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Background Reversible ε‐amino acetylation of lysine residues regulates transcription as well as metabolic flux; however, roles for specific lysine acetyltransferases in skeletal muscle physiology and function are unknown. In this study, we investigated the role of the related acetyltransferases p300 and cAMP response element‐binding protein‐binding protein (CBP) in skeletal muscle transcriptional homeostasis and physiology in adult mice. Methods Mice with skeletal muscle‐specific and inducible knockout of p300 and CBP (PCKO) were generated by crossing mice with a tamoxifen‐inducible Cre recombinase expressed under the human α‐skeletal actin promoter with mice having LoxP sites flanking exon 9 of the Ep300 and Crebbp genes. Knockout of PCKO was induced at 13–15 weeks of age via oral gavage of tamoxifen for 5 days to both PCKO and littermate control [wildtype (WT)] mice. Body composition, food intake, and muscle function were assessed on day 0 (D0) through 5 (D5). Microarray and tandem mass tag mass spectrometry analyses were performed to assess global RNA and protein levels in skeletal muscle of PCKO and WT mice. Results At D5 after initiating tamoxifen treatment, there was a reduction in body weight (−15%), food intake (−78%), stride length (−46%), and grip strength (−45%) in PCKO compared with WT mice. Additionally, ex vivo contractile function [tetanic tension (kPa)] was severely impaired in PCKO vs. WT mice at D3 (~70–80% lower) and D5 (~80–95% lower) and resulted in lethality within 1 week—a phenotype that is reversed by the presence of a single allele of either p300 or CBP. The impaired muscle function in PCKO mice was paralleled by substantial transcriptional alterations (3310 genes; false discovery rate < 0.1), especially in gene networks central to muscle contraction and structural integrity. This transcriptional uncoupling was accompanied by changes in protein expression patterns indicative of impaired muscle function, albeit to a smaller magnitude (446 proteins; fold‐change > 1.25; false discovery rate < 0.1). Conclusions These data reveal that p300 and CBP are required for the control and maintenance of contractile function and transcriptional homeostasis in skeletal muscle and, ultimately, organism survival. By extension, modulating p300/CBP function may hold promise for the treatment of disorders characterized by impaired contractile function in humans.

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

Heat shock protein (hsp) 90 inhibition attenuates NF-κB activation and blocks inflammation. However, the precise mechanism of NF-κB regulation by hsp90 in the endothelium is not clear. We investigated the mechanisms of hsp90 inhibition by 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) on NF-κB activation by LPS in primary human lung microvascular endothelial cells. Transcriptional activation of NF-κB was measured by luciferase reporter assay, gene expression by real-time RT-PCR, DNA binding of transcription factors by chromatin immunoprecipitation assay, protein–protein interaction by coimmunoprecipitation/immunoblotting, histone deacetylase (HDAC)/histone acetyltransferase enzyme activity by fluorometry, and nucleosome eviction by partial microccocal DNase digestion. In human lung microvascular endothelial cells, 17-AAG–induced degradation of IKBα was accomplished regardless of the phosphorylation/ubiquitination state of the protein. Hence, 17-AAG did not block LPS-induced NF-κB nuclear translocation and DNA binding activity. Instead, 17-AAG blocked the recruitment of the coactivator, cAMP response element binding protein binding protein, and prevented the assembly of a transcriptionally competent RNA polymerase II complex at the κB elements of the IKBα (an NF-κB–responsive gene) promoter. The effect of LPS on IKBα mRNA expression was associated with rapid deacetylation of histone-H3(Lys9) and a dramatic down-regulation of core histone H3 binding. Even though treatment with an HDAC inhibitor produced the same effect as hsp90 inhibition, the effect of 17-AAG was independent of HDAC. We conclude that hsp90 inhibition attenuates NF-κB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells.

