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EVOLUTIONARY HOMOLOGS

Table of contents

c-Myb, through interaction with p300, controls the proliferation and differentiation of hematopoietic stem and progenitor cells

Precise control of hematopoietic stem cell (HSC) proliferation and differentiation is needed to maintain a lifetime supply of blood cells. Using genome-wide ENU mutagenesis and phenotypic screening, a mouse line has been identified that harbors a point mutation in the transactivation (TA) domain of the transcription factor c-Myb (M303V) that reduces c-Myb-dependent TA by disrupting its interaction with the transcriptional coactivator p300. The biological consequences of the c-Myb^M303V/M303V mutation include thrombocytosis, megakaryocytosis, anemia, lymphopenia, and the absence of eosinophils. Detailed analysis of hematopoiesis in c-Myb^M303V/M303V mice reveals distinct blocks in T cell, B cell, and red blood cell development, as well as a remarkable 10-fold increase in the number of HSCs. Cell cycle analyses show that twice as many HSCs from c-Myb^M303V/M303V animals are actively cycling. Thus c-Myb, through interaction with p300, controls the proliferation and differentiation of hematopoietic stem and progenitor cells (Sandberg, 2005).

The present study supports a critical role for c-Myb-p300 interaction throughout the hematopoietic hierarchy. Importantly, c-Myb is not required at every step and for every lineage; rather, there are distinct steps and lineages that are critically dependent on c-Myb function. These include negative regulation of HSC proliferation, megakaryocyte numbers, and essential roles in erythropoiesis, eosinophil, T, and B cell development. Although previous studies have provided clues that c-Myb may play a role in some of these processes, the severe phenotype of c-Myb^−/− animals has precluded a detailed examination of c-Myb function in adult animals (Sandberg, 2004).

Several lines of evidence support a critical role for c-Myb in directing megakaryocyte and erythrocyte development. (1) c-Myb^−/− mice die of anemia in utero at day 15 due to a failure to initiate fetal liver hematopoiesis. megakaryocyte/erythrocyte progenitors (MEPs) are likely formed in c-Myb^−/− mice, since megakaryocytes are present in the fetal liver of c-Myb^−/− animals. c-Myb^−/− animals contain low numbers of definitive stem/progenitor cells that can give rise to early thymic precursors and initiate myeloid cell development. Thus, the failure to initiate fetal erythropoiesis is likely to result from an inability of later progenitor cells to differentiate into RBCs. (2) Neonatal mice containing ∼20% the normal amount of c-Myb also have anemia and elevated numbers of megakaryocytes and platelets. (3) The c-Myb^M303V/M303V mice and other ENU-induced alleles of c-Myb contain elevated numbers of platelets and CD41⁺ megakaryocytes and reduced numbers of RBCs. Thus, multiple independent alleles of c-Myb give an identical phenotype: reduced numbers of RBCs and elevated numbers of megakaryocytes. Since these two cell types arise from a common precursor, the simplest explanation is that c-Myb is required for the MEP to initiate the RBC program and also participates in turning off the megakaryocyte program. In support of this view, only modest reductions in the number of MEPs are found in c-Myb^M303V/M303V animals compared to normal littermates; however, these cells are unable to make RBCs following culture. Thus, these bipotential cells are present, but their differentiation potential is limited. Collectively, these data suggest that c-Myb normally limits the ability of the HSC to differentiate toward the megakaryocyte lineage while promoting the RBC program (Sandberg, 2004).

The low numbers of B and T cells in the blood of c-Myb^−/− mice suggest that c-Myb may be regulating both B and T cell development. c-Myb^M303V/M303V mice contained normal numbers of phenotypic Lin⁻Kit⁺Sca1⁺IL7R⁺ common lymphoid progenitors (CLPs), suggesting that these early precursor cells are present in normal numbers. Consistent with this observation, normal numbers of the earliest pre-pro B cells were found. However, the numbers of pro-B cells were reduced 3-fold, and the numbers of pre-B cells were down more than 10-fold in c-Myb^M303V/M303V mice. Importantly, the percentage of B cells at subsequent stages of development was not different, although the absolute numbers were reduced in c-Myb^M303V/M303V mice, and their functional capacity remains to be tested. Reduced numbers of pro-B and pre-B cells in c-Myb^M303V/M303V mice suggest that c-Myb may play a role in directing IgH chain rearrangement or in the expansion of those cells that have successfully rearranged IgH (Sandberg, 2004).

The ability of c-Myb to regulate T cell development is well established; however, identification of the precise points that c-Myb regulates has been controversial. Early studies demonstrated a requirement for c-Myb at the pre-DN1 stage of T cell development, while later studies with a dominant-negative form of c-Myb showed a role for c-Myb in the regulation of DN3 and DN4 cell expansion post-TCR β chain selection. A recent study with neonatal mice with partial reductions in c-Myb levels showed that in fetal thymus development c-Myb is required for DN1 to DN2 maturation and the transition from DN2 to DN3, with the former step requiring higher threshold levels of c-Myb. Very recent data generated with conditional null alleles in which Myb is deleted early or late in T cell development in the thymus support a roll for c-Myb in directing the DN3-to-DN4 transition, survival of preselection double-positive cells, and differentiation of CD4 SP cells. In agreement with these data, the c-Myb^M303V/M303V mice described in this study show 2- to 10-fold reductions in thymocyte number with 3-fold reductions in the number of both DN1 and DN4 cells in mature animals. Interestingly, competitive bone marrow reconstitution experiments show that cells harboring the c-Myb^M303V/M303V allele contain normal numbers of DN1 cells and more than 4-fold reductions in DN4 cells. Based on the current work and previous studies, it seems that c-Myb can regulate T cell development at many points; however, the mechanistic details remain an area of active research (Sandberg, 2004).

Reconstitution studies with mixed bone marrow chimeras show a 10- to 20-fold increase in the number of functional HSCs in bone marrow from c-Myb^M303V/M303V mice, and 5-fold elevations in the absolute number of HSCs in primary recipients; thus, these phenotypic HSCs are functional. Secondary and tertiary transplant experiments of these bone marrow chimeras show that these HSCs are capable of self-renewal and long-term multilineage reconstitution, thus confirming that these represent bona fide long term HSCs. These experiments also reveal that the defects in hematopoiesis in c-Myb^M303V/M303V mice are cell intrinsic; c-Myb^M303V/M303V bone marrow gives rise to normal numbers of myeloid and granulocytes, while the numbers of T and B cells are decreased. Importantly, WT cells cotransplanted into these same animals differentiate normally, showing that the defects in hematopoiesis result from direct effects of c-Myb^M303V/M303V on the development of HSC and progenitors and not from secondary effects of the lymphopenia, anemia, or megakaryocytosis. HSCs from c-Myb^M303V/M303V mice contain an increase in the number of actively cycling cells, providing a mechanistic framework for the elevated numbers of HSCs. Thus, c-Myb-p300 normally acts to repress the proliferation of HSCs or links proliferation with subsequent differentiation (Sandberg, 2004).

Previous data have shown that c-Myb is required to generate definitive HSCs, that c-Myb^−/− animals die at E14.5 as a result of a lack of definitive erythropoiesis, and that few HSCs are found in early embryos. In contrast, partial loss of c-Myb results in increased numbers of fetal liver progenitor cells expressing CD34⁺ or Sca-1⁺, supporting a negative role for c-Myb in the control of early progenitors. Interestingly, many of the phenotypes described in the c-Myb^M303V/M303V mice are also found in mice homozygous for a triple point mutation in the KIX domain of the transcriptional coactivator p300 (p300^KIX/KIX) that disrupts the association of p300 with c-Myb. It will interesting to determine if the p300^KIX/KIX and other c-Myb mutants have similar alterations in the number of HSCs and progenitor cells (Sandberg, 2004).

