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CtBP family proteins are unique in animals and in plants. The invertebrates and plants contain a single CtBP family gene while vertebrates have two genes. Genetic studies in and in mice indicate that CtBPs play pivotal roles in animal development. The vertebrate CtBPs (CtBPl and CtBP2) are highly related and are functionally redundant for certain developmental processes and non redundant for others. The vertebrates code two isoforms of each CtBPl and CtBP2. The animal CtBPs exhibit a highly conserved sequence and structural similarity to D-isomer specific 2-hydroxy acid dehydrogenases (D2-HDH). Structural and molecular modeling studies indicate that CtBPl is a dehydrogenase and could also bind with acyl-CoA under a different configuration. The CtBP family members function predominantly as transcriptional corepressors in the nucleus in conjunction with a number of different DNA binding repressors. The transcriptional regulatory activity of CtBPs appears to be regulated by NAD(H)-binding and the metabolic status of the cell. The compressor complex of CtBPl contains enzymatic constituents that mediate coordinated histone modification by deacetylation and methylation of histone H3-K9 and demethylation of histone H3-K4. In the cytosol, they perform diverse functions associated with membrane trafficking, central nervous system synapses and in regulation of the microtubule cytoskeleton. The mammalian CtBPs modulate oncogenesis by regulating the activities of tumor suppressor genes and cellular and viral oncogenes, consistent with a role in tumor suppression as well as in tumor promotion. The CtBPs promote tumorigenesis by repressing transcription of several critical pro-apoptotic genes and by inhibiting genes involved in the regulation of epithelial to mesen-chymal transition. This Chapter presents a comprehensive general review of the CtBP field and highlights contents of the individual Chapters of this book which contain detailed discussions on structure and functions of animal and plant CtBP family proteins.

Transcriptional repression is essential for patterning gene expression in the early embryo. Biochemical and genetic studies on C-terminal binding protein (dCtBP) have provided solid evidence that dCtBP acts as a corepressor for several transcriptional repressors. Similarly to mammalian CtBPs, dCtBP interacts with a short peptide motif, PxDLS, or related motifs. It appears that dCtBP is essential for short-range transcriptional repression in the early embryo. In contrast, it has been recendy reported that dCtBP participates in Polycomb-mediated long-range repression. In this chapter, we will review how the dCtBP corepressor functions, from the biochemical, developmental, and genetic point of views.

The C-terminal binding proteins (CtBPs) are ubiquitous corepressors that recruit histone-modifying enzymes to a variety of sequence specific DNA-binding proteins and other transcriptional regulators. CtBPs appear to play an important role in mediating repression and transforming activities of a variety of hematopoietic transcription factors such as Basic Krüppel-like Factor/Krüppel-like Factor 3 (BKLF/KLF3), Friend of GATA (FOG), Evi-1 and members of the Ikaros family. Mice lacking CtBPs die during embryonic development and exhibit defects in a wide range of developmental processes, including aberrant heart formation and absence of blood vessels in the yolk sac. The ongoing identification of repressed target genes and interacting transcriptional partners will help to unravel the contributions of CtBP proteins to hematopoiesis.

Adenovirus E1a proteins are potent and ubiquitously acting tumor suppressors in human tumor cells. Through interaction with CtBP (as well as other mechanisms), E1a protein sensitizes cells to several apoptotic responses including anoikis. This interaction also induces the expression of certain epithelial cell adhesion and cytoskeletal genes in various tumor cell lines. Functionally analogous results are observed in mouse embryo fibroblasts lacking CtBP1 and CtBP2 genes. These results implicate CtBP as a potential modulator of the epithelial-to-mesenchymal transition (EMT) as well as apoptosis.

C-terminal binding protein (CtBP) associates with adenovirus early region 1A (AdE1A) proteins through a highly conserved PXDLS motif located very close to its C-terminus in conserved region 4. To try to understand the importance of this interaction for the virus a point mutation in the CtBP binding site of Adl2ElA (P→S at amino acid 255) was engineered. The mutant Ad12E1A DNA (Ad12E1A6f) encoded a protein temperature sensitive (ts) for transformation of baby rat kidney cells when in combination with Ad12ElB. At 33°C transformation frequency was comparable to . At 37° and 38.5° transformants appeared as larger epithelioid cells and colonies senesced relatively rapidly. When the Ad12 6f AdE1A was incorporated into a mutant virus it caused a marked reduction in its ability to replicate with only Ad12E1A and Ad12ElB19K being expressed at early times. It was observed that 6fElA bound to CtBP very inefficiently. Ad12E1 transformed rat cell lines, carrying the 6f mutation were established from the 33°C transformants but failed to express the Ad12E1B54K protein. After a number of weeks in culture the cells developed a mesenchymal character; expression of proteins such as E-cadherin, P-cadherin and γ catenin was much reduced and expression of fibronection increased. These observations are consistent with inhibition of CtBP activity in Ad12E1 transformants but not in the 6f transformed cells. In a complementary study the effect of down-regulation of CtBP expression (using siRNA protocols) was examined. Consistent with results obtained with the 6f virus it was observed that reduction in expression of CtBP1 and CtBP2 facilitated viral infection and this effect was enhanced when expression of C-terminal interacting protein (CTIP) was also reduced.

