The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear.in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors.We report the first genome-wide identification and characterization of TR4 Our data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases. Elucidating the position and activities of individual cis modules using reporter genes is time consuming and expensive. With recent advances in DNA sequencing technology, it is now feasible to generate global protein-DNA interaction profiles by chromatin immunoprecipitation (ChIP) followed by ultra-high-throughput sequencing [Cis modules can then often be identified by applying bioinformatics searches for one or more cis motifs recognized by unrelated alternative factors near the binding sites of the factor analyzed by ChIP-seq or by the co-localization of bound sites for two or more unrelated different site-specific factors.There are an estimated 1400 site-specific DNA binding factors encoded in the human genome . These rquencing . Cis modNuclear receptors (NRs) represent a special class of transcription factors that direct target gene transcription in a ligand-dependent fashion. NRs contain a DNA-binding domain that recognizes a specific DNA sequence, as well as a ligand binding domain that renders these factors environmentally-dependent regulators via interaction with distinct cognate ligands . The grein vitro is equivalent to that of the TR2:TR4 heterodimer [in vivo genome-wide binding patterns of, this broadly expressed nuclear receptor are obscure. We therefore chose to initially investigate genome wide TR4 binding anticipating that these studies might reveal some common, but also perhaps some tissue-specific, metabolic processes to which this factor contributes.NR2C2  belongs to the nuclear receptor superfamily and is termed an orphan receptor due to the fact that no ligand has been discovered -8. TR4 werodimer , and TR4in vivo binding in multiple cell types, including those in which TR4 has been suggested to be an activator (liver) and cells in which TR4 has been suggested to be a repressor (blood). Using ChIP-seq, we determined TR4 in vivo binding in four human ENCODE cell lines: K562 erythroleukemia cells, HepG2 liver carcinoma, HeLa cervical carcinoma, and GM12878 immortalized lymphoblast cells. TR4 binding patterns identified in the four diverse cell lines suggest that this factor controls cell metabolism by binding to the proximal promoter regions that are common to several hundred genes. Motif analysis shows that TR4 strongly prefers a DR1 sequence to all other categories of repeat elements in vivo. By integration of TR4 binding data with histone modification patterns and other genomic structures, we predict, and then experimentally test, putative cis modules.In this study we investigated the first genome-wide identification of cellular targets of TR4 and preliminary characterization of TR4 + cells, but only after in vitro differentiation for 11 days. To further elucidate biological roles for TR4, we set out to identify in vivo TR4 binding sites throughout the entire human genome using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). We wanted to compare its binding profiles in cells derived from different tissue types. We chose to identify TR4 targets in cell types selected by the ENCODE Consortium (http://www.genome.gov/10005107), including human chronic myelogenous leukemia cells (K562), human cervical carcinoma cells (HeLa), lymphoblastoid cells (GM12878), and hepatocellular carcinoma cells (HepG2). By characterizing its binding in these cell lines, we could compare TR4 binding sites with other transcription factor binding sites and histone marks determined by other ENCODE groups examining these same cell types. We first validated the presence of TR4 protein in these cell lines by Western Blot analysis . Only scells K56, human cThe position to which a transcription factor binds relative to the start site of transcription can provide insight into how the factor regulates transcription. For example, E2F family members bind to core promoter regions and are thought to stimulate transcription by interaction with the basal transcription machinery ,23. In cA significant fraction of TR4 binding sites was shared among cell types Figure . For exahttp://conceptgen.ncibi.org/core/conceptGen/index.jsp; [As shown above, the majority of TR4 targets are shared between different cell types. To shed light on the common function of genes targeted by TR4, gene ontology analysis was performed using ConceptGen  to idendex.jsp; . Furtherdex.jsp;  suggestiIn recent years it has become evident that transcription factors often play dual roles, affecting activation as well as repression of target genes. Previous studies have implicated TR4 in both activation and repression of cellular target genes . TR4 binhttp://chipseq.genomecenter.ucdavis.edu/cgi-bin/chipseq.cgi; [The correlation between TR4 binding and expression of target genes suggests that TR4 binds to open accessible chromatin regions. To test this hypothesis, we examined the epigenetic signature at TR4 binding sites using ChIP-seq data of various histone marks in K562 cells. Overlap of TR4 binding sites with histone marks typical for open and repressed chromatin was determined using the gffOverlap tool from Sole-search . A distseq.cgi; ,31. To dIn vitro experiments have shown that TR4 binds to the direct repeat (DR) of AGGTCA, which is the consensus binding site for a number of nuclear hormone receptors including estrogen receptor alpha and PPAR. Further studies have indicated that TR4 can bind to direct repeats separated by zero to five nucleotides (DR0 - DR5) [in vitro assays. We used the de novo motif discovery program MEME to identify motifs overrepresented in TR4 binding sites to determine if TR4 has the same specificity in vivo. To allow identification of DR elements and its spacing and flanking nucleotides, the minimum motif length was set between 12  and 20 nucleotides . The canonical DR motif with one nucleotide spacing (DR1) was significantly overrepresented in all four cell types with the preferred spacing nucleotide being an A or G  ,13,17,32G Figure . The cancis regulatory modules [de novo motif discovery programs such as NHR-Scan [Transcription factors often regulate expression of nearby genes in combination with other transcription factors through complex  modules . Our ini modules . The ETSNHR-Scan  and W-ChNHR-Scan .cis regulatory network involving TR4 and ETS family members.It has been postulated that the true binding site for transcription factors should be located under the center of the peak . We analin vivo using ChIP-seq. Motif analysis implicates the ETS family, but does not provide information as to which family member might bind to TR4 target sites. There is a high degree of functional redundancy between different members of the ETS transcription factors. Comparison of ELK1 and GABPA binding regions revealed redundant as well as unique targets between the two ETS family members [We wanted to test the hypothesis that TR4 and a member of the ETS family co-localize with TR4  members ,38. It h members ,39. ChIPcis module that accounts for 30% of TR4 binding sites. These sites are characterized by overrepresentation of the ETS motif in 96% of the sites and the lack of a DR1 element typically thought to recruit TR4. Therefore, TR4 does not directly bind to DNA via a DR1 element at these sites, but appears to be recruited through an ETS factor. We also analyzed the localization of binding relative to gene structure and found that TR4 and ELK4 display very similar patterns, with maximum binding between 500 bp upstream and downstream of a transcription start site  at endogenous target sites in all four cell types examined. Using de novo motif discovery, we found that the ETS motif CCGGAA was significantly overrepresented in TR4 binding sites, suggesting a role for ETS family members in TR4 action. To confirm the co-occurrence of these two factors in vivo, we performed ChIP-seq for the ETS transcription factor ELK4 and we found that about one third of TR4 target sites were indeed bound by ELK4. Sites that are bound by both factors contain an ETS motif, but lack the DR1 element typically thought to recruit TR4. These data suggest that TR4 may regulate specific subsets of target genes through ETS dependent as well as ETS independent pathways. Future studies will focus on the interdependence of these two transcription factors. Thus our approach of defining genome-wide binding patterns for a factor, followed by motif analysis to suggest possible cis modules, and then genome-wide analysis of the putative co-localizing factor has worked well to identify a TR4-ELK4 cis module.While it had been established that TR4 plays a critical role in embryonic development, differentiation and lipid metabolism, the modes by which it functions were previously unclear. To obtain a better understanding of the TR4 modes of action, we used ChIP-seq technology to identify TR4 target genes Interestingly, we identified TR4 target genes that are common to quite diverse cell types . These genes were involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, TR4 also binds near genes that are highly cell type-specific. For example, in HepG2 cells TR4 binds near genes that are involved in organic acid, lipid and carbohydrate metabolism. TR4 knockout mice show insulin hypersensitivity  and TR4 K562, HeLa, HepG2, and GM12878 cells for ChIP-seq were grown and crosslinked by the National Cell Culture Center (NCCC) as part of the ENCODE project. K562 and GM12878 cells were grown in RPMI supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 U/mL penicillin-streptomycin. HeLa and HepG2 cells were grown in DMEM medium supplemented with 10% FBS, 2 mM L-Glutamine, 100 U/mL penicillin-streptomycin. Cells were either processed for RNA isolation or crosslinked 10 minutes at a concentration of 1% formaldehyde, snap frozen and stored at -80C.8 cells was diluted with 5 volumes IP dilution buffer  igepal, 0.25% (w/v) deoxycholic acid, 1 mM EDTA pH8) and incubated at 4C over night with either 50 μl of rabbit anti-TR4 antibody [http://www.genomecenter.ucdavis.edu/farnham/pdf/FarnhamLabChIP%20Protocol.pdf). For sequencing experiments, StaphA cells were only blocked with BSA and the preclearing step was omitted. After reversal of crosslinks and RNase treatment, ChIP DNA was purified and used directly for library preparation.ChIP assays and the libraries for Illumina sequencing were prepared as described in detail in O'Geen et al. 2010 . Brieflyhttp://genomecenter.ucdavis.edu/dna_technologies/). The ChIP-seq data has been deposited in the NCBI Gene Expression Omnibus (accession number GSE24685). In addition, all TR4 ChIP-seq data can be visualized and downloaded from the UCSC browser at http://www.genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=169984430&c=chr9&g=wgEncodeYaleChIPseq. Peaks were called using the Sole-search software with default parameters  using sequenced libraries of matched Input DNA for each cell type [Libraries were sequenced using the Illumina GA2 platform by the DNA Technologies Core Facility at the University of California-Davis . Unbiased motif analysis was performed using MEME to identify statistically overrepresented motifs in the TR4 peak sequences present in 4 cell types. The following parameters were used "-dna -nmotifs 5 -mod zoops -minw 12 -maxw 20 -maxsize 2000000 -revcomp", which specify the number of motifs to search for, the zoops assumption (zero or one occurrence per peak sequence), the minimum motif length of 12 , the maximum motif length of 20 , the maximum dataset size of 2,000,000 characters. Sequences were searched in forward and reverse orientation.6 HeLa or HepG2 cells using Invitrogen Trizol according to the manufacture's recommendations. The Illumina TotalPrep RNA amplification kit from Ambion (AMIL1791) was used to generate biotinylated, amplified RNA for hybridization with the Illumina Sentrix Expression Beadchips, HumanHt-12. The Sentrix gene expression beadchips used for this study consisted of a 12-array, 2 stripe format comprising approximately 48 k probes/array. In this collection 24,000 probes were from RefSeq sequences and 24,000 from other Genbank sequences (see http://www.illumina.com/pages.ilmn?ID=197 for more details). Arrays were processed as per manufacturer's instructions, scanned at medium PMT settings as recommended by the manufacturer, and analyzed using Bead Studio Software v. 2.3.41. Data was normalized using the "average" method, which simply adjusts the intensities of two populations of gene expression values such that the means of the populations become equal. Relative expression values were calculated using an algorithm provided by Bead Studio. The expression array data has been deposited in the NCBI Gene Expression Omnibus (accession numbers GSE24419 for HepG2 and GSE19146 for HeLa data).RNA was prepared from three independent cultures of 107 cells were used per ChIP experiment and adjusted amounts of the same antibodies and pre-immune serum (rabbit IgG) as described above. Immunoprecipitated DNA was purified and eluted in 50 μl water. 