The currently accepted model in the field is that KAP1 is brought to DNA via interaction of the N-terminal RBCC domain with members of the site-specific DNA binding KRAB ZNF family. Once recruited to DNA, KAP1 is thought to serve as a scaffold protein for the assembly of a large repression complex that functions via SETDB1-mediated methylation of histone H3 on lysine 9, leading to the silencing of nearby promoters. We have tested several aspects of this model in our current study, including the role of the RBCC domain in recruiting KAP1 to the genome and the functional consequences of KAP1 binding to promoters, within genes, and intergenic regions. Our studies have provided the first in vivo support for the hypothesis that KAP1 is recruited to the human genome via interaction with KRAB ZNFs. Surprisingly, we show that this recruitment mechanism mediates the binding of KAP1 only to the 3′ ends of ZNF genes and is not involved in the recruitment of KAP1 to promoter regions. Thus, we suggest that several aspects of the current model should be reconsidered.
Using ChIP-seq, we have identified thousands of binding sites for KAP1 in the human genome. To test the first aspect of the model (i.e., that KAP1 is recruited to promoter regions via a KRAB ZNF), we used ChIP-seq to monitor the genome-wide binding pattern of a mutant KAP1 protein that lacks the N-terminal RBCC domain. Surprisingly, we found that the mutant KAP1 protein that lacks the ability to bind to KRAB ZNF transcription factors can still be recruited to promoter regions. However, this mutant protein completely loses the ability to bind to intragenic regions (i.e., the 3′ coding exons of ZNF genes). The requirement for the RBCC domain for binding to the 3′ coding exons of ZNFs may explain why these peaks are by far the strongest in the ChIP-seq data. Others have previously shown that the RBCC domain binds to KRAB domains of ZNFs as a heterotrimer (21
). Perhaps a heterotrimeric KAP1 complex is recruited to ZNF 3′ ends, making them more efficient ChIP targets. Because the promoter targets can still be bound by KAP1 lacking the RBCC domain, the complexes at promoters may include only one KAP1 protein.
In an attempt to define the domains of KAP1 that are required for promoter-specific localization, we performed ChIP-seq for three additional mutant KAP1 proteins, including constructs lacking the HP1 binding domain, the C-terminal PHD and bromodomain, and both the N-terminal RBCC and the C-terminal PHD and bromodomain. However, none of these previously identified protein interaction domains were critical for the recruitment of KAP1 to promoter regions. We next turned to motif analyses of the KAP1-bound promoter regions with the goal of identifying binding sites for potential KAP1-recruiting DNA binding proteins. We found that an ETS family motif, in particular, an ELK4 motif, was highly enriched in the top-ranked set of KAP1-bound promoters. To test this bioinformatic prediction that ELK4 might be involved in KAP1 recruitment, we performed ChIP-seq for ELK4 and found that 30% of the KAP1 promoter targets were also bound by ELK4. Further studies, such as targeted knockdown of ELK4 and mass spectroscopy, are required to determine if ELK4 is critical for the recruitment of KAP1.
The second aspect of the model that we have tested in this study was focused on the consequences of having KAP1 bound to a genomic location. We examined cellular RNA in cells stably expressing shRNAs targeting KAP1 and found that depletion of KAP1 has very minor effects on the transcriptome of HEK293 cells. Strikingly, the few genes that are responsive to reduction of KAP1 are not in the set of strong KAP1 binding sites. For example, the strongest KAP1 binding sites are ZNF 3′ exons, followed by ZNF promoters and promoters of other genes. However, the set of ~90 genes that are both bound by and regulated by KAP1 show KAP1 binding at intergenic regions located quite far from the transcription start site. These results suggest that KAP1 may regulate the transcriptional output of few, if any, genes in the human genome.
