SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

Abstract

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

KAP1 Is a Host Restriction Factor That Promotes Human Adenovirus E1B-55K SUMO Modification

“…Primary antibodies specific for the viral proteins used in this study included E1A mouse monoclonal antibody (MAb) M73 (35), E1B-55K mouse MAb 2A6 (36), E2A-72K mouse MAb B6-8 (37), E4orf6 mouse MAb RSA3 (38), L4-100K rat MAb 6B-10 (39), E4orf3 rat MAb 6A11 (40), and HAdV type 5 (HAdV5) rabbit polyclonal serum L133 (34). Primary antibodies specific for cellular proteins included KAP1 rabbit polyclonal antibody H-300 (Santa Cruz Biotechnology), phospho-KAP1 (S824) rabbit polyclonal antibody (Bethyl Laboratories, Inc.), SPOC1 rat monoclonal antibody 6F6 (8,10), GAPDH 2542 KAP1S824A fwd 5=-CTGGCCTGAGTGCCCAGGAGCTG-3= 2543 KAP1S824A rev 5=-CAGCTCCTGGGCACTCAGGCCAG-3= 2548 KAP1K554A fwd 5=-GTCTCCTCCTCCGCGACAATGG-3= 2549 KAP1K554A rev 5=-CCATTGTCGCGGAGGAGGAGAC-3= 2550 KAP1K779A fwd 5=-TGCACGTCTGCCGCGTCCTCAG-3= 2551 KAP1K779A rev 5=-CTGAGGACGCGGCAGACGTGCA-3= 2552 KAP1K804A fwd 5=-ACAGCAGAGAACGCGGTGTCACC-3= 2553 KAP1K804A rev 5=-GGTGACACCGCGTTCTCTGCTGT-3= 2645 KAP1K676A fwd 5=-CATCCTCCTCCGCCAGGTCAGG-3= 2646 KAP1K676A rev 5=-CCTGACCTGGCGGAGGAGGATG-3=…”

“…Another well-studied SPOC1-interacting protein is the hetero-chromatin-associated transcription factor KAP1 (Kruppel-associated box [KRAB]-associated protein 1)/transcriptional intermediary factor 1␤ (TIF1␤)/KRAB-interacting protein 1 (KRIP1)/ tripartite motif containing 28 (TRIM28) (9,10). Recruitment of this protein to genetic loci increases H3K9me2/3 repressive histone marks, induces the formation of heterochromatin, and blocks gene expression (8).…”

“…KAP1 contains an N-terminal RING, B-box, and coiled-coil domain (RBCC)/tripartite interaction motif (TRIM) domain, which is responsible for the interaction with KRAB domain-containing zinc finger (KRAB ZNF) transcription factors (20) and SPOC1 (10). The heterochromatin protein 1 (HP1) box, a central PXVXL pentapeptide region within the KAP1 protein, mediates the interaction with HP1 (21), and the C-terminal PHD domain facilitates the recruitment of the Mi2␣ subunit within the NuRD histone deacetylase complex (22) and the H3K9-specific HMT SETDB1 (23).…”

Interaction between p53 and estradiol pathways in transcriptional responses to chemotherapeutics

“…H3K9me2 can also be associated with openness/ gene activity when present at the 5' region of a gene 47 and can reflect changes elicited by DNA damage responses. 48,49 p53 and ER have been functionally and physically related to proteins involved in chromatin methyl mark changes, such as G9a and LSD1. [50][51][52][53][54][55] However, the outcome of the induced epigenetic changes is variable.…”

PHF13: A new player involved in RNA polymerase II transcriptional regulation and co-transcriptional splicing

“…Interestingly, PHF13 has been previously shown to play a role in DNA damage response (DDR) and DNA repair and likewise to interact with several proteins important for DNA repair and surveillance. 26 One such factor, TRIM28 (also known as TIF1b and KAP1), has been recently shown to globally regulate RNAPII promoter proximal pausing and release, which hinged on whether or not it was phosphorylated by DNA-PK and ATM, two important DDR kinases. 27,28 PHF13 interacts with all of these proteins to modulate DDR, and we have recently shown that in the absence of induced DNA damage (albeit DNA damage and recovery is a continuous and naturally occurring process, even in the absence of DNA damage inducing agents) PHF13 interacts with TRIM28 and ATM.…”