scholarly journals Different Genetic Functions for the Rpd3(L) and Rpd3(S) Complexes Suggest Competition between NuA4 and Rpd3(S)

2008 ◽  
Vol 28 (14) ◽  
pp. 4445-4458 ◽  
Author(s):  
Debabrata Biswas ◽  
Shinya Takahata ◽  
David J. Stillman
Keyword(s):  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Histone Acetyltransferase ◽  
Strand Break ◽  
Triple Mutant ◽  
Growth Defects ◽  
Mutant Phenotypes ◽  
Synthetic Growth ◽  
Synthetic Phenotype

ABSTRACT Rpd3(L) and Rpd3(S) are distinct multisubunit complexes containing the Rpd3 histone deacetylase. Disruption of the GCN5 histone acetyltransferase gene shows a strong synthetic phenotype when combined with either an sds3 mutation affecting only the Rpd3(L) complex or an rco1 mutation affecting only Rpd3(S). However, these synthetic growth defects are not seen in a gcn5 sds3 rco1 triple mutant, suggesting that the balance between Rpd3(L) and Rpd3(S) is critical in cells lacking Gcn5. Different genetic interactions are seen with mutations affecting the FACT chromatin reorganizing complex. An sds3 mutation affecting only Rpd3(L) has a synthetic defect with FACT mutants, while rco1 and eaf3 mutations affecting Rpd3(S) suppress FACT mutant phenotypes. Rpd3(L) therefore acts in concert with FACT, but Rpd3(S) opposes it. Combining FACT mutations with mutations in the Esa1 subunit of the NuA4 histone acetyltransferase results in synthetic growth defects, and these can be suppressed by an rco1 or set2 mutation. An rco1 mutation suppresses phenotypes caused by mutations in the ESA1 and ARP4 subunits of NuA4, while Rco1 overexpression exacerbates these defects. These results suggest a model in which NuA4 and Rpd3(S) compete. Chromatin immunoprecipitation experiments show that eliminating Rpd3(S) increases the amount of NuA4 binding to the ARG3 promoter during transcriptional activation and to the sites of DNA repair induced by a double-strand break. Our results suggest that the Rpd3(L) and Rpd3(S) complexes have distinct functions in vivo and that the relative amounts of the two forms alter the effectiveness of other chromatin-altering complexes, such as FACT and NuA4.

scholarly journals In Vivo Role for Actin-regulating Kinases in Endocytosis and Yeast Epsin Phosphorylation

2001 ◽  
Vol 12 (11) ◽  
pp. 3668-3679 ◽  
Author(s):  
Hadiya A. Watson ◽  
M. Jamie T. V. Cope ◽  
Aaron Chris Groen ◽  
David G. Drubin ◽  
Beverly Wendland
Keyword(s):  
Consensus Sequence ◽  
Signaling Proteins ◽  
Wild Type ◽  
Cortical Actin ◽  
Growth Defects ◽  
Endocytic Protein ◽  
Mutant Phenotypes ◽  
Actin Localization ◽  
Synthetic Growth

The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins localized to cortical actin patches, which may be sites of endocytosis. Interactions between the endocytic proteins Pan1p and End3p may be regulated by Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence [L/I]xxQxTG. We identified two Prk1p phosphorylation sites within the Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate Prk1p-dependent phosphorylation of both threonines. Converting both threonines to either glutamate or alanine mimics constitutively phosphorylated or dephosphorylated Ent1p, respectively. Synthetic growth defects were observed in a pan1–20 ENT1EE double mutant, suggesting that Ent1p phosphorylation negatively regulates the formation/activity of a Pan1p–Ent1p complex. Interestingly,pan1–20 ent2Δ but not pan1–20 ent1Δdouble mutants had improved growth and endocytosis over thepan1–20 mutant. We found that actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that overexpressing either wild-type or alanine-substituted Ent1p partially suppressed phenotypes associated with loss of ARK kinases, including growth, endocytosis, and actin localization defects. Consistent with synthetic growth defects of pan1–20 ENT1EE cells, overexpressing glutamate-substituted Ent1p was deleterious to ARK mutants. Surprisingly, overexpressing the related Ent2p protein could not suppress ARK kinase mutant phenotypes. These results suggest that Ent1p and Ent2p are not completely redundant and may perform opposing functions in endocytosis. These data support the model that, as for clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein phosphorylation inhibits endocytic functions.

