MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6

<div><p>Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions.</p></div

Primers for RT-qPCR analyses.

<p>Primers for RT-qPCR analyses.</p

Mga, L3mbtl2 and Pcgf6 colocalize in mouse ESCs and repress genes involved in differentiation.

<p>(A) Venn diagrams representing the overlap of Mga, L3mbtl2 and Pcgf6 peaks in mouse ESCs. The total number of filtered (≥30 tags and ≥3-fold enrichment over IgG control) ChIP-seq peaks and their overlap is shown. (B) A heat map view of the distribution of the top 8000 union Mga-L3mbtl2-Pcgf6 peaks in mouse ES cells at +/- 2 kb regions centred over the MGA peaks. (C) Representative genome browser screenshot of a 100 kb region of chromosome 1 showing co-binding of Mga, L3mbtl2 and Pcgf6 to four promoter regions. (D) Sequence motifs enriched in Mga-L3mbtl2-Pcgf6 binding regions in mouse ESCs. Top, logos were obtained by running MEME-ChIP with 300 bp summits of the top 600 union Mga-L3mbtl2-Pcgf6 ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, the statistical significance (E-value) and the transcription factors that bind to the motif. Bottom, local motif enrichment analysis (CentriMo) showing central enrichment of the Mga/Max bHLH domain E-box binding motif and the motif that identified MEME Tomtom as a T-box as well as a E2f6 recognition sequence. The Nrf1 motif was not centrally enriched within the 300 bp peak regions. (E) Distribution of Mga, L3mbtl2 and Pcgf6 peaks relative to positions -2000 bp upstream to +2000 bp downstream of gene bodies. TSS, transcription start site; TES, transcription end site. (F) Middle panel, Venn diagram illustrating the overlap of PRC1.6-bound genes and genes up-regulated in Pcgf6<i>ko</i> cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref026" target="_blank">26</a>] and in L3mbtl2<i>ko</i> cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref013" target="_blank">13</a>]. Left panel, GO analyses of biological functions of PRC1.6-bound genes that were de-repressed ≥2-fold in Pcgf6<i>ko</i> cells. Right panel, GO analyses of biological functions of PRC1.6-bound genes that were de-repressed ≥2-fold in L3mbtl2<i>ko</i> cells. Enriched GO terms were retrieved using DAVID 6.8. (GOTERM_BP_DIRECT, Functional Annotation Chart). Benjamini values are plotted in log10 scale.</p

PRC1.6 binding sites partially overlap with cPRC1, PRC2 and ncPRC1.1 binding sites.

<p>(A) ChIP-seq heatmaps of Pcgf6, IgG control, Ring1b (GSM1041372) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Rybp (GSM1041375) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Cbx6-HA (GSM2610616) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref033" target="_blank">33</a>], Cbx7 (GSM2610619) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref033" target="_blank">33</a>], Pcgf2 (GSM1657387) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref056" target="_blank">56</a>], Suz12 (GSM1041374) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Kdm2b (GSM1003594) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref006" target="_blank">6</a>] and H3K27me3 (GSM1341951) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref010" target="_blank">10</a>] peaks in mESCs at +/- 2 kb regions centred over the Mga-L3mbtl2-Pcgf6 peaks. (B) Venn diagrams showing the overlap of high confidence Pcgf6 target genes (location of binding sites between -2.5 kb of TSS and TES) with those of Cbx7 (cPRC1), Suz12 and H3K27me3 (PRC2) and Kdm2b (ncPRC1.1). (C) Genome browser screenshots of ChIP-seq tracks at promoters of representative meiosis-related genes (<i>Dazl</i>, <i>Sycp3</i>, <i>Stk31 and Mei1</i>) (D) Genome browser screenshots of ChIP-seq tracks at cPRC1 target genes (<i>Nkx2-4</i> and <i>Hoxa7)</i>.</p

Model summarizing PRC1.6 targeting mechanisms.

<p>(1) PRC1.6 is recruited to a subset of target promoters by direct DNA binding of MGA/MAX to E-boxes (CACGTG) and/or T-boxes (TCACACCT). (2) Interaction of L3MBTL2 and HP1γ with methylated histones may promote binding site selection by facilitating and stabilizing binding of MGA/MAX. (3) MGA also acts as a scaffold tethering E2F6 that in turn mediates PRC1.6 binding to E2F6-recognition sites. (4) PCGF6 recruits RING1/2 that deposits the repressive histone mark H2AK119ub1.</p

L3MBTL2 and E2F6 contribute to chromatin binding of PRC1.6.

