NANOG, SOX2, cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes, including  OCT4 and NANOGEmbryonic stem cells (ESCs) are derived from blastocysts and are considered pluripotent since they have the potential to give rise to a myriad of cell types. For this reason they are of great therapeutic value. However, before stem cells or induced pluripotent stem (iPS) cells are taken to the clinic, a greater understanding of the basic biology of embryonic stem cells is needed. Embryonic stem cells have the ability to self-renew and proliferate indefinitely in culture and several studies have described the importance of the core regulatory circuitry that is comprised of NANOG, OCT4 and SOX2 proteins to maintain a pluripotent state Proteins like NANOG, OCT4 and SOX2 regulate gene expression in a classic manner, by binding to target gene promoters and facilitating transcriptional activation. Other components, such as insulator proteins, function differently from canonical transcription factors by segregating genomic loci into distinct domains to protect them from inappropriate activation or repression by adjacent DNA regions (reviewed in H19/IGF2 Imprinted Control Region (ICR) IGF2 promoter, thus silencing IGF2 on the maternal allele. However, on the paternal allele, CTCF binding is abrogated due to methylation of ICR thereby rendering IGF2 transcriptionally active. Furthermore, deletion of CTCF binding sites within this locus results in loss of enhancer-blocking activity p16 tumor suppressor gene, which segregates it from an adjacent region of heterochromatin. Intriguingly, aberrant epigenetic silencing of the p16 gene, which is widespread among human cancers, occurs when the boundary destabilizes upon loss of CTCF binding, and nearby heterochromatin spreads into the locus At the molecular level, episomal enhancer-blocking assays have led to the identification of several enhancer-blocking insulators including the 5′ hypersensitive site-4 site (5′HS-4) from the chicken β-globin locus and the Genome-wide CTCF binding analyses indicate that CTCF association with DNA is largely conserved across cell types including pluripotent and differentiated hESCs, even though gene expression patterns differ considerably among distinct tissues and species NANOG, SOX2, cMYC, KLF4 and LIN28, are affected. CTCF-depletion accelerates BMP4-induced loss of pluripotency. We find that CTCF associates with the distal ends of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus in hESCs and that each of these sites can serve as CTCF-dependent enhancer-blocking insulator in a heterologous assay. Interestingly, CTCF binding to the NANOG locus does not change upon BMP4-induced differentiation, however the interaction of CTCF cofactors is selectively modulated at particular CTCF-bound sites.In this study, we sought to bridge this gap. Our data show that depletion of CTCF in hESCs does not lead to spontaneous differentiation of hESCs although hESC proliferation and expression of certain genes implicated in pluripotency regulation, such as NANOG, OCT4, SOX2, DPPA3, GDF3, LIN28 and cMYC while increasing expression of a BMP4-responsive gene-ID3  hESCs grown under defined conditions, i.e, in Matrigel and TeSR (pluripotent hESCs) and (2) hESCs treated with BMP4 to induce differentiation (differentiated hESCs) gene-ID3  [22]–[24gene-ID3 . Finally lineage  [25], [2Genome-wide CTCF binding profiles have been elucidated in a variety of cell types including mouse and human ESCs; however, the significance of CTCF association with specific genomic regions remains unknown NANOG, OCT4, SOX2, DPPA3 and TERT to different degrees  upon loss of CTCF. cMYC has been implicated in maintaining proliferation of ESCs; therefore loss of proliferation is consistent with decreased cMYC mRNA upon CTCF knockdown , which drives cells towards the epithelial mesenchymal lineage  as described in CTCF is a diverse protein that can function as an activator, repressor or insulator, thus we hypothesized that CTCF may associate with and regulate critical stem cell genes/gene loci. Genome-wide CTCF binding studies have been conducted across terminally differentiated cell types  but not when placed upstream of the enhancer (PstI site) (APOBEC1 gene (We were intrigued by CTCF binding at tI site) . InteresEC1 gene . The actNANOG locus can function as enhancer-blocking insulators in a CTCF-dependent manner, the strongest of these being the outermost sites at –151 and +17 kb that flank the entire ∼160 kb domain.Next, we asked if the enhancer-blocking activity is CTCF-dependent. To test this, we ablated CTCF using siRNA before performing the heterologous enhancer-blocking assay. Upon CTCF depletion, the enhancer-blocking activity was abrogated by two-to three-fold . ImportaNANOG locus upon differentiation, we performed CTCF ChIP experiments in BMP4-treated hESCs. Interestingly, CTCF association does not significantly change even when the NANOG locus becomes transcriptionally repressed upon differentiation  Polymerase 1 (PARP1), TopoIIβ, Cohesin, Nucleophosmin and Nucleolin. Importantly, co-localization of some of these partners and post-translational modification by Poly(ADP-ribosyl)ation (PARlation) have been shown to be critical for CTCF-mediated insulator activity  TopoIIβ . Second, TopoIIβ . Third,  TopoIIβ . These oNANOG locus. In pluripotent hESCs, these sites were associated strongly with Rad21 (a subunit of Cohesin)  and TopoIIβ levels did not change : –146 and –112 within the mal site  and S9. p21, concomitant with decreased proliferation NANOG, OCT4 and SOX2 are essential genes for ESC pluripotency; NANOG participates in a positive regulatory feedback loop with OCT4 and SOX2 and plays a key role in activating other genes that are components of critical stem cell signaling pathways NANOG-DPPA3-GDF3 locus at −151, −146, −112, and +17 kb and that CTCF depletion negatively impacts NANOG and DPPA3 expression in hESCs (p<0.05 respectively at 96 hrs for both). Interestingly, mRNA levels of GDF3 increase upon CTCF knockdown possibly because this gene is transcribed in the opposite direction from NANOG and DPPA3 or a previously restricted enhancer within the locus has become available to the GDF3 promoter.CTCF is a ubiquitous protein that can regulate chromatin “barrier” boundaries or enhancer-blocking insulators in addition to functioning as a transcriptional activator or repressor . Several studies have shown that CTCF plays important roles in processes such as genetic imprinting, X-chromosome inactivation and preventing aberrant transcriptional silencing of tumor suppressor genes NANOG locus can each function as an enhancer-blocking insulator in a heterologous system. In particular, the sites flanking the NANOG locus at –151 and +17 kb have the highest affinity for CTCF, since these sites remain associated even upon siRNA-mediated CTCF ablation, and are the most effective as enhancer-blocking insulators. Thus, we hypothesize that the outermost CTCF recognition sites at –151 and +17 kb may segregate the multi-gene NANOG locus into a discrete chromosomal domain and shield it from the effect of surrounding regions by serving as barrier insulators.We further establish that the four CTCF binding sites within the NANOG-DPPA3-GDF3 genes has been reported in human embryonal carcinomas, seminomas and breast carcinomas β-globin locus NANOG transcription start site http://insulatordb.uthsc.edu, NANOG transcription start site; however, we did not detect CTCF enrichment by ChIP at these sites in hESCs (data not shown). Consistent with our data, global ChIP-seq analysis in hESCs did not reveal CTCF interaction in the vicinity around ∼50 kb upstream of the human NANOG locus NANOG gene locus or possible differences within the murine and human ES cell regulatory network.The human chr12p13 is a hotspot for teratocarcinomas and synchronized over-expression of the NANOG locus, we found that CTCF also associates with SOX2, cMYC and LIN28 loci. Interestingly, CTCF interaction with these genes varies in location: CTCF flanks the NANOG-DPPA3-GDF3 locus; CTCF binds to distal promoters of cMYC and SOX2, to proximal promoter of cMYC; and finally, CTCF associates within gene coding region of LIN28. This binding diversity may indicate that CTCF controls these genes by multiple mechanisms. We show that the four CTCF sites within the NANOG locus can function as enhancer-blocking insulators in a heterologous system. It is plausible that CTCF is an activator of cMYC transcription by associating with their proximal promoters. By contrast, at the cMYC and SOX2 distal promoters, CTCF may provide a boundary or insulator activity. Importantly, CTCF was originally characterized as a repressor of cMYC transcription. However, a recent study demonstrated that at the endogenous cMYC site, CTCF is required for cMYC transcription and that deletion of distal and proximal CTCF sites at the native locus results in progressive encroachment of DNA methylation LIN28 and is important for maintaining its proper expression since CTCF depletion reduces LIN28 mRNA levels. In fact, a large number of CTCF sites are intragenic and it has been suggested that these sites can regulate transcription by modulating RNA polymerase II progression In addition to the NANOG locus is largely unaltered upon BMP4-induced differentiation or in terminally differentiated fibroblasts  lines H9 (WA09) or H1 (WA01) were grown on Matrigel (BD Bioscience) in mTeSR1 (Stem Cell Technologies) or an in-house version . For differentiation, 200 ng/ml recombinant BMP4 (Stemgent) was added to TeSR for five days unless otherwise mentioned. Media was replenished every 24 hrs. For