1 μl of ChIP DNA or 3 ng of Input DNA were used for qPCR analysis. Quantitative PCR experiments were performed at least in duplicates, from at least two independent ChIP assays on a Bio-Rad DNA Engine Opticon Real-Time PCR System using SYBR® Green Master PCR Mix (SIGMA) according to the manufacturer's instructions. Results were analyzed relative to input. Each target site was calculated as 2 to the power of the cycle threshold (cT) difference between input DNA and ChIP samples. Enrichments at target sites are compared to negative/unbound control regions CDH1 and CDH10 (see Additional file To confirm targets identified by ChIP-seq, all ChIP assays were performed using StaphA cells. 10HOG designed, performed and analyzed experiments and drafted the manuscript. YHL performed bioinformatics analysis, provided figures and text for the manuscript. XX performed ChIP-seq experiments and data analysis. LE performed ChIP-seq experiments and data analysis. VMK helped with RNA and ChIP-seq data analysis and provided figures and text for the manuscript. DH performed QPCR experiments and data analysis. SF performed RNA experiments and data analysis. OT and LS generated and purified anti-TR4 antisera. MAS assisted with bioinformatics analysis. JDE and PJF helped with experimental design and writing of manuscript. All authors read and approved the final manuscript.Validation of TR4 expression in four different cell types. Western blot analysis to validate expression of TR4 protein. 10 μg nuclear extract were loaded per lane. Cell types used are indicated above each lane.Click here for filePrimer sequences for standard and qPCR validation of TR4 ChIP samples.Click here for filePCR anaslysis of three TR4 binding sites. ChIP assays were performed in K562 and HepG2 cells using TR4 antibody. PCR was performed using primers to TR4 binding sites identified by ChIP-seq (see Additional file Click here for fileChIP-seq data and analysis summaryClick here for file


Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate  that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. To evaluate these predictions, we analysed transcription through a nucleosome by different, structurally related RNA polymerases and mutant yeast Pol II having different histone-interacting surfaces that presumably stabilize the Ø-loop. The height of the nucleosomal barrier to transcription and efficiency of nucleosome survival correlate with the net negative charges of the histone-interacting surfaces. Molecular modeling and analysis of Pol II-nucleosome intermediates by DNase I footprinting suggest that efficient Ø-loop formation and nucleosome survival are mediated by electrostatic interactions between the largest subunit of Pol II and core histones. Other chimera/) . A T.th.ID: 2O5I ] to Pol ID: 2O5I  and dispThermus thermophilus (HB8 strain) genomic DNA (Refseq: NC_006461) has been used to PCR-amplify rpoA, rpoB, rpoC, rpoZ and rpoD genes and to clone them in pET series E. coli expression vectors. Plasmids pET28-TthA, pET28-TthB, pET28-TthC, pET28-TthZ and pET28-TthD overexpressing, respectively, untagged T. thermophilus RNAP α and β subunits, N-terminally hexahistidine-tagged β′, untagged ω and N-terminally hexahistidine-tagged σA subunits were constructed using routine cloning methods. Plasmid pET28-TthABCZ co-overexpressing RNAP subunits sufficient for the assembly of catalytically proficient T. thermophilus RNAP core enzyme was created in two steps by using the single-rpo-plasmids described above. In all the plasmids a T7 RNAP promoter precedes each of the rpo genes.Thermus thermophilus and Thermus aquaticus RNAPs and their sigma factors were purified as described (Yeast Pol II [wild-type (WT) and mutant (MU)] and core histones were purified using published protocols . Thermusescribed . The -H1escribed .Strain YDC412  has three point mutations in the RPB1 gene: E1404A (GAA to GCA), E1407A  and E1411A . YDC412 was obtained by transformation of BJ5464 Rpb3 His-Bio  with a STemplates: λ-603 and λ-603-49 contained λ promoter , a 50-bpin vitro transcription assay with Thermus’ RNAPs was similar to the published protocol (Thermus’ RNAPs were first activated at 65°C in transcription buffer (TB)  for 5 min and then shifted to 37°C. The elongation complexes containing 26-mer RNA (EC-17) on pre-assembled nucleosomal templates as was described . EC-17 was extended in the presence of 400 mM NTPs at 25°C in the TBs having different concentrations of KCl . In footprinting experiments with T. aquaticus RNAP, all steps were performed in solution. EC+49 complexes were formed in the presence of 1 µM ATP on the 603-49 templates. EC-5 was assembled first and then further extended in the presence of 300 µM CTP, UTP, GTP and 300 µM 3′dATP at 25°C for 4 min in TB300. Labeled DNA was purified from native PAGE and separated by denaturing PAGE. To analyse the nucleosome fate after transcription, the DNA-labeled templates were transcribed and analysed by native PAGE as described . Averages from three experiments and standard deviations were determined.The 0 buffer . EC-39 wOur previous studies have identified the Ø-loop-containing +49 elongation complex  as the key intermediate mediating nucleosome survival and a similar complex (EC+39) has been modeled using docking approach . The EC+o DNA bend present in the EC faces the octamer surface and allows formation of the Ø-loop. (iii) DNA-histone contacts with ∼20-bp DNA region behind the EC stabilize the Ø-loop. (iv) Displacement of ≥50 bp from the promoter-distal end of the nucleosome reduces the size of the DNA region interacting with histones in front of the enzyme from ∼100 to ≤50 bp. This likely facilitates further uncoiling of DNA from the octamer ahead of Pol II and transcription through the nucleosome (The EC+39 and EC+49 share the following properties : (i) TheSupplementary Table S1). Escherichia coli RNAP that utilizes the Pol II-type mechanism of transcription through chromatin   . Althouge charge B, there e charge . The dat5 region , a decreT. thermophilus and T. aquaticus RNAPs in vitro, the λ promoter has been placed upstream of the strong 603 NPSs . The pulse-labeled RNA was analysed by denaturing PAGE  of nucleosomal templates at 40 and 150 mM KCl . To analing PAGE A.FigureT. thermophilus and T. aquaticus RNAPs was much less pronounced and the yield of the run-off transcripts was more than 80%. In particular, only very limited nucleosome-specific pausing at the +45 region was observed during transcription by T. thermophilus and T. aquaticus RNAPs .To evaluate the fate of the nucleosome on transcription by T. aquaticus RNAP, as was determined by analysis by native PAGE  and nucleosome displacement during transcription by this enzyme (see Supplementary Discussion).Transcription by yeast Pol II results in displacement of the histone octamer from ∼20% of templates and conversion of remaining nucleosomes into the hexasomes remaining at the original position on the template . In contive PAGE B and C a pausing A and theT. aquaticus RNAP, the enzyme has been stalled at the position +49 . DNA both in front and behind of the enzyme is much more accessible to DNase I digestion in nucleosomal EC+49 than in the original nucleosome. Furthermore, the pattern of DNA digestion by DNase I downstream of the enzyme in the EC+49 is very similar with the pattern characteristic for histone-free DNA, indicating that in the majority of the complexes DNA downstream of the enzyme is displaced from the octamer. At the same time, ∼15-bp DNA region upstream of nucleosomal EC+49 is partially protected from DNase I, indicating binding of histones to a fraction of EC+49 complexes. Thus in the EC+49 complex formed by T. aquaticus RNAP all downstream and the majority of upstream DNA–histone interactions are disrupted. Core histones are at least partially displaced from the nucleosome by the enzyme and the canonical Ø-loop is not formed. Such disrupted DNA–histone interactions are not expected to cause strong pausing; further transcription may result in displacement of the entire histone octamer, as was indeed observed experimentally . The WT and the MU yPol II have similar processivities and transcribe DNA at similar rates , suggesting that these properties are not strongly affected by the mutations. The data suggest that higher negative charge of the ZLS Pol II region positively correlates with stronger +45 pausing, likely because it more efficiently interacts with the histone octamer and therefore facilitates efficient formation of the Ø-loop.Finally, the effect of neutralizing negative charge on the putative ZLS Pol II surface has been evaluated. To construct an MU Pol II having lower negative charge on the ZLS surface, three negative charged glutamate residues within the region 2  were substituted with non-polar alanines A. WT andThe data on the net charge of ZLS region, the height of +45 nucleosomal pausing and the nucleosome fate on transcription by different RNAPs are summarized in T. thermophilus and T. aquaticus RNAPs), nucleosomal +45 pausing is weak, the Ø-loop does not form, and nucleosomes are lost during transcription.Overall, the data suggest that the presence and net charge of the ZLS region in part determine the structure of the key intermediate formed at the position +49, and, in turn, the nucleosome fate on transcription. When the ZLS region is present and negatively charged (e.g. human and yeast Pol II), nucleosomal +45 pausing is strong, Ø-loop forms at the position +49 and nucleosomes efficiently survive during transcription. When the ZLS region is present, but does not contain negative charge  during transcription through chromatin has been evaluated using computational modeling and experimental analysis of the mechanism of transcription through chromatin by various RNAPs. Computational modeling suggested that in the elongation complex that determines nucleosome survival during transcription (EC+49) three ZLS regions are in close proximity to a positively charged region of the histone octamer and thus could stabilize the Ø-loop and facilitate nucleosome survival during transcription  and 2. Acription  and 5. Icription ,20,34. Tcription . Thus thHow can the presence and charge of the ZLS region affect these multiple aspects of transcription through chromatin? Previously we have proposed that the ZLS region could affect the structure/stability of the key Ø-loop-containing intermediate (EC+49) formed during transcription through a nucleosome . This inT. thermophilus and T. aquaticus RNAPs having uncharged ZLS region the Ø-loop (if formed) is unstable (complex 2′). Therefore transcription is accompanied by only minor nucleosomal pausing and by displacement of the histone octamer during further transcription through the nucleosome (3′). Thus the structures of the key intermediates formed during transcription through the +45 region of nucleosomal DNA likely dictate multiple aspects of transcription through chromatin, including nucleosomal pausing and the fate of histones on transcription. The presence and charge of the ZLS region are unlikely to be the only factors that affect the outcome of transcription through chromatin. Thus, the distance between ZLS regions and the histone octamer in the EC+49 is likely important for the stability of the Ø-loop.Based on our data, we propose the following model explaining the observed relationship between the +45 pausing, Ø-loop formation and nucleosome fate . As diffThe negatively charged ZLS region 2 of yeast Pol II is localized within the switch 2 domain and is likely important for separation of the DNA strands during transcription . It is aSupplementary Data are available at NAR Online, including [38–40].National Institute of Allergy and Infectious Diseases (NIAID) [R21-AI090558 to K.K.]; National Institutes of Health (NIH) [GM58650 to V.M.S.]; Russian Foundation for Basic Research (RFBR) [12-04-31942 to A.K.S.]; Intramural Research Program of the National Institutes of Health (NICHD) (to D.J.C.). Funding for open access charge: Grant of NIH [GM58650 to V.M.S.].Conflict of interest statement. None declared.


