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1
Stallcup, M. R., D. Chen, S. S. Koh, H. Ma, Y. H. Lee, H. Li, B. T. Schurter, and D. W. Aswad. "Co-operation between protein-acetylating and protein-methylating co-activators in transcriptional activation." Biochemical Society Transactions 28, no. 4 (August 1, 2000): 415–18. http://dx.doi.org/10.1042/bst0280415.
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Nuclear hormone receptors (NRs) activate transcription by binding to specific enhancer elements associated with target genes. Transcriptional activation is accomplished with the help of complexes of co-activator proteins that bind to NRs. p160 co-activators, a family of three related 160 kDa proteins, serve as primary co-activators by binding directly to NRs and recruiting additional secondary co-activators. Some of these (CBP/p300 and p/CAF) can acetylate histones and other proteins in the transcription complex, thus helping to modify chromatin structure and form an active transcription initiation complex. We recently discovered co-activator-associated arginine methyltransferase 1 (CARM1), which binds to p160 co-activators and thereby enhances transcriptional activation by NRs on transiently transfected reporter genes. CARM1 also methylates specific arginine residues in the N-terminal tail of histone H3 in vitro. A related arginine-specific protein methyltransferase, PRMT1, also binds p160 co-activators and enhances NR function. PRMT1 methylates histone H4 in vitro. The enhancement of NR function by CARM1, PRMT1 and p300 depends on their interactions with p160 co-activators. In the presence of p160 co-activators, some pairs of these three secondary co-activators provide a highly synergistic enhancement of NR function on transiently transfected reporter genes. We have also observed an enhancement of NR function on stably integrated reporter genes by these co-activators. We propose that the synergy of co-activator function between p300, CARM1 and PRMT1 is due to their different but complementary protein modification activities.
2
Chan, Ho Man, and Nicholas B. La Thangue. "p300/CBP proteins: HATs for transcriptional bridges and scaffolds." Journal of Cell Science 114, no. 13 (July 1, 2001): 2363–73. http://dx.doi.org/10.1242/jcs.114.13.2363.
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p300/CBP transcriptional co-activator proteins play a central role in co-ordinating and integrating multiple signal-dependent events with the transcription apparatus, allowing the appropriate level of gene activity to occur in response to diverse physiological cues that influence, for example, proliferation, differentiation and apoptosis. p300/CBP activity can be under aberrant control in human disease, particularly in cancer, which may inactivate a p300/CBP tumour-suppressor-like activity. The transcription regulating-properties of p300 and CBP appear to be exerted through multiple mechanisms. They act as protein bridges, thereby connecting different sequence-specific transcription factors to the transcription apparatus. Providing a protein scaffold upon which to build a multicomponent transcriptional regulatory complex is likely to be an important feature of p300/CBP control. Another key property is the presence of histone acetyltransferase (HAT) activity, which endows p300/CBP with the capacity to influence chromatin activity by modulating nucleosomal histones. Other proteins, including the p53 tumour suppressor, are targets for acetylation by p300/CBP. With the current intense level of research activity, p300/CBP will continue to be in the limelight and, we can be confident, yield new and important information on fundamental processes involved in transcriptional control.
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Manning, E. Tory, Tsuyoshi Ikehara, Takashi Ito, James T. Kadonaga, and W. Lee Kraus. "p300 Forms a Stable, Template-Committed Complex with Chromatin: Role for the Bromodomain." Molecular and Cellular Biology 21, no. 12 (June 15, 2001): 3876–87. http://dx.doi.org/10.1128/mcb.21.12.3876-3887.2001.
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ABSTRACT The nature of the interaction of coactivator proteins with transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this study, we used a biochemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in vitro transcription template competition assays, we observed that p300 forms a stable, template-committed complex with chromatin during the transcription process. The template commitment is dependent on the time of incubation of p300 with the chromatin template and occurs independently of the presence of a transcriptional activator protein. In studies examining interactions between p300 and chromatin, we found that p300 binds directly to chromatin and that the binding requires the p300 bromodomain, a conserved 110-amino-acid sequence found in many chromatin-associated proteins. Furthermore, we observed that the isolated p300 bromodomain binds directly to histones, preferentially to histone H3. However, the isolated p300 bromodomain does not bind to nucleosomal histones under the same assay conditions, suggesting that free histones and nucleosomal histones are not equivalent as binding substrates. Collectively, our results suggest that the stable association of p300 with chromatin is mediated, at least in part, by the bromodomain and is critically important for p300 function. Furthermore, our results suggest a model for p300 function that involves distinct activator-dependent targeting and activator-independent chromatin binding activities.
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MATT, Theresia, Maria A. MARTINEZ-YAMOUT, H. Jane DYSON, and Peter E. WRIGHT. "The CBP/p300 TAZ1 domain in its native state is not a binding partner of MDM2." Biochemical Journal 381, no. 3 (July 27, 2004): 685–91. http://dx.doi.org/10.1042/bj20040564.
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The transcriptional co-activator CBP [CREB (cAMP-response-element-binding protein)-binding protein] and its paralogue p300 play a key role in the regulation of both activity and stability of the tumour suppressor p53. Degradation of p53 is mediated by the ubiquitin ligase MDM2 (mouse double minute protein) and is also reported to be regulated by CBP/p300. Direct protein–protein interaction between a central domain of MDM2 and the TAZ1 (transcriptional adaptor zinc-binding domain) [C/H1 (cysteine/histidine-rich region 1)] domain of p300 and subsequent formation of a ternary complex including p53 have been reported previously. We expressed and purified the proposed binding domains of HDM2 (human homologue of MDM2) and CBP, and examined their interactions using CD spectroscopy. The binding studies were extended by using natively purified GST (glutathione S-transferase)–p300 TAZ1 and GST–p53 fusion proteins, together with in vitro translated HDM2 fragments, under similar solution conditions to those in previous studies, but omitting added EDTA, which causes unfolding and aggregation of the zinc-binding TAZ1 domain. Comparing the binding properties of the known TAZ1 interaction partners HIF-1α (hypoxia-inducible factor 1), CITED2 (CBP/p300-interacting transactivator with glutamic- and aspartic-rich tail) and STAT2 (signal transducer and activator of transcription 2) with HDM2, our data suggest that TAZ1 in its native state does not serve as a specific recognition domain of HDM2. Rather, unfolded TAZ1 and HDM2 proteins have a high tendency to aggregate, and non-specific protein complexes are formed under certain conditions.
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Li, Chia-Wei, Gia Khanh Dinh, Aihua Zhang, and J. Don Chen. "Ankyrin repeats-containing cofactors interact with ADA3 and modulate its co-activator function." Biochemical Journal 413, no. 2 (June 26, 2008): 349–57. http://dx.doi.org/10.1042/bj20071484.
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ANCO (ankyrin repeats-containing cofactor)-1 and ANCO-2 are a family of unique transcriptional co-regulators with dual properties: they interact with both the co-activators and the co-repressors [Zhang, Yeung, Li, Tsai, Dinh, Wu, Li and Chen (2004) J. Biol. Chem. 279, 33799–33805]. Specifically, ANCO-1 is thought to recruit HDACs (histone deacetylases) to the p160 co-activator to repress transcriptional activation by nuclear receptors. In the present study, we provide new evidence to suggest further that ANCO-1 and ANCO-2 also interact with the co-activator ADA3 (alteration/deficiency in activation 3). The interaction occurs between the conserved C-terminal domain of ANCO-1 and the N-terminal transactivation domain of ADA3. Several subunits of the P/CAF {p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein]-associated factor} complex, including ADA3, ADA2α/β and P/CAF, showed co-localization with ANCO-1 nuclear dots, indicating an in vivo association of ANCO-1 with the P/CAF complex. Furthermore, a transient reporter assay revealed that both ANCO-1 and ANCO-2 repress ADA3-mediated transcriptional co-activation on nuclear receptors, whereas ANCO-1 stimulated p53-mediated transactivation. These data suggest that ADA3 is a newly identified target of the ANCO proteins, which may modulate co-activator function in a transcription-factor-specific manner.
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Guidez, Fabien, Louise Howell, Mark Isalan, Marek Cebrat, Rhoda M. Alani, Sarah Ivins, Sarah Pierce, Philip A. Cole, Jonathan D. Licht, and Arthur Zelent. "Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein." Blood 104, no. 11 (November 16, 2004): 359. http://dx.doi.org/10.1182/blood.v104.11.359.359.
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Abstract The Promyelocytic Leukemia Zinc Finger (PLZF) gene was identified in a rare case of acute promyelocytic leukemia (APL) with translocation t(11;17)(q23;q21) and resistance to therapy with all-trans-retinoic acid. Recent studies have indicated a critical role of PLZF in maintenance of spermatogonial stem cells. Prominent expression of PLZF in hematopoietic stem cells, suggest that it may also play a similar role in this compartment. The wild type PLZF protein is a DNA sequence-specific transcription repressor containing nine Krüppel-like C2-H2 zinc fingers and an N-terminal BTB/POZ repression domain. Transcriptional repression by PLZF is mediated through recruitment of the nuclear receptor co-repressor (N-CoR/SMRT)/histone deacetylase (HDAC) complexes to its target genes, such as c-MYC and HOX genes. We now show that transcriptional repression by PLZF is surprisingly also dependent on the histone acetyl transferase (HAT) activity of the p300 protein. PLZF associates with p300 in vivo and its ability to repress transcription is specifically dependent on acetylation of PLZF on lysines in its C-terminal C2-H2 zinc-finger motifs. Acetylation of PLZF enhances its ability to bind its cognate DNA binding site in vitro as determined by EMSA and in vivo as measured by chromatin immunoprecipitation. An acetylation site mutant of PLZF fails to repress transcription despite retaining its abilities to interact with co-repressor/HDAC complexes, due to inefficient DNA binding. Inhibitors of p300 abolish transcriptional repression by PLZF and mutants of PLZF that mimic acetylation were insensitive to these inhibitory effects. Acetylation of PLZF by p300 was specific since over-expression of another HAT, p/CAF or the selective inhibition of p/CAF had no effect on PLZF activity despite the ability of the proteins to associate with each other. Taken together, our results indicate that a histone deacetylase dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a co-activator protein in transcriptional repression.
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Kim, Hee Eun, Eunju Bae, Deok-yoon Jeong, Min-Ji Kim, Won-Ji Jin, Sahng-Wook Park, Gil-Soo Han, George M. Carman, Eunjin Koh, and Kyung-Sup Kim. "Lipin1 regulates PPARγ transcriptional activity." Biochemical Journal 453, no. 1 (June 13, 2013): 49–60. http://dx.doi.org/10.1042/bj20121598.
