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Posttranslational modification of many transcription factors with the small Ubiquitin-like modifier SUMO is associated with transcriptional repression. The molecular mechanisms by which SUMO attachment represses transcription were largely unknown. By a genome-wide RNA interference screen our laboratory has recently identified a number of SUMO-dependent repression components using the SUMOylated transcription factor Sp3 as a paradigm. Based on the primary data of the screen, the involvement of 120 proteins in SUMO-mediated repression was verified. Knockdown of these proteins by specific double-stranded RNAs (dsRNAs) impaired Sp3-SUMO-dependent transcriptional repression, whereas the activity of a SUMOylation-deficient Sp3 mutant was not affected. A number of validation experiments revealed that the ATP-dependent chromatin remodeler Mi-2, the D. melanogaster ortholog of the C. elegans protein MEP-1 and the polycomb protein Sfmbt act as direct SUMO-dependent transcriptional corepressors: (1) Knockdown of these proteins did not impair SUMO conjugation demonstrating that they act downstream of SUMO attachment. (2) Mi-2, MEP-1 and Sfmbt interact with SUMO and SUMO-modified Sp3 and (3) are recruited to promoters in a SUMOylation-dependent manner. (4) Knockdown of Mi-2, MEP-1 and Sfmbt relieved also repression mediated by the SUMO-modified transcription factor Dorsal. These results suggest that Mi-2, MEP-1 and Sfmbt are part of a common repression complex established by DNA-bound SUMO-modified transcription factors. In order to analyze the chromatin changes established by SUMO-modified transcription factors in the mammalian system in detail a cell line with a stably integrated chromatinized Gal4-driven reporter gene was generated. On transfection, SUMOylated transcription factors (Gal4 fusions of wildtype Sp3 or steroidogenic factor 1) repressed transcription whereas the corresponding SUMOylation-deficient mutants activated transcription of the integrated reporter gene. Chromatin-immunoprecipitations revealed that the promoter-bound SUMO-modified transcription factors led to the establishment of local repressive chromatin with characteristics of compacted heterochromatin. SUMO-dependent heterochromatin formation included recruitment of the chromatin remodeler Mi-2, the MBT-domain proteins L3MBTL1 and L3MBTL2 involved in chromatin compaction, the heterochromatin protein HP1, and the histone methyltransferases SETDB1 and SUV4-20H accompanied by the establishment of repressive histone modifications such as H3K9 and H4K20 trimethylation. These heterochromatic proteins and repressive histone modifications were also present at the endogenous Sp3-regulated murine dihydrofolate-reductase promotor in mouse embryonic fibroblasts. The establishment of heterochromatic structures was Sp3-SUMO-dependent because they were found only in the presence of SUMOylated wildtype Sp3 but not in Sp3-/- cells expressing a SUMOylation-deficient Sp3 mutant. These results indicate that SUMOylation of transcription factors plays a crucial role in regulating gene expression by initiating chromatin structure changes that renders DNA inaccessible to the transcription machinery.