Rubinstein–Taybi Syndrome (RSTS, OMIM 180849) is a rare condition, which in 65% of cases is caused by haploinsufficiency of CREBBP (cAMP response element binding protein binding protein) localized to 16p13.3. A small subset of RSTS cases caused by 16p13.3 microdeletions involving neighboring genes have been recently suggested to be a true contiguous gene syndrome called severe RSTS or 16p13.3 deletion syndrome (OMIM 610543). In the present report, we describe a case of a 2-year-old female with RSTS who, besides most of the typical features of RSTS has corpus callosum dysgenesis and a Chiari type I malformation which required neurosurgical decompression. CGH microarray showed a ∼520.7 kb microdeletion on 16p13.3 involving CREBBP, ADCY9, and SRL genes. We hypothesize that the manifestations in this patient might be influenced by the haploinsufficiency for ADCY9 and SRL. © 2010 Wiley-Liss, Inc.

Previous studies have demonstrated that activation of thrombin receptor could promote endothelial progenitor cell (EPC) migration. As cAMP-response-element-binding-protein-binding protein (CBP) is involved in many cellular biological processes, we hypothesized that CBP mediates thrombin-induced EPC migration. In this study, we examined whether CBP silencing would affect EPC migration induced by thrombin using small interference RNA approach. EPC isolated from the bone marrow of femurs and tibias of Sprague-Dawley rats were cultured and identified, and then were treated by thrombin alone or combined with CBP-shRNA lentivirus. Transwell chamber assay was performed to measure EPC migration. Quantitative real-time polymerase chain reaction and Western blot were carried out to detect the expression of CBP and CXCR4. Thrombin induced CBP expression in a time- and dose-dependent manner. Small interference RNA for CBP downregulated thrombin-induced CBP expression. Thrombin-induced EPC migration was also attenuated by CBP downregulation. Western blot indicated that CXCR4 expression on EPC is upregulated by thrombin and this effect was blocked by CBP silencing. In conclusion, thrombin-induced EPC migration was inhibited by CBP silencing via downregulation of CXCR4 expression, indicating that CBP plays an important role in thrombin-induced EPC migration.

Casein kinase I (CKI)-ε and GSK-3β phosphorylate β-catenin at Ser45(β-cat45) and Thr41/Ser37,33(β-cat33,37,41) residues, thereby facilitating its ubiquitination and proteasomal degradation. We used a Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model to determine Ser/Thr phosphorylation and biological function of β-catenin during crypt hyperproliferation. TMCH was associated with 3-fold and 3.3-fold increases in CKI-ε cellular abundance and 2-fold and 1.8-fold increase in its activity at 6 and 12 days after infection, respectively. β-Catenin coimmunoprecipitated with both cellular and nuclear CKI-ε and cellular axin at these time points. Cellular β-catenin was constitutively phosphorylated at Ser45and underwent subcellular redistribution to cytoskeletal and nuclear fractions at days 6 and 12 of TMCH, respectively. β-cat33,37,41, however, exhibited only subtle changes in either phosphorylation status or subcellular distribution even after blocking proteasomal degradation in vivo. Interestingly, GSK-3β underwent increased phosphorylation at Ser9, leading to 40% and 70% decreases in its activity at these time points, respectively. Coimmunoprecipitation studies exhibited strong association of GSK-3β with PKC-ζ at either time point. Cellular β-cat45stabilized and, along with unphosphorylated β-catenin, underwent nuclear translocation, associated with nuclear accumulated Tcf-4 and cAMP response element binding protein binding protein, and was significantly acetylated, leading to increases in DNA binding. Priming of β-catenin at Ser45exists in vivo. However, β-cat45does not necessarily enter the degradation pathway. Impairment in linking β-cat45to subsequent GSK-3β-mediated phosphorylation and degradation may account for increased steady-state levels of both unphosphorylated as well as Ser45-phosphorylated β-catenin, which may be causally linked to increases in cell census during TMCH.

In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory mediators, many of which are regulated by the nuclear factor kappaB (NF-kappaB) family of transcription factors. Recently, we have provided evidence that the RelA NF-kappaB subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the Galphas/cAMP/protein kinase A pathway. TNFalpha is a potent activator of NF-kappaB, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNFalpha was observed to repress expression of Galphas while, at the same time, stimulating NF-kappaB activity. Furthermore, this effect could be replicated by exposure to bacterial lipopolysaccharide and exogenous expression of RelA. Moreover, TNFalpha was seen to repress endogenous Galphas mRNA expression as judged by RT-PCR analyses. Using the chromatin immunoprecipitation assay, we show that RelA did not bind directly to the Galphas promoter. Significantly, expression of a coactivator protein, cAMP response element binding protein binding protein, relieved RelA-induced down-regulation of Galphas expression. Together, these data suggest that, in human myometrium, repression of the Galphas gene by NF-kappaB occurs through a non-DNA binding mechanism involving competition for limiting amounts of cellular coactivator proteins including cAMP response element binding protein binding protein.