TPA-induced differentiation, 50 ng/ml TPA was added directly to TeSR. For retinoic acid induced differentiation, hESCs were grown in TeSR media minus FGF and TGFβ growth factors prior to differentiation using 1 µM retinoic acid. For Embryoid Body (EB) differentiation, cells were displaced by dispase treatment and re-suspended in EB media . Media was replenished every 48–72 hrs.t-test.hESCs, cultured in 10cm dishes, were cross-linked for 15 minutes by addition of formaldehyde  to TeSR. Cross-linking was stopped upon addition of Glycine to a final concentration of 125 mM for 5 minutes. After washing with 1X PBS thrice, cells were scraped and collected in RIPA  containing protease inhibitors. Lysates were subsequently sonicated and 500 µg of lysate was pre-cleared for 1 hour using 40 µl of a 50% slurry of 1∶1 protein A- and protein G-Sepharose beads . For immunoprecipitation, the indicated antibodies were added to pre-cleared lysates along with 40 µl of a 50% slurry of 1∶1 protein A/G beads and incubated at 4°C overnight. Antibodies used were: α-CTCF (Millipore), α-Rad21 (Abcam), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz) and α-PAR (Millipore). Catalog number and lot number information is included in For CTCF depletion, hESCs were collected as single cells using TrypLE (Gibco), washed and plated in TeSR containing ROCK inhibitor Y27632. The next day, cells were transfected with 100 nM scrambled siRNA (Ambion) or a pre-designed Silencer Select siRNA against CTCF  using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. 24 hours later, cells were transfected again. In For gene expression analysis, cells were collected by trypsinization and RNA was extracted using RNAeasy (Qiagen) and subjected to DNAse (Invitrogen) treatment according to the manufacturer’s instructions. cDNA was synthesized from 0.5 to 1 µg of RNA using Superscript III Reverse transcriptase (Invitrogen) according to the manufacturer’s protocol and subjected to qPCR using the primers listed in For immunoblotting, cells were lysed with RIPA containing protease inhibitors. 25 or 50 µg of protein was resolved on SDS gels, transferred to nitrocellulose membranes and proteins were recognized using the following antibodies: α-CTCF (BD Bioscience), α-Rad21 (Abcam), α-PARP (Santa Cruz), α-Nucleophosmin (Santa Cruz), α-TopoIIβ (Santa Cruz), α-Nanog (Abcam), α-Oct4 (Santa Cruz), α-Sox2 (Chemicon), α-Actin (Sigma). Catalog number and lot information are provided in 2, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.5% Triton-X-100) for 30 to 45 minutes on ice. Supernatant was recovered after centrifugation, 2 mg lysate was diluted 6X with IP buffer  and pre-cleared using 50 µl of protein-G Sepharose beads for 2 hours. To pre-cleared lysates, 2–5 µg of antibody was added and incubated overnight at 4°C. Subsequently, 50 µl of a 50% slurry of protein-G beads was added and incubated for 4 hours. Immunocomplexes were washed with IP buffer containing 0.5% Triton-X-100 thrice and once with IP buffer containing 0.1% Triton-X-100. 25 µl of 2X SDS loading buffer was added and boiled for 10 minutes prior to SDS-PAGE and Western blotting using the antibodies indicated above.For co-immunoprecipitation, cells were collected by trypsinization and lysed using twice the volume of whole cell extract lysis buffer  or α-Oct4 (Santa Cruz) antibodies overnight. Coverslips were washed with 1% BSA solution and incubated with secondary antibodies conjugated with FITC or Alexa568 fluorophores. DAPI (Sigma) staining was performed along with secondary antibody incubation. Coverslips were mounted on a glass slide using Vectashield (Vector laboratories) and visualized on a Leica SP2 confocal microscope. Image contrasts and brightness parameters were adjusted across all treatment groups where necessary. Images were acquired in a sequential mode of the fluorescent channels to prevent fluorescence bleed-through.5 cells were plated in 24-well dishes and transfected with 100 ng of plasmid when cells were 70% confluent (approximately 24 hours later) using lipofectamine 2000 (Invitrogen). Plasmids were linearized using an enzyme downstream of the luciferase reporter (KpnI or Apa1). 24 hours after transfection, luciferase activity was measured using the Dual-luciferase reporter assay system (Promega). Luciferase activity was normalized to a co-transfected renilla-luciferase reporter plasmid and represented relative to the luciferase/renilla activity of an empty pELuc plasmid set to 100%. Statistical analyses were performed using unpaired two-tailed student’s t-test.Enhancer-blocking assays were performed as described in Lunyak et al In 2500 cells were plated in a 96-well dish and siRNA transfections were performed as described above. Proliferation was measured using a BrdU cell proliferation assay kit  according to the manufacturer’s protocol. Briefly, approximately 48 hours after the first transfection, BrDU was pulsed overnight. Subsequently, cells were fixed and BrdU was detected using a α-BrdU antibody followed by a peroxidase-conjugated secondary antibody. Absorbance at 450 nm was measured on a plate reader upon addition of a substrate for peroxidase. BrdU incorporation in CTCF-depleted cells was normalized to that of control-transfected cells set to 1.For cell death assays, PI was added to trypsinized cells and extent of PI staining was measured on BD FACSCalibur flow cytometer.Spent media were collected from hESCs before and after differentiation. One-fifth of the spent media was used for each ELISA. hCG ELISA was performed using the hCG ELISA kit  as per manufacturer’s instructions.Figure S1Morphology of hESCs after CTCF knockdown. A. Western analyses showing kinetics of CTCF knockdown in H9 hESCs. Control si represents a scrambled siRNA control. Time points represent time after first siRNA transfection. For details regarding the experimental design, see (TIF)Click here for additional data file.Figure S2Confirmation of CTCF depletion phenotype using additional siRNAs. A. Western analysis of CTCF upon CTCF depletion using additional siRNAs (2) and (3) in comparison to a scrambled control (control si) in H9 hESCs. B. Phase contrast images of H9 hESCs at 4X magnification transfected with indicated siRNAs. C. mRNA levels of indicated genes at 48 hrs after CTCF knockdown in H9 hESCs. mRNA levels of indicated genes in control si and siCTCF were normalized to respective GAPDH levels. Subsequently, mRNA levels of siCTCF were normalized to control si set to 1. siRNA (1) represents siRNA used in (TIF)Click here for additional data file.Figure S3Cell death analysis of control si and siCTCF transfected cells. 72 hrs after siRNA transfection, attached cells and cells in supernatant were collected and pooled. Collected cells were stained with propidium iodide and analyzed by BD FACSCalibur. Following flow cytometry, cells were collected back, lysed and analyzed by western blot analysis. For a positive control, untransfected H9 hESCs were treated with 1 mM hydrogen peroxide for 4 hrs (bottom left panel).(TIF)Click here for additional data file.Figure S4Representative images of immunofluorescence analysis of NANOG and OCT4 proteins 96 hrs after CTCF depletion. Scale bar represents 150 µm.(TIF)Click here for additional data file.Figure S5Loss of pluripotency upon CTCF depletion depends on the differentiation protocol. RT-PCR analysis of indicated genes upon control si (black line) or siCTCF (red line) transfection followed by TPA treatment (50 ng/ml) for 2 days in H9. mRNA levels were analyzed at the indicated days and normalized to GAPDH and further normalized to vehicle (DMSO) control. **represents p<0.01, *represents p<0.05. A. RT-PCR analysis of indicated genes upon control si or siCTCF transfection followed by 1 µM Retinoic Acid treatment for 48 hrs in H9. mRNA levels are normalized to GAPDH and further normalized to vehicle (DMSO) control. *represents p<0.05. B. RT-PCR analysis of indicated genes upon control si or siCTCF transfection followed by embryoid body formation. mRNA levels of represented genes in both groups was analyzed on day 10 after embryoid body formation, normalized to GAPDH and further normalized to control si set to 1.(TIF)Click here for additional data file.Figure S6Consensus motifs of CTCF recognition sites in the NANOG locus. Top: CTCF consensus motif as identified in NANOG locus were identified using a prediction algorithm as described in http://insulatordb.uthsc.edu/. Asterisks indicate a perfect match of the denoted base to the consensus. Hyphen indicates lack of a match. A. Browser snapshot of CTCF ChIP-seq data from the NANOG-DPPA3-GDF3 locus in H1 hESCs from the UCSC genome browser. Asterisks represent the sites that we have identified and characterized in H9 hESCs in this study.(TIF)Click here for additional data file.Figure S7Different CTCF cofactors are present at distinct CTCF sites on several critical pluripotent genes. A. ChIP analyses as in (TIF)Click here for additional data file.Figure S8ChIP analyses of protein binding to the NANOG locus in H9 hESCs when transfected with control si or siCTCF. Statistical significance in panel C depicts fold enrichment of nucleophosmin at –146 over the negative control region.(TIF)Click here for additional data file.Figure S9ChIP analyses of CTCF binding at indicated loci in H9 hESCs when transfected with control si or siCTCF.(TIF)Click here for additional data file.Table S1Primer sequences for ChIP-qPCR and RT-qpCR experiments.(XLSX)Click here for additional data file.Table S2Antibodies used in this study, along with vendor name, catalog number and lot information (where available).(XLSX)Click here for additional data file.


