Acetylates histones, giving a specific tag for transcriptional activation. Also acetylates non-histone proteins, like NCOA3 and FOXO1. Binds specifically to phosphorylated CREB and enhances its transcriptional activity toward cAMP-responsive genes. Acts as a coactivator of ALX1 in the presence of EP300.
We have previously shown that interferon regulatory factor-2 (IRF-2) is acetylated by p300 and PCAF in vivo and in vitro. In this study we identified, by mass spectrometry, two lysine residues in the DNA binding domain (DBD), Lys-75 and Lys-78, to be the major acetylation sites in IRF-2. Although acetylation of IRF-2 did not alter DNA binding activity in vitro, mutation of Lys-75 diminished the IRF-2-dependent activation of histone H4 promoter activity. Acetylation of IRF-2 and IRF-2-stimulated H4 promoter activity were inhibited by the adenovirus E1A, indicating the involvement of p300/CBP. Mutation of Lys-78, a residue conserved throughout the IRF family members, led to the abrogation of DNA binding activity independently of acetylation. H4 is transcribed only in rapidly growing cells and its promoter activity is dependent on cell growth. Consistent with a role for acetylated IRF-2 in cell growth control, IRF-2 was acetylated only in growing NIH 3T3 cells, but not in growth-arrested counterparts. Chromatin immunoprecipitation assays showed that IRF-2 interacted with p300 and bound to the endogenous H4 promoter only in growing cells, although the levels of total IRF-2 were comparable in both growing and growth-arrested cells. These results indicate that IRF-2 is acetylated in a cell growth-dependent manner, which enables it to contribute to transcription of cell growth-regulated promoters.
The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity.
Proc. Natl. Acad. Sci. U.S.A. 95, 9855-9860 (1998)[PubMed:9707565]
Erythroid Krüppel-like factor (EKLF) is a red cell-specific transcriptional activator that is crucial for consolidating the switch to high levels of adult beta-globin expression during erythroid ontogeny. EKLF is required for integrity of the chromatin structure at the beta-like globin locus, and it interacts with a positive-acting factor in vivo. We find that EKLF is an acetylated transcription factor, and that it interacts in vivo with CBP, p300, and P/CAF. However, its interactions with these histone acetyltransferases are not equivalent, as CBP and p300, but not P/CAF, utilize EKLF as a substrate for in vitro acetylation within its trans-activation region. The functional effects of these interactions are that CBP and p300, but not P/CAF, enhance EKLF's transcriptional activation of the beta-globin promoter in erythroid cells. These results establish EKLF as a tissue-specific transcription factor that undergoes post-translational acetylation and suggest a mechanism by which EKLF is able to alter chromatin structure and induce beta-globin expression within the beta-like globin cluster.
The paired-like homeoprotein, Cart1, is involved in skeletal development. We describe here that the general coactivator p300/CBP controls the transcription activity of Cart1 through acetylation of a lysine residue that is highly conserved in other homeoproteins. Acetylation of this residue increases the interaction between p300/CBP and Cart1 and enhances its transcriptional activation. INTRODUCTION: Cart1 encodes a paired-like homeoprotein expressed selectively in chondrocyte lineage during embryonic development. Although its target gene remains unknown, gene disruption studies have revealed that Cart1 plays an important role for craniofacial bone formation as well as limb development by cooperating with another homeoprotein, Alx4. In this report, we study the functional involvement of p300/CBP, coactivators with intrinsic histone acetyltransferase (HAT) activity, in the transcriptional control of Cart1. METHODS: To study the transcription activity of Cart1, a reporter construct containing a putative Cart1 binding site was transiently transfected with the expression vectors of each protein. The interaction between p300/CBP and Cart1 was investigated by glutathione S-transferase (GST) pull-down, yeast two-hybrid, and immunoprecipitation assays. In vitro acetylation assay was performed with the recombinant p300-HAT domain and Cart1 in the presence of acetyl-CoA. RESULTS AND CONCLUSIONS: p300 and CBP stimulate Cart1-dependent transcription activity, and this transactivation is inhibited by E1A and Tax, oncoproteins that suppress the activity of p300/CBP. Cart1 binds to p300 in vivo and in vitro, and this requires the homeodomain of Cart 1 and N-terminal 139 amino acids of p300. Confocal microscopy analysis shows that Cart1 recruits overexpressed and endogenous p300 to a Cart1-specific subnuclear compartment. Cart1 is acetylated in vivo and sodium butyrate and trichostatin A, histone deacetylase inhibitors, markedly enhance the transcription activity of Cart1. Deletion and mutagenesis analysis identifies the 131st lysine that locates immediately adjacent to the homeodomain as a target of p300-HAT, and a point mutation to this residue attenuates the binding affinity to p300 as well as p300-dependent transcription activity. Together, these results indicate that p300/CBP acts as a cotransactivator to Cart1 through a direct interaction and specific lysine acetylation. In addition, because 131st lysine is highly conserved in other types of homeoprotein, this lysine may be a common target for HAT of p300/CBP for these proteins.
The AML1-CBF beta transcription factor complex 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. We report here that 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. On the other hand, 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.
Interacting selectively and non-covalently with chromatin, the network of fibers of DNA, protein, and sometimes RNA, that make up the chromosomes of the eukaryotic nucleus during interphase.
Interacting selectively and non-covalently with a sequence of DNA that is in cis with and relatively close to the core promoter. The transcribed region might be described as a gene, cistron, or operon.
The SP1/Krüppel-like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. The discovery of KLF1 (EKLF), a key regulator of HBB (β-globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ-globin) regulation by KLF4. siRNA-mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ-CACCC and β-CACCC probes demonstrated KLF4 preferentially binds the endogenous γ-CACCC, while CREB binding protein (CREBBP) binding was not selective. Co-immunoprecipitation studies confirmed protein-protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co-localization of both factors in the γ-CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans-activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans-factors in HBG regulation.
