Cloning and characterization of mCtBP2, a co-repressor that associates with basic Kruppel-like factor and other mammalian transcriptional regulators

Abstract

Basic Krü ppel-like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. We have studied the activity of BKLF and found that it is capable of repressing transcription, and have mapped its repression domain to the N-terminus. We carried out a two-hybrid screen against BKLF and isolated a novel clone encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein which binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. We show that mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA-binding domain, mCtBP2 functions as a potent repressor. Finally, we demonstrate that mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6, ZEB and FOG. These results establish a new member of the CtBP family, mCtBP2, as a mammalian co-repressor targeting diverse transcriptional regulators.

Sumoylation of MEL1S at lysine 568 and its interaction with CtBP facilitates its repressor activity and the blockade of G-CSF-induced myeloid differentiation

“…The GAL4 DBD-fused MEL1S expression constructs with various deletions and amino acid replacements were cloned into the pM expression vector (Clontech). The murine CtBP2 expression vector, pFLAG-CtBP2, contained a full-length murine CtBP2 (kindly provided by Dr Crossley; Turner and Crossley, 1998) cloned into the FLAG-epitope tag expression vector pFLAG-Mock. The human UBC9 expression vectors, pMyc-UBC9 and pMyc-UBC9DN, contain full-length human UBC9 and the dominant negative mutant (C93S), respectively, cloned into the Myc-epitope tag expression vector pMyc-Mock.…”

The mammalian zinc finger transcription factor Krüppel‐like factor 3 (KLF3/BKLF)

“…The molecular mechanisms by which KLF3 regulates transcription have been extensively analyzed. KLF3 recruits the transcriptional corepressor C-terminal binding protein (CtBP) to the promoters of target genes and is thus believed to act mainly as a repressor of transcription (5). In addition to the interaction with CtBP, full repression activity is dependent on sumoylation (6).…”

A Mechanism of COOH–Terminal Binding Protein–Mediated Repression

“…We limited the number of candidate proteins by examining only human proteins with established function in transcription regulation (Table 1). Among this list were many transcription factors known to act as negative regulators of transcription, most of which are already known to bind CtBP (25,27,28,30,(42)(43)(44)(45). Interestingly, we also identified several transcription corepressors, such as the proteins N-CoR, 5V-TG-3V-interacting factor, and methyl CpG -binding protein, proteins found in complexes with HDAC.…”

“…Recent work has shown that the CtBP protein, from either mammals or Drosophila, plays a role as a transcription corepressor through physical interactions with a variety of promoter-specific transcription factors (17,21,(23)(24)(25)(26)(27)(28)(29)(30). Indeed, expression of a Gal4 binding domain (Gal4BD)-CtBP fusion protein resulted in a substantial repression of the major late promoter (MLP) when assayed in C33A cells.…”

“…CtIP functions as a transcriptional corepressor in part by recruiting CtBP, a protein first identified as an adenovirus E1A-interacting protein (19,20). Other work has shown that CtBP functions as a short-range corepressor in Drosophila (21)(22)(23)(24), and numerous experiments have shown a role for CtBP as a transcriptional repressor in mammalian cells (17,(25)(26)(27)(28)(29)(30). Although the exact mechanism of transcriptional repression remains unclear, several reports have shown that CtBP has both HDACdependent and HDAC-independent mechanisms of repression (27,29,(31)(32)(33)(34).…”