In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP), a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1) of CRP, which interacts directly with the αCTD  of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed.The expression of σ AR3 is defined as containing both the activating  and inhibitory (residue 52 of CRP) determinants CRP, the cyclic AMP (cAMP) receptor protein, is one of the best studied transcriptional factor, which is responsible for the regulation of more than 100 genes mainly involved in catabolism of sugars, amino acids and nucleotides in 54-dependent dctA and glnAp2 promoters in Escherichia coliglnAp2 promoter glnHPQ operon and in turn de-activates glnAp2. Second, in vitro studies show that CRP can be recruited by Eσ54 to a site upstream of glnAp2 through the direct interaction between αCTD of Eσ54 and AR1 of CRP, preventing the activator protein from approaching the activator-accessible face of the promoter-bound Eσ54 closed complex E.coli glnAp2, glnHp2glnK (unpublished observations) and astABCDE promoters Klebsiella pneumoniae nifB, nifE, nifF, nifH, nifJ, nifLA and nifU promoters E.coli.Previous studies showed that CRP-cAMP down-regulated the σ54-dependent promoters as mentioned above, this study was undertaken to investigate the influence of CRP-cAMP on the Pu promoter expression in E. coli. Our data show that CRP had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner and the inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. This transcription program leads to the maximal production of toluene and xylenes degradation enzymes only in the absence of cAMP signal. Moreover, AR1 of CRP, which interacts directly with the αCTD of RNA polymerase, was found essential for this CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed.Based on the striking inhibitory effect of CRP-cAMP on the expression of σBacterial strains and plasmids used in this study are listed in M63 modified medium was prepared as previously described 4 footprinting experiment.Preparation of plasmid DNA, restriction enzyme digestions, ligations and horizontal agarose gel electrophoresis in Tris-borate-EDTA buffer were performed according to the standard methods xyl upper operon of TOL plasmid pWW0 was sequenced previously . In order to amplify the Pu promoter and its upstream sequence, two primers were synthesized: 5′-CCCAAGCTTAGCGCGATGAACCTTTTTTATCGC-3′  and 5′- CGGGATCCGAGTTGAGAAAATACAACATTG-3′ . Restriction sites present in oligonucleotide primers used for cloning are underlined. Polymerase Chain Reactions (PCRs) were carried out, and the entire Pu region and the first 7 codons of the xylU open reading frame (ORF) (from –200 to +50 of xylU) together with artificially introduced restriction sites was amplified, using TOL plasmid pWW0 from P. putida strain mt-2 as template and p1, p2 as primers. The DNA fragment was restricted with HindIII and BamHI, and cloned into pBluescript-SK. This resulted in pBS/Pu and its DNA sequence was verified. The 250 bp HindIII-BamHI fragment of pBS/Pu was subsequently inserted into pGD926 to produce an in frame Pu::lacZ fusion, plasmid pKU700. In order to amplify the P. putida crp gene, PCR was carried out by using P. putida strain mt-2 genome as template and oligonucleotides p3  and p4  as primers. The BamHI-SalI fragment was verified subsequently and inserted into pLG339 to gain the plasmid designated as pLG339PpCRP.The complete nucleotide sequence of 54-DNA open complex, E. coli strain carrying the indicated plasmids was pre-grown aerobically at 30°C to late-logarithmic phase in the LB medium, diluted into 10 mL of the same medium with an addition of 0.2 mmol/L m-methylbenzyl alcohol (mMBA) as the inducer at an initial OD600 of 0.05 and then grown out aerobically. At 0.9 OD600, each sample was treated with 40 µL of 50 mg mL−1 rifampicin (dissolved in methanol) for 5 min. The cells were immediately spun down, followed by resuspension in 5 mL of 0.09 mol/L KMnO4 for 2 min. The reaction was stopped by adding 100 µL of β-mercaptoethanol. The cells were spun down, and the plasmid DNA was isolated using SV DNA purification kit (Promega Corporation). 7 µL of 100 µL eluted plasmid DNA was analyzed by PCR amplified primer extension. 7 µL of DNA solution, 2 µL of 5× Taq polymerase buffer, 1.5 µL dNTP (2.5 mmol/L each), 0.25 µL of 5′-32P-labeled p3   and 0.25 µL of sequencing grade Taq polymerase  were mixed together and denatured at 94°C for 1 min, hybridized at 47°C for 30 sec, and extended 30 sec at 72°C. This cycle was repeated 35 times. The samples were analyzed on a 6% (w/v) polyacrylamide sequencing gel, calibrated with the corresponding sequencing reactions.To detect Eσ54 RNA polymerase to Pu promoter, E. coli strain carrying the indicated plasmids was grown aerobically at 30°C in 50 mL LB medium, supplemented with 40 mmol/L NH4+ , in the presence or absence of 2 mmol/L exogenous cAMP. During the late-logarithmic growth, DMS was added  from a fresh 2% (w/v) solution in saline phosphate (SP) buffer. Cells were incubated for 1 min, then spun down and washed twice with 50 mL SP buffer. The plasmid DNA was isolated using SV DNA purification kit (Promega Corporation). 75 µL of 100 µL eluted plasmid DNA was cleaved with 10% (v/v) piperidine at 90°C for 30 min. Following piperidine cleavage they are purified through a 1 mL Sephadex G-50 spin DNA column (Roche Diagnostics Corporation). 35 µL of DNA in distilled water, 1 µL of 5′-32P-labeled p3  for pKU700 or p4  (p4: 5′-GGTTTTCCCAGTCACGACGTTG-3′) for pKU101 containing a glnAp2::lacZ fusion in the reporter plasmid pGD9262O, 0.2 µL of Klenow DNA polymerase  was added and extended at 37°C for 45 min. The samples were analyzed on a 6% (w/v) polyacrylamide sequencing gel, calibrated with the corresponding sequencing reactions.To detect binding of EσP. putida Pu promoter was recreated in the heterologous host E. coli. A translational Pu::lacZ fusion was constructed in the 28 kb low-copy reporter plasmid pGD926 P. putida XylR activator Pu promoter in E. coli. Plasmid pKU700 was co-transformed with pTS174 into E. coli cya mutant TP2006 (unable to produce cAMP) and its isogenic wild type TP2101 separately. β-galactosidase activities were measured in the presence of different concentrations of mMBA, an effective aromatic inducer for XylR Pu promoter was observed in both E. coli strains (Pu was much higher in the cya mutant TP2006 than that in its isogenic wild type strain TP2101 (Pu activation in both E. coli strains (data not shown). These results suggest that the presence of cAMP affects Pu expression. Hence, it was thought necessary to examine whether the CRP-cAMP complex influenced Pu expression.In this study, XylR-mediated activation of the  strains . Howevern TP2101 . In addiPu output in E. coli, promoter activity was monitored in the E. coli wild-type strain TP2101, the cya mutant TP2006 and the cya crp double mutant TP2339-1 respectively. All strains harbor plasmids pTS174 and pKU700. In addition, low copy pLG339-derived plasmids E. coli crp gene (pLG339CRP), P. putida (pLG339PpCRP), or a deleted crp gene , were introduced into TP2339-1. Expression of Pu was monitored in the absence or presence of cAMP. In the absence of cAMP, the Pu promoter was expressed at high levels in TP2006 and TP2339-1  (Pu was comparatively lower than that in the absence of cAMP in TP2006 and TP2339-1 containing pLG339CRP (carrying the E.coli crp gene) or pLG339PpCRP (carrying the P. putida crp gene), but it remained constant at a high level in TP2339-1 containing pLG339ΔRS (E. coli and P. putida) had a cAMP-dependent inhibitory effect on Pu expression.To explore the role of CRP-cAMP in controlling ng cAMP) . In contng cAMP) . When exLG339ΔRS . These rPu operates at the transcriptional level, we detected the influence of CRP-cAMP on the open complex formation at Pu. The presence of open complexes may be probed by preventing transcription elongation with rifampicin to trap open complexes and then footprinting with potassium permanganate (KMnO4), an agent that primarily oxidizes T and C residues in single-stranded DNA of melted promoters E. coli cya mutant TP2006 containing plasmid pKU700 and pTS174 was chemically treated with KMnO4. The footprints obtained with the bottom strand of plasmid DNA pKU700 from intact cells are shown in 4, indicating that open complex formed at Pu  from solution, without a need of binding to the UAS. Plasmid pKU700 was introduced into the cya mutant TP2006, and β-galactosidase activities were measured in the presence or absence of exogenous cAMP under nitrogen-deficient growth conditions. Activation of the Pu promoter was observed in the absence of CRP-cAMP, while inhibition of Pu was observed in the presence of CRP-cAMP. In addition, along with the reduced nitrogen supplied in the growth medium (which means increasing amount of active NtrC in the cells), CRP-cAMP-mediated inhibition on Pu was strongly reduced  attack of critical guanines within the −12 and −24 regions of the promoter in vivo footprinting to analyze protection of the guanine residues at −14, −25 and −26 of Pu from DMS modification by bound Eσ54. In the absence or presence of 2 mmol/L exogenous cAMP, DMS footprints were conducted on plasmid pKU700 in the cya mutant TP2006. As controls, the E. coli rpoN mutant TH1 (unable to produce σ54) containing plasmid pKU700 was also treated with DMS. The footprints obtained with the bottom strand of plasmid DNA pKU700 from intact cells are shown in 54, the bands at both the −12 and −24 regions of Pu are approximately equal in intensity to the −18 reference band . The same result was obtained with no crp gene on the plasmid  54 RNA polymerase to Pu requires the interaction of the αCTD with the UP-like elements at PuPu promoter. The "Pu phenomenon" observed in this article could also be compared with what has been observed for the dctA and glnAp2 promoter. On the dctA promoter, binding of Eσ54 is not inhibited, but enhanced by the presence of CRP-cAMP in vitroin cis from remote sites and in trans with the Eσ54-dctA promoter closed complex, and such an interaction prevents activator-dependent transcriptional activation glnAp2 promoter, the CRP can be recruited by Eσ54 to a site upstream of glnAp2 through the direct interaction between αCTD of Eσ54 and AR1 of CRP, preventing the activator protein from approaching the activator-accessible face of the promoter-bound Eσ54 closed complex 54 in the closed complex is poised by CRP-cAMP through direct interaction between the AR1 of CRP and the αCTD of Eσ54.In this study, we find out that only the CRP mutants defective in AR1, but not in AR2 or AR3, lost the capability of inhibition on Pu , indicatPu was strongly reduced  or the activity (NtrC-phosphate) of the regulatory proteins was increased, CRP-cAMP-mediated inhibition on P. putida KT2440 crp gene  could function as a cyclic AMP receptor. When PpCRP were expressed in an E. coli cya crp minus strain and experiments concerning its role on the expression of lac promoter with exogenous cAMP were done, similar activation results were obtained from PpCRP and CRP from E. coliPu promoter in E. coli in the presence of exogenous cAMP (crp cya system is controlled in its host P. putida.To date, the product of ous cAMP . TherefoPu promoter in E. coli, and maybe similarly in P. putida. Our results also provided proof that, despite the existence of nonspecific activator, the expression of Pu promoter could be inhibited by CRP in cAMP-dependent manner in E. coli.Taken together, our data implicated that CRP can function on


