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BindDB: Oct4 Case Study
No full textEpigenomic Profiling of Oct4 PromoterDue to the flexibility of the BindDB platform, more general questions related to epigenetic regulation in the ESC context can be asked. For example, how are the alternative promoters of the stem cell specific transcription factor OCT4 regulated? In both mESCs and hESCs, two main isoforms of Oct4 exist (Oct4A, Oct4B), which differ by approximately 3kb in their 5' regions and have the potential to be regulated by two distinct promoter regions (Wang and Dai, 2010). Interestingly, Oct4A and Oct4B display different temporal and spatial expression patterns (Cauffman et al., 2006). It has been hypothesized that the Oct4A is the pluripotent promoter, whereas, Oct4B is related to stress responses (Wang et al., 2009).To study these promoters in greater depth, we queried ‘Pou5f1’ in BindDB (Figure 2A). The tool automatically discerns the presence of two alternative promoters while searching for the gene's genomic location and directs the user to a heatmap of per-promoter results. From the heatmap, it is apparent that both promoters share some of their epigenetic signature including H3K4me3, H3K27ac and H3K9ac active chromatin marks (Figure 2B). Also, the OCT4 protein itself binds both of its promoters, along with other pluripotent factors such as UTX and KLF4. Beyond this common epigenetic core, the two promoters differ widely: The Oct4A promoter of the longer isoform displays binding for a plethora of factors and evidence of RNAPII suggests that Oct4 is actively transcribed in ESCs. In general, this type of large-scale epigenetic profile is characteristic of active gene regulation and goes far beyond the simple histone modification code. On the other hand, the Oct4B promoter exhibits binding for an entirely different set of factors including SMC1, SMC3, SA1, SA2, RAD21, and CTCF, the main components of the structural Cohesin complex. It is also bound by ZC3H11A, which we found to cluster globally with the Cohesin complex (data not shown). Cohesin binding to the Oct4B promoter suggests that a specific enhancer may be driving the expression of this second isoform in ESCs. On the other hand, the mediator proteins MED1 and MED12 bind the first promoter and do not cluster with the Cohesin complex as they are proposed to do. These findings may be particularly insightful into discovering the molecular mechanisms governing alternative promoter regulation of Oct4 and genes in general
BindDB: LincRNAs Case Study
No full textEpigenetic Classification of Novel Genes Provides Insights into Functional SubgroupsThe introduction of NGS platforms has greatly facilitated the discovery of novel genes, especially non-coding genes, which were under the radar of the previous methodologies. By combining epigenetic analysis to the regions of novel transcription, 1000s of novel long intervening non-coding RNAs, or lincRNAs, residing in intergenic regions were discovered (Guttman et al., 2009; Khalil et al., 2009). The understanding that histone marks can point to areas of active and functional transcription enabled the use of a minimal epigenetic profile ("K4-K36"), to locate regions with H3K4me3 marking a promoter region, followed by H3K36me3 within the potential coding region. But, epigenetic regulation of active genes could be far more complex. We applied the BindDB analysis capabilities to this novel group of genes in order to ask whether their promoters are regulated similarly to protein-coding genes and test if we could expand it beyond the basic "K4-K36" profile. We uploaded a list of 2074 genomic regions (in BED format since many are not annotated with gene symbols) classified as lincRNAs in the Ensembl annotation (Hubbard et al., 2002) according to the algorithms in Guttman et al. (Guttman et al., 2009), yet also include strand information from which promoter regions can be deduced. These regions were merged by BindDB into 1729 non-overlapping proximal promoter regions. It is striking to see a noticeable enrichment for almost all factors beyond H3K4me3 (1.55-2.65-fold, log2 scale, see Supplemental Experimental Procedures) and H3K36me3 (0.04-1.25-fold) (FigureS3). The low enrichment of H3K36me3 persisted when we examined only lincRNA bodies compared to all gene bodies in a separate analysis (0.54-1.92), indicating that lincRNAs have similar levels of H3K36me3 as in protein-coding genes. The clustered heatmap view of all factors and histone modification along all lincRNA promoters (Figure1B) gives us two important insights into this large group. First, there is quite an extensive epigenetic profile for many of them involving active histone modification and transcription factors. Second, there are as much as 20% of lincRNA annotations that do not display such a profile (Figure1B, orange star). This group also exhibits none to low levels of RNA expression and may include lincRNAs specific to non-ESC types or false-positives resulting from the extensive statistical methods applied in search of the "K4-K36" profile along with some regions that have only the minimal "K4-K36" profile. We can speculate that this minimal profile may not be enough to support bona-fide regulated transcription, at least not in ESCs, but the deposit of histone modifications may be a byproduct of another process
BindDB: Pseudogenes Case Study
No full textAs a negative control for BindDB, we uploaded a list of 76 pseudogene names, identified by the 'ps' extension to the gene symbols in the Refseq annotation, to the multi-gene query form of BindDB and selected to query their proximal promoter regions. Overall, the epigenetic profile of these pseudogenes was that of inactive chromatin. Sorting by the "Enrichment" column of the positive results table shows depletion of epigenetic marks that are associated with active transcription such as H3K4me1, H3K36me3, H3K27ac and H3K79me2 (Figure S2A). In agreement, the heatmap shows very little evidence of factor binding to most of the pseudogenes and the prominent yellow color of the sidebar indicates that there is no evidence of RNA expression coming from these regions either(Figure S2B). Yet, a handful of pseudogenes, including Rpl34-ps and Pisd-ps1/2,display evidence of H3K4me3 and RNAP II bindingas well as RNA expression (Figure S2B, top cluster).These pseudogenes are also bound by many transcription factors, including those from the pluripotent network, such as OCT4, indicating that this small group of genes are likely expressed in ESCs and may be inaccurately annotated as "pseudogenes". In the case of Rpl34-ps, its annotation completely overlaps the non-pseudo gene version of the same gene, such that it shares its epigenetics with Rpl34 itself, a key ribosomal protein that is expressed in ESCs. For Pisd-ps1/2, the Refseq and Ensembl annotations have categorized it as a non-coding transcript, hence misleadingly termed a pseudogene. By clicking the gene name, Pisd-ps1, on the BindDB results page, the UCSC genome browser(Kent et al., 2002)opens up at the gene's genomic location along with the ENCODE/LICR Histone Mods(ENCODE, 2012) and RNA-seq tracks for E14 ESCs(Mortazavi et al., 2008) providing a more focused view on this gene (Figure S2C). Its active epigenetic profile, including OCT4 binding, makes it an interesting candidate for the study of ncRNA in ESCs. Running the same analysis on a larger cohort of ~5500 non-overlapping pseudogenes in the Ensembl annotation, reassuringly replicated the same general lack of factor binding and histone gene modifications at their promoters (data not shown)
BindDB: Bivalent genes Case Study
No full textValidate and Expand Established Epigenetic ProfilesIn order to validate the ability of BindDB to detect an already well-established epigenetic profile, we began with the large group of 'bivalent' genes coined on the premise of the epigenetic characteristics of their promoters. These 3913 genes in human and 2984 genes in mouse(Azuara et al., 2006; Bernstein et al., 2006; Li et al., 2013) have the H3K4me3 active promoter hallmark as well as the H3K27me3 repressive histone modification in close proximity to their transcriptional start sites and are lowly expressed in ESCs(Bernstein et al., 2006). The repressed nature of the bivalent promoters in ESCs is conferred by the Polycomb repressive complexes (PRC), which interact with the H3K27me3 mark. We uploaded the list of bivalent genes by gene name to BindDB, selected the 'proximal promoter (-1000, +1000) as the gene portion to query and within less than 5 minutes, could confirm all of the above. Enrichment score analysis (the ratio of factor binding to the queried gene promoters divided by the ratio of factor binding to all gene promoters) clearly indicates an enrichment in the characteristic "K4-K27" (Figure S1A, blue arrows) histone marking at bivalent gene promoters. In addition, the depletion of H3K36me3 indicates that these genes, although exhibiting an active chromatin mark, are not expressed at high levels. H3K36me3 is also depleted across the bivalent gene bodies when