In songbirds, the initiation of song behaviour and the neural substrate of this system are highly influenced by gonadal steroids. Receptors for gonadal steroid hormones, such as androgens and oestrogens, have been localized within select nuclei of the song system. An important step in steroid receptor action is the recruitment of nuclear receptor coactivators. The coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), has been implicated in both androgen and oestrogen receptor transactivation. Although the role of CBP in transcriptional mechanisms has been widely studied, little is known about CBP expression in the brain. The association between the distribution of CBP and oestrogen receptors in the hippocampus has been related to long-term memory. However, the distribution of brain CBP has not been related to the expression of gonadal steroid receptors in a system as relevant to reproductive behaviour as the avian song system. Western immunoblotting of European starling (Sturnus vulgaris) brain tissue reveals a band at 265 kDa. Immunohistochemical localization of CBP in starling brain indicates wide, but heterogeneous expression. CBP-immunoreactive (CBP-ir) cells define the boundaries of song control nuclei. In HVc (sometimes called the High Vocal Center) and the robust nucleus of the archistriatum (RA), there is a higher density of CBP-ir cells within the boundaries of these nuclei than in adjacent neostriatum or archistriatum, for HVc and RA, respectively. We also report that the distribution of CBP-ir cells varies among different nuclei within the song control system. CBP-ir cells within area X (also a part of the song system) and HVc are densely packed into clusters, whereas cells can be easily discriminated in RA. CBP is also highly expressed in hypothalamic areas, indicating that areas rich in steroid receptors also contain CBP. These data suggest that CBP is important for modulating transcriptional activities in the song system and other sites in the songbird brain that express gonadal steroid receptors.

Angiotensin II, a potent vasoactive peptide produced by proteolysis of the angiotensinogen (AGT) prohormone, plays a critical role in cardiovascular homeostasis. Recently we showed that IL-6 induces human (h)AGT transcription by activating the signal transducers and activators of transcription (STATs). Here we investigated the role of the coactivator p300/cAMP response element binding protein-binding protein (CBP) in STAT3-mediated hAGT gene expression. Overexpression of adenovirus 12S E1A, which binds and inactivates p300/CBP, strongly inhibited basal and stimulated hAGT transcription, whereas a mutant E1A defective in binding p300/CBP did not. Conversely, ectopic expression of p300 and CBP potentiated inducible hAGT promoter activity. Coimmunoprecipitation assays revealed STAT3-p300 interaction upon IL-6 stimulation. The STAT3-p300 association requires the STAT3 C-terminal transactivation domain, as STAT3 deleted of transactivation functions as a dominant-negative inhibitor and does not associate with p300/CBP. The observation that IL-6 stimulation increases histone H4 acetylation of the endogenous hAGT promoter, and expression of p300 deficient in histone acetyltransferase activity down-regulates hAGT promoter activity both suggest that p300 histone acetyltransferase activity is required for hAGT expression. Finally, treatment of HepG2 cells with a histone deacetylase inhibitor increased the hAGT mRNA abundance by 2- to 3-fold. Taken together, our results indicate that IL-6-inducible expression of the hAGT promoter is mediated by physical association of the COOH terminus of STAT3 with p300/CBP, the recruitment of which targets histone acetylation and results in chromatin remodeling.