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 Role of an ING1 Growth Regulator in Transcriptional Activation and Targeted Histone Acetylation by the NuA4 Complex

2001 ◽  
Vol 21 (22) ◽  
pp. 7629-7640 ◽  
Author(s):  
Amine Nourani ◽  
Yannick Doyon ◽  
Rhea T. Utley ◽  
Stéphane Allard ◽  
William S. Lane ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcription Regulation ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Yeast Cells ◽  
Growth Defect ◽  
Histone H4 ◽  
Phd Finger

ABSTRACT The yeast NuA4 complex is a histone H4 and H2A acetyltransferase involved in transcription regulation and essential for cell cycle progression. We identify here a novel subunit of the complex, Yng2p, a plant homeodomain (PHD)-finger protein homologous to human p33/ING1, which has tumor suppressor activity and is essential for p53 function. Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable stoichiometric association of this protein with purified NuA4. Yeast cells harboring a deletion of theYNG2 gene show severe growth phenotype and have gene-specific transcription defects. NuA4 complex purified from the mutant strain is low in abundance and shows weak histone acetyltransferase activity. We demonstrate conservation of function by the requirement of Yng2p for p53 to function as a transcriptional activator in yeast. Accordingly, p53 interacts with NuA4 in vitro and in vivo, an interaction reminiscent of the p53-ING1 physical link in human cells. The growth defect of Δyng2 cells can be rescued by the N-terminal part of the protein, lacking the PHD-finger. While Yng2 PHD-finger is not required for p53 interaction, it is necessary for full expression of the p53-responsive gene and other NuA4 target genes. Transcriptional activation by p53 in vivo is associated with targeted NuA4-dependent histone H4 hyperacetylation, while histone H3 acetylation levels remain unchanged. These results emphasize the essential role of the NuA4 complex in the control of cell proliferation through gene-specific transcription regulation. They also suggest that regulation of mammalian cell proliferation by p53-dependent transcriptional activation functions through recruitment of an ING1-containing histone acetyltransferase complex.

scholarly journals TFIIS Enhances Transcriptional Elongation through an Artificial Arrest Site In Vivo

2001 ◽  
Vol 21 (13) ◽  
pp. 4162-4168 ◽  
Author(s):  
Dmitry Kulish ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Analysis ◽  
Transcriptional Elongation ◽  
Specific Assay ◽  
Physical Evidence ◽  
Reduced Temperature

ABSTRACT Transcriptional elongation by RNA polymerase II has been well studied in vitro, but understanding of this process in vivo has been limited by the lack of a direct and specific assay. Here, we designed a specific assay for transcriptional elongation in vivo that involves an artificial arrest (ARTAR) site designed from a thermodynamic theory of DNA-dependent transcriptional arrest in vitro. Transcriptional analysis and chromatin immunoprecipitation experiments indicate that the ARTAR site can arrest Pol II in vivo at a position far from the promoter. TFIIS can counteract this arrest, thereby demonstrating that it possesses transcriptional antiarrest activity in vivo. Unexpectedly, the ARTAR site does not function under conditions of high transcriptional activation unless cells are exposed to conditions (6-azauracil or reduced temperature) that are presumed to affect elongation in vivo. Conversely, TFIIS affects gene expression under conditions of high, but not low, transcriptional activation. Our results provide physical evidence for the discontinuity of transcription elongation in vivo, and they suggest that the functional importance of transcriptional arrest sites and TFIIS is strongly influenced by the level of transcriptional activation.

scholarly journals Regulation of NuA4 Histone Acetyltransferase Activity in Transcription and DNA Repair by Phosphorylation of Histone H4