Regulatory DNA elements such as enhancers, silencers and insulators are embedded in metazoan genomes, and they control gene expression during development. Although they fulfil different roles, they share specific properties. Herein we discuss some examples and a parsimonious model for their function is proposed. All are transcription units that tether their target promoters close to, or distant from, transcriptional hot spots (or 'factories'). Silencein trans  and/or cin trans . The intin trans -9 fine-tin trans . Althougin trans . Herein in trans -15.cis or in trans) [Enhancers were characterised almost 30 years ago , but then trans) ,18, posin trans) . Althougn trans) . Within n trans) , are flan trans) . Attemptn trans) . These an trans) ,22. Mostn trans) ,24.cis-regulatory elements in this locus are also in close proximity, where they form an 'active chromatin hub' [The first and most studied example of gene regulation by an enhancer is provided by the β-globin locus; here, the locus control region (LCR) is located 40 to 60 kb upstream from the promoter it regulates. The two interact when the chromatin fibre forms new, or rearranges preexisting, loops ,25. All tin hub' ,26. An atin hub' . SimilarArc promoter depend on the activity of that promoter, as removing the promoter abolishes eRNA production [Enhancers are transcribed into RNAs (eRNAs) that do not encode proteins, run the length of the enhancer sequence and appear to stabilise enhancer-promoter interactions ,24,27-29oduction . β-globioduction ,30,31. Hbona fide enhancers) [HOTTIP (a lincRNA transcribed from the 5' end of the HOXA locus) coordinates the activation of several HOXA genes; chromatin looping brings HOTTIP close to its targets, and this drives H3K4 trimethylation and transcription [An additional class of ncRNAs longer than 200 nucleotides (long intergenic ncRNAs (lincRNAs)) were found in a survey of human transcripts, and some exhibited enhancer function . In diffhancers) . For exacription .At the opposite functional extreme lie silencers. They prevent gene expression during differentiation and progression through the cell cycle . This agAccumulating evidence supports a broad and general role of both long and short RNA molecules in transcriptional inhibition. Antigene RNAs (agRNAs) are small RNAs that target promoters and downstream regions . The expcis in CD4+ T-cells and ES cells  and the of XOXD ). In humsilenced . Furtherhylation ,47 and phylation . Here thhylation . Such eqFunctionally autonomous domains are strung along the chromatin fibre, and these need to be insulated from their neighbours to prevent the action of irrelevant enhancers and silencers. Insulator or boundary elements perform this task. These can be further categorised as enhancer blockers (when the insulator is located between a promoter and a cognate enhancer) and barriers (when located between a promoter and a silencer) . MutatinDrosophila Hox gene cluster, stalled polymerases, in conjunction with elongation factors DISF and NELF, insulate four of eight promoters from Hox enhancers, and this correlates with the rearrangement and/or de novo formation of chromatin loops [It has been suggested that insulators evolved from a class of promoters binding a specific subset of transcription factors that drive chromatin remodelling and long-range interactions . Many arin loops .Igf2-H19 imprinted locus, CTCF prevents activation of the maternal Igf2 allele by a distal enhancer. When its cognate binding site is lost, the gene is reactivated [H19 gene [renin, ETNK2 [CFTR [c-Myc genes [Perhaps the most abundant protein associated with insulator activity is CTCF. In the well-studied example of the ctivated . HoweverH19 gene . MoreoveH19 gene . The insH19 gene ,56. TheiH19 gene , as welln, ETNK2 , CFTR [5K2 [CFTR ,52 and cyc genes .Drosophila elements, additional factors, such as boundary element associated factor, GAGA and CP190, were used to identify and classify domain boundaries [However, the CTCF-cohesin duplet is characteristic of only one type of insulator or boundary. In a comprehensive mapping of such undaries . Again, undaries .trans [The following four models have been proposed to describe gene regulation by enhancers Figure . 1) Acc Acc1,7,6trans ,68 and itrans  and modetrans ,15.de novo formation of appropriately oriented loops efficiently insulate promoters from enhancer elements [gypsy insulators in Drosophila propagates between distant loci to be repressed via the organisation of local loops [Similarly, among the three major models proposed for insulator function , the topological loop model is best supported by experimental data: Rearrangement and/or elements . Note alal loops .Kit gene locus via specific loop formation and exchange with Gata-2 reforms the enhancer-promoter loop and reactivates expression [IgH locus is another example of how this might occur, because its approximately 2.7-Mbp region is reorganised spatially during activation [Gene regulation from distal regulatory elements via local looping or broader rearrangements in three-dimensional organisation is now widely accepted. For example, we have seen that the β-globin LCR loops back to its target promoter to activate it  through pression . The IgHtivation . Similartivation ; Gata-1,tivation ; CTCF [7tivation ,74; Ldb1tivation ; and cohtivation ,76. Knoctivation ,77,78.On a broader scale, the genome is organised nonrandomly in three-dimensional space ,6-10,15 How can these ideas be extended to explain the function of enhancers and silencers and/or insulators? As we have established, all share common features ; therefore, we propose that canonical regulatory elements are primarily transcription units Table  and thatex vivo [Does the number of factories in a given cell suffice to accommodate all transcription units, including enhancers and/or silencers? To date, the lowest estimate of about 200 factories concerns murine primary cells and comes from RNAPII immunostaining ex vivo . This suex vivo  or that ex vivo ,98. Moreex vivo ,99. The ex vivo ,98. But ex vivo ,97-99. N2) close to factory or hub A that contains the necessary machinery. As a result, the target promoter 2 will diffuse through the nucleoplasm and frequently collide with a polymerase in factory A to initiate transcription. Although promoter 3 is also tethered close to the same factory, it will initiate rarely (because factory A lacks the necessary transcription factors required by this particular promoter). Although promoter 3 can initiate in factory B (which contains high concentrations of the relevant factors), it will do so rarely, simply because it is tethered close to factory A and far from B. Next, transcription unit 1 acts as an enhancer of unit 2 and as a silencer of unit 3. The addition of histone modifications that mark the various units as active or inactive will now reinforce the status quo. After that, once unit 1 has been transcribed, these marks will make it more likely that unit 1 or unit 2 will reinitiate in factory A to create a virtuous cycle. Similarly, at another developmental stage, when a different set of transcription factors are expressed . As units 2 and 3 cannot stably interact with each other by binding to factory C, unit 1 now acts as an insulator or barrier. As before, histone marks will reinforce this different (virtuous) cycle.Now consider that an enhancer (transcription unit 1) Figure  tethers d Figure , unit 1 The model we propose here Figure  illustrabp: base pair; kb: kilobase; IL: interleukin; Mbp: megabase pair; miRNA: microRNA; RNAi: RNA interference; TNF: tumour necrosis factor.The authors declare that they have no competing interests.All authors structured, wrote and proofread the manuscript.


