A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing

Abstract

BackgroundKRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.MethodTo investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.ResultsWe found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.ConclusionTogether, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.

The Krüppel-associated Box Repressor Domain Can Induce Reversible Heterochromatization of a Mouse Locus in Vivo

“…S1). This indicates that the lack of complete repression in the transgenic mice was at least partly due to suboptimal levels of the trans-repressor, although we cannot exclude locus-specific cis-acting influences, which can sometimes interfere with KRAB/KAP1-mediated effects as we previously observed (25,26). Nevertheless, the experiments described here provide a robust demonstration of the attractiveness of the TrapSil system for controlling gene expression and chromatin states in vivo.…”

“…Ligation-mediated PCR (LM-PCR)-Integration sites were mapped by LM-PCR using a previously described protocol (26).…”

“…TetO-repeats inserted within the gene trap further allow us to monitor the effect of tTRKRAB-induced "silencing" on these genomic sites. Using this system at hundreds of trapped loci in cultured cells, we found that efficient KRAB/KAP1-mediated silencing preferentially takes place in gene-rich and active genomic environments (26). In addition, molecular analyses of cellular "TrapSil" clones revealed that, in contrast to former belief, KRAB/KAP1 can mediate reversible long-range transcriptional repression through the spread of H3K9me3-containing heterochromatin (25).…”

“…For this, we made use of a system, which we previously developed to investigate the effect of KRAB/KAP1 recruitment on euchromatic gene bodies in cell culture. Here, the ectopic tTRKRAB repressor is targeted to TetO-carrying gene traps in a dox-dependent way (25,26). This so-called "Trapping/Silencing" or "TrapSil" approach uses retroviral and LV vector-based gene traps, which carry a promoterless reporter gene.…”

KAP1 Represses Differentiation-Inducible Genes in Embryonic Stem Cells through Cooperative Binding with PRC1 and Derepresses Pluripotency-Associated Genes

“…The average change in transcription upon KAP1 fl/fl knockout was calculated for each group of genes on the basis of the microarray data and was plotted as a function of the average ChIP-seq tag enrichment or the change in ChIP-seq tag enrichment in each group. KAP1 also had distinct effects on transcription when they bound to reporter constructs that were integrated at different genomic locations in HeLa cells (51). The opposite effects of both KAP1 and REST on PRC1 binding and on transcription at different genes indicate that they regulate PRC1 binding through multiple mechanisms that have both positive and negative effects on PRC1 binding.…”