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CAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1).

ABSTRACT: 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.

SUBMITTER: Silverman ES

PROVIDER: S-EPMC1219856 | biostudies-other | 1998 Nov

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Reversible ?-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 ?-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.

Project description:Interactions between transforming growth factor-? (TGF-?) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated ?-catenin-dependent and transforming growth factor-?1 (TGF-?1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the ?-catenin/CBP (but not ?-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-?1-mediated ?-smooth muscle actin (?-SMA) and collagen induction in AEC. We now demonstrate that TGF-?1 induces LEF/TCF TOPFLASH reporter activation and nuclear ?-catenin accumulation, while LiCl augments TGF-?-induced ?-SMA expression, further confirming co-operation between ?-catenin- and TGF-?-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of ?-catenin and overexpression of ICAT abrogated effects of TGF-?1 on ?-SMA transcription/expression, indicating a requirement for ?-catenin in these Smad3-dependent effects. Following TGF-? treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and ?-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of ?-SMA via complex formation among Smad3, ?-catenin, and CBP. ICG-001 inhibited ?-SMA expression/transcription in response to TGF-? as well as ?-SMA promoter occupancy by ?-catenin and CBP, demonstrating a previously unknown requisite TGF-?1/?-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by ?-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically uncoupling CBP/catenin-dependent signaling downstream of TGF-?.

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CAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1).

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