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PPARγ (peroxisome-proliferator-activated receptor γ) is a master transcription factor involved in adipogenesis through regulating adipocyte-specific gene expression. Recently, lipin1 was found to act as a key factor for adipocyte maturation and maintenance by modulating the C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ network; however, the precise mechanism by which lipin1 affects the transcriptional activity of PPARγ is largely unknown. The results of the present study show that lipin1 activates PPARγ by releasing co-repressors, NCoR1 (nuclear receptor co-repressor 1) and SMRT (silencing mediator of retinoid and thyroid hormone receptor), from PPARγ in the absence of the ligand rosiglitazone. We also identified a novel lipin1 TAD (transcriptional activation domain), between residues 217 and 399, which is critical for the activation of PPARγ, but not PPARα. Furthermore, this TAD is unique to lipin1 since this region does not show any homology with the other lipin isoforms, lipin2 and lipin3. The activity of the lipin1 TAD is enhanced by p300 and SRC-1 (steroid receptor co-activator 1), but not by PCAF (p300/CBP-associated factor) and PGC-1α (PPAR co-activator 1α). The physical interaction between lipin1 and PPARγ occurs at the lipin1 C-terminal region from residues 825 to 926, and the VXXLL motif at residue 885 is critical for binding with and the activation of PPARγ. The action of lipin1 as a co-activator of PPARγ enhanced adipocyte differentiation; the TAD and VXXLL motif played critical roles, but the catalytic activity of lipin1 was not directly involved. Collectively, these data suggest that lipin1 functions as a key regulator of PPARγ activity through its ability to release co-repressors and recruit co-activators via a mechanism other than PPARα activation.
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Jethanandani, Poonam, and Randall H. Kramer. "α7 Integrin Expression Is Negatively Regulated by δEF1 during Skeletal Myogenesis." Journal of Biological Chemistry 280, no. 43 (August 28, 2005): 36037–46. http://dx.doi.org/10.1074/jbc.m508698200.
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α7 integrin levels increase dramatically as myoblasts differentiate to myotubes. A negative regulatory element with putative sites for δEF1 is present in the α7 proximal promoter region. To define the role of δEF1 in regulating α7 integrin expression, we overexpressed δEF1 in C2C12 myoblasts. This resulted in a major down-regulation of α7 protein expression. Promoter assays revealed that C2C12 myoblasts transfected with δEF1 showed a decrease in activity of the 2.8-kb α7 promoter fragment, indicating regulation of α7 integrin at the transcriptional level. We have identified two E-box-like sites for δEF1 in the negative regulatory region. Mutation of these sites enhanced α7 promoter activity, indicating that these sites function in repression. MYOD, an activator of α7 integrin transcription, can compete with δEF1 for binding at these sites in gel shift assay. By using chromatin immunoprecipitation, we demonstrated a reciprocal binding of δEF1 and MYOD to this regulatory element depending on the stage of differentiation: δEF1 is preferentially bound in myoblasts to this region, whereas MYOD is bound in myotubes. The N-terminal region of δEF1 is necessary for α7 repression, and this region also binds the co-activator p300/CBP. Importantly, we found that the p300/CBP co-activator can overcome repression by δEF1, suggesting that δEF1 can titrate limiting amounts of this co-activator. These findings suggest that δEF1 has a role in suppressing integrin expression in myoblasts by displacing MYOD and competing for p300/CBP co-activator.
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SILVERMAN, Eric S., Jing DU, Amy J. WILLIAMS, Raj WADGAONKAR, Jeffrey M. DRAZEN, and Tucker COLLINS. "cAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1)." Biochemical Journal 336, no. 1 (November 15, 1998): 183–89. http://dx.doi.org/10.1042/bj3360183.
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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.
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Hyndman, Brandy D., Richard Bayly, and David P. LeBrun. "Acetylation of a Conserved Lysine Residue within Activation Domain One of E2A Transcription Factors Plays a Role in Transcriptional Regulation." Blood 108, no. 11 (November 1, 2006): 2218. http://dx.doi.org/10.1182/blood.v108.11.2218.2218.
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Abstract The E2A locus encodes transcription factors, called E12 and E47, involved in lineage-specific cellular differentiation. The locus is also involved in chromosomal translocations associated with acute lymphoblastic leukemia, the most common of which results in expression of the oncoprotein E2A-PBX1. We showed recently that direct interaction between E2A and the histone acetyl-transferase (AT) and transcriptional co-activator proteins CBP/p300 is required in leukemogenesis by E2A-PBX1. E2A proteins have also been shown to interact with another AT/co-activator, p/CAF. Interaction with these AT proteins results in acetylation of E2A proteins themselves. Here we map the acetylated lysine residues within the oncogenic portion of E2A proteins and begin to elucidate some functional correlates of E2A acetylation. Our results indicate that the isolated AT domain of p/CAF as well as full-length p/CAF were capable of acetylating E2A. Interestingly, full-length p300 was capable of acetylating E2A while an isolated AT domain was unable to acetylate E2A, suggesting that additional domains of CBP/p300 are required to mediate E2A acetylation. We demonstrate that both p300 and p/CAF can interact directly with E2A, independent of the known interaction between p300 and p/CAF. These co-activators do, however, appear to co-operate to achieve maximal E2A acetylation. Mutagenesis-based mapping studies indicate that several lysines are substrates for acetylation. Of particular interest, a conserved lysine residue (K34) located within the N-terminal transcriptional activation domain (AD1) is acetylated in vitro by p/CAF. K34 is located within a GKXXP consensus sequence, suggested to be a recognition motif for acetylation by several AT enzymes including CBP/p300 and p/CAF. Conservative replacement of K34 with arginine (i.e., K43R) substantially impairs transcriptional activation of a luciferase reporter gene by E2A, suggesting that post-translational modification of this residue may play an important functional role. Consistent with a role for acetylation, relative to some other lysine-dependent modification, we were unable to demonstrate sumoylation or ubiquitination of the N-terminus of E2A. Therefore, we have found that acetylation by AT/co-activator proteins contributes to transcriptional regulation by the functionally critical N-terminal activation domain (AD1) of E2A proteins. The mechanisms by which this acetylation event is regulated and how it contributes to target gene induction by E2A are not clear. It seems plausible that the acetylation status of AD1 could be determined by upstream signaling events and acetylation of E2A could modulate interactions with transcriptional co-regulators, DNA or chromatin. Further studies to investigate these possibilities are underway. In particular, results using an amino acid substitution that mimics acetylation of AD1 (K34Q) will be presented.
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Qiu, Yi, Min Guo, Suming Huang, and Roland Stein. "Insulin Gene Transcription Is Mediated by Interactions between the p300 Coactivator and PDX-1, BETA2, and E47." Molecular and Cellular Biology 22, no. 2 (January 15, 2002): 412–20. http://dx.doi.org/10.1128/mcb.22.2.412-420.2002.
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ABSTRACT Pancreatic β-cell-type-specific expression of the insulin gene requires both ubiquitous and cell-enriched activators, which are organized within the enhancer region into a network of protein-protein and protein-DNA interactions to promote transcriptional synergy. Protein-protein-mediated communication between DNA-bound activators and the RNA polymerase II transcriptional machinery is inhibited by the adenovirus E1A protein as a result of E1A’s binding to the p300 coactivator. E1A disrupts signaling between the non-DNA-binding p300 protein and the basic helix-loop-helix DNA-binding factors of insulin’s E-element activator (i.e., the islet-enriched BETA2 and generally distributed E47 proteins), as well as a distinct but unidentified enhancer factor. In the present report, we show that E1A binding to p300 prevents activation by insulin’s β-cell-enriched PDX-1 activator. p300 interacts directly with the N-terminal region of the PDX-1 homeodomain protein, which contains conserved amino acid sequences essential for activation. The unique combination of PDX-1, BETA2, E47, and p300 was shown to promote synergistic activation from a transfected insulin enhancer-driven reporter construct in non-β cells, a process inhibited by E1A. In addition, E1A inhibited the level of PDX-1 and BETA2 complex formation in β cells. These results indicate that E1A inhibits insulin gene transcription by preventing communication between the p300 coactivator and key DNA-bound activators, like PDX-1 and BETA2:E47.
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FANIELLO, Maria C., Giuseppa CHIRICO, Barbara QUARESIMA, Giovanni CUDA, Giovanna ALLEVATO, Maria A. BEVILACQUA, Francesco BAUDI, et al. "An alternative model of H ferritin promoter transactivation by c-Jun." Biochemical Journal 363, no. 1 (March 22, 2002): 53–58. http://dx.doi.org/10.1042/bj3630053.
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c-Jun is a member of the activator protein 1 family, and its interaction with different nuclear factors generates a wide spectrum of complexes that regulate transcription of different promoters. H ferritin promoter transcription is tightly dependent on nuclear factor Y (NFY). Ferritin transcription is activated by c-Jun, although the promoter does not contain a canonical binding site. NFY, on the other hand, does not bind c-Jun in vitro, whereas in vivo c-Jun is found in the complex containing NFY. Moreover, a c-Jun—GCN4 chimaeric construct containing only the transactivation domain of Jun and the basic-region leucine-zipper domain of GCN4 stimulates the H ferritin promoter. A synthetic GAL4 promoter and the cognate activator, the fusion protein NFY—GAL4, are potently activated by c-Jun. Titration of p300 by co-expressing E1A abolishes the stimulatory effect. Moreover, another p300-dependent promoter, the cAMP-response element, can be superactivated by c-Jun using the same mechanism. These data indicate that c-Jun, when activated or overexpressed, is recruited to the H ferritin promoter by p300, which links NFY, bound to DNA, to the complex. These results add a new level of complexity to transcriptional regulation by c-Jun, which can activate p300-dependent promoters without binding directly to the target DNA.
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Zimmermann, Holger, Choon-Heng Koh, Roland Degenkolbe, Mark J. O’Connor, Andreas Müller, Gertrud Steger, Jason J. Chen, Yun Lui, Elliot Androphy, and Hans-Ulrich Bernard. "Interaction with CBP/p300 enables the bovine papillomavirus type 1 E6 oncoprotein to downregulate CBP/p300-mediated transactivation by p53." Journal of General Virology 81, no. 11 (November 1, 2000): 2617–23. http://dx.doi.org/10.1099/0022-1317-81-11-2617.