O(6)-Methylguanine-DNA methyltransferase (MGMT)(1), a ubiquitous DNA repair protein, removes O(6)-alkylguanine from DNA, including cytotoxic O(6)-chloroethylguanine induced by chemotherapeutic N-alkyl N-nitrosourea-type drugs, e.g. 1,3-bis(2-chloroethyl)-1-nitrosourea. Treating the pancreatic carcinoma cell line MIA PaCa-2 with trichostatin A (TSA), a specific inhibitor of histone deacetylase, increased MGMT mRNA and protein levels by 2-3-fold. Surprisingly, TSA treatment increased MGMT promoter-dependent luciferase activity by some 40-fold in a transient reporter expression assay. Deletion and point mutation analysis showed that two AP-1 binding sites in the MGMT promoter are involved in activation by TSA. Ectopic expression of the transcriptional coactivators cAMP response element-binding protein-binding protein (CBP) and p300, which have intrinsic histone acetyltransferase activity, enhanced luciferase expression. Overexpression of adenovirus E1A, which binds CBP/p300, strongly inhibited both basal and TSA-inducible MGMT promoter activity, while a mutant E1A, defective in binding CBP/p300, did not. Chromatin immunoprecipitation assays revealed that TSA treatment increased histone acetylation in the endogenous MGMT promoter region, which also showed association with CBP/p300. Taken together, our results indicate that targeted histone acetylation results in the remodeling of chromatin by recruitment of the coactivator CBP/p300, and constitutes an important step in regulating MGMT expression.

Transcription of pituitary alpha-glycoprotein hormone subunit (alpha-GSU) and thyrotropin beta subunit (TSH-beta) genes is stimulated by thyrotropin-releasing hormone (TRH). Since cAMP response element-binding protein (CREB)-binding protein (CBP) integrates a number of cell signaling pathways, we investigated whether CBP is important for TRH stimulation of the TSH subunit genes. Cotransfection of E1A in GH(3) cells completely blocked TRH stimulation of the TSH subunit genes, suggesting that CBP is a key factor for TRH signaling in the pituitary. CBP and Pit-1 acted synergistically in TRH stimulation of the TSH-beta promoter, and amino acids 1-450 of CBP were sufficient for the TRH effect. In contrast, on the human alpha-GSU promoter, CREB and P-Lim mediated TRH signaling. Intriguingly, CREB was phosphorylated upon TRH stimulation, leading to CBP recruitment to the alpha-GSU promoter. CBP also interacted with P-Lim in a TRH-dependent manner, suggesting that P-Lim is an important factor for non-cAMP response element-mediated TRH stimulation of this promoter. Distinct domains of CBP were required for TRH signaling by CREB and P-Lim on the alpha-GSU promoter, amino acids 450-700 and 1-450, respectively. Thus, the amino terminus of CBP plays a critical role in TRH signaling in the anterior pituitary via both Pit-1-dependent and -independent pathways, yielding differential regulation of pituitary gene products.

OCA-B was identified as a B cell-specific coactivator that functions with either Oct-1 or Oct-2 to mediate efficient cell type-specific transcription via the octamer site (ATGCAAAT) both in vivo and in vitro. Mice lacking OCA-B exhibit normal Ag-independent B cell maturation. In contrast, Ag-dependent functions, including production of secondary Ig isotypes and germinal center formation, are greatly affected. To better understand OCA-B expression and, ultimately, the defects observed in the OCA-B knockout mice, we have cloned the OCA-B promoter and examined its function in both transformed and primary B cells. We show here that the OCA-B promoter is developmentally regulated, with activity increasing throughout B cell differentiation. Through physical and functional assays, we have found an activating transcription factor/cAMP response element binding protein binding site (or cAMP response element) that is crucial for OCA-B promoter activity. Furthermore, we demonstrate that IL-4 and anti-CD40 induce both the OCA-B promoter and octamer-dependent promoters, thus implicating OCA-B in B cell signaling events in the nucleus.

