We recover previously known chromatin signaturesassociated with promoters and enhancers. We also observe several chromatinsignatures with strong enrichment of H3K36me3 marking exons. Closer examinationreveals that H3K36me3 is found on well-positioned nucleosomes at exon5′ ends, and that this modification is a global mark of exonexpression that also correlates with alternative splicing. Additionally, weobserve strong enrichment of H2BK5me1 and H4K20me1 at highly expressed exonsnear the 5′ end, in contrast to the opposite distribution ofH3K36me3-marked exons. Finally, we also recover frequently occurring chromatinsignatures displaying enrichment of repressive histone modifications. Thesesignatures mark distinct repeat sequences and are associated with distinct modesof gene repression. Together, these results highlight the rich informationembedded in the human epigenome and underscore its value in studying generegulation.Transcriptional regulation in human cells is a complex process involving amultitude of regulatory elements encoded by the genome. Recent studies haveshown that distinct chromatin signatures mark a variety of functional genomicelements and that subtle variations of these signatures mark elements withdifferent functions. To identify novel chromatin signatures in the human genome,we apply a  Recent studies have observed that histone tails can be modified in a variety ofways. Analyzing a collection of 21 histone modifications, we attempted todetermine what common signatures are associated with different classes ofregulatory elements and whether they mark places of distinct function. Indeed,at promoters, we identified a number of distinct signatures, each associatedwith a different class of expressed and functional genes. We also observedseveral unexpected signatures marking exons that directly correlate with theexpression of exons. Finally, we recovered many places marked by two distinctrepressive modifications, and showed that they mark distinct populations ofrepetitive elements associated with distinct modes of gene repression. Together,these results highlight the rich information embedded in the human epigenome andunderscore its value in studying gene regulation. The genome sequence is a static entity defining the possible transcriptional outputof every cell type in the human body Several classes of regulatory elements are marked by different chromatin signaturesThese observations prompted us to systematically examine the chromatin signaturesthat exist in known and putative regulatory elements in the human genome. Our goalis to explore whether other frequently occurring chromatin signatures exist, andwhether specific functions are associated with these signatures. Focusing on 21histone modifications mapped in CD4+ T cells We hypothesize that loci sharing common regulatory functions may share similarchromatin signatures. To systematically identify chromatin signaturesgenome-wide, we examine different classes of regulatory loci in turn. These locimay contain chromatin signatures, but they may not be aligned or even orientedin the same direction. We therefore apply an unbiased clustering and alignmentmethod called ChromaSig , Table S1). Promoters in the same cluster share a commonchromatin signature, and the chromatin signatures of different clusters aredistinct in apparent or subtle ways. For example, the P4 cluster contains strongenrichment for various H3K4 methylations while P2 lacks these modifications. Onthe other hand, P9 and P12 clusters contain enrichment for the same chromatinmodifications, but the pattern of enrichment is different, with P12 containingenrichment over a noticeably wider region. It is also evident that there is ahigh level of redundancy of histone modifications at promoters. Notably, H2AZ,H3K4me1, H3K4me2, H3K4me3, and H3K9me1 are either all found together or allabsent together at promoters, consistent with recent findings While chromatin signatures at promoters have been studied extensively, we stilldo not have a complete picture of all the distinct, commonly occurring chromatinsignatures spanning all promoters. As such, our understanding of how differentsignatures relate to gene expression is incomplete. To address this, we applyChromaSig to the chromatin modifications near the set of manually annotatedpromoters defined in the Refseq database Previous studies have shown that there are at least three different classes ofchromatin signatures at promoters: actively transcribed promoters marked byH3K4me3 but not H3K27me3, inactive promoters marked with H3K27me3 but notH3K4me3, and bivalent promoters having both these marks Next, we wondered if different signatures correspond to different gene expressionactivities. On the basis of gene expression CpG islands often mark the promoters of house-keeping genes that are ubiquitouslyexpressed In contrast, clusters P2,5,6,7,11 spanning 34% of all promoters aresignificantly depleted of CpG islands. Nearly half of these promoters are markedby H3K27me3 but not H3K4me3 in cluster P2. Consistent with previous studiessuggesting these promoters are inactive Together, these results show that ChromaSig can reliably detect distinctchromatin signatures at promoters with unique functional specificities.While transcriptional regulation occurs at the level of promoters, it is alsoclear that the action of promoter-distal regulatory elements is essential tocontrolling gene expression When active, enhancers are bound by transcription factors and co-activatorsto increase gene expression at promoters Fig.S4, TableS2). Like promoters, there also appears to be much redundancy ofchromatin modifications at enhancers. For example, all chromatin signaturesgenerally share enrichment for H2BK5me1, H3K4me2, H3K9me1, H3K27me1, andH3K36me1. Interestingly, the chromatin marks H2A.Z and H4K20me1 appear to beinversely correlated: E1-5 are enriched in H2A.Z but not H4K20me1, E6 hasenrichment of both marks, and E7–11 are enriched in H4K20me1 butnot H2A.Z.We have previously observed in 1% of the human genome (the ENCODEregions) that different variations of chromatin modifications exist atenhancers Fig.S5, TableS3). The only consistent feature of CTCF binding sites is enrichment ofH2A.Z, consistent with previous observations CTCF is an insulator binding protein in mammals, and when bound preventsenhancers from interacting with promoters, thereby preventing activationFig.S6, TableS4). However, chromatin signatures at 3′ ends are not as wellaligned as those at promoters, suggesting that these chromatin signaturesmay occur at some other genomic feature near 3′ ends, or that the3′ ends are not as well annotated as promoters.Transcription of pre-mRNA stops at the 3′ end of the gene. To findchromatin signatures at this genomic feature, we apply ChromaSig to 16,703Refseq gene 3′ ends distal to Refseq 5′ ends Fig.S7, TableS5). Clusters D1–D2 are only enriched in H3K27me1 andH3K36me3, resembling gene 3′ ends. Several signaturesD3–10 display characteristic enrichment of H3K4me1/2/3, which wehave observed at promoters and enhancers. These may be novel promoters orenhancers missed by the enhancer prediction method. For example, D3,6,9,10are clusters with the strongest enrichment of H3K4me3, and 31.2%of these loci are recovered by multiply-occurring CAGE tags Recently, Boyle et al mapped nearly 100,000 DNase I hypersensitive sitesgenome-wide in CD4+ T cells using DNase-Seq In eukaryotes, control of gene expression is a complex process involving thecoordinated action of a wide assortment of genomic regulatory elements. Of thefive classes of genomic regulatory elements examined here, the ones leaststudied and perhaps most important to controlling gene expression are enhancersand DNase I hypersensitive sites. To examine the potential regulatory roles ofthese genomic loci, we measure the enrichment of these loci near differentclasses of expressed genes as defined by the 14 clusters of promoter chromatinsignatures . At a large scale, we observe that inactive promoters P1–6generally lack enrichment for all the chromatin signatures cataloged here. Incontrast, CTCF-defined domains containing active promoters P8–14 areenriched in numerous chromatin signatures. Strikingly, different classes ofpromoters are enriched in different classes of enhancers. For example, the twomost highly expressed clusters P10 and P12 are uniquely enriched inE6–11. These enhancers are distinguished from other enhancer classesby strong enrichment of H3K9me1 and H4K20me1, indicating that these chromatinmarks may be an indicator of enhancer activity. Of these enhancers, the classthat most distinguishes highly active promoters from all other promoters is E9.This cluster may contain the most active enhancers, and its chromatin signaturemay be a general mark for highly active enhancers. In general, we observe weakerenrichment of the DNase I hypersensitive clusters within CTCF-defined blockscontaining highly expressed promoters, with the exception of D6–8which are likely enriched in novel promoters and enhancers missed by theenhancer prediction method.When a CTCF-bound insulator falls between a promoter and enhancer, the enhanceris blocked from activating the promoter , TableS6). The recovered signatures are distinct from the previously definedH3K4me3-rich promoter and H3K4me1-rich enhancer signatures −18 to<10−300) (TableS7).Having observed chromatin signatures at regulatory elements including promotersand enhancers, we next ask if other chromatin signatures exist that mark locidistal to known regulatory elements. By definition, places in the genome withchromatin signatures contain enrichment of histone modifications. Therefore, weidentify 85,318 loci with strong ChIP enrichment of histone modifications, ofwhich 50,183 are distal to promoters The most prominent chromatin feature of these clusters is H3K36me3, known to markthe 3′ ends of genes To gain an understanding of potential functions associated with the abovefrequently-occurring novel chromatin signatures, we compare the loci bearingeach signature to genomic annotations.Fig.S9), we ask if these sites are biased towards exons or introns. We observethat 57.9% of U1 sites and 63.8% of U2 sites are eitherinside exons or within 1-kb of exon ends, while at random only 26% ofthe genic regions of the genome match these criteria. To see if H3K36me3 marksexons, we examine the enrichment of this chromatin mark at exons . To examine only those exons unambiguously marked by a chromatinsignature, we only consider an exon if it is the only exon within 1-kb of acluster locus. We observe a striking enrichment of H3K36me3 at the 5′ends of exons unambiguously marked by U1, U2, and U4. This enrichment decreasessharply upstream of the 5′ end, but more gradually into the exon body.This observation also holds for exons larger than 1-kb , indicating that the result is not biased by the relatively small exonsizes in the human genome H3K36me3 is known to be enriched within the body of transcriptionally activegenes Fig.S8). Next, profiling H3K36me3 at a catalog of more than 250,000 distinctexons , S10). The presence of this chromatin mark also correlatesstrongly with exonic expression .In addition to exon 5′ ends, it also appears that the 3′ endsof expressed exons have