test the presence of H3K9 and H3K14 acetylations over the bivalent promoters, all the 27095 mouse promoters from UCSC genome browser were taken  and subjTo test the presence of H3K9ac and H3K14ac over bivalent promoters, we took a subset of randomly selected loci and successfully validated them by ChIP-qPCR FigureB. FurtheWe next analyzed co-localization of the H3K9 and H3K14 acetylation marks with various active and repressive histone modifications. The Pearson correlation coefficient was calculated for these modifications in a window of 2 kb upstream and downstream the TSSs of all the mouse refseq genes 27095). Heatmap of various histone modifications suggest that active histone marks have high correlation and are grouped together for efficient gene expression  and methylation (H3K4me3) provide a general signature for facilitating gene expression over active promoters. On the other hand we showed differential enrichment of H3K14ac over inactive promoters. Furthermore our analyses suggest correlation between acetylation of nucleosomes over the promoters and the underlying sequences and demonstrate the presence of H3K9ac and H3K14ac over enhancers, which can be used in the future studies to discriminate active enhancers from inactive/poised ones. In conclusion, our comprehensive study of H3K9ac and H3K14ac demonstrates a wider role for these to marks in gene regulation than originally thought.2) in DMEM (4.5 gm/lit), 15% FCS, leukemia inhibiting factor, penicillin/streptomycin, L-glutamine, and non-essential amino acids. At least three passages under feeder free conditions on 0.1% gelatin were used to exclude feeders.Wild type embryonic stem cells were derived from blastocysts (3.5 PC) and cultivated on feeder cells  was produced against the peptide “STGGK(ac)APRKC”. The characterization of the antibody is shown in Additional file mES cells were treated with 5 mg/ml sodium butyrate for 0 min, 5 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr and 12 hr. Cells were cross linked with 1% formaldehyde and ChIP was performed as described below. Level of Oct4 was monitored by Western blot to see the pluripotent state of the ES cells at various time points after sodium butyrate treatment  to obtain an average chromatin size of 200-500 bp. Chromatin was pre-cleared using 50 μl of a 50% protein A sepharose  slurry for 1 h at 4°C with gentle inverting. Immunoprecipitation were carried out in 10 ml of IP buffer . 15 μl of ZZZ3 and SPT20 serum antibodies were used. Input chromatin was obtained after pre-clearing, by de-cross linking and purifying input DNA using a Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitation were carried out with inverting at 4°C for 14–16 h. The samples were then incubated with 50 μL of a 50% Protein A sepharose slurry for 3 h at 4°C with gentle inverting. IP samples were reverse-cross linked and the DNA was purified using a Qiaquick column (Qiagen). Q-PCR using SYBR green was used to validate known target sites before and after sequencing. For sequential ChIP, at least four ChIP assays of 2X107 cells were used for the first IP (H3K9ac). Following standard washing, elution was performed with 10 mM DTT . The eluates from four ChIPs were combined, diluted at least 30 times with ChIP dilution buffer and secondary antibody (H3K14ac) was incubated overnight. The subsequent steps were performed as for regular ChIPs. Validation of ChIP-seq was performed by the assessment of the individual enrichment over the control genomic region by ChIP-qPCR in triplicate with primers specific for these regions using SYBR Green master mix (Roche).All ChIP experiments were carried out on 2X10We followed the manufacturer's (Solexa) protocol for creating genomic DNA libraries as previously described in . ChIP DNAdapters in excess were eliminated by using gel purification on a 2% agarose E-Gel (Invitrogen) for 20 min, together with Track-It 50 bp DNA ladder (Invitrogen). DNA fragments ranging from 150 base pairs to 500 base pairs were extracted and recovered in 28 μl EB with a QIAquick gel extraction kit (Qiagen). To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flow cell. The library was recovered in 20 μl EB using MinElute Gel Extraction kit (Qiagen). Finally, DNA concentrations and purities (A260/280 nm ratios) were measured on a Nanodrop spectrophotometer. Sequencing was carried out on the Illumina (Solexa) platform at a sequencing depth of 1 lane averaging 10 million reads, read length of 27 + bp, single-end reads and mapped to mouse genome build (mm9).ChIP-seq data were mapped using the ELAND software (Illumina) allowing one mismatch. Mapped read data were used as an input and to establish list of loci using MACS  . Input D′ end of each ChIP-seq read was extended to 200 bp in the direction of the reads. For average gene profiles, genes (+/-5000 bp from binding site) were divided in 100 bins of length relative to the gene length. Moreover 10 equally sized (50 bp) bins were created on the 5’ and 3’ of the gene and ChIP-seq densities were collected for each dataset in each bin. The average of enrichments over input in each bin was plotted for each dataset. For average TSS profile, the genes as above were aligned on their TSSs and densities were collected in 100 equally sized bins around the TSS (-/+5000 bp).We extracted the tag density in a 5 kb window surrounding the TSSs and gene body using the program seqMINER which generates heatmap as well as the profiles . The seqhttps://main.g2.bx.psu.edu/). All those regions, which do not correspond to the beforehand defined promoter regions, that are not gene bodies, and are not regions that are situated 2 kb downstream of the end of annotated genes, are considered as intergenic regions. Overlapping piece of intervals  with an overlap of at least 500 bps was used to distinguish peaks, which are intergenic for H3K9 and H3K14 acetylation.The list of refseq genes was obtained from UCSC table browser . Co-ordihttp://www.ncbi.nlm.nih.gov/gds) under the accession number: GSM307618 (H3K4me3-mES); GSM307619 (H3K27me3-mES); GSM307621 (H3K9me3-mES); GSM594577 (H3K4me1-mES); GSM594578 (H3K27ac-mES); GSM307623 (Pol II-mES); GSM307624 ; GSM307625 (input-mES); GSM699164 (p300-mES). The sequencing data we have generated is deposited in the GEO database under the accession number GSM775313 (H3K9ac-mES) and GSM775314 (H3K14ac-13HH3-1A5-mES). CpG island definition and locations of known genes were downloaded from UCSC genome browser [ChIP-seq datasets were downloaded from the public data bank Gene Expression Omnibus  density file creation script as described in . Reads ahttp://r-project.org/). The distribution of H3K9ac and H3K14ac in various genomic regions is calculated using the CEAS software [http://liulab.dfci.harvard.edu/CEAS/usermanual.html and [http://david.abcc.ncifcrf.gov/home.jsp).The scatter plots, k-means clustering and average gene profiles are created using seqMiner . Box plooftware . Brieflytml and ). Gene omES: Mouse embryonic stem cells; HAT: Histone acetyl transferase; HDAC: Histone deacetylases; ChIP: Chromatin immunoprecipitation; ChIP-seq: Chromatin immunoprecipitation coupled high throughput sequencing; mAbs: Monoclonal antibodies; TSSs: Transcription start sites; CpG: Cytosine-phosphate diester-guanine; PHD: Plant homeodomain; eRNA: Enhancer RNA; Pol II: RNA Polymerase II.The authors declare that they have no competing interests.KK generated ChIP-seq data and performed experiments. KK, ARK and LT analyzed data. MOA and HK generated antibodies. KK and LT wrote the manuscript. LT planned, coordinated and supervised the project. All authors read and approved the manuscript.Figure S1. The commercially available anti-H3K14ac antibody (Upstate (07-353)) cross-reacts with other peptides. Enzyme linked immunosorbent assay (ELISA) using various peptides such as H3K14 acetylated (H3K14ac) and non-acetylated (H3K14) and non specific histone H4 acetylated peptide (H4K5ac-K12ac) suggest that anti-H3K14ac antibody from Upstate (07-353) cross-reacts with non-acetylated H3K14 peptide as well as with H4K5andK12ac peptide.Click here for fileFigure S2. Characterization of mouse monoclonal anti-H3K14ac (13HH3-1A5) antibody. (A) Western blot analysis using the 13HH3-1A5 (anti H3K14ac antibody) on the recombinant E. coli expressed histone H3 and histones extracted by and acidic extraction protocol from human HeLa cells (right panel). Coomassie blue stained SDS-PAGE of the proteins used for the western blot analysis in a 100-fold dilution. (B) Enzyme linked immunosorbent assay (ELISA) using various peptides such as H3K14 acetylated (H3K14ac) and non-acetylated (H3K14), H3K14 dimethylated, H3S10 phosporylated and K14 dimethylated (H3pS10K14dimethyl), H3S10 phosporylated (H3pS10), H3K9 dimethylated (H3K9dimethyl), H3S10 phosporylated and K9 dimethylated (H3pS10K9dimethyl) and histone H4 acetylated peptide .Click here for fileFigure S3. Validation of (A) H3K9ac and (B) H3K14ac ChIP-seq in mouse embryonic stem cells by ChIP-qPCR. Peaks of local enrichment of H3K9 and H3K14 acetylation were determined after sequence alignment and normalization to input DNA. Tag density per peak (shown on the right of the graph) is plotted as fold enrichment over an arbitrarily chosen control genomic region. The list of primer is provided as Supplementary Table S1. These ChIP-qPCR results confirmed the specificity of the predicted peaks for H3K9 and H3K14 acetylation.Click here for fileTable S1. List of primers used for ChIP-qPCR validation.Click here for fileFigure S4. Enrichment of H3K9ac and H3K14ac over various genomic regions. Average enrichment profile of H3K9 and H3K14ac over the promoters, coding exons, introns and distal intergenic regions. Enrichment over distal intergenic and intronic sites is comparable to promoters.Click here for fileFigure S5. Correlation of H3K9ac (A) and H3K14ac (B) intergenic peaks with H3K4me1, H3K27ac, Pol II and p300. Heatmaps of the signal density using k-means clustering over H3K9ac and H3K14ac distal intergenic sites (-/+ 5 kb). Strong correlation with H3K4me1 and H3K27ac and presence of Pol II and p300 over H3K9ac and H3K14ac intergenic sites suggest that they may act as enhancers.Click here for fileFigure S6. Measurement of Oct4 level at various time points after the treatment with 5 mg/ml sodium butyrate in ES cells. Level of Oct4 was monitored by Western blot to see the pluripotent state of the ES cells at various time points after sodium butyrate treatment. The similar levels of Oct4 observed at different experimental time points suggest that the time points we have used for sodium butyrate treatment have no effect on pluripotency of the ES cells. Tubulin was used as a loading control.Click here for fileFigure S7. Ratio of tag density of least expressed to highest expressed gene promoters for H3K9 and H3K14 acetylation. H3K9 and H3K14 acetylation tag density were calculated over 500 least and highest expressed gene promoters based upon their RNA-sequencing expression profile. The ratio of tag density of least expressed to highest expressed plotted for H3K9 and H3K14 acetylation, which shows that H3K14ac is specifically enriched at inactive promoters as compared to H3K9ac.Click here for fileTable S2.Number of mapped reads for the data set used in this study.Click here for file


TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERα to the enhancer element. Our study defines the mechanism by which MLL1 nucleates histone H3K4 methylation marks in CpG-enriched regions to maintain permissive chromatin architecture and allow FOXA1 and estrogen receptor α binding to transcriptional regulatory sites in breast cancer cells.A number of genome-wide analyses have revealed that estrogen receptor α binding to and regulation of its target genes correlate with binding of FOXA1, a pioneer factor, to nearby DNA sites in MCF-7 breast cancer cells. The enhancer element-specific histone H3K4me1/2 mark is enriched at the specific FOXA1/ERα recruitment sites in chromatin, but the mechanism by which these enhancer marks are established in chromatin before hormone treatment is unclear. Here, we show that mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the  It is generally thought that the enhancer region is looped out or organized to permit enhancer–promoter interaction. This flexibility of enhancer element position relative to the transcription start site, however, impedes the comprehensive assignment of enhancer elements to the target genes they control. Recent genome-wide studies from independent laboratories have revealed several characteristic chromatin ‘signatures’ that define the enhancer elements in genes. For enhancer elements that are actively engaged or poised for activation, these signatures consist of high levels of p300 occupancy and H3K4me1 and a low level of H3K4me3  proteins are SET1 family members that play critical roles in tivation . In mammtivation ,9–11. Retivation . Recentltivation , suggestThe pioneer factor FOXA1 is a transcription factor that binds to thousands of sites across the human genome in breast and prostate cancer cells . FOXA1 iHOXA1 locus and regulates the expression of multiple transcripts -qPCR to assess chromatin conformation and nucleosome occupancy methylome–sequencing (NOMe-seq) to monitor nucleosome positioning, we investigated how the depletion of MLL1 affects the establishment of open (active) chromatin and the binding of the pioneer factor, FOXA1. These findings provide the first evidence of the molecular mechanisms of MLL1, which nucleates histone H3K4 methylation marks at CpG islands to maintain chromatin accessibility for binding of ERα and its pioneer factor FOXA1 in breast cancer cells.Considering that MLL1 binds to specific clusters of CpG residues within the nscripts , we hypoThe pSG5–2 × FLAG expression vectors encoding the following proteins were constructed by inserting the appropriate cDNA coding region into EcoRI and XhoI sites: pSG5–2 × FLAG-MLL1(CX) (a.a. 1067–1432) and pSG5–2 × FLAG-MLL1 (CXPB) (a.a. 1067–1989). The pTriEX-His-MLL1(CX) expression vector encoding His-tagged MLL1 (a.a. 1067–1432) was constructed by inserting the appropriate cDNA coding region into NcoI and XhoI sites. Point