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The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) can transform cells independently of p53 degradation. The precise mechanisms underlying this transformation are not yet completely understood. Here it is shown that BPV-1 E6 interacts with CBP/p300 in the same way as described for the E6 proteins of oncogenic human papillomaviruses. This interaction results in an inhibition of the transcriptional coactivator function of CBP/p300 required by p53 and probably by other transcription factors. The comparison of the CBP/p300-binding properties of BPV-1 E6 mutants previously characterized in transcription and transformation studies suggests (i) that the E6–CBP/p300 interaction may be necessary, but not sufficient, for cell transformation, and (ii) that the transcriptional activator function, inherent to the E6 protein, is not derived from forming a complex with CBP/p300.
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Robek, Michael D., and Lee Ratner. "Immortalization of T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Is Independent of the Tax-CBP/p300 Interaction." Journal of Virology 74, no. 24 (December 15, 2000): 11988–92. http://dx.doi.org/10.1128/jvi.74.24.11988-11992.2000.
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ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein is a 40-kDa nuclear phosphoprotein which functions in the viral replication cycle as a transcriptional trans-activator of the viral long terminal repeat. Tax interacts with a variety of different transcription factors, including the CREB binding protein (CBP)/p300 family of transcriptional accessory proteins. We demonstrate that a Tax mutant defective for the CBP/p300 interaction retains the capacity to immortalize primary human T lymphocytes when it is expressed from a functional molecular clone of HTLV-1. Thus, immortalization of HTLV-1-infected cells appears to be independent of Tax-induced alterations in CBP/p300 function.
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Cvijic, Helena, Kay Bauer, Dennis Löffler, Gabriele Pfeifer, Conny Blumert, Antje K. Kretzschmar, Christian Henze, Katja Brocke-Heidrich, and Friedemann Horn. "Co-activator SRC-1 is dispensable for transcriptional control by STAT3." Biochemical Journal 420, no. 1 (April 28, 2009): 123–32. http://dx.doi.org/10.1042/bj20081989.
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SRC (steroid receptor co-activator)-1 has been reported to interact with and to be an essential co-activator for several members of the STAT (signal transducer and activator of transcription) family, including STAT3, the major signal transducer of IL (interleukin)-6. We addressed the question of whether SRC-1 is crucial for IL-6- and STAT3-mediated physiological responses such as myeloma cell survival and acute-phase protein induction. In fact, silencing of SRC-1 by RNA interference rapidly induced apoptosis in IL-6-dependent INA-6 human myeloma cells, comparable with what was observed upon silencing of STAT3. Using chromatin immunoprecipitation at STAT3 target regions of various genes, however, we observed constitutive binding of SRC-1 that decreased when INA-6 cells were treated with IL-6. The same held true for STAT3 target genes analysed in HepG2 human hepatocellular carcinoma cells. SRC-1-knockdown studies demonstrated that STAT3-controlled promoters require neither SRC-1 nor the other p160 family members SRC-2 or SRC-3 in HepG2 cells. Furthermore, microarray expression profiling demonstrated that the responsiveness of IL-6 target genes is not affected by SRC-1 silencing. In contrast, co-activators of the CBP [CREB (cAMP-response element-binding protein)-binding protein]/p300 family proved functionally important for the transactivation potential of STAT3 and bound inducibly to STAT3 target regions. This recruitment did not depend on the presence of SRC-1. Altogether, this suggests that functional impairment of STAT3 is not involved in the induction of myeloma cell apoptosis by SRC-1 silencing. We therefore conclude that STAT3 transactivates its target genes by the recruitment of CBP/p300 co-activators and that this process generally does not require the contribution of SRC-1.
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Qiu, Yi, Arun Sharma, and Roland Stein. "p300 Mediates Transcriptional Stimulation by the Basic Helix-Loop-Helix Activators of the Insulin Gene." Molecular and Cellular Biology 18, no. 5 (May 1, 1998): 2957–64. http://dx.doi.org/10.1128/mcb.18.5.2957.
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ABSTRACT Pancreatic β-cell-type-specific and glucose-inducible transcription of the insulin gene is mediated by the basic helix-loop-helix factors that bind to and activate expression from an E-box element within its enhancer. The E-box activator is a heteromeric complex composed of a β-cell-enriched factor, BETA2/NeuroD, and ubiquitously distributed proteins encoded by the E2A and HEB genes. Previously, we demonstrated that the adenovirus type 5 E1A proteins repressed stimulation by the E-box activator in β cells. In this study, our objective was to determine how E1A repressed activator function. The results indicate that E1A reduces activation by binding to and sequestering the p300 cellular coactivator protein. Thus, we show that expression of p300 in β cells can relieve inhibition by E1A, as well as potentiate activation by the endogenous insulin E-box transcription factors. p300 stimulated activation from GAL4 (amino acids 1 to 147) fusion constructs of either BETA2/NeuroD or the E2A-encoded E47 protein. The sequences spanning the activation domains of BETA2/NeuroD (amino acids 156 to 355) and E47 (amino acids 1 to 99 and 325 to 432) were required for this response. The same region of BETA2/NeuroD was shown to be important for binding to p300 in vitro. The sequences of p300 involved in E47 and BETA2/NeuroD association resided between amino acids 1 and 1257 and 1945 and 2377, respectively. A mutation in p300 that abolished binding to BETA2/NeuroD also destroyed the ability of p300 to activate insulin E-box-directed transcription in β cells. Our results indicate that physical and functional interactions between p300 and the E-box activator factors play an important role in insulin gene transcription.
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Debes, Jose D., Thomas J. Sebo, Hannelore V. Heemers, Benjamin R. Kipp, De Anna L. Haugen, Christine M. Lohse, and Donald J. Tindall. "p300 Modulates Nuclear Morphology in Prostate Cancer." Cancer Research 65, no. 3 (February 1, 2005): 708–12. http://dx.doi.org/10.1158/0008-5472.708.65.3.
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Abstract Alterations in nuclear structure distinguish cancer cells from noncancer cells. These nuclear alterations can be translated into quantifiable features by digital image analysis in a process known as quantitative nuclear morphometry. Recently, quantitative nuclear morphometry has been shown to predict metastasis and biochemical recurrence of prostate cancer. However, little is known about the cellular mechanisms underlying these nuclear morphometric changes. Alterations of nuclear matrix proteins are frequently involved in changes of nuclear structure. A number of co-activators interact with these nuclear structure–related proteins, suggesting that they might be involved in quantitative nuclear morphometry changes. We have shown previously that the transcriptional co-activator p300 is involved in prostate cancer progression. However, the ability of a transcriptional regulator like p300 to modulate nuclear morphology has not been described previously. In the present study, we show that p300 expression in prostate cancer biopsy tissue from 95 patients correlates with quantifiable nuclear alterations. Moreover, we show that transfection of p300 into prostate cancer cells in culture induces quantifiable nuclear alterations, such as diameter, perimeter, and absorbance among others, as assessed by digital image analysis. These alterations correlate individually with aggressive features in prostate cancer, such as expression of the proliferation marker Ki-67 and extraprostatic extension of the tumor. Finally, we found that transfection of p300 into prostate cancer cells specifically increases mRNA and protein levels of nuclear matrix peptides lamins A and C, suggesting that these proteins mediate the p300-induced effects. These findings reveal a new insight into the transcriptional and structural regulation of prostate cancer.
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Schmidt, Thomas J., and Karl-Heinz Klempnauer. "Natural Products with Antitumor Potential Targeting the MYB-C/EBPβ-p300 Transcription Module." Molecules 27, no. 7 (March 23, 2022): 2077. http://dx.doi.org/10.3390/molecules27072077.
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The transcription factor MYB is expressed predominantly in hematopoietic progenitor cells, where it plays an essential role in the development of most lineages of the hematopoietic system. In the myeloid lineage, MYB is known to cooperate with members of the CCAAT box/enhancer binding protein (C/EBP) family of transcription factors. MYB and C/EBPs interact with the co-activator p300 or its paralog CREB-binding protein (CBP), to form a transcriptional module involved in myeloid-specific gene expression. Recent work has demonstrated that MYB is involved in the development of human leukemia, especially in acute T-cell leukemia (T-ALL) and acute myeloid leukemia (AML). Chemical entities that inhibit the transcriptional activity of the MYB-C/EBPβ-p300 transcription module may therefore be of use as potential anti-tumour drugs. In searching for small molecule inhibitors, studies from our group over the last 10 years have identified natural products belonging to different structural classes, including various sesquiterpene lactones, a steroid lactone, quinone methide triterpenes and naphthoquinones that interfere with the activity of this transcriptional module in different ways. This review gives a comprehensive overview on the various classes of inhibitors and the inhibitory mechanisms by which they affect the MYB-C/EBPβ-p300 transcriptional module as a potential anti-tumor target. We also focus on the current knowledge on structure-activity relationships underlying these biological effects and on the potential of these compounds for further development.
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Bernat, Agnieszka, Paola Massimi, and Lawrence Banks. "Complementation of a p300/CBP defective-binding mutant of adenovirus E1a by human papillomavirus E6 proteins." Journal of General Virology 83, no. 4 (April 1, 2002): 829–33. http://dx.doi.org/10.1099/0022-1317-83-4-829.
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Previous studies have shown that the human papillomavirus type 16 (HPV-16) E6 protein binds to p300/CBP and abrogates its transcriptional co-activator function. However, there is little information on the biological consequences of this interaction and discrepancy as to whether the interaction is high-risk E6 specific or not. We performed a series of studies to compare the interactions of HPV-18 and HPV-11 E6 with p300, and showed that both high- and low- risk E6 proteins bind p300. In addition, using a transformation-deficient mutant of adenovirus E1a, which cannot interact with p300, we demonstrated that HPV-16, HPV-18 and, to a lesser extent, HPV-11 E6, can complement this mutant in cell transformation assays. In contrast, a mutant of HPV-16 E6 which does not bind p300 failed to rescue the E1a mutant. These results suggest that the E6–p300 interaction may be important for the ability of HPV E6 to contribute towards cell transformation.
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SCHMID, Tobias, Jie ZHOU, Roman KÖHL, and Bernhard BRÜNE. "p300 relieves p53-evoked transcriptional repression of hypoxia-inducible factor-1 (HIF-1)." Biochemical Journal 380, no. 1 (May 15, 2004): 289–95. http://dx.doi.org/10.1042/bj20031299.
