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Distinct isoforms of the Drosophila Brd4 homologue are present at enhancers, promoters and insulator sites.

Kellner WA, Van Bortle K, Li L, Ramos E, Takenaka N, Corces VG - Nucleic Acids Res. (2013)

Bottom Line: Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph.Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present.The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Emory University, Atlanta, GA 30322, USA and Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

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Sites of Fs(1)h-S correlate with gene expression and show a different binding profile than Fs(1)h-L. (A) TSSs were sorted by expression level from highest to lowest and given up/downstream orientation according to strand. The total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream from each TSS and viewed in Java Treeview. (B) Peaks unique to Fs(1)h-S and Fs(1)h-L data sets present around TSSs were plotted in 20 bp bins, and the average profile was plotted covering 200 bp upstream and downstream from the TSS and compared with the average profile of H3K4me3. Read counts within the window were normalized to the total reads within the window and plotted as a fold enrichment over the baseline calculated, as the average number of reads across the window to compare between data sets. (C) Example of a gene with an Fs(1)h-L&S peak shows that the Fs(1)h-L summit is found slightly upstream of the TSS, and the Fs(1)h-S summit is found slightly downstream of the TSS but still has enrichment upstream from the Fs(1)h-L cross-reactivity. (D) Enhancers identified by H3K4me1 enrichment were used as anchors around which the total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream. The enrichments were then sorted by Fs(1)h-L&S intensity and viewed in Java Treeview.
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gkt722-F3: Sites of Fs(1)h-S correlate with gene expression and show a different binding profile than Fs(1)h-L. (A) TSSs were sorted by expression level from highest to lowest and given up/downstream orientation according to strand. The total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream from each TSS and viewed in Java Treeview. (B) Peaks unique to Fs(1)h-S and Fs(1)h-L data sets present around TSSs were plotted in 20 bp bins, and the average profile was plotted covering 200 bp upstream and downstream from the TSS and compared with the average profile of H3K4me3. Read counts within the window were normalized to the total reads within the window and plotted as a fold enrichment over the baseline calculated, as the average number of reads across the window to compare between data sets. (C) Example of a gene with an Fs(1)h-L&S peak shows that the Fs(1)h-L summit is found slightly upstream of the TSS, and the Fs(1)h-S summit is found slightly downstream of the TSS but still has enrichment upstream from the Fs(1)h-L cross-reactivity. (D) Enhancers identified by H3K4me1 enrichment were used as anchors around which the total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream. The enrichments were then sorted by Fs(1)h-L&S intensity and viewed in Java Treeview.

Mentions: To further investigate the role of Fs(1)h-S present in regions enriched in H3K4me3 presumed to be promoters, TSSs from a Kc cell expression array (18,19) were sorted by expression level and given strand-specific orientation. The enrichment of Fs(1)h-L&S follows the same pattern as H3K4me3, a well-characterized histone modification correlated with transcription levels (Figure 3A). As expected, the binding profile of Fs(1)h-L is different from that of Fs(1)h-L&S. The heatmap in Figure 3A indicates that some sites of Fs(1)h-L are present at TSSs, but when present close to promoters, the long Fs(1)h isoform appears to bind at a slightly different location than Fs(1)h-S and does not correlate as well with transcription levels (Figure 3A and B). Fs(1)h-L binds on average at −20 bp with respect to the TSS, in what is characterized as the histone-free region of the promoter. Conversely, Fs(1)h-S is present at +140 bp with respect to the TSS at the same location as the peak of H3K4me3, a histone modifications known to occur at promoters along with hyperacetylated histone H3 and H4 to which Fs(1)h is reported to bind during transcription activation (Figure 3B). The location of the summit of Fs(1)h-L is slightly upstream from the TSS, whereas the Fs(1)h-S summit is slightly downstream at promoters having both isoforms (Figure 3C), supporting the hypothesis that the two isoforms perform different functions. The results also suggest that, if the bromodomains of the two isoforms are involved in their targeting to chromatin, they may do so by recognizing acetylated residues in different proteins.Figure 3.


Distinct isoforms of the Drosophila Brd4 homologue are present at enhancers, promoters and insulator sites.

