The oligomeric state of CtBP determines its role as a transcriptional co-activator and co-repressor of Wingless targets

Abstract

C‐terminal‐binding protein (CtBP) is a well‐characterized transcriptional co‐repressor that requires homo‐dimerization for its activity. CtBP can both repress and activate Wingless nuclear targets in Drosophila. Here, we examine the role of CtBP dimerization in these opposing processes. CtBP mutants that cannot dimerize are able to promote Wingless signalling, but are defective in repressing Wingless targets. To further test the role of dimerization in repression, the positions of basic and acidic residues that form inter‐molecular salt bridges in the CtBP dimerization interface were swapped. These mutants cannot homo‐dimerize and are compromised for repression. However, their co‐expression leads to hetero‐dimerization and consequent repression of Wingless targets. Our results support a model where CtBP is a gene‐specific regulator of Wingless signalling, with some targets requiring CtBP dimers for inhibition while other targets utilize CtBP monomers for activation of their expression. Functional interactions between CtBP and Pygopus, a nuclear protein required for Wingless signalling, support a model where monomeric CtBP acts downstream of Pygopus in activating some Wingless targets.

Nicotinamide Adenine Dinucleotide-induced Multimerization of the Co-repressor CtBP1 Relies on a Switching Tryptophan

“…We previously observed that a soluble CtBP1(A52D) may act as a dominant negative mutant in a cellular model of p21 expression (21), further suggesting that CtBP1 can interact with its binding partners when not bound to chromatin. Interestingly, the possibility of a functional monomeric CtBP pool has been proposed (18,30). Taken together, these results suggest that the activities of CtBP1 are not exclusively downstream of NAD ϩ binding and that CtBP1 can interact with its targets without dimerizing, potentially having a regulatory role as a monomer or dimer.…”

“…6) clearly show intra-nuclear self-association, but how CtBP1 maintains a pool of monomers or if CtBP1 is immediately directed to associate by NAD(H) or responds to varying intra-nuclear dinucleotide concentrations (7,14) has not been definitively described using a cellular model of gene expression. CtBP1 monomers may have important biologic activities as recently shown in Drosophila for Wingless targets (18) and the ability of CtBP1 to recruit chromatin-modifying enzymes as a monomer may have important regulatory functions.…”

“…Some studies (7) have used CtBP1(G183) to determine that dinucleotide binding was not required for co-repressor activity in plasmid-based assays, but we and others (18) have found this protein to be unstable in transfected cells. Differences in the expression system for purified CtBP (bacterial versus insect cells) and in vitro versus cellbased assays are likely to account for other experimental discrepancies.…”

“…8) (27, 28), suggesting both that monomeric CtBP1 (18) or directed assembly itself may have regulatory roles in gene expression. Targeting a critical switch for the assembly of a functional co-repressor unit may be another useful strategy to disrupt CtBP function.…”

“…NAD(H) binding initiates dimerization and may influence dehydrogenase activity (14 -16) although the substrate(s) and reaction are unknown. Recent Drosophila studies suggest that residues with high homology to dehydrogenase catalytic subunits (17) and the oligomeric state (18) are critical for CtBP function during development, although these studies also do not clarify the substrate. Prior reports suggests that NAD(H) binding is necessary for PXDLS target binding (7), although others observe relatively high affinity of CtBP for the PXDLS target protein E1A in vitro in the absence of dinucleotide binding (16,19).…”