Chromatin regulators and their impact on DNA repair and G2 checkpoint recovery.

Chromatin plays a pivotal role in regulating the DNA damage response and during DNA double-strand break repair. Upon the generation of DNA breaks, the chromatin structure is altered by post-translational modifications of histones and chromatin remodeling. How the chromatin structure, and the epigenetic information that it carries, is reestablished after the completion of DNA break repair remains unclear though. Also, how these processes influence recovery of the cell cycle remains poorly understood. We recently performed a reverse genetic screen for novel chromatin regulators that control checkpoint recovery after DNA damage. Here we discuss the implications of PHD finger protein 6 (PHF6) and additional candidates from the NuA4 ATPase-dependent chromatin-remodeling complex and the Cohesin complex, required for sister chromatid cohesion, in DNA repair and checkpoint recovery in more detail. In addition, the potential role of this novel function of PHF6 in cancer development and treatment is reviewed.

PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks.

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.

PHF6 promotes non-homologous end joining and G2 checkpoint recovery.

The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Börjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint.

Control of DNA Replication Initiation by Ubiquitin.

Eukaryotic cells divide by accomplishing a program of events in which the replication of the genome is a fundamental part. To ensure all cells have an accurate copy of the genome, DNA replication occurs only once per cell cycle and is controlled by numerous pathways. A key step in this process is the initiation of DNA replication in which certain regions of DNA are marked as competent to replicate. Moreover, initiation of DNA replication needs to be coordinated with other cell cycle processes. At the molecular level, initiation of DNA replication relies, among other mechanisms, upon post-translational modifications, including the conjugation and hydrolysis of ubiquitin. An example is the precise control of the levels of the DNA replication initiation protein Cdt1 and its inhibitor Geminin by ubiquitin-mediated proteasomal degradation. This control ensures that DNA replication occurs with the right timing during the cell cycle, thereby avoiding re-replication events. Here, we review the events that involve ubiquitin signalling during DNA replication initiation, and how they are linked to human disease.

USP7 controls Chk1 protein stability by direct deubiquitination.

Chk1, an essential checkpoint kinase in the DNA damage response pathway (DDR), is tightly regulated by both ATR-dependent phosphorylation and proteasome-mediated degradation. Here we identify ubiquitin hydrolase USP7 as a novel regulator of Chk1 protein stability. USP7 was shown before to regulate other DDR proteins such as p53, Hdm2 and Claspin, an adaptor protein in the ATR-Chk1 pathway required for Chk1 activation. Depletion or inhibition of USP7 leads to lower Chk1 levels. The decreased Chk1 protein after USP7 knock down cannot be rescued by simultaneously elevating Claspin levels, demonstrating that the effect of USP7 on Chk1 is independent of its known effect on Claspin. Conversely, overexpression of USP7 wild type, but not a catalytic mutant version, elevates Chk1 levels and increases the half-life of Chk1 protein. Importantly, wild type, but not catalytic mutant USP7 can deubiquitinate Chk1 in vivo and in vitro, confirming that USP7 directly regulates Chk1 protein levels. Finally we show that USP7 catalytic mutant is (mono-)ubiquitinated, which suggests auto-deubiquitination by this ubiquitin hydrolase, possibly important for its regulation.