SIN3A binding at the two classes of HDAC2 target genes in <i>HDAC2</i> nulls.

<p><b>(A-B)</b> ChIP-qPCR was conducted on crosslinked chromatin of WT, HDAC2 null clones #5, #14, and #15 with antibodies to SIN3A or IgG control and qPCR analysis was conducted to evaluate the binding of SIN3A at the promoter regions of HDAC2-repressed gene targets <b>(A)</b> or HDAC2-activated gene targets <b>(B).</b> Enrichment was calculated by normalization to input and IgG control samples and plotted as mean of n = 3 with error bars representative of S.E.M.</p

CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cells

<div><p>The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt <i>HDAC2</i> in human cells. We find that while <i>HDAC2</i> null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.</p></div

HDAC2-activated gene targets.

<p><b>(A)</b> qPCR validation of candidate targets of HDAC2 activation as determined by RNA-seq. WT cells or <i>HDAC2</i> null clonal lines #5, #14 and #15 cDNA were analyzed by qPCR analysis in triplicates for transcript levels of the indicated candidate target genes. Gene expression was internally normalized to GAPDH and represented relative to WT. All three clonal lines exhibited decreased target gene expression with p<0.05 for <i>RECQL4</i>, <i>SNX22</i>, <i>TP53BP1</i>, and p<0.001 for <i>CCT5</i> and <i>RPS6</i>. <b>(B-E)</b> ChIP-qPCR was conducted on crosslinked chromatin of WT and clones #5, #14, and #15 with antibodies to HDAC2 <b>(B)</b>, HDAC1 <b>(C)</b>, H3K9ac <b>(D)</b>, H3K9me3 <b>(E)</b> or IgG control and SYBR green qPCR analysis was conducted to evaluate the binding of HDAC2 or HDAC1 or enrichment of the specified histone marks at the promoter regions of the genes indicated. Enrichment was calculated by normalization to input and IgG control samples and samples are plotted as mean of n = 3 with error bars representative of S.E.M.</p

RNA-seq analysis of HDAC2 affected genes.

<p>RNA-seq was conducted on control and <i>HDAC2</i> null clonal lines to assess transcriptomic effects. <b>(A)</b> Heatmap plot of log normalized counts for significantly upregulated and downregulated genes found in the three lines of <i>HDAC2</i> nulls compared to WT cells (p<0.01). <b>(B)</b> Plot of overlap in transcriptomic changes between each of the three cell lines. The Y axis plots the number of genes that overlap for each comparison, with the color of the bar indicating the p-value, and the different gene set comparisons are indicated on the X axis by green circles. <b>(C)</b> Proportions of downregulated and upregulated genes in each <i>HDAC2</i> null cell line indicate approximately equal numbers of differentially expressed genes. <b>(D)</b> Gene ontology analysis of genes upregulated by <i>HDAC2</i> disruption. <b>(E)</b> Gene ontology analysis of genes downregulated by HDAC2 disruption.</p

An overall model of the two classes of HDAC2-target genes.

<p>HDAC2-repressed gene targets require the presence of HDAC2 for both HDAC2 and HDAC1 binding at gene targets as evident by the loss of enrichment of both with disruption of <i>HDAC2</i>. In the second class, novel HDAC2-activated target genes, HDAC2 is not necessary for the recruitment of HDAC1 to target genes. In both classes of gene targets, SIN3 enrichment is variably altered but not completely disrupted. Varying degrees of increases in histone acetylation, mainly at H3K9, at gene targets is observed with loss of <i>HDAC2</i>.</p

The sequence surrounding the core motif Mizm1 is dispensable for Miz-1 binding.

<p>When the sequence surrounding the Mizm1 motif is mutated (P1m3), the unlabeled probe retains its ability to compete with labeled probe P1 or P2 to bind MBP-Miz-1-FL (A-B) or MBP-Miz-1-ZF (C-D). Representative images are shown (A, C), along with quantification of three replicate experiments (B, D). * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Probe sequences used in EMSA experiments.

<p>The motifs Mizm1 (probe P1) and Mizm2 (probe P2) are underlined, while mutations are bolded.</p

Miz-1 Activates Gene Expression via a Novel Consensus DNA Binding Motif

<div><p>The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput <i>in vitro</i> technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences—ATCGGTAATC and ATCGAT (Mizm1 and Mizm2)—bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the <i>in vivo</i> relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed <i>in vivo</i> to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.</p></div

Luciferase reporter assays in HeLa cells demonstrate that Miz-1 activates gene expression via Mizm1.

<p>(A) Four luciferase reporter vectors were constructed: pGL3ec containing no putative Miz-1 binding motifs, pGL3e-MizM containing four repeats of both the Mizm1 and Mizm2 motifs upstream of the transcription start site, pGL3e-Mizm1 containing four repeats of the P1 probe sequence, and pGL3e-Mizm2 containing four repeats of the P2 probe sequence. (B) Miz-1 overexpression in HeLa cells produces a statistically significant increase in luciferase reporter activity with all of the three reporter vectors containing putative Miz-1 binding motifs. (C) Three mutant luciferase reporter vectors were constructed, containing two (Mizm1mut2), three (Mizm1mut3), or five (Mizm1mut5) changes in highly conserved bases of the motif. (D) Miz-1 overexpression produces a statistically significant increase in luciferase reporter activation in the presence of Mizm1, but the effect is eliminated by mutating as few as two bases in the motif. (E) Overexpression of c-Myc does not synergize with Miz-1; instead, c-Myc overexpression produces a statistically significant increase in luciferase activity for all conditions: with or without Miz-1 overexpression, and with or without the presence of Miz-1 binding motifs. Luciferase expression was normalized to expression of the Renilla luciferase control reporter vector and to luciferase expression in untreated HeLa cells. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. EV  =  empty vector control; RC  =  reverse complement.</p

Analysis of ChIP-seq peak locations with respect to genes.

<p>(A-B) Peaks lacking MDAm are highly concentrated within 1 kb of the TSS in the ChIP-seq data sets from MDA cells (A) and NPCs (B), while in both cases, peaks containing MDAm are less likely to be localized near the TSS. Density plots were generated in R using the ggplot2 package; peaks occurring more than 50 kb from the nearest TSS were plotted at +/−50 kb. (C-D) Homer annotations of peak locations for ChIP-seq peaks from MDA cells (C) and NPCs (D). The promoter is defined as −1 kb to +100 bp surrounding the TSS; TTS (transcription termination site) is defined as −100 bp to +1 kb surrounding the TTS. Peaks containing MDAm were identified using FIMO, and the distance to nearest TSS and gene-centered annotations were determined using Homer with all RefSeq human (A, C) or mouse (B, D) genes. *** <i>p</i><0.0001 (Chi-squared test).</p