The current study shows that the c-Myb-p300 interaction controls hematopoiesis at many distinct points, both promoting and repressing proliferation and differentiation and thus highlighting c-Myb as a key regulator of hematopoiesis. Determining how a single transcription factor is able to control the diverse processes of selfrenewal, proliferation, and differentiation at distinct points in hematopoiesis and defining the molecular interactions that control c-Myb activity remain important areas of future research (Sandberg, 2004).

Myc regulation by p300

The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 (see Drosophila Pcaf) and Tip60. This study reports a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independent of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene; also, p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. These results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation (Faiola, 2005).

Other CBP interactions

Growth factors such as epidermal growth factor (EGF) and insulin regulate development and metabolism via genes containing both POU homeodomain (Pit-1) and phorbol ester (AP-1) response elements. Although CREB binding protein (CBP) functions as a coactivator on these elements, the mechanism of transactivation has been unclear. CBP is recruited to these elements only after it is phosphorylated at serine 436 by growth factor-dependent signaling pathways. In contrast, p300, a protein closely related to CBP that lacks this phosphorylation site, binds only weakly to the transcription complex and in a growth factor-independent manner. A small region of CBP (amino acids 312-440), which has been termed the GF box, contains a potent transactivation domain and mediates this effect. Direct phosphorylation represents a novel mechanism controlling coactivator recruitment to the transcription complex (Zanger, 2001).

Previous in vitro studies have suggested that CBP and p300 interact constitutively with both c-Fos and c-Jun and mediate activation on an AP-1 element. These constitutive interactions are confirmed in this study, where both CBP and p300 activate the AP-1 element 2- to 3-fold in the absence of growth factor stimulation. CBP is found to bind to c-Jun through its CREB binding domain. Although the constitutive interactions between p300/CBP and c-Jun may be functionally significant on the AP-1 element, the growth factor-dependent recruitment of CBP to the AP-1 complex may prove to be quantitatively more important (Zanger, 2001).

The molecular mechanisms of transcriptional activation by beta-catenin are only poorly understood. The closely related acetyltransferases p300 and CBP potentiate beta-catenin-mediated activation of the siamois promoter, a known Wnt target. beta-catenin and p300 also synergize to stimulate a synthetic reporter gene construct, whereas activation of the cyclin D1 promoter by beta-catenin is refractory to p300 stimulation. Axis formation and activation of the beta-catenin target genes siamois and Xnr-3 in Xenopus embryos are sensitive to the E1A oncoprotein, a known inhibitor of p300/CBP. The C-terminus of beta-catenin interacts directly with a region overlapping the CH-3 domain of p300. p300 could participate in alleviating promoter repression imposed by chromatin structure and in recruiting the basal transcription machinery to promoters of particular Wnt target genes (Hecht, 2000).

The finding that p300/CBP serves as a coactivator for beta-catenin in vertebrates is unexpected given that in Drosophila dCBP has been shown to negatively regulate Wingless-signaling. The apparent discrepancy between the function of vertebrate p300/CBP and dCBP could be explained most easily if vertebrate CBP and p300, or dCBP and its mammalian orthologs, were functionally different from one another. Species-specific differences are known for certain aspects of Wnt signaling. Also, CBP and p300 are differentially engaged in retinoic acid and cAMP responses in mammalian cells. However, the results presented here show that CBP and p300 can both serve as cofactors for ß-catenin. An interaction between LEF-1 and the CH-3 domain of p300, which corresponds to the dCBP-2 region used in the Drosophila studies, could not be detected. Rather, ß-catenin can interact with p300 in the absence of TCFs (Drosophila homolog, Pangolin). These findings indicate that dCBP differs from p300 and CBP and that in vertebrates p300 or CBP enter the beta-catenin-TCF complex through an interaction with beta-catenin, whereas dCBP may be brought to a promoter by its interaction with dTCF (Hecht, 2000).

Aside from the difference in the TCF interaction, the genetic studies in Drosophila and the experiments in vertebrates may also reveal a more complicated involvement of p300/CBP in Wnt signaling. For example, upon binding to dTCF, Armadillo may form a ternary complex and reprogram the activity of dCBP that is already present. Alternatively, p300 or CBP may function as coactivators of beta-catenin only initially. Over time their activity could change and lead to the downregulation of target genes as reported for the interferon-beta enhanceosome, which eventually is destabilized and disassembled by CBP through acetylation of an architectural component, the HMG I(Y) protein. Similarly, coactivator complexes associated with the promoter-bound estrogen receptor are dissociated after acetylation by p300/CBP, which leads to the attenuation of the hormone response. Thus, one could hypothesize that p300 performs a stimulatory function and also provides a shut-off mechanism in Wnt signaling (Hecht, 2000).

ß-catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of ß-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of ß-catenin, a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane, was used. RRS is based on the ability of mammalian Ras to rescue the growth defect of the yeast temperature-sensitive cdc25-2 strain, in which the endogenous Ras is inactive at the nonpermissive temperature (37°C) due to the lack of a functional Cdc25 guanyl nucleotide exchange factor. For RRS screening, a bait protein of interest is fused at the COOH terminus of mammalian activated Ras. This activated Ras lacks the membrane localization signal [Ras(61)deltaF], whereas library cDNAs are fused to the v-Src myristoylation sequence targeted to the plasma membrane. A protein-protein interaction between the bait and library protein results in the recruitment of Ras to the membrane and complementation of the cdc25-2 mutation. Using this system, the CREB-binding protein (CBP) was isolated. From armadillo (arm) repeat 10 to the COOH terminus of ß-catenin is involved in binding to CBP, whereas ß-catenin interacts directly with the CREB-binding domain of CBP. ß-Catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, ß-catenin-dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between ß-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of ß-catenin (Takemaru, 2000).

ß-catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of ß-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of ß-catenin, a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane, was used. RRS is based on the ability of mammalian Ras to rescue the growth defect of the yeast temperature-sensitive cdc25-2 strain, in which the endogenous Ras is inactive at the nonpermissive temperature (37°C) due to the lack of a functional Cdc25 guanyl nucleotide exchange factor. For RRS screening, a bait protein of interest is fused at the COOH terminus of mammalian activated Ras. This activated Ras lacks the membrane localization signal [Ras(61)deltaF], whereas library cDNAs are fused to the v-Src myristoylation sequence targeted to the plasma membrane. A protein-protein interaction between the bait and library protein results in the recruitment of Ras to the membrane and complementation of the cdc25-2 mutation. Using this system, the CREB-binding protein (CBP) was isolated. From armadillo (arm) repeat 10 to the COOH terminus of ß-catenin is involved in binding to CBP, whereas ß-catenin interacts directly with the CREB-binding domain of CBP. ß-Catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, ß-catenin-dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between ß-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of ß-catenin (Takemaru, 2000).

