Browsing by Subject "SUMO-1 Protein"
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Item Functional Analysis of the Human SMC5/6 Complex in Homologous Recombination and Telomere Maintenance(2008-05-13) Potts, Patrick Ryan; Yu, HongtaoDNA repair is required for the genomic stability and well-being of an organism. The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex promotes HR by localizing to DSBs where it holds sister chromatids in close proximity to allow HR-induced strand invasion and exchange. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this has been unclear. We have characterized the role of the human SMC5/6 complex in HRmediated DNA damage repair. The yeast SMC5/6 complex has been shown to be composed of the SMC5-SMC6 heterodimer and six non-SMC element (NSE) proteins. We show that the human homolog of one of these NSE proteins, MMS21/NSE2, is a ligase for small ubiquitin-like modifier (SUMO). Depletion of MMS21 by RNA interference (RNAi) sensitizes cells toward DNA damageinduced apoptosis. This hypersensitization of MMS21-RNAi cells is not due to a defect in DNA damage-induced cell cycle checkpoint, but rather in the kinetics of DNA damage repair. Since the yeast SMC5/6 complex has been implicated in HR-mediated DSB repair, we investigated the role of the human SMC5/6 complex in HR-mediated DSB repair. RNAi-mediated knockdown of the SMC5/6 complex components specifically decreases sister chromatid HR, but not non-homologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. We show that one potential mechanism by which the SMC5/6 complex specifically promotes sister chromatid HR is by facilitating the recruitment of the SMC1/3 cohesin complex to DSBs. We next examined whether the SMC5/6 complex is also required for sister chromatid HR of telomeres. Specific types of cancer cells, known as alternative lengthening of telomeres (ALT) cells, rely on telomere recombination for telomere lengthening and unlimited replicative potential. We show that the SMC5/6 complex promotes telomere recombination and lengthening in ALT cells by MMS21-dependent sumoylation of telomere-binding proteins. Sumoylation of these telomere-binding proteins relocalizes telomeres to nuclear PML bodies where HR proteins facilitate telomere recombination. These studies identify the human SMC5/6 complex and SUMO modification as critical mediators of sister chromatid HR.Item Regulation of Chromatin-Associated Proteins by Sumoylation(2007-08-08) Gocke, Christian Burris; Yu, HongtaoSmall Ubiquitin-like Modifier (SUMO) regulates diverse cellular processes through its reversible, covalent attachment to target proteins. Many SUMO substrates are involved in transcription and chromatin structure. Sumoylation appears to regulate the functions of target proteins by changing their subcellular localization, increasing their stability, and/or mediating their binding to other proteins. Using an In Vitro Expression Cloning (IVEC) approach, we have identified 40 human SUMO1 substrates. We have validated the sumoylation of 24 substrates in living cells. We show that one of these substrates, Mef2C, is coordinately regulated by phosphorylation and sumoylation. The spectrum of human SUMO1 substrates identified in our screen suggests general roles of sumoylation in transcription, chromosome structure, and RNA processing. Moreover, multiple subunits of a given chromatin-associated complex are targets for SUMO-conjugation. For example, a substrate identified in our screen, lysinespecific demethylase 1 (LSD1), is part of a complex that also contains Histone Deacetylase 1 (HDAC1), that is a SUMO substrate. This prompted me to study the function of this complex and its regulation by SUMO. Histone methylation regulates diverse chromatin-templated processes, including transcription. Many transcriptional corepressor complexes contain LSD1 and CoREST that collaborate to demethylate mono- and di-methylated histone H3 lysine 4 (H3K4) of nucleosomes. We have determined the crystal structure of the LSD1-CoREST complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 recognizes a large segment of the H3 tail through a deep, negatively charged pocket at the active site and possibly a shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the SANT2-DNA interaction diminishes CoREST-dependent demethylation of nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent nucleosome-binding mode may apply to other chromatin-modifying enzymes that generally contain multiple nucleosomebinding modules. The core CoREST corepressor complex, consisting of CoREST, LSD1, and HDAC1/2, represses transcription by coordinately removing histone modifications associated with gene activation. ZNF198 and other MYM-type zinc-finger proteins are also components of this complex. ZNF198, HDAC1, and LSD1 are SUMO substrates, and ZNF198 binds to SUMO non-covalently. We show that ZNF198 and its homologues do not regulate REST-responsive genes. Consistently, binding of REST and ZNF198 to CoREST are mutually exclusive. However, these MYM-domain proteins are required for tethering LSD1 to nuclear compartments and for repression of E-cadherin, a non-REST responsive gene. ZNF198 interacts efficiently only with the intact LSD1-CoREST-HDAC1 ternary complex, but not its individual subunits. ZNF198 also binds specifically to sumoylated, but not unsumoylated HDAC1. These interactions are mediated by tandem zinc-fingers of ZNF198. HDAC1 activity is not stimulated by sumoylation or ZNF198 binding. Sumoylated HDAC1 does not interact with CoREST, and LSD1 sumoylation is inhibited by CoREST binding. Therefore, ZNF198, through its unique and diverse protein-protein interactions, helps to maintain the intact CoREST complex on specific promoters.