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TRIM28 Functions As the SUMO E3 Ligase for PCNA in Prevention of Transcription Induced DNA Breaks

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TRIM28 Functions As the SUMO E3 Ligase for PCNA in Prevention of Transcription Induced DNA Breaks TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks Min Lia, Xiaohua Xua, Chou-Wei Changa, and Yilun Liua,1 aDepartment of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA 91010-3000 Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved August 17, 2020 (received for review March 3, 2020) In human cells, the DNA replication factor proliferating cell nuclear SUMO2-PCNA is important for resolving TRC (17), which, if antigen (PCNA) can be conjugated to either the small ubiquitinlike not resolved, is a major cause of DNA breaks and instability at modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is common fragile sites (CFSs) (25, 26). SUMO2-PCNA achieves induced by transcription to facilitate resolution of transcription– TRC resolution by dissociating active RNAPII (RNAPIIo) via replication conflict (TRC). To date, the SUMO E3 ligase that pro- chromatin remodeling by enriching the histone chaperones vides substrate specificity for SUMO2-PCNA conjugation in re- CAF1 and facilitates chromatin transaction (FACT) at the rep- sponse to TRC remains unknown. Using a proteomic approach, lication fork (17). CAF1 deposits new histones that contain re- we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA pressive histone marks at the replication fork, and FACT conjugation. In vitro, TRIM28, together with the RNA polymerase II removes parental histones ahead of the replication fork (27, 28). (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA con- Through this mechanism, SUMO2-PCNA promotes chromatin jugation but inhibits SUMO1-PCNA formation. This activity re- remodeling to establish compact chromatin structure and de- quires a PCNA-interacting protein (PIP) motif located within the stabilize RNAPIIo from TRC sites to allow replication fork bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA progression (17). Because CAF1A, the catalytic subunit of on human chromatin is dependent on both transcription and CAF1, interacts with SUMO2 but not SUMO1 in vitro (29), this RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. preference for SUMO2 binding likely explains why TRC spe- As a consequence, TRIM28 depletion led to RNAPII accumulation at cifically induces SUMO2-PCNA conjugation to facilitate CAF1- TRC sites, and expression of a TRIM28 PIP mutant failed to sup- mediated chromatin remodeling. press TRC-induced DNA breaks. The distinct functions of SUMO1-PCNA and SUMO2-PCNA BIOCHEMISTRY indicate that these modifications are mediated by different sets TRIM28 | PCNA | SUMO2 | RECQ5 | DNA replication of enzymes. In general, SUMOylation is initiated by the conju- gation of a SUMO protein to the E1 ligase Sae1/2, which CNA is a DNA replication factor that forms a homotrimeric transfers SUMO to the E2 ligase UBC9. A SUMO E3 ligase then Pring on DNA and interacts with and anchors DNA poly- simultaneously binds SUMO-UBC9 and the target substrate to merases at the replication fork to enhance their processivity. In promote SUMO conjugation of the substrate (30). Although addition to its role in normal DNA replication, PCNA also or- cells contain only one SUMO E1 and E2, there are several E3 chestrates several cellular processes to regulate the cell cycle and ligases, which provide substrate specificity. Most likely, a distinct maintain genomic stability. These functions depend on distinct SUMO E3 ligase and/or its cofactor(s) is required to provide the posttranslational modifications of PCNA lysine residue 164 specificity needed to conjugate SUMO2 but not SUMO1 to (K164). For example, in undamaged cells, PCNA is mono- PCNA in a transcription-dependent manner. Even though the ubiquitinated at K164 by the ubiquitin E3 ligase CRL4(CDT2) to antagonize the ubiquitin hydrolase USP1 (1) and facilitate Significance proteasome-dependent degradation of p21 and CDT1 to ensure proper cell cycle progression (2). In cells treated with DNA PCNA is an essential protein in DNA replication and repair, and damaging agents, monoubiquitination of PCNA K164, mediated these functions rely on multiple posttranslational modifica- by the ubiquitin E3 ligase RAD18, recruits error-prone DNA tions, including small ubiquitin-like modifiers SUMO1 and polymerases to the stalled replication fork and bypasses DNA SUMO2. SUMO2-conjugated PCNA has a distinct function in lesions (3). K164 can also be polyubiquitinated to recruit maintaining genomic stability from SUMO1-conjugated PCNA. ZRANB3 for DNA damage bypass through template switching Therefore, different SUMO E3 ligases are needed to provide – (4 6). Posttranslational modification of PCNA K164 is not lim- the specificity to covalently attach either SUMO1 or SUMO2 to ited to ubiquitination (7). In both yeast and human cells, PCNA in response to different replication stress. However, even SUMOylation of PCNA K164 recruits antirecombination heli- though SUMO-conjugated PCNA molecules have been ob- cases (i.e., Srs2, PARI, FBH1, and RTEL) to the replication fork served in human cells since 2012, to date, the SUMO E3 ligases – to suppress homologous recombination (8 14). for PCNA have yet to be identified. This paper reports a SUMO Unlike yeast, which only contains one SUMO gene, there are E3 ligase that directly conjugates SUMO2 to PCNA with high – four SUMO paralogs in human cells (i.e., SUMO1 4). Both specificity to prevent transcription induced DNA breaks in SUMO1- and SUMO2-conjugated PCNA are found in human human cells. cell extracts (12, 15, 16). SUMO1-PCNA recruits the helicase PARI to suppress unwanted homologous recombination at Author contributions: M.L. and Y.L. designed research; M.L., X.X., and C.