Regulation of Chromatin-Associated Proteins by Sumoylation



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Small 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.

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SUMO-1 Protein, Protein Processing, Post-Translational, Sumoylation


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