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates viral transcription through interaction with the cellular transcription factor CREB (cyclic AMP response element [CRE] binding protein). Although Tax stabilizes the binding of CREB to the Tax-responsive viral CREs in the HTLV-1 promoter, the precise molecular mechanism by which Tax mediates strong transcriptional activation through CREB remains unclear. In this report, it is shown that Tax promotes high-affinity binding of the KIX domain of CREB binding protein (CBP) to CREB-viral CRE complexes, increasing the stability of KIX in these nucleoprotein complexes by up to 4.4 kcal/mol. Comparable KIX binding affinities were measured for both phosphorylated and unphosphorylated forms of CREB; in all cases high-affinity binding is dependent upon both Tax and the viral CRE. Tax also promoted association of KIX to a truncated form of CREB containing only the 73-amino-acid basic leucine zipper (bZIP) domain, indicating that the entire amino-terminal CBP-interacting domain of CREB is nonessential in the presence of Tax. Functional studies upholds the binding studies, as expression of the bZIP domain of CREB is sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Transfection of a KIX expression plasmid, which lacks activation properties, inhibits Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely to be a cofactor in mediating Tax stimulation of HTLV-1 transcription. Together, these data support a model in which Tax anchors CBP to the HTLV-1 promoter, with strong transcriptional activation resulting from the CBP-associated activities of nucleosome remodeling and recruitment of the general transcription machinery (Giebler, 1997).

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. A nuclear histone acetyltransferase activity is shown to associate with Tat. Intracellularly, Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR is linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, a requirement for the HAT component was delineated from the latter but not the former in Tat function. Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR (Benkirane, 1998).

Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II-dependent transcription. p300/CBP and PCAF (see Drosophila Pcaf) histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1alpha, and participate in transcription by forming an activation complex and by promoting histone acetylation. The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex. E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo. Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation. These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation (Chakravarti, 1999).

The implications of these studies are several. (1) These results provide a novel mechanism of E1A action and delineate a novel transcriptional repressor function of the carboxyl half of the protein. (2) Perhaps more importantly, the mechanism of action of repression is novel and establishes a block of the HAT activity as an additional mode of RNA polymerase II control. (3) This study provides evidence to explain the inhibitory activity of E1A for transcription factors that do not depend on both p300/CBP and PCAF-HAT activity. These results help explain why E1A blocks Myo D function during muscle differentiation, a process that does not require the p300/CBP-HAT activity. (4) The ability of E1A to disrupt activation complexes and to inhibit HAT activity are the properties that map to separable domains. Thus, E1A mutants can be used as valuable probes to study both HAT activity-dependent and -independent transcriptional processes. At this stage the mechanism of inhibition of the HAT activity remains obscure. It is possible that the regulation of acetylases would be analogous to that of kinases by cellular kinase inhibitors. However, since there are no obvious structural similarities among the tails of histones, p53, and E1A, this possibility remains purely speculative. In the future, it will be important to determine whether other coactivation/HAT complexes, including the SAGA, p300, and TFIID complexes as well as other nonhistone substrates, are also sensitive to E1A inhibition. Additionally, it is suggested that other viral proteins (e.g., SV40 large T antigen) could have similar activity, which leads to the intriguing speculation that cellular factors with corresponding activities may exist. In conclusion, this work demonstrates that the acetyltransferase activity of coactivators can be modulated and thus reveals a novel pathway for controlling hormonal signaling by modulating RNA polymerase II transcription (Chakravarti, 1999).

A novel cofactor, ACTR, directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR has sequence homology to SRC-1 and TIF2, factors that associate with members of the nuclear receptor family and augment the transcription activity of steroid receptors such as glucocorticoid receptor and estrogen receptor. ACTR possesses an N-terminal PAS/bHLH domain, a central receptor-interaction domain able to interact with RAR, RXR (Drosophila homolog: Ultraspiracle) and TR (See Ecdysone Receptor for more information) in a hormone dependent fashion, and a C-terminal histone acetyl transferase domain. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. ACTR is able to recruit P/CAF. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex. Interestingly, while all currently identified histone acetyltransferases (including ACTR) are capable of acetylating core histones H3 and H4, ACTR displays an unusual additional activity as an effective acetylator of mononucleosomes, similar only to that observed in CBP/p300 (Chen, 1997).

The mechanism has been examined by which growth factor-mediated induction of the Ras pathway interferes with signaling via the second messenger cAMP. Activation of cellular Ras with insulin or NGF stimulates recruitment of the S6 kinase pp90RSK to the signal-dependent coactivator CBP. Formation of the pp90RSK-CBP complex occurs with high stoichiometry and persists for 6-8 hr following growth factor addition. pp90RSK specifically recognizes the E1A-binding domain of the coactivator CBP. In addition, like E1A, binding of pp90RSK to CBP is sufficient to repress transcription of cAMP-responsive genes via the cAMP-inducible factor CREB. By contrast with its effects on the cAMP pathway, formation of the pp90RSK-CBP complex is required for induction of Ras-responsive genes. These results provide a demonstration of cross-coupling between two signaling pathways that occurs at the level of a signal-dependent coactivator (Nakajima, 1996).

Cells respond to either viral infection or exposure to double-stranded RNA by carrying out the transcriptional induction of a subset of alpha/beta interferon-stimulated genes, employing a pathway distinct from the interferon signal pathway. The transcriptional induction is mediated through a DNA sequence containing the alpha/beta interferon-stimulated response element (ISRE). A novel transcription factor recognizes this response element and has been designated double-stranded RNA-activated factor 1 (DRAF1). The DNA-binding specificity of DRAF1 correlates with transcriptional induction, thereby distinguishing it as a positive regulator of alpha/beta interferon-stimulated genes. Two of the components of DRAF1 have now been identified as interferon regulatory factor 3 (IRF-3) and the transcriptional coactivator CREB-binding protein (CBP)/p300. IRF-3 preexists in the cytoplasm of uninfected cells and translocates to the nucleus following viral infection. Translocation of IRF-3 is accompanied by an increase in serine and threonine phosphorylation. Coimmunoprecipitation analyses of endogenous proteins demonstrate an association of IRF-3 with the transcriptional coactivators CBP and p300 only subsequent to infection. Antibodies to the IRF-3, CBP, and p300 molecules react with DRAF1 bound to the ISRE target site of induced genes. The cellular response that leads to DRAF1 activation and specific gene expression may serve to increase host survival during viral infection (Weaver, 1998).

The AML1 transcription factor and the transcriptional coactivators p300 and CBP are the targets of chromosome translocations associated with acute myeloid leukemia and myelodysplastic syndrome. In the t(8;21) translocation, the AML1 (CBFA2/PEBP2alphaB) gene becomes fused to the MTG8 (ETO) gene. The terminal differentiation step leading to mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF) is inhibited by the ectopic expression of the AML1-MTG8 fusion protein in L-G murine myeloid progenitor cells. Overexpression of normal AML1 proteins reverses this inhibition and restores the competence to differentiate. Immunoprecipitation analysis shows that p300 and CREB-binding protein (CBP) interact with AML1. The C-terminal region of AML1 is responsible for the induction of cell differentiation and for the interaction with p300. Overexpression of p300 stimulates AML1-dependent transcription and the induction of cell differentiation. These results suggest that p300 plays critical roles in AML1-dependent transcription during the differentiation of myeloid cells. Thus, AML1 and its associated factors p300 and CBFbeta, all of which are targets of chromosomal rearrangements in human leukemia, function cooperatively in the differentiation of myeloid cells (Kitabayashi, 1998).