well-positioned nucleosomes . Forspliced in exons, we observe that the enrichment of H3K36me3 increases graduallyfrom upstream to alternatively spliced to downstream exons  . U3, which contains no H3K36me3 enrichment , is a negative control containing no preference ofexon inclusion. Interestingly, exons marked by U4, which are enriched in therepressive H3K9me3 modification, are preferentially excluded from mature mRNAs. Taken together, these results suggest that severaldistinct chromatin signatures are found at exon 5′ ends, that somesignatures mark exons for preferential inclusion, and others for preferentialexclusion. These different functional specificities may be attributed tospecific differences in chromatin signatures . These two chromatin signatures are distinct, with U5 having strongerenrichment of repressive modifications H3K9me2 and H3K9me3. This subtledifference prompted us to ask if these signatures mark distinct regions of thegenome. Indeed, we find that only 23.3% of U5 loci are intragenic, anotable depletion over the expected value of about 40% . In contrast, U6 loci are closer to the expected value at36.3% intragenic.In addition to chromatin signatures U1–4, ChromaSig also identifies twonew chromatin signatures, U5–6, having strong enrichment of repressivehistone modifications , only U6 is notablyenriched in MIR repeats(p = 2.31E–177). Similarly, L2 LINErepeats and simple repeats are notably more enriched in U6 loci than U5 loci.These results suggest that U5 and U6 have different genic distributions and markdistinct sequences of the genome.Additional analysis suggests that the sequences underlying U5 and U6 fragmentsare associated with distinct properties. First, we compare to the PhastConsdatabase containing over 2 million conserved elements in the human genomeconserved over 28 mammalian genomes Fig.S8). In contrast, U5 is enriched near promoters in cluster P6, which aredepleted of U6 elements . These results further underscore the notion that these elementsrepress the genome in distinct ways.We next examine whether the different genic distributions and sequencepreferences of U5 and U6 relate to gene expression. It is thought that thegenome is organized into different domains of transcriptional activity, with theinsulator binding protein CTCF defining the boundaries of these domains enrichment<1E–300), compared to only37.4% for U6 loci(pdepletion = 1.57E–10), previouslymapped in fibroblast cells and known to contain repressed genes and genedeserts. Indeed, more than 60% of U5 loci are in LADs we searched within a region±500-bp around the sites, but for less-defined loci  we relaxed the search to a ±1-kb region. To focus onlyon the most frequently-occurring chromatin signatures, we analyzed only thoseclusters output having at least 500 loci and an average normalized enrichmentgreater than 0.25 for at least one modification.Chromatin signature significance. For a given cluster of size N, wedefined the motif h,im to be the mean normalizedenrichment of the aligned loci at a specified position i formodification h. Well-aligned motifs have higher values ofenrichment. For each motif, we computed the score:Higher values of S indicate more significant motifs. To assess significance ofobserving a motif spanning N loci with score S or greater, we randomly sampled 100sets of clusters with random alignment offsets  and orientations (positive or negative strand), computed S scores foreach random set, and modeled the random distribution of S scores as a Guassiandistribution to allow for calculation of significance. We performed thisrandomization either within loci in the same cluster as the original motif or overloci from all clusters.Heatmaps. All heatmaps consist of normalized data over 100-bp bins (seeabove), and were visualized using Java TreeView Expression data. Transcript and exon expression data were measured inCD4+ T cells by Crawford et al Randomization. To determine enrichment for a given cluster, we comparedto 100 random clusters. Each random cluster contains the same number of loci as theoriginal cluster and follows the same chromosomal distribution. Random sampling islimited to bins containing ChIP-Seq reads.Statistical tests. To assess significance of overlap with known genomeannotations, we assume that the overlap statistics for 100 random clusters follows aGaussian distribution. To assess significance of exon inclusion for marked versusunmarked exons, we use a two-sided Wilcoxon rank sum test to compare the median exonexpression of the two sets. To assess that U5 and U6 are enriched near differentclasses of expressed genes, we use the paired two-sided Wilcoxon signed rank test tocompare the enrichment profiles.Figure S1U1, U2, and U4 mark exon 5′ ends. An exon is unambiguously markedif it is the only exon within 1-kb of a genomic locus. We profiled chromatinenrichment relative to the 5′ ends of unambiguously marked exonsfor clusters (a) U1, (b) U2, (c), U3, and (d) U4. The top panels are heatmaps representing the H3K36me3 enrichment in a 10-kb region surrounding the5′ ends of unambiguously marked exons. The bottom panels representthe average profiles of the heat maps. U3 is the negative control.(0.89 MB TIF)Click here for additional data file.Figure S2U1 and U2 mark the 5′ ends of exons greater than 1-kb in length. Anexon is unambiguously marked if it is the only exon within 1-kb of a genomiclocus. We profiled chromatin enrichment relative to the 5′ ends ofunambiguously marked exons of length >1-kb for clusters U1 and U2.The top panels are heat maps representing the H3K36me3 enrichment in a 10-kbregion surrounding the 5′ ends of unambiguously marked exons. Thebottom panels represent the average profiles of the heat maps. Only a smallnumber of U3- and U4-marked unambiguous exons are larger than 1-kb, and soare not shown here.(0.76 MB TIF)Click here for additional data file.Figure S3Chromatin signatures associated with preferential inclusion and exclusion ofexons into mature mRNAs. (a) Schematic of a gene containing an exon markedby a chromatin signature in pink and an unmarked alternatively spliced exonin green. After transcription and splicing, mature mRNAs either have oneexon or the other. We compared exonic expression for marked exons in pinkversus unmarked alternatively spliced exons in green for (b) U1, (c) U2, (d)U3, and (e) U4. The overlap is in brown. Wilcoxon rank sum p-values areindicated. Red p-values indicate enrichment of marked over unmarked exons,while green p-values indicate enrichment of unmarked over marked exons. U3is the negative control.(0.85 MB TIF)Click here for additional data file.Figure S4Distinct chromatin signatures spanning predicted enhancers. On the basis of apreviously published enhancer chromatin signature having strong H3K4me1enrichment but weak H3K4me3 enrichment, we predicted 32,237 promoter-distalenhancers. Applying ChromaSig to these loci using the full panel ofchromatin modifications mapped by Barski et al., we recovered 11 clusters.The heat map represents the enrichment of H2AZ, 20 histone modifications,CTCF, and RNA polymerase II in the 10-kb region surrounding each enhancerprediction. To organize these clusters visually, we performed hierarchicalclustering on the average profiles using a Pearson correlation distancemetric (left).(3.73 MB TIF)Click here for additional data file.Figure S5Distinct chromatin signatures spanning promoter-distal and enhancer-distalCTCF binding sites. We used MACS (1.75 MB TIF)Click here for additional data file.Figure S6Distinct chromatin signatures spanning Refseq 3′ ends distal toRefseq promoters. Applying ChromaSig to the histone modifications near16,703 Refseq gene 3′ ends that are distal to Refseq TSSs, werecover 12 clusters. The heat map represents the enrichment of H2AZ, 20histone modifications, CTCF, and RNA polymerase II in the 10-kb regionsurrounding each Refseq gene 3′ end. To organize these clustersvisually, we performed hierarchical clustering on the average profiles usinga Pearson correlation distance metric (left).(1.71 MB TIF)Click here for additional data file.Figure S7Distinct chromatin signatures spanning DNase I hypersensitive sites.Previously, Boyle et al mapped 95,709 DNase I hypersensitive sites inCD4+ T cells, 31,824 of which are distal to Refseq TSSs, CTCFbinding sites, and enhancer predictions. We applied ChromaSig to thechromatin modifications around these loci, recovering 13 clusters. The heatmap represents the enrichment of H2AZ, 20 histone modifications, CTCF, andRNA polymerase II in the 10-kb region surrounding each distal DNase Ihypersensitive site. To organize these clusters visually, we performedhierarchical clustering on the average profiles using a Pearson correlationdistance metric (left).(3.28 MB TIF)Click here for additional data file.Figure S8Chromatin signatures of distal regulatory elements correlate with differentclasses of promoters. We partitioned the genome into CTCF-defined domainsand counted the number of predicted enhancers and DNase I hypersensitivesites in each promoter-containing domain. To calculate enrichment, wecompared to distributions of 100 sets of randomly placed loci see .(0.72 MB TIF)Click here for additional data file.Figure S9Distinct genomic distributions of chromatin signatures. The percentage eachcluster within the 5′ and 3′ ends of genes (black), ascompared to random sites (grey). The error bars indicate 1 standarddeviation.(0.19 MB TIF)Click here for additional data file.Figure S10The distribution of H3K36me3 reads within exon and introns. The number ofreads found within introns and exons, normalized by the total size of each.(0.04 MB TIF)Click here for additional data file.Figure S11The distribution of H3K36me3 reads at long exon 5′ and 3′ends. The top panel shows the enrichment of H3K36me3 within 5-kb from (left)exon 5′ ends and (right) 3′ ends, for the longest 30,000exons sorted by length (far right). The bottom panel is the average H3K36me3enrichment profile of the shortest, middle, and longest third of exons fromthe top panel.(1.41 MB TIF)Click here for additional data file.Table S1Locations of clusters recovered when applying ChromaSig to Refseq promoters.(0.31 MB TXT)Click here for additional data file.Table S2Locations of clusters recovered when applying ChromaSig to predictedenhancers.(0.51 MB TXT)Click here for additional data file.Table S3Locations of clusters recovered when applying ChromaSig to CTCF bindingsites.(0.27 MB TXT)Click here for additional data file.Table S4Locations of clusters recovered when applying ChromaSig to Refseq gene3′ ends.(0.25 MB TXT)Click here for additional data file.Table S5Locations of clusters recovered when applying ChromaSig to DNase Ihypersensitive sites.(0.48 MB TXT)Click here for additional data file.Table S6Locations of clusters recovered when applying ChromaSig to ChIP-enrichedsites distal to Refseq promoters, Refseq gene 3′ ends, predictedenhancers, CTCF binding sites, and DNase I hypersensitive sites.(0.80 MB TXT)Click here for additional data file.Table S7Statistical significance of observed chromatin signatures. Significance foreach cluster is calculated by comparing to random sets of clusters sampledfrom within the cluster or over all clusters.(0.30 MB PDF)Click here for additional data file.


