scholarly journals The N-Terminal Domain of p73 Interacts with the CH1 Domain of p300/CREB Binding Protein and Mediates Transcriptional Activation and Apoptosis

2000 ◽  
Vol 20 (4) ◽  
pp. 1299-1310 ◽  
Author(s):  
Xiaoya Zeng ◽  
Xiaorong Li ◽  
Ashley Miller ◽  
Zhimin Yuan ◽  
Wuchao Yuan ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Binding Protein ◽  
Small Cell Lung Carcinoma ◽  
Creb Binding Protein ◽  
Cell Lung Carcinoma ◽  
Cell Extracts ◽  
N Terminus ◽  
Mcf 7

ABSTRACT The newly identified p53 homolog p73 mimics the transcriptional function of p53. We have investigated the regulation of p73's transcriptional activity by p300/CREB binding protein (CBP). p73-p300 complexes were identified in HeLa cell extracts by cofractionation and coimmunoprecipitation assays. The p73-p300 interaction was confirmed in vitro by glutathione S-transferase–protein association assays and in vivo by coimmunoprecipitating the overexpressed p300 and p73 in human p53-free small-cell lung carcinoma H1299 or osteosarcoma Saos-2 cells. The N terminus but not the N-terminal truncation of p73 bound to the CH1 domain (amino acids [aa] 350 to 450) of p300/CBP. Accordingly, this p73 N-terminal deletion was unable to activate transcription or to induce apoptosis. Overexpression of either p300 or CBP stimulated transcription mediated by p73 but not its N-terminally deleted mutant in vivo. The N-terminal fragment from aa 19 to 597, but not the truncated fragment from aa 242 to 1700 of p300, reduced p73-mediated transcription markedly. p73-dependent transcription or apoptosis was partially impaired in either p300- or CBP-deficient human breast carcinoma MCF-7 or H1299 cells, suggesting that both coactivators mediate transcription by p73 in cells. These results demonstrate that the N terminus of p73 directly interacts with the N-terminal CH1 domain of p300/CBP to activate transcription.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

scholarly journals Trichostatin A downregulates bromodomain and extra-terminal proteins to suppress osimertinib resistant non-small cell lung carcinoma

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yuting Meng ◽  
Xixi Qian ◽  
Li Zhao ◽  
Nan Li ◽  
Shengjie Wu ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Small Cell Lung Carcinoma ◽  
Cell Types ◽  
Inhibitory Effects ◽  
Cell Lung Carcinoma ◽  
Growth Inhibitory ◽  
Terminal Proteins ◽  
P Cells

Abstract Background The third-generation epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have shown significant therapeutic effects on patients with non-small cell lung carcinoma (NSCLC) who carry active EGFR mutations, as well as those who have developed acquired resistance to the first-generation of EGFR-TKIs due to the T790M mutation. However, most patients develop drug resistance after 8–10 months of treatment. Currently, the mechanism has not been well clarified, and new therapeutic strategies are urgently needed. Methods Osimertinib resistant cell lines were established by culturing sensitive cells in chronically increasing doses of osimertinib. The anticancer effect of reagents was examined both in vitro and in vivo using the sulforhodamine B assay and a xenograft mouse model. The molecular signals were detected by western blotting. The combination effect was analyzed using CompuSyn software. Results We found that bromodomain and extra-terminal proteins (BETs) were upregulated in osimertinib resistant (H1975-OR) cells compared with those in the paired parental cells (H1975-P), and that knockdown of BETs significantly inhibited the growth of H1975-OR cells. The BET inhibitor JQ1 also exhibited stronger growth-inhibitory effects on H1975-OR cells and a greater expression of BETs and the downstream effector c-Myc than were observed in H1975-P cells. The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) showed stronger growth suppression in H1975-OR cells than in H1975-P cells, but vorinostat, another HDAC inhibitor, showed equal inhibitory efficacy in both cell types. Consistently, downregulation of BET and c-Myc expression was greater with TSA than with vorinostat. TSA restrained the growth of H1975-OR and H1975-P xenograft tumors. The combination of TSA and JQ1 showed synergistic growth-inhibitory effects in parallel with decreased BET and c-Myc expression in both H1975-OR and H1975-P cells and in xenograft nude mouse models. BETs were not upregulated in osimertinib resistant HCC827 cells compared with parental cells, while TSA and vorinostat exhibited equal inhibitory effects on both cell types. Conclusion Upregulation of BETs contributed to the osimertinib resistance of H1975 cells. TSA downregulated BET expression and enhanced the growth inhibitory effect of JQ1 both in vitro and in vivo. Our findings provided new strategies for the treatment of osimertinib resistance.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex

1998 ◽  
Vol 18 (10) ◽  
pp. 5861-5867 ◽  
Author(s):  
Philip B. Komarnitsky ◽  
Edward R. Klebanow ◽  
P. Anthony Weil ◽  
Clyde L. Denis
Keyword(s):  
Transcriptional Activation ◽  
Yeast Cells ◽  
Binding Studies ◽  
Whole Cell ◽  
Transactivation Domains ◽  
Cell Extracts ◽  
Genes Expression ◽  
Tfiid Complex

ABSTRACT The yeast transcriptional activator ADR1, which is required forADH2 and other genes’ expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reducedADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

scholarly journals Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

1992 ◽  
Vol 12 (8) ◽  
pp. 3490-3498 ◽  
Author(s):  
N Hosokawa ◽  
K Hirayoshi ◽  
H Kudo ◽  
H Takechi ◽  
A Aoike ◽  
...  
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Mobility Shift ◽  
Human Colon Cancer ◽  
Heat Shock Element ◽  
Mrna Accumulation ◽  
Cell Extracts

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3

2010 ◽  
Vol 431 (3) ◽  
pp. 391-402 ◽  
Author(s):  
Boon Shang Chew ◽  
Wee Leng Siew ◽  
Benjamin Xiao ◽  
Norbert Lehming
Keyword(s):  
Mutant Strain ◽  
Transcriptional Activation ◽  
Glutathione Transferase ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Mutant Protein ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)–Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

scholarly journals A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

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 The Gcn4p Activation Domain Interacts Specifically In Vitro with RNA Polymerase II Holoenzyme, TFIID, and the Adap-Gcn5p Coactivator Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1711-1724 ◽  
Author(s):  
Connie M. Drysdale ◽  
Belinda M. Jackson ◽  
Richard McVeigh ◽  
Edward R. Klebanow ◽  
Yu Bai ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Adapter Proteins ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Cell Extracts ◽  
Tata Box Binding Protein ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathioneS-transferase (GST)–Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

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