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HIF-1 (hypoxia-inducible factor-1), a heterodimeric transcription factor comprising HIF-1α and HIF-1β subunits, serves as a key regulator of metabolic adaptation to hypoxia. HIF-1 activity largely increases during hypoxia by attenuating pVHL (von Hippel–Lindau protein)-dependent ubiquitination and subsequent 26 S-proteasomal degradation of HIF-1α. Besides HIF-1, the transcription factor and tumour suppressor p53 accumulates and is activated under conditions of prolonged/severe hypoxia. Recently, the interaction between p53 and HIF-1α was reported to evoke HIF-1α degradation. Destruction of HIF-1α by p53 was corroborated in the present study by using pVHL-deficient RCC4 (renal carcinoma) cells, supporting the notion of a pVHL-independent degradation process. In addition, low p53 expression repressed HIF-1 transactivation without affecting HIF-1α protein amount. Establishing that p53-evoked inhibition of HIF-1 reporter activity was relieved upon co-transfection of p300 suggested competition between p53 and HIF-1 for limiting amounts of the shared co-activator p300. This assumption was confirmed by showing competitive binding of in vitro transcription/translation-generated p53 and HIF-1α to the CH1 domain of p300 in vitro. We conclude that low p53 expression attenuates HIF-1 transactivation by competing for p300, whereas high p53 expression destroys the HIF-1α protein and thereby eliminates HIF-1 reporter activity. Thus once p53 becomes activated under conditions of severe hypoxia/anoxia, it contributes to terminating HIF-1 responses.
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Felzien, Lisa K., Susan Farrell, Jonathan C. Betts, Rashid Mosavin, and Gary J. Nabel. "Specificity of Cyclin E-Cdk2, TFIIB, and E1A Interactions with a Common Domain of the p300 Coactivator." Molecular and Cellular Biology 19, no. 6 (June 1, 1999): 4241–46. http://dx.doi.org/10.1128/mcb.19.6.4241.
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ABSTRACT The p300 and CREB binding protein (CBP) transcriptional coactivators interact with a variety of transcription factors and regulate their activity. Among the interactions that have been described, the COOH-terminal region of p300 binds to cyclin E-cyclin-dependent kinase 2 (cyclin E-Cdk2) and TFIIB, as well as to the E1A gene products of adenovirus. Inhibition of Cdk activity by Cdk inhibitors, such as p21 or p27, potentiates NF-κB activity and provides a mechanism to coordinate cell cycle progression with the transcription of genes expressed during growth arrest. In this report, we analyze the specific domains of p300 required for the binding of p300 to cyclin E-Cdk2, TFIIB, and E1A and the ability of these proteins to interact with p300, alone or in combination. 12S E1A, an inhibitor of p300-dependent transcription, reduces the binding of TFIIB, but not that of cyclin E-Cdk2, to p300. In contrast, 13S E1A, a pleiotropic transcriptional activator, does not inhibit TFIIB binding to p300, although it enhances the interaction of cyclin E-Cdk2 with p300. Modification of cyclin E-Cdk2 is most likely required for association with p300 since the interaction is observed only with cyclin E-Cdk2 purified from mammalian cells. Domain swap studies show that the cyclin homology domain of TFIIB is involved in interactions with p300, although the homologous region from cyclin E does not mediate this interaction. These findings suggest that p300 or CBP function is regulated by interactions of various proteins with a common coactivator domain.
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Nanan, Kyster, and David P. LeBrun. "Identification and Characterization By ChIP-Seq Of Genomic Sites Bound By E2A-PBX1 In Acute Lymphoblastic Leukemia Demonstrates Associations With p300 Recruitment, Transcriptionally Active Chromatin and Abundant Transcription." Blood 122, no. 21 (November 15, 2013): 2501. http://dx.doi.org/10.1182/blood.v122.21.2501.2501.
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Abstract E2A-PBX1 (EP1) is a chimeric oncogenic transcription factor expressed consequent to the 1;19 chromosomal translocation in cases of acute lymphoblastic leukemia (ALL). EP1 can induce transcription of reporter genes and EP1-driven oncogenesis requires direct binding of EP1 with the transcriptional co-activator and histone acetyltransferase p300. Therefore, we hypothesized that EP1 recruits p300 and other co-activators to cis-acting regulatory elements throughout the genome thereby inducing or maintaining transcription of target genes some of which contribute to the neoplastic phenotype. Here we have used chromatin immunoprecipitation followed by next generation DNA sequencing (ChIP-seq) to identify and characterize EP1-bound sites across the genome of the t(1;19)-associated, ALL-derived cell line RCH-ACV. ChIP was performed with an anti-FLAG antibody using sheared chromatin prepared from RCH-ACV cells that stably expressed FLAG-tagged EP1; ChIP from parent RCH-ACV cells not expressing FLAG-EP1 served as a negative control for peak calling. Parallel immunoprecipitations were performed with antibodies for p300 and the chromatin marks H3K4me3, H3K4me1 and H3K27me3. Sequencing of DNA purified from the immunoprecipitated material and of total RNA (RNA-seq) was carried out commercially by BGI whereas bioinformatic analyses were performed in-house. Bioinformatic analysis of data from replicate samples identified 3166 EP1 binding peaks across the RCH-ACV genome (irreproducible discovery rate threshold <0.01). Most EP1 binding sites were located in intronic (1408 sites) or intergenic (1346 sites) regions. Binding site consensus analysis showed overrepresentation of binding motifs for REST, CTCF, MYC, PAX5 and other transcription factors suggesting indirect recruitment of EP1 to DNA mediated by protein-protein interactions. EP1-bound regions were enriched for p300 binding (Figure 1), consistent with the documented importance of p300 recruitment in EP1 oncogenesis. A particular association with H3K4me3 relative to H3K4me1 or H3K27me3 (Figure 2) suggested association with active promoters. Three hundred and forty-two genes had EP1 binding sites within 1000 bp of their transcriptional start sites and these genes were associated with differentially abundant transcription (Figure 3, P<0.001). Querying the online Mammalian Phenotype Ontology tool with genes associated with EP1 binding generated terms that were obviously rich in phenotypes pertaining to B-lymphopoiesis. In summary, our results suggest that EP1 recruits p300 and other co-activators to transcriptionally active chromatin in ALL cells. Results from studies currently underway to confirm the dependency of target gene expression and p300 recruitment upon binding of EP1 at specific binding sites will be presented. Disclosures: No relevant conflicts of interest to declare.
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TRAN, Nhu Q., and David L. CROWE. "Regulation of the human involucrin gene promoter by co-activator proteins." Biochemical Journal 381, no. 1 (June 22, 2004): 267–73. http://dx.doi.org/10.1042/bj20031653.
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Stratified squamous epithelial cells undergo an orderly process of terminal differentiation that is characterized by specific molecular and morphological changes, including expression of the cornified envelope protein involucrin. Significant progress has been made in characterizing the upstream regulatory region of the involucrin gene. Binding sites for AP-1 (activator protein 1) and Sp1 transcription factors were shown to be important for involucrin promoter activity and tissue-specific expression. Defective terminal differentiation is often characterized by decreased or lack of involucrin expression. Recently, a dominant-negative construct of the transcriptional co-activator P/CAF [p300/CBP-associated factor, where CBP stands for CREB (cAMP-response-element-binding protein)-binding protein] was shown to inhibit involucrin expression in immortalized keratinocytes [Kawabata, Kawahara, Kanekura, Araya, Daitoku, Hata, Miura, Fukamizu, Kanzaki, Maruyama and Nakajima (2002) J. Biol. Chem. 277, 8099–8105]. Loss of expression or inactivation of other co-activators has also been demonstrated [Suganuma, Kawabata, Ohshima, and Ikeda (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 13073–13078]. In the present study, we re-expressed CBP and P/CAF in immortalized keratinocyte lines that had lost expression of these co-activator proteins. Re-expression of these proteins restored calcium- and RA (retinoic acid)-responsive involucrin expression in these cells. RA and calcium signalling induced exchange of CBP and P/CAF occupancy at the AP-1 sites of the involucrin promoter. CBP and P/CAF inductions of the involucrin expression were not dependent on MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), p38, protein kinase C or CaM kinase (calcium/calmodulin-dependent kinase) signalling. Kinase-induced changes in involucrin promoter activity directly resulted from changes in AP-1 protein expression. We concluded that CBP and P/CAF are important regulators of involucrin expression in stratified squamous epithelial cells.
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Campbell, K. J., N. R. Chapman, and N. D. Perkins. "UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation." Biochemical Society Transactions 29, no. 6 (November 1, 2001): 688–91. http://dx.doi.org/10.1042/bst0290688.
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The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.
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Felzien, L. K., C. Woffendin, M. O. Hottiger, R. A. Subbramanian, E. A. Cohen, and G. J. Nabel. "HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator." Proceedings of the National Academy of Sciences 95, no. 9 (April 28, 1998): 5281–86. http://dx.doi.org/10.1073/pnas.95.9.5281.
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Lundblad, James R., Roland P. S. Kwok, Megan E. Laurance, Marian L. Harter, and Richard H. Goodman. "Adenoviral ElA-associated protein p300 as a functional homologue of the transcriptional co-activator CBP." Nature 374, no. 6517 (March 1995): 85–88. http://dx.doi.org/10.1038/374085a0.
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Rehtanz, Manuela, Hanns-Martin Schmidt, Ursula Warthorst, and Gertrud Steger. "Direct Interaction between Nucleosome Assembly Protein 1 and the Papillomavirus E2 Proteins Involved in Activation of Transcription." Molecular and Cellular Biology 24, no. 5 (March 1, 2004): 2153–68. http://dx.doi.org/10.1128/mcb.24.5.2153-2168.2004.
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ABSTRACT Using a yeast two-hybrid screen, we identified human nucleosome assembly protein 1 (hNAP-1) as a protein interacting with the activation domain of the transcriptional activator encoded by papillomaviruses (PVs), the E2 protein. We show that the interaction between E2 and hNAP-1 is direct and not merely mediated by the transcriptional coactivator p300, which is bound by both proteins. Coexpression of hNAP-1 strongly enhances activation by E2, indicating a functional interaction as well. E2 binds to at least two separate domains within hNAP-1, one within the C terminus and an internal domain. The binding of E2 to hNAP-1 is necessary for cooperativity between the factors. Moreover, the N-terminal 91 amino acids are crucial for the transcriptional activity of hNAP-1, since deletion mutants lacking this N-terminal portion fail to cooperate with E2. We provide evidence that hNAP-1, E2, and p300 can form a ternary complex efficient in the activation of transcription. We also show that p53 directly interacts with hNAP-1, indicating that transcriptional activators in addition to PV E2 interact with hNAP-1. These results suggest that the binding of sequence-specific DNA binding proteins to hNAP-1 may be an important step contributing to the activation of transcription.