The complex between AML1 (Runx1) and CBFß is the most frequent target of specific chromosome translocations in human leukemia. The MOZ gene, which encodes a histone acetyltransferase (HAT), is also involved in some leukemia-associated translocations. MOZ is part of the AML1 complex and strongly stimulates AML1-mediated transcription. The stimulation of AML1-mediated transcription is independent of the inherent HAT activity of MOZ. Rather, a potent transactivation domain within MOZ appears to be essential for stimulation of AML1-mediated transcription. MOZ, as well as CBP and MOZ-CBP, can acetylate AML1 in vitro. The amount of AML1-MOZ complex increases during the differentiation of M1 myeloid cells into monocytes/macrophages, suggesting that the AML1-MOZ complex might play a role in cell differentiation. However, the MOZ-CBP fusion protein, which is created by the t(8;16) translocation associated with acute monocytic leukemia, inhibits AML1-mediated transcription and differentiation of M1 cells. These results suggest that MOZ-CBP might induce leukemia by antagonizing the function of the AML1 complex (Kitabayashi, 2001).

p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. The vitamin D receptor (VDR) is a member of the steroid and nuclear hormone receptor superfamily of eukaryotic transcription factors and binds target DNA, or response elements, as a homodimer or heterodimer with the 9-cis retinoid X receptor (RXR). In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D₃ via the vitamin D₃ receptor complex. Therefore, p300 control of basal and vitamin D₃-enhanced activity of the OC promoter was addressed. Transient overexpression of p300 was found to result in a significant dose-dependent increase of both basal and vitamin D₃-stimulated OC gene activity. This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element. In addition, by coimmunoprecipitation, it has been shown that the endogenous Runx2/Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations. It has also been demonstrated, by chromatin immunoprecipitation assays, that p300, Runx2/Cbfa1, and 1alpha,25-dihydroxyvitamin D₃ receptor interact with the OC promoter in intact osteoblastic cells expressing this gene. The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, since a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300. On the basis of these results, it is proposed that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D₃ (Sierra, 2003).

The Ets-1 transcription factor plays a critical role in cell growth and development, but the means by which it activates transcription remain unclear. It has been shown that Ets-1 binds the transcriptional coactivators CREB binding protein (CBP) and the related p300 protein (together referred to as CBP/p300) and that this interaction is required for specific Ets-1 transactivation functions. The Ets-1- and c-Myb-dependent aminopeptidase N (CD13/APN) promoter and an Ets-1-dependent artificial promoter are repressed by adenovirus E1A, a CBP/p300-specific inhibitor. Ets-1 activity is potentiated by CBP and p300 overexpression. The transactivation function of Ets-1 correlates with its ability to bind an N-terminal cysteine- and histidine-rich region spanning CBP residues 313 to 452. Ets-1 also binds a second cysteine- and histidine-rich region of CBP, between residues 1449 and 1892. Both Ets-1 and CBP/p300 form a stable immunoprecipitable nuclear complex, independent of DNA binding. This Ets-1-CBP/p300 immunocomplex possesses histone acetyltransferase activity, consistent with previous findings that CBP/p300 is associated with such enzyme activity. These results indicate that CBP/p300 may mediate antagonistic and synergistic interactions between Ets-1 and other transcription factors that use CBP/p300 as a coactivator, including c-Myb and AP-1 (Yang, 1998).

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT, mSin3A/B and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, the histone acetyltransferase functions of CBP or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors (L. Xu, 1998).

The transcriptional coactivators CBP and P/CAF are required for activation of transcription from the IFN beta enhanceosome. CBP and P/CAF acetylate HMG I(Y), the essential architectural component required for enhanceosome assembly. Acetylation takes place at distinct lysine residues, causing distinct effects on transcription. Thus, in the context of the enhanceosome, acetylation of HMG I by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly. Acetylation of HMG I(Y) by CBP is essential for turning off IFN beta gene expression. The acetyltransferase activities of CBP and P/CAF modulate both the strength of the transcriptional response and the kinetics of virus-dependent activation of the IFN beta gene (Munshi, 1998).

Xenopus NF-Y is identified as a key regulator of acetylation responsiveness for the Xenopus hsp70 promoter within chromatin assembled in Xenopus oocyte nuclei. Y-box sequences are required for the assembly of DNase I-hypersensitive sites in the hsp70 promoter, and for transcriptional activation both by inhibitors of histone deacetylase and by the p300 acetyltransferase. The viral oncoprotein E1A interferes with both of these activation steps. Xenopus NF-YA, NF-YB and NF-YC have been cloned and NF-Y has been established as the predominant Y-box-binding protein in Xenopus oocyte nuclei. NF-Y interacts with p300 in vivo and is itself a target for acetylation by p300. Transcription from the hsp70 promoter in chromatin can be enhanced further by heat shock factor. Two steps in chromatin modification at the Xenopus hsp70 promoter are suggested: first the binding of NF-Y to the Y-boxes to pre-set chromatin and second the recruitment of p300 to modulate transcriptional activity (Li, 1998).

The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73, and consequently, AP-1 activation as well, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. It is shown here that dexamethasone, a glucocorticoid receptor agonist, and two other nuclear hormone receptors, the retinoic acid receptor and the thyroid hormone receptor, also block c-Jun activation by a mechanism that is (1) independent of the c-Jun DNA binding domain and is one which (2) relies specifically on the c-Jun amino-terminal phosphorylation step. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite for interaction with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents. Nuclear receptor interference would rely on the inhibition of MEKK activity or a downstream step in the pathway. Dexamethasone can also inhibit a constitutively active MAPK pathway (Caelles, 1997).

Calcium is the principal second messenger in the control of gene expression by electrical activity in neurons. Recruitment of the coactivator CREB-binding protein, CBP, by the prototypical calcium-responsive transcription factor, CREB and stimulation of CBP activity by nuclear calcium signals is one mechanism through which calcium influx into excitable cells activates gene expression. Another CBP-interacting transcription factor, c-Jun, can mediate transcriptional activation upon activation of L-type voltage-gated calcium channels. Calcium-activated transcription mediated by c-Jun functions in the absence of stimulation of the c-Jun N-terminal protein kinase (JNK/SAPK1) signaling pathway and does not require c-Jun amino acid residues Ser63 and Ser73, the two major phosphorylation sites that regulate c-Jun activity in response to stress signals. Similar to CREB-mediated transcription, activation of c-Jun-mediated transcription by calcium signals requires calcium/calmodulin-dependent protein kinases and is dependent on CBP function. These results identify c-Jun as a calcium-regulated transcriptional activator and suggest that control of coactivator function (i.e. recruitment of CBP and stimulation of CBP activity) is a general mechanism for gene regulation by calcium signals (Cruzalegui, 1999).

p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series of cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex (Dallas, 1998).

Using the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor, was identifed. Nmi interacts with all STATs except Stat2. Two cytokine systems, IL-2 and IFNgamma, were evaluated and Nmi was demonstrated to augment STAT-mediated transcription in response to these cytokines. Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 augments IL-2- and IFNgamma-dependent transcription. Therefore, these data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors (Zhu, 1999).

Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. The cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML in vitro and is recruited to the PODs in vivo. Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate that a fraction of CBP is compartmentalized to the POD through its association with PML and thus suggest that PML and other POD-associated proteins may play an unexpectedly broad role in aspects of transcriptional regulation and human disease (Doucas, 1999).

