Suz12 binds to silenced regions of the genome in a cell-type-specific manner

Abstract

Suz12 is a component of the Polycomb group complexes 2, 3, and 4 (PRC 2/3/4). These complexes are critical for proper embryonic development, but very few target genes have been identified in either mouse or human cells. Using a variety of ChIP-chip approaches, we have identified a large set of Suz12 target genes in five different human and mouse cell lines. Interestingly, we found that Suz12 target promoters are cell type specific, with transcription factors and homeobox proteins predominating in embryonal cells and glycoproteins and immunoglobulin-related proteins predominating in adult tumors. We have also characterized the localization of other components of the PRC complex with Suz12 and investigated the overall relationship between Suz12 binding and markers of active versus inactive chromatin, using both promoter arrays and custom tiling arrays. Surprisingly, we find that the PRC complexes can be localized to discrete binding sites or spread through large regions of the mouse and human genomes. Finally, we have shown that some Suz12 target genes are bound by OCT4 in embryonal cells and suggest that OCT4 maintains stem cell self-renewal, in part, by recruiting PRC complexes to certain genes that promote differentiation.

Figure 1.

Identification of Suz12 target genes. (A) The number of genes commonly found on the two biological replicates of the Suz12 ChIP-chip experiments were compared in bins of 200, through the top 10,000 ranked genes on each list (Suz12). Also shown are the number of genes found to be in common using randomized Suz12 data sets (Random). Finally, the number of genes in common at each point in the comparison of the randomized data sets was subtracted from the number of genes in common in the two ranked Suz12 ChIP-chip data sets (Corrected). (B) PCR confirmations (using a third biological replicate of Suz12 amplicons) of Suz12 binding to a set of promoters identified in the ChIP-chip assays. All enrichments were normalized to the enrichment at the Wnt1 promoter. For these experiments, the number of PCR cycles was kept low to ensure that the signals were within the linear range of the assay, providing a semiquantitative analysis.

Figure 2.

Suz12 target genes are cell type specific. Shown are the categories of Suz12 target genes, as determined using the program DAVID, for (A) the 1076 set of F9 Suz12 targets, (B) the top 1000 Suz12 targets in mES cells, (C) the top 600 SUZ12 targets from Ntera2 cells, (D) the top 600 SUZ12 targets from SW480 cells, (E) the top 600 SUZ12 targets from MCF7 cells, and (F) a set of 1000 randomly chosen promoters. Shown in parentheses are the P-values, which indicate the probability that the category has been identified by random chance. The black bars indicate Suz12 target genes that are stem cell or germ cell tumor specific, the hatched bars indicate Suz12 target genes that are common to all cell types, and the open bars indicate the most common categories of genes in a randomly chosen set of 1000 promoters.

Figure 3.

Analysis of SUZ12 target promoters. Shown are the SUZ12 binding patterns on four top ranked SUZ12 target promoters from SW480 (left panel) and Ntera2 (right panel) cells. The black bar indicates the 5-kb region of each promoter that is tiled by the oligomer probes.

Figure 4.

PRC complexes can silence large regions in a cell-type-specific manner. (A) SUZ12 ChIP-chip analysis of a 500-kb region of the HOXA cluster, using three different cell lines. (B) SUZ12, EZH2, and H3me3K27 ChIP-chip analysis of a 500-kb region of the HOXA cluster, using human Ntera2 cells. The coding regions of the indicated genes are shown by the black boxes.

Figure 5.

Custom ChIP-chip analysis of mouse F9 cell Suz12 target promoters. Custom ChIP-chip analysis using mouse F9 cells of (A) a 10-kb region of a Suz12 target gene, (B) a 10-kb region of the negative control RNAPII promoter, or (C) a 100-kb region of the HoxA cluster.

Figure 6.

Most Suz12 target genes are bound by Eed1. Shown is the percentage of Suz12 targets found in the top 5000 (in bins of 1000) Eed1 target genes in F9 and mES cells (indicated as F9 and mES). Also shown is the “corrected” percentage of Suz12 target genes found in the top 5000 Eed1 target genes for both mouse F9 and mES cells (indicated as F9 Corrected and mES Corrected). The “corrected” percentage was calculated by subtracting the percentage of Suz12 target genes that was found in a randomized set of promoters (indicated as random) from the number found in the ranked set of Eed1 targets for F9 or mES cells.

Figure 7.

Different PRC complexes regulate different types of Suz12 targets. Shown are the categories, as determined using the program DAVID, for (A) F9 cell Suz12⁺, Eed1⁺ and (B) F9 cell Suz12⁺, Eed1⁻ target genes. The black bars indicate Suz12 target genes that are stem cell or germ cell tumor specific, and the hatched bars indicate Suz12 target genes that are common to all cell types. Shown in parentheses are the P-values, which indicate the probability that the category has been identified by random chance. For the Suz⁺, Eed1⁺ targets, the median Suz12 value was 1.816 (log₂), and the median Eed1 value was 0.762 (log₂). For the Suz12⁺, Eed1⁻ targets, the median Suz12 value was 0.91 (log₂), and the median Eed1 value was 0.205 (log₂). (C) PCR confirmations were performed to confirm the existence of two classes of target genes, those bound by Suz12, Ezh2, and Eed1 and those bound by Suz12 and Ezh2, but not by Eed1.

Figure 8.

Promoters bound by both SUZ12 and OCT4 in Ntera2 cells. DAVID analysis of the set of (A) OCT4⁺ RNAPII⁺ and (B) OCT4⁺ RNAPII⁻ promoters in Ntera2 cells. Shown in parentheses are the P-values, which indicate the probability that the category has been identified by random chance. The entire list of OCT4 targets (having known function) can be found as Supplemental Table S8. (C) PCR analysis of amplicons prepared from Ntera2 ChIP samples obtained using antibodies to the indicated proteins. The NANOG promoter was used for a known OCT4 target gene; NANOG was not expected to be bound by SUZ12 because it is expressed at high levels in Ntera2 cells (i.e., it is an OCT4⁺ RNAPII⁺ promoter target). (D) PCR analysis of amplicons prepared from SUZ12 ChIP samples after siRNA-mediated knockdown of OCT4 (hatched bars) or introduction of a control siRNA (black bars) in Ntera2 cells. The horizontal black bar indicates the SUZ12/Total ratio that is expected for nontarget promoters such as NANOG (nontarget genes should show a ratio of ∼1 when equivalent amounts of amplicons prepared from ChIP and total samples are analyzed).