Kellner WA, Van Bortle K, Li L, Ramos E, Takenaka N, Corces VG - Nucleic Acids Res. (2013)

Sites of Fs(1)h-S correlate with gene expression and show a different binding profile than Fs(1)h-L. (A) TSSs were sorted by expression level from highest to lowest and given up/downstream orientation according to strand. The total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream from each TSS and viewed in Java Treeview. (B) Peaks unique to Fs(1)h-S and Fs(1)h-L data sets present around TSSs were plotted in 20 bp bins, and the average profile was plotted covering 200 bp upstream and downstream from the TSS and compared with the average profile of H3K4me3. Read counts within the window were normalized to the total reads within the window and plotted as a fold enrichment over the baseline calculated, as the average number of reads across the window to compare between data sets. (C) Example of a gene with an Fs(1)h-L&S peak shows that the Fs(1)h-L summit is found slightly upstream of the TSS, and the Fs(1)h-S summit is found slightly downstream of the TSS but still has enrichment upstream from the Fs(1)h-L cross-reactivity. (D) Enhancers identified by H3K4me1 enrichment were used as anchors around which the total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream. The enrichments were then sorted by Fs(1)h-L&S intensity and viewed in Java Treeview.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814382&req=5

gkt722-F3: Sites of Fs(1)h-S correlate with gene expression and show a different binding profile than Fs(1)h-L. (A) TSSs were sorted by expression level from highest to lowest and given up/downstream orientation according to strand. The total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream from each TSS and viewed in Java Treeview. (B) Peaks unique to Fs(1)h-S and Fs(1)h-L data sets present around TSSs were plotted in 20 bp bins, and the average profile was plotted covering 200 bp upstream and downstream from the TSS and compared with the average profile of H3K4me3. Read counts within the window were normalized to the total reads within the window and plotted as a fold enrichment over the baseline calculated, as the average number of reads across the window to compare between data sets. (C) Example of a gene with an Fs(1)h-L&S peak shows that the Fs(1)h-L summit is found slightly upstream of the TSS, and the Fs(1)h-S summit is found slightly downstream of the TSS but still has enrichment upstream from the Fs(1)h-L cross-reactivity. (D) Enhancers identified by H3K4me1 enrichment were used as anchors around which the total number of reads is plotted for each ChIP-seq data set in 20 bp bins covering 1 kb upstream and downstream. The enrichments were then sorted by Fs(1)h-L&S intensity and viewed in Java Treeview.
Mentions: To further investigate the role of Fs(1)h-S present in regions enriched in H3K4me3 presumed to be promoters, TSSs from a Kc cell expression array (18,19) were sorted by expression level and given strand-specific orientation. The enrichment of Fs(1)h-L&S follows the same pattern as H3K4me3, a well-characterized histone modification correlated with transcription levels (Figure 3A). As expected, the binding profile of Fs(1)h-L is different from that of Fs(1)h-L&S. The heatmap in Figure 3A indicates that some sites of Fs(1)h-L are present at TSSs, but when present close to promoters, the long Fs(1)h isoform appears to bind at a slightly different location than Fs(1)h-S and does not correlate as well with transcription levels (Figure 3A and B). Fs(1)h-L binds on average at −20 bp with respect to the TSS, in what is characterized as the histone-free region of the promoter. Conversely, Fs(1)h-S is present at +140 bp with respect to the TSS at the same location as the peak of H3K4me3, a histone modifications known to occur at promoters along with hyperacetylated histone H3 and H4 to which Fs(1)h is reported to bind during transcription activation (Figure 3B). The location of the summit of Fs(1)h-L is slightly upstream from the TSS, whereas the Fs(1)h-S summit is slightly downstream at promoters having both isoforms (Figure 3C), supporting the hypothesis that the two isoforms perform different functions. The results also suggest that, if the bromodomains of the two isoforms are involved in their targeting to chromatin, they may do so by recognizing acetylated residues in different proteins.Figure 3.

Bottom Line: Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph.Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present.The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Emory University, Atlanta, GA 30322, USA and Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

Show MeSH
Related in: MedlinePlus