mutations in MLL1 were introduced by QuikChange site-directed mutagenesis kit (Stratagene). pF-MLL1 was kindly provided by Jay Hess (University of Michigan). MCF-7, MDA-MB231 and 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum.Supplementary data. Total RNA was isolated from MCF-7 cells with Trizol (Invitrogen) after hormone treatment as indicated and subjected to reverse transcription (RT) by iScript cDNA synthesis kit (Bio-Rad) in a total volume of 50 µl. In all, 2 µl of RT product was used for qPCR analysis with the primers shown in Supplementary data. Relative expression levels were normalized to GAPDH mRNA levels. Results shown are mean and range of variation of duplicate PCR reactions performed on the same cDNA sample; the results are from a single experiment that is representative of at least two independent experiments.Small interfering RNA experiments were performed according to previously published methods . Transfe5 cells/well). The next day, the cells were transfected with siRNA and then cultured for 3 days in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were trypsinized on indicated days, and total live cells were counted after Trypan blue staining.MCF-7 cells were plated into six-well plates , and molecules were cloned using the Topo TA Kit (Invitrogen) according to the manufacturers’ instructions.Escherichia coli strain BL21(DE3), purified by incubation with Ni-NTA agarose beads and eluted with phosphate-buffered saline (PBS) containing 250 mM imidazole. Point mutations in MLL1 were introduced by QuikChange site-directed mutagenesis kit (Stratagene). The CpG-rich enhancer (approximately −10 kb from TSS) or control region (approximately −5 kb from TSS) of the TFF1 gene was amplified using 5′-biotinylated oligonucleotide primers. Biotinylated PCR products were immobilized on streptavidin-agarose beads and incubated with MLL1 proteins. Immunoblotting was performed as described previously . MLL1 cleavage by taspase generates N-terminal 320 kDa and C-terminal 180 kDa fragments, which form a stable complex that localizes to a subnuclear compartment , the estradiol (E2)-induced expression of promised A when MLTFF1, GREB1, CCND1 and PgR after 8 h, and most of this increase was eliminated by depletion of MLL1 (P < 0.01)  and pre-mRNA (data not shown) of ERαtarget genes by using a second siRNA that targeted a different part of the MLL1 coding region.We also measured the pre-mRNA levels of four ERα target genes, which is a more reliable reflection of the rapid changes in transcriptional rates, by using PCR primers spanning an exon/intron junction. E2 treatment increased the pre-mRNA levels of  of MLL1 C, indica < 0.01) D, confirTFF1 gene  was highly occupied by MLL1 and that the recruitment of MLL1 to this region was further enhanced after E2 treatment in MCF-7 cells  is the major ER binding site and considered to be the major enhancer element. Our chromatin immunoprecipitation (ChIP) assay showed that the enhancer region of the -7 cells A. We valreatment B.FigureHOXA9 gene  bound to the CpG-rich ERE3-region (In vitro methylation of the CpG-rich region by the CpG-specific methyltransferase (M.SssI) inhibited most of the binding of MLL1-CX  of the TFF1 gene, indicating that the 40 kDa MLL1-CX fragment is sufficient for recruitment to the enhancer region in vivo .In MCF-7 cells, H3K4me1 and me2 are detected at estrogen responsive enhancers before E2 stimulation and ERα binding, reminiscent of FOXA1 recruitment . We havereatment A and is reatment . We hypo at ERE3 A, indicaTFF1 gene. We validated these findings in independent experiments . In contrast, depletion of FOXA1 by specific siRNA did not affect MLL1 occupancy of TFF1 ERE3 , even though E2-induced ERα binding to ERE3 was almost eliminated . These data support the notion that MLL1 is the key molecule that regulates chromatin occupancy by FOXA1 both before and after E2 stimulation, and also regulates the subsequent E2-induced ERα recruitment to the TFF1 enhancer.In the absence of E2, FOXA1 occupancy at ERE3 was 7.8-fold higher than that in the −5 kb control region, and increased 5.2-fold after estrogen treatment in MCF-7 cells. Surprisingly, specific silencing of MLL1 resulted not only in a 2-fold decrease of the pre-existing FOXA1 occupancy  coupled with qPCR. Hormone-deprived MCF-7 cells were transfected with siNS or siMLL1 and treated with ethanol or E2 for 1 h. siNS-transfected and ethanol-treated MCF-7 cells showed weak FAIRE signals at the proximal and distal ERα binding sites  ; in contrast, ERα binding to the c11orf49 gene that is not occupied by MLL1 was not affected by MLL1 depletion. These data suggest that MLL1 is required to maintain the optimal chromatin structure at the enhancers of many estrogen target genes.Given that the enhancer region of the activity  and 4, wng sites A. We obsed cells A, suggesand MYC) B. In addTFF1 enhancer even before hormone stimulation, we were unable to observe any significant changes in the FAIRE signal by MLL1 silencing in ethanol-treated cells. A possible explanation for this may be the limited resolution level of the assay. To further understand the local nucleosome dynamics, we performed NOMe-seq, a method that measures the methylation of GpC in isolated chromatin by M.CviPI DNA methyltransferase, which allows high-resolution determination of nucleosome occupancy (TFF1 promoter is largely occupied by nucleosome (indicated by pink bars showing regions inaccessible to the GpC methyltransferase), it is somewhat accessible to enzyme , indicating the presence of a minimal nucleosome-depleted region (NDR) in some of the cells that may allow the binding of basal transcription factors . One of the most highly studied gene systems is the TFF1 gene and its response to estradiol; the timing of specific occupancy by many coregulators and histone modifications has been carefully documented  project has systematically mapped regions of transcription, chromatin structure and histone modification, providing new insights into the mechanisms of gene regulation  was sufficient to bind to the CpG region of the TFF1 gene in vitro and in vivo, although the inclusion of PHD fingers and bromodomain further increased MLL1 binding efficiency (in vitro methylation of the CpG-rich TFF1 enhancer fragment by the CpG Methyltransferase (M.SssI) reduced the binding of MLL1-CX  suggests that MLL1 occupancy is required for the transcriptional activity of MLL1 in ERα-mediated gene expression , and human CpG-binding protein (hCGBP) . In MLL1pression ,33,41. WFF1 gene . IntroduFF1 gene E and H.  in vivo . A fragmficiency F and G. ficiency D, and in MLL1-CX D. Thus,  by MLL1 ; and thepression .Previous studies have shown that FOXA1 is colocalized to the majority of ERα binding sites in MCF-7 cells ,19. MoreTFF1 enhancer region  and establishes the histone H3K4 methylation marks  in the RET gene locus  ; National Cancer Institute provided the Cancer Center Support Grant [P30CA014089 to the University of Southern California]. Funding for open access charge: NIH [DK043093].Conflict of interest statement. None declared.