The AML1-CBF beta transcription factor complex 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. We report here that 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. On the other hand, 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.
Interacting selectively and non-covalently with Myogenic Regulatory Factor (MRF), a member of the basic Helix-Loop-Helix (bHLH) superfamily of transcription factors.
J. Biol. Chem. 271, 9009-9013 (1996)[PubMed:8621548]
Human p300 protein is a cellular target of adenoviral E1A oncoprotein and a potential transcriptional coactivator. Both p300 and Rb family protein-binding regions of E1A are required for the repression of muscle gene expression, which is regulated by MyoD family transactivators. This implies that p300 is involved in MyoD-dependent transactivation. We show that the repression of MyoD-mediated E box (MyoD consensus) reporter activity by E1A is correlated with its interaction with p300, indicating that p300 participates in MyoD-dependent transactivation. In addition, p300 is able to interact both in vivo and in vitro with MyoD through a portion at the carboxyl-terminal cysteine/histidine-rich domain and associates with the components of the basal transcriptional complex through its two separate transactivation domains at the amino and carboxyl termini. Consistent with its role as a coactivator, p300 potentiates MyoD-activated transcription.
The adenovirus E1A and SV40 large-T-antigen oncoproteins bind to members of the p300/CBP transcriptional coactivator family. Binding of p300/CBP is implicated in the transforming mechanisms of E1A and T-antigen oncoproteins. A common region of the T antigen is critical for binding both p300/CBP and the tumour suppressor p53, suggesting a link between the functions of p53 and p300. Here we report that p300/CBP binds to p53 in the absence of viral oncoproteins, and that p300 and p53 colocalize within the nucleus and coexist in a stable DNA-binding complex. Consistent with its ability to bind to p300, E1A disrupted functions mediated by p53. It reduced p53-mediated activation of the p21 and bax promoters, and suppressed p53-induced cell-cycle arrest and apoptosis. We conclude that members of the p300/CBP family are transcriptional adaptors for p53, modulating its checkpoint function in the G1 phase of the cell cycle and its induction of apoptosis. Disruption of p300/p53-dependent growth control may be part of the mechanism by which E1A induces cell transformation. These results help to explain how p53 mediates growth and checkpoint control, and how members of the p300/CBP family affect progression from G1 to the S phase of the cell cycle.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
Genetic studies have demonstrated that the basic helix-loop-helix protein E2A is an essential transcription factor in B lymphocyte lineage commitment and differentiation. However, the mechanism underlying E2A-mediated transcription regulation is not fully understood. Here, we investigated the physical and genetic interactions between E2A and co-activators histone acetyltransferases (HATs) in B cells. Gel filtration analysis of human pre-B cell nuclear extract showed that E2A co-elutes with the HATs p300, CBP, and PCAF. A co-immunoprecipitation assay further demonstrated that a fraction of endogenous E2A proteins is associated with each of the three HATs. We show that these HATs acetylate E2A in vitro, enhance E2A-mediated transcription activity, and promote nuclear retention of E2A proteins. A catalytic mutation of p300 completely abrogates the ability of p300 to acetylate E2A and to promote E2A nuclear retention in 293T cells. A breeding test between E2A heterozygous mice and p300 heterozygous mice demonstrated that these two genes interact for proper B cell development. Collectively, these results suggest that E2A and HATs collaboratively regulate B cell development.
Evidence
2:
Inferred from Physical InteractionIntAct
Activating transcription factor (ATF) 3 plays a role in determining cell fate and generates a variety of alternatively spliced isoforms in stress response. We have reported previously that splice variant ATF3deltaZip2, which lacks the leucine zipper region, is induced in response to various stress stimuli. However, its biological function has not been elucidated. By using cells treated with tumor necrosis factor-alpha and actinomycin D or cells overexpressing ATF3deltaZip2, we showed that ATF3deltaZip2 sensitizes cells to apoptotic cell death in response to tumor necrosis factor-alpha, at least in part through suppressing nuclear factor (NF)-kappaB-dependent transcription of anti-apoptotic genes such as cIAP2 and XIAP. ATF3deltaZip2 interacts with a p65 (RelA)-cofactor complex containing CBP/p300 and HDAC1 at NF-kappaB sites of the proximal promoter region of the cIAP2 gene in vivo and down-regulates the recruitment of CBP/p300. Our study revealed that ATF3deltaZip2 counteracts anti-apoptotic activity of NF-kappaB, at least in part, by displacing positive cofactor CBP/p300 and provides insight into the mechanism by which ATF3 regulates cell fate through alternative splicing in stress response.
Evidence
3:
Inferred from Physical InteractionUniProtKB
Epithelium-specific ETS (ESE)-1 is a prototypic member of a novel subset of the ETS transcription factor family that is predominantly expressed in cells of epithelial origin but can also be induced in other cell types including vascular endothelial and smooth muscle cells in response to inflammatory stimuli. To further define the molecular mechanisms by which the transcriptional activity of ESE-1 is regulated, we have focused our attention on identifying proteins that interact with ESE-1. We have determined that Ku70, Ku86, p300, and CREB-binding protein (CBP) are ESE-1 interacting proteins. The Ku proteins have previously been shown to bind to breaks in DNA where they function to recruit additional proteins that promote DNA repair. Interestingly, Ku70 and Ku 86 negatively regulate the transcriptional activity of ESE-1. Using a series of deletion constructs, we have determined that the Ku proteins bind to the DNA-binding domain of ESE-1. The Ku proteins inhibit the ability of ESE-1 to bind to oligonucleotide probes in gel mobility shift assays. The finding that Ku proteins can interact with other transcription factors and block their function has not been previously demonstrated. In contrast, co-transfection of p300 and CBP with ESE-1 enhances the transcriptional activity of ESE-1. Moreover, the induction of ESE-1 in response to inflammatory cytokine interleukin-1 is associated with a parallel increase of the expression of p300 in vascular endothelial cells, suggesting that in the setting of inflammation, the transcriptional activity of ESE-1 is positively modulated by interaction with the transcriptional co-activator p300. In summary, our results demonstrated that the activity of ESE-1 is positively and negatively modulated by other interacting proteins including Ku70, Ku86, p300, and CBP.