IGF2), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the IGF2 gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II.Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 . A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications  or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation. Allele-specific gene expression is an integral component of cellular programming and development and contributes to the diversity of cellular phenotypes ,2. AllelIGF2) and H19 genes [IGF2 promoter with enhancer elements downstream of the H19 gene [IGF2 gene.The best-characterized locus with strict monoallelic imprinted gene expression is the region containing the insulin-like growth factor 2 . In contrast, the unmethylated allele is characterized by permissive histone markers, including histone H3 trimethylated at lysine 4 . ColocalIGF2 gene prompted us to test whether the presence of CTCF and H3K9me3 specifies a chromatin arrangement which demarcates random monoallelically expressed alleles. Using array-based chromatin immunoprecipitation (ChIP-chip), we identified 293 loci displaying these chromatin markers. We selected the IGF2BP1 gene locus to further examine whether the presence of CTCF and H3K9me3 comprises a necessary chromatin arrangement for a specific expression profile analogous to the monoallelic behavior observed at the IGF2/H19 locus. Surprisingly, colocalization of CTCF and H3K9me3 does not provide a reliable measure of monoallelic binding of CTCF at the IGF2BP1 gene. Our studies included allele-specific sequencing of immunoprecipitated chromatin to demonstrate that chromatin at each IGF2BP1 allele is bivalent. Importantly, both alleles recruit RNA polymerase II, suggesting that silencing of one IGF2BP1 allele occurs after transcription initiation. By establishing which epigenetic configurations are involved in governing monoallelic gene expression, we will broaden the understanding of epigenetic mechanisms as they relate to cancer progression and cellular differentiation.Despite the importance of monoallelic expression in cellular development and differentiation, little is known about the establishment and maintenance of random monoallelic expression. The link between allele-specific binding of CTCF and monoallelic expression of the IGF2 gene and correlates with differential cytosine methylation and the presence of H3K9me3 [IGF2/H19 locus. Using ChIP-chip, we identified CTCF binding sites by tiling through the nonrepetitive portion of the genome in 100-bp intervals. Genomic sites bound by CTCF were assembled on a condensed array set that tiled through 9,823 sites using overlapping probes, and replicate ChIP experiments were performed. By using conservative criteria  in this analysis, we identified 8,462 loci that interact with CTCF. To identify the subset of sites that associate with both CTCF and H3K9me3, we tested the association of these 8,462 loci with H3K9me3 using the condensed DNA array set. These analyses revealed 293 loci that are both bound by CTCF and marked by H3K9me3  of 4,000 human genes examined were subject to random monoallelic expression, with a majority of the latter being capable of biallelic expression [Genes classified as "monoallelically expressed" encompass both imprinted genes, such as the than 300 .5% of 4,et al. [To examine whether CTCF binding at sites marked by H3K9me3 is indicative of monoallelic expression, we first compared the genomic positions of our 293 loci with the list of genes expressed in a random allele-specific manner. Only a small number of genes (8 of 293 loci) were common to both the monoallelically expressed cohort described by Gimelbrant et al.  and our DIAPH1, FUS1, PKP1, ARFGAP2, PCDHGA, MTHFR, LAIR1, GPR3, ARMET, NPR1, NHLRC1 and IGF2BP1) to search lymphoblastoid cell lines (LCLs) derived from a pedigree from the Center d'Etude du Polymorphisme Humaine (CEPH) for SNPs in exonic and 3'-UTR regions. The monoclonality of LCLs was confirmed by analysis of their immunoglobulin heavy chain (IgH) gene rearrangement  to cluster sequences of a specific length (reads) from a set of input sequences (such as enriched genomic loci identified by ChIP-chip experiments). Motif alignment using STAMP [To precisely determine the DNA sequence required for CTCF binding at the SOMBRERO , a de nong STAMP  and compng STAMP  identifing STAMP . The clung STAMP , our freIGF2BP1 gene locus enriched in our ChIP-chip experiments. Two of the putative binding sites, Y and Z, accumulated a significant number of matches to motif models. However, only one of the three putative CTCF binding sites belongs to the group of high-confidence binding sites (site Y)  Figure  within t) Figure . In suppZ Figure , suggestin vitro using immobilized template assays   Figure . In conts Figure . TargeteIGF2BP1 gene contains a single CpG residue adjacent to the 14-bp core sequence of CTCF  and monoallelically (GM6989) expressing cells by using bisulfite sequencing  and biallelically (GM7057) expressing individuals were heterozygous at SNP site rs11870560 at the CTCF site  and 7057  were heterozygous at SNP rs9890278 located upstream of the transcription initiation site, whereas GM7007  was heterozygous for SNP rs4794017 located 1 kb downstream of the transcription initiation site. To address whether active and silent alleles in these cell lines are distinguished by specific histone markers, we sequenced SNPs rs9890278 and rs4794017 in gDNA recovered from ChIP experiments using anti-H3K4me3 and anti-H3K27me3 antibodies. The results revealed that both H3K4me3 and H3K27me3 are detected on both alleles in a bivalent fashion . This protocol allowed detection of pre-mRNA free of gDNA contamination  lines. However, while only three hESC lines were informative (heterozygous at SNP rs11655950), all three expressed IGF2BP1 in a biallelic manner. Although the number of available and informative hESC lines is not sufficient to clearly define a role for allele-specific elongation in early developmental stages, we believe that it is unlikely that this mechanism is restricted to cell types with low levels of IGF2BP1 expression. Control of transcriptional activity through promoter proximal pausing or premature termination of transcription is not restricted to specific gene classes characterized by low levels of transcriptional activity [The expression of activity . We specIGF2BP1 gene is highly expressed during embryonic development and is required for the regulation of mRNA stability of several genes involved in growth regulation, including the IGF2, β-catenin and MYC genes [IGF2BP1 gene is downregulated in differentiated cell types, and overexpression of IGF2BP1 is known to occur in multiple human cancers, including breast, lung and colon [IGF2BP1 expression through silencing of only one allele could provide a safeguard against pathogenesis and disease.The YC genes -25. Consnd colon -52. ThusIGF2/H19 locus, raising the question whether these epigenetic markers are useful for identifying both imprinted and random monoallelically expressed genes throughout the genome. In this study, we have demonstrated that colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. In addition, we conclude that allele-specific binding of CTCF requires methylation of very specific cytosine residues within the target motif, effectively limiting the number of CTCF binding sites potentially affected by allele-specific binding. In addition, the active and inactive alleles of random monoallelically expressed genes do not necessarily correlate with active or inactive histone markers. Remarkably, the selection of individual alleles for expression at the IGF2BP1 locus occurs during early stages of transcription elongation.Allele-specific gene expression is common in the human genome and is thought to contribute to phenotypic variation. The allele-specific association of CTCF, H3K9me3 and DNA methylation is a characteristic marker of imprinted gene expression at the The amplification and preparation of immunoprecipitated DNA derived from HBL100 cells for hybridization to ENCODE arrays  was performed essentially as described previously . Sample 2 ratio values above a specified cutoff value. The cutoff value was calculated separately for each chromosome. The cutoff value is a given percentage of the value (mean + 6 × standard deviation) of the log2 ratio values of all the probes covering the chromosome. The possible binding sites thus detected are called peaks. To calculate the false-positive rate (FPR) by data permutation, the log2 ratio values among probes were scrambled to generate a randomized data set for each individual chromosome. Multiple repetitions of this process generated 20 randomized data sets for each chromosome. Subsequently, the peak detection algorithm described above was applied to count the average number of peaks in the 20 randomized data sets using the same cutoff. The ratio of that number to the number of peaks from the nonrandomized data set is the FPR. The FPR is associated with the threshold setting, which is indicated by the value of cutoff P. Peak detection and randomization of data sets were repeated for different threshold settings of P. The corresponding FPRs were calculated and assigned to peaks. The FPR value assigned to the individual peaks is the value associated with the cutoff P at which the peak is first detected.A genomic sequence was considered a possible CTCF-binding site if there were at least four probes among the sequence probe and the flanking probes within a window covering 250 bp on both sides of the probe had log2 ratio values were reassigned to probes in a random fashion, allowing selection of stringency and specificity levels. To define sites of CTCF interaction with high confidence, peaks were required to be present in all three biological replicates and to be generated at a FPR < 0.05.Peak discovery was performed using chromatin immunoprecipitate:input ratios combined from adjacent oligonucleotides within 250-bp regions. The FPR of detection was estimated by permutation analyses in which the experimentally determined log7 cells was immunoprecipitated with anti-CTCF antibody , anti-H3K9me3 antibody , anti-trimethyl K4-histone H3 antibody , anti-trimethyl K27-histone H3 antibody  or anti-RNA polymerase II antibody . Immunoprecipitates were washed, the DNA protein cross-links were reversed and the recovered DNA was tested by performing conventional quantitative PCR as described previously [Chromatin was prepared for immunoprecipitation as described previously  by crosseviously . RNA poleviously . SequencSynthesis of cDNA was carried out according to the manufacturer's instructions  using 1 μg of total RNA. For detection of pre-mRNA, RNA preparations were pretreated with TURBO DNase I (Ambion/Applied Biosystems) as described in the manufacturer's protocol. RT was carried out at 37°C for one hour.Cell lines were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine and the antibiotics penicillin (50 U/mL) and streptomycin.IGF2BP1 CTCF region . The amplified products were purified by gel electrophoresis and sequenced. Each case was scored as methylated or unmethylated, and the percentage of methylation was calculated using BiQ Analyzer software [gDNA was treated with sodium bisulfite using the EZ DNA Methylation Kit  according to the manufacturer's instructions. PCR amplification of bisulfite-treated DNA was performed using ZymoTaq DNA Polymerase  and conversion-specific primers targeted to the software .TaqMan allelic discrimination assays were performed according to the manufacturer's instructions with the following adjustments: cDNA from B lymphoblasts was preamplified for 14 cycles. PCR products were gel-purified and subsequently used as templates in the genotyping of samples. The specific primer sequences used are avaliable upon request.9 Jurkat cells grown in growth media  according to methods described previously [IGF2BP1 intronic region using a biotinylated/nonbiotinylated primer combination. The specific primer sequences are available upon request. For each binding reaction, 1 pM biotinylated DNA template was coupled to 50-μg streptavidin-linked magnetic beads . Templates immobilized to magnetic beads were washed three times in B&W buffer , 1 M NaCl) and resuspended in Jurkat nuclear extract. After a two-hour incubation at 4°C, immobilized templates were washed three times in Dignam buffer D (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.9, 20% glycerol, 0.1 M KCl, 1 mM EDTA, 0.1 mM ethylene glycol tetraacetic acid, 1% Nonidet P-40, 1 mM dithiothreitol) containing protease