Nucleosomes often undergo extensive rearrangement when genes are activated for transcription. We have shown previously, using paired-end sequencing of yeast nucleosomes, that major changes in chromatin structure occur when genes are activated by 3-aminotriazole (3AT), an inducer of the transcriptional activator Gcn4. Here, we provide a global analysis of these data. At the genomic level, nucleosomes are regularly phased relative to the transcription start site. However, for a subset of 234 strongly induced genes, this phasing is much more irregular after induction, consistent with the loss of some nucleosomes and the re-positioning of the remaining nucleosomes. To address the nature of this rearrangement, we developed the inter-nucleosome distance auto-correlation (DAC) function. At long range, DAC analysis indicates that nucleosomes have an average spacing of 162 bp, consistent with the reported repeat length. At short range, DAC reveals a 10.25-bp periodicity, implying that nucleosomes in overlapping positions are rotationally related. DAC analysis of the 3AT-induced genes suggests that transcription activation coincides with rearrangement of nucleosomes into irregular arrays with longer spacing. Sequence analysis of the +1 nucleosomes belonging to the 45 most strongly activated genes reveals a distinctive periodic oscillation in the A/T-dinucleotide occurrence that is present throughout the nucleosome and extends into the linker. This unusual pattern suggests that the +1 nucleosomes might be prone to sliding, thereby facilitating transcription. The nucleosomal organization of eukaryotic chromatin is critical for gene regulation . The higNucleosome positioning is characterized by two parameters: rotational positioning, defined by the side of the DNA helix that faces the histones, and translational positioning, defined by the nucleosome midpoint (or dyad) with regard to the DNA sequence . The strRecently, using genome-wide paired-end sequencing of yeast nucleosomes, we showed that nucleosomes occupy one of several mutually exclusive, overlapping positions, termed a position cluster, which often includes a dominant position . Thus, aHere, we analyze the nucleosomal relocations that occur upon induction of transcription by 3AT at the genomic level, using the DAC function. To eliminate the ‘noise’ in the datasets caused by ‘contamination’ of core DNA by the fragments much longer and much shorter than 147 bp, we focus on the nucleosomal DNA whose lengths are around 147 bp. Our analysis shows that the prevalent distance between nucleosomes is 162 bp, consistent with the nucleosome repeat length (NRL) in yeast, which is 160–165 bp . In addiSaccharomyces cerevisiae genome using ELAND (Illumina). Only the reads uniquely aligned to the genome with no mismatch were selected.The paired-end nucleosome sequence data from 3AT-treated cells and control yeast cells (CC) have been described previously ,27. The Supplementary Figure S1). The highest peak in both distributions occurs in the interval from 147 to 152 bp. Therefore, all DNA fragments of 147–152 bp were selected for further analysis . Note that the length of DNA in the highest resolution crystal structure of the NCP is 147 bp  and coordinate +140 of the gene. The sequences were oriented according to the direction of transcription.Supplementary Figure S2). For each pair of NCP sequences, the distance between the NCP start locations was calculated. Then the occurrences of all distances are summed up for both strands. Note that multiple occurrences of nucleosomes in the same position were counted multiple times—that is, if two specific nucleosome sequences occur 5 and 10 times, respectively, the corresponding auto-correlation will be calculated as 5 × 10 = 50. In this regard, our DAC differs from the ‘coincidence number’ as a function of ‘start-to-start distance’ used by Valouev et al.  function , we used the distance cross-correlation (DCC) function. It is similar to the auto-correlation function, except that the distances were calculated between the start location of each nucleosome in one dataset and all of the start locations of nucleosomes in the other dataset.in vivo map (P < 10−15) between the CC map and the YPD map . Comparison of the nucleosome organization around the 5′-end of genes (The CC and 3AT datasets contain ∼5.4 and 3.9 million DNA fragments of core particle length 147–152 bp), respectively. These NCP sequences were used to build nucleosome maps centered on the 5′-ends (TSSs) of all yeast genes. Both the CC and 3AT nucleosome maps are similar to the vivo map  obtainedof genes A reveals–152 bp, of genes : nucleosof genes , the+1 nof genes A.FigureSupplementary Figure S2). This function exhibits a series of well-defined peaks separated by 150–170 bp . The NRL is determined from the sizes of the oligo-nucleosomal DNA fragments in the ‘ladder’ observed after separation in an agarose gel. Each oligo-nucleosomal band represents a population of chromatin fragments with a certain number of nucleosomes on the same DNA molecule, which cannot overlap . The NRL that is inferred from these bands therefore describes the relationship between physically connected nucleosomes. In contrast, the DAC function . Therefore the DAC function describes the distance relationship between all nucleosomes, not just those on the same DNA molecule. That the two repeat lengths are