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Kaida, Atsushi, Yasuo Ariumi, Keiko Baba, Masami Matsubae, Toshifumi Takao, and Kunitada Shimotohno. "Identification of a novel p300-specific-associating protein, PRS1 (phosphoribosylpyrophosphate synthetase subunit 1)." Biochemical Journal 391, no. 2 (October 10, 2005): 239–47. http://dx.doi.org/10.1042/bj20041308.
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CBP [CREB (cAMP-response-element-binding protein)-binding protein] and p300 play critical roles in transcriptional co-activation, cell differentiation, proliferation and apoptosis. Multiple transcription factors associate with CBP/p300. With the exception of the SYT oncoprotein, no proteins have been identified that specifically associate with p300, but not CBP. In the present study, we isolated a novel p300-associated protein for which no interaction with CBP was observed by GST (glutathione S-transferase) pull-down assay using Jurkat cell lysates metabolically labelled with [35S]methionine. This protein bound the KIX (kinase-inducible) domain of p300. Following resolution by two-dimensional acrylamide gel electrophoresis, we identified the KIX-domain-bound protein by MS analysis as PRS1 (phosphoribosylpyrophosphate synthetase subunit 1), a protein essential for nucleoside biosynthesis. This is the first report to demonstrate the existence of a p300 KIX-domain-specific-interacting protein that does not interact with CBP. Thus p300 may play a role in the regulation of DNA synthesis through interactions with PRS1.
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Saint Just Ribeiro, Mariana, Magnus L. Hansson, and Annika E. Wallberg. "A proline repeat domain in the Notch co-activator MAML1 is important for the p300-mediated acetylation of MAML1." Biochemical Journal 404, no. 2 (May 14, 2007): 289–98. http://dx.doi.org/10.1042/bj20061900.
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Ligand activation of Notch leads to the release of Notch IC (the intracellular receptor domain), which translocates to the nucleus and interacts with the DNA-binding protein CSL to control expression of specific target genes. In addition to ligand-mediated activation, Notch signalling can be further modulated by interactions of Notch IC with a number of other proteins. MAML1 has previously been shown to act co-operatively with the histone acetyltransferase p300 in Notch IC-mediated transcription. In the present study we show that the N-terminal domain of MAML1 directly interacts with both p300 and histones, and the p300–MAML1 complex specifically acetylates histone H3 and H4 tails in chromatin. Furthermore, p300 acetylates MAML1 and evolutionarily conserved lysine residues in the MAML1 N-terminus are direct substrates for p300-mediated acetylation. The N-terminal domain of MAML1 contains a proline repeat motif (PXPAAPAP) that was previously shown to be present in p53 and important for the p300–p53 interaction. We show that the MAML1 proline repeat motif interacts with p300 and enhances the activity of the MAML1 N-terminus in vivo. These findings suggest that the N-terminal domain of MAML1 plays an important role in Notch-regulated transcription, by direct interactions with Notch, p300 and histones.
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CHEN, Feifei, Kenji OGAWA, Xubao LIU, Teresa M. STRINGFIELD, and Yan CHEN. "Repression of Smad2 and Smad3 transactivating activity by association with a novel splice variant of CCAAT-binding factor C subunit." Biochemical Journal 364, no. 2 (June 1, 2002): 571–77. http://dx.doi.org/10.1042/bj20011703.
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Activation by transforming growth factor-β (TGF-β)/activin receptors leads to phosphorylation of Smad2 (Sma- and Mad-related protein 2) and Smad3, which function as transcription factors to regulate gene expression. Using the MH2 domain (Mad homologue domain of Smad proteins 2) of Smad3 in a yeast two-hybrid screening, we isolated a novel splice variant of CAATT-binding factor subunit C (CBF-C), designated CBF-Cb, that associated with Smad3. CBF-C is one of the subunits that form a heterotrimeric CBF complex capable of binding and activating the CAATT motif found in the promoters of many eukaryotic genes. CBF-Cb is 62 amino acids shorter than the wild-type CBF-C in the N-terminal region. In addition, CBF-Cb is expressed ubiquitously in various mouse tissues. By an immunoprecipitation assay, we detected an in vivo association of CBF-Cb with Smad2 and Smad3, independent of signalling by activated TGF-β type I receptors. In transient transfection experiments, overexpression of CBF-Cb was able to repress the transactivating activity of Smad2 and Smad3, mediated either by direct binding to the Smad-responsive element or through their association with the Smad-interacting transcription factor FAST-2 (forkhead activin signal transducer-2). The Smad-mediated transcriptional response after TGF-β receptor activation was also inhibited by overexpression of unspliced CBF-C. In addition, the repressive activity of CBF-Cb on Smad2- and Smad3-mediated transcriptional regulation was abrogated by co-expression of the general transcription activator p300. The association of CBF-Cb with Smad2 was competitively inhibited by overexpression of p300. These data indicate a novel mechanism for modulation of the transcriptional activity of Smad proteins, whereby the interaction of CBF-Cb, as well as canonical CBF-C, with the MH2 domain of Smads may prevent the association of Smads with transcriptional co-activators.
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Su, Mack, Laurie A. Steiner, Hannah Bogardus, Vincent P. Schulz, Ross C. Hardison, and Patrick G. Gallagher. "Identification of Biologicaly Relevant Enhancers in Human Erythroid Cells." Blood 120, no. 21 (November 16, 2012): 368. http://dx.doi.org/10.1182/blood.v120.21.368.368.
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Abstract Abstract 368 Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Recent studies utilizing genomic methodologies have shown that enhancers are frequently associated with biologically relevant and disease-associated genetic variants. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulate gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, in primary human erythroid cells. These data were combined with parallel gene expression analyses and candidate enhancers identified. Cell and tissue-type specific enhancers act over distances of tens to hundreds of kilobases, thus bona fide erythroid enhancers are expected to be enriched in the genomic vicinity of genes expressed and functional in erythroid cells. Sites of occupancy were correlated with levels of gene expression. Promoter sites within 2kb of annotated transcriptional start sites (TSS) were excluded. Consistent with their predicted function, there was significant enrichment of p300 peaks within 2–50kb of the TSS of genes highly expressed in erythroid cells c.f. peaks >100kb of a TSS. There was also significant enrichment of combinations of 2, 3, and 4 co-localizing erythroid transcription factor peaks within 2–50kb of the TSS of genes highly expressed in erythroid cells. In contrast, similar to other cell type-specific enhancers, there was no enrichment of p300 or erythroid transcription factor sites within 2–50kb of genes highly expressed in nonerythroid cells. When analyses were performed comparing a set of erythroid-specific genes vs. a random set of genes, there was significant enrichment of combinations of 2, 3, and 4 co-localizing erythroid transcription factor binding sites, but not p300, within 2–50kb of the TSS of erythroid-specific genes. Evolutionary analyses revealed high conservation between man and chimp for p300 and erythroid transcription factors. However, there was a very large falloff between human and mouse, with. Disclosures: No relevant conflicts of interest to declare.
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Resendes, Karen K., and Alan G. Rosmarin. "GABP (GA Binding Protein) Is Required for Formation of a Retinoic Acid-Induced Myeloid Enhanceosome on the CD18 Promoter." Blood 106, no. 11 (November 16, 2005): 1731. http://dx.doi.org/10.1182/blood.v106.11.1731.1731.
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Abstract CD18, the β chain of the leukocyte integrins, mediates cell-cell and cell-matrix interactions of white blood cells and is required for normal innate immunity. Retinoic acid (RA) induces maturation of myeloid cells; mutations in retinoic acid receptors (RARs) account for acute promyelocytic leukemia. CD18 is transcriptionally regulated by RA in myeloid cells. The proximal promoter of CD18 is transcriptionally regulated by RA but, strikingly, this region is not bound or activated by retinoic acid receptors. Rather, the RA-responsiveness of the CD18 proximal promoter is dependent on ets sites that are bound by the ets transcription factor, GA-binding protein (GABP). The transcriptional co-activator, p300, further increases the RA-responsiveness of CD18. We show that p300 and GABPα, the ets DNA binding subunit of GABP, physically interact in myeloid cells. This interaction uses the cysteine/histidine regions of p300, and the pointed domain (PNT) of GABPα. This demonstrates p300 as the first known interaction partner of the GABPα PNT domain. We prepared a GABPα PNT domain construct for expression in mammalian cells and showed that this PNT construct inhibited the GABPα:p300 interaction and dramatically decreased the RA-responsiveness of the CD18 proximal promoter. We used Chromatin immunoprecipitation (ChIP) to demonstrate that RA induces formation of an enhanceosome on the CD18 promoter; thus, GABPα and p300 on the CD18 proximal promoter recruit RAR/RXR bound to a distal enhancer in the presence of RA. We prepared a dominant negative p300 construct and showed that its expression blocks the formation of the CD18 enhanceosome. These results demonstrate that GABP is required for formation of an enhanceosome that mediates responsiveness of CD18 to RA in myeloid cells. Dominant negative forms of GABPα and p300 interfere with RA responsiveness in myeloid cells and disrupt this enhanceosome. The CD18 enhanceosome is the first known RA-induced enhanceosome and demonstrates its role in mediating retinoid responsiveness in myeloid cells.
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Dubuissez, Marion, Ingrid Loison, Sonia Paget, Han Vorng, Saliha Ait-Yahia, Olivier Rohr, Anne Tsicopoulos, and Dominique Leprince. "Protein Kinase C-Mediated Phosphorylation of BCL11B at Serine 2 Negatively Regulates Its Interaction with NuRD Complexes during CD4+T-Cell Activation." Molecular and Cellular Biology 36, no. 13 (May 9, 2016): 1881–98. http://dx.doi.org/10.1128/mcb.00062-16.
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The transcription factor BCL11B/CTIP2 is a major regulatory protein implicated in various aspects of development, function and survival of T cells. Mitogen-activated protein kinase (MAPK)-mediated phosphorylation and SUMOylation modulate BCL11B transcriptional activity, switching it from a repressor in naive murine thymocytes to a transcriptional activator in activated thymocytes. Here, we show that BCL11B interacts via its conserved N-terminal MSRRKQ motif with endogenous MTA1 and MTA3 proteins to recruit various NuRD complexes. Furthermore, we demonstrate that protein kinase C (PKC)-mediated phosphorylation of BCL11B Ser2 does not significantly impact BCL11B SUMOylation but negatively regulates NuRD recruitment by dampening the interaction with MTA1 or MTA3 (MTA1/3) and RbAp46 proteins. We detected increased phosphorylation of BCL11B Ser2 uponin vivoactivation of transformed and primary human CD4+T cells. We show that following activation of CD4+T cells, BCL11B still binds toIL-2andId2promoters but activates their transcription by recruiting P300 instead of MTA1. Prolonged stimulation results in the direct transcriptional repression ofBCL11Bby KLF4. Our results unveil Ser2 phosphorylation as a new BCL11B posttranslational modification linking PKC signaling pathway to T-cell receptor (TCR) activation and define a simple model for the functional switch of BCL11B from a transcriptional repressor to an activator during TCR activation of human CD4+T cells.