inhibitor . To recover template-bound proteins, beads were incubated in elution buffer  including protease inhibitors. After a 5-minute incubation, the eluate was removed and transferred into a fresh tube. The presence of CTCF in the eluate was determined using standard Western blot analysis protocols.Crude nuclear extract was prepared from 1 × 10eviously . BiotinyFCS: fetal calf serum; PCR: polymerase chain reaction; RT: reverse transcriptase; SNP: single-nucleotide polymorphism.The authors declare that they have no competing interests.AK conceived of and designed the study. BJT, EDR and AK performed the experiments. PÓB, AAG, JMG and NK provided bioinformatics support and carried out the statistical analyses. PW and KB contributed the samples. BJT, PW, AAG and AK drafted the paper. All authors read and approved the final manuscript.Table S1. Genomic coordinates of 293 genomic sites that are marked by both CTCF and H2K9me3. Table S2. List of genes tested for monoallelic expression in lymphoblastoid cell lines.Click here for filechip experimentsFigure S1. Detection and colocalization of CTCF and H3K9me3 at the human IGF2-H19 ICR locus by ChIP-. Top: Enrichment of CTCF binding sites. Middle: Results of large-scale array-based chromatin immunoprecipitation (ChIP-chip) survey of histone H3 trimethylated at lysine 9 (H3K9me3) binding. Bottom: H19 exons demonstrating positions of CTCF binding and histone modifications relative to exons. Figure S2. Analysis of the clonal status of lymphoblastoid cell lines used in this study. Following the protocol described in [Figure S3. Sequencing results give results identical to those derived from the TaqMan allelic discrimination assay. (A) Standard sequencing results of two individuals at SNP site rs9904288. (B) TaqMan allelic discrimination assay confirms the heterozygosity of GM7057 and the homozygosity of GM6990. Figure S4. Quantitative assessment of TaqMan genotyping using specific probe set at SNP rs11655950. The 3'-UTR of the IGF2BP1 gene was amplified using primers given in Supplemental Table 2. This segment contains an A/G SNP. The PCRs included a FAM-labeled probe for the A allele and a VIC-labeled probe for the B allele. After PCR amplification, an end point fluorescence reading was taken on the ABI PRISM 7700 with SDS version 1.4 software (Applied Biosystems). The determination of the quantitative assignment of known genotypes is plotted. Concentration dilutions were created using known homozygous cell lines. Preparations of gDNA samples shown represent the following allele B/allele A ratios: 100:0, 80:20, 60:40, 50:50, 40:20, 20:80 and 0:100. Heterozygosity was based on the fluorescence intensity of FAM, VIC or both dyes together. Error bars indicate 5% of triplicate sample value. Allele A curve yields y = 0.0102x + 0.0415 with R2 = 0.98934. Allele B curve yields y = -0.0085x + 0.9796 with R2 = 0.98196. Figure S5. Phylogenetic tree of motifs determined from motif analysis of the 8,462 loci derived from the ChIP-chip analysis using STAMP. All members of the highlighted group have matches identical to the canonical CTCF motif model as part of the JASPAR transcription factor binding site database. The resulting familial binding profile for all 68 such models is displayed. Figure S6. Fine mapping of CTCF motifs in sequences enriched in ChIP-chip experiments. Motif reads were mapped onto the genomic loci defined by ChIP-chip for CTCF binding. The extent of the ChIP-enriched sequences is indicated by red bar. Several read clusters are apparent and vary in depth and spatial extent (green areas). Figure S7. Frequency distribution of cluster depth for all motif clusters. A power law is apparent for clusters of depth ≤ 10 with evident deviation in the population and a maximum of about 40. The vertical green line demarcates the low and high confidence clusters. Figure S8. Discrimination between high- and low-confidence sites. The region shown in Supplemental Figure S6 is annotated by overlaying enriched sequences with high- and low-confidence tracks. in vitro binding experimentsFigure S9. Sequences of immobilized templates used in . CTCF core motifs Y and Z are underlined. Site-specific mutations in either the Y or Z motif are highlighted in yellow. In Ymut chFII and Ymut mmR3, site-specific mutations (highlighted in green) were introduced to generate CTCF motifs identical to the chicken HS4 FII site and the mouse imprinting control region R3. The IGF2 wild-type huB1 sequence is derived from the human IGF2 imprinting control region containing the methylation-sensitive CTCF binding site B1.ribed in , PCR ampClick here for file


RET) is the major HSCR gene and its expression is crucial for ENS development. We have previously reported that (i) HOXB5 transcription factor mediates RET expression, and (ii) mouse with defective HOXB5 activity develop HSCR phenotype. In this study, we (i) elucidate the underlying mechanisms that HOXB5 mediate RET expression, and (ii) examine the interactions between HOXB5 and other transcription factors implicated in RET expression. We show that human HOXB5 binds to the promoter region 5′ upstream of the binding site of NKX2-1 and regulates RET expression. HOXB5 and NKX2-1 form a protein complex and mediate RET expression in a synergistic manner. HSCR associated SNPs at the NKX2-1 binding site , which reduce NKX2-1 binding, abolish the synergistic trans-activation of RET by HOXB5 and NKX2-1. In contrast to the synergistic activation of RET with NKX2-1, HOXB5 cooperates in an additive manner with SOX10, PAX3 and PHOX2B in trans-activation of RET promoter. Taken together, our data suggests that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis- or trans-regulation of RET by HOXB5 could lead to reduction of RET expression and contribute to the manifestation of the HSCR phenotype.The enteric nervous system (ENS) regulates peristaltic movement of the gut, and abnormal ENS causes Hirschsprung's disease (HSCR) in newborns. HSCR is a congenital complex genetic disorder characterised by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene ( In mammalian embryos, neural crest cells (NCC) migrate from the neural tube, enter the foregut and colonize the gut, where they differentiate into neurons and glia of the enteric nervous system The receptor tyrosine kinase gene RET encodes a receptor for glial cell-line derived neurotrophic factor (GDNF) on NCC and is crucial for ENS development HOX) gene HOXB5 is expressed in both migrating NCC and the gut mesenchyme, and the expression pattern of HOXB5 is associated with the migration and differentiation of NCC, suggesting a unique role of HOXB5 in ENS and gut musculature development Hoxb5 function in ENS development and to circumvent the problem of functional redundancy, we generated transgenic mice that can be induced by Cre to express a chimeric protein (enb5) in NCC. In this, the DNA-binding homeodomain of Hoxb5 is linked to the repressor domain of the Drosophila engrailed protein. This chimeric enb5 repressor competes with wild-type Hoxb5 for binding to target genes and inhibits transcription, thereby blocking the developmental pathways that normally require Hoxb5. We showed that the Hoxb5 trans-activated Ret expression, and that perturbation of Hoxb5 function caused down-regulation of Ret in NCC resulting in defective NCC migration in the developing gut and HSCR phenotype in enb5/Cre transgenic mice Ret is a downstream target of Hoxb5 whose perturbation causes reduced Ret expression, impairs NCC migration and leads to ENS phenotypes. In addition, our case-control study suggests HOXB locus may affect the penetrance of the HSCR RET expression to unravel the roles of HOXB5 in the etiology of HSCR.In human and mouse embryonic guts, Homeobox . Therefore, SK-N-SH cell was chosen to examine the regulatory role of HOXB5 on RET gene. HOXB5 displayed a 7-fold induction of transcription from human RET promoter, as compared to empty vector control pRC/CMV , and this binding was abolished when 100-fold molar excess of cold probe was added (Overlapping PCR fragments spanning the promoter region (−1205 to −177) were tested for HOXB5 trans-activation to demarcate the HOXB5-responsive element. It was found that HOXB5 induced luciferase activity from D2 fragment (spanning −932 to −666) with heterologous SV40 promoter ; D2-Luc.as added . ChIP anas added . Furtheras added . HOXB5 was added .RET promoter and is important for the expression of RETRET promoter, suggesting that these transcription factors may cooperate in the regulation of RET transcription. HOXB5 was co-transfected with NKX2-1 into SK-N-SH cells to examine the cooperation between these two transcription factors in the trans-activation of RET promoter. The inductions were calculated as fold activations relative to the empty vector pRc/CMV. HOXB5 showed a 7.3-fold induction, and NKX2-1 displayed a 4.5-fold induction from the RET promoter. In contrast, 14.5-fold induction was observed when HOXB5 and NKX2-1 were co-transfected, which was significantly higher (p<0.05) than the sum of trans-activation of HOXB5 and NKX2-1 alone     resulted; n = 3) . Synergipromoter .RET promoter, and HOXB5 synergized NKX2-1 in the RET transcription, prompted us to investigate if HOXB5 physically associates with NKX2-1. Using antiserum against HOXB5 for pull-down and anti-NKX2-1 for the detection of NKX2-1 protein in the protein complex, NKX2-1 was co-precipitated with HOXB5 in the Co-IP experiment  of the human RET gene, and deletion of the binding sequence abolishes HOXB5 trans-activation. HOXB5 and NKX2-1 form protein complex binding to the RET promoter, and work in a synergistic manner in mediating RET expression. Down-regulation of NKX2-1 by siRNA suppresses HOXB5 trans-activation of RET promoter, indicating that association with NKX2-1 is essential for HOXB5 in the regulation of RET expression. NKX2-1 binds at the region overlapping with the transcription start site, and mediates RET transcription RETRET transcription, indicating that binding of NKX2-1 to the promoter is required for HOXB5 and NKX2-1 to work synergistically in mediating RET expression. NKX2-1 is a developmentally regulated homeodomain transcription factor RET promoter without affecting its binding onto the RET promoter, suggesting that the Gly→Ser mutation may interfere with the protein-protein interactions of NKX2-1 with other transcription factors HOXB5 binds to the HOX consensus sequence at 875bp 5′ upstream of transcription start of RET promoter containing the SOX10/PAX3-responsive element. In line with a lack of synergistic interactions between HOXB5 with SOX10 and PAX3, HOXB5 exhibits comparable trans-activation from the full-length promoter and truncated promoter (Del.D construct) in which the SOX10/PAX3-responsive element is deleted. These data indicate that HOXB5 mediates transcription of RET independent of SOX10 or PAX3.Our data show only an additive effect between HOXB5 and SOX10 on the trans-activation from the full-length SOX10, PAX3, HOXB5, NKX2-1 and RET, co-expression of these transcription factors and RET is not always found in all SOX10-, PAX3- or HOXB5-expressing tissues RET and NKX2-1, but not SOX10, PAX3 and HOXB5RET transcription RET is regulated by a number of transcription factors including HOXB5, NKX2-1, SOX10, PAX3 and PHOX2B, and that none of these transcription factors is necessary or sufficient for RET transcription. Instead, these transcription factors work either in a synergistic and/or additive manner to mediate RET expression.Although NCC express RET is repressed in most tissues and cells, and its expression is associated with a transition from a repressive chromatin to an open, permissive chromatin RET promoter bringing transcription factors in close proximity of minimal promoter facilitating the recruitment of basal transcription factors mediating RET transcription.RET regulatory region and affects RET expression; ii) HOXB5 synergizes with NKX2-1 transcription factor, and iii) HOXB5 regulatory activity is compromised by sequence variants within RET regulatory regions. Taken altogether, our data suggests that HOXB5,in coordination with NKX2-1, plays an essential role in RET expression. Future studies shall focus in delineating the interplay of molecular components implicated in the regulation of RET expression, which will provide important mechanistic insights into the pathogenesis of HSCR.In conclusion, we showed that i) HOXB5 binds to crucial 2. Trypsin-EDTA solution  was used for trypsinization of cells. Cells were stored in 5% (v/v) DMSO in liquid nitrogen.Human neuroblastoma cell