C-terminal binding protein
Transcriptional repression is based on the selective actions of recruited corepressor complexes, including those with enzymatic activities. One well-characterized developmentally important corepressor is the C-terminal binding protein (CtBP). Although intriguingly related in sequence to D2 hydroxyacid dehydrogenases, the mechanism by which CtBP functions remains unclear. Biochemical and crystallographic studies reveal that CtBP is a functional dehydrogenase. In addition, both a cofactor-dependent conformational change, with NAD+ and NADH being equivalently effective, and the active site residues are linked to the binding of the PXDLS consensus recognition motif on repressors, such as E1A and nuclear receptor interacting protein RIP140. Together, these data suggest that CtBP is an NAD+-regulated component of critical complexes for specific repression events in cells (Kumar, 2002).
The dehydrogenase domain alone is sufficient to bind the PXDLS motif. This domain is highly conserved within CtBP family members from C. elegans to vertebrates. However the C terminus extension (C') is highly variable with no predicted secondary structure. It is possible that C' is a regulatory region, likely to mediate CtBP function after recruitment to a PXDLS motif. In Drosophila, there are three splice variants of CtBP differing only in the C', and the shortest of these splice variants is essentially only composed of the dehydrogenase domain. It was initially speculated, based on the crystal structure, that the PXDLS motif might bind in a cavity near the entrance to the active site cleft. However, mutations in this cavity do not disrupt E1A interactions in vitro or the repression function in vivo. Unexpectedly, mutation of the active site residues do affect E1A binding, suggesting that the PXDLS motif interacts with these residues at the periphery of the active site cleft. The cleft is walled off by a loop extending from the 2-fold related subunit that may provide additional interactions with the PXDLS sequence. Indeed, CtBP may be a simple dehydrogenase that has evolved or gained an extra ability to bind a PXDLS recognition motif (Kumar, 2002).
Nuclear receptor interaction protein p140 is highly recruited to ligand receptors on cognate DNA sites. An interaction between CtBP and RIP140 has also been reported, which is intriguing because RIP140 is recruited to nuclear receptors in response to ligand based on RIP140 LXXLL motifs; it competes with other coactivators. While at ambient levels of CtBP, liganded retinoic acid receptor induces recruitment of coactivators, including CBP and p160 co-activator factors; it is hown here that increased expression of CtBP completely blocks RAR activation, an effect entirely dependent on RIP140. Again, this effect requires specific CtBP catalytic residues, consistent with the expected role for these residues in stabilizing binding to interacting cofactors. This also implies that, in the presence of ligand, regulation of CtBP can be a key component to the nature of the transcriptional response (Kumar, 2002).
The finding that E1A-CtBP requires NAD+ has interesting implications. In particular, it raises the possibility that alterations in NAD+ levels might modulate the binding of CtBP to specific repressor complexes, as well as regulating its own enzymatic activity. Alterations in NAD+ level has been documented in response to DNA damage, and the reported associations between CtBP and p130/Rb complex, BRCA1, and KU70 may be critical regulatory components of cellular homeostasis. The ratio of NAD+/NADH can vary in response to activation of metabolic dehydrogenases during day-night periods of food intake and starvation, and rhythmic cycles in the cellular redox state have been shown to regulate DNA binding of Clock and NPAS2 heterodimeric transcription factors (Kumar, 2002).
Using CtBP prepared and expressed in a mammalian system, no evidence was found that E1A-CtBP interaction is regulated differently by NADH versus NAD+. Intriguingly, these results differ from those reported for a bacterially expressed CtBP, where NADH is two to three orders of magnitude more effective than NAD+ in stimulating CtBP-E1A interaction. This discrepancy may reflect different sources of CtBP; since in vitro transcribed and translated (TnT) CtBP was used in this study. The rabbit reticulocyte lysate used for the TnT reaction is known to posttranslationally modify proteins, including phosphorylation, acetylation, and isoprenylation. It is possible that one or more of these modifications dampen the differential effect observed with bacterial CtBP. In all, it is not easy to see from the structure how NADH could be up to three orders of magnitude more effective than NAD+ in stimulating E1A-CtBP interaction, considering that the two cofactors differ chemically by only a hydrogen atom on the nicotinamide ring. This may be further clarified by comparing the structure described in this study to a complex of CtBP with NADH (Kumar, 2002).
CtBP is not the only transcription corepressor to bind NAD+. The Sir2 family of transcriptional corepressors also binds NAD+ as a cofactor for histone deacetylation reactions, and furthermore, there is direct evidence that activity of NAD+-dependent Sir2 repressors can regulate life span in C. elegans. Whether levels of nuclear NAD+ vary during development, viral infection, or transcriptional silencing remains to be determined, but it marks an intriguing new direction for future research (Kumar, 2002).