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), plays an important role in the development of the adrenal gland and in sexual differentiation. SF-1 regulates the transcription of variety of genes, including several steroidogenic enzymes, Mullerian inhibiting substance, and gonadotropin genes. Attempts have been made to identify domains in SF-1 that are required for transactivation and to determine whether SF-1 interacts with a subset of known coactivators. Natural variants of the FTZ-F1 locus include embryonal long terminal repeat-binding protein (ELP)-1, ELP-2, and SF-1, all of which share the DNA-binding domain. Analyses of the transcriptional activity of these variants reveal that the activity of ELP-2 and SF-1 is much greater than ELP-1, which contains a distinct carboxy terminus. Further studies were performed using GAL4-SF-1 fusion proteins that were constructed by replacement of the zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding domain of GAL4. Elimination of the putative AF-2 domain at the carboxy terminus of GAL4-SF-1 proteins results in a complete loss of transactivation. Several lines of evidence demonstrate that SF-1 interacts with steroid receptor coactivator-1 (SRC-1). Full-length SRC-1 enhances GAL4-SF-1-mediated transactivation, whereas a dominant negative form of SRC-1, consisting of its interaction domain alone, inhibits the activity of GAL4-SF-1. In mammalian two-hybrid assays, fusion of the VP16 activation domain to the interaction domain of SRC-1 confirms the interaction between SRC-1 and GAL4-SF-1 and demonstrates that the AF-2 domain is required for interaction with SRC-1. Furthermore, SRC-1, together with the cAMP responsive element binding protein (CBP) or a closely related factor, p300, synergistically enhance transcriptional activity of GAL4-SF-1. It is concluded that the carboxy-terminal AF-2 region of SF-1 functions as an activation domain and that SRC-1 and CBP/p300 are components of the coactivator complex with SF-1 (Ito, 1998)

The co-activators CBP and p300 are important for normal cell differentiation and cell cycle progression and are the targets for viral proteins that dysregulate these cellular processes. The E6 protein from the oncogenic human papillomavirus type 16 (HPV-16) binds to three regions (C/H1, C/H3 and the C-terminus) of both CBP and p300. The interaction of E6 with CBP/p300 is direct and independent of proteins known to bind the co-activators, such as p53. The E6 protein from low-risk HPV type 6 does not interact with C/H3 or the C-terminus but associates with the C/H1 domain at 50% of the level of HPV-16. HPV-16 E6 inhibits the intrinsic transcriptional activity of CBP/p300 and decreases the ability of p300 to activate p53- and NFkappa-B-responsive promoter elements. Interestingly, some mutations in HPV-16 E6 abrogate C/H3-E6 interactions, but do not alter the ability of E6 to associate with the C/H1 domain, suggesting that these modified proteins could be used to delineate the functional significance of the C/H1 and C/H3 domains of CBP/p300 (Patel, 1999).

What are the advantages for the virus to target CBP/p300? The human papillomaviruses infect basal epithelial cells, which are destined to terminally differentiate. Papillomaviruses have a small coding capacity and do not code for any of the replicative machinery; differentiating cells contain very little or none of the cell's replicative components. Therefore the virus needs to stimulate cells into S-phase to have on hand a supply of replicative enzymes. CBP/p300 is known to be important for cell differentiation, and transcription factors such as members of the AP-1 family, which are thought to be important for keratinocyte differentiation, bind to the regions bound by E6. Therefore disruption of AP-1 transactivation may have an effect on keratinocyte differentiation. In the natural infection HPV-16 causes disruption of differentiation and hyperproliferation of the stratum spinosum and granulosum. In addition, HPV-16 E6 and E7 proteins can independently modulate the ability of primary human keratinocytes to differentiate. In contrast, HPV-6 does not disrupt differentiation, but does cause a hyperproliferation of the epithelial cells. The HPV-16 E6 protein may therefore have a role in inhibiting differentiation, resulting in hyperproliferation. The papillomavirus group cause persistent infections with lesions remaining for months, even years. It has been suggested that the viruses can down-regulate the local immune response to allow persistence of the infection. In fact studies have shown a reduced class I MHC expression and reduced numbers of Langerhans and T cells in the immediate vicinity of the lesion. The fact that HPV-16 E6 can inhibit the activation of NF-kappaB by CBP/p300 may help to explain these findings, since this transcription factor is activated by a number of stimuli, including viral infection. In addition, binding sites for NF-kappaB are found in a number of promoters including those for class I MHC, cytokine modulators of the immune response including IL-2, IL-6, IL-8 and GM-CSF. Keratinocytes synthesize a variety of these chemotactic factors, including IL-6, IL-8 and GM-CSF, when stimulated by viral infection. Therefore inhibition of the co-activation of NF-kappaB may help the virus escape immune recognition in the epithelium. In summary, E6 of HPV-16 binds to three regions of CBP/p300, resulting in the abrogation of the co-activation functions. These co-activators are important for cell differentiation and cell cycle control and are central to the activation of a number of genes that modulate immune responses. Therefore the outcome of the interaction between E6 and CBP/p300 may involve the inhibition of differentiation of the epithelial cells harboring the virus and the down-regulation of the immune recognition machinery to permit the persistence of the viral infection (Patel, 1999).

It was asked whether the acetylase CREB-binding protein (CBP) can acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-alpha) and importin-alpha7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, G/SK (in the single-letter amino acid code) was identified as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impairs Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, it was show that Rch1 is acetylated at Lys22 in vivo and that CBP or p300 can mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-beta. Acetylation of Lys22 promotes interaction with importin-beta in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import (Bannister, 2000).

The CCAAT displacement protein/cut homolog (CDP/cut) is a divergent homeodomain protein that is highly conserved through evolution and has properties of a potent transcriptional repressor. CDP/cut contains three conserved cut-repeat domains and a conserved homeobox, each involved in directing binding specificity to unique nucleotide sequence elements. Furthermore, CDP/cut may play a role as a structural component of chromatin through its direct interaction with nucleosomal DNA and association with nuclear matrix attachment regions. CDP/cut is cell-cycle regulated through interactions with Rb, p107, specific kinases and phosphatases directing the transcriptional activity of CDP/cut on such genes encoding p21^WAF1,CIP1, c-myc, thymidine kinase, and histones. CDP/cut is associated with histone deacetylase activity and is associated with a corepressor complex through interactions with histone deacetylases. The interaction of CDP/cut with CBP and p300/CREB-binding protein-associated factor (PCAF) is reported along with the modification of CDP/cut by the histone acetyltransferase PCAF. Acetylation of CDP/cut by PCAF is directed at conserved lysine residues near the homeodomain region and regulates CDP/cut function. These observations are consistent with the ability of CDP/cut to regulate genes as a transcriptional repressor, suggesting acetylation as a mechanism that regulates CDP/cut function (Li, 2000).

Complexes containing p300, but not CBP, and the nuclear proto-oncoprotein SYT were detected in confluent cultures of G1-arrested cells but not in sparse cells or during S or G2. SYT sequences constitute the N-terminal segment of a fusion oncogene product, SYT-SSX, routinely detected in synovial sarcoma, an aggressive human tumor. SYT/p300 complex formation promotes cell adhesion to a fibronectin matrix, as reflected by compromise of this process in cells expressing SYT deletion mutants that retain p300 binding activity and in the primary fibroblasts of p300 but not CBP heterozygous null mice. The mechanism linking the action of SYT/p300 complexes to adhesion function is, at least in part, transcription activation-independent and results in proper activation of beta1 integrin, a major adhesion receptor (Eid, 2000).

Indirect evidence, based on studies of E1A and SV40 T Ag function, implies that p300/CBP actively suppresses the emergence in cultured cells of multiple neoplastic characteristics, one of which is loss of contact inhibition of cell growth. Results presented here reveal an unexpected function of p300. Both p300 and a known human proto-oncoprotein, SYT, interact in the nucleus. Formation of SYT/p300 complexes is confined to G1-arrested, confluent cells, is absent in sparse G1 cells, and appears to translate into proper cell adhesion to a fibronectin matrix. What seems likely to be a failure to form a sufficient quantity of these nuclear complexes, as in p300+/- embryonic cells and in cells synthesizing certain dominant negative SYT mutants, leads to a major defect in cell adhesion/spreading. Taken together, these results are consistent with the hypothesis that SYT/p300 complex formation participates in the control of contact inhibition of cell growth, a G1-specific phenomenon, and an adhesion-dependent process (Eid, 2000).