The differentiation and maintenance of a neurotransmitter phenotype is guided by the interaction of exogenous cues with intrinsic genetic machinery. For the noradrenergic phenotype, these influences combine to activate the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). In this study, we evaluate the molecular mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual homeodomain binding sites in the rat DBH promoter region and find that all are essential for both basal and cAMP-dependent protein kinase A (PKA)-stimulated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core recognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PKA in SHSY-5Y neuroblastoma and HepG2 hepatoma cells. Intracellular Arix-Arix interactions are evident and may contribute to the interdependence of homeodomain binding sites. Analysis of functional domains of Arix reveals an N-terminal activation domain and a C-terminal repression domain. The N terminus of Arix contains an amino acid motif similar to a region in Brachyury and Pax9 transcription factors. The N-terminal activation domain of Arix interacts with the transcriptional co-activator, cAMP-response element-binding protein-binding protein, which potentiates transcription from the DBH promoter in a PKA-dependent manner. The present study supports the hypothesis that the paired-like homeodomain protein, Arix, acts as a critical phenotype-specific regulator of the DBH promoter by serving as an integrator of signal-dependent transcription activators within the network of the general transcription machinery.

The TATA-less murine Msx1 promoter contains two Msx1-binding motifs, located at -568 to -573 and +25 to +30, and is subject to potent autorepression [Takahashi, Guron, Shetty, Matsui and Raghow (1997) J. Biol. Chem. 272, 22667-22678]. To investigate the molecular mechanism by which Msx1 represses the activity of its own promoter, we transfected C2C12 myoblasts with Msx1-promoter-luciferase constructs and assessed reporter gene activity, with and without the exogenous expression of Msx1. We demonstrate that Msx1-mediated autorepression remained unaffected, regardless of the presence or absence of the Msx1 recognition motifs on the promoter. Furthermore, graded exogenous expression of TATA-binding protein (TBP), Sp1 or cAMP-response-element-binding protein-binding protein (CBP/p300) could counteract the autoinhibitory activity of Msx1. Finally, we demonstrate that Msx1 protein can be immunoprecipitated in a multiprotein complex containing TBP, Sp1 and CBP/p300. We hypothesize that the interaction of Msx1 protein with one or more ubiquitous or tissue-restricted transcription factors mediates transcriptional autorepression of the Msx1 gene.

Altered gene transcription has been implicated in the pathogenesis of polyglutamine-dependent neurodegeneration. In this issue of Neuron, Sopher et al. demonstrate that androgen receptors containing expanded polyglutamine cause decreased expression of vascular endothelial growth factor (VEGF) by interfering with cAMP response element binding protein binding protein (CBP), thereby contributing to the motor neuron degeneration in spinal and bulbar muscular atrophy.

Several basic leucine zipper (B-ZIP) transcription factors have been implicated in cancer, substance abuse, and other pathological conditions. We previously identified arylstibonic acids that bind to B-ZIP proteins and inhibit their interaction with DNA. In this study, we used electrophoretic mobility shift assay to analyze 46 arylstibonic acids for their activity to disrupt the DNA binding of three B-ZIP [CCAAT/enhancer-binding protein α, cyclic AMP-response element-binding protein (CREB), and vitellogenin gene-binding protein (VBP)] and two basic helix-loop-helix leucine zipper (B-HLH-ZIP) [USF (upstream stimulating factor) and Mitf] proteins. Twenty-five arylstibonic acids showed activity at micromolar concentrations. The most active compound, P6981 [2-(3-stibonophenyl)malonic acid], had half-maximal inhibition at ∼5 nM for CREB. Circular dichroism thermal denaturation studies indicated that P6981 binds both the B-ZIP domain and the leucine zipper. The crystal structure of an arylstibonic acid, NSC13778, bound to the VBP leucine zipper identified electrostatic interactions between both the stibonic and carboxylic acid groups of NSC13778 [(E)-3-(3-stibonophenyl)acrylic acid] and arginine side chains of VBP, which is also involved in interhelical salt bridges in the leucine zipper. P6981 induced GFP-B-ZIP chimeric proteins to partially localize to the cytoplasm, demonstrating that it is active in cells. P6981 inhibited the growth of a patient-derived clear cell sarcoma cell line whose oncogenic potential is driven by a chimeric protein EWS-ATF1 (Ewing9s sarcoma protein-activating transcription factor 1), which contains the DNA binding domain of ATF1, a B-ZIP protein. NSC13778 inhibited the growth of xenografted clear cell sarcoma, and no toxicity was observed. These experiments suggest that antimony containing arylstibonic acids are promising leads for suppression of DNA binding activities of B-ZIP and B-HLH-ZIP transcription factors.