Evidence
4:
Inferred from Physical InteractionUniProtKB
DNA damage response (DDR) acts as a tumorigenesis barrier, and any defects in the DDR machinery may lead to cancer. SOX4 expression is elevated in many types of tumors; however, its role in DDR is still largely unknown. Here, we show that SOX4, a new DNA damage sensor, is required for the activation of p53 tumor suppressor in response to DNA damage. Notably, SOX4 interacts with and stabilizes p53 protein by blocking Mdm2-mediated p53 ubiquitination and degradation. Furthermore, SOX4 enhances p53 acetylation by interacting with p300/CBP and facilitating p300/CBP/p53 complex formation. In concert with these results, SOX4 promotes cell cycle arrest and apoptosis, and it inhibits tumorigenesis in a p53-dependent manner. Therefore, these findings highlight SOX4 as a potential key factor in regulating DDR-associated cancer.
Evidence
5:
Inferred from Physical InteractionIntAct
We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptor-interacting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors.
Evidence
6:
Inferred from Physical InteractionBHF-UCL
HAND proteins are tissue-restricted members of the basic helix-loop-helix transcription factor family that play critical roles in cell differentiation and organogenesis including placental, cardiovascular, and craniofacial development. Nevertheless, the molecular basis underlying the developmental action of HAND proteins remains undefined. Within the embryo, HAND1 is first detected in the developing heart where it becomes restricted to the atrial and left ventricular compartments, a pattern identical to that of the Nppa gene, which encodes atrial natriuretic factor, the major secretory product of the heart. We hereby report that the cardiac atrial natriuretic factor promoter is directly activated by HAND1, making it the first known HAND1 transcriptional target. The action of HAND1 does not require heterodimerization with class I basic helix-loop-helix factors or DNA binding through E-box elements. Instead, HAND1 is recruited to the promoter via physical interaction with MEF2 proteins. MEF2/HAND1 interaction results in synergistic activation of MEF2-dependent promoters, and MEF2 binding sites are sufficient to mediate this synergy. MEF2 binding to DNA is not enhanced in the presence of HAND1. Instead, cooperativity likely results from corecruitment of co-activators such as CREB-binding protein. The related HAND2 protein can also synergize with MEF2. Thus, HAND proteins act as cell-specific developmental co-activators of the MEF2 family of transcription factors. These findings identify a novel mechanism for HAND action in the heart and provide a general paradigm to understand the mechanism of HAND action in organogenesis.
Evidence
7:
Inferred from Physical InteractionUniProtKB
J. Biol. Chem. 274, 22563-22568 (1999)[PubMed:10428834]
Targeted recruitment of histone acetyltransferase (HAT) activities by sequence-specific transcription factors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), has been proposed to lead to destabilization of nucleosomal cores by acetylation of core histones. However, biochemical evidence indicates that destabilization and depletion of linker H1 histones must also occur at the promoter regions of actively transcribing genes. Mechanisms by which nuclear receptors and other transcription factors affect the removal of histone H1 from transcriptionally silent chromatin have not been previously described. In this report, we show that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromatin. We further show that HMG-I and a novel related protein, HMG-R, also interact with other transcription factors. Using sense and antisense constructs of HMG-I/R in transient transfection assays with a retinoid responsive reporter, we also demonstrate that HMG-I/R is important for retinoid dependent transcriptional activity of RAR. These findings suggest a step wise mechanism by which RARs and other transcription factors can cause a targeted unfolding of compact chromatin as a first step in transcriptional activation, which would then be followed by recruitment of HAT activity and subsequent events.
Evidence
8:
Inferred from Physical InteractionIntAct
Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/beta-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.
Evidence
9:
Inferred from Physical InteractionUniProtKB
Malignant glioma is a consistently fatal brain cancer. The tumor invades the surrounding tissue, limiting complete surgical removal and thereby initiating recurrence. Identifying molecules critical for glioma invasion is essential to develop targeted, effective therapies. The expression of astrocyte elevated gene-1 (AEG-1) increases in malignant glioma and AEG-1 regulates in vitro invasion and migration of malignant glioma cells by activating the nuclear factor-kappaB (NF-kappaB) signaling pathway. The present studies elucidate the domains of AEG-1 important for mediating its function. Serial NH(2)-terminal and COOH-terminal deletion mutants were constructed and functional analysis revealed that the NH(2)-terminal 71 amino acids were essential for invasion, migration, and NF-kappaB-activating properties of AEG-1. The p65-interaction domain was identified between amino acids 101 to 205, indicating that p65 interaction alone is not sufficient to mediate AEG-1 function. Coimmunoprecipitation assays revealed that AEG-1 interacts with cyclic AMP-responsive element binding protein-binding protein (CBP), indicating that it might act as a bridging factor between NF-kappaB, CBP, and the basal transcription machinery. Chromatin immunoprecipitation assays showed that AEG-1 is associated with the NF-kappaB binding element in the interleukin-8 promoter. Thus, AEG-1 might function as a coactivator for NF-kappaB, consequently augmenting expression of genes necessary for invasion of glioma cells. In these contexts, AEG-1 represents a viable potential target for the therapy of malignant glioma.
Evidence
10:
Inferred from Physical InteractionUniProtKB
The lasting effects of neuronal activity on brain development involve calcium-dependent gene expression. Using a strategy called transactivator trap, we cloned a calcium-responsive transactivator called CREST (for calcium-responsive transactivator). CREST is a SYT-related nuclear protein that interacts with adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB)-binding protein (CBP) and is expressed in the developing brain. Mice that have a targeted disruption of the crest gene are viable but display defects in cortical and hippocampal dendrite development. Cortical neurons from crest mutant mice are compromised in calcium-dependent dendritic growth. Thus, calcium activation of CREST-mediated transcription helps regulate neuronal morphogenesis.