Drosophila, the MSL  complex up regulates transcription of active genes on the single male X-chromosome to equalize gene expression between sexes. One model argues that the MSL complex acts upon the elongation step of transcription rather than initiation. In an unbiased forward genetic screen for new factors required for dosage compensation, we found that mutations in the universally conserved transcription elongation factor Spt5 lower MSL complex dependent expression from the miniwhite reporter gene in vivo. We show that SPT5 interacts directly with MSL1 in vitro and is required downstream of MSL complex recruitment, providing the first mechanistic data corroborating the elongation model of dosage compensation.In  Spt5 gene that encodes a universally conserved elongation factor. SPT5 closes the RNA polymerase II clamp around the DNA template to prevent pausing or premature termination. We find that the dosage compensation complex genetically and physically interacts with SPT5 on actively transcribed genes providing direct molecular support for the elongation model of dosage compensation.Drosophila males hypertranscribe most of the genes along their single X chromosome to match the output of females with two X chromosomes. It had been difficult to imagine how the MSL dosage compensation complex could impose a modest, but essential, ∼two-fold increase by interacting with hundreds of different factors that control transcription initiation for such a diverse collection of genes. An alternative model proposed that dosage compensation instead acted at some step of transcription elongation common to all genes. We performed a genetic screen for mutations that subtly reduce dosage compensation and recovered mutations in the  Drosophila dosage compensation is widely used as a model system to investigate how transcription is regulated by large scale chromatin modifications roX (RNA on X) RNAs A long-standing puzzle is the biochemical mechanism by which the MSL complex up regulates X-linked genes, each of which is controlled by different transcription factors. An elegant model that solves this problem posits that MSL complex does not act with diverse gene-specific transcription factors to alter initiation, but rather at the elongation step of transcription common to all genes Spt5, a universally conserved transcription elongation factor. We found that SPT5 is required for dosage compensation in males and extensively colocalizes with the MSL complex on the X-chromosome. Moreover, we found that SPT5 and MSL1 directly interact with each other. We propose that SPT5 is required downstream of MSL complex recruitment to stimulate transcription elongation. The identification of SPT5 is strong mechanistic evidence supporting the elongation model of dosage compensation.To search for new factors involved in dosage compensation we performed an unbiased forward genetic screen that relies on a sensitive eye pigmentation reporter of MSL complex activity. This approach was designed to recover heterozygous mutations in genes that are essential for general transcription in both sexes but play an additional role in male dosage compensation. We recovered multiple alleles of roX1 transgenic males is a sensitive reporter of MSL activity roX1 transgenes occasionally land in repressive chromatin, the miniwhite marker is epigenetically silenced so that females have solid white eyes. Males have spotted eyes because the MSL complex binds the autosomal roX1 transgene and locally modifies the chromatin allowing miniwhite expression in a fraction of cells. We have previously described a strategy for isolating mutations that increased local MSL activity in vivo, but may associate with the MSL complex too weakly or transiently to copurify with MSL proteins. Second, our genetic strategy retains a wild type allele of the relevant gene allowing us to capture factors that have additional essential functions. Homozygous mutations in such factors would be lethal to both sexes and thus would have been missed in earlier genetic screens based on the male specific lethal phenotype.The eye color of miniwhite reporter linked to roX1. Mutants that were also recessive lethal to both sexes were placed in complementation groups (roX1 transgenes inserted in distinct repressive locations reasoning that those were more likely to affect dosage compensation rather than the particular silencing factors acting on flanking chromatin (miniwhite gene in our roX1 reporters. We tested the effect of the new mutants on m4In(1)w which displays classic position effect variegation in both sexes. Most of the candidate modifiers of dosage compensation did not affect pigmentation in m4In(1)w wm4  arguing Spt5 and Elongin-C are located . However, when we tested the MGE-3Spt5 mutation Complementation group C was chosen for detailed analysis. Meiotic recombination placed the locus near the polytene bands 56C-F but none of the available chromosome deficiencies uncovered the mutation  located . AvailabroX1 lines just like Spt5 mutations had. We crossed six different roX1 mosaic lines that carry roX1 transgenes in diverse chromatin environments to 190 deficiencies. The idea was that any deficiency that affected the eye coloration of multiple roX1 reporter lines was more likely to affect some aspect of dosage compensation rather than the particular repressive environment surrounding the different inserts. We found that only 10 intervals reduced MSL complex reporter activity . This shows that a 50% reduction in SPT5 levels does not alter the phenotype from hypomorphic white alleles or miniwhite. We conclude that Spt5 mutations dramatically affected the probability that males overcome silencing not because of global reduction of transcription across the genome or the white promoter itself, but rather because SPT5 plays some role in dosage compensation to which our roX1 reporter is responsive.We were still concerned that the entation . We alsoSpt5 affected dosage compensation of the X chromosome in addition to the roX1 eye color reporter transgene. Because SPT5 is essential for most transcription, homozygous null animals die early in development. The viability of Spt5/+ males demonstrates that dosage compensation must be adequate even with reduced SPT5 levels. The same is true for any of the msl/+ heterozygotes. However, males with limiting MSL complex might be more sensitive to reduced levels of SPT5. Males missing either roX1 or roX2 are alive but males missing both roX RNAs have greatly reduced male viability roX1 roX2 double mutant males are completely lethal but can be rescued by an autosomal roX transgene msl1 or mle showed reduced viability when roX1 RNA is also limiting roX1 RNA was limiting consistent with a role in dosage compensation S192, a mutation in the elongation factor ELL did reduce male viability. Others have reported that ELL RNAi lines display male specific lethality consistent with a role in dosage compensation roX1 RNA levels.We used a sensitized genetic background to see if ensation . We assaiability . Howevermsl2 mRNA. Ectopic dosage compensation can be induced in females by artificial expression of MSL2 by the [H83M2] transgene that escapes SXL regulation Spt5 weakens dosage compensation then that might reduce the toxic effects of inappropriate dosage compensation in [H83M2] overexpression females. When [H83M2] females also carried a mutation in Spt5, the female-specific developmental delay was modestly rescued  of most Drosophila genes, with additional binding across the transcribed regions In order to place SPT5 in the dosage compensation pathway, we focused on two of its most intensively studied roles. First, unphosphorylated SPT5 binds to and pauses RNA polymerase over the transcription start site (TSS). Release from this 5′ pause requires phosphorylation by P-TEFb at multiple sites at the C-termini of both RNA polymerase large subunit and SPT5 Spt5 null tissue. However, we can approximate that condition by using elongation inhibitors DRB and flavopiridol that block P-TEFb phosphorylation of RNAPII CTD Ser2 and SPT5 that are necessary for pause-release and entry into elongation The Pause Release model is less appealing because it calls for MSL action at the TSS, when MSL complex is instead predominantly located farther downstream Modified histone H3K36me3 is found within active genes with a 3′ bias similar to the MSL complex. This modification may provide one component of MSL targeting specificity through the MSL3 chromodomain To examine MSL binding at a higher resolution than is possible with polytenes, we turned to ChIP analysis of male S2 cells. We measured MSL1 binding to the 5′ and 3′ ends of two highly validated target genes after treatment with flavopiridol. If the scarcity of MSL complex at the 5′ ends of X-linked genes was caused by released RNAPII/SPT5 complex quickly carrying it into the body of genes, we might be able to trap MSL complex over the TSS by treatment with P-TEFb kinase inhibitors. The Pause Release Model predicts that flavopiridol treatment should cause the MSL signal to accumulate at the 5′ end of genes with a corresponding loss at the 3′ end. However, just as was seen with the polytene experiments, flavopiridol treatment did not alter MSL complex distribution as measured by ChIP . These rroX RNAs with regions of SPT5 in vivo, the data show that SPT5 and MSL complex have the ability to interact via MSL1 PEHE.We tested whether the genetic interactions observed between SPT5 and dosage compensation might arise from direct physical contacts. Early attempts to purify intact MSL complex did not recover SPT5 as a partner suggesting that if such interactions occur, they are transient Spt5, we wondered if the other modifier mutations found in the screen might identify a new class of factors needed for dosage compensation. We were able to map a few modifiers to previously characterized genes. In the case of deficiencies, we tested whether point mutations of candidate genes could recapitulate the effect of deficiency. That approach showed that Chromator was the relevant gene that dominantly suppresses the MSL complex dependent reporter expression in Df(3L)BSC21. CHRO is a chromodomain protein that localizes specifically to the interband regions and is implicated in maintaining chromosome structure Df(2R)vg-C vg-C . This inroX RNAs, and the fact that active MSL complex is tightly associated with transcribed chromatin. Extraction methods strong enough to release soluble MSL complex from chromatin may destroy critical contacts with key partners. Genetic approaches also face limitations. If an important partner performs additional functions beyond dosage compensation, mutations would likely be lethal to both sexes masking its interaction with the MSL complex.It is possible that dosage compensation in Drosophila is entirely a consequence of the known histone modifications carried out by its subunits, H4K16ac (MOF) and H2BK34ub (MSL2). However, if additional factors are required, new approaches