The level of ribosomal gene transcription has been shown to be finely regulated in response to changes in cell growth rate and the state of differentiation. This regulation is believed, at least in part, to be due to a change in the number of actively transcribed genes. Transcription of the ribosomal genes by RNA polymerase I (PolI) is activated both in vitro and in vivo by UBF. The recruitment of this protein to the PolI promoter is in fact the first step in ribosomal gene activation, permitting the subsequent association of the TATA-binding protein (TBP)-containing complex SL-1, and hence of the polymerase. UBF contains multiple tandem homologies to the DNA binding domain of high mobility group 1 (HMG-1), the HMG box, and loops approximately 140 bp of ribosomal DNA into a single turn, a structure that has been called the ribosomal enhancesome. Data on the promotion of PolI transcription in vertebrates are compatible with the formation of two precisely juxtaposed enhancesomes on the PolI promoter as a prerequisite to promoter recognition by SL-1. Mammalian and Xenopus UBFs are functionally interchangeable for this task in vivo. However, enhancesome formation is clearly incompatible with the nucleosomal chromatin structure of the inactive genes. Therefore, the transition from the inactive to active ribosomal gene state requires the replacement of one or more nucleosomes with enhancesomes. Chromatin remodeling has been shown to be facilitated by the recruitment of co-activators with acetyltransferase activity. Further, the HMG box of Drosophila TCF/LEF functionally recruits the histone acetyltransferase (HAT) CREB-binding protein (CBP). The potential of CBP to activate ribosomal transcription by PolI has been investigated (Pelletier, 2000 and references therein).

In the canonical Wnt signaling pathway, ß-catenin activates target genes through its interactions with Tcf/Lef-family transcription factors and additional transcriptional coactivators. The crystal structure of ICAT, an inhibitor of ß-catenin-mediated transcription, bound to the armadillo repeat domain of ß-catenin, has been determined. ICAT contains an N-terminal helilical domain that binds to repeats 11 and 12 of ß-catenin, and an extended C-terminal region that binds to repeats 5-10 in a manner similar to that of Tcfs and other ß-catenin ligands. Full-length ICAT dissociates complexes of ß-catenin, Lef-1, and the transcriptional coactivator p300, whereas the helical domain alone selectively blocks binding to p300. The C-terminal armadillo repeats of ß-catenin may be an attractive target for compounds designed to disrupt aberrant ß-catenin-mediated transcription associated with various cancers (Daniels, 2002).

RNA polymerase I (PolI) transcription is activated by the HMG box architectural upstream binding factor (UBF), which loops approximately 140 bp of DNA into the enhancesome, necessitating major chromatin remodeling. The acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation 'flip-flop.' Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb (Pelletier, 2000).

Rb suppresses ribosomal transcription in vitro via a direct interaction with UBF. An interaction has been identified in vitro between Rb aa 379-928 and the HMG boxes 1 and/or 2 of mUBF. Other data further suggest that the Rb 'pocket' (aa 379-792) is sufficient for transcriptional suppression in vitro and for the Rb-UBF interaction in vivo. Since HMG boxes 1 and 2 also bind CBP, it is possible that Rb and CBP binding to UBF are exclusive events. If this is the case, suppression by Rb and activation by CBP would also be exclusive (Pelletier, 2000).

Rb suppresses ribosomal transcription in vivo. Consistent with a role for the Rb pocket in this suppression, it has also been found that the Rb-related pocket protein p107 is equally effective in this suppression. It was next determined if the pocket domain of Rb (aa 379-792) could bind HMG boxes 1 and/or 2 of xUBF. Rb(379-792) binds a polypeptide containing HMG boxes 1 and 2, but does not bind to sequences C-terminal of HMG box 2. Thus the pocket region of Rb is sufficient to bind xUBF. It was also found that the individual HMG boxes 1 and 2 bind the Rb pocket region less efficiently than does the box12 combination. Rb and CBP were then placed in competition for xUBF. Preincubation of full-length xUBF with the interaction domain CBP2 is sufficient to inhibit subsequent binding to Rb(379-792). Conversely, pre-incubation of xUBF with Rb(379-792) inhibits its subsequent binding to CBP2. These data strongly suggest that suppression of PolI transcription by Rb results, at least in part, from its capacity to interfere with the recruitment of CBP to UBF. However, in other systems, Rb has also been shown to suppress transcription by the recruitment of histone deacetylase 1 (HDAC1). Hence, Rb could also potentially reverse the catalytic effects of recruiting CBP to UBF. It was therefore asked if HDAC1 deacetylates xUBF acetylated with the CBP-HAT domain and whether the presence of Rb enhances this deacetylation. The rate of deacetylation of xUBF by HDAC1 in the presence of Rb(379-792) is nearly two times more rapid than in its absence. Suppression of PolI transcription in vivo by Rb (and p107) is at least partly relieved by inhibiting deacetylation with TSA. The fact that Rb suppression can not be reversed with TSA alone is consistent with Rb also preventing CBP recruitment to UBF. In fact, the Rb-induced suppression of PolI transcription can be completely relieved, and indeed reversed, by the coexpression of CBP in combination with TSA treatment (Pelletier, 2000).

The recruitment of CBP to UBF could activate transcription (1) by the acetylation of UBF, (2) by the acetylation of local chromatin, (3) by displacing Rb, or (4) by a combination of these effects. The fact that Rb can cooperate in the deacetylation of UBF suggests that acetylated UBF can effectively bind Rb and this was confirmed in pulldown experiments. The role of CBP, Rb, and acetylation in DNA binding by UBF was also investigated. Neither an excess of CBP2 nor saturation acetylation of UBF with the HAT domain of CBP has any detectable effect on UBF's capacity to bind the ribosomal promoter DNA. Rb(379-928), [or the pocket domain Rb(379-792) or GST-Rb(379-928)], has no effect on DNA binding by UBF (Pelletier, 2000).

Since the DNA binding of UBF is unaffected by CBP or Rb binding or by acetylation, it was asked if acetylated UBF is necessary for transcription activation in vitro. Bacterially produced UBF, which is necessarily unacetylated, has been found in many laboratories to be refractory for in vitro transcription. However, UBF produced in mammalian and insect cells or by in vitro translation has been found to be functional. This suggests that post-transcriptional modification of UBF may be important, and indeed this has been shown to be the case for UBF phosphorylation. Rat and mouse nuclear extracts were therefore depleted of endogenous UBF and used to study the capacity of bacterially expressed (i.e., unacetylated) UBF to activate transcription from the rat or mouse PolI promoters. Bacterially produced rUBF and xUBF were either acetylated with matrix-immobilized active CBP HAT domain or mock acetylated (unacetylated) with the immobilized inactive HAT domain and then the HAT protein was removed by centrifugation. UBF has been shown both to derepress PolI transcription in vitro as well as to activate it. The derepression properties of rUBF in the rat extract was investigated in competition with added histone H1. As expected, addition of H1 to the rat extract represses transcription of the rat promoter, and this repression is even more pronounced after UBF depletion. Addition of unacetylated rUBF does not relieve H1 repression, and even increases it somewhat. However, the acetylated rUBF relieves H1 repression and gives about a 2-fold increase in transcription (or more than 4-fold the level observed in the presence of the same amount of unacetylated UBF). The capacity of UBF to activate transcription from the mouse promoter was also tested in a UBF-depleted mouse nuclear extract. Here the unacetylated rUBF gives a small degree of transcription activation (1.7 times), but the acetylated rUBF activates much more effectively (3.5 times). Xenopus UBF has been shown to activate the mouse promoter in vivo, although it has also been shown to be ineffective in activating the rat or human promoters in vitro. Bacterial unacetylated xUBF has a clear repressive (0.5 times) effect on the mouse promoter in vitro, but after acetylation, this repression is completely relieved and transcription is somewhat activated (Pelletier, 2000).