-W.C. performed stalled replication forks (12, 15). However, whether SUMO2- research; M.L. contributed new reagents/analytic tools; M.L. and Y.L. analyzed data; and PCNA plays a redundant role to SUMO1-PCNA in suppressing Y.L. wrote the paper. homologous recombination for maintaining replication fork The authors declare no competing interest. stability was not clear until our recent study, which showed that This article is a PNAS Direct Submission. SUMO2-conjugated PCNA but not SUMO1-conjugated PCNA Published under the PNAS license. was induced by transcription during DNA replication (17). 1To whom correspondence may be addressed. Email: [email protected]. SUMO2-PCNA conjugation is dependent on RECQ5 (17), a This article contains supporting information online at https://www.pnas.org/lookup/suppl/ DNA helicase that functions as a tumor suppressor and is a doi:10.1073/pnas.2004122117/-/DCSupplemental. member of the RNAPII complex (18–24). Transcription-induced www.pnas.org/cgi/doi/10.1073/pnas.2004122117 PNAS Latest Articles | 1of9 Downloaded by guest on September 23, 2021 presence of SUMO1- and SUMO2-conjugated PCNA in human A cell extracts has been reported since 2012 (12, 15, 16), to date, E the E3 ligases for these different PCNA SUMO modifications remain unclear. In the current study, we describe our identification of TRIM28, also known as KRAB-associated protein 1 (KAP1), as B the E3 ligase that specifically conjugates SUMO2 but not SUMO1 to PCNA. TRIM28 interacts with PCNA through its PIP motif to promote SUMO2 conjugation, and this interaction depends on both transcription and RECQ5, which may act as a sensor for TRC by interacting with both components of the replication and transcriptional complexes when they are in F proximity. Our identification of TRIM28 as the SUMO2-PCNA E3 ligase is further supported by our demonstration that TRIM28 depletion leads to the accumulation of RNAPII and C DNA damage at TRC sites, and that DNA breaks formed in the absence of TRIM28 are suppressed by the expression of a SUMO2-PCNA fusion protein to bypass the requirement for TRIM28 in SUMO2-PCNA conjugation. Results TRIM28-PCNA Interaction Takes Place during DNA Replication and Is Dependent on Transcription. The SUMO E3 ligase that promotes SUMO2 conjugation of PCNA is expected to interact with PCNA. Therefore, we searched for the presence of any known SUMO E3 ligase(s) in our mass spectrometric analysis of FLAG- tagged PCNA protein complexes purified from human chroma- tin prepared from HEK293T cells (17). Our mass spectrometric D GH analysis detected the presence of the E3 ligases RANBP2 (20 peptides) and TRIM28 (16 peptides) in the purified FLAG- PCNA complex (Fig. 1A) as well as limited PIAS1 (1 peptide) and PIAS2 (1 peptide). Western blot analysis of the purified FLAG-PCNA complex confirmed the presence of the two top candidates, RANBP2 and TRIM28 (SI Appendix, Fig. S1A). Each of these SUMO E3 ligases is capable of conjugating SUMO2 to its substrate and is a potential candidate for the SUMO2-PCNA conjugation reaction (31–34). RECQ5 is a critical factor for preventing TRC-induced DNA breaks in multiple human cell lines, including HEK293T cells, Fig. 1. TRIM28-PCNA interaction on human chromatin is dependent on and the level of SUMO2-PCNA on human chromatin is de- transcription. (A) Molecular weight (MW) and number of peptides detected pendent on RECQ5 (17). Importantly, SUMO2 conjugation of by mass spectrometry for each of the indicated proteins that copurified with PCNA requires a direct interaction between RECQ5 and PCNA FLAG-tagged PCNA isolated from the chromatin-bound (CB) fraction of (17). Therefore, to narrow down the potential SUMO E3 ligase HEK293T cells as performed previously (17). (B) MW and number of peptides detected by mass spectrometry for each of the indicated proteins that candidates, we determined which of these SUMO E3 ligases was copurified with FLAG-tagged RECQ5 isolated from the CB fraction of also associated with RECQ5 in an abundant amount on human HEK293T cells. (C) Western blot analysis of the indicated proteins in the CB chromatin. For this, we immunopurified FLAG-tagged RECQ5 fractions (Left) and the FLAG-RECQ5 complexes immunopurified from CB from the CB fraction of HEK293T cells and identified RECQ5- fractions (Right) prepared from HEK293T cells with or without exogenously associated proteins using mass spectrometry (SI Appendix, Table expressed FLAG-RECQ5.
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