may be needed to identify them. Biochemical purification is challenging due to the very large size of the MSL complex, the presence of the noncoding Spt5, a universally conserved transcription processivity factor for RNAPs, in the MSL pathway msl1, mle and ChroWe developed an unbiased forward genetic screen able to detect subtle changes in MSL activity that are not large enough to prevent dosage compensation of the male X, but sufficient to alter a sensitive eye pigmentation reporter. This screen implicated Spt5 mutations on the white eye color reporter was entirely dependent upon the adjacent roX1 locus that can recruit soluble MSL complex to any location in the genome. Spt5 mutations had no effect on white or miniwhite gene expression when not linked to roX1. Spt5 mutations acted on all mosaic roX1 reporter transgenes regardless of the chromatin environment surrounding the inserts. Interactions of Spt5 mutants and gain of function msl1 alleles suggest that SPT5 acts between MSL complex and RNA polymerase. Mutations in Spt5 selectively reduced male viability under limiting roX RNA conditions in a manner comparable to the effect of mle mutations. Additionally mutations in Spt5 partially suppressed the toxic effects of ectopic dosage compensation in females. An independent screen of the Drosophila deficiency collection showed that the Spt5 phenotype is rare. Removing one allele of almost any transcription related factor had no effect on the eye pigmentation levels of mosaic roX1 reporters arguing that dosage compensation is particularly sensitive to SPT5 protein levels. Finally, we found that the most ancient and conserved segment of the MSL1 protein physically binds to two different regions of the SPT5 protein consistent with the largely overlapping patterns of chromatin occupancy across the body of X-linked genes.Multiple lines of evidence support a role for SPT5 in dosage compensation. The effect of In eukaryotes SPT5 along with SPT4 forms the DSIF  hsp70 gene from Drosophila While regulated release from pause was originally described using the highly inducible MSL complex mediated H4K16ac is enriched within the body of genes and drives decondensation of chromatin possibly facilitating easier passage of RNAPII Spt5 in this report, our genetic approach also yielded additional candidates. So far, we have mapped two of these to Chro and Sin3A. CHRO, a chromodomain protein copurifies with the MSL complex While we have focused on the analysis of in vivo genetic strategy.An independent RNAi screen using an MSL complex dependent luciferase expression as a reporter in S2 cells also identified a role for CHRO and SIN3A in dosage compensation in vivo support for the elongation model of dosage compensation by linking the SPT5 elongation factor to the MSL complex Drosophila males have ∼1.4 fold more transcriptionally engaged RNAPII at the distal ends of X-linked genes as compared to autosomes also supports the idea of increased elongation Our results provide direct Mammals also contain a version of the MSL complex composed of MSL1, 2, 3 and MOF, but apparently lacking a large noncoding RNA component and RNA helicase + GMroX1-58D], [w+ GMroX1-60F], [w+ GMroX1-69C], [w+ GMroX1-75C], [w+ GMroX1-99F][w and + GMroX1-102C]y w roX1Mutagenesis was performed as described Plasmids for bacterial expression of MBP fusion SPT5 protein fragments, SPT5-N (aa 112–393), M (aa 389–733) and C (aa 732–1054) were a kind gift from Dr. John Lis Polytene squashes were prepared as described in Detailed protocol can be found in MBP-SPT5N, MBP-SPT5M, MBP-SPT5C, the unrelated protein MBP-MCP (MS2 phage coat protein), GST-MSL1 C-terminal domain fusion protein and GST were expressed and isolated from bacteria. Equivalent molar concentrations of MBP proteins bound to amylose beads were incubated with GST proteins. After three washes with 150 mM NaCl, 0.1% NP40 and 20 mM Tris for 10 mins at 4°C, proteins were eluted by boiling in SDS-loading buffer and separated on 8% SDS-polyacrylamide gels. Westerns were performed as described Figure S1spt5.A forward genetic screen to identify modifiers of MSL complex activity. (A) Approximately 16,000 males were screened and 48 modifier lines established. (B) Mutations were placed into complementation groups based on recessive lethality. The mutations scored as single hits are recessive lethals, which could be due to the modifier allele or an EMS induced secondary mutation. The mutants scored as viable suppressors lower MSL complex dependent red pigmentation and are homozygous viable. We identified 5 alleles of (TIF)Click here for additional data file.Figure S2Spt5 consistently lower mosaic eye pigmentation independent of autosomal roX1 insertion site. Shown above are flies hemizygous for the [GMroX1]/+ transgene at different positions in the genome shown on the side. Flies on the left are wildtype whereas flies on the right are heterozygous for S14FSpt5. All flies shown are males.Mutations in (TIF)Click here for additional data file.Figure S3Spt5 do not affect the white promoter. Males and female eye pigmentation is shown for the hypomorphic wa and we alleles with and without Spt5 mutations. Although Spt5/+ heterozygous males show dramatic pigment reductions from the dosage compensation [w+GMroX1] mosaic transgenes .Mutations is ansgenes , the sam(TIF)Click here for additional data file.Figure S4Validation of anti SPT5 antibodies. (A) Polytene chromosomes stained with anti-SPT5 antibodies. SPT5 is widely distributed on many sites on the genome. (B) Immunodepletion of anti-SPT5 antibodies with SPT5 fragments- SPT5N, SPT5M and SPT5C results in loss of signal indicating that the antibodies predominantly recognize SPT5. (C) Polytene chromosome spreads prepared from males subjected to 30 min heat shock at 37°C were stained with anti-SPT5 antibodies. In agreement with previous reports (TIF)Click here for additional data file.Figure S5SPT5 and MSL1 colocalize extensively on the X-chromosome. Polytene chromosome spreads were stained with antibodies against (A) MSL1 (green) and (B) SPT5 (red). As expected of a general transcription elongation factor SPT5 binds all over the genome. (C) Colocalization of MSL1 and SPT5.  A closer look at two regions of the X-chromosome. Arrow heads indicates MSL1 only (no SPT5) binding and arrows indicate SPT5 only (no MSL1) binding.(TIF)Click here for additional data file.Figure S6Treatment with Flavopiridol does not affect H3K36me3. (A) Polytene chromosome spreads prepared from salivary glands treated with either DMSO (control panel) or 500 nM Flavopiridol for 30 minutes. Chromosomes were stained with antibodies against H3K36me3. (B) Ser5P RNAPII and SPT5 colocalize on polytenes. After treatment with Flavopiridol, SPT5 is greatly reduced but low levels of SPT5 are found at polytene bands that are positive for Ser5 Phosphorylated RNAPII. (C) A close-up of Ser5P RNAPII and SPT5 colocalization in control and flavopiridol treated samples.(TIF)Click here for additional data file.Table S1roX1 lines were assayed in the deficiency screen (Bloomington Deficiency collection) and the deficiencies that dominantly suppressed the red pigmentation across more than four different mosaic roX lines are indicated. Candidate genes that have been mapped within those deficiencies are also shown. Only ten intervals (14/190 deficiencies) assayed had an effect on the eye phenotype indicating that a general suppression of transcription does not lower the expression of the dosage compensation reporter. Spt5 would be an eleventh locus, but is not uncovered in the Df collection.Results of Deficiency screen. Six mosaic (DOC)Click here for additional data file.Table S2Spt5 decreases male viability. In the absence of both roX1 and roX2 males are dead. The male specific lethality can be rescued by an autosomal GMroX1 transgene. In this assay roX1-,roX2-; [GMroX1-75C] virgins were crossed to balanced males carrying the indicated mutations. The male progeny of this cross that are roX1-,roX2-/Y;mutation/+;[GMroX1-75C]/+ are compared to the nonbalanced female progeny. All the flies therefore carry only one copy of the indicated mutation. The statistical significance was determined by Fisher exact fit test. Lowering (DOC)Click here for additional data file.Text S1Supplementary materials and methods.(DOC)Click here for additional data file.


Saccharomyces cerevisiae can occur in the context of little, if any, covalent histone modification. Using a SIR-regulated heat shock-inducible transgene, hsp82-2001, and a natural drug-inducible subtelomeric gene, YFR057w, as models we demonstrate that substantial transcriptional induction (>200-fold) can occur in the context of restricted histone loss and negligible levels of H3K4 trimethylation, H3K36 trimethylation and H3K79 dimethylation, modifications commonly linked to transcription initiation and elongation. Heterochromatic gene activation can also occur with minimal H3 and H4 lysine acetylation and without replacement of H2A with the transcription-linked variant H2A.Z. Importantly, absence of histone modification does not stem from reduced transcriptional output, since hsp82-ΔTATA, a euchromatic promoter mutant lacking a TATA box and with threefold lower induced transcription than heterochromatic hsp82-2001, is strongly hyperacetylated in response to heat shock. Consistent with negligible H3K79 dimethylation, dot1Δ cells lacking H3K79 methylase activity show unimpeded occupancy of RNA polymerase II within activated heterochromatic promoter and coding regions. Our results indicate that large increases in transcription can be observed in the virtual absence of histone modifications often thought necessary for gene activation.It is widely accepted that transcriptional regulation of eukaryotic genes is intimately coupled to covalent modifications of the underlying chromatin template, and in certain cases the functional consequences of these modifications have been characterized. Here we present evidence that gene activation in the silent heterochromatin of the yeast  The proper regulation of gene expression is of fundamental importance in the maintenance of normal growth and development. Misregulation of genes can lead to such outcomes as cancer, diabetes and neurodegenerative disease. A key step in gene regulation occurs during the transcription of the chromosomal DNA into messenger RNA by the enzyme, RNA polymerase II. Histones are small, positively charged proteins that package genomic DNA into arrays of bead-like particles termed nucleosomes, the principal components of chromatin. Increasing evidence suggests that nucleosomal histones play an active role in regulating transcription, and that this is derived in part from reversible chemical  modifications that take place on their amino acids. These histone modifications create novel surfaces on nucleosomes that can serve as docking sites for other proteins that control a gene's expression state. In this study we present evidence that contrary to the general case, covalent modifications typically associated with transcription are minimally used by genes embedded in a specialized, condensed chromatin structure termed heterochromatin in the model organism baker's yeast. Our observations are significant, for they suggest that gene transcription