The cells were transfected with control or Phf5a siRNA and then 24? h later were stimulated with 1?mM OHT for another 24?h. of Phf5a severely impairs AID\induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ\dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID\ and by incorporating biotin\16\dUTP and TdT. The DNA was fragmented and labeled fragments were enriched by streptavidin pull\down, followed by quantification by qPCR. The signal from the ?\Macroglobulin (?m) locus served as a negative control. The values are presented as mean??SD (NHEJ repair factor, but also other DNA repair\associated factors like Mre11, CtIP, and Exo1, all of which are known to be involved in DNA end processing prior to end joining and in CSR (Dinkelmann and H2A.Z facilitates the recruitment of Ku complex to the site of DSBs on chromatin, which further promotes the recruitment of other NHEJ factors, such as DNA\PKcs and the Xrcc4CXlfCDNA ligase 4 complex, to complete the DNA end joining process (Xu H2A.Z increases the chromatin accessibility to DNA end\processing enzymes such as Exo1 (Adkins the presence of H2A.Z flanking DSB has been CD8B suggested to determine the upstream and downstream boundaries of the DNA break, which in turn may protect DNA ends from excessive resection to promote NHEJ. Consistent with the DSB resolution defect, (Rac)-VU 6008667 increased insertions and deletions (indels) were evident in both CSR and em I\Sce /em I\cleaved junctions upon Phf5a depletion. Indeed, KD of either Phf5a or H2A.Z reduced the NHEJ efficiency dramatically (Fig?4), and the frequency of indels at the DNA repaired junctions was significantly increased in either Phf5a\ or H2A.Z\deficient cells (Fig?4; Appendix Fig S5), suggesting their involvement in a common pathway during NHEJ (Ogiwara & Kohno, 2011). The high incidence of long insertions and deletions may (Rac)-VU 6008667 reflect an alternative mode of DNA repair such as homology\directed or copy\paste\mediated mechanism while attempting to rescue the resected DNA break ends (Onozawa em et al, /em 2014; Iliakis em et al, /em 2015). We envisage that the loss of Phf5a/p400 leads to impaired (Rac)-VU 6008667 DDR and consequentially the recruitment defect of crucial DSB end shielding factors like Ku80, 53BP1, and others (Fig?5 and unpublished). Moreover, CtIP level remains unchanged, which might promote DNA end resection directly or indirectly with the available MRN/DNA2 complex, especially when the S region DSBs are no longer flanked by sufficient H2A.Z (Sartori em et al, /em 2007; Xu em et al, /em 2012). Activation of AIDER, which induces DNA breaks in the S regions, led to the accumulation of H2AX, Ku80, and Exo1, but not of H2AX or H2A.Z at these sites (Appendix Fig S13A). Using AID\deficient CH12F3\2A cells, we further confirmed that Phf5a, but not AID activation, regulates H2A.Z and p400 level in the IgH locus (Appendix Fig S13B). The finding may suggest that the S regions are preloaded and/or continuously supplied with histone H2A variants. Indeed, CSR is also strongly affected by the KD of H2A.Z chaperone p400. Unexpectedly, H2AX was found to interact with p400, and its recruitment was reduced upon p400 or Phf5a KD, suggesting that p400 is also involved in H2AX deposition in the S region. Although p400\dependent deposition of the H3.3 variant has been reported, we were unable to detect any alteration of H3.3 level in the S region by depleting Phf5a or p400. However, Phf5a or p400 KD affected the deposition of two additional H2A variants, H2A.Bbd and macroH2A (Tolstorukov em et al, /em 2012; Gaspar\Maia em et al, /em 2013). These observations collectively suggest that the Phf5a\p400 axis modulates the H2A variant stoichiometry in the S region. We revealed that Phf5a promotes stabilization of the p400 histone chaperone complex in the S region. Based on an extensive mutagenesis study, we also concluded that the zinc\finger motifs of Phf5a are critically involved in CSR through interaction with p400 and Sf3b components (Fig?7; Appendix Fig S12). The unique trefoil knot structure of Phf5a may play a role in its association with large, multi\protein complexes. However, the interaction of Phf5a with certain protein complexes such as the RNA methylation complex, Paf1/Ski8, or senataxin was found to be irrelevant in the context of CSR and H2A variant regulation. Although Phf5a strongly interacts.