[PMC free article] [PubMed] [Google Scholar] 66. DSB repair is vital for cell survival, genome stability, and tumor suppression. In contrast to NHEJ, HR relies on extensive homology and templated DNA synthesis to restore the sequence surrounding the break site. We report a new role for the multifunctional protein CCCTC-binding factor (CTCF) in facilitating HR-mediated DSB repair. CTCF is usually recruited to DSB through its zinc finger domain name independently of poly(ADP-ribose) polymers, known as PARylation, catalyzed by poly(ADP-ribose) polymerase 1 (PARP-1). CTCF ensures proper DSB repair kinetics in response to -irradiation, and the loss of CTCF compromises HR-mediated repair. Consistent with its role in HR, loss of CTCF results in hypersensitivity to DNA damage, inducing brokers and inhibitors of PARP. Mechanistically, CTCF acts downstream of BRCA1 in the HR pathway and associates with BRCA2 in a PARylation-dependent manner, enhancing BRCA2 recruitment to DSB. In contrast, CTCF does not influence the recruitment of the NHEJ protein 53BP1 or LIGIV to DSB. Together, our findings establish for the first time that CTCF is an important regulator of the HR pathway. locus in the normal mammary epithelial cell line MCF10A using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Constitutive deletion of in mice leads to embryonic lethality, which likely explained why we were unable to generate a full CTCF knockout cells. However, we obtained single-allele knockouts of CTCF in MCF10A (CTCF+/?), with each clone being derived from a single-cell expansion. We observed a significant reduction of CTCF protein in three distinct clones, ranging from approximately 20 to 50% of controls, dependent on the clone being studied (Fig. 2A). Next, we uncovered cells to 2-Gy (gray) -irradiation and monitored the repair of DSB over time under control conditions or in three impartial Bifendate CTCF+/? clones. The repair kinetics of DSB, as determined by the disappearance of H2AX foci (Fig. 2B), is usually significantly slowed in the three CTCF+/? clones. We observed that H2AX foci persist significantly in CTCF-depleted cells (Fig. 2, B and C). It is unlikely Mouse monoclonal antibody to PYK2. This gene encodes a cytoplasmic protein tyrosine kinase which is involved in calcium-inducedregulation of ion channels and activation of the map kinase signaling pathway. The encodedprotein may represent an important signaling intermediate between neuropeptide-activatedreceptors or neurotransmitters that increase calcium flux and the downstream signals thatregulate neuronal activity. The encoded protein undergoes rapid tyrosine phosphorylation andactivation in response to increases in the intracellular calcium concentration, nicotinicacetylcholine receptor activation, membrane depolarization, or protein kinase C activation. Thisprotein has been shown to bind CRK-associated substrate, nephrocystin, GTPase regulatorassociated with FAK, and the SH2 domain of GRB2. The encoded protein is a member of theFAK subfamily of protein tyrosine kinases but lacks significant sequence similarity to kinasesfrom other subfamilies. Four transcript variants encoding two different isoforms have been foundfor this gene that this defect is due to altered cell cycle kinetics because CTCF+/? cells showed similar proliferation profiles as control cells (fig. S3A). We validated this experiment in the breast Bifendate cancer cell line MCF7 using two impartial short hairpin RNA (shRNA) constructs to knock down CTCF (Fig. 2, D to F, and fig. S3, B and D). Again, after exposure to 2-Gy (Fig. 2D) or 5-Gy (fig. S3, B and D) irradiation, the kinetics of H2AX foci resolution was delayed in CTCF knockdown cells. These data suggest that CTCF depletion impairs DSB repair. In support of this possibility, we quantified the resolution of DSBs upon irradiation of MCF7 control or CTCF knockdown cells using the neutral comet assay. Again, we observed the persistence of comet tails at 24 hours after irradiation in CTCF knockdown cells, whereas control cells showed almost complete resolution of comet tails by this time period (Fig. 2, G and H). -IrradiationCinduced DSBs may be repaired by the NHEJ or HR pathways. Therefore, we monitored the disappearance of the key NHEJ factor 53BP1 at DSBs under the same conditions described above using the MCF7 knockdown cells. In contrast to what we observe for H2AX, CTCF knockdown has little impact on the timing of 53BP1 foci dissolution (fig. S3, C and E). Overall, these data support the conclusion that CTCF plays a role in the repair of DSBs, likely impartial of NHEJ. Open in a separate window Fig. 2 CTCF knockdown leads to altered DNA damage repair kinetics.(A) Western blotting analyses for CTCF using lysates from three MCF10A clones Bifendate with heterozygous deletion of CTCF (CTCF+/?) were subjected to immunoblotting with the indicated antibodies. Bar graph represents the quantification of CTCF signal. (B) MCF10A WT and three CTCF heterozygote (CTCF+/?) clone cell lines were fixed at the indicated times after irradiation (2 Gy) and stained with an anti-H2AX antibody. (C) Quantification of the percent of cells with more than 10 H2AX foci. Error bars correspond to means SEM (= 3; *** 0.005, ** 0.01, 2 test). (D) MCF7 cells were infected with Ctl shRNA.