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Geiger, Timothy R., Neelam Sharma, Young-Mi Kim, and Jennifer K. Nyborg. "The Human T-Cell Leukemia Virus Type 1 Tax Protein Confers CBP/p300 Recruitment and Transcriptional Activation Properties to Phosphorylated CREB." Molecular and Cellular Biology 28, no. 4 (December 10, 2007): 1383–92. http://dx.doi.org/10.1128/mcb.01657-07.
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ABSTRACT The human T-cell leukemia virus-encoded oncoprotein Tax is a potent activator of viral transcription. Tax function is strictly dependent upon the cellular transcription factor CREB, and together they bind cAMP response elements within the viral promoter and mediate high-level viral transcription. Signal-dependent CREB phosphorylation at Ser133 (pCREB) correlates with the activation of transcription. This activation has been attributed to recruitment of the coactivators CBP/p300 via physical interaction with the KIX domain. Here we show that the promoter-bound Tax/pCREB complex strongly recruits the recombinant, purified full-length coactivators CBP and p300. Additionally, the promoter-bound Tax/pCREB (but not Tax/CREB) complex recruits native p300 and potently activates transcription from chromatin templates. Unexpectedly, pCREB alone failed to detectably recruit the full-length coactivators, despite strong binding to KIX. These observations are in marked contrast to those in published studies that have characterized the physical interaction between KIX and pCREB and extrapolated these results to the full-length proteins. Consistent with our observation that pCREB is deficient for binding of CBP/p300, pCREB alone failed to support transcriptional activation. These data reveal that phosphorylation of CREB is not sufficient for CBP/p300 recruitment and transcriptional activation. The regulation of transcription by pCREB is therefore more complex than is generally recognized, and coregulators, such as Tax, likely play a critical role in the modulation of pCREB function.
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Kim, Jung Ha, Kabsun Kim, Bang Ung Youn, Hye Mi Jin, Ji-Young Kim, Jang Bae Moon, Aeran Ko, Sang-Beom Seo, Kwang-Youl Lee, and Nacksung Kim. "RANKL induces NFATc1 acetylation and stability via histone acetyltransferases during osteoclast differentiation." Biochemical Journal 436, no. 2 (May 13, 2011): 253–62. http://dx.doi.org/10.1042/bj20110062.
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NFATc1 (nuclear factor of activated T-cells c1), a key transcription factor, plays a role in regulating expression of osteoclast-specific downstream target genes such as TRAP (tartrate-resistant acid phosphatase) and OSCAR (osteoclast-associated receptor). It has been shown that RANKL [receptor activator of NF-κB (nuclear factor κB) ligand] induces NFATc1 expression during osteoclastogenesis at a transcriptional level. In the present study, we demonstrate that RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via HATs (histone acetyltransferases), such as p300 and PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor], thereby stabilizing NFATc1 proteins. PCAF physically interacts with NFATc1 and directly induces NFATc1 acetylation and stability, subsequently increasing the transcriptional activity of NFATc1. In addition, RANKL-mediated NFATc1 acetylation is increased by the HDAC (histone deacetylase) inhibitors sodium butyrate and scriptaid. Overexpression of HDAC5 reduces RANKL- or PCAF-mediated NFATc1 acetylation, stability and transactivation activity, suggesting that the balance between HAT and HDAC activities might play a role in the regulation of NFATc1 levels. Furthermore, RANKL and p300 induce PCAF acetylation and stability, thereby enhancing the transcriptional activity of NFATc1. Down-regulation of PCAF by siRNA (small interfering RNA) decreases NFATc1 acetylation and stability, as well as RANKL-induced osteoclastogenesis. Taken together, the results of the present study demonstrate that RANKL induces HAT-mediated NFATc1 acetylation and stability, and subsequently increases the transcriptional activity of NFATc1 during osteoclast differentiation.
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Sánchez-Molina, Sara, José Luis Oliva, Susana García-Vargas, Ester Valls, José M. Rojas, and Marian A. Martínez-Balbás. "The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway." Biochemical Journal 398, no. 2 (August 15, 2006): 215–24. http://dx.doi.org/10.1042/bj20060052.
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The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators.
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Rozovski, Uri, David M. Harris, Ping LI, Zhiming Liu, Alessandra Ferrajoli, Jan A. Burger, Philip Thompson, et al. "P300 Acetylates STAT3 and Increases Its Constitutive Transcriptional Activity in CLL Cells." Blood 126, no. 23 (December 3, 2015): 4143. http://dx.doi.org/10.1182/blood.v126.23.4143.4143.
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Abstract Introduction: Signal transducer and activator of transcription (STAT)-3 is thought to promote oncogenesis by modulating the expression of genes that are required for tumor cell survival1. Following translation STAT3 may undergo several modifications such as phosphorylation or acetylation which modulate its activity. These modifications are typically cytokine-dependent but may also be cytokine-independent or constitutive. In chronic lymphocytic leukemia (CLL) STAT3 is constitutively phosphorylated (p) on serine-727 residues2. Serine pSTAT3 forms heterodimers, shuttles to the nucleus and induces transcription of STAT3 targeted genes. The transcriptional activity of STAT3 is also increased if STAT3 undergoes acetylation3. The conserved bormodomain motif of the acetyltransferase p300 binds and acetylates lysine residues of several proteins including STAT34. Whether STAT3 is acetylated in CLL cells is currently unknown. We sought to determine whether p300 acetylates STAT3 and affects its transcriptional activity in CLL cells. Methods and Results: Using western immunoblotting of the peripheral blood (PB) lymphocytes from 16 CLL patients we found that STAT3 is constitutively phosphorylated on lysine 685 residues. As previously reported2,5 we also detected serine pSTAT3 in all samples. Flow cytometry analysis revealed that a significant number of CD19+ CLL cells are constitutively phosphorylated on serine 727 and acetylated on lysine 685 residues. Immunoprecipitation studies confirmed these data. Protein extract of PB from 8 CLL patients were immunoprecipitated either with STAT3 or serine pSTAT3 antibodies and acetyl-STAT3 was detected in all immunoprecipitates, suggesting that the activated form of STAT3 is also acetylated in CLL cells. Because previous studies suggested that p300 acetylates STAT3 we obtained PB samples from 5 CLL patients and using western immunobloting and detected high levels of p300 in all patients but not in B lymphocytes from 2 healthy individuals. Then, to determine whether p300 affects the transcriptional activity of STAT3, we transfected CLL cells with p300 small-interfering (si) RNA. With a transfection efficiency of 30%, p300 transcripts and protein levels and acetyl-STAT3 levels were significantly reduced. Transfection with p300-siRNA reduced STAT3-DNA binding, as assessed by an electormobility shift assay (EMSA), decrease the levels of the STAT3-target gene p21 as assessed by chromatin immunoprecipitation (ChIP), and the levels of several STAT3-induced genes including STAT3, VEGF-c, c-Myc and p21, as assessed by RT-PCR. In addition, using Annexin V/PI we detected high apoptosis rates in p300-siRNA- transfected cells. Taken together these data suggest that the transcriptional activity of STAT3 is enhanced by p300 and STAT3 acetylation provides the cells with survival advantage. Conclusion: Our findings suggest that the aberrant expression of p300 induces acetylation and increases the transcriptional activity of STAT3 in CLL. Whether targeting STAT3 acetylation could be effective therapeutic modality in CLL remains to be determined. References 1. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nature reviews. Cancer. 2009;9(11):798-809. 2. Hazan-Halevy I, Harris D, Liu Z, et al. STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells. Blood. 2010;115(14):2852-2863. 3. Yuan ZL, Guan YJ, Chatterjee D, Chin YE. Stat3 dimerization regulated by reversible acetylation of a single lysine residue. Science. 2005;307(5707):269-273. 4. Wang R, Cherukuri P, Luo J. Activation of Stat3 sequence-specific DNA binding and transcription by p300/CREB-binding protein-mediated acetylation. J Biol Chem. 2005;280(12):11528-11534. 5. Frank DA, Mahajan S, Ritz J. B lymphocytes from patients with chronic lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues. J Clin Invest. 1997;100(12):3140-3148. Disclosures Rozovski: Novartis: Other: Advisory board. Wierda:Genentech: Consultancy; AbbVie and Genentech: Research Funding.
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Wang, Zhongyan, and Peter A. Burke. "Modulation of hepatocyte nuclear factor-4α function by the peroxisome-proliferator-activated receptor-γ co-activator-1α in the acute-phase response." Biochemical Journal 415, no. 2 (September 25, 2008): 289–96. http://dx.doi.org/10.1042/bj20080355.
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HNF-4α (hepatocyte nuclear factor-4α) is a key regulator of liver-specific gene expression. To understand the mechanisms governing the regulation of HNF-4α function during the APR (acute-phase response), the effects of transcription co-activators, including p300, PGC-1α (peroxisome-proliferator-activated receptor-γ co-activator-1α) and SRC (steroid receptor co-activator)-1α were investigated in an injury cell model. We have shown previously that the HNF-4α-sensitive APR genes ApoB (apolipoprotein B), TTR (transthyretin) and α1-AT (α1-antitrypsin) were regulated at the DNA binding and transcriptional levels after cytokine stimulation. We now show that co-activators have a differential impact on the transactivation of HNF-4α-sensitive genes via HNF-4α-binding sites in ApoB, TTR or α1-AT promoters. PGC-1α strongly enhances the transactivation of ApoB and α1-AT and, to a lesser extent, of TTR, whereas SRC-1α and p300 only have a weak or no effect on these three genes. More importantly, it was found that PGC-1α has a novel role in the modulation of the binding ability of HNF-4α in response to cytokine treatment. Using in vitro and in vivo approaches, electrophoretic mobility-shift and chromatin immunoprecipitation assays, we demonstrate that the reduced HNF-4α–DNA binding ability induced by cytokines is eliminated by overexpression of PGC-1α. Cytokine treatment does not significantly alter the protein levels of HNF-4α and PGC-1α, but it does reduce the recruitment of PGC-1α to HNF-4α-binding sites and thereby decreases transcriptional activity. These results establish the importance of PGC-1α for HNF-4α function and describe a new HNF-4α-dependent regulatory mechanism that is involved in the response to injury.