The protein acetyltransferases p300 and cAMP response element-binding protein binding protein (CBP) are homologous, ubiquitously expressed proteins that interact with hundreds of proteins involved in transcriptional regulation and are involved globally as transcriptional coregulators. Although these two proteins acetylate and interact with overlapping sets of proteins, we found that p300 and CBP contribute to androgen-induced regulation of distinct sets of genes in C4-2B prostate cancer cells, a model of advanced prostate cancer. CBP cannot compensate for the loss of p300 to support androgen-induced expression of many genes, such as TMPRSS2 and PSA. Global gene expression analysis indicated that 47% of androgen-regulated genes are p300-dependent in these cells, whereas, surprisingly, only 0.3% of them are CBP-dependent. Chromatin immunoprecipitation analysis after depletion of cellular p300 indicated that p300 is required for androgen-induced acetylation of histones H3 and H4, methylation of histone H3 at Lys-4, and recruitment of TATA box binding protein (TBP) and RNA polymerase II, but not recruitment of the androgen receptor, on the TMPRSS2 gene in response to androgen. Thus, p300 is the dominant coregulator of the CBP/p300 pair for androgen-regulated gene expression in C4-2B cells. p300 is required at an early stage of chromatin remodeling and transcription complex assembly after binding of androgen receptor to the gene but before many critical histone modifications occur.

Cyclic AMP-regulated gene expression frequently involves a DNA element known as the cAMP-regulated enhancer (CRE). Many transcription factors bind to this element, including the protein CREB, which is activated as a result of phosphorylation by protein kinase A. This modification stimulates interaction with one or more of the general transcription factors or, alternatively, allows recruitment of a co-activator. Here we report that CREB phosphorylated by protein kinase A binds specifically to a nuclear protein of M(r) 265K which we term CBP (for CREB-binding protein). Fusion of a heterologous DNA-binding domain to the amino terminus of CBP enables the chimaeric protein to function as a protein kinase A-regulated transcriptional activator. We propose that CBP may participate in cAMP-regulated gene expression by interacting with the activated phosphorylated form of CREB.

Hes6 is a basic helix-loop-helix transcription factor that functions in the differentiation of pluripotent progenitor cells and during tumorigenesis. However, the molecular mechanism for its function is largely unknown. Here we show that Hes6 is a component of the promyelocytic leukemia nuclear body (PML-NB) complex in the nuclei and that Hes6 inhibits cell proliferation through induction of p21 cyclin-dependent kinase inhibitor. We further show that Hes6 directly interacts with CREB-binding protein (CBP), one of the key components of PML-NB, via its basic domain. This association is critical for p21 induction through multiple mechanisms, including chromatin remodeling and p53 acetylation. Taken together, these results suggest that the Hes6-CBP complex in PML-NB may influence the proliferation of cells via p53-dependent and -independent pathways.

Fatty acids in milk are thought to play an important role in intestinal maturation and gene expression in the rat small intestine during the suckling – weaning period. In the present study, we determined the jejunal mRNA level of the cAMP-response-element-binding-protein-binding protein (CBP)/p300, which is one of the chromatin remodelling factors and regulates histone acetylation, during the postnatal period in rats. The mRNA level of CBP/p300 was high during the suckling and middle of the weaning period (day 5 to 20) and then declined sharply to a low level at the end of the weaning period and after weaning. In situ hybridisation also showed that CBP/p300 mRNA levels in the villus as well as the basal membrane clearly decreased after weaning. Rat pups at age 17 d, weaned to a high-fat diet, showed higher levels of CBP/ p300 mRNA than those weaned to a low-fat diet. Oral administration of caprylic acid, oleic acid and linoleic acid, which are major fatty acid components in milk, induced jejunal CBP/p300 gene expression. The present results suggest that fatty acids in components of milk enhance expression of the CBP/p300 genes in the small intestine. CBP/p300: Fatty acids: Postnatal development: Small intestine Rat pups ingest fatty acids from milk as major nutrients during the 4-week period following birth which corresponds to the suckling –weaning period. Several studies have shown that fatty acids in milk are important for the development of the small intestine in rat pups during the suckling–weaning period 1–4 . During the transition period from suckling to weaning (13–27 d after birth), the small intestine matures dramatically to its adult form as an adaptation to altered nutrition associated with the change from a diet rich in fat (milk) to one rich in carbohydrate (solid food). The expression of many genes related to digestion or absorption, such as sucrase –isomaltase (SI) 5 , GLUT5 6 , cellular retinol-binding protein type II (CRBPII) 7 , and liver-type and intestinal-type fatty acid-binding proteins (L-FABP and I-FABP) 8 , increase during this period. Many studies, including some from our own laboratory, indicate that putative transcriptional factors for these intestinal genes are also up regulated during this period. Our previous research indicates that caudal-related homeobox 2 (Cdx-2) and hepatocyte nuclear factor (HNF)-1, which are transcriptional factors for SI gene expression, are highly expressed during the suckling –weaning transitional

