title: Sumoylation of the transcriptional intermediary factor 1beta (TIF1beta), the Co-repressor of the KRAB Multifinger proteins, is required for its transcriptional activity and is modulated by the KRAB domain.
abstract: Small ubiquitin-related modifier (SUMO) has emerged as a key post-translational modulator of protein functions. Here we show that TIF1beta, a developmental regulator proposed to act as a universal co-repressor for the large family of KRAB domain-containing zinc finger proteins, is a heavily SUMO-modified substrate. A combined analysis of deletion and punctual mutants identified TIF1beta as a multilysine acceptor for SUMO which specifically targets six lysine residues (Lys(554), Lys(575), Lys(676), Lys(750), Lys(779), and Lys(804)) within the TIF1beta C-terminal repressive region. Reporter gene assays indicate that TIF1beta requires SUMO-modification for its repressive activity. Indeed, sumoylation-less mutants failed to recapitulate TIF1beta-dependent repression. TIF1beta homodimerization properties and interaction with the KRAB domain are preserved in the mutants with lysine to arginine substitutions as confirmed by in vivo bioluminescence resonance energy transfer (BRET). Using histone deacetylase (HDAC) inhibitors, we also demonstrate that TIF1beta sumoylation is a prerequisite for the recruitment of HDAC and that TIF1beta SUMO-dependent repressive activity involves both HDAC-dependent and HDAC-independent components. Finally, we report that, in addition to relying on the integrity of its PHD finger and on its self-oligomerization, TIF1beta sumoylation is positively regulated by its interaction with KRAB domain-containing proteins. Altogether, our results provide new mechanistic insights into TIF1beta transcriptional repression and suggest that KRAB multifinger proteins not only recruit TIF1beta co-repressor to target genes but also increase its repressive activity through enhancement of its sumoylation.
authors.affiliation: Department of Biochemistry, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors.forename: Xavier H
authors.initials: XH
authors.lastname: Mascle
authors.forename: Delphine
authors.initials: D
authors.lastname: Germain-Desprez
authors.forename: Phuong
authors.initials: P
authors.lastname: Huynh
authors.forename: Patricia
authors.initials: P
authors.lastname: Estephan
authors.forename: Muriel
authors.initials: M
authors.lastname: Aubry
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006655:Histone Deacetylases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D000076183:Tripartite Motif-Containing Protein 28
chemical_list: D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D001120:Arginine; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28; D006655:Histone Deacetylases; D008239:Lysine