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Kino, Tomoshige, Alexander Gragerov, Olga Slobodskaya, Maria Tsopanomichalou, George P. Chrousos, and George N. Pavlakis. "Human Immunodeficiency Virus Type 1 (HIV-1) Accessory Protein Vpr Induces Transcription of the HIV-1 and Glucocorticoid-Responsive Promoters by Binding Directly to p300/CBP Coactivators." Journal of Virology 76, no. 19 (October 1, 2002): 9724–34. http://dx.doi.org/10.1128/jvi.76.19.9724-9734.2002.
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ABSTRACT The accessory Vpr protein of human immunodeficiency virus type 1 (HIV-1) is a promiscuous activator of viral and cellular promoters. We report that Vpr enhances expression of the glucocorticoid receptor-induced mouse mammary tumor virus (MMTV) promoter and of the Tat-induced HIV-1 long terminal repeat promoter by directly binding to p300/CBP coactivators. In contrast, Vpr does not bind to p/CAF or to members of the p160 family of nuclear receptor coactivators, such as steroid receptor coactivator 1a and glucocorticoid receptor (GR)-interacting protein 1. Vpr forms a stable complex with p300 and also interacts with the ligand-bound glucocorticoid receptor in vivo. Mutation analysis showed that the C-terminal part of Vpr binds to the C-terminal portion of p300/CBP within amino acids 2045 to 2191. The same p300 region interacts with the p160 coactivators and with the adenovirus E1A protein. Accordingly, E1A competed for binding to p300 in vitro. Coexpression of E1A or of small fragments of p300 containing the Vpr binding site resulted in inhibition of Vpr's transcriptional effects. The C-terminal part of p300 containing the transactivating region is required for Vpr transactivation, whereas the histone acetyltransferase enzymatic region is dispensable. Vpr mutants that bind p300 but not the GR did not activate expression of the MMTV promoter and had dominant-negative effects. These results indicate that Vpr activates transcription by acting as an adapter linking transcription components and coactivators.
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Michael, Laura F., Hiroshi Asahara, Andrew I. Shulman, W. Lee Kraus, and Marc Montminy. "The Phosphorylation Status of a Cyclic AMP-Responsive Activator Is Modulated via a Chromatin-Dependent Mechanism." Molecular and Cellular Biology 20, no. 5 (March 1, 2000): 1596–603. http://dx.doi.org/10.1128/mcb.20.5.1596-1603.2000.
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ABSTRACT Cyclic AMP (cAMP) stimulates the expression of numerous genes via the protein kinase A (PKA)-mediated phosphorylation of CREB at Ser133. Ser133 phosphorylation, in turn, promotes recruitment of the coactivator CREB binding protein and its paralog p300, histone acetyltransferases (HATs) that have been proposed to mediate target gene activation, in part, by destabilizing promoter bound nucleosomes and thereby allowing assembly of the transcriptional apparatus. Here we show that although histone deacetylase (HDAC) inhibitors potentiate target gene activation via cAMP, they do not stimulate transcription over the early burst phase, during which CREB phosphorylation and CBP/p300 recruitment are maximal. Rather, HDAC inhibitors augment CREB activity during the late attenuation phase by prolonging CREB phosphorylation on chromosomal but, remarkably, not on extrachromosomal templates. In reconstitution studies, assembly of periodic nucleosomal arrays on a cAMP-responsive promoter template potently inhibited CREB phosphorylation by PKA, and acetylation of these template-bound nucleosomes by p300 partially rescued CREB phosphorylation by PKA. Our results suggest a novel regulatory mechanism by which cellular HATs and HDACs modulate the phosphorylation status of nuclear activators in response to cellular signals.
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Khurana, Simran, Sharmistha Chakraborty, Xuan Zhao, Yu Liu, Dongyin Guan, Minh Lam, Wei Huang, Sichun Yang, and Hung-Ying Kao. "Identification of a Novel LXXLL Motif in α-Actinin 4-spliced Isoform That Is Critical for Its Interaction with Estrogen Receptor α and Co-activators." Journal of Biological Chemistry 287, no. 42 (August 20, 2012): 35418–29. http://dx.doi.org/10.1074/jbc.m112.401364.
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α-Actinins (ACTNs) are a family of proteins cross-linking actin filaments that maintain cytoskeletal organization and cell motility. Recently, it has also become clear that ACTN4 can function in the nucleus. In this report, we found that ACTN4 (full length) and its spliced isoform ACTN4 (Iso) possess an unusual LXXLL nuclear receptor interacting motif. Both ACTN4 (full length) and ACTN4 (Iso) potentiate basal transcription activity and directly interact with estrogen receptor α, although ACTN4 (Iso) binds ERα more strongly. We have also found that both ACTN4 (full length) and ACTN4 (Iso) interact with the ligand-independent and the ligand-dependent activation domains of estrogen receptor α. Although ACTN4 (Iso) interacts efficiently with transcriptional co-activators such as p300/CBP-associated factor (PCAF) and steroid receptor co-activator 1 (SRC-1), the full length ACTN4 protein either does not or does so weakly. More importantly, the flanking sequences of the LXXLL motif are important not only for interacting with nuclear receptors but also for the association with co-activators. Taken together, we have identified a novel extended LXXLL motif that is critical for interactions with both receptors and co-activators. This motif functions more efficiently in a spliced isoform of ACTN4 than it does in the full-length protein.
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Zeng, Xiaoya, Lihong Chen, Christine A. Jost, Ruth Maya, David Keller, Xinjiang Wang, William G. Kaelin, Moshe Oren, Jiandong Chen, and Hua Lu. "MDM2 Suppresses p73 Function without Promoting p73 Degradation." Molecular and Cellular Biology 19, no. 5 (May 1, 1999): 3257–66. http://dx.doi.org/10.1128/mcb.19.5.3257.
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ABSTRACT The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73α and p73β stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.
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McConnell, Melanie-Jane, Emma Langley, Yolanda Martinez-Martinez, Tony Kouzarides, and Jonathan D. Licht. "Both the Activity and Stability of the Transcriptional Repressor PLZF Are Modified by the Class III Histone Deacetylase SIRT1." Blood 104, no. 11 (November 16, 2004): 360. http://dx.doi.org/10.1182/blood.v104.11.360.360.
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Abstract The transcription factor PLZF is expressed in hematopoietic development and rearranged in t(11;17) acute promyelocytic leukemia (APL). PLZF expression is high in the quiescent progenitor CD34+ cell, and declines during differentiation along myeloid and erythroid lineages. PLZF encodes a BTB-Zinc finger transcriptional repressor that inhibits the cell cycle through inhibition of targets genes such as cyclin A and c-myc through the recruitment of histone deacetylase complexes. However, PLZF itself is regulated by acetylation. In a separate study we demonstrate that acetylation of PLZF by p300/CBP enhances the transcriptional repression activity of PLZF. Through a yeast two-hybrid study we found that PLZF associates with the protein deacetylase SIRT1. SIRT1 is a member of the sirtuin family of class III histone deacetylases. In model organisms such as yeast, worms and flies, sirtuins play a common role in lifespan extension. The interaction between PLZF and SIRT1 was confirmed by co-precipitation of endogenous proteins and localized to the zinc fingers of PLZF, the region targeted for acetylation by p300/CBP. Acetylation of PLZF mediated by p300/CBP was reversed by SIRT1. Furthermore while acetylation of PLZF enhances its ability to repress transcription, co-expression of SIRT1 decreased PLZF transcriptional repression activity, consistent with loss of acetylation. Conversely, inhibition of SIRT1 activity with nicotinamide enhanced both PLZF acetylation and transcriptional repression of PLZF on its endogenous target gene c-myc. Further, increasing PLZF acetylation by inhibition of SIRT1 was associated with decreased PLZF stability. Acetyl-PLZF levels were stabilized by inhibition of the proteosomal degradation machinery, together implying that PLZF acetylation and activation results in increased protein turnover. These data point to the increasing complexity of the role of acetylation in transcriptional regulation and stand in contrast with data for the related Bcl6 repressor where acetylation of the protein inhibits repression. These data also indicate a novel function for sirtuins in regulation of transcriptional repression.
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Shima, Yutaka, Takito Shima, Tomoki Chiba, Tatsuro Irimura, and Issay Kitabayashi. "PML Protects HIPK2 and p300 from SCF-Mediated Ubiquitin-Dependent Degradation To Activate Transcription." Blood 110, no. 11 (November 16, 2007): 2653. http://dx.doi.org/10.1182/blood.v110.11.2653.2653.
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Abstract The Pml gene is the target of t(15;17) chromosome translocation in acute promyelocytic leukemia. PML protein is known to localize in discrete nuclear speckles, named PML nuclear bodies (NBs). In NBs, PML interacts with several transcription factors, such as p53 and AML1, and their co-activators, such as HIPK2 and p300. PML activates transcription of their target genes. PML is thought to stabilize transcription factor complex and function as a mediator in transcription activation, but little is known about the molecular mechanism by which PML activates transcription. To clarify the role of PML in transcription regulation, we purified the PML complex and identified a novel F-box protein (FBP), Skp1, and Cullin1 (Cul1) in the PML complex by LC/MS/MS analysis. FBPs form SCF ubiquitin ligase complexes with Skp1, Cul1 and ROC1 and mediate recognition of specific substrates for ubiquitination. We found that the FBP that we identified here also forms a SCF complex with Skp1, Cul1 and ROC1. To identify substrates for the SCF complex, we tested several proteins that could bind to PML, and found that the FBP promotes degradation of HIPK2 and p300. These degradations were inhibited in the presence of a proteasome inhibitor, MG132. The FBP stimulated ubiquitination of HIPK2. These results suggest that the SCF promotes degradation of these proteins by the ubiquitin-proteasome pathway. The fact that the SCF is a part of the PML complex suggests that PML plays a role in the SCF-mediated degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. In order to clarify the role of PML in degradation of HIPK2 and p300, we tested effects of PML on the degradation and found that PML inhibited the SCF-mediated degradation of HIPK2 and p300 without inhibition of ubiquitination. To clarify roles of HIPK2, PML IV and the FBP in p53-dependent transcription, we performed reporter analysis using the MDM2 promoter in H1299 cells. Since the FBP promotes degradation of HIPK2, we initially thought that the FBP might inhibit activation of p53-dependent transcription by HIPK2 and PML IV. However, the FBP, HIPK2 and PML synergistically stimulated the p53-dependent transcriptional activation. Taken together our data suggest that the SCF-induced ubiquitination of transcription co-activators HIPK2 and p300 plays a critical role in transcriptional regulation, and that PML stimulates transcription by protecting HIPK2 and p300 from ubiquitin-dependent degradation.