Evidence
11:
Inferred from Physical InteractionUniProtKB
Recruitment of p300/CBP by the hypoxia-inducible factor, HIF-1, is essential for the transcriptional response to hypoxia and requires an interaction between the p300/CBP CH1 region and HIF-1alpha. A new p300-CH1 interacting protein, p35srj, has been identified and cloned. p35srj is an alternatively spliced isoform of MRG1, a human protein of unknown function. Virtually all endogenous p35srj is bound to p300/CBP in vivo, and it inhibits HIF-1 transactivation by blocking the HIF-1alpha/p300 CH1 interaction. p35srj did not affect transactivation by transcription factors that bind p300/CBP outside the CH1 region. Endogenous p35srj is up-regulated markedly by the HIF-1 activators hypoxia or deferoxamine, suggesting that it could operate in a negative-feedback loop. In keeping with this notion, a p300 CH1 mutant domain, defective in HIF-1 but not p35srj binding, enhanced endogenous HIF-1 function. In hypoxic cells, p35srj may regulate HIF-1 transactivation by controlling access of HIF-1alpha to p300/CBP, and may keep a significant portion of p300/CBP available for interaction with other transcription factors by partially sequestering and functionally compartmentalizing cellular p300/CBP.
Evidence
12:
Inferred from Physical InteractionIntAct
In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.
Evidence
13:
Inferred from Physical InteractionUniProtKB
EVI1 is a very complex protein with two domains of zinc fingers and is inappropriately expressed in many types of human myeloid leukemias. Using reporter gene assays, several investigators showed that EVI1 is a transcription repressor, and recently it was shown that EVI1 interacts with the co-repressor carboxyl-terminal binding protein 1 (CtBP1). Earlier, we showed that the inappropriate expression of EVI1 in murine hematopoietic precursor cells leads to their abnormal differentiation and to increased proliferation. Using biochemical assays, we have identified two groups of transcription co-regulators that associate with EVI1 presumably to regulate gene expression. One group of co-regulators includes the CtBP1 and histone deacetylase. The second group includes the two co-activators cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF), both of which have histone acetyltransferase (HAT) activity. All of these proteins require separate regions of EVI1 for efficient interaction, and they divergently affect the ability of EVI1 to regulate gene transcription in reporter gene assays. Confocal microscopy analysis shows that in the majority of the cells, EVI1 is nuclear and diffused, whereas in about 10% of the cells EVI1 localizes in nuclear speckles. However, in the presence of the added exogenous co-repressors histone deacetylase or CtBP1, all of the nuclei have a diffuse EVI1 staining, and the proteins do not appear to reside together in obvious nuclear structures. In contrast, when CBP or P/CAF are added, defined speckled bodies appear in the nucleus. Analysis of the staining pattern indicates that EVI1 and CBP or EVI1 and P/CAF are contained within these structures. These nuclear structures are not observed when CBP is substituted with a point mutant HAT-inactive CBP with which EVI1 also physically interacts. Finally, we show that the interaction of EVI1 with either CBP or P/CAF leads to acetylation of EVI1. These results suggest that the assembly of EVI1 in nuclear speckles requires the intact HAT activity of the co-activators.
Evidence
14:
Inferred from Physical InteractionIntAct
Pax5 (BSAP) is essential for B cell development and acts both as a transcriptional activator and a repressor. Using a yeast two-hybrid assay to identify potential coregulators of Pax5, we identified Daxx, a protein that is highly conserved, ubiquitously expressed, and essential for embryonic mouse development. The interaction between Pax5 and Daxx involves the partial homeodomain of Pax5 and the C-terminal fragment of Daxx. A component of promyelocytic leukemia protein nuclear bodies, Daxx has been implicated in apoptosis and characterized as a transcriptional corepressor. Upon transient transfection assay of Daxx in B cells expressing endogenous Daxx and Pax5, we observed not only transcriptional corepression but also, unexpectedly, coactivation in M12.4.1 and A20 mouse B cell lines. Pax5 domains required for coactivation were identified using 293T cells. Coactivation apparently involves recruitment of the CREB binding protein (CBP), because we precipitated complexes containing Pax5, Daxx, and CBP in B cell lines. These data suggest that Daxx can affect Pax5's roles as an activator or repressor in B cells and describe a role for Daxx as a transcriptional coactivator.
Evidence
15:
Inferred from Physical InteractionUniProtKB
The adenoviral oncoprotein E1A induces progression through the cell cycle by binding to the products of the p300/CBP and retinoblastoma gene families. A new cellular p300/CBP-associated factor (P/CAF) having intrinsic histone acetylase activity has been identified that competes with E1A. Exogenous expression of P/CAF in HeLa cells inhibits cell-cycle progression and counteracts the mitogenic activity of E1A. E1A disturbs the normal cellular interaction between p300/CBP and its associated histone acetylase.
Evidence
16:
Inferred from Physical InteractionIntAct
Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.
Evidence
17:
Inferred from Physical InteractionIntAct
Longevity regulatory genes include the Forkhead transcription factor FOXO and the NAD-dependent histone deacetylase silent information regulator 2 (Sir2). Genetic studies demonstrate that Sir2 acts to extend lifespan in Caenorhabditis elegans upstream of DAF-16, a member of the FOXO family, in the insulin-like signaling pathway. However, the molecular mechanisms underlying the requirement of DAF-16 activity in Sir2-mediated longevity remain unknown. Here we show that reversible acetylation of Foxo1 (also known as FKHR), the mouse DAF-16 ortholog, modulates its transactivation function. cAMP-response element-binding protein (CREB)-binding protein binds and acetylates Foxo1 at the K242, K245, and K262 residues, the modification of which is involved in the attenuation of Foxo1 as a transcription factor. Conversely, Sir2 binds and deacetylates Foxo1 at residues acetylated by cAMP-response element-binding protein-binding protein. Sir2 is recruited to insulin response sequence-containing promoter and increases the expression of manganese superoxide dismutase and p27(kip1) in a deacetylase-activity-dependent manner. Our findings establish Foxo1 as a direct and functional target for Sir2 in mammalian systems.