line SK-N-SH (#HTB-11)  was cultured in Eagle's minimal essential medium (EMEM)  supplemented with 10% fetal bovine serum (FBS)  without antibiotics at 37°C and 5% CORET full-length promoter , the 372 bp minimal promoter and the mutated RET full-length promoter containing -5G>A and -1A>C or -1A>C alleles, were generated by PCR and cloned upstream of the luciferase reporter in the pXP1 vector  RET gene. A 1.4 kb 5′ deletion construct (Del.D), was generated by cloning of the PCR fragment (-1205 to 195) of RET gene upstream of the luciferase reporter in the pXP1 vector. Furthermore, D2A-Luc construct was generated by cloning of the PCR fragment (-932 to -666) of RET gene upstream of the luciferase reporter in the pGL3 vector .The 3.7 kb HOXB5 and/or NKX2-1 expression vectors with Lipofectamine 2000 . Cell lysate was analyzed 24 hours by Dual-Luciferase Reporter Assay System  on MicroLumatPlus LB 96V instrument. At least two independent triplicate or quintupled experiments were performed, and the luciferase activity was presented as relative luciferase unit (RLU) normalized with the Renilla luciferase internal control.SK-N-SH cells were seeded on 24-well 18 hours prior to transfection. Cells were co-transfected with pRL internal control, reporter constructs and appropriate amount NKX2-1 siRNA  and negative control  were purchased from Invitrogen .Transfection of small interfering RNA (siRNA) was performed using Lipofectamine 2000 reagent  according to the siRNA-plasmid co-transfection protocol from Invitrogen. Human Real-time PCR was performed on 7900HT Fast Real-Time PCR System  using Fast SYBR Green Master Mix  according to the manufacturer's protocol.NKX2-1/HOXB5 co-transfected SK-N-SH cells was prepared by NE-PER Nuclear and Cytoplasmic Extraction Reagent , and pre-cleared with 50%-slurry of Protein G-Agarose. After pre-clearing, the slurry was incubated with mouse anti-HOXB5 antibody  or non-immunized mouse IgG control  at 4°C for 3 hours. The immune-precipitated protein complexes were resolved in a 8% SDS-Polyacrylamide gel before electro-transferred onto PVDF membrane. Mouse anti-NKX2-1 primary antibody  and goat anti-mouse HRP-conjugated secondary antibody  were used in western blot detection. Signals were visualized by chemiluminescence using ECL Plus Western Blotting Detection Kit .Nuclear protein from HOXB5 transfected SH-N-SH cells and HOXB5-GST fusion protein  were used in this study. End-to-end overlapping PCR fragments . In brief, 25 fmole of Biotin-labeled DNA fragment was incubated with nuclear extract (7 µg) or HOXB5-GST protein (0.25 µg) with or without the addition of unlabeled fragment as specific competitor (300-fold in excess) in binding buffer  at room temperature for 25 minutes. Protein-DNA complex was resolved by electrophoresis on a 4.5% non-denaturing polyacrylamide gel, before being electro-transferred onto PVDF membrane, and signals were detected by chemiluminescence.Nuclear extract from 6HOXB5/NKX2-1 transfected SK-N-SH cells were cross-linked in 1% formaldehyde and chromatin DNA was sheared by sonication  to generate fragments of around 500 bp in length. Dynabeads® were pre-coupled with anti-HOXB5 , anti-NKX2-1 , mouse or rabbit IgG. Sheared chromatin DNA was incubated with corresponding antibody-coupled beads at 4°C with rotation for 3 hours. After pull-down and reversion of cross-linking, chromatin DNA was purified. Binding of D2 to HOXB5 and NKX2-1 was quantified by real-time quantitative PCR analysis.Chromatin immunoprecipitation (ChIP) was performed using MAGnify™ ChIP System  according to the manufacturer's instruction. Briefly, 3×10Figure S1A, Schematic diagram of the human RET promoter. “+1” denoted transcription start of RET gene, and the first ATG of RET gene was indicated. B, Overlapping DNA fragments (D1 to D4) were generated by PCR and labeled with biotin for EMSA. Only D2 was able to bind to HOXB5 as shown by the retarded migration of the probe (arrow). C, Trans-activation of HOXB5 from D1 to D4 fragments were assayed by luciferase activity. Luciferase activity was normalized with Renilla luciferase to obtain relative luciferase unit. Fold increase (mean±SD) was determined relative to luciferase unit of empty vector (SV40-LUC) which was arbitrarily regarded as 1.(TIF)Click here for additional data file.Figure S2No synergistic interaction between HOXB5 and PHOX2B, SOX10 or PAX3 in the trans-activation of RET promoter. Wild-type full-length RET promoter was transfected with expression constructs of HOXB5, SOX10, PAX3 and PHOX2B either alone or in combination into SK-N-SH cells. Relative luciferase units for each combinations were determined, and fold increase (mean±SD) was calculated as compared to luciferase unit of empty vector (pXP1 Basic), which was arbitrarily regarded as 1.(TIF)Click here for additional data file.Figure S3SK-N-SH cells express RET and NKX2-1, but not SOX10, PAX3 and HOXB5. Total RNA was isolated from SK-N-SH cells for RT-PCR analysis to assay for the expression of RET, NKX2-1, SOX10, PAX3 and HOXB5. RT-PCR for β-actin was included as a positive control to test the integrity of the RNA and the RT-PCR reaction. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.(TIF)Click here for additional data file.


ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations.The LIM homeodomain gene Islet-1 ( Congenital heart malformations occur in approximately 3 per 1000 births, more than half of which are potentially lethal malformations of the outflow tract (OFT) Two spatially distinct groups of myocardial progenitors, located in the first and the second heart fields, contribute to the definitive heart pump Isl1 is necessary for multipotent cardiovascular progenitors within the second heart field to proliferate, survive, and migrate into the forming heart. Isl1 is highly conserved over chordate evolution in this role Isl1-null mice die at mid-gestation from gross cardiac malformations, notably the lack of the OFT and right ventricle myocardium Coordination between these separate but adjacent mesodermal primordia is orchestrated by signaling events that converge on a common palette of transcription factors necessary for the site-appropriate differentiation of the multiple cell types present in a mature heart. The LIM homeodomain transcription factor Islet-1 (Isl1) is one of these. In the developing heart, these combinatorial partners include members of the tinman (Nkx), GATA-binding and T-box (Tbx) families Mef2c and Nkx2-5 during cardiac development via elements in their promoters that also contain nearby, active GATA-binding sites GATA4 promoter to positively control its transcription during fetal cardiomyocyte differentiation Isl1 and Nkx2.5 expression in murine mesoderm not normally fated to integrate the heart, leading to cardiac transdifferentiation For example, murine Isl1 directly controls the expression of the early mesodermal transcription factors ISL1 coding mutations have been identified in humans, probably because of an embryonic lethal phenotype for complete inactivation and no gross effect of haploinsufficiency, as seen for murine Isl1ISL1 mutations have not directly been reported to cause conotruncal cardiopathies either, although a block of single nucleotide polymorphisms around and within ISL1 have indeed been found to be in linkage disequilibrium with a risk for complex congenital heart phenotypes involving “developmental structures aberrantly formed as derivatives of the secondary [sic] heart field.” No Isl1 homozygous knockout mice, the residual hearts no longer express certain bone morphogenetic protein (Bmp) or Wnt family members, Fgf8 or Fgf10, and are missing the OFT entirely Fgf10 expression in the OFT through a 5′ enhancer element in silico within a 0.4 kb Fgf10 promoter element that is highly conserved among amniotes and capable of directing expression to the otic anlage In Fgf10-null mice demonstrates the irreplaceable role of Fgf10 in epithelial-mesenchymal interactions needed for the development of many organ systems, including but not restricted to endodermal organs and glands of the head and neck FGF10 lead to isolated or syndromic aplasia of the lacrimal and salivary glands and ducts ISL1, no biallelic inactivation of FGF10 has been found to date in human disease Fgf10+/− phenotypes have only been described progressively over the years since the first murine knockout models.The phenotype of ISL1 has recently been demonstrated to be compatible with the existence of a subset of embryonic progenitors that would contribute specifically to the inflow and outflow tracts, as in animal models ISL1 is co-expressed with other transcription factors in the cardiac primordium, but that in vivo it directly binds and positively regulates the transcription of FGF10. ISL1 exerts this effect through an enhancer within the FGF10 first intron that is evolutionarily conserved among mammals, becomes additionally responsive to ISL1 in vitro in the presence of GATA and TBX factors, and is capable of responding to endogenous cardiac OFT transcription factors in a transgenic mouse reporter.The spatiotemporal expression of human FGF10 and ISL1 in a region probably corresponding to a second heart field in human embryos at appropriate and similar stages of morphogenesis FGF10 locus to search for putative highly conserved ISL1 consensus binding sites with the sequence YTAATGR, using rVista 2.0 (http://rvista.dcode.org) http://ecrbrowser.dcode.org) FGF10 promoter FGF10-Pr2; another, within the first intron of FGF10, was termed FGF10-Int1. A third promoter region, without an ISL1 consensus binding site, was designated as FGF10-Pr1. A non-canonical (i.e. 5′-TGATTA-3′) potential binding site for GATA-type transcription factors FGF10-Int1 and these sites were nearly identical in nucleotide composition and distance from one another between mice and humans  of microdissected embryonic human hearts, we demonstrated that at Carnegie stages 14–15 (33–36 dpf), ISL1 bound to and enriched a 327 bp fragment . In conte stages  [43].FGF10-Int1 element in developing human hindlimb buds, since FGF10 and ISL1 are co-transcribed at foot plate stages at CS16-17 . In contrast, these genes were no longer transcribed at CS16 (37–40 dpf), despite continued expression of the ubiquitous ACTB  transcription factor FGF10-Int1 fragment located 3′ to the luciferase sequence, mimicking the endogenous location of this regulatory element relative to the initiation site for FGF10 transcription. Co-transfection into mesenchymal 10T1/2 cells of a GATA4 or ISL1 expression construct, together with the FGF10-Int1-luciferase reporter, indeed resulted in robust activation of luciferase activity  and with prior approval of the protocol (to M.V.) from the Necker ethical review committee. All mice used in this study were housed under specific pathogen-free conditions at the mouse genetics engineering center (C.I.G.M.) of the Pasteur Institute, Paris, under authorization number A75-15-09 from the Paris Departmental Directorate for the Protection of Populations and handled in accordance with French and European directives.ChIP was carried out as previously described, starting from nuclear isolation ISL1 and GATA4 in situ hybridizations were performed using transverse sections of normal human embryos from CS12 to 15. Tissue fixation, sectioning, and in situ hybridization were carried out as previously described TBX20 cDNA and a fragment of ISL1 cDNA with the N-terminal 142 amino acids removed GATA4 cDNA was purchased from GenScript (GN026113). HeLa cells were transfected with these constructs, and nuclear protein extracts were made using standard protocols. The LightShift Chemiluminescent EMSA Kit (Pierce) was used as specified. Primers are listed in Supplementary Human TBX20 and ISL1 expression vectors were generated. Full-length FGF10 reporter construct (LUC-FGF10-Int1), 1047 bp of the FGF10 first intron (NCBI36/hg18 chromosome 5:44421556–44422602) were subcloned into the BamHI site 3′ to luc+ in pGL3 (Promega). Mouse 10T1/2 cells For the FGF10-Int1 fragment as in the transactivation assays was subcloned into the BamHI site of the pSKT-TK-nLacZ plasmid The same 1047 bp Figure S1Composite image of embryonic hearts at stages ranging from the beginning of the fourth to the ninth week of human gestation . Rostral to top. Congenital heart and great vessel malformations arise during this time window when molecular signaling between cardiac progenitors and their environment is impaired.(TIF)Click here for additional data file.Table S1Primer sequences for PCR and EMSA.(DOC)Click here for additional data file.