How can our current results be reconciled with previous studies that suggest that KAP1 functions as a repressor of transcription? In previous work, KAP1 has been studied using artificial systems (1
). For example, several investigations have used drug-regulated artificial fusion proteins (e.g., the KRAB domain of KOX1 fused to the PAX3 DNA binding domain, the GAL4 DNA binding domain, or the Tet repressor DNA binding domain) and a reporter construct containing multiple binding sites for the fusion protein for transcriptional analyses. These experimental designs did, in fact, show KAP1-mediated transcriptional repression. Such studies suggest that perhaps KAP1 has the ability to act as a transcriptional repressor under certain conditions (such as if multiple complexes are recruited via high-affinity DNA binding proteins right at transcriptional start sites), but they do not actually address the role of KAP1 in regulating endogenous cellular genes. A recent study used shRNAs targeting KAP1 to analyze a small number of mRNAs in HeLa cells (12
). Using PCR assays, they observed increases in mRNA levels for three ZNF genes in the KAP1 knockdown cells and concluded that KAP1 is involved in repressing these cellular genes. However, the effects were quite modest (generally less than a 10 to 20% increase in transcript levels of the “repressed genes” in the KAP1 knockdown cells). Examination of these same ZNF transcripts in our array data revealed that they were in the “not expressed” category in both the control and KAP1 knockdown cells. The most convincing effect of KAP1 on transcript levels comes from a study of mouse embryonic stem cells. Rowe et al. found robust upregulation of ERV repetitive elements (in particular, the IAP type) in mouse embryonic stem cells lacking KAP1 (25
). Interestingly, this observed increase of retrotransposon RNAs was not seen in mouse embryonic fibroblasts lacking KAP1, suggesting that perhaps KAP1 may function in a cell type-specific manner.
We have used ChIP-seq to perform a genome-wide functional analysis of KAP1 protein mutants, identifying thousands of intragenic sites (mainly 3′ coding exons of ZNF genes) to which KAP1 is recruited via its RBCC domain (and thus likely by a KRAB ZNF) and thousands of intergenic sites (mainly promoter regions of ZNF and non-ZNF genes) to which KAP1 is recruited via a mechanism distinct from RBCC-KRAB ZNF interactions. Interestingly, although the identified binding sites are quite strong and found in multiple cell types, KAP1 does not regulate the expression of most genes that are near its binding sites. However, we have identified a small set of genes that are both responsive to loss of KAP1 and identified as the “nearest” gene to a KAP1 binding site. KAP1 recruitment to some, but not all, of these putative target genes requires the RBCC domain, suggesting that KRAB ZNFs and other site-specific factors may be involved in mediating KAP1-dependent long-range regulation of transcription. However, because the effects of loss of KAP1 on the human transcriptome are very small, we hypothesize that the main role of KAP1 may not lie in transcriptional repression. A striking feature of the KAP1 genomic binding pattern is its enrichment at KRAB ZNF genes. This family of transcription factors is noted for its rapid expansion in recent evolution; there are more than 300 KRAB ZNF genes in the human genome (5
). The DNA binding domains of the KRAB ZNFs are encoded in the 3′ exons and consist of multiple copies of highly related zinc fingers. It is possible that the function of the KAP1/SETDB1 complex at these 3′ exons is to deposit H3K9me3 and heterochromatin protein 1 and thus maintain a heterochromatic state that reduces recombination-mediated deletion at the KRAB ZNF gene clusters (3
). Also, KAP1 colocalizes with DNA damage response factors at DNA lesions (37
), suggesting that KAP1 may provide an important link between heterochromatin and the recognition and repair of DNA by the cellular DNA damage response machinery (10
). For example, Ziv et al. (10
) have shown that phosphorylation of KAP1 is required for global chromatin decondensation in response to double-strand breaks. Although their studies suggest that a KAP1-mediated conversion of heterochromatin to open chromatin is important in the cellular response to DNA damage, the mechanism by which phosphorylation of KAP1 mediates chromatin decondensation is still unknown. Clearly, further studies focusing on the role of KAP1 in the regulation of genomic integrity are needed.