Evidence
18:
Inferred from Physical InteractionIntAct
The tumour suppressor p53 is a transcriptional regulator whose ability to inhibit cell growth is dependent upon its transactivation function. Here we demonstrate that the transcription factor CBP, which is also implicated in cell proliferation and differentiation, acts as a p53 coactivator and potentiates its transcriptional activity. The amino-terminal activation domain of p53 interacts with the carboxy-terminal portion of the CBP protein both in vitro and in vivo. In transfected SaoS-2 cells, CBP potentiates activation of the mdm-2 gene by p53 and, reciprocally, p53 potentiates activation of a Gal4-responsive target gene by a Gal4(1-147)-CBP(1678-2441) fusion protein. A double point mutation that destroys the transactivation function of p53 also abolishes its binding to CBP and its synergistic function with CBP. The ability of p53 to interact physically and functionally with a coactivator (CBP) that has histone acetyltransferase activity and with components (TAFs) of the general transcription machinery indicates that it may have different functions in a multistep activation pathway.
Evidence
19:
Inferred from Physical InteractionUniProtKB
The proto-oncoprotein c-Myc functions as a transcriptional regulator that controls different aspects of cell behavior, including proliferation, differentiation, and apoptosis. In addition, Myc proteins have the potential to transform cells and are deregulated in the majority of human cancers. Several Myc-interacting factors have been described that mediate part of Myc's functions in the control of cell behavior. Here, we describe the isolation of a novel 150 kDa protein, designated PARP-10, that interacts with Myc. PARP-10 possesses domains with homology to RNA recognition motifs and to poly(ADP-ribose) polymerases (PARP). Molecular modeling and biochemical analysis define a PARP domain that is capable of ADP-ribosylating PARP-10 itself and core histones, but neither Myc nor Max. PARP-10 is localized to the nuclear and cytoplasmic compartments that is controlled at least in part by a Leu-rich nuclear export sequence (NES). Functionally, PARP-10 inhibits c-Myc- and E1A-mediated cotransformation of rat embryo fibroblasts, a function that is independent of PARP activity but that depends on a functional NES. Together, our findings define a novel PARP enzyme involved in the control of cell proliferation.
Evidence
20:
Inferred from Physical InteractionIntAct
Cytokine-activated receptors initiate intracellular signaling by recruiting protein kinases that phosphorylate the receptors on tyrosine residues, thus enabling docking of SH2 domain-bearing activating factors. Here we report that in response to type 1 interferon (IFNalpha), IFNalpha receptors recruit cytoplasmic CREB-binding protein (CBP). By binding to IFNalphaR2 within the region where two adjacent proline boxes bear phospho-Ser364 and phospho-Ser384, CBP acetylates IFNalphaR2 on Lys399, which in turn serves as the docking site for interferon regulatory factor 9 (IRF9). IRF9 interacts with the acetyl-Lys399 motif by means of its IRF homology2 (IH2) domain, leading to formation of the ISGF3 complex that includes IRF9, STAT1, and STAT2. All three components are acetylated by CBP. Remarkably, acetylation within the DNA-binding domain (DBD) of both IRF9 and STAT2 is critical for the ISGF3 complex activation and its associated antiviral gene regulation. These results have significant implications concerning the central role of acetylation in cytokine receptor signal transduction.
Evidence
21:
Inferred from Physical InteractionIntAct
To systematically investigate innate immune signaling networks regulating production of type I interferon, we analyzed protein complexes formed after microbial recognition. Fifty-eight baits were associated with 260 interacting proteins forming a human innate immunity interactome for type I interferon (HI5) of 401 unique interactions; 21% of interactions were modulated by RNA, DNA, or LPS. Overexpression and depletion analyses identified 22 unique genes that regulated NF-κB and ISRE reporter activity, viral replication, or virus-induced interferon production. Detailed mechanistic analysis defined a role for mind bomb (MIB) E3 ligases in K63-linked ubiquitination of TBK1, a kinase that phosphorylates IRF transcription factors controlling interferon production. Mib genes selectively controlled responses to cytosolic RNA. MIB deficiency reduced antiviral activity, establishing the role of MIB proteins as positive regulators of antiviral responses. The HI5 provides a dynamic physical and regulatory network that serves as a resource for mechanistic analysis of innate immune signaling.
Erratum in:
Immunity. 35(4), 647-8 (2011 Oct 28)
Evidence
22:
Inferred from Physical InteractionIntAct
Viral infection induces type I interferons (IFN-alpha and IFN-beta) that recruit unexposed cells in a self-amplifying response. We report that the transcription factor MafB thwarts auto-amplification by a metastable switch activity. MafB acted as a weak positive basal regulator of transcription at the IFNB1 promoter through activity at transcription factor AP-1-like sites. Interferon elicitors recruited the transcription factor IRF3 to the promoter, whereupon MafB acted as a transcriptional antagonist, impairing the interaction of coactivators with IRF3. Mathematical modeling supported the view that prepositioning of MafB on the promoter allows the system to respond rapidly to fluctuations in IRF3 activity. Higher expression of MafB in human pancreatic islet beta cells might increase cellular vulnerability to viral infections associated with the etiology of type 1 diabetes.