Gli3 expression must be subject to a tight spatiotemporal regulation. However, the genetic mechanisms and the cis-acting elements controlling the expression of Gli3 remained largely unknown.The zinc-finger transcription factor GLI3 is an important mediator of Sonic hedgehog signaling and crucial for patterning of many aspects of the vertebrate body plan. In vertebrates, the mechanism of SHH signal transduction and its action on target genes by means of activating or repressing forms of GLI3 have been studied most extensively during limb development and the specification of the central nervous system. From these studies it has emerged, that GLI3-intronic conserved non-coding sequence elements (CNEs) autonomously control individual aspects of Gli3 expression. Their combined action shows many aspects of a Gli3-specific pattern of transcriptional activity. In the mouse limb bud, different CNEs enhance Gli3-specific expression in evolutionary ancient stylopod and zeugopod versus modern skeletal structures of the autopod. Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos. Three of these elements govern central nervous system specific gene expression during mouse embryogenesis, each targeting a subset of endogenous Gli3 transcription sites. Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly. During evolution, ancient broad-range regulatory elements within Gli3 attained higher specificity, critical for patterning of more specialized structures, by abolishing the potential for redundant expression control.Here, we demonstrate in chicken and mouse transgenic embryos that human Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates.These results not only demonstrate the high level of complexity in the genetic mechanisms controlling  Zinc-finger proteins of the GLI family, GLI1, GLI2, and GLI3 act as transcriptional mediators integrating various upstream patterning signals in a context dependent combinatorial and cooperative fashion to direct a multitude of developmental programs. GLI2 and GLI3 can serve both as transcriptional activators or repressors, whereas GLI1, whose expression is transcriptionally regulated by GLI2 and GLI3, appears to play a secondary role, e.g. in potentiating response to the secreted protein sonic hedgehog (SHH)  containing 0.1% X-gal at 37°C. The staining reaction was stopped by washing the embryos repeatedly in PBS. The embryos were postfixed overnight in 0.5% glutaraldehyde at 4°C. To analyze the distribution of reporter gene-expressing cells, embryos were dehydrated, embedded in paraffin wax, sectioned at 10-40 μm, deparaffinized, mounted for histological analysis, following standard protocols.Stable expression of the Fertilized White Leghorn eggs were obtained from H. Stewart  and incubated at 39°C. 1 μg/μl of GFP reporter construct containing a selected CNE was co-electroporated with 1 μg/μl of RFP expression vector  was compared with orthologous sequences from multiple placental mammalian species (22 species) by using the PhyloP [Fugu gli3 by using chain and net alignments [http://genome.ucsc.edu. Human GLI3 genomic sequence was used as baseline and annotated by using exon/intron information available at UCSC.Approximately 276 kb of human e PhyloP  and alsoignments  availablhttp://rvista.dcode.org[Within human CNE1, CNE6, CNE9 and CNE11 genomic intervals the conserved transcription factor binding sites were identified with rVISTA. 2.0 dcode.org searchesdcode.org derived Wet lab experiments were designed and performed by K-HG, AAA, ZP, JL-R, SM, and FB with advice in histological analysis by AS, and SK. Computational analyses were performed by AAA. The manuscript was written by K-HG and AAA. All authors read and approved the final publication.GLI3 Selected for Functional Analysis in Transgenic mice assay.Table S1: Tetrapod-Teleost Conserved Non-Coding elements (CNEs) from Introns of Human Click here for fileFigure S1: ClustalW-derived multiple alignment of CNE6 sequence across a diverse set of mammalian species. Star symbols underneath represent nucleotide positions conserved in all species. Conserved putative transcription factor binding sites (TFBSs) are enclosed in rectangles. ALX4, aristaless-like homeobox 4; SOX5, SRY (sex determining region Y)-box 5; PITX2, paired-like homeodomain transcription factor 2; LHX3, LIM homeobox protein 3; HOXD13, homeobox D13; PITX1, paired-like homeodomain transcription factor 1; GLI, GLI family zinc finger; HOXD11, homeobox D11; HOXA7, homeobox A7; dHAND, basic helix-loop-helix transcription factor; MSX1, msh homeobox 1; PBX1, pre-B-cell leukemia homeobox 1.Click here for fileFigure S2: ClustalW-derived multiple alignment of CNE11 sequence across a diverse set of mammalian species. Star symbols underneath represent nucleotide positions conserved in all species. Conserved putative transcription factor binding sites (TFBSs) are enclosed in rectangles. HOXA13, homeobox 13; HOXD13, homeobox 13; dHAND, basic helix-loop-helix transcription factor; TBX3, T-box 3; HOXA3, homeobox 3; PITX2, paired-like homeodomain transcription factor 2.Click here for fileFigure S3: ClustalW-derived multiple alignment of CNE1 sequence across a diverse set of amniotic vertebrate species. Star symbols underneath represent nucleotide positions conserved in all species. Conserved putative transcription factor binding sites (TFBSs) are enclosed in rectangles. MEIS1, Meis homeobox 1; PAX3, paired box 3; FOXM1, forkhead box M1; SOX5, SRY (sex determining region Y)-box 5; HOXA4, homeobox 4; FOXP3, forkhead box P3; dHAND, basic helix-loop-helix transcription factor; MSX1, msh homeobox 1; HOXA3, homeobox 3; NFKB1, nuclear factor kappa-B; GATA3, GATA binding protein 3; CDX2, caudal type homeobox 2; OCT4, POU domain, class 5, transcription factor1; TBX5, T-box 5; CHX10, visual system homeobox 2; SP1, Sp1 transcription factor; PBX1, pre-B-cell leukemia homeobox 1; TCF. Transcription factor; LEF, lymphoid enhancer binding factor.Click here for fileFigure S4: ClustalW-derived multiple alignment across a diverse set of amniotic vertebrate species reveals highly conserved putative TFBSs within the core of the CNE9 interval. Star symbols underneath represent nucleotide positions conserved in all species. Conserved putative transcription factor binding sites are enclosed in rectangles. HOXA3, homeobox 3; TBX5, T-box 5; AP2REP, Kruppel-like factor 12; OCT1, POU domain transcription factor; ZEB1, zinc finger E-box binding homeobox 1.Click here for fileGLI3 CNEs, tested functionally in transgenic mice assay.Dataset S1: Genomic sequences of intra-Click here for file


Here we show that GABP binds to more than 80% of divergent promoters in at least one cell type. Furthermore, we demonstrate that GABP binding is correlated and associated with bidirectional transcriptional activity in a luciferase transfection assay. In addition, we find that the addition of a strict consensus GABP site into a set of promoters that normally function in only one direction significantly increases activity in the opposite direction in 67% of cases. Our findings demonstrate that GABP regulates the majority of divergent promoters and suggest that bidirectional transcriptional activity is mediated through GABP binding and transactivation at both divergent and nondivergent promoters.Approximately 10% of genes in the human genome are distributed such that their transcription start sites are located less than 1 kb apart on opposite strands. These divergent gene pairs have a single intergenic segment of DNA, which in some cases appears to share regulatory elements, but it is unclear whether these regions represent functional bidirectional promoters or two overlapping promoters. A recent study showed that divergent promoters are enriched for consensus binding sequences of a small group of transcription factors, including the ubiquitous  Surveys of the locations of genes in the human genome have revealed that a surprising number of genes, greater than 10%, have transcription start sites within 1 kb of one another on opposite strands. These divergent gene pairs, sometimes referred to as bidirectional genes, are common in organisms such as bacteria and yeast, but it is unknown why such an arrangement exists in large, mammalian genomes. Recently, it has become apparent that the promoters of these divergent genes are regulated by a subset of transcription factors, and we have focused on one of these, GA-binding protein (GABP). We find that it regulates a large number of human genes, including the majority of divergent genes, and that its binding is associated with, correlated with, and sufficient for bidirectional transcriptional activity. Although clearly GABP is a major regulator of divergent genes, which carry out a variety of roles critical for the function and survival of the cell, these data also propose novel roles for GABP as a transcription factor. For example, the ability of GABP to promote bidirectional transcription may prove to be biologically relevant in generating many of the transcripts that have been observed outside of protein coding genes. Since the discovery that more than 10% of the protein-coding genes in the human genome are located on opposite strands with transcription start sites less than 1 kb away from each other, there has been considerable interest in determining why such an arrangement exists . StudiesA likely contributor to coregulation of divergent gene pairs is the less than 1-kb-long intergenic region between divergent transcription start sites of an annotated gene on the plus strand and another on the minus strand. Efforts to determine whether differences exist between these promoters and those that do not lie between start sites for two annotated divergent genes  have yielded mixed results. As a group, divergent promoters exhibit increased colocation with CpG islands, a paucity of TATA elements, and an enriched subset of transcription factor binding sites ,9. Theseets family transcription factor, was of particular interest because the presence or absence of its binding site in a set of divergent promoters explained much of the variation in activity observed for a set of reporter deletion constructs [Our recent analysis of 376 promoters showed that five motifs are over-represented in divergent gene pairs . These mnstructs . Additioets-family transcription factors as it functions as a heterodimer composed of an α and a β subunit. The α subunit, encoded by the GABPA gene, contains the ets DNA-binding domain and is widely expressed from a divergent promoter that it self-regulates [cis-elements [GABP is a key transcriptional regulator of myeloid, cellular respiration (reviewed in ), and riegulates . The β selements , indicatelements . These aIn this paper, we report our studies of the relationships between GABP, divergent gene promoters, and bidirectional transcriptional activity. First, we used ChIP to measure GABP occupancy at 412 randomly selected promoters from divergent and general genes in three cell types. We then compared binding results for a subset of these promoters in a single cell type to the transient promoter activity in both directions for a representative promoter fragment to test whether binding and bidirectional transcriptional activity are correlated. On the basis of these results, we assayed whether the introduction of a single GABP site into a nondivergent promoter was sufficient for bidirectional transcriptional activity. Our data indicate that GABP not only binds the majority of divergent promoters, but that GABP binding correlates with bidirectional transcriptional activity and that