The C-terminal binding protein 2 (CtBP2) is a 48 kDa phosphoprotein reported
to function as a co-repressor for a growing list of transcriptional repressors. CtBP is a dimeric NAD+-regulated
D-isomer-specific 2-hydroxy acid dehydrogenase. However, the specific
substrate(s) of CtBP enzymatic activity and the relationship of this activity to
its co-repression function remain unknown. The ability of a human CtBP to bind
and serve as a co-repressor of E1A has been shown to be regulated by nuclear
NADH levels. This study extends the functional characterization of CtBP by
demonstrating that amino acid substitutions at Gly189 in the conserved
NAD+-binding fold both abrogate the ability of CtBP2 to homodimerize and are
associated with a dramatic loss of co-repressor activity. Consistent with the
known enzymatic activity of CtBP2, mutations at Arg272 in the substrate-binding
domain and at His321 in the catalytic domain result in significant loss of CtBP2
transcriptional co-repressor activity. High resolution serial C-terminal
deletion analysis of CtBP2 also reveals a novel N-terminal repression domain
that is distinct from its dehydrogenase domain. These results suggest a model in
which CtBP2 co-repressor function is regulated, at least in part, through the
effect of NADH on CtBP2 homodimerization (Thio, 2004).
It is proposed that the CtBP2 homodimerization function serves as a cellular redox sensor for its co-repressor function. In such a role, CtBPs can detect the
readiness of a cell to proceed with various cellular processes by the levels of
cellular NAD+/NADH present. The ability of CtBP co-repressors to sense the
cellular redox state is likely to be specific to NAD+/NADH. It has been
reported that the binding of hCtBP1 to E1A was insensitive to NADP+, NADPH and
FAD+. This is not surprising since the NAD+-binding domain of CtBPs contain
the conserved GXGXXG motif (where X is any amino acid), characteristic of
enzymes that use NADH as a cofactor. The nucleotide-binding domains of enzymes
that use NADPH as a cofactor contain a distinct GXGXXA motif. The
nucleotide-binding domain in the 2HAD [PDB] family of bacterial enzymes
moderates enzyme activity by determining the availability of the active site.
The active site is found in a crevice between the nucleotide-binding and
substrate-binding domains, which are connected by a flexible hinge region.
Cofactor binding causes a conformational change of the dimer, thus closing the
active site cleft. This effectively brings the NAD+ cofactor into a favorable
position relative to the substrate. Once the hydride transfer from substrate to
NAD+ is completed, the cleft opens again to allow the release of the enzymatic
product. The inability to bind NAD+/NADH, as in the case of the Gly189
mutation in mCtBP2, would leave the active site closed once the substrate is
bound, thus rendering the enzyme incapable of carrying out any further catalytic
reactions (Thio, 2004).
This proposal is supported by a well-established paradigm of redox-regulated
transcriptional regulation. The silent information regulator 2 protein (Sir2) in
yeast mediates transcriptional silencing by promoting the deacetylation of
histones in a reaction that absolutely depends on the presence of NAD+. A
detailed mechanism for the reaction catalyzed by Sir2 has been proposed.
The proto-oncogenes c-fos and c-jun are known to be regulated by the
cellular redox state. Their protein products, Fos and Jun, function
cooperatively by forming a heterodimeric complex known as activator protein-1
(AP-1). AP-1 binds to the DNA regulatory element known as the AP-1-binding site.
The ability of AP-1 to bind DNA is regulated by the reduction-oxidation of a
single conserved cysteine residue of the DNA-binding domain of these two
proteins. The reduction of this residue is carried out by redox factor-1
(Ref-1), which is itself subject to redox-mediated regulation. The oxidized
or reduced state of cells would thus trigger a redox cascade involving Ref-1,
AP-1 and possibly many other proteins, which would eventually lead to the
transcriptional repression or activation of downstream target genes. NADPH
has been reported to reverse the oxidation of the yeast homolog of AP-1 (YAP-1).
The demonstration that CtBP is an NAD+-regulated dehydrogenase that mediates
transcriptional repression, and the finding that the level of NADH controls
and regulates CtBP2 co-repressor function through its effect on CtBP2
homodimerization, provides further evidence for a broader functional role of
enzymatic activity in general, and NAD+-dependent dehydrogenase activity in
particular, in gene transcriptional regulation (Thio, 2004).
The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras
cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, termed C-terminal binding protein (CtBP). The cDNA for CtBP was cloned via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain reveals that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation (Schaeper, 1995).
A region of the C-terminus of adenovirus type 2/5 E1A protein has been associated with negative modulation of tumorigenicity, as well as the extent of oncogenic transformation. In contrast with the N-terminus of the E1A protein, which has been extensively characterized and shown to associate with a number of cellular proteins, the function of the C-terminus is poorly understood. To date, a single 48-kDa protein, CTBP1, has been shown to associate with this region. Human (CTBP2) and mouse (Ctbp2), both highly related to CTBP1, have been identification and sequenced and these also are likely to bind to the E1A protein. CTBP2 is expressed in all tissues tested, with a higher level of expression in the heart, skeletal muscle, and pancreas. CTBP1 and CTBP2 map to human chromosomes 4p16 and 21q21.3, respectively (Katsanis, 1998).
Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is
dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in
tumorigenesis-restraining activity, cellular proteins were sought that complex with CtBP. A 125-kDa protein, CtIP, has been cloned and characterized that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. These results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif (Schaeper, 1998).
