Resumen de Role of the tousled like kinases in maintaining genome and epigenome stability
Histone deposition during DNA replication, transcription and repair ensures the faithful maintenance of genetic and epigenetic information. The histone H3-H4 chaperone ASF1 participates in both replication dependent and independent nucleosome assembly. While much attention has been focused on the role of histone modifications, the significance of histone chaperones and their regulation in chromatin maintenance remains relatively unexplored. The Tousled like kinases 1 and 2 (TLK1 and TLK2) are evolutionarily conserved Ser/Thr kinases that regulate ASF1. TLKs have been implicated in DNA repair and chromosome stability but their cellular functions remain poorly defined. Apart from ASF1, the DNA damage signaling protein RAD9 is another proposed target of TLK activity but the extent to which they both rely on TLK1/2 has not been clearly established. The goal of this thesis is to characterize the relative functions of TLK1 and TLK2, identify their cellular targets, modes of regulation and consequences of their deficiency during development and in cancer. To this end, we have generated conditional mouse models with loss of function alleles, studied mouse-derived cells from homeostatic and cancerous tissues, and performed siRNA mediated depletion of TLK1/2 in multiple human cell lines. In vivo, TLK1 and TLK2 play largely redundant roles in genome maintenance and cell viability in tissue homeostasis, with the exception of a placental specific requirement for TLK2. Depletion of TLK activity in cells leads to reduced histone deposition, DNA replication stress, extensive DNA damage accumulation and arrest or cell death. In addition, TLK deficient cells exhibit synthetic lethality with several DNA damage checkpoint inhibitors, consistent with a role for TLK activity in replication stress tolerance. Genome wide analysis indicated that TLK activity is required for heterochromatin maintenance, particularly in regions of repetitive DNA. This is consistent with defects in histone variant deposition and increased non-coding RNA transcription at these sites. While the phenotypes of TLK depletion support a major role in ASF1-mediated histone deposition, our data indicates that they likely control additional targets and suggests that the identification of small molecule inhibitors for these kinases could suppose a valuable target for cancer therapy to augment existing strategies for cancer treatment.