2005 ◽  
Vol 25 (18) ◽  
pp. 8179-8190 ◽  
Author(s):  
Rhea T. Utley ◽  
Nicolas Lacoste ◽  
Olivier Jobin-Robitaille ◽  
Stéphane Allard ◽  
Jacques Côté
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Chromatin Immunoprecipitation ◽  
Time Course ◽  
Histone Acetyltransferase ◽  
Strand Break ◽  
Histone H4 ◽  
Normal Gene ◽  
Chromatin Acetylation ◽  
Histone Acetyltransferase Activity

ABSTRACT The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair.

scholarly journals Simultaneous Recruitment of Coactivators by Gcn4p Stimulates Multiple Steps of Transcription In Vivo

2005 ◽  
Vol 25 (13) ◽  
pp. 5626-5638 ◽  
Author(s):  
Chhabi K. Govind ◽  
Sungpil Yoon ◽  
Hongfang Qiu ◽  
Sudha Govind ◽  
Alan G. Hinnebusch
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Tata Binding Protein ◽  
Time Intervals ◽  
Pol Ii ◽  
Reading Frame ◽  
Induction Process ◽  
Multiple Stages

ABSTRACT Transcriptional activation by Gcn4p is dependent on the coactivators SWI/SNF, SAGA, and Srb Mediator, which are recruited by Gcn4p and stimulate assembly of the preinitiation complex (PIC) at the ARG1 promoter in vivo. We show that recruitment of all three coactivators is nearly simultaneous with binding of Gcn4p at ARG1 and is followed quickly by PIC formation and elongation by RNA polymerase II (Pol II) through the open reading frame. Despite the simultaneous recruitment of coactivators, rapid recruitment of SWI/SNF depends on the histone acetyltransferase (HAT) subunit of SAGA (Gcn5p), a non-HAT function of SAGA, and on Mediator. SAGA recruitment in turn is strongly stimulated by Mediator and the RSC complex. Recruitment of Mediator, by contrast, occurs independently of the other coactivators at ARG1. We confirm the roles of Mediator and SAGA in TATA binding protein (TBP) recruitment and demonstrate that all four coactivators under study enhance Pol II recruitment or promoter clearance following TBP binding. We also present evidence that SWI/SNF and SAGA stimulate transcription elongation downstream from the promoter. These functions can be limited to discrete time intervals, providing evidence for multiple stages in the induction process. Our findings reveal a program of coactivator recruitment and PIC assembly that distinguishes Gcn4p from other yeast activators studied thus far.

scholarly journals Zfp281 is essential for mouse epiblast maturation through transcriptional and epigenetic control of Nodal signaling

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Xin Huang ◽  
Sophie Balmer ◽  
Fan Yang ◽  
Miguel Fidalgo ◽  
Dan Li ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Transcriptional Activation ◽  
Molecular Model ◽  
Embryo Implantation ◽  
Cell Lineage ◽  
Nodal Signaling ◽  
Epigenetic Control ◽  
Basic Body ◽  
Mutant Phenotypes

Pluripotency is defined by a cell's potential to differentiate into any somatic cell type. How pluripotency is transited during embryo implantation, followed by cell lineage specification and establishment of the basic body plan, is poorly understood. Here we report the transcription factor Zfp281 functions in the exit from naive pluripotency occurring coincident with pre-to-post-implantation mouse embryonic development. By characterizing Zfp281 mutant phenotypes and identifying Zfp281 gene targets and protein partners in developing embryos and cultured pluripotent stem cells, we establish critical roles for Zfp281 in activating components of the Nodal signaling pathway and lineage-specific genes. Mechanistically, Zfp281 cooperates with histone acetylation and methylation complexes at target gene enhancers and promoters to exert transcriptional activation and repression, as well as epigenetic control of epiblast maturation leading up to anterior-posterior axis specification. Our study provides a comprehensive molecular model for understanding pluripotent state progressions in vivo during mammalian embryonic development.