the introduction of a GABP site into a promoter fragment is sufficient for such activity. These findings provide a mechanism for the regulation of bidirectional transcription at divergent and general promoters by GABP.p < 0.0001 for all cell types; χ2). Overall, 105/121 (87%) of the divergent promoters were bound by GABP in at least one cell type, which was significantly greater than 89/291 (31%) general promoters that were bound in at least one cell type  (We performed ChIP followed by quantitative PCR (QPCR) in Jurkat, K562, and HeLa cells with a monoclonal antibody that specifically recognizes GABPα to determine the fraction of divergent and general promoters bound by this factor. We tested for GABP enrichment in ChIP QPCR assays of 121 divergent and 50 general promoters that we previously studied . We also001; χ2) .p < 0.0001 for all cell types; χ2).As was previously noted , divergep = 0.0011; χ2)  . Accordi011; χ2) . We founets-family transcription factors all recognize very similar binding sites [ets-family member, ETS1, by ChIP to exclude the possibility that divergent genes were not regulated more generally by ets factors. We tested for ETS1 enrichment at a subset of divergent and general promoters exhibiting a range of GABP enrichment values and cell-line specificity. For divergent promoters, ETS1 bound 19/44 (43%), 3/44 (7%), and 1/47 (2%) of targets in Jurkat, K562, and HeLa cells, respectively, using the same 3-fold enrichment cutoff as was used for GABP . We looked for overlap between ETS1 and GABP binding at the same promoter and found 17/44 (37%), 3/44 (7%), and 1/47 (2%) divergent promoters were bound by both factors in the three tested lines. For general promoters, 18/33 (55%), 19/32 (59%), and 18/34 (53%) of selected targets were bound by GABP in the three tested cell lines, compared to 9/33 (27%), 2/32 (6%), and 2/34 (6%) promoters that were bound by ETS1. For all three cell lines, GABP bound a significantly greater number of general promoters than ETS1 . Despite ETS1 binding to a large number of divergent promoters in Jurkat cells, GABP bound a greater number of targets than ETS1 for all tested cell lines and promoter types.Given that ng sites , we testfor GABP . For the2 ratio of luciferase activity in the forward direction over the reverse direction (log2 Fwd/Rev). As the forward direction was arbitrarily chosen for divergent promoters, we chose to reflect expression activity as the log2 ratio of the higher luciferase activity over the lower of the two orientations for a given promoter (log2 H/L). All promoters used in this study demonstrated activity above the mean plus three standard deviations of a set of negative controls in at least one direction and no general promoters were observed with reverse activity greater than forward.A previous study reported that many general promoters are capable of bidirectional transcriptional activity in a transient reporter assay . This fip < 0.0001; χ2). Previously, the majority of divergent promoters were observed to direct luciferase expression to similar levels in both directions (observed log2 H/L ratio of less than 2), a phenomenon we call “balanced bidirectional transcription.” To test whether GABP binding and balanced bidirectional activity are associated, we performed another categorical analysis, this time classifying promoter activity as balanced bidirectional or otherwise. For comparison, the percentage of all promoters with a log2 H/L ratio of less than 2 was lower than the percentage of promoters with activity above background in both directions (57% versus 80%). For promoters of divergent genes (n = 117), 80% exhibited balanced expression while 93% demonstrated promoter activity above background in both directions, compared to 34% and 64% of general promoters (n = 116), respectively . Thus, GABP binding is very strongly associated with transcriptional activity above background in both directions as well as with balanced bidirectional activity.We tested for an association between GABP binding and promoter activity in both directions by first classifying each promoter as bound (ChIP enrichment greater than 3-fold) or unbound. We then scored each promoter for whether its representative fragment was capable of directing luciferase activity above background in both directions or only one. We considered all promoters together, regardless of annotation, and compared the number of promoters that were or were not bound and did or did not demonstrate luciferase activity in both directions above background . We founectively . This mo2 H/L ratios (median = 1.02), indicating similar levels of luciferase activity in each direction. General promoters showed a much broader range of expression ratios (median = 2.81), as well as a trend for lower GABP enrichment values (median = 1.66). Across all promoters examined, GABP binding and log2 H/L are significantly anticorrelated . Thus, regardless of the promoter's annotation, promoters bound by GABP  tended to exhibit balanced bidirectional transcriptional activity (low log2 H/L ratio).Lastly, we examined the correlation between GABP enrichment at each promoter region and its promoter activity ratio . In addi2 H/L ratio indicative of balanced bidirectional activity. In contrast, the minimum log2 H/L ratio before the introduction of the GABP site was 3.6. Notably, only one of the six promoters demonstrated a significant increase in forward transcriptional activity. Of the two promoters without significant increases in expression, one exhibited significantly decreased expression, while the other was unchanged. In the majority of tested promoters, the addition of a single GABP site was sufficient to increase transcriptional activity in the reverse orientation.Having shown that GABP binding and bidirectional transcriptional activity are correlated, we tested whether the introduction of a GABP site into a promoter is sufficient to induce bidirectional transcriptional activity. We selected six functional promoters that were not bound by GABP in any tested cell type and had little or no expression in the reverse direction, and introduced a single, strict consensus GABP site, CCGGAAGTG, into each promoter through site-directed mutagenesis. Four of six promoters (67%) demonstrated significantly increased luciferase activity in the reverse direction . The aveWe tested 121 and 291 randomly selected promoters from divergent and general genes, respectively, by ChIP in three cell lines to determine the percentage of genes from each class that were bound by GABP. Using an enrichment threshold of 3-fold for classifying a promoter as bound, we found that 86.8% of divergent promoters and 30.6% of general promoters were bound in at least one cell type. The significantly larger fraction of divergent promoters bound by GABP was also observed when comparing a subset of the two promoter types with matched CpG content. Furthermore, most divergent promoters were bound in all three cell types, suggesting that GABP regulates these promoters in a wide variety of tissues. This is not surprising given the broad expression of GABP and its tendency to regulate ubiquitously and broadly expressed genes, a class to which many divergent gene pairs belong [The finding that GABP binds to such a large percentage of divergent gene promoters is surprising, but not unexpected. Our previous study estimated that the transcription factor binds to 57% of divergent gene promoters based upon observed binding frequencies, using a 5-fold enrichment cutoff, for promoters containing a high, medium, or low scoring motif . In thisets-family transcription factors and the similarity in their binding sites led us to test whether ETS1, another ubiquitously expressed family member, might also be involved in the regulation of divergent genes [ets-family member in this cell line [ets transcription factors, it would appear that GABP regulates divergent promoters on a widespread basis at a basal level with ETS1 and/or other family members, perhaps serving to modify this activity in a cell-specific manner.The broad expression of many nt genes . Interesell line . Howeverp < 0.0001; χ2) and with balanced bidirectional transcription , supporting a model whereby GABP directs bidirectional transcription. Furthermore, GABP enrichment was significantly anticorrelated with the log2 ratio of promoter activity for each direction of the corresponding promoter fragment . The low correlation coefficient can be partly explained by recognizing that we would not expect a perfect correlation between the degree of enrichment and a lower ratio of activities in each direction. Although the sum of motif occurrence scores correlates with the degree of enrichment [In light of the observation that many general promoters are capable of balanced bidirectional transcriptional activity, we explored the relationship between GABP binding and bidirectional transcriptional activity at all promoters . We founrichment , there iDespite strong statistical evidence that GABP binding and bidirectional activity are both correlated and associated, we observed a number of unbound promoters that demonstrated balanced bidirectional activity as well as examples of bound promoters with activity primarily or only in one direction. Many of the former cases likely represent promoters that are bound in vivo but were not scored as bound because they fell below 3-fold enrichment threshold. HeLa cells, in addition to having the lowest percentage of bound divergent promoters that are bound by GABP, had enrichment values on average half that of those observed in K562 and Jurkat cells for those promoters that were bound in all three cell types (unpublished data). In addition, there are almost certainly mechanism(s) for bidirectional activity that are not dependent on GABP binding. For example, five adjacent Sp1 sites were capable of directing bidirectional transcription in vitro . We comp2 H/L expression ratios of less than 2. Although this definition of balanced bidirectional transcriptional activity encompasses the majority of divergent promoters, it may exclude many biologically relevant bidirectionally active promoters. In addition, unknown regulatory element(s) may modify the bidirectional activity conveyed by GABP binding. For example, TATA elements are known to convey tissue and start-site specificity and may also influence the directionality of transcription [2 H/L ratio among all promoters in this study and observed a significant, positive relationship , although, in another study, the introduction of a TATA and/or Inr element into the mouse thymidylate synthase promoter had no effect on its bidirectional activity [There are several possible interpretations for GABP bound promoters that do not conform to our definition of balanced bidirectional transcription. First, not all divergent promoters have logcription . We testactivity . In genep < 0.0001). However when we compared the luciferase activity values for general promoters that were categorized as having balanced bidirectional activity and those that did not, there was no significant difference . This suggests that although there may be some expression in the reverse direction due to a strong promoter, this was not sufficient to result in balanced bidirectional activity. Another likely explanation is that the approximately 500-bp promoter fragment we cloned may not have contained all the regulatory sequences necessary for its proper function in the genome. Doubtless other transcription factor(s) and/or boundary elements such as insulators play a role in controlling the level and