PMC

A model depicting the role of SIN3 isoforms in the regulation of gene expression

Genome-wide occupancy profile of SIN3 isoforms. a The chromosomal distribution of SIN3 isoforms across the Drosophila melanogaster genome as determined by the cis-regulatory element annotation system (CEAS). The P-value represents the significance of relative enrichment over genome. It was calculated using a one-sided binomial test. b The metagene analysis of all peaks showing the enrichment of SIN3 isoforms around the transcription start site (TSS) and the transcription end site (TES). c Bar plot representing the enrichment of SIN3 isoforms over genomic features

SIN3 isoform binding sites largely overlap. a Binding of SIN3 187HA and SIN3 220HA are depicted as standard genomic tracks on the integrated genomic viewer. Peaks were aligned to the built-in Drosophila melanogaster gene annotation track, shown in blue. Exons are shown in solid blue, introns as blue lines and arrows indicate the directionality of genes. SIN3 187HA and SIN3 220HA peaks as called by MACS2 are displayed in dark green and dark orange bars, respectively, below their enrichment tracks. Input tracks are shown in green for SIN3 187HA and orange for SIN3 220HA. All peaks shown are highly significant, P-value <1e-20. b Venn diagram showing the overlap of peaks between the SIN3 187HA and the SIN3 220HA ChIP-seq data. A minimum of 50 % overlap between peaks of the two SIN3 isoforms was considered

SIN3 187 distinctly regulates differentiation related genes. a Average ChIP signal of SIN3 187HA over the genes directly regulated by SIN3 187. b qRT-PCR verification of genes identified by RNA-seq to change in expression after overexpression of SIN3 187 versus control (left) but not affected upon Sin3A knockdown versus GFP RNAi (right). P-value * <0.05, *** <0.0001. c Gene ontology analysis of the Class C genes identified in Fig.  are represented as bar plots. The Y-axis lists the broad GO categories. Red: categories of genes that are directly repressed. Blue: categories of genes that are directly activated. P-value (gene ontology categories) <0.05. Biological processes labelled in bold indicate the unique processes that are overrepresented for genes distinctly regulated by SIN3 187

SIN3 187 regulates distinct as well as common genes as that of SIN3 220. a Scatter plots showing the correlation between genes bound and that change in expression upon overexpression of SIN3 187. Green dots denote genes regulated by SIN3 187 but not bound. Red dots indicate genes that are regulated by SIN3 187 and are bound by both SIN3 isoforms. Black dots refer to genes uniquely bound and regulated by SIN3 187. b Venn diagrams showing the comparison of genes directly regulated by SIN3 isoforms. Different classes of genes are labelled as A, B, C and D. r, repression. a, activation. c Schematic showing the regulation of Class A, B and C genes by the SIN3 isoforms. The middle column shows the genes bound by SIN3 220 in wild type cells. Gene expression upon Sin3A (SIN3 220) knockdown is depicted in the left column and gene expression upon ectopic expression of SIN3 187HA is represented in the right column

ChIP-seq and transcriptome analysis reveal the genes directly regulated by SIN3 220. a Western blot for SIN3 of whole cell extracts prepared from GFP RNAi or Sin3A knockdown (KD) cells. β-Actin was used as a loading control. b Scatter plot showing the correlation between genes bound by SIN3 220HA and those that change in expression following Sin3A knockdown. Green dots denote genes regulated by SIN3 220 but not bound. Red dots indicate genes that are regulated by SIN3 220 and bound by both SIN3 isoforms. Black dots refer to genes uniquely bound and regulated by the SIN3 220 isoform. c Average ChIP signal of SIN3 220HA over the genes as identified in (b). d qRT-PCR verification of genes identified by RNA-seq to change in expression upon Sin3A knockdown versus GFP RNAi. P-value ** <0.01, *** <0.0001. e Gene-ontology analysis of the genes identified in (b) are represented as bar plots. The Y-axis lists the broad GO categories. Red: categories of genes that are directly repressed. Blue: categories of genes directly activated. P-value (gene ontology categories) <0.05

Expression of SIN3 187 affects levels of SIN3 220. a Schematic representing the different cell lines used in this study as well as the SIN3 isoform expressed in that line. b Western blot analysis of whole cell extract prepared from S2 cells, SIN3 220HA (left) and SIN3 187HA (right) cell lines. The expression of SIN3 220 with a C-terminal HA tag was driven by an inducible metallothionein promoter. Due to leakiness of the metallothionein promoter, the maximum level of expression of SIN3 220HA was achieved without induction. The SIN3 187HA cell line was treated with 0.07 M of CuSO₄ to induce the transgene. Blots were probed with antibodies listed at the right. SIN3 PAN antibody recognizes all SIN3 isoforms. * denotes degradation product of SIN3 220HA. β-Actin or α-Tubulin was used as a loading control. c, d ChIP was performed on chromatin prepared from S2 (control), SIN3 220HA (c) or SIN3 187HA (d) cells using antibody against the HA tag. Immunoprecipitated DNA was quantified by quantitative PCR (qPCR). PCR amplification of ChIP DNA was carried out using primers designed within 500 bp upstream or downstream of the transcription start site (TSS) of all genes. Enrichment of SIN3 isoforms at gene targets is represented as a mean value of the percent of input ± standard error of the mean from three independent biological replicates