These data strongly support a 'flip-flop' model for the regulation of ribosomal transcription by CBP and Rb-HDAC1. (The term 'flip-flop' is used to describe a system with two alternative semistable states, here CBP-bound or Rb-bound UBF.) The formation of a UBF-CBP complex activates transcription by acetylation of UBF itself, and perhaps also by opening up the adjacent ribosomal chromatin, allowing further UBF ingression and gene activation. Excess Rb prevents formation of a UBF-CBP complex and, by recruiting HDAC1, catalyses UBF deacetylation and hence its inactivation. Acetylation of UBF significantly enhances its ability to activate PolI transcription in vitro. Although changes in DNA binding and the ability of UBF to bind Rb have been excluded, the mechanism by which UBF acetylation functions remains unknown. One possible explanation being actively pursued is that acetylation induces a structural change in UBF. Quite possibly Rb recruitment to UBF has roles other than just to promote UBF deacetylation. Rb can inhibit SL-1 recruitment to UBF. Rb may also cooperate in deacetylation of adjacent histones. Enhancesome structure, with its single 140 bp loop of DNA, could accommodate the core histones in a weak association with the DNA. Yet, xUBF can also associate stably with nucleosomes. Thus, the CBP/Rb flip-flop could catalyze the transition between a predominantly nucleosomal and a predominantly enhancesomal gene state, the transition not necessarily requiring complete displacement of either core histones or UBF. It has in fact been observed that the core histones remain associated with the active ribosomal genes, but only via their N-terminal domains. Whether a nucleosome-enhancesome transition is facilitated by UBF acetylation, histone acetylation, or a combination of the two must now be determined (Pelletier, 2000).

Recent data suggest that both acetylation and phosphorylation can cooperate to activate transcription in vivo. UBF is known to be multiply phosphorylated, mainly within the C-terminal acidic domain but also in HMG box 5. Each of these modifications has been shown to activate transcription in vitro and, in the case of the acidic domain, phosphorylation has been shown to enhance recruitment of SL-1. Here acetylation is also important for UBF function. In future work attempts will be made to test whether a functional link exists between the phosphorylation and the acetylation of UBF (Pelletier, 2000).

Spinal and bulbar muscular atrophy (SBMA) is one of eight inherited neurodegenerative diseases known to be caused by CAG repeat expansion. The expansion results in an expanded polyglutamine tract, which likely confers a novel, toxic function to the affected protein. Cell culture and transgenic mouse studies have implicated the nucleus as a site for pathogenesis, suggesting that a critical nuclear factor or process is disrupted by the polyglutamine expansion. In this report evidence is presented that CREB-binding protein (CBP), a transcriptional co-activator that orchestrates nuclear response to a variety of cell signaling cascades, is incorporated into nuclear inclusions formed by polyglutamine-containing proteins in cultured cells, transgenic mice and tissue from patients with SBMA. CBP incorporation into nuclear inclusions form in a cell culture model of another polyglutamine disease, spinocerebellar ataxia type 3. Evidence is presented that soluble levels of CBP are reduced in cells expressing expanded polyglutamine despite increased levels of CBP mRNA. Finally, it is demonstrated that over-expression of CBP rescues cells from polyglutamine-mediated toxicity in neuronal cell culture. These data support a CBP-sequestration model of polyglutamine expansion disease (McCampbell, 2000).

Huntington's Disease (HD) is caused by an expansion of a polyglutamine tract within the huntingtin (htt) protein. Pathogenesis in HD appears to include the cytoplasmic cleavage of htt and release of an amino-terminal fragment capable of nuclear localization. Potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) have been investigated including aggregation, protein-protein interactions, and transcription. httex1p coaggregates with p53 in inclusions generated in cell culture and interacts with p53 in vitro and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters, p21(WAF1/CIP1) and MDR-1. httex1p also interacts in vitro with CREB-binding protein (CBP) and mSin3a, and CBP to localize to neuronal intranuclear inclusions in a transgenic mouse model of HD. These results raise the possibility that expanded repeat htt causes aberrant transcriptional regulation through its interaction with cellular transcription factors that may result in neuronal dysfunction and cell death in HD (Steffan, 2000).

Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. CBP is depleted from its normal nuclear location and os present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescues polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders (Nicifora, 2001).

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. The Caenorhabditis elegans protein DAF-16 binds the IGFBP-1-IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX (Drosophila homolog: Foxo), activate transcription through the IGFBP-1.IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1.IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, it is concluded that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site (Nasrin, 2000).

The IFN-beta enhanceosome activates transcription by directing the ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. The enhanceosome is assembled in the nucleosome-free enhancer region of the IFN-beta gene, leading to the modification and remodeling of a strategically positioned nucleosome that masks the TATA box and the start site of transcription. Initially, the GCN5 complex is recruited, which acetylates the nucleosome, and this is followed by recruitment of the CBP-PolII holoenzyme complex. Nucleosome acetylation in turn facilitates SWI/SNF recruitment by CBP, resulting in chromatin remodeling. This program of recruitment culminates in the binding of TFIID to the promoter and the activation of transcription (Agalioti, 2002)

Hypoxia-inducible factor 1alpha (HIF1alpha) plays a pivotal role in embryogenesis, angiogenesis, and tumorigenesis. HIF1alpha-mediated transcription requires the coactivator p300, at least in part, through interaction with the cysteine- and histidine-rich 1 domain of p300. To understand the molecular basis of this interaction, a random mutagenesis screen in yeast has been employed for efficient identification of residues that are functionally critical for protein interactions. As a result, four residues (Leu-795, Cys-800, Leu-818, and Leu-822) in the C-terminal activation domain of HIF1alpha have been identified as crucial for HIF1 transactivation in mammalian systems. Moreover, data from residue substitution experiments indicate the stringent necessity of leucine and hydrophobic cysteine for C-terminal activation domain function. Likewise, Leu-344, Leu-345, Cys-388, and Cys-393 in the cysteine- and histidine-rich 1 domain of p300 have also been shown to be essential for the functional interaction. It is proposed that hypoxia-induced HIF1alpha-p300 interaction relies upon a leucine-rich hydrophobic interface that is regulated by the hydrophilic and hydrophobic sulfhydryls of HIF1alpha Cys-800 (Gu, 2001).

DNA repair in chromatin is subject to topological constraints, suggesting a requirement for chromatin modification and remodeling activities. Thymine DNA glycosylase (TDG) initiates repair of G/T and G/U mismatches, commonly associated with CpG islands, by removing thymine and uracil moieties. TDG associates with transcriptional coactivators CBP and p300 and the resulting complexes are competent for both the excision step of repair and histone acetylation. Furthermore, TDG stimulates CBP transcriptional activity in transfected cells and reciprocally serves as a substrate for CBP/p300 acetylation. Remarkably, this acetylation triggers release of CBP from DNA ternary complexes and also regulates recruitment of repair endonuclease APE. These observations reveal a potential regulatory role for protein acetylation in base mismatch repair and a role for CBP/p300 in maintaining genomic stability (Tini, 2002).

