Studies on KRAB/KAP1-Mediated Transcriptional Repression

Anna Claire Groner
EPFL, 2011
EPFL thesis

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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Anna Claire Groner
EPFL, 2011
EPFL thesis

Abstract

Official source

https://infoscience.epfl.ch/record/162118?ln=en

About this result

Related concepts (23)

Related concepts

Related publications (107)

Related publications

Genomic Context and KRAB/KAP1-mediated Transcriptional Repression

Sylvain Meylan

Chromatin structure and its impact on transcriptional activity represent one of the major revolutions in the biomedical field over the passed decade. The implications concern as much fundamental as clinically-oriented research; diseases and therapeutical strategies are being approached from a new point of view (Mackay et al., 2008; Richman et al., 2009). The current work dissects the different susceptibilities of genes in the human genome and how these react to the changes in chromatin landscape upon the recruitment of a transcriptional corepressor. The KRAB-Zinc finger proteins are the largest family of transcriptional repressors in the human genome. These repressors use the KRAB domain as a docking site for KAP-1 which in turn recruits a macromolecular chromatin-remodelling complex leading to heterochromatin formation. Yet current knowledge of their impact on gene regulation is limited. By artificially recruiting a KRAB-repressor in the context of genes, our lab has previously shown that this repression can act over several tens of kilobases (Groner et al., 2010). Specifically, transcriptional silencing depends on the spreading of repressive marks to the upstream promoter. However, such spreading to the trapped promoter is not systematic, suggesting potential counter-acting influences. To identify the determinants of repression, we designed a high-throughput approach of our experimental system to accumulate thousands of KRAB docking sites for which the trapped promoter is regulable and docking sites for which the promoter is immune to repression. Firstly, our data indicate that KRAB/KAP-1-repression can act efficiently over distances of 20 kb albeit in a very variable fashion, but is limited beyond that. Second, propagation of repression in the transcribed regions was favoured in most open chromatin. Third, we observed that the broader context of genes sustaining repression was that of most euchromatic regions, recapitulating the genetic context of natural targets of KAP1 repression. On the other hand, we have no evidence that barrier elements such as CTCF counteract this KRAB-mediated repression within genes. Finally, work on cases where repression acts over distances greater than 40 kb hints at the regulation of enhancers by our KRAB repressor rather than promoter. In conclusion, this work broadens the knowledge in the emerging field of gene regulation and provides further insight on the complexity of chromatin architecture and its meaning at a functional level.

EPFL2010

KRAB zinc finger protein-mediated control of transposon-embedded regulatory sequences from human germline to early embryos

Alexandra Iouranova

Transposable elements (TEs), also called jumping genes, are genetic elements capable of changing their position within the genome of their host. They make up large fractions of genomes, including 45% of human DNA content, according to current estimates. Some of these elements are of retroviral origin and are known as endogenous retroviruses (ERVs). Long terminal repeats (LTRs) of ERVs, like other TEs, influence host gene expression in various ways. They can for example create new promoters, enhancers or imprinted regions upon integration. As ERVs derive from a germline infection in the common ancestor of a species, they are often restricted to a particular evolutionary clade. While rewiring by TEs can harm a host genome, some of the features of TE invasions can increase host fitness and appear to be universally exploited by various organisms. For instance, TE insertions happen faster than protein-coding genes evolve. They can therefore be coopted for a species-specific evolutionary remodeling of transcriptional networks. Specifically, the LTR12/ERV9 TE family has colonized primate genomes over the past 30 million years and has been widely reported to contribute regulatory elements to the human genome. In this work, we set out to establish the features that made this family successful, and to assess the prevalence of LTR12-derived regulatory elements in early development. We observe the hominoid-restricted LTR12C subfamily to be a driver of transcription in both gametogenesis and early embryogenesis. LTR12C loci have rewired genes involved in ciliogenesis and flagellogenesis that are critical for male fertility. Relatedly, they also present unusual behavior during the epigenetic reprogramming characteristic of these periods by resisting genome-wide demethylation in primordial germ cell (PGC) development. To identify the determinants of the epigenetic status of LTR12C elements that made them attractive for cooption, we turn to KRAB zinc finger proteins (KZFPs). TEs appear to be prominent targets of this large family of epigenetic repressors. Through functional studies, we determine ZNF676 is a regulator of LTR12C, with its targets resisting genome-wide demethylation. This resistance possibly pre-marks them for zygotic rather than maternal expression. We show that ZNF676 levels influence the expression of some cilium-related genes and are able to disrupt ciliogenesis. We conclude this KZFP likely coevolved with its target ERVs, which carried its binding sites into the vicinity of multiple genes, including those important for male fertility. KZFPs can therefore become regulators of genes with a common function along with the transposable elements they target. More generally, in our study of TEs expressed in human embryonic development, we find the expression of some TE families to be a hallmark of a particular development stage, an indicator of the current chromatin state. These markers provide a criterion that can be used to assess the similarity of embryonic development models to real embryos. Transcription of some marker TEs anticorrelates with that of their putative KZFP regulators, implying proper KZFP expression to be important to properly recapitulate embryonic development. Our results provide insight into the TE- and KZFP-mediated rewiring of transcriptional networks related to the germline and early development, and suggest that the improper expression of a KZFP can have consequences ranging up to infertility.