direction(s) of promoter activity in their endogenous genomic context. Finally, many of the general promoters for which we observed bidirectional activity may actually belong to an unannotated divergent gene pair. A subset of these may prove to be functional bidirectional promoters with a protein-coding gene in one orientation and some other type of transcript in the other. Several recent studies have reported that a much larger percentage of the genome is transcribed than was previously thought, much of this occurring outside of annotated genes [In this study, we observed balanced bidirectional transcriptional activity at 34% of general promoters. One possible explanation for this observation is that a strong promoter contains elements sufficient for leaky expression in the other direction when reversed in our reporter plasmids. To investigate this possibility, we looked for a correlation between activities in each direction for general promoters and found a significant relationship  indicated that although GABPα has an ortholog in Drosophila, the most distant GABPβ ortholog has been observed in Danio rerio (Fugu is not currently represented in HomoloGene). The coincident emergence of conserved divergent gene pairs and a GABP heterodimer capable of bidirectional transcriptional activity reinforces the finding that GABP is the major regulator of divergent genes.While our data do not suggest how the paired gene arrangement came to exist, they are consistent with a role for GABP regulation in maintaining this relationship. In this model, unknown forces would bring two critical genes together, each with its own promoter, and GABP would reinforce this relationship through its ability to direct balanced, bidirectional transcriptional activity. Given its ability to be sufficient for such activity, the presence of a GABP site could make other factor binding sites redundant over evolutionary time. Thus, the short intergenic region between the gene pair would be free to accumulate mutations, each gene losing its independent regulators and becoming part of an inseparable, truly bidirectional promoter. This supports the conclusion that the promoters of divergent gene pairs do not consist of two overlapping promoters, and that in addition to the previously noted characteristics of these promoters, their bidirectional character is in large part conveyed by GABP binding. Analyses of transcription start site distributions in species from fish to mammals have revealed that although a bimodal distribution, indicative of an abundance of divergent gene pairs, seems to exist only in mammals, 83 divergent pairs have been maintained in proximity and orientation as far separated from humans as Fugu ,4. A seaIn summary, we showed that GABP binds the majority of divergent promoters and is correlated with and sufficient for bidirectional activity. We have established its role as a major regulator of divergent genes, which carry out a variety of activities critical for the function and survival of many different cell types. In addition, GABP binds a large number of nondivergent genes, and further study will be needed to ascertain whether its ability to promote bidirectional transcription genes has widespread biological consequences through the generation of novel and/or noncoding transcripts.2 analysis in the Prism 4.0c (GraphPad Software) software package. We also determined the number of promoters that were bound in at least one cell type and compared this to those that were bound in no cell types for each type of promoter. We also compared the number of promoters that were and were not bound by GABP and ETS1 again for each cell line independently. We considered only promoters with binding information for both factors in each cell type. However, as divergent and general promoters were not randomly sampled for ETS1, we did not compare the fraction of promoters bound for each type of promoter.ChIP was performed as described  in HeLa,We attempted to extract the largest possible set of CpG-content matched divergent and general promoters from our dataset. We calculated CpG percentage based upon the cloned promoter sequence, and then for each cell type, averaged CpG content for promoters for which QPCR data was available. Promoters from each group were then removed until the average CpG content for the two groups was within 0.1% for equal sample sizes. For divergent promoters, the average CpG contents were 31.2%, 31.1%, and 31.0% for Jurkat, K562, and HeLa, respectively. For general promoters, CpG contents were 31.1%, 31.1%, and 31.0% for promoters sampled from those three cell lines.MluI and BglII (New England BioLabs), and then recloned them into a pGL3 Basic vector with a reversed MCS, yielding a luciferase reporter plasmid with a reversed promoter sequence. A total of 50 ng of each promoter construct was cotransfected with 5 ng of pRLTK (Promega), a Renilla luciferase-expressing transfection control, using FuGene (Roche). Transfections were performed in 96-well plates seeded approximately 24 h earlier with 5,000 HeLa cells in each well. Cells were lysed 24 h after transfection and assayed for luminescence on a Victor Light luminometer (Perkin Elmer). The ratio of luciferase to Renilla luminescence was calculated for all fragments and compared to a panel of 24 negative controls. Sample ratios that were lower than the mean plus three standard deviations were considered to not be expressed above background and were recorded as 0.1 to permit the calculation of directional activity ratios. Only promoters with activity above background in at least one direction were subjected to further analyses. For 88 promoters we calculated the log2 ratio of the promoter orientation with the higher promoter activity over the lower (log2 H/L). In all cases, the higher activity of the two orientations corresponded to the sense orientation relative to the annotated gene product. We also calculated log2 H/L ratios for 117 divergent and 28 general genes from a previous study [Renilla ratios for each direction and the log2 H/L ratio for promoters in this study can be found in Dataset 1. We plotted the activity ratio for each promoter against its corresponding GABP enrichment score and then performed a nonparametric correlation on the data using the Spearman method in the Prism 4.0c software package. We also categorized each promoter as either having luciferase activity above background in one or both promoter orientations. We then compared the number of promoters that were or were not bound by GABP and did or did not demonstrate luciferase activity above background using χ2 analysis in Prism 4.0c. We also performed a similar χ2 analysis, but this time compared the number of bound and unbound promoters with log2 H/L ratios below two, indicating balanced bidirectional activity, or above two.The 241 general promoters tested for GABP ChIP enrichment were previously cloned into the plasmid pGL3 Basic (Promega) and verified to be functional promoters as part of the ENCODE project . We amplhttp://bioinformatics.org/primerx/), to introduce one to four substitutions around an existing GGAA sequence in the wild-type promoter resulting in a GABP consensus site, CCGGAAGTG. Mutagenesis reactions were performed with Quikchange mutagenesis kit (Stratagene) in accordance with the manufacturer's protocol. The mutant sequences, once confirmed by sequence in each orientation, were then transfected into HeLa cells three times independently in sextuplicate alongside wild-type controls as above. The original cloned promoter sequence, mutagenesis primers, and luciferase to Renilla ratios for each direction for mutated and wild-type promoters can be found in Dataset 2. Luciferase to Renilla ratios were pooled across the three independent experiments and wild-type ratios were compared to those of the mutant using a two-tailed Welch's t-test in Prism 4.0c.We selected six sequences with experimentally confirmed unidirectional activity and no evidence for GABP binding in any cell type. We then designed primers, using the web-based PrimerX tool (Dataset S1(516 KB XLS)Click here for additional data file.Dataset S2(37 KB XLS)Click here for additional data file.Table S1(24 KB DOC)Click here for additional data file.Table S2(28 KB DOC)Click here for additional data file.Table S3(28 KB DOC)Click here for additional data file.Table S4(32 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/RefSeq/) accession numbers for the genes and gene products discussed in this paper are: GABPA and ETS1 . GABPB2 is listed under accession numbers NM_181427, NM_002041, NM_016655, NM_005254, and NM_016654.The RefSeq (


Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.Previous studies in  Saccharomyces cerevisiae, when particular factors are defective, cryptic promoters within several coding regions become active and produce shorter transcripts corresponding to the 3′ portions of genes. (Transcription proceeds from the 5′ end of genes to the 3′ end.) A comprehensive analysis of cryptic transcription identified the factors that normally repress this event. We find that at least 50 factors, many involved in chromatin structure and transcription, are required to repress cryptic transcription. Other results suggest that the potential for cryptic transcription is widespread, initiating from at least 1,000 locations across the S. cerevisiae genome. In mutants in which cryptic transcripts are produced, some of the transcripts are translated into proteins not normally made in unmodified, wild-type cells. Finally, in wild-type cells, a subset of cryptic transcripts is transiently induced following a nutritional shift, suggesting a possible role for cryptic transcription. Taken together, our results demonstrate that the normal pattern of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.Recent studies have shown that much more of the eukaryotic genome is transcribed into RNA than previously thought. In  S. cerevisiae, which is normally repressed by many chromatin- and transcription-related factors, has the potential to be widespread and to express alternative genetic information.Transcription from within open reading frames in Drosophila melanogaster, recent studies estimate that 85% of the genome is transcribed, with extensive intergenic transcription and multiple transcription start sites , which affects the fidelity of translational termination and thus allows for the possible production of novel protein products [PSI+] can acquire complex phenotypic traits distinct from [psi−] strains, and when outcrossed to wild-type strains, these phenotypic traits can sometimes be maintained even after treatment to remove [PSI+]  ,71. A poe [PSI+] . Third, g mating . In thisg mating . Finallyg mating . In suchg mating . Given tS. cerevisiae strains are listed in GAL2+ derivative of S288C dup duplication allele was constructed by integrating plasmid pFW216 (a derivative of pRS306 containing SPT10 and URA3) at the ura3-52 genomic locus [hht2-S57P allele, a 1.9-kb SmaI-EcoRI fragment containing HHT2 and HHF2 from plasmid pDM18 was ligated into vector pRS306 (URA3) [HHT2 (relative to the +1 ATG start codon) was mutated to a CCT codon (Pro) by site-directed mutagenesis . The resulting plasmid was used to replace the wild-type HHT2 allele in strain FY2716 by two-step gene replacement. The presence of the mutant hht2-S57P allele in the genome was verified by sequencing. Strain FY2724 contains a point mutation in HHT2 changing the GAA codon (Glu) at base pair +316 to an AAA codon (Lys) and was created by UV mutagenesis of strain FY2713 and verified