Nuclear receptors activate transcription by recruiting multiple coactivators to the promoters of specific target genes. The functional synergy of the p160 coactivators [steroid receptor coactivator-1, glucocorticoid receptor interacting protein (GRIP1), or the activator for thyroid hormone and retinoid receptors], the histone acetyltransferases cAMP response element binding protein binding protein (CBP) and p300 and the histone methyltransferase coactivator-associated arginine methyltransferase (CARM1) depends on the methyltransferase activity of CARM1. CARM1 methylates histone H3 and other factors including the N-terminal region of p300. Here, we report that CARM1 also methylates Arg-2142 within the C-terminal GRIP1 binding domain (GBD) of p300. In the GBD, both Arg-2088 and Arg-2142 are important for binding GRIP1. Methylation of Arg-2142 inhibits the bimolecular interaction of GRIP1 to p300 in vitro and in vivo . This methylation mark of p300 GBD is removed by peptidyl deiminase 4, thereby enhancing the p300–GRIP1 interaction. These methylation and demethylimination events also alter the conformation and activity of the coactivator complex and regulate estrogen receptor-mediated transcription, and they thus represent unique mechanisms for regulating coactivator complex assembly, conformation, and function.

The p160 co-activators, SRC1 (steroid receptor co-activator 1), GRIP1 (glucocorticoid-receptor-interacting protein 1) and ACTR (activator for thyroid hormone and retinoid receptors), have two ADs (activation domains), AD1 and AD2. AD1 is a binding site for the related co-activators, CBP (cAMP-response-element-binding protein-binding protein) and p300, whereas AD2 binds to another co-activator, co-activator-associated arginine methyltransferase 1 (CARM1). Here, we identified two CBP-interacting sites [amino acids 1075–1083 (site I) and 1095–1106 (site II)] in a so-called CBP-dependent transactivation domain (AD1; amino acids 1057–1109) of GRIP1. Site I was the major site for CBP-dependent AD1 transactivation activity of GRIP1 whereas, following the deletion of site II, full or partial transactivation activity was retained without the recruitment of CBP in yeast, HeLa, human embryonic kidney 293 and CV-1 cells. GRIP1 (with a deletion of site II) expressed stronger co-activator activity than that of wild-type GRIP1 in the TR (thyroid receptor) and the AR (androgen receptor), but not the ER (oestrogen receptor), systems in HeLa cells. We also demonstrated that these CBP-binding sites of GRIP1 are not the only functional domains for its AD1 function in TR, AR and ER systems in HeLa cells by the exogenous overexpression of one E1A mutant, which led to a lack of CBP-binding ability. Our results suggest that these two CBP-interacting sites in the GRIP AD1 domain not only determine its AD1 activity, but are also involved in its co-activator functions in some nuclear receptors.