The fundamental question facing developmental biology is how the diversity of cell and tissue types that comprise a vertebrate organism can be generated from a single fertilized egg. A critical aspect of the developmental process is setting up and maintaining the differential gene expression that is required to establish the variety of cell lineages present in the adult organism. Thus, an important aspect of understanding development is understanding how early asymmetries in the transcriptome of various cell types are established and, once established, how they are maintained or modified through subsequent generations and differentiation events. This process is carried out by the combined activities of both sequence specific DNA binding factors and their associated coactivators and compressors that act on either the general transcriptional machinery or the histone component of chromatin. CtBP proteins comprise one branch of corepressors that get recruited to DNA via sequence-specific DNA binding proteins and regulate gene expression. In mice, the CtBP family proteins are encoded by two loci, and . The transcripts encoding the CtBP1 and CtBP2 proteins are widely expressed and exhibit both unique and shared expression domains in the developing embryo. Genetic analysis of mice harboring mutations in and indicate that the proteins they encode likely have redundant functions during embryogenesis but are differentially required for specific developmental processes. This analysis shows that CtBP proteins are important in the formation of the placenta and tissues derived from all three germ layers, including muscles, skin, neural ectoderm, and intestinal epithelium. This chapter focuses on the roles of CtBPl and CtBP2 proteins in vertebrate development, with an emphasis on the genetics of and , the possible pathways that utilize CtBP proteins during embryogenesis, and the evidence that CtBP proteins could be implicated in multiple developmental processes linked to human diseases.

The corepressor CtBP (carboxyl-terminal binding protein) is involved in transcriptional pathways important for development, cell cycle regulation, and transformation. We demonstrate that CtBP binding to transcription repressors is stimulated by NAD and NADH, with NADH being two to three orders of magnitude more effective. Fluorescence resonance energy transfer studies of CtBP show a > 100-fold higher affinity for NADH than NAD, in agreement with the tighter interaction observed in the crystal structure of NADH-bound CtBP. Levels of free nuclear nicotinamide adenine dinucleotides, determined using two-photon microscopy, correspond to the concentrations required for half-maximal CtBP binding. Free cellular NAD concentration greatly exceeds that of NADH and the redox changes are mainly reflected by NADH levels. Agents increasing NADH levels stimulate CtBP binding to its partners in vivo and potentiate CtBP-mediated repression. These findings suggest that the transcriptional corepressor CtBP may serve as a redox sensor to provide a link between gene expression and metabolism.

Recent biochemical and proteomic approach has identified a CtBP super complex consisting of a host of chromatin modifying enzymes. Analysis of this complex has led to the appreciation that enzymes that mediate deacetylation and histone H3 lysine 9 methylation are present in the same biochemical complex, which facilitates coordinated histone modifications important for establishing repressive chromatin. Importandy, studies of this complex also resulted in the finding of the first histone demethylase LSDI, which represses transcription by demethylating histone K4, where methylation is linked to active transcription. It is anticipated that additional important new insights will be gained from further investigation of this unusual transcriptional repression machine.

CtBP is a transcriptional corepressor and is one of the three main transcriptional cofactors that are directly targeted by the viral oncoprotein E1A during oncogenic transformation. To explore mechanisms by which CtBP mediates transcriptional repression, a biochemical approach was taken to isolate proteins that are associated with CtBP. This effort has led to the identification of a CtBP super-complex, consisting of, among others, six potential enzymatic activities. While the exact composition of this super-complex may differ in different cell types, characterization of these enzymatic functions in HeLa cells has already provided significant insight into mechanism of action of CtBP and eukaryotic gene regulation. Below we provide a brief discussion of the enzymatic components of the CtBP complex and our current understanding of their individual as well as coordinated enzymatic actions in transcriptional repression. While other aspects of CtBP are covered in other chapters, this chapter is largely confined to the CtBP super complex.

The structural characteristics of the CtBP family of transcriptional corepressors suggest an additional role for coenzyme nicotinamide adenine dinudeotide in the repression of gene expression. Remarkably, CtBP orthologues are unique among transcriptional regulators in that they display striking primary sequence and structural similarity to the D-isomer specific 2-hydroxyacid dehydrogenase class of enzymes. Recent structural studies of rat CtBP/BARS and human CtBPl provide insight into the role of pyridine dinucleotide binding in regulation of CtBP quaternary structure, and corepression activity through association with -PXDLS-containing targets.

CtBP3/BARS was the third protein of the CtBP (C-terminal binding protein) family to be identified. It was initially isolated as a 50-kDa cytosolic protein during the characterisation of the molecular targets of the toxin brefeldin A (BFA). As this protein is a substrate of BFA-dependent ADP-ribosylation, it was initially named BARS-50 (BFA-dependent ADP-ribosylation substrate), or BARS. After its purification and cloning, the protein was shown to be the third member (hence CtBP3/BARS) of the CtBP transcription corepressor family of proteins, sharing a high degree of aminoacid identity with CtBPl (97%). CtBP3/BARS induces membrane fission in isolated Golgi membranes and is necessary for the fragmentation of the Golgi complex that occurs at the beginning of mitosis; its direct role in transcription regulation has not yet been specifically investigated. The CtBPs are thus a multifunctional protein family that can modulate both nuclear and cytosolic functions.