Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest

Abstract

Edited by Patrick SungThe WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN-or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1-3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress.Replication stress, defined as disturbances to normal progression rate, density, or distribution of replication forks, is a major driver of genomic instability and carcinogenesis (1-3). Replication stress caused by fluctuations in cellular pools of NTPs and dNTPs is highly relevant to the understanding of the mechanisms of oncogene-driven mutagenesis and chemosensitivity (1-3). Hydroxyurea (HU), 4 a ribonucleotide reductase inhibitor, depletes dNTP pools in a dose-dependent manner to cause a reversible global reduction in replication fork progression rate. Slowing or stalling of forks in HU and subsequent reactivation of normal fork progression after HU are highly regulated processes, which protect forks from inactivation and ensure faithful and complete replication of the genome. This includes preserving the ability of forks to resume DNA synthesis after conditions normalize as well as preventing excessive truncation of nascent DNA strands at the fork and involves coordinated activities of many proteins, including checkpoint effectors and mediators, exonucleases, helicases, ATPases, low fidelity DNA polymerases, and proteins of homologous recombination machinery (4, 5). Nonetheless, prolonged stalling eventually leads to development of double strand breaks and activation of the DNA damage response (6 -8).We and others have shown that the human RECQ helicases WRN and BLM are among the proteins that are important for normal progression of replication forks as ...

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

“…Consistent with this, loss of H3 lysine 56 acetylation and CAF-1 have similar effects on DNA replication in S. cerevisiae (49). Histone deacetylases, HDAC1 and HDAC2, which have been proposed to deacetylate newly synthesized histones following their assembly into chromatin, have been shown to be important for replication fork function and for the stabilization of stalled replication forks in conjunction with the WRN helicase (52)(53)(54)(55)(56).…”

Osteosarcoma: Molecular Pathogenesis and iPSC Modeling

“…WRN phosphorylation at S1133 by CDK1 favors WRN-DNA replication helicase/nuclease 2 (DNA2)-dependent long-range DNA resection and promotes homologous recombination in human fibroblasts [66]. WRN can also interact with HDAC1 and 2 to promote replication restart following replication stress in mammalian cells [67]. Although loss of WRN in WS may directly lead to cancer development through increased chromosome instability [68], its overall function in cancer is not clear.…”

An RNAi screen in human cell lines reveals conserved DNA damage repair pathways that mitigate formaldehyde sensitivity

“…A custom-designed RNAi library was used that consisted of a pool of four siRNAs targeting each of 320 genes representing all major DNA repair pathways with additional genes involved in the DNA damage response, cell cycle regulation, and mitotic cell division (54). All siRNAs were synthesized on a 0.25 µmol scale, and then each gene-specific set of 4 siRNAs was pooled in a single master plate well (Qiagen).…”

“…Each gene-specific pool was tested in triplicate on separate plates to establish experimental variability, statistical validity, and to identify potential batch effects. The full 320 DDR RNAi library and sequences of gene-specific and control siRNAs are provided in Supplementary Table 1 of Kehrli et al 2016 (54). This siRNA library is available for both academic and commercial use as the 320 DDR (DNA 7 Damage Repair) Library through the University of Washington Quellos High Throughput Screening Core (http://depts.washington.edu/iscrm/quellos/rnai-screens).…”

“…We transfected each cell line, 24 h prior to formaldehyde exposure with our pooled siRNA library (54). Cell proliferation was assessed by CTG assay after 5 days of continuous formaldehyde exposure.…”