The adenovirus E1A-243R protein has the ability to force a resting cell into uncontrolled proliferation by modulating the activity of key targets in cell cycle control. Most of these regulatory mechanisms are dependent on activities mapping to conserved region 1 (CR1) and the non-conserved N-terminal region
of E1A. CR1 functions as a very patent transactivator when it is tethered to a promoter through a heterologous DNA binding domain. However, artificial DNA binding is not sufficient to convert full-length E1A-243R to a transactivator. Thus, an additional function(s) of the E1A-243R protein modulates the effect of CR1 in transcription regulation. A 44 amino acid region at the extreme C-terminus of ElA inhibits transactivation by a Gal4-CR1 fusion protein. Inhibition correlates with binding of the nuclear 48 kDa C-terminal binding protein
(CtBP), which has been implicated in E1A-mediated suppression of the metastatic potential of tumou cells. This suggests that CtBP binding can regulate E1A-mediated transformation by modulating CR1-dependent control of transcription (Sollerbrant, 1998).
Because of the high sequence conservation from human to Drosophila CtBP, it was expected that Drosophila CtBP would also interact with adenovirus E1a. dCtBP indeed interacts strongly with the C-terminus of the Ad2 E1a protein in a directed two-hybrid assay. Full-length E1a fused to LexA is lethal to yeast cells and could not be tested. Point mutations within a six amino acid motif in the E1a C-terminus, PXDLSX, eliminate or attenuate CtBP binding. A similar sequence was sought within the sufficient 25 amino acid interaction region of Hairy; the five amino acid PLSLV sequence identified from full-length Hairy abolishes interaction with dCtBP while still retaining Hairy's ability to interact with other proteins, including Groucho, which binds to the adjacent WRPW sequence. The dCtBP interaction domain for E(spl)mdelta was also mapped. 16 amino acids (143-158) are found to be sufficient for this interaction. Deletion of five amino acids similar to the Hairy consensus from full-length E(spl)mdelta abolishes interaction with dCtBP, while deletion of an adjacent five amino acids has no effect (Sollerbrant, 1998).
Adenovirus E1A mediates its effects on cellular transformation and transcription
by interacting with critical cellular proteins involved in cell growth and
differentiation. The amino terminus of E1A binds to CBP/p300 and associated
histone acetyltransferases such as P/CAF. The carboxyl terminus binds to the
carboxyl-terminal binding protein (CtBP), which associates with histone
deacetylases. 12S E1A can be acetylated by p300 and P/CAF, and one of the acetylation sites maps to Lys-239. This Lys residue is adjacent to the
consensus CtBP binding motif, PXDLS. Mutation of Lys-239 to Gln or Ala blocks
CtBP binding in vitro and disrupts the E1A-CtBP interaction in vivo. Peptide
competition assays demonstrate that the interaction of E1A with CtBP is also
blocked by Lys-239 acetylation. Supporting a functional role for Lys-239 in CtBP
binding, mutation of this residue to Ala decreases the ability of E1A to block
cAMP-regulated enhancer (CRE)-binding protein (CREB)-stimulated gene expression.
Lys-239 is acetylated in cells as detected by using an antibody
directed against an acetyl-Lys-239 E1A peptide. CtBP interacts with a wide
variety of other transcriptional repressors through the PXDLS motif, and, in
many instances, this motif is followed by a Lys residue. It is suggested that
acetylation of this residue by histone acetyltransferases, and the consequent
disruption of repressor complexes, might be a general mechanism for gene
activation (Zhang, 2000).
CtBP has been shown to be a highly conserved corepressor of transcription. E1A
and all the various transcription factors to which CtBP binds contain a
conserved PLDLS CtBP-interacting domain, and EBNA3C includes a PLDLS motif
(amino acids [aa] 728 to 732). EBNA3C binds to CtBP both in
vitro and in vivo and the interaction requires an intact PLDLS. The C
terminus of EBNA3C (aa 580 to 992) has modest trans-repressor activity when it
is fused to the DNA-binding domain of Gal4, and deletion or mutation of the
PLDLS sequence ablates this and unmasks a transactivation function within the
fragment. However, loss of the CtBP interaction motif has little effect on the
ability of full-length EBNA3C to repress transcription. A striking correlation
between CtBP binding and the capacity of EBNA3C to cooperate with (Ha-)Ras in
the immortalization and transformation of primary rat embryo fibroblasts has
also been revealed (Touitou, 2001).
PDZ domains function in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, affinity chromatography was performed of brain extract with the nNOS PDZ domain. The carboxyl-terminal-binding protein (CtBP) was identified as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DXL, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, these data suggest a new function for nNOS as a regulator of CtBP nuclear localization (Riefler, 2001).
The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. CtBP1 is SUMOylated and its SUMOylation profoundly affects its subcellular localization. SUMOylation occurs at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacks the SUMOylation site and is not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifts CtBP1 from the nucleus to the cytoplasm, while it has little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolishes the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 is inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression (Lin, 2003).
In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation,
requires a blood meal. Consequently, mosquitoes are vectors of many devastating
human diseases. An important adaptation for anautogenicity is the
previtellogenic arrest (the state of arrest) preventing the activation of the
yolk protein precursor (YPP) genes Vg and VCP prior to blood feeding. A novel
GATA factor (AaGATAr) that recognizes GATA binding motifs (WGATAR) in the
upstream region of the YPP genes serves as a transcriptional repressor at the
state of arrest. Importantly, AaGATAr can override the 20-hydroxyecdysone
transactivation of YPP genes, and its transcriptional repression involves the
recruitment of CtBP, one of the universal corepressors. AaGATAr transcript is
present only in the adult female fat body. Furthermore, in nuclear extracts of
previtellogenic fat bodies with transcriptionally repressed YPP genes, there is
a GATA binding protein forming a band with mobility similar to that of AaGATAr.
