2014-10-01 DNA Damage • DNA is continuously damaged by endogenous and/or exogenous insults Novel Role of RYBP in DNA Damage • This results in a variety of DNA lesions: Repair; Implications in Cancer Therapy – Damaged or modified bases – Inter-strand cross links Faculty Research Seminar – Single strand breaks September 30, 2014 – Double strand breaks (DSB) • DSB is the most cytotoxic type of DNA damage Mohammad A.M. Ali, PhD DNA Damage Repair Pathways DNA Damage • Cell responds rapidly to DSB by recruiting DNA • DSB is repaired mainly by non-homologous end damage sensors/repairs factors into and joining (error-prone) or homologous surrounding the damage site (foci) recombination (error-free) (S/G2) NHEJ HR 53BP1 BRCA Rad51 1 2014-10-01 DNA Damage Response Signaling DNA Damage Repair Pathways • DSB repair is crucial to maintain the genome MRN Histone H2AX Phosphorylation integrity of the cell ATM Histone H2AX pSer139 • Radiation and chemo-therapy overwhelm this MDC1 repair system to induce DSBs in cancer cells PRC1 RNF8 K63-chain K48-chain Polyubiquitin • DSB repair signaling is termed DNA Damage decoration Response (DDR) which includes highly uH2AK119 coordinated signaling cascades to detect, VCP/p97 RAP80/BRCA1, 53BP1 transduce and repair the DSB displacement HR pathway NHEJ pathway Polycomb Repressive Complex 1 Transcriptional repression by PRC1 • PRC1 proteins are chromatin modifiers first PRC1 discovered as transcriptional repressors Non-canonical Canonical – Regulation of morphogenesis RYBP RYBP – Cell identity – Proliferation – Differentiation – Stem cell self-renewal and carcinogenesis • PRC1 exists mainly in two complexes canonical J. Cell. Sci. 1(125), 2012. - mono-Ub of H2AK119 PRC1 and non-canonical PRC1 - Chroman compac;on 2 2014-10-01 RYBP and Cancer: Oncogenic or RING1-YY1 Binding Protein (RYBP) Tumor suppressor ? • First discovered as a putative component of PRC1 • RYBP is overexpressed in classical Hodgkin’s lymphoma complex (yeast two hybrid screens)- binds to RING1 (HL) and adult T-cell leukemia/lymphoma • RYBP is 60% homologous to YAF-2 • High level of RYBP is correlated to poor prognosis in HL and • RYBP binds to a wide range of proteins: adult T-cell lymphoma – Transcription factors; YY1, E2F6, E4TF1, E2F2/3 • Loss of RYBP is observed in prostate cancer and cervical cancer – Death effector domains; caspase-8/10, FADD, DEDD • In prostate cancer; RYBP loss was associated with ERG – Pro-apoptotic proteins: Apoptin, Hippi fusion and poor prognosis – Ubiquitin and ubiquitinated proteins • Low RYBP level in cervical cancer was associated with poor • RYBP seems to mediate opposing functions according to outcome after chemo/radiotherapy cellular context • In breast cancer, the cell killing effect of HDAC inhibitors was associated with high RYBP levels Laser Micro-irradiation Time-lapse Aim of the Work Microscopy Technique Damage sites Investigate the potential role of the non- canonical PRC1 protein (RYBP) in DNA damage repair Cell. 127(3), 2006 Recruitment of CBX7 to DSB sites 3 2014-10-01 RYBP is displaced from the sites of DNA damage induced by laser micro-irradiation RYBP displacement is RNF8-dependent GFP-RYBP Before 30 s 60 s 180 s GFP-RYBP Before 30 s 60 s 180 s Control WT MEF +MG132 RNF8-/- +DBeQ RYBP displacement is mediated by K48 polyubiquitin chain RYBP Domain Structure 1 228 NZF NLS NLS C-terminus RYBP Ub-WT RYBP Ub-noK RYBP Ub-K48 180 s 180 s 180 s - Ubiquitin binding - Transcriptional repression - RING1A, RING1B - E4TF1 - YY1 - E2F 2/3 and 6 - Apoptin - DED 4 2014-10-01 The NZF-domain of RYBP is required for its The NZF-domain of RYBP is required for its displacement for DNA damage sites displacement for DNA damage sites WT_RYBP WT_RYBP dNZF_RYBP NZF NLS NLS C-terminus dNZF_RYBP NLS NLS C-terminus TF-AA_RYBP dC_RYBP TF-AA_RYBP TF-AA NZF NLS NLS C-terminus dC_RYBP NZF NLS NLS RYBP abrogates the ubiquitin-dependent RYBP abrogates the ubiquitin-dependent recruitment of HR repair proteins recruitment of HR repair proteins DAPI RYBP DAPI RYBP γ-H2AX BRCA1 γ-H2AX Rad51 5 2014-10-01 Effect of RYBP on the recruitment of Clinical Implications NHEJ repair protein • In 2005, Helleday and Ashworth’s groups unraveled DAPI RYBP specific killing of breast cancer cells of BRCA mutations with PARP-inhibitors (Synthetic Lethality) • BRCA mutations compromise the homologous- recombination DSB repair and make PARP inhibition lethal to cancer cells γ-H2AX 53BP1 • RYBP may sensitize breast caner cells to PARP inhibitors in the absence of BRCA mutations? (therapeutic_value) • RYBP levels may predict the response to radiation/ chemo-therapy in breast cancer? (prognostic_value) ACKNOWLEDGEMENTS The Hendzel Lab Dr. Michael Hendzel Ismail Hassan Ismail, Ph.D. Hilmar Strickfaden, Ph.D. Darin McDonald, M.Sc. Cross Cancer Institute Cell Imaging Facility Dr. Xuejun Sun and Gerry Barron THANK YOU Reagents: Dr. Gordon Chan’s lab Funding 6 .