so close indicates that yeast nucleosome positioning is characterized by a strong long-range order which is systematically reproduced in all cells, genome-wide.Note that the two periods mentioned above have essentially different meanings , but not with the set of NCP sequences having rigorously defined lengths of 147–152 bp  A second explanation implies that indeed, the yeast nucleosomes are in general more regularly positioned than in nematode. This is a reasonable possibility, given that C. elegans has many cell types and thus, its chromatin organization is likely to be more heterogeneous than in yeast. To clarify this issue, the lengths of the nematode nucleosomal DNA sequences are needed.The sub-peaks we observed at 10, 20, 30 bp, etc. B appear 7–152 bp B. HoweveHIS3, for example, 3AT induction results in the disruption of two nucleosome position clusters (D3 and D4)   and the PET56–HIS3 chromatin rigorously, we calculated the DAC function for nucleosome sequences in the CC and 3AT sets  that are induced >2-fold . The regular nucleosome pattern downstream of the 5′-end of these genes was clearly disrupted in the 3AT set compared to the CC set . For the CC set, the profile of the function gradually decreases in the interval from 10 to 80 bp and then increases in the interval from 90 to 140 bp, so that a continuous envelope curve can be drawn through the peaks of the function C. In thehromatin C. The sthromatin D is qualhromatin C.The most likely interpretation of the discontinuity observed at a distance of 90 bp in the 3AT set D is thatTo compare nucleosome positions directly (on the 234 genes induced by 3AT) in the CC and 3AT sets, we calculated the cross-correlation function (see ‘Methods’ section). This function has the highest value at position 0 . Also, the fine structures of the two DAC functions look very similar in terms of the overall trend displayed by their profiles . Note that at the two positions denoted T|T above, thymines occur in ∼50% of all sequences, whereas occurrence of W (either A or T) at these positions is as high as 83 and 93% (see positions 0 and +1 in Supplementary Table S1). However, this consensus is very weak because it describes <5% of the actual cleavage sites. A much more predictive consensus sequence is W|W, which accounts for ∼80% of cleavage sites, and agrees with that reported by Field et al. . Both datasets exhibit the well-known sinusoidal WW and SS patterns with ∼10-bp periodicity and shifted from each other by ∼5 bp  on the observed periodic sequence patterns generally believed to guide nucleosome positioning, at least at the level of dimeric steps. A recent study of nucleosomes reconstituted on genomic DNA reached the same conclusion with regard to MNase bias .in vivo has been described in many studies . Interestingly, this effect is rather strong for the top-45 set , but not for the bottom-45 set . Thus, we predict that the +1 nucleosomes in the top-45 set should slide downstream more easily than their counterparts in the bottom-45 set. All these factors taken together suggest that the +1 nucleosomes in the bottom-45 group will be less easily shifted, which correlates with the weaker induction of these genes.In addition, we analyzed WW profiles that are not symmetrized with respect to the dyad axis and found that for the 234 genes activated by 3AT, the periodic peaks extend into the downstream linker , indicating that the unusual DNA sequence patterns described above exist only in +1 nucleosomes.In summary, we see that there is a clear negative correlation between the 3AT-induced activation of gene transcription and the strength of +1 nucleosome positioning, as deduced from the periodic patterns of WW peaks. This correlation is not found in the −1 and +2 nucleosomes . In this study, we have introduced the DAC function, which takes into account the frequency of occurrence of nucleosomes at a given position. Accordingly, the variation of our DAC value is larger: the peak-to-trough ratio is 3.25/1.5 (=2.17) .Valouev et al.  introducHIS3 chromatin, which undergoes substantial changes upon 3AT induction  rendered no periodic pattern in the WW distribution . The WW periodicity was detected only after we re-aligned the nucleosome sequences using the WW, SS and other sequence patterns critical for nucleosome positioning , see Supplementary Figure S7B. In contrast, the strong periodic WW pattern is clearly visible in the raw data from Cole et al. . We obtained a similar result for the nucleosome positions reported by Henikoff and co-authors . Apparently, these +1 nucleosomes might easily slide by ∼10 or 20 bp (particularly downstream from the TSS) and still retain the same orientation of the DNA helix relative to the histone core. Importantly, this pattern exists in the +1 nucleosomes before activation . Here, the +1 nucleosomes are distributed so broadly that there is no gap between nucleosomes +1 and +2. In the bottom-45 genes, on the contrary, the peaks for nucleosomes +1 and +2 are clearly separated from each other by a very deep and wide gap, both in 3AT-activated and control cells . Apparently, these +1 nucleosomes are well positioned, in accord with the unusually strong WW rotational positioning signal revealed in this case . Funding for open access charge: The Intramural Research Program of the National Institutes of Health.Conflict of interest statement. None declared.


Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes.The principal virulence determinant of  Mycobacterium tuberculosis (Mtb), is the ESX-1 secretion system. It has been postulated that the DNA-binding protein EspR controls the virulence of Mtb by specifically regulating expression of the exported EspA protein, which is required for ESX-1 to function. Previous structural studies indicated that EspR forms dimers capable of multimerizing on DNA and forming loop structures, thus bringing together otherwise distant chromosomal regions. Such characteristics are reminiscent of nucleoid-associated proteins (NAPs), the histone equivalent in bacteria. Here we use ChIP-Seq technology to map EspR binding sites on the Mtb chromosome in living bacterial cells. Genome-wide analysis of EspR identified hundreds of binding-sites, with almost equal inter- and intra-genic distribution, and mostly found in proximity to genes associated with cell wall function. We validated a subset of EspR-binding sites experimentally and identified a consensus motif required for optimal binding affinity. Moreover, our study reveals that EspR expression varies with bacterial growth and that intracellular levels are not linked to EspR secretion. These findings corroborate the NAP nature of EspR and its dual roles, architectural and regulatory, that impact the Mtb chromosome and pathogenesis globally rather than the ESX-1 loci specifically.A major infection mechanism employed by the causative agent of tuberculosis,  Mycobacterium tuberculosis (Mtb) by specifically regulating expression of EspA, an exported protein Mtb but functional information is scarce for all of them, although ESX-1 is by far the most studied Mtb since it secretes the EsxAB (ESAT-6 and CFP10) proteins and ESX-1 secretion-associated proteins (Esps) Details of the genetic control mechanisms governing the pathogenicity of the etiological agent of human tuberculosis are starting to emerge espACD locus espA locus, where DNA bending and bridging resulted in loop formation Structural studies revealed that EspR is a homodimer with two domains: an N-terminal DNA-binding domain with a helix-turn-helix (hth) motif and C-terminal domain that mediates dimerization Mtb H37Rv genome was performed. Unlike cDNA hybridization to micro-arrays, ChIP-Seq generates quantitative information with single nucleotide resolution. The complete repertoire of EspR-binding sites across the chromosome was established with the majority of the gene targets appearing to impact cell wall function generally rather than ESX-1 expression in particular. The ChIP-Seq findings were confirmed independently by ChIP followed by quantitative real-time PCR (ChIP-qPCR), by gel shift and DNase I footprinting assays, as well as by transcript analysis, and a binding motif was deduced using bioinformatics. Finally, the expression levels of EspR and its subcellular localization were monitored and quantified during the growth cycle. Altogether, these data provide a genome-wide view of EspR regulatory functions and place EspR firmly in the NAP family.To test the hypothesis that EspR might be a NAP and to gain more insight into the extent of EspR-mediated regulation in living mycobacteria, ChIP-Seq analysis of the Mtb strain H37Rv during exponential growth by ChIP-Seq, chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing Mtb H37Rv genome. Based on the peak detection criteria, we identified 165 enriched loci harboring 582 EspR-binding peaks , and some of these, such as ppe58 (rv3426), also bind EspR at their 5′-ends. Binding sites were present within genes that are thought to have been acquired by horizontal transfer rv0986-rv0989c region.Diverse functions are encoded by genes where EspR bound upstream and classification by functional category reveals over-representation of cell wall/cell processes and the surface-exposed PE/PPE proteins , encoding a lipid esterase, and rv3488, whereas the last peak of the top ten ChIP-Seq list was found at the 3′-end of fadB2 (rv0468) encoding a beta-hydroxybutyryl-CoA dehydrogenase and upstream of umaA (rv0469) coding for a mycolic acid synthase. EspR binds to multiple sites in the ESX-1, ESX-2 and ESX-5 loci  distributed among 80% of the EspR peak sequences analyzed. Of these motifs, 59% were localized within open reading frames (ORF). The entire Mtb genome sequence was searched for potential EspR-binding sites using the TTTGC[TC][GA] consensus sequence and 199 putative intergenic and 827 intragenic sites were identified, of which 163 (43 intergenic and 120 intragenic) correspond to known ChIP-Seq peak sites. Further experimental support for the consensus sequence is available from footprinting studies of the espAespR promoter regions (see below), which revealed EspR protection from DNase I digestion at sites comprising at least one TTTGC-like motif.An EspR consensus sequence was identified in the ChIP-Seq peaks using the MEME suite in vivo dataset, we initially focused on the EspR-dependent espACD operon in vitro work revealed two EspR binding sites separated by 19 bp and located between 506 and 444 bp upstream of espACDespA . While this work was in progress, another report of the presence of two additional sites upstream of espACD appeared espACD promoter fragment espACD upstream region. The 5′-end of the espA mRNA was located 66 bp upstream of the translation start codon using 5′ RACE (To obtain independent confirmation for selected parts of the  5′ RACE . Consequrv0888 and sigA ORF) as controls. All of the selected EspR-binding regions exhibited enrichment comparable to that observed from ChIP-Seq analysis  and two non-peak regions  using ∼100 bp DNA sequences covering the top five binding sites  sequence 144 bp upstream of the translational start codon. The promoter is therefore situated in a region between peaks a and b so simultaneous occupation by EspR of both the a and b sites might form a repression loop. Expression data presented below indicate a negative effect of EspR on its own transcription.5′ RACE analysis was employed to localize the espR conditionally in Mtb , is known to occur early in the growth cycle. To determine whether levels were constant or variable during growth and to estimate the number of EspR molecules per cell, kinetic experiments were performed. We monitored EspR protein levels by quantitative Western blotting at different time points corresponding to the early-log (day 2), mid-log (day 3) and stationary (days 4 and 5) phases of growth. Analysis of equivalent cell numbers showed that the intracellular concentration of EspR increases throughout the bacterial growth cycle, ranging from ∼20,000 molecules at early log-phase (day 2) to ∼100,000 molecules per cell at stationary phase (day 5)  . Peak ce (day 5) . To detee points . InteresMtb H37Rv and Mtb H37RvΔRD1 cultured in Sauton's medium to mid-log phase. Under these conditions, we were unable to detect EspR among the culture filtrate (CF) proteins in either case, whereas EsxA was present in the CF of Mtb H37Rv, as expected, but not in CF from the ESX-1 mutant H37RvΔRD1 that lacks esxA among other genes  was fractionated by ultracentrifugation into the cell wall/cell membrane (W/M) and cytosolic (CYT) components. Since the chromosome is