The specific repression of YPP genes by AaGATAr in the fat body of the female
mosquito during the state of arrest represents an important molecular adaptation
for anautogenicity (Martin, 2001).
Basic Kruppel-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. BKLF is capable of repressing transcription, and its
repression domain has been mapped to the N-terminus. A two-hybrid screen against BKLF was carried out, and a
novel clone was isolated encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human
CtBP, a cellular protein that binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral
oncoprotein, E1a. 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. mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6,
ZEB and FOG (Drosophila homolog: U-shaped). These results establish a new member of the CtBP family, mCtBP2, as a mammalian
co-repressor targeting diverse transcriptional regulators (Turner, 1998).
GATA4 is a transcriptional activator of cardiac-restricted promoters and is
required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a
multizinc finger protein that associates with GATA4 and represses
GATA4-dependent transcription. To better understand the transcriptional
repressor activity of FOG-2 a functional analysis of the FOG-2
protein was performed. The results demonstrate that (1) zinc fingers 1 and 6 of FOG-2 are each
capable of interacting with evolutionarily conserved motifs within the
N-terminal zinc finger of mammalian GATA proteins; (2) a nuclear localization
signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of
FOG-2, and (3) FOG-2 can interact with the transcriptional co-repressor,
C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS).
Surprisingly, however, this interaction with C-terminal-binding protein-2 is not
required for FOG-2-mediated repression of GATA4-dependent transcription.
Instead, a novel N-terminal domain of FOG-2 (amino acids
1-247) has been identifed that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent
transcription and thereby form the foundation for genetic studies designed to
elucidate the role of FOG-2 in cardiac development (Svensson, 2000).
CACCC-boxes are recognized by transcription factors of the Sp/Kruppel-like
Factor (Sp1/KLF) family. Described here is one member of this family,
KLF8/ZNF741/BKLF3 (KLF8). KLF8 contains a characteristic C-terminal DNA-binding
domain comprised of three Kruppel-like zinc fingers, but it also has limited
homology to another family member, KLF3/Basic Kruppel-like Factor (KLF3/BKLF),
in its N-terminus. Most significantly, it shares with KLF3/BKLF a
Pro-Val-Asp-Leu-Ser/Thr motif. In KLF3/BKLF this motif mediates contact with the
co-repressor protein C-terminal Binding Protein (CtBP). The
KLF8 Pro-Val-Asp-Leu-Ser motif also contacts CtBP. The N-terminus
of KLF8 functions as a repression domain and its activity relies on the
integrity of the CtBP recognition motif. The zinc fingers of
KLF8 recognize CACCC elements in DNA, and full-length KLF8 can repress a
CACCC-dependent promoter. KLF8 is broadly expressed in
human tissues. These results establish KLF8 as a CACCC-box binding protein that
associates with CtBP and represses transcription (van Vliet, 2000).
deltaEF1, a representative of the zinc finger-homeodomain protein family, is a
transcriptional repressor that binds E2-box (CACCTG) and related sequences and
counteracts the activators through transrepression mechanisms. It has been shown
that the N-proximal region of the protein is involved in the transrepression.
deltaEF1 has a second mechanism of transrepression,
recruiting CtBP1 or CtBP2 as its corepressor. A two-hybrid screen of mouse cDNAs
with various portions of deltaEF1 has identified these proteins, which bind to
deltaEF1 in a manner dependent on the PLDLSL sequence located in the short
medial (MS) portion of deltaEF1. CtBP1 is the mouse ortholog of human CtBP,
known as the C-terminal binding protein of adenovirus E1A, while CtBP2 is the
second homolog. Fusion of mouse CtBP1 or CtBP2 to Gal4DBD (Gal4 DNA binding
domain) makes these proteins Gal4 binding site-dependent transcriptional repressors in
transfected 10T1/2 cells, indicating their involvement in a transcriptional
repression mechanism. When the MS portion of deltaEF1 is fused to Gal4DBD and
used to transfect cells, a strong transrepression activity is generated, but
this activity is totally dependent on the PLDLSL sequence that serves as the
site for interaction with endogenous CtBP proteins, indicating that CtBP-1 and -2
can act as corepressors. Exogenous CtBP1/2 significantly enhances
transcriptional repression by deltaEF1, and this enhancement is lost if the
PLDLSL sequence is altered, demonstrating that CtBP1 and -2 act as corepressors
of deltaEF1. In the mouse, CtBP1 is expressed from embryo to adult, but CtBP2 is
mainly expressed during embryogenesis. In developing embryos, CtBP1 and CtBP2
are expressed broadly with different tissue preferences. Remarkably, their high
expression occurs in subsets of deltaEF1-expressing tissues, e.g., cephalic and
dorsal root ganglia, spinal cord, posterior-distal halves of the limb bud
mesenchyme, and perichondrium of forming digits, supporting the conclusion that
CtBP1 and -2 play crucial roles in the repressor action of deltaEF1 in these
tissues (Furusawa, 1999).
Previous work has demonstrated the critical role for transcription repression in quiescent cells through the action of E2F-Rb or
E2F-p130 complexes. Recent studies have shown that at least one mechanism for this repression involves the recruitment of
histone deacetylase. Nevertheless, these studies also suggest that other events likely contribute to E2F/Rb-mediated repression.