Evidence
23:
Inferred from Physical InteractionUniProtKB
J. Biol. Chem. 275, 8825-8834 (2000)[PubMed:10722728]
The MSG1 nuclear protein has a strong transcriptional activating activity but does not bind directly to DNA. When cotransfected, MSG1 enhances transcription mediated by the Smad transcription factors in mammalian cells in a manner dependent on ligand-induced Smad hetero-oligomerization. However, the mechanism of this MSG1 effect has been unknown. We now show that MSG1 directly binds to the p300/cAMP-response element-binding protein-binding protein (CBP) transcriptional coactivators, which in turn bind to the Smads, and enhances Smad-mediated transcription in a manner dependent on p300/CBP. The C-terminal transactivating domain of MSG1 is required for binding to p300/CBP and enhancement of Smad-mediated transcription; the viral VP16 transactivating domain could not substitute for it. In the N-terminal region of MSG1, we identified a domain that is necessary and sufficient to direct the specific interaction of MSG1 with Smads. We also found that the Hsc70 heat-shock cognate protein also forms complex with MSG1 in vivo, suppressing both binding of MSG1 to p300/CBP and enhancement of Smad-mediated transcription by MSG1. These results indicate that MSG1 interacts with both the DNA-binding Smad proteins and the p300/CBP coactivators through its N- and C-terminal regions, respectively, and enhances the functional link between Smads and p300/CBP.
Evidence
24:
Inferred from Physical InteractionIntAct
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. We have investigated potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) including aggregation, protein-protein interactions, and transcription. httex1p was found to coaggregate with p53 in inclusions generated in cell culture and to interact with p53 in vitro and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters, p21(WAF1/CIP1) and MDR-1. httex1p was also found to interact 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 which may result in neuronal dysfunction and cell death in HD.
Interacting selectively and non-covalently with an RNA polymerase II transcription activating factor, a protein involved in positive regulation of transcription.
RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity involved in negative regulation of transcriptiondefinition[GO:0001078]
Interacting selectively and non-covalently with a sequence of DNA that is in cis with and relatively close to a core promoter for RNA polymerase II (RNAP II) in order to stop, prevent, or reduce the frequency, rate or extent of transcription from an RNA polymerase II promoter.
The SP1/Krüppel-like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. The discovery of KLF1 (EKLF), a key regulator of HBB (β-globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ-globin) regulation by KLF4. siRNA-mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ-CACCC and β-CACCC probes demonstrated KLF4 preferentially binds the endogenous γ-CACCC, while CREB binding protein (CREBBP) binding was not selective. Co-immunoprecipitation studies confirmed protein-protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co-localization of both factors in the γ-CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans-activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans-factors in HBG regulation.
Interacting selectively and non-covalently with an RNA polymerase II (RNAP II) regulatory transcription factor and also with the RNAP II basal transcription machinery in order to increase the frequency, rate or extent of transcription. Cofactors generally do not bind DNA, but rather mediate protein-protein interactions between activating transcription factors and the basal RNAP II transcription machinery.
Interacting selectively and non-covalently with an RNA polymerase II transcription factor, any protein required to initiate or regulate transcription by RNA polymerase II.
Evidence
1:
Inferred from Physical InteractionBHF-UCL
The SP1/Krüppel-like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. The discovery of KLF1 (EKLF), a key regulator of HBB (β-globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ-globin) regulation by KLF4. siRNA-mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ-CACCC and β-CACCC probes demonstrated KLF4 preferentially binds the endogenous γ-CACCC, while CREB binding protein (CREBBP) binding was not selective. Co-immunoprecipitation studies confirmed protein-protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co-localization of both factors in the γ-CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans-activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans-factors in HBG regulation.
RNA polymerase II transcription factor binding transcription factor activity involved in negative regulation of transcriptiondefinition[GO:0001191]
Interacting selectively and non-covalently with an RNA polymerase II transcription factor, which may be a single protein or a complex, in order to stop, prevent, or reduce the frequency, rate or extent of transcription from an RNA polymerase II promoter. A protein binding transcription factor may or may not also interact with the template nucleic acid (either DNA or RNA) as well.
The SP1/Krüppel-like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. The discovery of KLF1 (EKLF), a key regulator of HBB (β-globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ-globin) regulation by KLF4. siRNA-mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ-CACCC and β-CACCC probes demonstrated KLF4 preferentially binds the endogenous γ-CACCC, while CREB binding protein (CREBBP) binding was not selective. Co-immunoprecipitation studies confirmed protein-protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co-localization of both factors in the γ-CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans-activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans-factors in HBG regulation.
Interacting selectively and non-covalently with a specific DNA sequence in order to modulate transcription. The transcription factor may or may not also interact selectively with a protein or macromolecular complex.
The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.
Conveys a signal across a cell to trigger a change in cell function or state. A signal is a physical entity or change in state that is used to transfer information in order to trigger a response.
A number of signalling pathways stimulate transcription of target genes through nuclear factors whose activities are primarily regulated by phosphorylation. Cyclic AMP regulates the expression of numerous genes, for example, through the protein kinase-A (PKA)-mediated phosphorylation of transcription factor CREB at Ser 133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA-binding affinity, CREB belongs to a class of proteins whose phosphorylation appears specifically to enhance their trans-activation potential. Recent work describing a phospho-CREB binding protein (CBP) which interacts specifically with the CREB trans-activation domain prompted us to examine whether CBP is necessary for cAMP regulated transcription. We report here that microinjection of an anti-CBP antiserum into fibroblasts can inhibit transcription from a cAMP responsive promoter. Surprisingly, CBP also cooperates with upstream activators such as c-Jun, which are involved in mitogen responsive transcription. We propose that CBP is recruited to the promoter through interaction with certain phosphorylated factors, and that CBP may thus play a critical role in the transmission of inductive signals from cell surface receptor to the transcriptional apparatus.
The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.
Interacting selectively and non-covalently with a activating transcription factor and also with the basal transcription machinery in order to increase the frequency, rate or extent of transcription. Cofactors generally do not bind DNA, but rather mediate protein-protein interactions between activating transcription factors and the basal transcription machinery.
Evidence
1:
Inferred from Physical InteractionUniProtKB
The adenoviral oncoprotein E1A induces progression through the cell cycle by binding to the products of the p300/CBP and retinoblastoma gene families. A new cellular p300/CBP-associated factor (P/CAF) having intrinsic histone acetylase activity has been identified that competes with E1A. Exogenous expression of P/CAF in HeLa cells inhibits cell-cycle progression and counteracts the mitogenic activity of E1A. E1A disturbs the normal cellular interaction between p300/CBP and its associated histone acetylase.