The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. The KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Another domain of CBP is specifically methylated by CARM1 on conserved arginine residues both in vitro and in vivo. Functional evidence is provided that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, these data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity (Chevillard-Briet, 2002).

What then could be the biochemical consequence of CBP methylation? One obvious possibility is that CBP methylation regulates its HAT activity. However, significant change was found in CBP HAT activity following CBP methylation by CARM1. Another interesting possibility is that methylation could change CBP conformation and/or regulate some protein-protein interactions responsible for CBP-mediated transcriptional activation on steroid hormone receptors. Interestingly, methylation of two proteins has been shown to link arginine methylation to the regulation of protein-protein interaction. To date, no protein has been shown to bind directly to the domain of CBP that is methylated by CARM1 (Chevillard-Briet, 2002).

p300 and CREB binding protein can both activate and repress transcription. The CRD1 transcriptional repression domain has been localized between residues 1017 and 1029 of p300. This region contains two copies of the sequence ψKxE that are modified by the ubiquitin-like protein SUMO-1. Mutations that reduce SUMO modification increase p300-mediated transcriptional activity and expression of a SUMO-specific protease or catalytically inactive Ubc9 relieves repression, demonstrating that p300 repression is mediated by SUMO conjugation. SUMO-modified CRD1 domain binds HDAC6 in vitro, and p300 repression is relieved by histone deacetylase inhibition and siRNA-mediated ablation of HDAC6 expression. These results reveal a mechanism controlling p300 function and suggest that SUMO-dependent repression is mediated by recruitment of HDAC6 (Girdwood, 2003).

Transcriptional coactivators showing physical and functional interactions with PPARgamma include the protein acetyl transferase p300, the TRAP/Mediator complex that interacts with the general transcription machinery, and the highly regulated PGC-1alpha. PGC-1alpha directly interacts with TRAP/Mediator, through the PPARgamma-interacting subunit TRAP220, and stimulates TRAP/Mediator-dependent function on DNA templates. Further, while ineffective by itself, PGC-1alpha stimulates p300-dependent histone acetylation and transcription on chromatin templates in response to PPARgamma. These functions are mediated by largely independent PPARgamma, p300, and TRAP220 interaction domains in PGC-1alpha, whereas p300 and TRAP220 show ligand-dependent interactions with a common region of PPARgamma. Apart from showing PGC-1gamma functions both in chromatin remodeling and in preinitiation complex formation or function (transcription), these results suggest a key role for PGC-1gamma, through concerted but dynamic interactions, in coordinating these steps (Wallberg, 2003).

Nuclear receptors are ligand-inducible transcription factors that specifically regulate the expression of target genes involved in metabolism, development, and reproduction. After binding to their cognate DNA response elements, ligand-bound nuclear receptors activate target gene transcription through interactions with various coactivators and/or components of the basal transcription machinery. Coactivators are thought either to effect chromatin remodeling or to act as a bridge between gene-specific activators and the general transcription machinery (RNA polymerase II and cognate initiation or elongation factors) (Wallberg, 2003 and references therein).

Cofactors that modify chromatin structure fall mainly into two broad classes: those that regulate accessibility of nucleosomal DNA in an ATP-dependent manner and those that alter nucleosome/chromatin structure through covalent modifications (including acetylation, methylation, and phosphorylation) of the N-terminal tails of histones. Of relevance here, the latter category includes the well-characterized histone acetyltransferases (HATs) CBP/p300, PCAF/GCN5, and members of the p160/SRC family. These proteins have been shown to mediate transcriptional activity of many nuclear hormone receptors, and were originally isolated as targets of the ligand binding domains of nuclear receptors (Wallberg, 2003 and references therein).

Coactivators that act more directly on the general transcription machinery are evidenced by their ability to stimulate transcription from naked DNA templates. Among such coactivators, the phylogenetically conserved Mediator complex appears to provide the main conduit for communication between DNA-bound activators and the general transcriptional machinery. Several of the subunits in the Mediator complex have been shown to be direct targets for different activators. The human TRAP/Mediator complex (essentially equivalent to later-described complexes such as SMCC, DRIP, ARC, and NAT) was first identified through ligand-dependent interactions with thyroid hormone receptor, and subsequently shown to mediate the function of a number of nuclear receptors and other activators. Relevant to the present study, the TRAP220 subunit was shown to interact, through two NR boxes (containing LXXLL motifs), with a number of receptors that include TR and PPARγ. The involvement of TRAP220 (via the TRAP/Mediator) in TR and PPARγ function was shown most convincingly in cell-free systems reconstituted with purified factors and by assays in TRAP220 null fibroblasts (Wallberg, 2003 and references therein).

PGC-1α, now documented as a coactivator for many nuclear hormone receptors, was originally identified as a PPARγ-interacting coactivator in brown adipose tissue and has been implicated in diverse physiological processes related, in large part, to cellular metabolism. Moreover, it is highly regulated, being induced by cold in brown fat and by fasting or diabetes in the liver. PGC-1α in turn induces tissue-specific programs of metabolic control, including thermogenesis in brown fat, hepatic gluconeogenesis, and fiber-type switching in skeletal muscle. The PGC-1α interaction with PPARγ is ligand independent and mediated through an N-terminal domain (residues 200-400) and an LXXLL motif, whereas its interactions with other nuclear receptors are ligand dependent and mediated through the LXXLL motif (Wallberg, 2003 and references therein).

Although the precise mechanisms involved in its transcriptional coactivator function are not clear, PGC-1α binds, through its N-terminal 200 amino acids, to both p300/CBP and SRC-1. The PGC-1α C-terminal domain has been implicated in coupling of transcription and RNA processing on PGC-1α target genes, through interactions with splicing factors and an elongating form of RNA polymerase II, suggesting that PGC-1α might reside in both transcription preinitiation and transcription elongation complexes. However, it has not been clear whether the TRAP/Mediator complex, which also interacts with RNA polymerase II, might also play some role in facilitating PGC-1α function and, related, whether PGC-1α, like TRAP/Mediator, might act more directly to stimulate transcription by RNA polymerase II (Wallberg, 2003 and references therein).

The present study of coactivator functions in PPARγ-dependent transcription demonstrates direct interactions of PGC-1α with TRAP/Mediator via the TRAP220 subunit, TRAP/Mediator-dependent effects of PGC-1α on transcription from DNA templates, and p300-dependent effects of PGC-1α on histone acetylation and transcription from chromatin templates. These results document functions for PGC-1α, in both chromatin modification and preinitiation complex assembly/function steps, and further suggest that PGC-1α plays a key role in coordinating these events (Wallberg, 2003).

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, the molecular interactions among the encoded proteins was examined. Eya interacts directly with Six but never with Dach. Dach transactivates a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach is repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template is dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. These results demonstrate that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis (Ikeda, 2002).

Cell signaling affects gene expression by regulating the activity of transcription factors. Mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2 occurs at a conserved site N terminal to their Pointed (PNT) domains. This results in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. This phosphorylation-augmented interaction was discovered in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins has demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain are required for the interaction. Minimal regions that are competent for induced CBP/p300 binding in vitro also support MAPK-enhanced transcription in vivo. CBP coexpression potentiates MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interact in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, these findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis (Foulds, 2004).

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