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Abstract

The sequencing of the human genome revealed that the C2H2-zinc finger proteins (ZFPs) are the largest family of human transcription factors. Around 300 members of this family contain, in addition to their zinc finger motifs, a KRAB domain that has been implicated in transcriptional repression. It is thought that KRAB-ZFPs mediate this effect through the recruitment of the corepressor KAP1, which induces facultative heterochromatin through the activity of several associated chromatin-modifying factors. Our studies focused on the elucidation of the molecular mechanisms of transcriptional repression induced by KRAB-ZFPs and KAP1. For this, we made use of the conditional binding activity of the KRAB-containing repressor tTRKRAB. This ectopic protein serves as a paradigm for endogenous KRAB-ZFP functions, as it depends on cellular KAP1 for transcriptional repression. tTRKRAB only binds to heterologous TetO sites in the absence of doxycycline, thus making repressor activity drug-regulated. When we targeted the repressor to euchromatic sites modified with TetO, we found that it induced increased levels of H3K9me3 and HP1β at the genes it bound to. We also monitored the effect of repressor binding on transcription by assessing the activity of TetO-containing reporter genes that were driven by endogenous promoters and that were initially used to create euchromatic tTRKRAB binding sites. This analysis revealed that KRAB/KAP1 mediated long-range heterochromatin spreading, which only resulted in transcriptional repression if repressive marks reached the promoter and impaired transcriptional initiation. The efficiency of this process was highest in an environment of very strong gene activity, as indicated by the levels of active histone modifications. We found that KRAB-ZFP genes themselves are probably targets of KRAB/KAP1-mediated repression and elucidated their transcriptional regulation and chromatin structure. KAP1 was localized to the promoter and to the 3' end of KRAB-ZFP genes, suggesting self-regulatory loops. However, only KAP1 localized to the 3' end of KRAB-ZFP genes mediated increased levels of H3K9me3 and HP1β along the gene body, without affecting the promoter. Therefore, KAP1 is involved in controlling the spread of heterochromatin at these endogenous sites, largely without regulating transcription. The function of this heterochromatic chromatin landscape may lie in the protection from chromosomal rearrangements between highly homologous KRAB-ZFP genes. Despite this repressive structure, KRAB-ZFP genes are still expressed, as high levels of transcription-associated H3K36me3 accompany H3K9me3 throughout the gene bodies. Other projects described in this thesis include the elucidation of the role of KAP1 in behavior and the assessment of KRAB/KAP1-mediated repression in the context of episomal structures. Both of these areas are of scientific and biomedical interest and address basic principles of the molecular mechanisms of KRAB/KAP1-mediated transcriptional repression.

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