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Lemasson, Isabelle, Nicholas J. Polakowski, Paul J. Laybourn, and Jennifer K. Nyborg. "Transcription Factor Binding and Histone Modifications on the Integrated Proviral Promoter in Human T-cell Leukemia Virus-I-infected T-cells." Journal of Biological Chemistry 277, no. 51 (October 16, 2002): 49459–65. http://dx.doi.org/10.1074/jbc.m209566200.
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The human T-cell leukemia virus (HTLV-I)-encoded Tax protein is a potent transcriptional activator that stimulates expression of the integrated provirus. Biochemical studies indicate that Tax, together with cellular transcription factors, interacts with viral cAMP-response element enhancer elements to recruit the pleiotropic coactivators CREB-binding protein and p300. Histone acetylation by these coactivators has been shown to play a major role in activating HTLV-I transcription from chromatin templatesin vitro. However, the extent of histone modification and the precise identity of the cellular regulatory proteins bound at the HTLV-I promoterin vivois not known. Chromatin immunoprecipitation analysis was used to investigate factor binding and histone modification at the integrated HTLV-I provirus in infected T-cells (SLB-1). These studies reveal the presence of Tax, a variety of ATF/CREB and AP-1 family members (CREB, CREB-2, ATF-1, ATF-2, c-Fos, and c-Jun), and both p300 and CREB-binding protein at the HTLV-I promoter. Consistent with the binding of these coactivators, we observed histone H3 and H4 acetylation at three regions within the proviral genome. Histone deacetylases were also present at the viral promoter and, following their inhibition, we observe an increase in histone H4 acetylation on the HTLV-I promoter and a concomitant increase in viral RNA. Together, these results suggest that a variety of transcriptional activators, coactivators, and histone deacetylases participate in the regulation of HTLV-I transcription in infected T-cells.
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Cougot, Delphine, Yuanfei Wu, Stefano Cairo, Julie Caramel, Claire-Angélique Renard, Laurence Lévy, Marie Annick Buendia, and Christine Neuveut. "The Hepatitis B Virus X Protein Functionally Interacts with CREB-binding Protein/p300 in the Regulation of CREB-mediated Transcription." Journal of Biological Chemistry 282, no. 7 (December 11, 2006): 4277–87. http://dx.doi.org/10.1074/jbc.m606774200.
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The hepatitis B virus infects more than 350 million people worldwide and is a leading cause of liver cancer. The virus encodes a multifunctional regulator, the hepatitis B virus X protein (HBx), that is essential for virus replication. HBx is involved in modulating signal transduction pathways and transcription mediated by various factors, notably CREB that requires the recruitment of the co-activators CREB-binding protein (CBP)/p300. Here we investigated the role of HBx and its potential interaction with CBP/p300 in regulating CREB transcriptional activity. We show that HBx and CBP/p300 synergistically enhanced CREB activity and that CREB phosphorylation by protein kinase A was a prerequisite for the cooperative action of HBx and CBP/p300. We further show that HBx interacted directly with CBP/p300 in vitro and in vivo. Using chromatin immunoprecipitation, we provide evidence that HBx physically occupied the CREB-binding domain of CREB-responsive promoters of endogenous cellular genes such as interleukin 8 and proliferating cell nuclear antigen. Moreover expression of HBx increased the recruitment of p300 to the interleukin 8 and proliferating cell nuclear antigen promoters in cells, and this is associated with increased gene expression. As recruitment of CBP/p300 is known to represent the limiting event for activating CREB target genes, HBx may disrupt this cellular regulation, thus predisposing cells to transformation.
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Zhao, Xinyang, Animesh Parkanani, Jin Zhang, Richard Dunne, Andrew Xiao, C. David Allis, and Stephen Nimer. "Arginine Methylation of Runx1 Regulates Its Biological and Transcriptional Activities." Blood 104, no. 11 (November 16, 2004): 2041. http://dx.doi.org/10.1182/blood.v104.11.2041.2041.
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Abstract The AML1/Runx1 protein is required for definitive hematopoiesis and for the maturation of adult megakaryocytic cells. Alterations of the Runx1 gene by mutations, deletions and chromosome translocations are associated with several types of acute leukemia. Runx1 functions as both an activator and repressor of gene transcription with promoter and cell type context dependency. Likely, this relates to the ability of Runx1 to interact with a variety of transcription factors such as MEF, C/EBPa, Ets-1 and GATA-1 and also with repressor proteins such as Groucho, mSin3 and HDACs. Runx1 is post-translationally modified through acetylation and phosphorylation, and the acetylable and phosphorylable forms of Runx1 can activate transcription to higher level in Runx1 dependent reporter assays when HATs or Erk2 are coexpressed. Runx1 has also been shown to be methylated on lysine residues by SUV39H1 methyltransferase in fibroblasts. Based on the presence of a SGRGK motif in the runt domain of Runx1, we have been examining whether Runx1 is methylated on arginine residues by the protein arginine methyltransferases (PRMT). We have found that PRMT1 and PRMT5 are associated with Runx1 in AML cells by co-immunoprecipitation assays and using in vitro by GST-pulldown assays with in vitro translated PRMT(s) have shown that the interactions are direct. Using a luciferase gene reporter assay, we show that PRMT1 acts synergistically with p300 to activate Runx1 mediated transcription in response to cell proliferation signals. We have mapped the arginine methylation sites in Runx1 using GST-Runx1 fusion proteins, site-specific mutagenesis and mass spectrometry analysis. We have found three potential arginine methylation sites, one in the Runt domain, and two in the Runx1 carboxy-terminal region. Interestingly, one of these sites is in the region shown to interact with both the mSIN3A transcriptional repression complex and with p300. This suggests that arginine methylation of Runx1 may affects its transcriptional activating and repressing functions. Chromatin immunoprecipitation assays are underway to show how arginine methylation of Runx1 affects its activities in hematopoietic cells. Additional studies examining the effects of cross-talk between arginine methylation, lysine acetylation and serine phosphorylation has on Runx1 functions (biological and biochemical) will be presented.
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Marcello, Alessandro, Paola Massimi, Lawrence Banks, and Mauro Giacca. "Adeno-Associated Virus Type 2 Rep Protein Inhibits Human Papillomavirus Type 16 E2 Recruitment of the Transcriptional Coactivator p300." Journal of Virology 74, no. 19 (October 1, 2000): 9090–98. http://dx.doi.org/10.1128/jvi.74.19.9090-9098.2000.
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ABSTRACT Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1- and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300.
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Jin, Huajun, Arthi Kanthasamy, Vellareddy Anantharam, Ajay Rana, and Anumantha G. Kanthasamy. "Transcriptional Regulation of Pro-apoptotic Protein Kinase Cδ." Journal of Biological Chemistry 286, no. 22 (April 5, 2011): 19840–59. http://dx.doi.org/10.1074/jbc.m110.203687.
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We previously demonstrated that protein kinase Cδ (PKCδ; PKC delta) is an oxidative stress-sensitive kinase that plays a causal role in apoptotic cell death in neuronal cells. Although PKCδ activation has been extensively studied, relatively little is known about the molecular mechanisms controlling PKCδ expression. To characterize the regulation of PKCδ expression, we cloned an ∼2-kbp 5′-promoter segment of the mouse Prkcd gene. Deletion analysis indicated that the noncoding exon 1 region contained multiple Sp sites, including four GC boxes and one CACCC box, which directed the highest levels of transcription in neuronal cells. In addition, an upstream regulatory region containing adjacent repressive and anti-repressive elements with opposing regulatory activities was identified within the region −712 to −560. Detailed mutagenesis studies revealed that each Sp site made a positive contribution to PKCδ promoter expression. Overexpression of Sp family proteins markedly stimulated PKCδ promoter activity without any synergistic transactivating effect. Furthermore, experiments in Sp-deficient SL2 cells indicated long isoform Sp3 as the essential activator of PKCδ transcription. Importantly, both PKCδ promoter activity and endogenous PKCδ expression in NIE115 cells and primary striatal cultures were inhibited by mithramycin A. The results from chromatin immunoprecipitation and gel shift assays further confirmed the functional binding of Sp proteins to the PKCδ promoter. Additionally, we demonstrated that overexpression of p300 or CREB-binding protein increases the PKCδ promoter activity. This stimulatory effect requires intact Sp-binding sites and is independent of p300 histone acetyltransferase activity. Finally, modulation of Sp transcriptional activity or protein level profoundly altered the cell death induced by oxidative insult, demonstrating the functional significance of Sp-dependent PKCδ gene expression. Collectively, our findings may have implications for development of new translational strategies against oxidative damage.
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ZHANG, Xian, Roger J. A. GRAND, Christopher J. McCABE, Jayne A. FRANKLYN, Phillip H. GALLIMORE, and Andrew S. TURNELL. "Transcriptional regulation of the human glycoprotein hormone common α subunit gene by cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and p53." Biochemical Journal 368, no. 1 (November 15, 2002): 191–201. http://dx.doi.org/10.1042/bj20020634.
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We have investigated the functional interactions between adenovirus early region 1A (AdE1A) protein, the co-activators cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and SUG1, and the transcriptional repressor retinoblastoma (Rb) in mediating T3-dependent repression. Utilizing the human glycoprotein hormone common α-subunit (α-subunit) promoter and AdE1A mutants with selective binding capacity to these molecules we have determined an essential role for CBP/p300. In normal circumstances, wild-type 12S AdE1A inhibited α-subunit activity. In contrast, adenovirus mutants that retain both the SUG1- and Rb-binding sites, but lack the CBP/p300-binding site, were unable to repress promoter activity. We have also identified a role for the tumour-suppressor gene product p53 in regulation of the α-subunit promoter. Akin to 12S AdE1A, exogenous p53 expression repressed α-subunit activity. This function resided in the ability of p53 to interact with CBP/p300; an N-terminal mutant incapable of interacting with CBP/p300 did not inhibit α-subunit activity. Stabilization of endogenous p53 by UV irradiation also correlated positively with reduced α-subunit activity. Intriguingly, T3 stimulated endogenous p53 transcriptional activity, implicating p53 in T3-dependent signalling pathways. These data indicate that CBP/p300 and p53 are key regulators of α-subunit activity.