can occur in a living cell in the virtual absence of covalent modification of the chromatin template. Transcription in eukaryotes occurs in the context of chromatin. At most genes, transcriptional activation is accompanied by alterations to the chromatin template, exemplified by the enhanced DNase I sensitivity of coding regions and the presence of associated DNase I hypersensitive sites at linked regulatory elements INO1 promoter in the budding yeast S. cerevisiae where phosphorylation of the Ser10 residue of H3 (H3S10) by the Snf1 kinase triggers acetylation of the Lys14 residue of H3 (H3K14) by the Gcn5 acetyltransferase, and subsequent transcriptional activation Covalent histone modifications can also create novel nucleosomal surfaces that serve as recognition sites for effector proteins. Histone PTMs, acting singly or in combination, may therefore control the ultimate expression state of a gene, or the ability of the underlying DNA to be repaired, recombined or replicated. This concept has been termed the histone code An alternative view is that histone modifications and the enzymes that impart them are not regulatory; that is, they do not play a causative role in transcription In addition to the precise role(s) played by covalent histone modifications, it is unclear whether the dynamic properties of euchromatin are shared with heterochromatin, the compartment of the nucleus that remains condensed throughout interphase, replicates late, is resistant to recombination, and contains relatively few transcribed genes. In multicellular organisms, heterochromatin can exist in both constitutive and facultative  forms. Constitutive heterochromatin is enriched in HP-1  and SU(VAR)3–9 (an H3K9 methyltransferase) in organisms ranging from fission yeast to mammals, and is characteristic of telomeric and pericentric regions, repetitive DNA elements, and other DNA sequences critical to genomic stability. In contrast, facultative heterochromatin is characteristic of reversibly silenced genes such as X-linked genes in female mammals and genes encoding key developmental regulators, is enriched in H3K27me3- and ubiquitylated H2A-containing nucleosomes, and is under regulation of Polycomb chromatin modification complexes HML and HMR silent mating loci HM locus. Absence of any one of the Sir proteins prevents the assembly of silent chromatin +-dependent lysine deacetylase that deacetylates histones with a preference for H4K16 and H3K56 Budding yeast, although lacking HP-1 and Polycomb proteins, contains specialized chromatin structures that functionally resemble the Polycomb-regulated facultative heterochromatin of insects and vertebrates light gene, and at least eight others in Drosophila, depend on a heterochromatic location for normal expression Xist gene is 100-fold enhanced on the heterochromatic, inactive X chromosome relative to its euchromatic counterpart S. cerevisiae. We found that large increases in the transcription of disparate heterochromatic genes occur in the absence (or near absence) of covalent histone modifications. Strikingly, when the same genes are placed in a euchromatic context, they heavily utilize such modifications.While heterochromatin is generally repressive of transcription, hundreds of genes are localized within this nuclear compartment in protozoa, insects, plants and animals. The HSP82 gene and chromosomal HSP82 alleles flanked by integrated copies of the HMRE mating-type silencer that differ in their dosage and arrangement  conditions, and at levels that roughly correlate with the extent of silencing see . Sir3 wacriteria , closelyhsp82-2001 locus was only slightly reduced (hsp82-2001 promoter and recent observations of an activated subtelomeric URA3 gene In response to a 20 min heat shock (HS), all three transgenes were strongly activated , right. SIR4 deletion restored transcript levels of each transgene to WT levels (hsp82-1001 sir4Δ strain and data not shown). This indicates that cis-acting HMRE silencers (and the sequence-specific proteins bound to them) do not affect HSP82 regulation; assembly of the Sir protein complex is required. Consistent with this, the promoter chromatin structure of the three transgenes in a sir4Δ background is indistinguishable from the euchromatic HSP82 gene, based on nucleotide-resolution DNase I, MNase and dimethyl sulfate genomic footprint analyses Disruption of silent chromatin through HSP82hsp82-2001 gene, exhibiting a 75% reduction within 60 sec and >90% reduction within 5 min (sir4Δ samples). Consistent with diminished transcriptional activity during chronic heat shock HSP82 into heterochromatin not only resulted in at least a twofold increase in nucleosomal density throughout the gene , but also restricted the dynamic nature of the chromatin as the extent of nucleosomal disassembly was substantially reduced. Virtually identical results were seen when abundance of either myc-H4 or H2A was evaluated (data not shown); therefore, large (>200-fold) increases in expression can take place in the context of relatively modest changes in nucleosome occupancy, consistent with the retention of the Sir2/3/4 complex described above.We next investigated the effect of the Sir proteins on nucleosome density and stability. As previously seen for wild-type hsp82-2001 transgene. Consistent with earlier observations of SIR-silenced genes hsp82-2001, albeit at a reduced level . Importantly, its occupancy was substantially enhanced (10- to 15-fold) by heat shock. We additionally examined occupancy of the capping enzyme Cet1. Previous analysis of the HMLα1/α2 and HMRa1 silent mating genes indicated that occupancy of Cet1 was strongly restricted under SIR-silencing conditions SIR elicits silencing, at least in part, by targeting steps downstream of PIC assembly. In support, we found that under non-inducing conditions Cet1 was at near-background levels at all three heterochromatic hsp82 transgenes  were both H3 di-acetylated and H4 tetra-acetylated under NHS (−) conditions, mimicking the wild-type HSP82 gene SIR-silenced hsp82-2001 gene were negligibly acetylated under the same conditions. Thus, hsp82-2001 conforms to the general notion of a histone code, in which absence of N-tail histone acetylation codes for inactivation. The less efficiently silenced transgenes were also impoverished in both di-acetylated H3 and tetra-acetylated H4, but to a lesser degree. Notably, upon heat shock (+), the euchromatic transgene was enriched in both acetylated isoforms (once again resembling +HSP82) yet there was no detectable enrichment of either at the hyperrepressed hsp82-2001 gene. Similarly, the moderately silenced hsp82-1001 transgene, despite >60-fold increase in expression in response to heat shock, showed no enrichment in acetylation. By contrast, the weakly silenced hsp82-201 gene was H3 hyperacetylated, resembling its euchromatic counterpart.Previous work has shown that euchromatic R mutant . We therhsp82-2001 remains unaltered following a 20 min heat shock. However, as transcript accumulation is evident within the first 60 sec (see +SIR samples), it was possible that transient histone acetylation may have occurred. To address this, we assayed H3 and H4 acetylation of hsp82-2001 30 sec following an instantaneous heat shock, and at 15- to 30-sec intervals thereafter, to obtain ‘snapshots’ of the H3/H4 acetylation state within this gene. Despite a 30-fold increase in hsp82 transcript level within the first 60–90 sec of heat shock, neither di-acetylated H3 nor tetra-acetylated H4 was detectably increased (+SIR background). This again contrasts with the euchromatic state where promoter-associated nucleosomes, already enriched in acetylated H3 and H4 molecules, showed a further ∼30% increase.The foregoing analysis indicates that the minimal acetylation state of heterochromatic in vitro+SIR). This finding suggests that Sir2 may actively deacetylate H4K16 to suppress the extent of chromatin unfolding and, as a consequence, diminish gross transcriptional output. We also examined H4K12 acetylation, which has been implicated in telomeric heterochromatin formation and function in S. cerevisiaehsp82-2001, the hyperrepressed hsp82-2001 transgene was assembled in nucleosomes containing only background levels of H4K12ac, with little if any enrichment upon heat shock .Since the above results argue that heterochromatic transcription is uncoupled from histone N-terminal lysine acetylation, we asked whether acetylation of K56, located within the globular core of H3, or methylation of K36, located at the junction between the H3 N-terminus and the globular domain, accompanies transcriptional activation. H3K56 acetylation is predicted to break a histone-DNA interaction, potentially destabilizing the nucleosome  ∼2-fold . More st regions , sir4Δ.hsp82-2001 takes place in the context of minimal H3K36 trimethylation  gene promoters 001 gene . This inS. cerevisiae). Replacement of canonical H2A with H2A.Z at the +1 and or −1 nucleosome poises promoters for transcriptional activation as H2A.Z-containing nucleosomes are more susceptible to disassembly than canonical nucleosomes hsp82 promoter, consistent with prior findings hsp82-1001 sir4Δ, black bars). As expected, H2A.Z abundance was reduced at the SIR-repressed transgenes. At hsp82-201, its abundance was reduced 40% at the promoter, while at hsp82-1001, H2A.Z levels were altered only at the 3′-end. At both genes, promoter-associated H2A.Z was drastically reduced concomitant with transcriptional induction (hsp82-2001 gene is nearly bereft of H2A.Z in non-induced cells (∼90% reduced within both promoter and coding region) and this reduced level remained unchanged during activation. Consistent with this observation, deletion of the gene encoding H2A.Z did not impair hsp82-2001 mRNA induction, and may have even enhanced it (+ htz1ΔSIR samples).We next examined the role of the histone variant H2A.Z , which forms a heterodimer with Pdr1. Pdr1 is known to activate other genes involved in pleiotropic drug resistance YFR057w was strongly induced by exposure of cells to 200 µg/ml cycloheximide , one in which cells remained fully viable  is unlikely to be a primary consequence of cycloheximide-mediated mRNA stabilization sir2Δ counterpart showed just a several-fold increase over the same time frame  and dynamic occupancy of the transcriptional machinery. Activation with minimal chromatin modification is observed at genes regulated by disparate activators and is seen in distinct genetic backgrounds. It is not the consequence of insubstantial transcription, since the euchromatic gene, hsp82-ΔTATA, despite being expressed at a level of only 20–30% of heterochromatic hsp82-2001, exhibits striking chromatin modification during its activation including promoter-proximal H3 hyperacetylation and histone displacement. Moreover, at least in the case of HSP82, it is not due to a lack of a requirement for covalent histone modifications. Previous work has shown a clear dependence of euchromatic hsp82 alleles on the histone acetylases Gcn5 and Esa1 present in SAGA and NuA4, respectively HSP82 and hsp82-ΔTATA are extensively acetylated upon heat shock activation and HSP82 expression is reduced in an H4 N-terminal tail mutant. A similar requirement for histone acetylation likely