EPFL2021

KAP1 is an enigmatic regulatory protein, first described some twenty years ago, shown to be involved in multiple and diverse cellular functions. Specifically, it mediates tasks critical to cell growth and differentiation, pluripotency, apoptosis, gene silencing, DNA repair and genomic integrity maintenance. In line with these findings, Kap1 deletion results in embryonic lethality, and its conditional ablation impairs crucial processes such as erythropoiesis, hepatic metabolism homeostasis and stress response. Initially named also TIF1ðœ, transcriptional intermediary factor 1ðœ, KAP1 was thought to mediate mostly chromatin-related functions. Accordingly, global maps of genomic recruitment of KAP1, generated with the advent of high-throughput technologies, display binding of this protein to thousands of loci in a multiplicity of cell types. Coupling such maps with data of transcriptional perturbations detected upon Kap1 depletion, and with the protein complexes associated with KAP1, proved to be a highly efficient approach to elucidate the molecular functions of KAP1. In particular, such studies unveiled the mechanisms of KAP1-mediated repression of different classes of transposable elements (TEs), and led to important evolutionary considerations on the strategies evolved by higher organisms to restrict the activity of these entities. Through its multiple functional domains, KAP1 interacts with a broad range of proteins, and our first goal was to establish the complete set of such potential cofactors. We found marked enrichment of KAP1 in the chromatin fraction of the nucleus, and accordingly, among its associated proteins, a strong preponderance of nucleic-acid contacting proteins. We revealed that KAP1 is part of a variety of relatively low molecular weight complexes, co-existing at comparable abundances. Study of the molecular functions of the hits suggested that KAP1 could partake in processes thus far unexplored in this context, such as DNA replication and active transcription. We then pursued with the thorough characterization of genome-wide KAP1 binding sites, and found significant fractions of RNA polymerase II (PolII)-dependent promoters and RNA polymerase III (PolIII)-transcribed elements, for the majority enriched with euchromatic marks, to be targeted by KAP1. Using high-throughput techniques, we uncovered that KAP1 variably affected the PolII pausing index of defined sets of genes, and downregulated transcription of tRNA genes. Our analyses of KAP1 genomic recruitments further led us to the discovery of novel genetic entities in the mouse genome, highly related to KRAB zinc finger protein (KZFP) genes. We described possible mechanisms underlying the evolution and amplification of these sequences and more broadly of the KZFP genes family. Furthermore, our data supported the existence of a KAP1-dependent regulatory circuit taming TE-contained enhancers located in the vicinity of these genetic units, thereby regulating their expression. We explored the proposed models of KAP1 functions in euchromatic regions of the genome, and with our results we provided the most complete analysis of the interactome of KAP1 thus far reported. Moreover, we annotated new potentially protein-coding transcripts belonging to the biggest single family of transcription factors encoded in the murine and human genomes. In sum, this work brings novel pieces of information that complement the current knowledge in the field, opening the way to further molecular studies clarifying the physiological roles of KAP1 in higher vertebrates.

EPFL2017