KRAB-containing zinc finger proteins (KRAB-ZFPs) are early embryonic controllers of transposable elements (TEs), which they repress with their cofactor KAP1 through histone and DNA methylation, a process thought to result in irreversible silencing. Using a target-centered functional screen, we matched murine TEs with their cognate KRAB-ZFP. We found the paralogs ZFP932 and Gm15446 to bind overlapping but distinguishable subsets of ERVK (endogenous retrovirus K), repress these elements in embryonic stem cells, and regulate secondarily the expression of neighboring genes. Most importantly, we uncovered that these KRAB-ZFPs and KAP1 control TEs in adult tissues, in cell culture and in vivo, where they partner up to modulate cellular genes. Therefore, TEs and KRAB-ZFPs establish transcriptional networks that likely regulate not only development but also many physiological events. Given the high degree of species specificity of TEs and KRAB-ZFPs, these results have important implications for understanding the biology of higher vertebrates, including humans.

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression.

During cell division, maintenance of chromatin features from the parental genome requires their proper establishment on its newly synthetized copy. The loss of epigenetic marks within heterochromatin, typically enriched in repetitive elements, endangers genome stability and permits chromosomal rearrangements via recombination. However, how histone modifications associated with heterochromatin are maintained across mitosis remains poorly understood. KAP1 is known to act as a scaffold for a repressor complex that mediates local heterochromatin formation, and was previously demonstrated to play an important role during DNA repair. Accordingly, we investigated a putative role for this protein in the replication of heterochromatic regions. We first found that KAP1 associates with several DNA replication factors including PCNA, MCM3 and MCM6. We then observed that these interactions are promoted by KAP1 phosphorylation on serine 473 during S phase. Finally, we could demonstrate that KAP1 forms a complex with PCNA and the histone-lysine methyltransferase Suv39h1 to reinstate heterochromatin after DNA replication.

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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.