applies to the euchromatic YFR057w gene as its expression is reduced in an H4K16R mutant  moments after they appear. However, our inability to detect PTMs is unlikely to be due to masking of stable modifications. We utilized antibodies specific to multiple epitopes within the histone octamer – globular domain of H3, N-terminal epitope of Myc-H4, acidic patch of H2A – and all were readily detectable in SIR-mediated heterochromatin.hsp82-2001 gene is only partially and transiently depleted of the H3/H4 tetramer during heat shock. This is in contrast to euchromatic hsp82-2001 that sustains a >90% loss of H2A, H3 and H4 during the early stages (5–25 min) of heat shock induction  versus heterochromatic (+SIR) states, modification of the heterochromatic gene is maximally one PTM (of a given type) for every 5–10 nucleosomes. Since the upstream regulatory region of HSP82 spans only two nucleosomes hsp82-2001 promoter. This, combined with the fact that there is no evidence for epigenetic variegation of hsp82-2001 expression  in recruiting transcriptional co-regulators It is possible that the minimal changes in histone modification state that are detected at silent URA3 transgene under control of the Sir2/3/4 chromatin modification complex, both in cell populations in which it was expressed (cells grown in synthetic medium lacking uracil), as well as those in which it was repressed (cells grown in rich medium containing 5-FOA). As was the case here, the expressed state was characterized by substantial levels of Sir3 and deacetylated H3 and H4. In contrast to our findings, however, other histone modifications – in particular, H3K4me3, H3K36me3 and both mono- and di-methylated forms of H3K79 – were abundant URA3-TEL gene, disrupted its physiological interaction with the underlying nucleosomes, thereby accounting for the transcriptionally permissive template hsp82-2001 and YFR057w can be transcribed essentially in the absence of H3K4, H3K36 and H3K79 methylation, an obligatory role for these histone modifications in promoting Pol II transcription of heterochromatic genes seems unlikely.Our findings also provide an interesting contrast to those of Grunstein, Carey and coworkers S. cerevisiae which showed that despite the widespread localization of H3K4me, H3K36me and H3K79me marks, deletion of the enzymes responsible for imparting these marks – Set1, Set2 and Dot1 – had very specific effects with the expression of most genes unaffected Drosophila mutants in which essentially normal transcriptional regulation can occur in the complete absence of H3K4 methylation Instead, our results are more consistent with a large scale mutational analysis in hsp82-2001 and YFR057w is reduced several-fold as a consequence of their location within SIR-heterochromatin. Therefore, at least in these two cases, histone modifications may play a role in increasing transcriptional output. Other explanations for how Sir2/3/4 proteins reduce transcription of these genes, including direct or indirect roles in enhancing nucleosome stability Arabidopsis occurs without reversal of repressive chromatin marks such as DNA methylation and H3K9 and H3K27 methylation, and without the appearance of activating modifications such as H3 and H4 acetylation We have demonstrated that in yeast heterochromatin, substantial increases in transcription can take place in the absence (or near-absence) of chromatin alterations often thought necessary for activation. Nonetheless, gross transcriptional output of hsp82 transgenic strains (SLY101 background) were generated by integrating HMRE silencers (144 bp modules [separated by 6 bp spacers in the case of tandem integrants]) both 5′ and 3′ of the chromosomal HSP82 gene using two-step gene transplacement methods myc-HHF2 gene borne on plasmid pNOY436 (TRP1-CEN6-ARS4) as previously described Strains used in this study are listed in 600 = 0.3 to 0.7) in either rich yeast extract-peptone-glucose broth supplemented with 0.03 mg/ml adenine or synthetic complete medium lacking tryptophan (SDC-Trp). Heat shock induction was achieved by transferring the culture  to a vigorously shaking 39°C water bath; once the temperature reached 39°C, incubation was allowed to continue for an additional 20 min before addition of either sodium azide ) or formaldehyde ). For time course assays, instantaneous 30° to 39°C upshift was achieved by rapidly mixing equal volumes of 30°C culture and pre-warmed medium (55°C) and then incubating with rapid shaking at 39°C for the times indicated. Induction was terminated through addition of sodium azide.Yeast strains were cultivated at 30°C to early log phase (A600 ∼0.6) were made 200 µg/ml in cycloheximide through a 1∶100 dilution of a 20 mg/ml stock solution prepared in DMSO. Cells were then incubated with agitation at 30°C for various times  prior to addition of 20 mM sodium azide. Cells were then harvested, washed in 20 mM azide, and stored at −80°C for subsequent chromatin or RNA isolation.For drug induction, 100 ml early log cultures (A600 ∼0.5) were cross-linked with 1% formaldehyde for 10 min, then converted to spheroplasts with lyticase . Spheroplasts were lysed using one volume of 0.5 mm glass beads for 30 min at 4°C on an Eppendorf 5432 mixer. Chromatin was sheared to a mean size of ∼0.5 kb with a Branson 250 sonifier equipped with a microtip using three 25 s pulses at constant power and an output setting of 22 watts. The clarified supernatant  was used in immunoprecipitations (IPs) that were typically achieved by adding 2–5 µl antiserum to 300 µl of chromatin lysate. Signal quantification was done using a Storm 860 PhosphorImager (Molecular Dynamics) and ImageQuant 5.2 software. To calculate the relative abundance of a given gene sequence present in an IP, we used the following formula: Qgene = IPgene/Inputgene. In most cases, the abundance of each test locus was expressed relative to that of either PHO5 or ARS504, which served as internal recovery controls. In the case of Sir3, abundance at a given locus was quantified relative to its abundance at HMRa1. To reduce background, we subtracted the signal arising from a mock IP (-Ab) for the histone covalent modification, Htz1 and Cet1 ChIPs, and the signal arising from pre-immune serum for the Pol II (Rpb1) ChIPs. For the nonspecific ARS504 IP signal, only gel background was subtracted.End point (gel-based) ChIP-PCR analysis , 7A, S1A2 qPCR SYBR Green/ROX MasterMix . Through use of a standard curve specific for each amplicon, the quantity of DNA present in each IP was determined, and background signal was subtracted. In the case of Myc ChIPs, the background was the signal arising from chromatin isolated in a parallel culture of an isogenic strain lacking Myc-tagged H4; for histone PTM ChIPs, background was the signal arising from a beads alone control. For Pol II ChIP, background was the signal arising from pre-immune serum. To normalize for variation in sample recovery, abundance of a non-transcribed region on chromosome V (ARS504) was determined for each ChIP DNA sample, and HSP82/ARS504 and YFR057w/ARS504 quotients were derived. In certain cases (see figure legends), normalization to the PHO5 promoter was done instead. Finally, to account for nucleosome loss, all PTM data are presented as (histone PTM)/Myc-H4 or (histone PTM)/H3 quotients.Real Time (ChIP-qPCR) analysis , S3 was The following antibodies were used: Myc ; H3 globular domain ; H3 K9ac, K14ac ; H3 K18ac ; H3 K56ac ; H4 K16ac ; H4 K12ac ; H4 tetra-acetylated  ; H2A acidic patch ; H3 K4me3 ; H3 K4me2,3 ; H3 K36me3 ; H3 K79me2 ; Htz1 ; Cet1 ; Sir3 ; Pol II, rabbit antiserum raised in our laboratory against a recombinant GST-CTD polypeptide bearing 52 heptad repeats of the mouse large subunit HSP82 promoter, −401 to −34; HSP82 ORF, +1248 to +1444; HSP82 3′-UTR, +1883 to +2155; ARS504, coordinates 9746 to 9817; PHO5 promoter, −507 to −33; CIN2 5′ ORF, +149 to +427; CIN2 3′-UTR, +523 to +797; YAR1 ORF, +3 to +200; YAR1 3′-UTR, +1109 to +1297); IQG1 5′ ORF, +1016 to +1211; IQG1 3′ ORF, +2007 to +2289; SUI3, +451 to +748; YPL236c, +71 to +229; HMRa1, −80 to +50. For Real Time PCR, the amplicons were: HSP82 UAS, −227 to −140; HSP82 promoter, −157 to −88 or −227 to −88 : 7 to −88 , 6C–E; HACT1 probe as done previously HSP82, +2167 to +2228; CIN2, +149 to +427; YAR1, +3 to +200; SUI3, +451 to +748; IQG1, +1016 to +1211; and ACT1, +606 to +1000.For the expression analyses summarized in 600 of ∼0.5 in a 125 ml culture at 30°C and 15 ml aliquots were removed and subjected to an instantaneous 30 to 39°C upshift for the indicated times. Heat shock induction was terminated through addition of 20 mM sodium azide, and RNA was isolated as above. For induction of YFR057w , followed by phenol/chloroform extraction. 0.5 µg purified RNA was used in each cDNA synthesis with ProtoScript RT-PCR Kit (NEB #E6400S). Oligo(dT) primers were used in cDNA synthesis for quantification of Pol II gene transcripts; random primers were used in cDNA synthesis for quantification of Figure S1hsp82-1001 and hsp82-2001, while it is restricted to ∼1.5 kb at hsp82-201 that bears tandem silencers upstream of the gene. (a) Sir3 occupancy within each transgenic locus relative to its occupancy at HMRa1. ChIP analysis of +SIR strains bearing the indicated hsp82 allele was conducted and quantified as described in YAR1, CIN2, SUI3 and IQG1 in the parent HSP82+ strain (SLY101) and the indicated +SIR and sir4Δ transgenic strains. Mean transcript abundance of each gene  is quantified relative to that present in the parent strain, which was arbitrarily set to100 (N = 2).The domain of SIR silent chromatin spans at least 4 kb at the silencer-bracketed transgenes (PDF)Click here for additional data file.Figure S2+SIR and sir2Δ cells  were grown to mid-log (A600 = 0.3) in rich YPDA medium, then cycloheximide was added to a final concentration of 200 µg/ml and cells were cultivated at 30°C for the indicated times. Aliquots were removed, diluted 1∶400 in sterile water, and 10 µl were spread onto YPDA plates. Cells were incubated at 30°C for 2.5 days.Cycloheximide viability assay. Isogenic (PDF)Click here for additional data file.Figure S3YFR057w gene occurs without detectable nucleosomal disruption or H3/H4 acetylation. (a) H3 occupancy of the YFR057W promoter in sir2Δ and +SIR cells at the indicated times following addition of 200 µg/ml CX to mid-log cultures. H3 levels were normalized to those at PHO5. Depicted are means ± S.D. . (b) H2A occupancy of the YFR057W promoter conducted and quantified as in A. (c) H3K18ac, H4K16ac and H4 tetra-acetylated ChIP analysis of the YFR057w promoter and ORF in sir2Δ or +SIR cells exposed to CX for the indicated times. H3 and each PTM were normalized to their occupancy at ARS504, and then the PTM/H3 quotient of the non-induced (0 min) sir2Δ sample was set to 1.0. Depicted are means ± S.D. .Activation of the heterochromatic (PDF)Click here for additional data file.Figure S4YFR057w gene is impeded in an H4 K16R mutant. Depicted is YFR057w transcript abundance in sir2Δ derivatives of MSY529 and MSY541. Cells were incubated for the indicated times in the presence of cycloheximide (CX), RNA isolated and YFR057w RNA levels determined as in Transcriptional activation of the euchromatic (TIF)Click here for additional data file.