Using a yeast two-hybrid screen to identify proteins that specifically interact with the Rb-related p130 protein, it has been demonstrated
that p130, as well as Rb, interacts with a protein known as CtIP. This interaction depends on the p130 pocket domain, which is
important for repression activity, as well as an LXCXE sequence within CtIP, a motif previously shown to mediate interactions of viral proteins with Rb. CtIP
interacts with CtBP, a protein named for its ability to interact with the C-terminal sequences of adenovirus E1A. Recent work has demonstrated that the Drosophila homolog of CtBP is a transcriptional corepressor for Hairy, Knirps, and Snail. Both CtIP and CtBP can efficiently repress transcription when
recruited to a promoter by the Gal4 DNA binding domain, thereby identifying them as corepressor proteins. Moreover, the full repression activity of CtIP requires a PLDLS domain that is also necessary for the interaction with CtBP. It is proposed that E2F-mediated repression involves at least two events, either the recruitment of a histone deacetylase or the recruitment of the CtIP/CtBP corepressor complex (Meloni, 1999).
The homeodomain protein TGIF represses transcription in part by recruiting
histone deacetylases. TGIF binds directly to DNA to repress transcription or
interacts with TGF-beta-activated Smads, thereby repressing genes normally
activated by TGF-beta. Loss of function mutations in TGIF result in
holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single
amino acid substitution in a conserved PLDLS motif within the amino-terminal
repression domain. TGIF interacts with the corepressor
carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first
identified by its ability to bind the adenovirus E1A protein, interacts both
with gene-specific transcriptional repressors and with a subset of polycomb
proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is
dependent on interaction with CtBP, and TGIF is able to recruit
CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF
abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF
to repress transcription. Thus, at least one HPE mutation in TGIF appears to
prevent CtBP-dependent transcriptional repression by TGIF, suggesting an
important developmental role for the recruitment of CtBP by TGIF (Melhuish, 2000).
Ectopic production of the EVI1 transcriptional repressor zinc finger protein (Drosophila homolog: CG10568) is seen in 4%-6% of human acute myeloid leukemias. Overexpression also transforms
Rat1 fibroblasts by an unknown mechanism, which is likely to be related to its
role in leukemia and which depends upon its repressor activity. Mutant murine Evi-1 proteins, lacking either the N-terminal zinc finger DNA
binding domain or both DNA binding zinc finger clusters, function as dominant
negative mutants by reverting the transformed phenotype of Evi-1 transformed
Rat1 fibroblasts. The dominant negative activity of the non-DNA binding mutants
suggests sequestration of transformation-specific cofactors and recruitment
of these cellular factors might mediate Evi-1 transforming activity. C-terminal
binding protein (CtBP) co-repressor family proteins bind PLDLS-like motifs. The murine Evi-1 repressor domain has two such sites, PFDLT (site a,
amino acids 553-559) and PLDLS (site b, amino acids 584-590), which
independently can bind CtBP family co-repressor proteins, with site b binding
with higher affinity than site a. Functional analysis of specific CtBP binding
mutants show site b is absolutely required to mediate both transformation of
Rat1 fibroblasts and transcriptional repressor activity. This is the first
demonstration that the biological activity of a mammalian cellular
transcriptional repressor protein is mediated by CtBPs. Furthermore, it suggests
that CtBP proteins are involved in the development of some acute leukemias and
that blocking their ability to specifically interact with EVI1 might provide a
target for the development of pharmacological therapeutic agents (Palmer, 2001).
Evi-1 is a zinc finger nuclear protein whose inappropriate expression leads to
leukemic transformation of hematopoietic cells in mice and humans. This expression blocks the antiproliferative effect of transforming growth
factor beta (TGF-beta). Evi-1 represses TGF-beta signaling by direct interaction
with Smad3 through its first zinc finger motif.
Evi-1 represses Smad-induced transcription by recruiting C-terminal binding
protein (CtBP) as a corepressor. Evi-1 associates with CtBP1 through one of the
consensus binding motifs, and this association is required for efficient
inhibition of TGF-beta signaling. A specific inhibitor for histone deacetylase
(HDAc) alleviates Evi-1-mediated repression of TGF-beta signaling, suggesting
that HDAc is involved in the transcriptional repression by Evi-1. This
identifies a novel function of Evi-1 as a member of corepressor complexes and
suggests that aberrant recruitment of corepressors is one of the mechanisms for
Evi-1-induced leukemogenesis (Izutsu, 2001).
ZEB is an active transcriptional repressor that regulates lymphocyte and muscle differentiation in vertebrates. Its homolog in Drosophila (Zfh-1) is also essential for differentiation of somatic and cardiac muscle. ZEB and Zfh-1 are shown to interact with the corepressor CtBP to repress transcription. ZEB and Zfh-1, both contain the sequence PLDLS in the same region of the repressor domain, and this sequence is shown to bind CtBP-1 and -2. In vertebrate species, ZEB contains two additional CtBP-like binding sites (variations of the PLDLS sequence) that also bind CtBP proteins and are required for full repressor activity. The three sites have an additive effect, and mutation of all three sites is necessary to abolish both binding to CtBP and repressor activity. Finally, the interaction of CtBP with ZEB at the promoter is shown to be necessary for repressor activity (Postigo, 1999).
