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BMI1–UBR5 Axis Regulates Transcriptional Repression at Damaged Chromatin

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BMI1–UBR5 Axis Regulates Transcriptional Repression at Damaged Chromatin BMI1–UBR5 axis regulates transcriptional repression at damaged chromatin Anthony Sancheza, Angelo De Vivoa, Nadima Upretyb, Jonghwan Kimb, Stanley M. Stevens Jr.a, and Younghoon Keea,1 aDepartment of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL 33620; and bDepartment of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712 Edited by Terry Magnuson, University of North Carolina at Chapel Hill, Chapel Hill, NC, and accepted by Editorial Board Member James E. Cleaver August 15, 2016 (received for review July 6, 2016) BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1), at damaged chromatin (11). How BMI1 or BMI1-induced H2AK119- which plays a key role in maintaining epigenetic silencing during de- Ub modulates transcriptional output upon DNA damage remains velopment. BMI1 also participates in gene silencing during DNA dam- incompletely understood. age response, but the precise downstream function of BMI1 in gene Here we found that the chromatin localization of the HECT E3 silencing is unclear. Here we identified the UBR5 E3 ligase as a down- ubiquitin ligase UBR5 is largely dependent on the PRC1 com- stream factor of BMI1. We found that UBR5 forms damage-inducible ponents BMI1, RNF1, and RNF2. Similar to BMI1 and PRC1 nuclear foci in a manner dependent on the PRC1 components BMI1, components, UBR5-depleted cells fail to repress transcription at RNF1 (RING1a), and RNF2 (RING1b). Whereas transcription is re- damaged chromatin. We further show that BMI1- and UBR5- pressed at UV-induced lesions on chromatin, depletion of the PRC1 mediated transcription repression involves the FACT histone members or UBR5 alone derepressed transcription elongation at chaperon complex. Our findings altogether suggest that UBR5 is a these sites, suggesting that UBR5 functions in a linear pathway with downstream effector of the PRC1 components in transcription PRC1 in inducing gene silencing at lesions. Mass spectrometry (MS) silencing at damaged chromatin. analysis revealed that UBR5 associates with BMI1 as well as FACT components SPT16 and SSRP1. We found that UBR5 localizes to the Results UV-induced lesions along with SPT16. We show that UBR5 ubiquiti- UBR5 Chromatin Localization Is Dependent on BMI1, RNF1, and RNF2. nates SPT16, and depletion of UBR5 or BMI1 leads to an enlargement During the course of our studies, we found that endogenous UBR5 of SPT16 foci size at UV lesions, suggesting that UBR5 and BMI1 re- proteins form distinct foci in the nucleus, which can be enhanced press SPT16 enrichment at the damaged sites. Consistently, depletion upon various DNA damaging agent treatments (Fig. 1A). Treat- of the FACT components effectively reversed the transcriptional de- ment of two independent siRNAs against UBR5 effectively elimi- repression incurred in the UBR5 and BMI1 KO cells. Finally, UBR5 and nated the foci, indicating that the foci represent the bona fide BMI1 KO cells are hypersensitive to UV, which supports the notion UBR5 proteins (knockdown efficiency is shown in Fig. 1B). The that faulty RNA synthesis at damaged sites is harmful to the cell foci partially overlap with ubiquitin aggregate (FK2 antibody) and fitness. Altogether, these results suggest that BMI1 and UBR5 repress γH2AX, markers of DNA damage (Fig. 1C); however, a significant the polymerase II (Pol II)-mediated transcription at damaged sites, by portion of the foci do not overlap, suggesting that UBR5 in chro- negatively regulating the FACT-dependent Pol II elongation. matin may have both DNA damage-dependent and -independent functions. The Pearson’s overlap coefficient is quantified using Polycomb | BMI1 | UBR5 | FACT | transcription ImageJ software and plotted (Fig. 1D), where the 53BP1–γH2AX overlap is used as a positive control (1 indicates a complete positive erturbation of chromatin structures can cause inappropriate relationship), and the MED12–γH2AX overlap as a negative Pgene expression and loss of genome integrity. Polycomb pro- control (−1 indicates a complete negative relationship). Consistent teins are recognized in all metazoans for their conserved tran- with the immunofluorescence (IF) data, a biochemical fractionation scriptional repressive function. The canonical Polycomb Repressive Complex 1 (PRC1) contains BMI1, RNF1 (RING1a), RNF2 Significance (RING1b), core components (PC), Polyhomeotic (PH), and CBX proteins (1, 2). BMI1 serves as a key regulatory component of the The BMI1-containing Polycomb Repressive Complex is an impor- PRC1complex,whichisrequiredtomaintainthetranscriptionally tant gene silencer during development, stem cell maintenance, and repressed state of many genes throughout development via chro- cancer progression. BMI1 is also involved in DNA damage response, matin remodeling and histone modification (1). The only known but its downstream mechanism