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 Interaction and Functional Cooperation between the LIM Protein FHL2, CBP/p300, and β-Catenin

2004 ◽  
Vol 24 (24) ◽  
pp. 10689-10702 ◽  
Author(s):  
Charlotte Labalette ◽  
Claire-Angélique Renard ◽  
Christine Neuveut ◽  
Marie-Annick Buendia ◽  
Yu Wei
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Transcriptional Activation ◽  
Ternary Complex ◽  
Histone Acetyltransferase ◽  
Lim Protein ◽  
Transactivation Activity ◽  
Exogenous Expression ◽  
Acetyltransferase Domain

ABSTRACT Transcriptional activation of gene expression by Wnt signaling is driven by the association of β-catenin with TCF/LEF factors and the recruitment of transcriptional coactivators. It has been shown that the LIM protein FHL2 and the acetyltransferase CBP/p300 individually stimulate β-catenin transactivating activity and that β-catenin is acetylated by p300. Here, we report that FHL2 and CBP/p300 synergistically enhanced β-catenin/TCF-mediated transcription from Wnt-responsive promoters and that the acetyltransferase activity of CBP/p300 was involved in the cooperation. CBP/p300 interacted directly with FHL2, predominantly through the CH3 domain but not the histone acetyltransferase domain, and different regions of CBP/p300 were involved in FHL2 and β-catenin binding. We provided evidence for the formation of a ternary complex by FHL2, CBP/p300, and β-catenin and for colocalization of the three proteins in the nucleus. In murine FHL2−/− embryo fibroblasts, the transactivation activity of β-catenin/TCF was markedly reduced, and this defect could be restored by exogenous expression of FHL2. However, CBP/p300 were still able to coactivate the β-catenin/TCF complex in FHL2−/− cells, suggesting that FHL2 is dispensable for the coactivator function of CBP/p300 on β-catenin. Furthermore, we found that FHL2 significantly increased acetylation of β-catenin by p300 in vivo. Finally, we showed that FHL2, CBP/p300, and β-catenin could synergistically activate androgen receptor-mediated transcription, indicating that the synergistic coactivator function is not restricted to TCF/LEF.

scholarly journals PA5339, a RidA Homolog, Is Required for Full Growth inPseudomonas aeruginosa

2018 ◽  
Vol 200 (22) ◽  
Author(s):  
Jessica Irons ◽  
Kelsey M. Hodge-Hanson ◽  
Diana M. Downs
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Salmonella Enterica ◽  
Metabolic Stress ◽  
Content Type ◽  
Growth Defects ◽  
Collective Data ◽  
Domains Of Life ◽  
Mutant Phenotypes

ABSTRACTThe Rid protein superfamily (YjgF/YER057c/UK114) is found in all domains of life. The archetypal protein, RidA fromSalmonella enterica, is a deaminase that quenches the reactive metabolite 2-aminoacrylate (2AA). 2AA deaminase activity is conserved in RidA proteins from humans, plants, yeast, archaea, and bacteria. Mutants ofSalmonella enterica,Escherichia coli, andSaccharomyces cerevisiaethat lack a functional RidA exhibit growth defects, suggesting that 2AA metabolic stress is similarly conserved. The PubSEED database showsPseudomonas aeruginosa(PAO1) encodes eight members of the Rid superfamily. Mutants ofP. aeruginosaPAO1 lacking each of five Rid proteins were screened, and the mutant phenotypes that arose in the absence of PA5339 were dissected. A PA5339::Tn mutant has growth, motility, and biofilm defects that can all be linked to the accumulation of 2AA. Further, the PA5339 protein was demonstrably a 2AA deaminasein vitroand restored metabolic balance to aS. enterica ridAmutantin vivo. The data presented here show that the RidA paradigm inPseudomonas aeruginosahad similarities to those described in other organisms but was distinct in that deleting only one of multiple homologs generated deficiencies. Based on the collective data presented here in, PA5339 was renamed RidA.IMPORTANCERidA is a widely conserved protein that prevents endogenous metabolic stress caused by 2-aminoacrylate (2AA) damage to pyridoxal 5′-phosphate (PLP)-dependent enzymes in prokaryotes and eukaryotes. The framework for understanding the accumulation of 2AA and its consequences have largely been defined inSalmonella enterica. We show here that inP. aeruginosa(PAO1), 2AA accumulation leads to reduced growth, compromised motility, and defective biofilm formation. This study expands our knowledge how the metabolic architecture of an organism contributes to the consequences of 2AA inactivation of PLP-dependent enzymes and identifies a key RidA protein inP. aeruginosa.

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