Balancing signals derived from the TGFbeta family are crucial for regulating cell proliferation and differentiation, and in establishing the embryonic axis during development. TGFbeta/BMP signaling leads to the activation and nuclear translocation of Smad proteins, which activate transcription of specific target genes by recruiting P/CAF and p300. The two members of the ZEB family of zinc finger factors (ZEB-1/deltaEF1 and ZEB-2/SIP1) regulate TGFbeta/BMP signaling in opposite ways: ZEB-1/deltaEF1 synergizes with Smad-mediated transcriptional activation, while ZEB-2/SIP1 represses it. These antagonistic effects by the ZEB proteins arise from the differential recruitment of transcriptional coactivators (p300 and P/CAF) and corepressors (CtBP) to the Smads. Thus, while ZEB-1/deltaEF1 binds to p300 and promotes the formation of a p300-Smad transcriptional complex, ZEB-2/SIP1 acts as a repressor by recruiting CtBP. This model of regulation by ZEB proteins also functions in vivo, where they have opposing effects on the regulation of TGFbeta family-dependent genes during Xenopus development (Postigo, 2003).
CtBP (carboxyl-terminal binding protein) participates in regulating cellular development and differentiation by associating with a diverse array of transcriptional repressors. Most of these interactions occur through a consensus CtBP-binding motif, PXDLS, in the repressor proteins. The CtBP-binding motif in E1A is flanked by a Lys residue and it has been suggested that acetylation of this residue by the p300/CBP-associated factor P/CAF disrupts the CtBP interaction. The interaction between CtBP and the nuclear hormone receptor corepressor RIP140 has been shown to be regulated similarly, in this case by p300/CBP itself. CtBP interacts with RIP140 in vitro and in vivo through a sequence, PIDLSCK, in the amino-terminal third of the RIP140 protein. Acetylation of the Lys residue in this motif, demonstrated in vivo by using an acetylated RIP140-specific antibody, dramatically reduces CtBP binding. Mutation of the Lys residue to Gln results in a decrease in CtBP binding in vivo and a loss of transcriptional repression. It is suggested that p300/CBP-mediated acetylation disrupts the RIP140-CtBP complex and derepresses nuclear hormone receptor-regulated genes. Disruption of repressor-CtBP interactions by acetylation may be a general mode of gene activation (Vo, 2001).
The transcription factor p53 lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. p53 is inactive in proliferating cells, largely because of negative regulation by the Hdm2/Mdm2 oncoprotein, with which it physically associates. Release from this negative regulation is sufficient to activate p53 and can be triggered in cells by multiple stimuli through diverse pathways. This diversity is achieved in part because Hdm2 uses multiple mechanisms to inactivate p53; it targets p53 for ubiquitination and degradation by the proteosome, shuttles it out of the nucleus and into the cytoplasm, prevents its interaction with transcriptional coactivators, and contains an intrinsic transcriptional repressor activity. Hdm2 can also repress p53 activity through the recruitment of a known transcriptional corepressor, hCtBP2. This interaction, and consequent repression of p53-dependent transcription, is relieved under hypoxia or hypoxia-mimicking conditions that are known to increase levels of intracellular NADH. CtBP proteins can undergo an NADH-induced conformational change, which results in a loss of their Hdm2 binding ability. This pathway represents a novel mechanism whereby p53 activity can be induced by cellular stress (Mirnezami, 2003).
The recruitment of hCtBP1 by proteins containing a PXDLS motif is regulated by changes in cellular redox potential. The central dehydrogenase domain of hCtBP1 contains a high-affinity binding site for NADH (GXGXXG), occupation of which induces a conformational change in the hCtBP1 molecule and an increase in binding to proteins such as E1A and ZEB. A mutation in hCtBP1 in the GXGXXG motif (G183A) abolishes NADH responsiveness. This site in hCtBP2 is conserved (amino acids 187-192): it was asked whether NADH could regulate the Hdm2:hCtBP2 interaction. NADH concentrations (0.01 to 1 mM) known to promote the interaction of hCtBP1 with PXDLS motif proteins inhibit binding of full-length GST-hCtBP2 to Hdm2. This inhibition did not occur when either GST-hCtBP2(1-110), lacking the dehydrogenase domain, or hCtBP2(G189A), containing a mutation in the NADH binding site, were used in the assays. Therefore, in contrast to interactions with PXDLS motif proteins, the conformational changes induced by NADH binding to the CtBP dehydrogenase domain result in a reduced affinity of hCtBP2 for Hdm2. Exposure of cells in culture to CoCl2 can be used as a model for the induction of a hypoxia-like stress response. CoCl2 treatment (200 μM) induces an increase in the cellular NADH/NAD+ ratio sufficient to promote binding of CtBP proteins to PXDLS motif proteins in the cell. 200 μM CoCl2 reduces the formation of Hdm2:hCtBP2 complexes in MCF-7 cells. Hypoxia, which has a greater effect on the cellular NADH/NAD+ ratio than CoCl2, is more effective than CoCl2 in reducing the Hdm2:hCtBP2 interaction. These data demonstrate, therefore, that the NADH-induced regulation of the Hdm2:hCtBP2 interaction also occurs in vivo (Mirnezami, 2003).
The distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, binds two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppresses binding of both SRF and YY1 complexes and abolishes DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoD-CArG sequence specifically and rapidly suppress MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and beta-galactosidase is induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter is no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration (L'honore, 2003).