is unclear. Here we identified UBR5 enzymatic activity of the BMI1-containing PRC1 complex is to as a downstream factor whose chromatin recruitment is regulated monoubiquitinate histone H2A at Lys-119 (K119) residue, which is by BMI1. We showed that BMI1 and UBR5 repress the RNA poly- associated with transcriptional repression (1, 3, 4). However, the merase II (Pol II) elongation and nascent RNA synthesis at UV- E3 ubiquitin ligase activity of RNF2 or the H2AK119-Ub is dis- induced DNA lesions. UBR5 associates with and regulates the pensable for repression of canonical PRC1 target genes during abundance of the histone chaperon complex FACT at damaged mouse or Drosophila embryonic development, respectively (5, 6), chromatin and regulates elongation of Pol II at the lesions. Thus, suggesting that the PRC1 complex may also induce gene silencing our studies identify a pathway that regulates the damage-induced through other mechanisms (7). A series of studies has suggested transcriptional repression that is governed by BMI1. that multiple distinct forms of the PRC1 complex with varying Author contributions: Y.K. designed research; A.S., A.D.V., and N.U. performed research; J.K. components could exist, and each of these may have distinct modes and S.M.S. contributed new reagents/analytic tools; A.S., A.D.V., J.K., S.M.S., and Y.K. ana- of regulation and functions (reviewed in ref. 2). lyzed data; and Y.K. wrote the paper. In addition to its well-known role as an oncogene, recent evi- The authors declare no conflict of interest. dence suggests that BMI1 participates in the DNA damage re- This article is a PNAS Direct Submission. T.M. is a Guest Editor invited by the Editorial sponse and genome integrity maintenance. BMI1 is known to Board. localize to DNA double-strand break (DSB) sites and facilitates 1To whom correspondence should be addressed. Email: [email protected]. – CELL BIOLOGY DNA repair (8 10). Additionally, consistent with its role in gene This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. silencing, BMI1 represses local elongation of RNA polymerase II 1073/pnas.1610735113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1610735113 PNAS | October 4, 2016 | vol. 113 | no. 40 | 11243–11248 Downloaded by guest on September 27, 2021 Etoposide UBR5 in the UBR5-knockdown cells (siUBR5). Fig. 3A, Right show A 80 Bsi: c #1 #2 NT UV - siUBR5 60 measurements of relative fluorescent intensity (RFI) along the UV 40 UBR5 250 spots, which highlight that there is no repression of Pol II elon- 20 50 Tubulin gation at the γH2AX spots in the knockdown cells. The same % cells with >5 UBR5 foci 0 - 1 2 3 NT UV MMC phenotypes were observed in BMI1 and UBR5 KO HeLa cells siUBR5#1siUBR5#2 (Fig. 3B; shown as Pearson’s correlation). A chemical probe, C D Etoposide E UBR5 FK2 Merge DAPI NT UV 5-ethynyl uridine (5-EU), is frequently used for detecting global S P S P 1 nascent RNA synthesis status using the Click-IT labeling technol- UBR5 0.5 250 ogy; whereas the 5-EU stain is excluded from the γH2AX spots, 0 SPT16 the 5-EU stain becomes overlapped in both BMI1 and UBR5 γ 100 UBR5 H2AX Merge DAPI -0.5 C D X knockdown cells (Fig. 3 ), or the KO cells (Fig. 3 ). These results Pearson’s correlation Pearson’s A AX H2A Ab pair: H2AX H2 15 suggest that RNA synthesis at damaged sites has indeed occurred γ ANCD2 UBR5/FK2 1 2 3 4 when UBR5 or BMI1 is depleted. 5-EU was added to dividing cells 53BP1/γUBR5/ UBR5/FMED12/γH2 immediately following UV irradiation, to specifically monitor the Fig. 1. UBR5 chromatin recruitment is enhanced upon DNA damage. (A) UBR5 nascent RNA synthesis that occurred after the damaging event. forms distinct nuclear foci in HeLa (shown), U2OS, and HCT116 cells in SI Ap- Similar phenotypes were observed when the WT, UBR5 KO, and pendix,Fig.S2following UV (30 J/m2, 1-h recovery), etoposide treatment (2 μM, BMI1 KO cells were globally irradiated with UV; the γH2AX and 12 h), or mitomycin C (MMC) (2 μM, 12 h). n = 80. (B) Confirmation of UBR5 P-Ser2 overlap is shown in SI Appendix,Fig.S5,andtheγH2AX knockdown. (C) The foci partially colocalize with ubiquitin aggregates (FK2) and 5-EU overlap is shown in SI Appendix, Fig. S6. Knockdown of and γH2AX. (D)Pearson’s correlation for overlap from each Ab pair is measured RNF2 also led to a near-identical phenotype (Fig. 3E), suggesting = + (n 100). (E) UBR5 becomes enriched in P fraction after UV (S, cyto nucleo- that Pol II elongation at damaged sites is regulated by the PRC1 plasm; P, chromatin enriched). complex activity. Knockdown of RNF20 (which induces H2B ubiquitination) or RNF8 (which induces H1 and H2A ubiquitina- assay shows that a fraction of UBR5 is present in the chromatin- tion) (13) did not show noticeable effects (Fig. 3E). The Pol II enriched fraction (P), which is increased upon UV damage (Fig. 1E). To identify the upstream regulator(s) for the foci formation, we screened siRNAs targeting known regulators of chromatin me- 100 BMI1 siC A B siBMI1#1 tabolism and DNA damage response (Fig.
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