scholarly journals Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation.

1997 ◽  
Vol 17 (1) ◽  
pp. 519-527 ◽  
Author(s):  
L Wang ◽  
C Mizzen ◽  
C Ying ◽  
R Candau ◽  
N Barlev ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Evolutionary Conservation ◽  
Enzymatic Modification ◽  
Basal Transcriptional Machinery ◽  
Physical Interactions ◽  
Substitution Mutation ◽  
Histone Acetyltransferase Activity

Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo and may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery. Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation. In this report, we demonstrate that hGCN5 is also an HAT and has the same substrate specificity as yGCN5. Since hGCN5 does not complement functional defects caused by deletion of yGCN5, we constructed a series of hGCN5-yGCN5 chimeras to identify human regions capable of activity in yeast. Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complemented gcn5- cells for growth and transcriptional activation. Moreover, an amino acid substitution mutation within the HAT domain reduced both HAT activity in vitro and transcription in vivo. These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation.

scholarly journals PCAF Interacts with Tax and Stimulates Tax Transactivation in a Histone Acetyltransferase-Independent Manner

1999 ◽  
Vol 19 (12) ◽  
pp. 8136-8145 ◽  
Author(s):  
Hua Jiang ◽  
Hanxin Lu ◽  
R. Louis Schiltz ◽  
Cynthia A. Pise-Masison ◽  
Vasily V. Ogryzko ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Independent Manner ◽  
Cell Leukemia Virus Type ◽  
Knock Out ◽  
Human T Cell

ABSTRACT Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

scholarly journals Polyomavirus Large T Antigen Binds the Transcriptional Coactivator Protein p300

1999 ◽  
Vol 73 (2) ◽  
pp. 1734-1739 ◽  
Author(s):  
Maria Nemethova ◽  
Erhard Wintersberger
Keyword(s):  
Viral Protein ◽  
Histone Acetyltransferase ◽  
T Antigen ◽  
Binding Motif ◽  
Large T Antigen ◽  
Transcriptional Coactivator ◽  
Coactivator Protein ◽  
Histone Acetyltransferase Activity

ABSTRACT Using coimmunoprecipitation and glutathioneS-transferase pulldown experiments, we found that polyomavirus large T antigen binds to p300 in vivo and in vitro. The N-terminal region of the viral protein, including the pRB binding motif, was dispensable for this interaction, which involved several regions within the C-terminal half of the large T antigen. Interestingly, anti-T antibody coimmunoprecipitated a subspecies of p300 which has high histone acetyltransferase activity.

scholarly journals Akt Phosphorylation of p300 at Ser-1834 Is Essential for Its Histone Acetyltransferase and Transcriptional Activity

2005 ◽  
Vol 25 (15) ◽  
pp. 6592-6602 ◽  
Author(s):  
Wei-Chien Huang ◽  
Ching-Chow Chen
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Histone Acetyltransferase ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Necrosis Factor Alpha ◽  
Akt Phosphorylation ◽  
Basal Transcriptional Machinery

ABSTRACT The PI3K/Akt pathway plays a critical role in the regulation of gene expression induced by numerous stimuli. p300, a transcriptional coactivator, acts in concert with transcription factors to facilitate gene expression. Here, we show that Akt is activated and translocated to the nucleus in response to tumor necrosis factor alpha. Nuclear Akt associates with p300 and phosphorylates its Ser-1834 both in vivo and in vitro. The phosphorylation induces recruitment of p300 to the ICAM-1 promoter, leading to the acetylation of histones in chromatin and association with the basal transcriptional machinery RNA polymerase II. These two events facilitate ICAM-1 gene expression and are abolished by the p300 S1834A mutant, inhibitors of PI3K/Akt, or small interfering RNA of Akt. Histone acetylation is attributed to the Akt-enhanced intrinsic histone acetyltransferase (HAT) activity of p300 and its association with another HAT, p/CAF. Our study provides a new insight into the molecular mechanism by which Akt promotes the transcriptional potential of p300.

scholarly journals Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

2004 ◽  
Vol 3 (2) ◽  
pp. 264-276 ◽  
Author(s):  
Qi Fan ◽  
Lijia An ◽  
Liwang Cui
Keyword(s):  
Plasmodium Falciparum ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Northern Analysis ◽  
Binding Assays ◽  
Core Histones ◽  
Chromatin Remodeling Complexes

ABSTRACT The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodium falciparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.

scholarly journals Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1349-1358 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Vladimir Poltoratsky ◽  
Tom Owen-Hughes ◽  
Carol Ying ◽  
Lin Liu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Dependent Protein Kinase ◽  
Nuclear Extracts ◽  
Dependent Protein ◽  
Endogenous Proteins ◽  
Hybrid Screening

ABSTRACT GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity.

scholarly journals Histone Acetyltransferase Complexes Can Mediate Transcriptional Activation by the Major Glucocorticoid Receptor Activation Domain

1999 ◽  
Vol 19 (9) ◽  
pp. 5952-5959 ◽  
Author(s):  
Annika E. Wallberg ◽  
Kristen E. Neely ◽  
Jan-Åke Gustafsson ◽  
Jerry L. Workman ◽  
Anthony P. H. Wright ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Saga Complex ◽  
Activation Domain ◽  
Transactivation Activity