compared to all gene bodies (data not shown).This finding complies with the hypothesis that PHF19, a component of the Polycomb complex, recruits histone-lysine demethylase NO66 in order to reduce levels of H3K36me3 at bivalent genes(Brien et al., 2012) and is strengthened by the enrichment of H3K36me2 instead, and PHF19 itself at these promoters.In addition to PHF19, many other components of the Polycomb complexes can be found as well, including EZH1 and EZH2, SUZ12, JARID2, and RING1 proteins. The presence of KDM2A and KDM2B may imply that these proteins are involved in the demethylation of either lysine 4 or lysine 27 during cell fate determination. Alternatively, KDM2B (FBXL10) along with CBX7, which also shows enrichment in the BindDB analysis (Figure S1A, left), have been shown to be involved in the recruitment of the PRC1 complex to the H3K27me3 histone mark (He et al., 2013; Morey et al., 2012). Strikingly, hardly any significant enrichment of transcription factors could be detected at bivalent genes in ESCs (Figure S1B, filtered heatmap), except for OCT4 and SOX2, in line with previous findings that these key ESC transcription factors bind approximately one third of PRC2-occupied genes that also encode developmental transcription factors (Boyer et al., 2006; Lee et al., 2006). This phenomenon is unique to this group of genes (see subsequent examples) and signifies the important role of epigenetic regulation on these 'poised', yet inactive genes in ESCs
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
BindDB: Bivalent genes Case Study
No full textValidate and Expand Established Epigenetic Profiles
In order to validate the ability of BindDB to detect an already well-established epigenetic profile, we began with the large group of 'bivalent' genes coined on the premise of the epigenetic characteristics of their promoters. These 3913 genes in human and 2984 genes in mouse(Azuara et al., 2006; Bernstein et al., 2006; Li et al., 2013) have the H3K4me3 active promoter hallmark as well as the H3K27me3 repressive histone modification in close proximity to their transcriptional start sites and are lowly expressed in ESCs(Bernstein et al., 2006). The repressed nature of the bivalent promoters in ESCs is conferred by the Polycomb repressive complexes (PRC), which interact with the H3K27me3 mark. We uploaded the list of bivalent genes by gene name to BindDB, selected the 'proximal promoter (-1000, +1000) as the gene portion to query and within less than 5 minutes, could confirm all of the above. Enrichment score analysis (the ratio of factor binding to the queried gene promoters divided by the ratio of factor binding to all gene promoters) clearly indicates an enrichment in the characteristic "K4-K27" (Figure S1A, blue arrows) histone marking at bivalent gene promoters. In addition, the depletion of H3K36me3 indicates that these genes, although exhibiting an active chromatin mark, are not expressed at high levels. H3K36me3 is also depleted across the bivalent gene bodies when compared to all gene bodies (data not shown).This finding complies with the hypothesis that PHF19, a component of the Polycomb complex, recruits histone-lysine demethylase NO66 in order to reduce levels of H3K36me3 at bivalent genes(Brien et al., 2012) and is strengthened by the enrichment of H3K36me2 instead, and PHF19 itself at these promoters.
In addition to PHF19, many other components of the Polycomb complexes can be found as well, including EZH1 and EZH2, SUZ12, JARID2, and RING1 proteins. The presence of KDM2A and KDM2B may imply that these proteins are involved in the demethylation of either lysine 4 or lysine 27 during cell fate determination. Alternatively, KDM2B (FBXL10) along with CBX7, which also shows enrichment in the BindDB analysis (Figure S1A, left), have been shown to be involved in the recruitment of the PRC1 complex to the H3K27me3 histone mark (He et al., 2013; Morey et al., 2012). Strikingly, hardly any significant enrichment of transcription factors could be detected at bivalent genes in ESCs (Figure S1B, filtered heatmap), except for OCT4 and SOX2, in line with previous findings that these key ESC transcription factors bind approximately one third of PRC2-occupied genes that also encode developmental transcription factors (Boyer et al., 2006; Lee et al., 2006). This phenomenon is unique to this group of genes (see subsequent examples) and signifies the important role of epigenetic regulation on these 'poised', yet inactive genes in ESCs
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