Infoscience

Thesis

Genomic targets and molecular partners of the chromatin regulator KAP1

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 TIF1𝛜, transcriptional intermediary factor 1𝛜, 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.

Fulltext

Related material