Recently, Sirtuins have been shown to play important roles, both direct and indirect, in transcriptional regulation. This transcriptional control, through incorporation of Sirtuins into transcription complexes and deacetylation of histones locally at gene promoters, or direct interaction with specific transcription factors, is central to the participation of Sirtuins in multiple diverse processes, including aging, apoptosis, hormone responses, stress tolerance, differentiation, metabolism and development. Here we review the contribution of the Sirtuin family, at multiple molecular levels, to transcriptional regulation.Sirtuins are NAD The modification of histones and transcription factors by acetylation and deacetylation play a central role in the control of transcription. Acetylation of histones is postulated to increase DNA accessibility through the neutralization of the positive charges of lysine residues. This modification correlates generally with transcriptional activation. Histone acetylation is catalyzed by histone acetyl transferases (HATs), whereas the reverse reaction is performed by histone deacetylases (HDACs). HATs and HDACs participate in the genome-wide turnover of acetyl groups on histones and, in addition, on many transcription factors. Through their physical interactions with DNA sequence-specific transcription factors, they are also targeted to specific promoters, where they locally modify histones or transcription factors or transcriptional co-activators/co-repressors, and thus can regulate gene transcription at multiple molecular levels.+-dependent histone deacetylases, wherein their deacetylase activity is controlled by the cellular [NAD+]/[NADH] ratio. The mammalian homologues of the Sirtuin family proteins consist of seven members, SIRT1—SIRT7 [HATs and HDACs are classified into numerous families that are often conserved from yeast to humans. Histone deacetylases (HDACs) are divided into three classes. Class I members  are transcriptional co-repressors homologous to yeast RPD3 . Class IT1—SIRT7 . SIRT1  –10, p53 Numerous mammalian transcription factors and their co-regulatory proteins have been identified as targets of the Sirtuins gene products . The deaNucleocytoplasmic shuttling of transcription factors is an important mechanism in transcription regulation. The acetylation or deacetylation of a transcription factor can directly regulate the accessibility of nuclear localization signals (NLSs) and nuclear export signals (NESs) to nuclear import and export proteins. SIRT1 is implicated in regulating the sub-cellular localization of several transcription factors, including members of the FOXO transcription factor family. However, the precise molecular mechanism regulating the shuttling of FOXO by Sirtuins is not yet clear.Caenorhabditis elegans DAF-16, are emerging as a central family of proteins that modulate the expression of genes involved in apoptosis, cell cycle progression, DNA damage repair, responses to oxidative stress, cell differentiation, glucose metabolism and other cellular functions [Forkhead box O (FOXO) transcription factors, including FOXO1, FOXO3a, FOXO4 and FOXO6, the mammalian orthologs of unctions , 19. FOXunctions , 21. In unctions . FOXO1 iunctions . -induced apoptosis in renal glomerular mesangial cells . SIRT1 rvia reversible changes in their sub-cellular localization, the stability of FOXO proteins represents an additional and irreversible level of regulation for this family of transcription factors. Degradation of FOXO transcription factors is mediated by the ubiquitin-proteasome pathway. In addition to phosphorylation modifications, acetylation also plays a role in the control of ubiquitin-mediated FOXO protein degradation. Acetylation of FOXO1 prevents its ubiquitin-dependent degradation [While FOXO transcription factors are regulated by Sirtuins predominantly radation . Our unpThe androgen receptor (AR) is a ligand-regulated modular nuclear receptor governing prostate cancer cellular proliferation, differentiation, and apoptosis in response to androgens. SIRT1 binds to, and deacetylates, the androgen receptor at a conserved lysine motif, down-regulating its levels in the cell, and repressing androgen-induced AR transcriptional activity .SIRT1 gene is reciprocally regulated by p53, as discussed below.The protein level of the p53 tumor suppressor is regulated by SIRT1. p53 is stabilized by acetylation of Lys-382, and this residue is a known target of SIRT1-mediated deacetylation . As p53 via a NAD+-dependent activity. This effect is reversed by the proteasome inhibitor epoxomicin, suggesting a Sir2α-mediated ubiquitin/proteasome-dependent mechanism for degradation of H2A.z. Sir2α directly deacetylates H2A.z, and Lys-115 and a conserved, ubiquitinatable Lys-121 are critical for Sir2α-mediated degradation. Sir2α is therefore involved in the deacetylation of H2A.z, leading to the ubiquitination of Lys-115 and Lys-121 and the consequent degradation of H2A.z via a ubiquitin/proteasome-dependent pathway [One mechanism of transcriptional regulation involves the replacement of core histones with more specialized variants, and Sirtuins has a role in regulating the stability of variant histones . H2A.z i pathway .The binding of transcription factors to gene promoters can be regulated by Sirtuins-mediated acetylation. As one example, the acetylation of lysine residues in the C-terminal regulatory domain of p53 enhances the sequence-specific binding of p53. SIRT1 deacetylates p53 at Lys-382 in the C-terminal domain and represses its transcriptional activity , 37. As SIRT1 gene is a transcriptional target of E2F1, this may provide some auto-regulation of SIRT1 levels in the cell, with SIRT1 repressing its own transcription through E2F (while stimulating its own transcription through inhibition of p53 activity).Binding of the central transcription factor and tumor suppressor E2F1 to its cognate gene promoter elements and their subsequent transcriptional activation, are stimulated by acetylation through PCAF. SIRT1 deacetylates E2F and suppresses its transcriptional activity, most likely through suppression of its DNA-binding activity . As the SIRT1 physically interacts with the RelA/p65 subunit of NFκB and inhibits NFκB-induced transcription, by deacetylating RelA/p65 at Lys-310. Increases in SIRT1 activity delays TNFα-induced recruitment of NFκB complexes containing RelA/p65 and p50 proteins to NFκB-regulated gene promoters .in vitro and in vivo deacetylase for the Rb protein [SIRT1 is a binding partner for the retinoblastoma tumor suppressor protein (Rb) and its family members, p107 and p130, and SIRT1 is an  protein . Active Sirtuins have been shown to directly deacetylate members of the FOXO transcription factor family and alter their transcriptional activity. Since many FOXO acetylation sites are located within the DNA-binding domain of the molecule, it is possible that acetylation of FOXO interferes with FOXO binding to DNA and thereby inhibits FOXO-dependent transcription. For example, SIRT1 has been shown to preferentially deacetylate FOXO1 at K242, K245, and K262, thereby altering its DNA-binding ability, and activating the transcriptional function of FOXO1 in a deacetylase-activity dependent manner , 42. As rRNA gene promoters and induces rRNA gene transcription in mammals. Sir2a deacetylates TAFI68 and decreases the binding of TAFI68 to rDNA promoters and represses rDNA gene transcription [Sirtuins can also regulate transcription by affecting the activity of the basal transcription machinery. Acetylation of TAFI68, a subunit of the RNA polymerase I complex, enhances the binding of TAFI68 to cription . Thus, TIn addition to regulating transcription factors at the levels of the sub-cellular localization, protein stability and DNA-binding ability, SIRT1 can also modulate intrinsic transcription factor activity through alteration of the affinity of the factors for their co-activators, co-repressors or the general transcription machinery. Sirtuins could modulate such interactions directly, by deacetylating residues on the transcription factors or the co-regulatory molecules which form parts of interacting surfaces, thereby blocking or facilitating interactions, or indirectly, with deacetylation inducing a conformational change in another part of the molecule which unmasks or masks binding surfaces.SIRT1-mediated deacetylation enhances FOXO1 and CCAAT/enhancer-binding protein alpha (C/EBPα) interactions, thereby promoting the transcription of adiponectin, an adipose tissue-derived hormone which plays an important role in maintaining energy homeostasis . C/EBPα via enhancing PPARγ docking with two of its co-repressors, NcoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor). Hence, SIRT1 acts as a co-repressor of PPARγ-mediated transcription, attenuating adipogenesis [Transcription factor PPARγ is a key regulator of adipogenesis and fat storage, controlling the expression of many adipocyte-specific genes. SIRT1 actively represses PPARγ activity ogenesis .The androgen receptor (AR) is a ligand-regulated modular nuclear receptor governing prostate cancer cellular proliferation, differentiation, and apoptosis in response to androgens. We have demonstrated ligand-dependent recruitment of SIRT1 as a co-repressor into a transcription complex , the firIn addition to direct regulation of transcription factors themselves, increasing evidence suggests that co-regulatory factors, including p300, PGC1 and CTIP1/CTIP2, are also directly targeted by Sirtuins.The p300 protein serves as a rate-limiting transcriptional co-integrator of diverse transcription factors, either activating or repressing transcription through its modular subdomains. SIRT1 physically interacts with, and represses transactivation by, p300. Two residues within the CRD1 domain of p300 (Lys-1020 and Lys-1024) are required for repression by SIRT1 and serve as substrates for SIRT1 deacetylation. Because p300 is a gating transcriptional cofactor, deacetylation and repression of p300 by SIRT1 may serve as an important integration point during metabolic processes and cellular differentiation .via deacetylation of PGC-1α, activates gluconeogenesis and represses glycolysis in the liver [The transcriptional co-activator PGC-1α plays an important role in hepatic and systemic glucose and lipid metabolism and respiration, as well as caloric restriction. In mammals, maintenance of energy and nutrient homeostasis during food deprivation is accomplished through an increase in mitochondrial fatty acid oxidation in peripheral tissues. PGC-1α is an important component driving this cellular oxidative process, and SIRT1 interacts with PGC-1α and deacetylates it at multiple lysine residues, to activate PGC-1α-mediated transcription. Fasting induces PGC-1α deacetylation in skeletal muscle and SIRT1 deacetylation of PGC-1α is required for activation of mitochondrial fatty acid oxidation genes. SIRT1 therefore serves as a functional regulator of PGC-1α regulating a metabolic gene transcription program of mitochondrial fatty acid oxidation. SIRT1, he liver , 48; prohe liver ; promotehe liver  and reguhe liver . Many ofhe liver  -interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-independent binding to a GC-rich response element. SIRT1 interacts directly with CTIP1/CTIP2 and is recruited to specific promoter elements in a CTIP1/CTIP2-dependent manner. SIRT1 enhances the deacetylation of template-associated histones H3/H4 in CTIP-transfected cells (see below), and stimulates CTIP-dependent transcriptional repression , 54.Recent reports now clearly demonstrate that SIRT1 can form complexes with certain transcription