Progression of human prostate cancer toward therapy resistance occurs in the presence of wild- type or mutated androgen receptors (ARs) that, in some cases, exhibit aberrant activation by various steroid hormones and anti-androgens. The AR associates with a number of co-activators that possess histone acetylase activity and act as bridging molecules to components of the transcription initiation complex. In previous reports, it was shown that the transcriptional co- activator CREB (cAMP response element-binding protein)-binding protein (CBP) enhances AR activity in a ligand-dependent manner. In the present study, we have investigated whether CBP modifies antagonist/agonist balance of the nonsteroidal anti-androgens hydroxyflutamide and bicalutamide. In prostate cancer DU-145 cells, which were transiently transfected with CBP cDNA, hydroxyflutamide enhanced AR activity to a greater extent than bicalutamide in the presence of either wild-type or the mutated AR 730 val-->met. In two sublines of LNCaP cells that contain the mutated AR 877 thr-->ala and overexpressed CBP, increase in AR activity was observed after treatment with hydroxyflutamide but not with bicalutamide. Anti- androgens did not influence AR expression in cells transfected with CBP cDNA, as judged by Western blot analysis. Endogenous CBP protein was detected by Western blot in nuclear extracts from the three prostate cancer cell lines, LNCaP, PC-3, and DU- 145, all derived from therapy-resistant prostate cancer. In addition, CBP was expressed in both basal and secretory cells of benign prostate epithelium, high-grade prostate intraepithelial neoplasia, and prostate cancer clinical specimens, as evidenced by immunohistochemical staining. Taken together, our findings demonstrate the selective enhancement of agonistic action of the anti-androgen hydroxyflutamide by the transcriptional co-activator CBP, which is a new, potentially relevant mechanism contributing to the acquisition of therapy resistance in prostate cancer.

A multifunctional transcription co-activator, cAMP response element-binding protein-binding protein (CBP)interacts with a number of cellular factors and participates in cell growth, transformation, and development. It is also targeted by many viral proteins for their transcriptional activity or for the regulation of cellular processes. Here, we report that the C/H3 region of CBP is targeted by the latency associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV). LANA interferes with the interaction between CBP and c-Fos, a representative C/H3 region binding, cellular transcription factor, in vivo and in vitro. In addition, we found that LANA inhibits the transcriptional activity and the in vitro histone acetyltransferase activity of CBP, suggesting that LANA modulates the global transcriptional activities of infected cells through the interaction with CBP. These results indicate that KSHV follows one of the conserved strategies, which other viruses utilize for influencing the cellular processes.

cAMP response element-binding protein-binding protein (CBP) is a eucaryotic transcriptional co-activator that contains multiple protein-protein interaction domains for association with various transcription factors, components of the basal transcriptional apparatus, and other co-activator proteins. Here, we report that CBP is also a co-activator of the human papillomavirus (HPV) E2 protein, which is a sequence-specific transcription/replication factor. We provide biochemical, genetic, and functional evidence that CBP binds directly to HPV E2 in vivo and in vitro and activates E2-dependent transcription. Mutations in an amphipathic helix within HPV-18 E2 abolish its transcriptional activation properties and its ability to bind to CBP. Furthermore, the binding of CBP to E2 was shown to be necessary for E2-dependent transcription. Interestingly, the histone acetyltransferase activity of CBP plays a role in CBP activation of E2-dependent transcription.

Egr-1 (early-growth response factor-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important for cell growth, development and the pathogenesis of disease. The transcriptional co-activators CBP (cAMP-response-element-binding-protein-binding protein) and p300 interact with sequence-specific transcription factors as well as components of the basal transcription machinery to facilitate RNA polymerase II recruitment and transcriptional initiation. Here we demonstrate a unique way in which Egr-1 physically and functionally interacts with CBP/p300 to modulate gene transcription. CBP/p300 potentiated Egr-1 mediated expression of 5-lipoxygenase (5-LO) promoter–reporter constructs, and the degree of trans-activation was proportional to the number of Egr-1 consensus binding sites present in wild-type and naturally occurring mutants of the 5-LO promoter. The N- and C-terminal domains of CBP interact with the transcriptional activation domain of Egr-1, as demonstrated by a mammalian two-hybrid assay. Direct protein–protein interactions between CBP/p300 and Egr-1 were demonstrated by glutathione S-transferase fusion-protein binding and co-immunoprecipitation/Western-blot studies. These data suggest that CBP and p300 act as transcriptional co-activators for Egr-1-mediated gene expression and that variations between individuals in such co-activation could serve as a genetic basis for variability in gene expression.