Role of the PLDLS-Binding Cleft Region of CtBP1 in Recruitment of Core and Auxiliary Components of the Corepressor Complex

Abstract

C-terminal binding protein (CtBP) family proteins CtBP1 and CtBP2 are highly homologous transcriptional corepressors and are recruited by a large number of transcription factors to mediate sequence-specific transcriptional repression. In addition to DNA-binding repressors, the nuclear protein complex of CtBP1 consists of enzymatic constituents such as histone deacetylases (HDAC1/2), histone methyl transferases (HMTases; G9a and GLP), and the lysine-specific demethylase (LSD1). Additionally, CtBPs also recruit the components of the sumoylation machinery. The CtBPs contain two different unique structural elements, a hydrophobic cleft, with which factors that contain motifs related to the E1A PLDLS motif bind, and a surface groove that binds with factors containing motifs related to the sequence RRTGXPPXL (RRT motif). By structure-based functional dissection of CtBP1, we show that the PLDLS-binding cleft region functions as the primary recruitment center for DNA-binding factors and for the core and auxiliary enzymatic constituents of the CtBP1 corepressor complex. We identify HDAC1/2, CoREST/LSD1, and Ubc9 (E2) as the core constituents of the CtBP1 complex, and these components interact with the PLDLS cleft region through non-PLDLS interactions. Among the CtBP core constituents, HDACs contribute predominantly to the repression activity of CtBP1. The auxiliary components include an HMTase complex (G9a/Wiz/CDYL) and two SUMO E3 ligases, HPC2 and PIAS1. The interaction of auxiliary components with CtBP1 is excluded by PLDLS (E1A)-mediated interactions. Although monomeric CtBP1 is proficient in the recruiting of both core and auxiliary components, NAD(H)-dependent dimerization is required for transcriptional repression. We also provide evidence that CtBP1 functions as a platform for sumoylation of cofactors.

CtBPs promote mitotic fidelity through their activities in the cell nucleus

“…Structural and mutational analysis of CtBPs have identified discreet functional domains within the proteins; the N terminus forms a hydrophobic cleft that contacts the PxDLS motif in many of its partner transcriptional regulators, and the core central domain undergoes NAD þ /NADH-dependent dimerization, which is required for the assembly of the chromatinmodifying complex (Nardini et al, 2003;Quinlan et al, 2006;Chinnadurai, 2007;Kuppuswamy et al, 2008). CtBP chromatin-modifying complexes are readily detectable in proliferating cancer cells in culture (Shi et al, 2003), and there is evidence that their formation is increased under conditions of increased glycolysis, for example, in response to hypoxia (Zhang et al, 2002).…”

“…CtBP chromatin-modifying complexes are readily detectable in proliferating cancer cells in culture (Shi et al, 2003), and there is evidence that their formation is increased under conditions of increased glycolysis, for example, in response to hypoxia (Zhang et al, 2002). The C-terminal domain makes secondary interactions with some PxDLS-containing factors (Kuppuswamy et al, 2008) and is also the minimal lipid interaction domain (Yang et al, 2008). Dimerization of CtBP molecules is required for the formation of an active chromatin-modifying complex (Kuppuswamy et al, 2008); we reasoned, therefore, that an N-terminal fragment of CtBP lacking the dimerization domain would act as a dominant negative by binding components of the CtBP repression complex and preventing their interaction with CtBP dimers.…”

“…The C-terminal domain makes secondary interactions with some PxDLS-containing factors (Kuppuswamy et al, 2008) and is also the minimal lipid interaction domain (Yang et al, 2008). Dimerization of CtBP molecules is required for the formation of an active chromatin-modifying complex (Kuppuswamy et al, 2008); we reasoned, therefore, that an N-terminal fragment of CtBP lacking the dimerization domain would act as a dominant negative by binding components of the CtBP repression complex and preventing their interaction with CtBP dimers. This region does not bind lipids (Yang et al, 2008), so would not be predicted to affect CtBP-dependent Golgi fission.…”

“…To test this assumption, we generated GST-CtBP DN V72R. V72 in CtBP2 is a critical residue in the PxDLS-binding domain, and its mutation to arginine (or mutation of the equivalent residue in CtBP1) has been clearly demonstrated by independent research groups to abrogate binding to PxDLS-containing proteins, including both chromatin-modifying enzymes and DNA-binding transcription factors (Nardini et al, 2003;Quinlan et al, 2006;Kuppuswamy et al, 2008). Thus, this mutant of CtBP DN would be unable to effectively compete with CtBPs for binding to PxDLS-motif-containing proteins.…”

“…CtBPs function in the nucleus to form a chromatinmodifying complex, which includes HDACs 1 and 2 amongst its core constituents (Shi et al, 2003;Kuppuswamy et al, 2008). Experimentally, the use of HDAC inhibitors has shown the ability of CtBPs to repress transcription to be at least partially HDAC dependant (Shi et al, 2003).…”