by sequencing.All of S288C . Strainsof S288C . The sptpt6-1004 ), spt16-pt16-197 ,  . The TCTkanMX-GAL1pr-flo8-HIS3 reporter, a 2-kb cassette containing the kanMX marker and the GAL1 promoter was amplified by PCR from plasmid pFA6a-kanMX6-PGAL1 [FLO8 promoter, replacing base pairs −1,147 to −1 (relative to the FLO8 +1 ATG start codon), to create strain FY2174. The HIS3 ORF (663 bp) was amplified by PCR from plasmid pRS403 [FLO8 locus, replacing the 3′ end of the FLO8 ORF and the first 105 bp of the 3′ UTR . Successful transformants were selected on SC-His medium and verified by PCR. The HIS3 ORF is inserted out-of-frame with respect to the FLO8 ORF and is inserted 3′ of both the internal FLO8 TATA element  and the cryptic transcription initiation sites of the FLO8 short transcript  [flo8-HIS3 reporter, the HIS3 ORF was transformed into strain FY2425 and inserted at the FLO8 genomic locus as described above.To construct the X6-PGAL1 . This cad pRS403  and tran +1,685) . To consAPM2-TAP::His3MX, DDC1-TAP::His3MX, OMS1-TAP::His3MX, PUS4-TAP::His3MX, and SYF1-TAP::His3MX alleles are from the S. cerevisiae TAP-tagged library [apm2–1-TAP::His3MX allele contains a point mutation in the in-frame ATG codon at base pair position +1,420 of APM2 (relative to the +1 ATG start codon), changing it to a TTG codon (Leu). The apm2–2-TAP::His3MX allele contains three point mutations at the +1 ATG start codon of APM2, changing it to a CGT codon. The apm2–3-TAP::His3MX allele contains both the +1 ATG and the +1,420 ATG mutations in APM2. The pus4-1-TAP::His3MX allele contains a point mutation in the in-frame ATG codon at base pair +478 of PUS4 (relative to the +1 ATG start codon), changing it to a GTG codon . The pus4–2-TAP::His3MX allele contains three point mutations at the +1 ATG start codon of PUS4, changing it to a CGT codon. The pus4-3-TAP::His3MX allele contains both the +1 ATG and the +478 ATG mutations in PUS4. All ATG mutations were constructed by a two-step gene replacement using a previously described method [The  library . The apmd method  and veriFor liquid cultures, strains were grown in either YPD rich medium  or SD minimal medium  as indicated. Synthetic complete media plates (SC) and synthetic complete drop-out media plates (SC-His) were made as previously described . SC + GaS. cerevisiae genome nonessential deletion set [kanMX-GAL1pr-flo8-HIS3 reporter. Parental strains were grown overnight in 5-ml YPD cultures at 30 °C, washed twice in water, and then either 1 × 107 cells or 1 × 108 cells from each culture were plated on SC-His media plates containing 0, 1, 2, 3, 4, 5, or 10 mM 3AT. Plates were either UV-irradiated  or left untreated, and then grown at 30 °C to select for His+ mutants. Potential His+ cryptic initiation mutants were single-colony purified and retested to verify their His phenotype. Mutant genes were identified by diploid complementation, plasmid complementation, linkage analysis, and cloning by plasmid complementation with an S. cerevisiae genomic library [SPT21, SPT10, HTA1-HTB1, HHT1, HHT2, HIR1, HIR2, HIR3, HPC2, and mutations linked to the kanMX-GAL1pr-flo8-HIS3 reporter. SGA analysis was performed as previously described [Cryptic initiation mutants were isolated using the following three methods: spontaneous mutant selection, synthetic genetic array (SGA) analysis with the tion set ,92, and  library . A totalS. cerevisiae genome were submitted to Agilent Technologies for microarray production. Each ORF was represented by six 60-mer probes spaced evenly along its coding sequence, with the most-5′ probe beginning at base pair position +1 (relative to the +1 ATG start codon) and the most-3′ probe ending at the final stop codon. Strains FY80 and FY2425 were used for four independent spt6 microarray experiments, and strains FY70 and FY347 were used for two independent spt16 microarray experiments. Experimental pairs were performed in dye reversal. Wild-type and mutant cells were grown in YPD medium at 30 °C to mid-log phase (1–3 × 107 cells/ml), shifted to 37 °C for 80 min, and then harvested as previously described [spt6 or the spt16 mutant using the following criteria. The mutant/wild-type ratio was calculated for each probe on the microarray using the normalized spot fluorescent intensity values. For each ORF, the 3′/5′ ratio was calculated by dividing the mutant/wild-type ratio of the most-3′ probe by the mutant/wild-type ratio of the most-5′ probe. Genes with high 3′/5′ ratios were predicted to express short transcripts, whereas genes with low 3′/5′ ratios (close to 1.0) were predicted to not express short transcripts. The location of the internal transcription start site for genes generating short transcripts was estimated by calculating the mutant/wild-type ratio of each probe in the gene relative to the corresponding ratio of the most-5′ probe. Based on the microarray results for five genes previously known to express short transcripts in an spt6 mutant  [spt6 microarray experiments or both spt16 microarray experiments were predicted to express a short transcript. Using this criterion, 960 genes were predicted to express short transcripts in an spt6 mutant, and 1,130 genes were predicted to express short transcripts in a spt16 mutant. It is likely, though, that even more cryptic promoters exist, as the method of calculation likely and unavoidably discriminates against the identification of cryptic transcripts from highly transcribed genes. This discrimination arises from the fact that the hybridization signal from the 3′ probe is the sum of the signals for both the full-length and cryptic transcripts. Thus, for genes with a high level of the full-length transcript, the level of a cryptic transcript would need to also be high to be detectable. The 3′/5′ ratio from the microarray results are shown plotted according to expression levels in Probe sequences corresponding to 5,869 ORFs of the escribed  Sample pescribed ,95. Micrd VPS72) , a 3′/5′7 cells/ml). For temperature shift experiments, cells were grown in YPD to mid-log phase at 30 °C and then shifted to 37 °C for 80 min. For media shift experiments, cells were grown in YPD to mid-log phase at 30 °C, washed twice with SD, and then grown in an equivalent volume of SD at 30 °C for the indicated times. Double-stranded northern probes were amplified by PCR from genomic DNA and were designed to hybridize to the 3′ ends of FLO8 , SPB4 , STE11 , APM2 , DDC1 , OMS1 , PUS4 , SYF1 , and CHS6 . A probe for SNR190 (+1 to +190) was used as a loading control for all northern analyses. Because the probes are double stranded, they could anneal to either sense or antisense transcripts. The base pair positions given for each probe is relative to the +1 ATG start codon of the respective gene.mRNA preparation and northern hybridization analysis were performed as previously described . Unless For Western analysis of histone H3 and H3 K36 methylation, whole-cell protein extracts were prepared as previously described . The pro7 cells/ml) and then shifted to 37 °C for 80 min. Cells were washed twice with wash buffer  and resuspended in 400 μl of lysis buffer . One milliliter of glass beads was added, and cells were lysed by vortexing in an Eppendorf multihead shaker 5432 for 40 min at 4 °C. The cell lysate was spun out through a hole punctured in the bottom of the tube, by spinning for 2 min at 1,000 rpm. The lysate was spun for 5 min at 14,000 rpm, and the supernatant was saved and spun again for 15 min at 14,000 rpm. The supernatant was saved from this final spin and used for western analysis. Total protein concentration of extracts was determined by Bradford assay (Bio-Rad). Equal amounts of whole-cell extracts were separated on 8% acrylamide SDS-PAGE gels, transferred to immobilon-P membrane (Millipore), and analyzed by immunoblotting as previously described [For western analysis of TAP-tagged proteins, whole-cell protein extracts were prepared as follows: 50 ml of cells were grown in YPD at 30 °C to mid-log phase  and mapped them onto the proteins encoded by genes with cryptic transcription initiation. We considered the first ATG after the most-5′ limit of the cryptic transcript as a conservative limit for the length of the short protein being produced; i.e., cases in which a minimal number of residues would be lost. A domain was called to be absent if the position of the ATG was downstream of the domain start site. Using published data on protein domains that are at the physical interface of the interacting partners [To examine which protein domains are present and lost, we obtained data on proteins from SGD .The microarray data, accession number GSE12272, can be found at GEO  for ind(49 KB PDF)Click here for additional data file.Figure S2spt6-1004 mutant; (B) spt16-197 mutant.Cryptic transcripts may be less likely to be detected for genes with abundant transcripts because the abundance of cryptic transcripts does not scale with that of the full transcript. The ratio of 3′ to 5′ of hybridization intensity is shown as a function of transcript abundance. The ratio was calculated as defined in the text. In grey are ratios of intensities for probe 6 and 5 and in black for probes 6 and 1. The red line indicates the cutoff for calling the presence of a clear cryptic transcript (2.5). (A) (7.41 MB PDF)Click here for additional data file.Figure S3spt6-1004 mutant; (B) spt16-197 mutant.The presence of a TATA motif in the coding sequence of a gene increases the probability of cryptic initiation of transcription in that gene, independent of the size of the gene. Genes were separated by size classes corresponding to each of the ten intervals corresponding to the ten quantiles of the size distribution. The expected number of genes was calculated as the product of the fraction of genes with a TATA motif in this size interval times the number of genes with cryptic transcripts in that interval. The observed number represents the fraction of genes with a TATA motif in the coding sequence that produce cryptic transcript. (A) (466 KB PDF)Click here for additional data file.Figure S4spt6-1004 mutant; (B) spt16-197 mutant.Genes with a TATA box in their promoter are less likely to produce cryptic transcripts, and the occurrence of a TATA box occurs independently from transcript abundance. Genes were separated by expression classes corresponding to each of the ten intervals corresponding to the ten quantiles of the distribution. The expected number of genes with a TATA promoter was calculated as the product of the fraction of genes with a TATA promoter for this expression level interval times the number genes with cryptic transcript in that interval. If the presence of a TATA promoter was independent from the production of a cryptic transcript, the fraction of genes that produce a cryptic transcript and that have a TATA promoter should be proportional to the fraction of genes with a TATA promoter in this size interval. (A) (517 KB PDF)Click here for additional data file.Figure S5Shown are proteins found to be expressed from cryptic promoters . The gra(172 KB PDF)Click here for additional data file.Table S1(22 KB DOC)Click here for additional data file.Table S2(20 KB XLS)Click here for additional data file.Table S3(5.62 MB XLS)Click here for additional data file.Table S4(5.62 MB XLS)Click here for additional data file.Table S5(22 KB DOC)Click here for additional data file.Table S6(1.01 MB XLS)Click here for additional data file.Table S7(22 KB DOC)Click here for additional data file.Table S8(1.34 MB XLS)Click here for additional data file.Table S9(33 KB DOC)Click here for additional data file.


Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration.Depending on the carbon source, 13C flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. In vivo abundances of 40 respiratory enzymes were quantified by GFP-fusions under each condition. During growth on the partly and fully respired substrates galactose and pyruvate, several TCA cycle and respiratory chain enzymes were significantly up-regulated. From these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. The most probable transcription factors to regulate the different degrees of respiration were Gcr1p, Cat8p, the Rtg-proteins and the Hap-complex. For the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains.We address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. Specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. Based on Cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. The Rtg-proteins and the Hap-complex are essential for wild-type like respiration under partially respiratory conditions. Under fully respiratory conditions, the Hap-complex, but not the Rtg-proteins are essential for respiration. Saccharomyces cerevisiae adjusts its energy metabolism in a process that is generally referred as carbon catabolite repression. Two extreme cases are exponential growth on glucose and on ethanol, which lead to almost exclusive fermentation with extensive secretion of ethanol and exclusive respiration to carbon dioxide, respectively.Depending on the environmental conditions, and in particular the nature of carbon substrates, Both, fully respiratory and fermentative energy production is mediated, to a large extent, by differentially active transcription factors. During fermentative metabolism, mainly the transcription factor complex of Tup1p, Ssn6p and Mig1p repress the expression of respiratory, gluconeogenic and alternative carbon source utilization genes . Minimal13C flux analysis  labeled glucose  or galactose  to better resolve the pentose phosphate pathway.For flux analysis experiments, FY4 was freshly plated from a glycerol stock on a YPD  plate. The liquid pre-culture was inoculated from the YPD plates. For the experiment cultures, minimal medium containing 10 g/l of either glucose, mannose, galactose or pyruvate as sole carbon source was used. FY4 was cultured in 96-deep-well plates   as batchFor the protein expression experiments, GFP-tagged strains and the TAP-tagged reference strain were plated from glycerol stocks on minimal medium plates supplemented with leucine (0.24 g/l), methionine (0.115 g/l) and uracil (0.05 g/l). Liquid pre-cultures of minimal medium containing leucine (0.24 g/l), methionine (0.115 g/l) and uracil (0.05 g/l) were inoculated from the minimal medium plates with 10 g/l glucose. The pre-cultures were cultivated in 96-deep-well plates with the same conditions as explained above. For the experimental culture, additionally histidine (0.025 g/l) was added to the minimal media already containing leucine, methionine and uracil, the carbon source was either 10 g/l glucose, mannose, galactose or pyruvate. The experiment cultures were cultivated in 96-microtiterplates in an incubator with online monitoring of biomass and fluorescence signals  at 30°C and 800 rpm and a shaking diameter of 3 mm.13C] experiments were performed.For the transcriptional regulation experiment, the transcription factor deletion strains were freshly plated from glycerol stocks on YPD plates containing 300 μg/ml geneticin (G418) . The liquid pre-cultures were inoculated from the YPD plates. For the liquid cultures yeast minimal medium was used. The pre-cultures were cultivated in 96-deep-well plates with 10 g/l glucose as described above. Medium and cultivation conditions were identical to the flux experiment described above. Only  experiment except for the carbon sources glucose and galactose where the ratio for the split between glycolysis and the pentose phosphate pathway was obtained from a [C1-13C] experiment. The gluconeogenic ratio varied dependent on the tuning of the MS instrument therefore a flux ratio range was used for the later calculation.Flux ratios were determined from the mass isotopomer distribution of the protein-bound amino acids with the software FiatFlux  using thNet fluxes ,23 were HO-TAP::HIS3 was used as reference to correct for auto fluorescence. Thus, the slope between the GFP and the biomass signal, calculated for the reference was subtracted from the slope calculated for the GFP-fusion strains. The slope, corrected for the autofluorescence, quantifies the enzyme abundances. Significant changes are assigned for p-values of 0.12 or lower which ensures that the error ranges between enzyme expression during growth on glucose compared to each other carbon source do not overlap and thus truly differential expressed enzymes are further investigated.The enzyme expression level was calculated from the slope between the biomass signal (light scattering)  excitat. HO-TAP:et al. [et al. [Transcription factors are more often associated with a subset of differentially expressed proteins than expected by chance were determined by a statistical analysis adopted from Boyle et al.  as outli [et al. : The likf : probability value of a transcription factorski: number of interactions between a transcription factor and the differential expressed enzymesm : total number of interactions between a transcription factor and all investigated enzymesn : number of differential expressed enzymesN : total number of enzymesf gives the probability that the transcription factor has at least the observed number of interactions with the subset of differential expressed enzymes by chance. When the probability value f is low we conclude that the considered transcription factor is likely to be involved in the regulation of the observed differential expressed enzymes. The transcription factor interactions were from the yeastract database (literature curated once) [f value  . The difed once) , wherease Figure . Thus wecdw: cell dry weight; CoA: coenzyme A; cyt: cytosolic; dd: double distilled; DMF: dimethyl-formamide; gal: galactose; GC: gas chromatograph; glc: glucose; man: mannose; mit: mitochondrial; MS: mass spectrometry; OD: optical density; pyr: pyruvate; TCA: tri-carboxylic-acid; UV: ultra violet.AU: arbitrary units; SMF designed the study, performed all experiments and drafted the manuscript. US conceived and supervised the study and revised the manuscript. All authors read and approved the final manuscript.Ratios and net fluxes. Flux rations and absolute metabolic (net) fluxes for yeast grown on either, glucose, mannose, galactose or pyruvate.Click here for fileFunction of investigated enzymes. Description of the functions of all investigated enzymes as listed on http://www.yeastgenome.org.Click here for file


Hes genes are the main effectors of this pathway, acting in neural progenitors during the lateral inhibition process to repress proneural genes and inhibit differentiation. However, Hes6 genes seem to behave differently: they are expressed in differentiating neurons and facilitate the activity of proneural genes in promoting neurogenesis. Still, the molecular mechanisms underlying this unique function of Hes6 genes are not yet understood.Notch signalling plays a central role in the mechanisms regulating neuronal differentiation in the vertebrate nervous system. The transcriptional repressors encoded by Hes6 genes that seem conserved in most vertebrate species and characterize a novel Hes6 gene in chicken: cHes6-1. The embryonic expression pattern of cHes6-1 suggests roles for this gene in the formation of the pancreas, nervous system and in the generation of body asymmetry. We show that cHes6-1 is negatively regulated by Notch signalling in the developing embryonic spinal cord and in pancreatic progenitors, but requires Notch for the observed asymmetric expression at the lateral mesoderm. Functional studies by ectopic expression in the chick embryonic neural tube revealed that cHES6-1 up-regulates the expression of cDelta1 and cHes5 genes, in contrast with overexpression of cHES6-2, which represses the same genes. We show that this activity of cHES6-2 is dependent on its capacity to bind DNA and repress transcription, while cHES6-1 seems to function by sequestering other HES proteins and inhibit their activity as transcriptional repressors.Here, we identify two subgroups of Hes genes, cHES6-1 acts later, sequestering HES proteins. Together, the two cHES6 proteins progressively shut down the Notch-mediated progenitor program and ensure that neuronal differentiation can proceed.Our results indicate that the two chick HES6 proteins act at different phases of neuronal differentiation, contributing to the progression of neurogenesis by different mechanisms: while cHES6-2 represses the transcription of  Hes genes (Drosophila Hairy and Enhancer of Split homologues), which encode basic helix-loop-helix (bHLH) transcriptional repressors , near the lumen of the tube. Progenitor cells have attachments at the apical and basal sides of the neuroepithelium and their nuclei show a characteristic interkinetic nuclear movement, with mitotic nuclei being always located apically . After dXenopus and in vitro have shown that one particular HES protein, HES6, acts differently, promoting neurogenesis when ectopically expressed Hes6 promoter Although vertebrate HES proteins usually function as Notch effectors to inhibit neuronal differentiation in the developing nervous system, studies in mouse, Multiple mechanisms have been shown to contribute to the inhibitory activity of HES6 on anti-neurogenic HES factors, but these do not seem to involve a DNA-binding dependent transcriptional activity of HES6. Instead, the mouse HES6 protein was shown to form heterodimers with HES1 and prevent recruitment of the transcriptional co-repressor TLE, necessary for HES1 repressive activity Hes6 genes are expressed and the product of one of them, cHES6-2, has been shown to cooperate with the proneural proteins to promote neuronal differentiation Hes6-1 is asymmetrically expressed in the mesoderm lateral to the primitive streak, suggesting a role for this gene in the formation of body asymmetry. cHes6-1 expression is negatively regulated by Notch signalling in the embryonic spinal cord and pancreas. However, in the mesoderm lateral to the primitive streak, cHes6-1 expression requires Notch activity, suggesting different regulation of this gene in distinct tissues. In the embryonic spinal cord, the two chick Hes6 genes are transiently expressed in differentiating neurons and function in the genetic cascade regulating neuronal differentiation, acting by different mechanisms. While cHES6-2 functions as a transcriptional repressor, cHES6-1 functions by sequestering and inhibiting other HES proteins. Together, the two chick HES6 proteins regulate neurogenesis by contributing to release differentiating neurons from Notch signalling.In the chick embryo, two Hes6-like genes in the chick genome, cHes6-1 and cHes6-2Period2 gene (Ensemble ref. ENSGALG00000005521)  . We founfeatures . These iHes6-1 throughout chick embryonic development was determined by in situ hybridization on whole-mount embryos and cryostat sections. At HH4  include the neural folds and neural tube, the cranial placodes, the infundibulum and the prospective heart  Hes6-1 expression. In the absence of Notch activity, cHes6-1 expression in the lateral mesoderm is strongly reduced  to mark cells in S-phase. Following immunohistochemistry for BrdU and in situ hybridization to detect cHes6-1 expression, we found that cells expressing cHes6-1 do not incorporate BrdU    , indicats5 genes  and we oComparison of the two subgroups of HES6 proteins reveals that a major structural difference is the size of the loop region in the bHLH domain, with the HES6-1 subgroup including proteins with shorter loops than HES6-2 proteins. The chick HES6-1 protein is particular in this aspect, as it contains the shortest loop (6 amino acids only) among all known vertebrate HES6 proteins . This unin vitro transcription assays Two other structural features might contribute to the different activities of cHES6 proteins. First, there is a specific serine residue in cHES6-1 (position 175), conserved in the HES6-1 subgroup but absent from HES6-2 proteins. Previous work on mHES6 has shown that phosphorylation of this serine  by Casein Kinase 2 leads to proteolytic degradation of the mHES6-mHES1 heterodimer, thereby contributing to the assayed neurogenic ability of mHES6 Hes5 genes, while cHES6-1 would instead work by forming inactive heterodimers with HES5 proteins. These latter proteins are known to bind DNA and repress their own transcription Hes5 gene transcription.The short loop of cHES6-1, together with the presence of serine 175 and the absence of the lysine involved in transcriptional repression, suggest that cHES6-1 does not bind DNA to repress transcription, but rather interacts with other HES