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 ...

Newly synthesized histone H4 that is incorporated into chromatin during DNA replication is acetylated on lysines 5 and 12. Histone deacetylase 1 (HDAC1) and HDAC2 are responsible for reducing H4 acetylation as chromatin matures. Using CRISPR-Cas9-generated hdac1- or hdac2-null fibroblasts, we determined that HDAC1 and HDAC2 do not fully compensate for each other in removing de novo acetyls on H4 in vivo. Proteomics of nascent chromatin and proximity ligation assays with newly replicated DNA revealed the binding of ATAD2, a bromodomain-containing posttranslational modification (PTM) reader that recognizes acetylated H4. ATAD2 is a transcription facilitator overexpressed in several cancers and in the simian virus 40 (SV40)-transformed human fibroblast model cell line used in this study. The recruitment of ATAD2 to nascent chromatin was increased in hdac2 cells over the wild type, and ATAD2 depletion reduced the levels of nascent chromatin-associated, acetylated H4 in wild-type and hdac2 cells. We propose that overexpressed ATAD2 shifts the balance of H4 acetylation by protecting this mark from removal and that HDAC2 but not HDAC1 can effectively compete with ATAD2 for the target acetyls. ATAD2 depletion also reduced global RNA synthesis and nascent DNA-associated RNA. A moderate dependence on ATAD2 for replication fork progression was noted only for hdac2 cells overexpressing the protein.

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