ABSTRACT Previous studies have shown that the Ada adapter proteins are important for glucocorticoid receptor (GR)-mediated gene activation in yeast. The N-terminal transactivation domain of GR, τ1, is dependent upon Ada2, Ada3, and Gcn5 for transactivation in vitro and in vivo. Using in vitro techniques, we demonstrate that the GR-τ1 interacts directly with the native Ada containing histone acetyltransferase (HAT) complex SAGA but not the related Ada complex. Mutations in τ1 that reduce τ1 transactivation activity in vivo lead to a reduced binding of τ1 to the SAGA complex and conversely, mutations increasing the transactivation activity of τ1 lead to an increased binding of τ1 to SAGA. In addition, the Ada-independent NuA4 HAT complex also interacts with τ1. GAL4-τ1-driven transcription from chromatin templates is stimulated by SAGA and NuA4 in an acetyl coenzyme A-dependent manner. Low-activity τ1 mutants reduce SAGA- and NuA4-stimulated transcription while high-activity τ1 mutants increase transcriptional activation, specifically from chromatin templates. Our results demonstrate that the targeting of native HAT complexes by the GR-τ1 activation domain mediates transcriptional stimulation from chromatin templates.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jin-Chun Qi ◽  
Zhan Yang ◽  
Tao Lin ◽  
Long Ma ◽  
Ya-Xuan Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Activation ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Biological Effects ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Positive Feedback Loop

Abstract Background Both E2F transcription factor and cyclin-dependent kinases (CDKs), which increase or decrease E2F activity by phosphorylating E2F or its partner, are involved in the control of cell proliferation, and some circRNAs and miRNAs regulate the expression of E2F and CDKs. However, little is known about whether dysregulation among E2Fs, CDKs, circRNAs and miRNAs occurs in human PCa. Methods The expression levels of CDK13 in PCa tissues and different cell lines were determined by quantitative real-time PCR and Western blot analysis. In vitro and in vivo assays were preformed to explore the biological effects of CDK13 in PCa cells. Co-immunoprecipitation anlysis coupled with mass spectrometry was used to identify E2F5 interaction with CDK13. A CRISPR-Cas9 complex was used to activate endogenous CDK13 and circCDK13 expression. Furthermore, the mechanism of circCDK13 was investigated by using loss-of-function and gain-of-function assays in vitro and in vivo. Results Here we show that CDK13 is significantly upregulated in human PCa tissues. CDK13 depletion and overexpression in PCa cells decrease and increase, respectively, cell proliferation, and the pro-proliferation effect of CDK13 is strengthened by its interaction with E2F5. Mechanistically, transcriptional activation of endogenous CDK13, but not the forced expression of CDK13 by its expression vector, remarkably promotes E2F5 protein expression by facilitating circCDK13 formation. Further, the upregulation of E2F5 enhances CDK13 transcription and promotes circCDK13 biogenesis, which in turn sponges miR-212-5p/449a and thus relieves their repression of the E2F5 expression, subsequently leading to the upregulation of E2F5 expression and PCa cell proliferation. Conclusions These findings suggest that CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 is responsible for PCa development. Targeting this newly identified regulatory axis may provide therapeutic benefit against PCa progression and drug resistance.

scholarly journals NR5A2 transcriptional activation by BRD4 promotes pancreatic cancer progression by upregulating GDF15

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Feng Guo ◽  
Yingke Zhou ◽  
Hui Guo ◽  
Dianyun Ren ◽  
Xin Jin ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Transcriptional Activation ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Cancer Cells ◽  
Gene Sets ◽  
And Migration

AbstractNR5A2 is a transcription factor regulating the expression of various oncogenes. However, the role of NR5A2 and the specific regulatory mechanism of NR5A2 in pancreatic ductal adenocarcinoma (PDAC) are not thoroughly studied. In our study, Western blotting, real-time PCR, and immunohistochemistry were conducted to assess the expression levels of different molecules. Wound-healing, MTS, colony formation, and transwell assays were employed to evaluate the malignant potential of pancreatic cancer cells. We demonstrated that NR5A2 acted as a negative prognostic biomarker in PDAC. NR5A2 silencing inhibited the proliferation and migration abilities of pancreatic cancer cells in vitro and in vivo. While NR5A2 overexpression markedly promoted both events in vitro. We further identified that NR5A2 was transcriptionally upregulated by BRD4 in pancreatic cancer cells and this was confirmed by Chromatin immunoprecipitation (ChIP) and ChIP-qPCR. Besides, transcriptome RNA sequencing (RNA-Seq) was performed to explore the cancer-promoting effects of NR5A2, we found that GDF15 is a component of multiple down-regulated tumor-promoting gene sets after NR5A2 was silenced. Next, we showed that NR5A2 enhanced the malignancy of pancreatic cancer cells by inducing the transcription of GDF15. Collectively, our findings suggest that NR5A2 expression is induced by BRD4. In turn, NR5A2 activates the transcription of GDF15, promoting pancreatic cancer progression. Therefore, NR5A2 and GDF15 could be promising therapeutic targets in pancreatic cancer.

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