Homeodomain-interacting protein kinase-2 (HIPK2) is a serine/threonine kinase
involved in transcriptional regulation and apoptosis. The transcriptional
corepressor CtBP (carboxyl-terminal binding protein) also plays a fundamental
role in these processes. HIPK2 has been shown to participate
in a pathway of UV-triggered CtBP clearance that results in cell death. HIPK2
phosphorylates CtBP at Ser-422 in vitro. A Ser-422 phospho-specific
antibody was developed to demonstrate that CtBP is phosphorylated on this residue in response
to UV irradiation. HIPK2 knock-down blocks the UV-induced Ser-422
phosphorylation and degradation. The proteasomal inhibitor MG-132 treatment
increases levels of ubiquitinated CtBP induced by UV. Interference
with HIPK2 function via the kinase-dead mutant decreases CtBP ubiquitination.
Furthermore, a phosphopeptide spanning Ser-422 blocks UV-triggered CtBP
degradation, confirming that Ser-422 phosphorylation marks CtBP for clearance.
Consequently, interference with HIPK2 action in H1299 cells rescues UV-triggered
apoptosis (Zhang, 2004).
HIPK2 is a Ser/Thr kinase that has been shown previously to be activated by UV
irradiation, TGF- ß treatment, Wnt signaling, and TAK1 action. It
plays important roles in transcriptional regulation and promotes cell growth
arrest and apoptosis. HIPK2 activation triggers Ser-46 phosphorylation on p53
and prevents MDM2-mediated ubiquitination and degradation of p53,
resulting in the apoptotic response. A recent report also demonstrated that
HIPK2 controls sensory neuron survival by suppressing the Brn3a-dependent
transcription of Brn3a, TrkA, and Bcl-xL. Trigeminal sensory neurons, which
are especially susceptible to HIPK2-induced apoptosis, express the highest
levels of HIPK2 during the peak of apoptosis in vivo, supporting the idea that
HIPK2 participates in programmed cell death in the developing peripheral nervous
system. This study confirmed that the transcriptional corepressor CtBP is a
HIPK2 target. In addition to stabilizing p53, HIPK2 phosphorylates Ser-422 on
the antiapoptotic factor CtBP. This action destabilizes CtBP, thus providing an
alternative pathway for programmed cell death. Of note, this pathway should be
functional in p53-deficient cells, which includes most human tumors (Zhang, 2004).
Precisely how Ser-422 phosphorylation marks CtBP for clearance is uncertain.
This modification on Ser-422 must be read and interpreted by the cellular
machinery. It is likely that CtBP phosphorylation causes the recruitment of a
particular E3 ligase that triggers ubiquitination. Alternatively, novel
enzyme(s) and bridging factor(s) might play critical roles in CtBP clearance.
Elucidating the mechanism of the phosphorylation-dependent degradation of CtBP
is essential for understanding CtBP regulation (Zhang, 2004).
It was interesting that Ser-422 phosphorylation and ubiquitination was observed
even in the absence of UV stimulation, suggesting that these modifications are a
part of CtBP homeostasis. This finding is in agreement with the finding
that the Ser-422 mutant of CtBP is detected at a higher level than the
wild-type protein when both were expressed in a CtBP-null background.
Whether basal activity of HIPK2 or other kinases are responsible for this action
is unknown (Zhang, 2004)
Mice harboring mutations in both Ctbp1 and Ctbp2 were generated to address the in vivo function of CtBPs during vertebrate development. Ctbp1 mutant mice are small but viable and fertile, whereas Ctbp2-null mice show defects in axial patterning and die by E10.5 due to aberrant extraembryonic development. Mice harboring various combinations of Ctbp1 and Ctbp2 mutant alleles exhibit dosage-sensitive defects in a wide range of developmental processes. The strong genetic interaction, as well as transcription assays with CtBP-deficient cells, indicates that CtBPs have overlapping roles in regulating gene expression. It is suggested that the observed phenotypes reflect the large number of transcription factors whose activities are compromised in the absence of CtBP (Hildebrand, 2002).
Binding of the C-terminal binding protein, CtBP, to the adenovirus E1A moiety of a Gal4-E1A fusion protein abolishes conserved region (CR) 1-dependent transcription activation. In contrast, a non-promoter targeted E1A peptide, capable of binding CtBP, can induce transcription from the proliferating cell nuclear antigen (PCNA) promoter (See Drosophila PCNA). CtBP is shown here to bind the histone
deacetylase HDAC1, suggesting that a promoter targeted CtBP-HDAC1 complex can silence transcription from the PCNA promoter through a deacetylation mechanism. Expression of the CtBP binding domain of E1A is sufficient to alleviate repression, possibly due to the displacement of the CtBP-HDAC1 complex from the promoter (Sundqvist, 1998).
Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity
during Drosophila and vertebrate development. To identify proteins that interact with vertebrate Pc homologs,
two-hybrid screens were performed with Xenopus Pc (XPc) and human Pc2 (HPC2).
C-terminal binding protein (CtBP) interacts with XPc and HPC2; CtBP and HPC2 coimmunoprecipitate,
and CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with
unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A
protein. Also, the Drosophila CtBP homolog interacts, through this conserved amino acid motif, with several
segmentation proteins that act as repressors. Similarly, it is found that CtBP binds with HPC2 and XPc through
the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog,
M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, CtBP is shown to be a transcriptional repressor. The results are discussed in terms of a model that brings
together PcG-mediated repression and repression systems that require corepressors such as CtBP (Sewalt, 1999).