factors bound to promoter elements, and regulate their transcriptional activity. The biochemical mechanisms underlying of transcription regulation by SIRT1 in these complexes are not clearly defined in every case, and may include local histone deacetylation at the promoter, direct deacetylation of other members of the complex, or a change the ratio of co-activators/co-repressors within the transcription complex (and more than one such SIRT1-mediated mechanism may be simultaneously operative within the same complex).in vitro and in vivo, and enhances the transcriptional repression mediated by these bHLH repressors [The basic helix—loop—helix (bHLH) superfamily of transcription factors comprises a large number of proteins which play important roles as activators or repressors in diverse aspects of development. SIRT1 physically associates with the human bHLH repressor proteins hHES1 and hHEY2, both pressors .SIRT1 gene, one result of SIRT1 interactions with HIC1-containing transcriptional repressor complexes is down-regulation of SIRT1 gene expression [Tumor suppressor HIC1 (hypermethylated in cancer 1) encodes a zinc-finger transcription factor that is essential for mammalian development, which is epigenetically silenced in many human tumors and is involved in a complex pathway regulating p53 tumor suppression activity. Two deacetylases, SIRT1 and HDAC4, interact and form a dual-deacetylase complex with the transcriptional repressor HIC1, enhancing the transcriptional suppressor effect of HIC1 . The actpression .SIRT1 acts predominantly as a co-repressor of gene expression through protein deacetylation. However, there is evidence that SIRT1 can also serve as a co-activator, positively-regulating gene expression. As one example, the human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional co-activator p300. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional co-activator during Tat transactivation . Tat andIn addition to its pleiotropic effects on specific non-histone target proteins, SIRT1 also regulates transcription by deacetylating histones and interacting with chromatin-remodeling complexes.in vitro and in vivo [A diverse array of post-translational modifications within the tail domains of histones have been shown to regulate chromatin structure and hence regulate gene transcription. SIRT1 preferentially deacetylates H3-K9, H3-K14 and H4-K16  in vivo .Certain transcription factors, including CTIP1, CTIP2, and AR, can recruit SIRT1 to target gene promoters, with resulting local deacetylation of either histones H3 or H4, thereby repressing gene transcription.via interaction with the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor II (COUP-TF II). SIRT1 interacts directly with CTIP1 and CTIP2 and is recruited to the promoter template in a CTIP1/CTIP2-dependent manner, leading to transcriptional repression. Chromatin immunoprecipitation assays revealed that expression of CTIP1 or CTIP2 in mammalian cells results in deacetylation of histones H3 and/or H4 associated with the promoter region, in a SIRT1-dependent mechanism. CTIP-mediated transcriptional repression, as well as deacetylation of histone H3/H4 in CTIP1-transfected cells, is partially reversed by nicotinamide, an inhibitor of class III histone deacetylases [As discussed above, CTIP1 (BCL11A) and CTIP2 (BCL11B) are transcriptional repressors which bind directly to a GC-rich motif and are also indirectly recruited to promoter elements etylases , 54.In addition to modulating the levels of AR by direct deacetylation of this transcription factor, discussed above, ligand-dependent recruitment of SIRT1 into AR transcriptional repressor complexes on AR-responsive promoters results in the deacetylation of histone H3 locally .SIRT2 also plays a role in histone deacetylation, in particular at histone residue H4-K16. SIRT2 is predominantly localized to the cytoplasmic compartment, where it serves to deacetylate α-tubulin. During mitosis, however, SIRT2 shuttles to the nucleus and deacetylates histone H4-K16 to promote chromatin condensation .in vivo and repress gene transcription [SIRT3 can also deacetylate Histone H4-K16 and repress transcription in a gene-specific manner. SIRT3 exists in two forms, a full-length protein and a processed polypeptide lacking 142 amino acids at the N-terminus. Both the full-length and processed forms of SIRT3 deacetylate H4-K16 cription .Tumor suppressor gene (TSG) silencing is a hallmark of cancer. Epigenetic mechanisms of silencing of tumor suppressor genes in some cases involves dense hypermethylation of 5′ CpG islands and local hypoacetylation of Lys-9 and -14 on histone H3 . Recently, SIRT1 has been shown to localize to the promoters of several aberrantly-silenced TSGs in which 5′ CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Inhibition of SIRT1 in breast and colon cancer cells causes increases in H4-K16 and H3-K9 acetylation at endogenous promoters and re-expression of silenced TSGs, despite full retention of promoter DNA hypermethylation. SIRT1, therefore, comprises a new component of epigenetic TSG silencing that may potentially link some of the epigenetic changes associated with aging with those found in cancers, and provides new directions for potential therapeutic targeting these important genes for re-expression .in vivo at Lys-26 and can be deacetylated by SIRT1. SIRT1 physically interacts with histone H1, and this interaction recruits histone H1 to establish repressive chromatin domains. Upon induction of SIRT1 activity, there is a reduction in H4-K16 acetylation in euchromatic chromosomal regions. This effect is accompanied by an enrichment of histone H1 at specific promoters, the spreading of heterochromatin marks like tri-Me H3-K9 and mono-Me H4-K20 throughout the coding region, and a decrease in the levels of methylation of H3-K79. Thus, a proposed model for SIRT1-mediated heterochromatin formation includes deacetylation of histone tails, recruitment and deacetylation of histone H1, and spreading of H3-K79 hypomethylation with resultant silencing.In addition to deacetylation of H3 and H4, the linker histone H1 may also serve as a target of SIRT1 in genome-wide transcriptional repression . HistoneIn addition to changes in acetylation in the N-terminal domains of histone polypeptides, methylation modification in histone tails also plays an important role in the transition between open and compacted chromatin states. SIRT1 has been shown to interact with a number of methyltransferase/chromatin-remodeling complexes, suggesting a role of SIRT1 at this level of transcriptional regulation.Methylation at K27 of histone H3 is a repressive “mark,” catalyzed by the SET-domain containing Enhancer of Zeste protein-2, Ezh2, in Polycomb Repressive Complexes (PRCs). Ezh2 is a histone-lysine methyltransferase with activity dependent upon its association with other components of the PRCs. Recently, SIRT1 has been found to form a complex with isoform 2 of the PRC component Eed . The assCooperation of deacetylases with other enzymes or other co-repressor proteins to remodel chromatin is suggested by the altered recruitment of methyl-CpG-binding proteins to a methylated CpG island after treatment of tumor cells with HDAC inhibitors. Human cells lacking DNA methyltransferase 1 (Dnmt1), but not those lacking Dnmt3b, demonstrate a loss of DNA methylation and an increase in the acetylation levels of Lys-16 on histone H4 at the rRNA genes. SIRT1, which demonstrates a preference for Lys-16 H4, interacts with Dnmt1, and SIRT1 recruitment to the rRNA genes is abrogated in Dnmt1-knockout cells . The obsH3-K79 hypomethylation is found in transcriptionally-repressed regions in mammal genomes. SIRT1 over-expression results in the reduction of methylated histone H3-K79 at promoters and within coding regions. H3-K79 methylation restricts the spreading of Sir2p in yeast, and thus, methylation of this residue may constitute a “boundary mark,” separating heterochromatic from euchromatic regions. These and other findings suggest that acetylation/deacetylation and methylation of chromatin can be coordinately regulated .Most recently, SIRT1 has been found to directly interact with and deacetylate the histone methyl-transferase SUV39H1 (suppressor of variegation 3–9 homologue 1), increasing its activity. Loss of SIRT1 strongly affects SUV39H1-dependent generation of H3K9me3 and impairs localization of heterochromatin protein 1 .UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which SIRT1 levels are reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. This up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knock-down of UCP2 restores insulin secretion in cells with reduced SIRT1, indicating that the defect in glucose-stimulated insulin secretion is due to induction of UCP2 by SIRT1 depletion [Several reports have shown that SIRT1 can suppress gene transcription by binding directly at gene promoters, but the molecular mechanism underlying this activity are not clear. For example, SIRT1 represses the uncoupling protein (UCP) gene epletion .Over-expression of SIRT7 increases rRNA transcription, whereas its down-regulation decreases rRNA transcription. Interestingly, SIRT7 expression is enriched in tissues with a high proliferative potential, such as liver, spleen, and testis. Conversely, tissues with a low cellular turnover rate, such as skeletal and heart muscle and brain, express low levels of SIRT7. SIRT7 appears to drive ribosome biogenesis in dividing cells, and has been associated with thyroid and breast cancers .Since the initial identification of p53 in 2001 as the first transcription factor substrate of SIRT1, multiple diverse transcription factors and co-activator/co-repressor proteins have been identified as targets of SIRT1, enabling direct control of transcription by the Sirtuin family members . SIRT1 a3Kinase activity alters SIRT1 subcellular localization [3Kinase or by other post-translational modification, with respect to compartmental localization or protein levels, and its ultimate effects on transcriptional activity, are unknown. Whether post-translational modifications of Sirtuins affect the acetylation status of promoter elements in a gene-specific manner also remains to be determined. Certainly, identification of physiologically-relevant signaling pathways which induce the activation or the inactivation of Sirtuins, and their consequences on transcription, will be important to our understanding of this central transcriptional regulator.In this review, we have discussed the molecular regulatory events targeted by Sirtuins in gene transcriptional regulation. However, Sirtuins is subject to multiple post-translational modifications, the molecular roles of which remain largely uncharacterized. Furthermore, as these post-translational modifications are effected by various signaling pathways, this presents yet another level of control of Sirtuins on transcription that remains to be elucidated in detail. As examples, phosphorylation of HuR by Chk2 regulates SIRT1 expression. Depending on its phosphorylation state, HuR can cause SIRT1 mRNA to either be stabilized or degraded , 72. Ovelization  and SIRTAnother perhaps surprising but major question which remains unanswered is whether SIRT1 is a tumor suppressor or, instead, an oncogene. While multiple lines of evidence suggest that Sirtuins protect against cancer development , 17, 74,Poised as they are, at the critical intersection of processes as diverse as aging, cancer susceptibility and metabolic regulation, a clearer mechanistic understanding of the central role that Sirtuins play at multiple levels in transcriptional control is of great importance.