What role does sequence-dependent positioning play? In this paper, using a curvature profile model, nucleosomes are predicted in the human genome and patterns of nucleosomes near some key sites are investigated.Nucleosome positioning has an important role in gene regulation. However, dynamic positioning Curvature profiling revealed that in the vicinity of a transcription start site, there is also a nucleosome-free region. Near transcription factor binding sites, curvature profiling showed a trough, indicating nucleosome depletion. The trough of the curvature profile corresponds well to the high binding scores of transcription factors. Moreover, our analysis suggests that nucleosome positioning has a selective protection role. Target sites of miRNAs are occupied by nucleosomes, while single nucleotide polymorphism sites are depleted of nucleosomes.The results indicate that DNA sequences play an important role in nucleosome positioning, and the positioning is important not only in gene regulation, but also in genetic variation and miRNA functions. Nucleosome positioning refers to the position of a DNA helix with respect to the histone core . PositioCaenorhabditis elegans [At the same time, computational predictions using DNA sequence information have also advanced. Since the report of the nucleosome positioning code (an ~10 bp repeating pattern of dinucleotides AA-TT-TA/GC) in yeast , some mo elegans .in vivo cast doubt on the reliability of predictions based on DNA sequence characteristics [S. pombe and S. cerevisiae [Such variations of nucleosome positions eristics ,6. Moreoeristics . Also, trevisiae . Nucleosrevisiae .C. elegans, flies, and humans [However, some factors should be noted. Firstly, the ~10-bp periodicity of dinucleotides AA-TT-TA/GC, which is identified as a positioning code in yeast, is also found in d humans ,20-24. Sd humans ,10,11. Ld humans ,9). Third humans ,11. In ad humans . All of In this paper, using the curvature profile, a new model based on the curvature pattern of nucleosomal DNA, nucleosomes positions were predicted for the human genome. Patterns of nucleosomes near interesting sites, including TSSs, TFBSs, single nucleotide polymorphism (SNP) sites, and target sites of miRNAs, were thoroughly investigated. The results also demonstrated the important roles of DNA sequences in determining nucleosomes. Moreover, we revealed that nucleosomes are not only functional in gene regulation, but also in genetic variation.et al.'s experimental dataset [Nucleosomes positions were predicted by recognizing the curvature pattern of the core DNA helix (see methods). The reconstructed curvature pattern of a nucleosome is shown in Figure  dataset . An averet al.'s predictions, the averaged centre-to-centre distance of neighbour nucleosomes is ~190 bp . Importantly, both the curvature profile and Kaplan et al.'s model has a much higher matching ratio for the experimental data than random positioning does. In comparison with the model nu-Score [+ T cells  . More than 53% of the experimentally determined nucleosomes were predicted by curvature profile, with a 40-bp deviation Figure . Using tnu-Score  on a 50kiation < 5 bp. Impiation < 5 bp. ImpFigure in vivo is based on this default arrangement.The curvature profile is based on the curvature characteristics of nucleosomal DNA, and is therefore sequence dependent. Subsequently, the curvature-dependent degree of nucleosome positioning was estimated using the nucleosome occupancy ratio of hexanucleotides in the whole of human chromosome 20. The correlation between occupancy ratio of the predictions and the experimental data was 0.6123 Figure . It shouNucleosome positioning at promoters has been extensively investigated because of its role in occluding binding sites. A typical NFR locates around a TSS, which allows the binding sites to be exposed to the pre-initiation complex (PIC) ,9,11,16.et al.'s experimental data in activated CD4+ T cells  and 2491 nucleosome-free TSSs (class II). Both the curvature profile and the experimental data are consistent with nucleosome depletion at class II TSSs  dinucleotides decreased in a broad range near the TSS  in promoters ,11. Thisin vivo [Recent findings suggest that the mechanisms by which DNA sequences affect nucleosome positioning is distinct in some species ,18,19,27in vivo ,7-13. DNin vivo -24. Thesin vivo . Taken tOryzias latipes, genetic variation downstream of a TSS occurs with an ~200-bp periodicity, and the insertion/deletion of more than 1 bp frequently occurs in the linker-DNA region [Patterns of nucleosomes near SNP sites were investigated . Therefore, genome sequences partly encode nucleosomes, and the latter allow mutations to occur at some sites and protect other special sites. Thus, nucleosome positioning has roles in genetic variation.In vivo, the TSSs of some genes are occupied by nucleosomes, and changes to nucleosome positioning will affect the genes' expression levels. In promoters, nucleosomes are depleted near TFBSs, and the distribution of TFBSs corresponds well with the NFRs. Moreover, a selective protection role of nucleosomes was revealed. SNP sites are enriched in NFRs, and miRNA target sites are associated with well-positioned nucleosomes. Our results indicate the vital role of the DNA sequence in encoding nucleosomes, and that the functions of nucleosome positioning are probably involved in further biological processes.In this study, human nucleosome positions were predicted with a curvature-dependent model; the curvature profile. The results indicate that the importance of DNA sequences in determining nucleosome positions, providing a default pattern. DNA sequence partly encodes an NFR near a TSS. In our previous work , we founC (eq.1), which is calculated with a matrix of roll ρ and tilt τ angles  were extracted from the crystal structure dataset of the nucleosomal DNA and histone proteins . The numbers (n2-n1) represent the integration steps. The modulus of the vector represents the deviation from B-DNA. The results generated a uniform pattern of curvature for nucleosomal sequences  (build 36.1), and nucleosomes were mapped using the curvature profile. To evaluate the predictions, the experimentally determined nucleosomes dataset of activated and resting CD4+ T-cells was downloaded from Zhao's website (http://dir.nhlbi.nih.gov/papers/lmi/epigenomes/hgtcellnucleosomes.aspx)[et al.'s dataset contains two columns for each chromosome, the first column indicates the genomic positions and the second shows scores of nucleosomes. A method based on wavelet transformation was used to detect the exact nucleosome positions from the series of scores (see below). Kaplan et al.'s predicted nucleosome dataset was downloaded from website (http://genie.weizmann.ac.il/software/nucleo_genomes.html) [et al.'s model were carried out on human chromosome 20.Human genomic DNA sequences were retrieved from NCBI (mes.aspx). The dates.html) . Compariet al.'s predictions, and the curvature profile, the dataset are a series of numbers. Thus, it is important to identify peaks positions to perform a quantitative comparison. Here, the maximal spectrum of continuous wavelet transformation (MSCWT) [In the experimentally determined datasets, Kaplan  (MSCWT)  . Information on miRNA promoters was extracted from the literature [in vivo, protein-coding TSSs were separated into two classes by a k-means clustering method using Zhao et al.'s experimental data [+ T cell. One class (class I) contained TSSs that are occupied by nucleosomes; the other (class II) contained nucleosome-free TSSs. A dataset of mRNA levels [More attention was paid to nucleosomes around special sites within the genome. Upstream and downstream sequences of TSSs, TFBSs , SNPs 3 and targterature . Detailstal data  in a ranA levels  was usedTo test whether nucleosomes limit the binding of transcription factors, the distribution of TFBSs was computed for both protein-coding promoters   and indehttp://www.gri.seu.edu.cn/icons).An online-prediction tool for the curvature profile is provided , single-nucleotide polymorphism (SNP) sites, target sites of miRNAs, start and stop codons, and boundaries of histone modifications. Table s4 - Three types of miRNAs in humans. Table s5 - Sixty-four human transcription factors used in scanning. Table s6 - Comparison of performances of the curvature profile and nu-Score. Table s7 - Prediction performance of the curvature profiles in Figure 3 and s5. Table s8 - Top 20 6-mer nucleotides that are favorable for nucleosomes and nucleosome-free regions. Figure s1 - Curvature pattern derived from 634 well-positioned nucleosome DNA sequences in the experimental dataset. Figure s2 - Identification of nucleosome dyad positions in DNA sequences from 8 k bp to 28 k bp of human chromosome 20. Figure s3 - Distribution of centre-to-centre distance of nucleosomes. Figure s4 - Predictions of nucleosomes for the segment from 90798 k bp to 90801 k bp of human chromosome 13. Figure s5 - Predictions of nucleosomes for a segment of human chromosome 17 (52269 k-52289 k bp). Figure s6 - (A) Faction distributions of WW (W = A ot T) dinucleotides and SS (S = G or C) dinucleotides near 3571 transcription start sites; (B), fraction of poly (dA) and poly (dT); (C), fraction of poly (dG) and poly (dC). Figure s7-Gene expression levels (mRNA levels) for the occupied-TSS genes (class I) and the open-TSS (class II) in activated CD4+ T cells, the gene expression data is from Zhao et al's experiment . Figure s8- Patterns of nucleosomes near SNP sites in the dog genome.Click here for file