Ikaros can repress transcription through the
recruitment of histone deacetylase complexes. Ikaros can also repress transcription through its interactions with the
co-repressor, C-terminal binding protein (CtBP). CtBP interacts with Ikaros
isoforms through a PEDLS motif present at the N terminus of these proteins but
not with homologs like Aiolos, which lack this motif. Mutations in Ikaros that
prevent CtBP interactions reduce its ability to repress transcription. CtBP
interacts with Sin3A but not with the Mi-2 co-repressor and it represses
transcription in a manner that is independent of histone deacetylase activity.
These data strongly suggest that CtBP contributes to a histone deacetylase
activity independent mechanism of repression by Ikaros. The viral oncoprotein E1A, which also binds to CtBP, shows a strong association
with Ikaros. This Ikaros-E1A interaction may underlie Ikaros's decreased ability
to repress transcription in E1A transformed cells (Koipally, 2000).
The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. The amino-terminal
regions of MITR and class II HDACs interact with the transcriptional
corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif
(P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence
in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the
ability of MITR to inhibit MEF2-dependent transcription. The residual repressive
activity of MITR mutants that fail to bind CtBP can be attributed to association
with other HDAC family members. These findings reveal CtBP-dependent and
-independent mechanisms for transcriptional repression by MITR and show that
MITR represses MEF2 activity through recruitment of multicomponent corepressor
complexes that include CtBP and HDACs (Zhang, 2001).
The transcriptional co-repressor CtBP (C-terminal binding protein)
is implicated in tumorigenesis because it is targeted by the
adenovirus E1A protein during oncogenic transformation. Genetic
studies have also identified a crucial function for CtBP in
animal development. CtBP is recruited to DNA by transcription
factors that contain a PXDLS motif, but the detailed molecular
events after the recruitment of CtBP to DNA and the mechanism
of CtBP function in tumorigenesis are largely unknown. A CtBP complex has been identified that contains the
essential components for both gene targeting and coordinated
histone modifications, allowing for the effective repression of
genes targeted by CtBP. Inhibiting the expression of CtBP and
its associated histone-modifying activities by RNA-mediated
interference results in alterations of histone modifications at
the promoter of the tumor invasion suppressor gene E-cadherin
and increased promoter activity in a reporter assay. These
findings identify a molecular mechanism by which CtBP mediates
transcriptional repression and provide insight into CtBP
participation in oncogenesis (Shi, 2003).
To understand how CtBP represses transcription,
the biochemical purification of CtBP was undertaken. Stable HeLa
cells were generated expressing human CtBP1 tagged with both the Flag and
hemagglutinin (HA) epitopes at its amino terminus. Nuclear extracts from these cells were
subjected to sequential purification with anti-Flag and anti-HA
antibody columns. About 20 polypeptides were
found to be specifically associated with the tagged CtBP1, whose identities were determined by mass
spectrometry. These CtBP1-
associated proteins can be grouped into at least four classes based on
their functions, which include the following: the DNA-binding
proteins such as ZEB 1, which have been shown to interact
with CtBP and to repress transcription in a CtBP-dependent manner;
the histone-modifying enzymes represented by the histone
deacetylases HDAC1 and 2 and the related histone methyltransferases
(HMTs) G9a and Eu-HMTase1 (EuHMT), and two chromodomain-containing proteins HPC2 and CDYL. The last group
includes CoREST (a protein found as a co-repressor to the
transcription factor REST), a CoREST-related protein (KIAA1343)
and a protein (KIAA0601) that shares sequence homology with
polyamine oxidases, which is referred to as NPAO (nuclear polyamine
oxidase). The presence of CoREST and its associated proteins in
the CtBP complex suggests a possible interplay between these two
co-repressors (Shi, 2003).
It is proposed that the action of the
CtBP complex is initiated by the DNA-binding repressors that
anchor the CtBP complex to its target promoters. This is followed
by the action of HDAC1/2, which removes the acetyl group from the
histone tails of transcriptionally active chromatin, allowing Lys 9 of
histone H3 to be methylated by G9a and EuHMT. The methylated
Lys 9 (and perhaps Lys 27) are then recognized by HPC2 and/or
CDYL, which might contribute to the formation of a local repressive
chromatin structure. These coordinated biochemical and enzymatic
events help convert an active chromatin environment to one that is
conducive to transcriptional repression. Biologically, the identification of the proteins involved in tumorigenesis in the CtBP
complex, and the finding that CtBP and the associated
HMTs repress transcription of the tumor invasion suppressor
E-cad, shed significant light on the role of CtBP in tumorigenesis.
Last, it is noted that the CtBP complex might contain additional
enzymes. Both CtBP complex and
recombinant CtBP possess dehydrogenase activity. In addition, NPAO is related to polyamine oxidases, and
CDYL shares homology with enoyl CoA isomerases/hydratases. It is
speculated that, whereas HDACs and HMTs are involved in histone
modifications, the other putative enzymes might also have a role in
CtBP-regulated transcription, perhaps by targeting non-histone
components of the chromatin. Future studies aimed at delineating
their functions and possible relationships with histone-modifying
enzymes in the CtBP complex are likely to provide new insight into
eukaryotic gene regulation (Shi, 2003).
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