known to be attached to the plasma membrane b Erdman . We thenb Erdman .Mtb and the remarkable, and most unusual, property of being secreted by the very secretion system ESX-1 whose expression it controls The EspR protein has attracted considerable interest because of its role in the regulation of virulence in lipF and the PDIM locus. Surprisingly, the latter harbored the major peak of EspR binding in the genome whereas the previously reported site preceding the espACD operon was less prominent  were involved in cell envelope functions. Among them were genes that contribute to ESX-1, ESX-2 and ESX-5-related activities, and others that contribute to mycobacterial virulence, such as rominent .in vitro, from a combination of studies performed with highly purified EspR  or traps RNAP that has already bound. In ChIP-Seq experiments performed with RNAP-antibodies, a major polymerase binding site was localized (data not shown) that partially overlaps EspR peak a  H37Rv and Erdman were grown in 7H9 Broth (Difco) supplemented with 0.2% glycerol, Middlebrook albumin-dextrose-catalase (ADC) enrichment and 0.05% Tween 80 or on solid Middlebrook 7H11 medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC). Mtb cells prepared for secretion analysis and cell fractionation were grown in Sauton liquid medium. Escherichia coli BL21(DE3) was used for expression of His9-tagged EspR as described Mtb H37Rv cultures (50 ml) grown to OD600 0.4–0.6 were treated with formaldehyde  for 10 min at 37°C. Cross-linking was quenched by addition of glycine to a final concentration of 125 mM. Harvested cells were washed twice with Tris-buffered saline , re-suspended in 600 µl Immunoprecipitation (IP) buffer ) and sonicated to shear DNA to an average size of 100–500 bp. Insoluble cellular debris was removed by centrifugation and the supernatant used as input sample in IP experiments. 300 µl of input was incubated with either no antibody (mock-IP) or 20 µl of serum containing rat anti-EspR polyclonal antibodies (kindly generated by Ida Rosenkrands) and then with 50 µl of Dynabeads sheep anti-rat IgG  pre-saturated with 0.1 mg/ml salmon sperm DNA and 1 mg/ml Bovine Serum Albumin (BSA) in IP buffer. Washing and crosslink reversal of protein-DNA complexes, as well as purification of the resulting DNA was carried out as previously described Chromatin immunoprecipitation was performed essentially as previously described DNA fragments (150 to 250 bp) were selected for library construction and sequencing libraries prepared using the ChIP-Seq Sample Preparation Kit  according to the protocol supplied with the reagents. Prior and post library construction, chromatin immunoprecipitation products were quantified using the Qubit fluorometer . One lane of each library was sequenced on the Illumina Genome Analyzer IIx using the Single-Read Cluster Generation Kit v4 and 36 Cycle Sequencing Kit v4. Data were processed using the Illumina Pipeline Software v1.60.Mtb H37Rv genome (NCBI accession NC_000962.2) using Bowtie Mtb H37Rv genome to identify sequencing artifacts and calculate enrichment values. Since comparison of the two ChIP-Seq datasets showed excellent correlation in binding signals , diluted to OD600 = 0.1 (day 0) and split into two 30 ml volumes prior to addition of 0 or 2 µg/ml pristinamycin IA. Cells were harvested at day 3.Construction of the pMYespR vector for pristinamycin IA-inducible overexpression of For whole cell Western blot analysis, bacteria were pelleted, washed twice in PBS containing 0.05% Tween 80 and resuspended in lysis buffer  prior to sonication (15 min at 4°C). Cell debris was then pelleted and the supernatant filter sterilized. After total protein quantification using Bradford reagent (Sigma), 10 µg of cell lysates were separated by SDS-PAGE, and specific proteins visualized by immunoblot as described below.Proteins were separated on NuPAGE Novex 4–12% bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. Membranes were incubated in TNT blocking buffer  with 5% w/v skim milk powder for 2 h prior to incubation with primary antibodies diluted in TNT with 1% BSA overnight. Membranes were washed in TNT five times, then incubated with secondary antibodies for 1 h before washing. Immunoblotting were performed with rat polyclonal anti-EspR antibodies (kindly provided by Ida Rosenkrands), mouse monoclonal anti-EsxA antibodies (Hyb 76-8), mouse monoclonal anti-GroEL2 antibodies (IT-70), rabbit polyclonal anti-EsxB and anti-SodA antibodies. All antibodies were used at a dilution 1∶1,000 except for anti-GroEL2 , anti-EsxB (1∶500) and anti-SodA (1∶50). Horseradish peroxidase (HRP) conjugated rabbit polyclonal anti-rat IgG, rabbit polyclonal anti-mouse IgG or goat polyclonal anti-rabbit IgG secondary antibodies (Sigma) were used at dilutions of 1∶100,000. Secondary antibodies were visualized using chemiluminescent substrates (Sigma).Quantitative RT-PCR reactions were performed with the 7900HT Fast Real-Time PCR System (Applied Biosystems) with the following parameters: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Melt curve analysis was used to confirm specific amplification for each primer pair. Threshold cycle values were determined automatically using the SDS software.Enrichment of ChIP DNA samples was measured by quantitative RT-PCR using peak-specific primer pairs , IP or msigA signals and are average of three biological replicates.For gene expression analysis, RNA was extracted with Trizol reagent (Invitrogen) and treated with DNase I (Roche) prior to generation of the cDNA template. cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's instructions using random hexamers primer. cDNA corresponding to 10 ng of input RNA was used in each RT-PCR reaction supplemented with specific primer pairs (200 nM each) listed in Mtb H37Rv cells were grown to OD600 0.6 and then diluted in 100 ml with a starting OD600 of 0.05 (day 0). At various time points corresponding to the early-log (day 2), mid-log (day 3) and stationary (days 4 and 5) phase of growth, the number of cells was estimated from OD600 and culture aliquots were pelleted by centrifugation for RNA and protein analysis. For protein analysis, 400 to 100 µl cell aliquots were resuspended in a 1× NuPAGE sample buffer (Invitrogen) and boiled for 45 min. For all time points, equivalent amounts of cells were subjected to SDS-PAGE together with purified EspR protein standards  and proteins detected by immunoblotting. Quantification was performed by densitometry of scanned blots using the ImageJ software. A standard curve was made from the intensity of the purified EspR bands and was shown to be linear in the 7.5 to 40 ng range. Quantification of expression levels at each time point was estimated from the standard curve after correction for small sample loading discrepancies using GroEL2 band intensity for normalization. The experiment was repeated four times and a typical immunoblot pattern of EspR expression is shown in Mtb starter cultures were first grown in complete 7H9 broth to late-logarithmic phase (OD600∼0.8–1). These were then used to inoculate Sauton's medium supplemented with 0.05% Tween-80, at starting OD600 of 0.1. Cells were grown to mid-log phase of growth (OD600∼0.6), centrifuged, washed once with