<p>(A) Western blot analysis of L3MBTL2, MGA, E2F6, PCGF6 and RING2 in L3MBTL2<i>ko</i>, E2F6<i>ko</i> and PCGF6<i>ko</i> cells. Re-probing for Tubulin (TUB) controlled loading of extracts. The Tubulin blots are related to the MGA blots. Uncropped versions of the MGA blots are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.s004" target="_blank">S4 Fig</a>. (B) Representative genome browser screenshots of a 0.5 Mb region of chromosome 19 showing reduced binding of PRC1.6 components to several promoters in L3MBTL2<i>ko</i> and in E2F6<i>ko</i> cells but not in PCGF6<i>ko</i> cells. (C) Heat map views of the distribution of MGA, L3MBTL2 and E2F6 peaks in wild type cells (n = 8342) and in MGA<i>ko</i>, L3MBTL2<i>ko</i>, E2F6<i>ko</i> and PCGF6<i>ko</i> cells at +/- 2 kb regions centred over the MGA peaks. (D) Scatter plots comparing the signal intensity of MGA, L3MBTL2 and E2F6 peaks in wild type cells with the signal intensity of corresponding peaks in L3MBTL2<i>ko</i> (left panels), E2F6<i>ko</i> (middle panels) or PCGF6<i>ko</i> cells (right panels). Normalized ChIP-seq read counts in MGA ChIP-seq peak regions of wild type cells were plotted against the normalized read counts in corresponding peak regions of L3MBTL2<i>ko</i>, E2F6<i>ko</i> or PCGF6<i>ko</i> cells. (E) Left panel, scatter plot showing the correlation between reduced MGA binding and reduced L3MBTL2 binding in E2F6<i>ko</i> cells. Right panel, scatter plot showing the correlation between reduced MGA binding and reduced E2F6 binding in L3MBTL<i>ko</i> cells. The top 500 ranked MGA binding sites were used to calculate the fold change of normalized ChIP-seq read counts in L3MBTL2<i>ko</i> and E2F6<i>ko</i> cells relative to wild type cells.</p

The role of PRC1.6 in HEK293 cell function.

<p>(A) Reduced proliferation of MGA<i>ko</i>, L3MBTL2<i>ko</i> and E2F6<i>ko</i> cells. Shown are growth curves of wildtype, MGA<i>ko</i>, L3MBTL2<i>ko</i>, E2F6<i>ko</i> and PCGF6<i>ko</i> HEK293 cells. Cells were seed at 3x10⁵, and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA<i>ko</i> cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA<i>ko</i> cells; right circle, genes with ≥2-fold increased transcript levels in MGA<i>ko</i> cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the <i>CNTD1</i> and <i>SMC1B</i> promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA<i>ko</i> cell clones (MGA<i>ko</i>_cl26, MGA<i>ko</i>_cl27 and MGA<i>ko</i>_cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA<i>ko</i>, E2F6<i>ko</i>, L3MBTL2<i>ko</i> and PCGF6<i>ko</i> cells. Transcript levels were normalized to <i>B2M</i> transcript levels, and are depicted relative to transcript levels in wild type cells.</p

Commercially available antibodies used in this study.

<p>Commercially available antibodies used in this study.</p

MGA promotes binding of PRC1.6 by DNA-binding-dependent and DNA-binding-independent mechanisms.

<p>(A) Expression of wild type MGA in MGA<i>ko</i> cells rescues binding of PRC1.6. ChIP-qPCR data showing binding of transiently expressed MGA and of endogenous L3MBTL2, E2F6, PCGF6, RING2 and MAX to representative PRC1.6 target promoters. The level of the H2AK119ub1 was not affected. Percent of input values represent the mean of at least three independent experiments +/- SD. (B) Schematic representation of the MGA ΔTbox and bHLH mutants. (C) Western blot analysis of wild type MGA and of the DNA-binding-deficient MGA mutants (ΔT-Box, bHLHmut and ΔTbHLHmut) expressed in MGA<i>ko</i> cells. The anti-Tubulin blot served as a loading control. (D) ChIP-qPCR analyses of MGA and L3MBTL2 binding to selected PRC1.6 target promoters in MGA<i>ko</i> cells and in MGA<i>ko</i> cells re-expressing wild type MGA (MGA WT) or DNA-binding-deficient MGA mutants (MGA ΔT-Box, MGA bHLHmut or MGA ΔTbHLHmut). The error bars denote SD; n = 3.</p

MGA is essential for genomic binding of PRC1.6.

<p>(A) Heat map view of the distribution of union MGA, L3MBTL2 and E2F6 peaks in wild type cells (n = 8342) and in MGA-depleted cells at +/- 2 kb regions centred over the MGA peaks. (B) Representative genome browser screenshots showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the <i>AEBP2</i>, <i>RPA2</i>, <i>RFC1</i> and <i>SPOP</i> promoters in wild type cells. MGA-depleted cells lack binding of L3MBTL2 and E2F6. (C) Western blot analysis of L3MBTL2, E2F6, PCGF6 and RING2 in wild type HEK293 cells and in two different MGA-depleted clones (cl26 and cl27). The anti-Tubulin blot served as a loading control. (D) <i>L3MBTL2</i>-, <i>E2F6</i>- and <i>PCGF6</i> transcripts were determined in wild type cells and in MGA-depleted cell clones by RT-qPCR analysis. <i>B2M</i> transcript levels were used to normalize the data across samples, and transcript levels in wild type cells were arbitrarily set to 1. Data represent the average of technical replicates ± SD. (E) ChIP-qPCR data showing lack of L3MBTL2, E2F6, PCGF6, MAX, RING2, RYBP and HP1γ binding to representative PRC1.6 target promoters in MGA<i>ko</i> cells, and diminished deposition of H2AK119ub1. The <i>CDC7</i> -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) PRC1.6 target promoters are not bound by PRC2 and lack H3K27me3. Local levels of EZH2 and H3K27me3 at selected PRC1.6 target promoters in wild type (WT) and in MGA<i>ko</i> cells (clones cl26 and cl27) were determined by ChIP-qPCR analysis. Genomic regions known to be bound by canonical PRC1 (<i>FUT9</i>, <i>MYT1</i> and <i>TSH2B</i>) served as positive control regions. These regions were not bound by MGA (right panel). Percent of input values represent the mean of at least three independent experiments +/- SD.</p