proteins to inactivate them. By contrast, the longer loop of cHES6-2, the absence of the serine, and the presence of lysine 59, all suggest that cHES6-2 functions by direct DNA binding and transcriptional repression. These observations raise the hypothesis that the functional differences between cHES6-1 and cHES6-2 in our assay arise from the different DNA-binding abilities of the two proteins, with cHES6-2 being able to bind DNA and acting as a transcriptional repressor of cHes5-1 as a read-out. The first variants to be tested involved the substitution of lysine 59 of cHES6-2 by an arginine, as a similar mutation in lysine 55 of mHES7 abolished its ability to repress transcription Hes5-1 , a phenotype similar to that obtained upon overexpression of normal cHES6-2 protein , similar to overexpression of normal cHES6-1     , clearly n = 63) , 7B. In  n = 19) . TogetheDrosophila Enhancer of Split proteins Hes5-1 transcription , again contrasting with the repressive activity of the normal cHES6-2 protein. In turn, electroporation of cHES6-1AQ leads to an increase in cHes5-1 expression , similar to what we observe when misexpressing unmodified cHES6-1 , an alteration that has been shown to abolish the DNA-binding activity of several bHLH proteins  cHES6-1 .Hes5-1 transcription  , which we named cHES6-1AQΔWRPW and cHES6-2AQΔWRPW. Their electroporation lead to similar results to those obtained with the previous variants containing single modifications, causing up-regulation of cAQΔWRPW) .Hes5 transcription in our assay, with cHES6-2 acting as a direct transcriptional repressor while cHES6-1 acts by preventing cHES5 proteins from repressing transcription of their own genes.Together, these results offer further support to the hypothesis that the two cHES6 proteins act through distinct molecular mechanisms to modulate cHes5-1 expression   .Hes5 expression, suggesting that it normally acts by directly sequestering the HES5 proteins. By contrast, DNA binding ability and recruitment of TLE are absolutely essential for cHES6-2 to function as a transcriptional repressor of cHes5 genes.Altogether, these results show that cHES6-1 does not require DNA binding nor the ability to recruit repressors for its function in regulating cHes genes, named Hes6, and identify two subgroups that are present in zebrafish, medaka, Xenopus, and chick, which we named Hes6-1 and Hes6-2 . This correlation between the expression of Nodal and cHes6-1 suggests that this gene is a downstream target of Nodal signalling in the left mesoderm and might play a role in left-right asymmetry, in agreement with previous results showing that Hes6 expression is downstream of Nodal signalling in Xenopus early gastrula embryos Characterization of cHes6-1 is expressed. Our results show that asymmetric expression of cHes6-1 in the lateral mesoderm is dependent on Notch activity, thus revealing that this signalling pathway continues to be active during the process of generating L-R asymmetry in the embryo.Notch signalling is also known to be an important player in L-R asymmetry, at the time when this asymmetry is first established around the Hensen's node  has been previously fate-mapped to be the main source of gonadal cells Hes6-1 expression in the lateral mesoderm might contribute to gonad asymmetry in chick development.The transient and asymmetric expression of cHes6-1 expression also occurs in the ventral endoderm in a salt-and-pepper pattern, starting at 6-7-somite stage. The position of cHes6-1-expressing cells suggests that they are pancreatic progenitors, although one of the first known markers of these cells, Pdx1, only starts to be expressed at the 9-10-somite stage Hes6-1 is an early marker for these cells, before Pdx1. This agrees with the findings that mouse Hes6 is also expressed in pancreatic cells and that its overexpression causes up-regulation of Pdx1 expression Early cDelta1 and cHes6-2 are also expressed in pancreatic progenitors at these early stages, suggesting that Notch signalling plays a role in the early stages of pancreatic cell fate specification. However, the finding that cHes6-1 expression is negatively regulated by Notch activity in prospective pancreatic cells indicates that this gene does not function as a Notch effector in the process. Instead, as suggested by studies of pancreatic cell differentiation at later stages Hes6-1 is likely to be downstream of NEUROG3, participating in the cascade of bHLH proteins that regulate pancreatic differentiation. Since Notch signalling leads to repression of Neurog3 expression during pancreas development Hes6-1 expression by Notch activity.We have found that cHes6-1 is transiently expressed during neuronal differentiation, in post-mitotic cells that co-express proneural transcription factors. Our expression analyses show that cHes6-1 and cHes6-2 are expressed at different steps of the neuronal differentiation cascade, with cHes6-2 being expressed before cDelta1, and cHes6-1 after, although co-expression of the two cHes6 genes is still observed in some cells. We further show that cHes6-1 expression is downstream of proneural transcription factors and is not a direct target of Notch signalling, being instead repressed by Notch activity, like in the prospective pancreas. Overall, our results suggest the existence of three consecutive stages in neuronal differentiation in the developing chick spinal cord: proneural proteins and cHES6-2 act first during the decision to commit to neurogenesis, followed by the expression of the Notch ligand cDelta1 to inhibit neighbouring progenitors from differentiating and, finally, by the expression of cHES6-1 and NEUROM to regulate subsequent differentiation steps , suggesting that cells do not progress further into differentiation. This is likely due to the fact that transcription of cHes5 genes is also up-regulated by cHes6-1 expression, thereby repressing the neuronal differentiation cascade. Furthermore, the high levels of proneural gene expression required to trigger neuronal differentiation are not yet reached in all neural progenitors at the embryonic stages used for electroporation, a fact that might also explain why these cells are not able to terminally differentiate when electroporated with cHes6-1.To investigate the functional role of cHES6-1 during neurogenesis, we used gain-of-function studies in the chick embryo, by ectopically expressing the protein in the spinal cord neuroepithelium. Our findings reveal that ectopic expression of cHES6-1 promotes commitment to neuronal differentiation, as suggested by the cell-autonomous up-regulation of cHes6-1 misexpression are in striking contrast to the activities of the closely related gene cHes6-2 in the same assay, which causes down-regulation of both cDelta1 and cHes5 expression. These functional differences are likely due to the unique structural features of each cHES6 protein, in particular the different sizes of the loop region within the bHLH domain, which is known to constrain the DNA-binding capacity of HES proteins The results of cHes5-1 transcription, implying that cHES6-2 works normally as a DNA-bound transcriptional repressor. In contrast, similar variants of cHES6-1, where the ability to bind DNA and/or to recruit co-repressors is missing, are still able to up-regulate cHes5-1 transcription, like normal cHES6-1, showing that this protein does not require DNA binding nor transcriptional repression activity to function. This is further supported by our finding that overexpression of mHES6, a known protein titrator that does not require DNA binding nor the WRPW domain for its neurogenic activity Hes5-1 transcription, like cHES6-1. However, the conserved serine residue in cHES6-1 is not necessary for its capacity to up-regulate cHes5 expression, suggesting that phosphorylation and proteolytic degradation of cHES6-1/cHES5 heterodimers is not an absolute requirement for HES6-1 activity. Although a similarly mutated form of mHES6 (mHES6S183A) revealed no pro-neurogenic activity in a different assay using in vitro culture of cortical progenitors, proteolytic degradation of HES1 could still be detected in the presence of mHES6S183A To test these hypotheses, we generated several variants of the two cHES6 proteins that predictably affect their DNA-binding capacity and/or their ability to repress transcription, and used these variants in the chick electroporation assay. Our results show that variants of cHES6-2 lacking the DNA binding ability, and/or the ability to recruit the co-repressor TLE, lose the capacity to repress cHes5-expressing neural progenitors, the ectopic expression of cHES6-1 (or mHES6) would prevent cHES5 proteins from repressing transcription of their own genes, breaking this negative feedback loop and causing an increase in cHes5 transcription.Together, our results support the hypothesis that, like its mouse counterpart mHES6 Hes5 transcription, strongly suggesting that this member of the HES6 family works as a classical DNA-bound transcriptional repressor. Thus, unlike the mouse, which contains a single HES6 gene, encoding a protein that works mainly by interfering with the anti-neurogenic activity of other HES proteins, the chick embryo contains two clustered HES6 genes that function through different but complementary mechanisms to regulate neuronal differentiation.The related cHES6-2 protein, however, requires the presence of the WRPW domain and an intact DNA-binding region to effectively repress cHes6-1 is expressed in differentiating neurons, which normally do not express cNotch1 or any cHes5 gene. However, it is possible that cHES5 proteins perdure in cells committing to differentiation, even if their mRNAs cannot be detected anymore, and cHES6-1 would ensure that any remaining cHES5 activity is blocked. Another possible target of cHES6-1 is cHES6-2, as the encoding mRNAs can be detected in the same cells during neuronal differentiation , distanced 4 mm apart, were placed parallel to the neural tube under the embryo. Using an Electro Square Porator™ ECM830 (BTX), we applied 4 pulses of 35 V for 50 ms. Embryos were incubated for 16 h or 24 h and then harvested.in situ hybridization and immunofluorescence. The results obtained after 16 h or 24 h were identical.For each construct, a minimum of 4 electroporated embryos were analysed by For the various HES6 plasmids, super-coiled DNA was injected into neural tubes of chicken embryos staged HH8-HH11 at a concentration of 1 µg/µl. Gold plated electrodes (BTX Genetrode 512), distanced 4 mm apart, were placed parallel to the neural tube on the top of the embryo. Using an Electro Square Porator™ ECM830 (BTX), we applied 4 pulses of 25 V for 50 ms. Embryos were incubated for 6 h and then harvested.The expression plasmids encoding NICD, CSLDN, NEUROG2 and cHES6-2 have been already described in ovo, 100 µl of the γ-secretase inhibitor LY411575, at 10 µM in PBS, were injected under 3–8 somite staged embryos, in three independent experiments. These embryos were harvested 7 h later. Control was done with similar injections of PBS alone . After 7 h of treatment, control embryos had an average increase of 5 somites, as expected. In contrast, LY411575-treated embryos only had an average increase of 3 somites. This significant difference in the rate of somite formation between control and LY411575 treated embryos , suggests that the drug was effective in inhibiting Notch signalling during somitogenesis.To chemically inhibit Notch activity in situ hybridizations were done as described in situ hybridization on cryostat sections was done with Digoxigenin (Dig)- and Fluorescein (Fluo)-labelled RNA probes. The Dig-labelled probe was first detected with AP-conjugated anti-DIG antibody  and developed with Fast-Red substract (Roche). After washing in PBS, sections were blocked and incubated with HRP-conjugated anti-Fluo antibody , followed by FITC-Tyramide amplification, as recommended by the manufacturer .Chicken embryos were collected and fixed in 4% paraformaldehyde in PBS overnight at 4°C. Whole-mount Delta1 and cHes5-1 riboprobes have been synthesized from specific PCR fragments, to avoid any plasmid regions in the probes and eliminate regions of putative cross reactivity between the probes and transgenes.For the analysis of HES6 electroporations in the neural tube, cin situ hybridization by immunohistochemistry using a rabbit polyclonal antibody against GFP . For BrdU treatment, 100 µL (12.5 mg/mL) were dropped onto E4 chick embryos, which were harvested and fixed 30 minutes later. Antigen retrieval for BrdU-treated embryos was done using HCl 2N for 30 min at 37°C. Primary antibodies used were mouse anti-BrdU  and mouse anti-Tuj-1 . Detailed protocols are available upon request. Photos were taken using the stereoscope LeicaZ6APO with a DFC490 camera attached, fluorescent microscopes Leica DMR or Leica DM5000B with cameras DC350F or DC500 attached, and the confocal Carl Zeiss 510 Meta.Electroporated cells were visualized after