Transcription of mitochondrial serine:pyruvate aminotransferase (SPT) mRNA (SPTm-mRNA) in rat liver is unique in that it occurs from the upstream site of the two transcription start sites within the first exon of the SPT gene and is selectively enhanced by cAMP via the protein kinase A (PKA) signaling pathway. In this study, we identified the DNA elements and nuclear factors responsible for the basal and PKA-induced activities of the upstream promoter. By using a luciferase reporter assay with HepG2 cells, DNase I footprinting analysis, and gel shift experiments, we identified the binding sites for Sp1 and AP-2 within the regions -125 to -89 and -14 to +10, respectively. Mutational analyses indicated that these regions are essential for the transcription factor binding and the SPT promoter activity. Expression of AP-2 caused a marked increase in the basal promoter activity to about the same level as that achieved by PKA. On the other hand, both the basal and PKA-induced activities were elevated by overexpression of Sp1, its effect on PKA-induced activity being more pronounced with coexpression of CBP and repressed by E1A oncoprotein. These results suggest that AP-2 and Sp1 regulate basal promoter activity, and Sp1 is also involved in PKA-mediated expression of the rat SPT gene in concert with the transcriptional coactivator CBP.
The paired-like homeoprotein, Cart1, is involved in skeletal development. We describe here that the general coactivator p300/CBP controls the transcription activity of Cart1 through acetylation of a lysine residue that is highly conserved in other homeoproteins. Acetylation of this residue increases the interaction between p300/CBP and Cart1 and enhances its transcriptional activation. INTRODUCTION: Cart1 encodes a paired-like homeoprotein expressed selectively in chondrocyte lineage during embryonic development. Although its target gene remains unknown, gene disruption studies have revealed that Cart1 plays an important role for craniofacial bone formation as well as limb development by cooperating with another homeoprotein, Alx4. In this report, we study the functional involvement of p300/CBP, coactivators with intrinsic histone acetyltransferase (HAT) activity, in the transcriptional control of Cart1. METHODS: To study the transcription activity of Cart1, a reporter construct containing a putative Cart1 binding site was transiently transfected with the expression vectors of each protein. The interaction between p300/CBP and Cart1 was investigated by glutathione S-transferase (GST) pull-down, yeast two-hybrid, and immunoprecipitation assays. In vitro acetylation assay was performed with the recombinant p300-HAT domain and Cart1 in the presence of acetyl-CoA. RESULTS AND CONCLUSIONS: p300 and CBP stimulate Cart1-dependent transcription activity, and this transactivation is inhibited by E1A and Tax, oncoproteins that suppress the activity of p300/CBP. Cart1 binds to p300 in vivo and in vitro, and this requires the homeodomain of Cart 1 and N-terminal 139 amino acids of p300. Confocal microscopy analysis shows that Cart1 recruits overexpressed and endogenous p300 to a Cart1-specific subnuclear compartment. Cart1 is acetylated in vivo and sodium butyrate and trichostatin A, histone deacetylase inhibitors, markedly enhance the transcription activity of Cart1. Deletion and mutagenesis analysis identifies the 131st lysine that locates immediately adjacent to the homeodomain as a target of p300-HAT, and a point mutation to this residue attenuates the binding affinity to p300 as well as p300-dependent transcription activity. Together, these results indicate that p300/CBP acts as a cotransactivator to Cart1 through a direct interaction and specific lysine acetylation. In addition, because 131st lysine is highly conserved in other types of homeoprotein, this lysine may be a common target for HAT of p300/CBP for these proteins.
Genetic studies have demonstrated that the basic helix-loop-helix protein E2A is an essential transcription factor in B lymphocyte lineage commitment and differentiation. However, the mechanism underlying E2A-mediated transcription regulation is not fully understood. Here, we investigated the physical and genetic interactions between E2A and co-activators histone acetyltransferases (HATs) in B cells. Gel filtration analysis of human pre-B cell nuclear extract showed that E2A co-elutes with the HATs p300, CBP, and PCAF. A co-immunoprecipitation assay further demonstrated that a fraction of endogenous E2A proteins is associated with each of the three HATs. We show that these HATs acetylate E2A in vitro, enhance E2A-mediated transcription activity, and promote nuclear retention of E2A proteins. A catalytic mutation of p300 completely abrogates the ability of p300 to acetylate E2A and to promote E2A nuclear retention in 293T cells. A breeding test between E2A heterozygous mice and p300 heterozygous mice demonstrated that these two genes interact for proper B cell development. Collectively, these results suggest that E2A and HATs collaboratively regulate B cell development.
The transcriptional co-activators and histone acetyltransferases p300/CREB-binding protein (CBP) interact with CITED2, a transcription factor AP-2 (TFAP2) co-activator. p300/CBP, CITED2, and TFAP2A are essential for normal neural tube and cardiac development. Here we show that p300 and CBP co-activate TFAP2A in the presence of CITED2. TFAP2A transcriptional activity was modestly impaired in p300(+/-) and CBP(+/-) mouse embryonic fibroblasts; this was rescued by ectopic expression of p300/CBP. p300, TFAP2A, and endogenous CITED2 could be co-immunoprecipitated from transfected U2-OS cells indicating that they can interact physically in vivo. CITED2 interacted with the dimerization domain of TFAP2C, which is highly conserved in TFAP2A/B. In mammalian two-hybrid experiments, full-length p300 and TFAP2A interacted only when CITED2 was co-transfected. N-terminal residues of TFAP2A, containing the transactivation domain, are both necessary and sufficient for interaction with p300, and this interaction was independent of CITED2. Consistent with this, N-terminal residues of TFAP2A were required for p300- and CITED2-dependent co-activation. A histone acetyltransferase-deficient p300 mutant (D1399Y) did not co-activate TFAP2A and did not affect the expression or cellular localization of TFAP2A or CITED2. In mammalian two-hybrid experiments p300D1399Y failed to interact with TFAP2A, explaining, at least in part, its failure to function as a co-activator. Our results suggest a model wherein interactions among TFAP2A, CITED2, and p300/CBP are necessary for TFAP2A-mediated transcriptional activation and for normal neural tube and cardiac development.
