Genome-Wide Analysis of KAP1 Binding Suggests Autoregulation of KRAB-ZNFs

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Abstract
We performed a genome-scale chromatin immunoprecipitation (ChIP)-chip comparison of two modifications (trimethylation of lysine 9 [H3me3K9] and trimethylation of lysine 27 [H3me3K27]) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger (ZNF) genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the Polycomb repressive complex 2 is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the Polycomb repressive complex 2, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 corepressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5-kb promoter arrays. We found that a large number of genes of ZNF transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38-array tiling set, identifying ~7,000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 ZNFs, especially those containing Krüppel-associated box (KRAB) domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1. Methylation of lysines 9 or 27 of histone H3 (H3me3K9 or H3me3K27, respectively) has been associated with silenced chromatin. However, a comprehensive comparison of the regions of the genome bound by these two types of modified histone H3 has not been performed. Therefore, we compared the binding patterns of H3me3K9 and H3me3K27 at ~26,000 human promoters in four different cell populations. Our studies indicated that the two marks segregate differentially with the two most common types of transcriptional regulators; H3me3K27 is highly enriched at homeobox genes and H3me3K9 is highly enriched at zinc-finger genes (ZNFs). We showed that many of the promoters bound by H3me3K9 are also bound by the corepressor KAP1. A genome-wide screen for KAP1 target genes revealed a difference in the location of KAP1 binding sites in the ZNF genes versus other targets. In general, KAP1 binding sites were localized to core promoter regions. However, KAP1 binding sites associated with ZNF genes are near the 3′ end of the coding region. Our results suggest that the KRAB-ZNF family members participate in an autoregulatory loop involving binding of the KAP1 protein to the 3′ end of the ZNF target genes, resulting in trimethylation of H3K9 and transcriptional repression.