phosphate buffered saline (PBS), resuspended in a volume of Sauton's medium without Tween-80 such that the OD600 was again ∼0.6 then grown further at 37°C with shaking. Cultures were harvested at required times post-transfer by centrifugation to obtain culture filtrates and cell pellets. Culture filtrates (CF) were filtered sequentially through 0.4 and 0.2 micron filters to remove any residual cells and concentrated in Vivaspin columns with 5-kDa molecular weight cut-off membranes . Cell lysates (CL) were prepared by resuspending cell pellets in lysis buffer (PBS containing Roche mini-protease inhibitor cocktail tablets) followed by bead beating with 100 micron glass beads and centrifuged. Total protein concentration of all preparations was determined using the BCA assays (Pierce) with BSA as the standard. Equivalent total protein concentrations of CF and CL were analyzed by SDS-PAGE and immunoblotting. For cell fractionation experiments, CL samples were split into two equivalent volumes and treated with either 50 µl PBS or 50 µl of 1,000 units/ml DNase I overnight at 4°C. CL samples were centrifuged at 200,000 g for 4 h at 4°C in a Beckman-Coulter ultracentrifuge (TLA-100.3 rotor) to obtain the cell wall/cell membrane (W/M) fraction. Supernatants containing the cytosolic (CYT) fractions were carefully recovered and the W/M fractions resuspended in PBS with 0.1% Triton X-100 and mini-protease inhibitor cocktail. Equivalent amounts of the different cell fractions were separated by SDS-PAGE and specific proteins were visualized by immunoblotting.Figure S1Correlation plot showing the reproducibility of EspR ChIP-Seq.(PDF)Click here for additional data file.Figure S2Mtb genome. (A) Venn diagram showing significant overlap between the gene targets of Lsr2 http://genome.ucsc.edu) view of selected binding profiles as determined by ChIP-Seq for EspR  and by ChIP-on-chip for Lsr2  the rv0986-7-8 operon region; (D) the espA-ephA intergenic region; (E) the ESX-1 (rv3864-3883c) and ESX-2 (rv3884c-rv3895c) regions; and (F) the PDIM/PGL locus (rv2928-rv2962c). The GC content of Mtb H37Rv genome (in 20 bp windows) normalized to the median GC content (65.6%) and the gene positions are indicated below.Comparison of EspR and Lsr2 binding sites on the sr2 red, . Shown a(PDF)Click here for additional data file.Figure S3espA promoter. RNA extracted from Mtb H37Rv at mid-log phase was used for the 5′ RACE. Translational starts are highlighted in red with +1 corresponding to the first base of the espA open reading frame. Transcriptional start mapped by 5′ RACE is boxed and indicated by a bent arrow. −10 and −35 positions for putative sigma factor binding sites are indicated. ChIP-Seq peak sequences are highlighted in gray.Analysis of the (PDF)Click here for additional data file.Figure S4sigA and rv0888 genes, showing no peak in EspR ChIP-Seq, were used as negative controls. Plot showing log2 enrichment calculated from ChIP-Seq and ChIP-qPCR experiments shows a good correlation between the output of the two experiments. The log2 enrichment values have also been indicated in the table below along with additional details about the peaks. The column “Peak feature” indicates where the EspR-binding site is located relative to the known gene annotation. “NA” denotes not applicable.Validation of 11 EspR-binding sites selected among a wide range of scores by quantitative RT-PCR. The (PDF)Click here for additional data file.Figure S5fadD26, rv1490, rv2929, pe_pgrs19, espA. A DNA fragment from within the espA coding region where no ChIP-Seq enrichment was observed was used as negative control.EMSA showing binding to the top five ChIP-Seq peak sequences related to the following genes: (PDF)Click here for additional data file.Figure S6espR promoter. Red and black peaks represent DNA incubated without or with 10 µM EspR proteins, respectively. Both reactions were partially digested with DNase I and analysed by capillary electrophoresis in a genetic analyser (Applied Biosystems 3130xl). The corresponding sequencing reaction of the DNA fragment is shown at bottom. Regions I and II protected from DNase I digestion by EspR and regions IIaΔ10 and IIbΔ10 protected from DNase I digestion by EspRΔ10 are denoted by square brackets. Positions indicated are relative to the translational start.DNase I footprint at peak “a” of the (PDF)Click here for additional data file.Figure S7Mtb Erdman wild-type (left) and 36–72  and 5 µg of cell lysates (CL) of (PDF)Click here for additional data file.Table S1List of primers used in this study.(PDF)Click here for additional data file.Table S2Mtb H37Rv genome sorted by score (highest to lowest). Peaks are numbered by order of appearance in the genome. The column “Feature” indicates where the EspR-binding site is located relative to the known gene annotation. The “Distance to feature” column designates the distance (in bp) from the midpoint position of the calculated peak to the closest translational start site. Values in the “Score” column are arbitrary units proportional to the peak density. The “Enrichment” column represents the ratio between the average ChIP-Seq read count at a particular locus (400 bp) and the input DNA read count at that locus.List of the EspR ChIP-Seq peaks on the (XLS)Click here for additional data file.


GAL1 promoter. The TAL-PrA fusion enabled chromatin affinity purification (ChAP) of a small section of native chromatin upstream from the GAL1 locus, permitting mass spectrometric (MS) identification of proteins and histone post-translational modifications regulating galactose-induced transcription. This TAL-ChAP-MS approach allows the biochemical isolation of a specific native genomic locus for proteomic studies and will provide for unprecedented objective insight into protein and epigenetic mechanisms regulating site-specific chromosome metabolism.Here, we describe an approach to isolate native chromatin sections without genomic engineering for label-free proteomic identification of associated proteins and histone post-translational modifications. A transcription activator-like (TAL) protein A fusion protein was designed to recognize a unique site in the yeast  Chromatin immunoprecipitation (ChIP) assays have traditionally been used to better understand genome-wide distributions of chromatin-associated proteins and histone post-translational modifications (PTMs) at the nucleosome level  effector proteins to site-specifically target a native section of a chromosome for purification and proteomic analysis. We term this approach TAL-ChAP-MS A. TAL eftivators . TALs cotivators ,17. Bindtivators . Thus, btivators ,19. To trevisiae B. We cho because  we previ because ; thus, aGAL1 promoter chromatin at levels sufficient for proteomic analysis  to bind a unique 18-nt sequence (GGGGTAATTAATCAGCGA) 193 base pairs upstream of the GAL1 open-reading frame  S. cerevisiae (W303 matA) cells were cultured to mid-log phase in 3% galactose-containing media, subjected to 1.25% formaldehyde cross-linking, cryogenically lysed and subjected to sonication to shear genomic DNA to ∼1 kb , [P30GM103450], [P20GM103429] and [UL1TR000039]. Funding for open access charge: NIH [R01GM106024 to A.J.T. and S.D.T.].Conflict of interest statement. None declared.