Mutations in the HNF1beta gene, encoding the dimeric POU-homeodomain transcription factor HNF1beta (TCF2 or vHNF1), cause various phenotypes including maturity onset diabetes of the young 5 (MODY5), and abnormalities in kidney, pancreas and genital tract development. To gain insight into the molecular mechanisms underlying these phenotypes and into the structure of HNF1beta, we functionally characterized eight disease-causing mutations predicted to produce protein truncations, amino acids substitutions or frameshift deletions in different domains of the protein. Truncated mutations, retaining the dimerization domain, displayed defective nuclear localization and weak dominant-negative activity when co-expressed with the wild-type protein. A frameshift mutation located within the C-terminal QSP-rich domain partially reduced transcriptional activity, whereas selective deletion of this domain abolished transactivation. All five missense mutations, which concern POU-specific and homeodomain residues, were correctly expressed and localized to the nucleus. Although having different effects on DNA-binding capacity, which ranged from complete loss to a mild reduction, these mutations exhibited a severe reduction in their transactivation capacity. The transcriptional impairment of those mutants, whose DNA-binding activity was weakly or not affected, correlated with the loss of association with one of the histone-acetyltransferases CBP or PCAF. In contrast to wild-type HNF1beta, whose transactivation potential depends on the synergistic action of CBP and PCAF, the activity of these mutants was not increased by the synergistic action of these two coactivators or by treatment with the specific histone-deacetylase inhibitor TSA. Our findings suggest that the complex syndrome associated with HNF1beta-MODY5 mutations arise from either defective DNA-binding or transactivation function through impaired coactivator recruitment.
The process, occurring in the embryo, by which the anatomical structures of the digit are generated and organized. A digit is one of the terminal divisions of an appendage, such as a finger or toe.
The AML1-CBF beta transcription factor complex 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. We report here that 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. On the other hand, 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.
Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.
Genetic studies have demonstrated that the basic helix-loop-helix protein E2A is an essential transcription factor in B lymphocyte lineage commitment and differentiation. However, the mechanism underlying E2A-mediated transcription regulation is not fully understood. Here, we investigated the physical and genetic interactions between E2A and co-activators histone acetyltransferases (HATs) in B cells. Gel filtration analysis of human pre-B cell nuclear extract showed that E2A co-elutes with the HATs p300, CBP, and PCAF. A co-immunoprecipitation assay further demonstrated that a fraction of endogenous E2A proteins is associated with each of the three HATs. We show that these HATs acetylate E2A in vitro, enhance E2A-mediated transcription activity, and promote nuclear retention of E2A proteins. A catalytic mutation of p300 completely abrogates the ability of p300 to acetylate E2A and to promote E2A nuclear retention in 293T cells. A breeding test between E2A heterozygous mice and p300 heterozygous mice demonstrated that these two genes interact for proper B cell development. Collectively, these results suggest that E2A and HATs collaboratively regulate B cell development.
The SP1/Krüppel-like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. The discovery of KLF1 (EKLF), a key regulator of HBB (β-globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ-globin) regulation by KLF4. siRNA-mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ-CACCC and β-CACCC probes demonstrated KLF4 preferentially binds the endogenous γ-CACCC, while CREB binding protein (CREBBP) binding was not selective. Co-immunoprecipitation studies confirmed protein-protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co-localization of both factors in the γ-CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans-activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans-factors in HBG regulation.
The AML1-CBF beta transcription factor complex 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. We report here that 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. On the other hand, 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.
The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.
Transcription of mitochondrial serine:pyruvate aminotransferase (SPT) mRNA (SPTm-mRNA) in rat liver is unique in that it occurs from the upstream site of the two transcription start sites within the first exon of the SPT gene and is selectively enhanced by cAMP via the protein kinase A (PKA) signaling pathway. In this study, we identified the DNA elements and nuclear factors responsible for the basal and PKA-induced activities of the upstream promoter. By using a luciferase reporter assay with HepG2 cells, DNase I footprinting analysis, and gel shift experiments, we identified the binding sites for Sp1 and AP-2 within the regions -125 to -89 and -14 to +10, respectively. Mutational analyses indicated that these regions are essential for the transcription factor binding and the SPT promoter activity. Expression of AP-2 caused a marked increase in the basal promoter activity to about the same level as that achieved by PKA. On the other hand, both the basal and PKA-induced activities were elevated by overexpression of Sp1, its effect on PKA-induced activity being more pronounced with coexpression of CBP and repressed by E1A oncoprotein. These results suggest that AP-2 and Sp1 regulate basal promoter activity, and Sp1 is also involved in PKA-mediated expression of the rat SPT gene in concert with the transcriptional coactivator CBP.
Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating lowered oxygen tension. Hypoxia, defined as a decline in O2 levels below normoxic levels of 20.8 - 20.95%, results in metabolic adaptation at both the cellular and organismal level.
Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.
Viral protein involved in a direct and specific interaction with a host macromolecule. Viruses interact with many cellular pathways to achieve their replication cycle. Entry into the host cell, transport to the viral replication sites or viral budding are all steps that require interaction between the host and the virus. Additionally, the evasion from the host immune response requires a lot of viral proteins to associate with and inhibit cellular proteins with antiviral functions.
Protein involved in the transfer of genetic information from DNA to messenger RNA (mRNA) by DNA-directed RNA polymerase. In the case of some RNA viruses, protein involved in the transfer of genetic information from RNA to messenger RNA (mRNA) by RNA-directed RNA polymerase.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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