scholarly journals Targeted Recruitment of the Sin3-Rpd3 Histone Deacetylase Complex Generates a Highly Localized Domain of Repressed Chromatin In Vivo

1998 ◽  
Vol 18 (9) ◽  
pp. 5121-5127 ◽  
Author(s):  
David Kadosh ◽  
Kevin Struhl
Keyword(s):  
Dna Binding ◽  
Histone Deacetylase ◽  
Chromatin Structure ◽  
Transcriptional Repression ◽  
Dna Binding Proteins ◽  
Histone Deacetylation ◽  
Yeast Cells ◽  
Multiprotein Complex ◽  
Eukaryotic Organisms

ABSTRACT Eukaryotic organisms contain a multiprotein complex that includes Rpd3 histone deacetylase and the Sin3 corepressor. The Sin3-Rpd3 complex is recruited to promoters by specific DNA-binding proteins, whereupon it represses transcription. By directly analyzing the chromatin structure of a repressed promoter in yeast cells, we demonstrate that transcriptional repression is associated with localized histone deacetylation. Specifically, we observe decreased acetylation of histones H3 and H4 (preferentially lysines 5 and 12) that depends on the DNA-binding repressor (Ume6), Sin3, and Rpd3. Mapping experiments indicate that the domain of histone deacetylation is highly localized, occurring over a range of one to two nucleosomes. Taken together with previous observations, these results define a novel mechanism of transcriptional repression which involves targeted recruitment of a histone-modifying activity and localized perturbation of chromatin structure.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 359-359
Author(s):  
Fabien Guidez ◽  
Louise Howell ◽  
Mark Isalan ◽  
Marek Cebrat ◽  
Rhoda M. Alani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Binding ◽  
Histone Deacetylase ◽  
Zinc Finger ◽  
Transcriptional Repression ◽  
Promyelocytic Leukemia ◽  
Promyelocytic Leukemia Zinc Finger ◽  
Hematopoietic Stem

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.

scholarly journals Targeted Recruitment of Rpd3 Histone Deacetylase Represses Transcription by Inhibiting Recruitment of Swi/Snf, SAGA, and TATA Binding Protein

2002 ◽  
Vol 22 (18) ◽  
pp. 6458-6470 ◽  
Author(s):  
Jutta Deckert ◽  
Kevin Struhl
Keyword(s):  
Dna Binding ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Associated Factors ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Histone Deacetylation ◽  
Nucleosome Remodeling ◽  
Activation Domains

ABSTRACT Certain DNA-binding repressors inhibit transcription by recruiting Rpd3 histone deacetylase complexes to promoters and generating domains of histone deacetylation that extend over a limited number of nucleosomes. Here, we show that the degree of Rpd3-dependent repression depends on the activator and the level of activation, not the extent of histone deacetylation. In all cases tested, activator binding is unaffected by histone deacetylation. In contrast, Rpd3-dependent repression is associated with decreased occupancy by TATA binding protein (TBP), the Swi/Snf nucleosome-remodeling complex, and the SAGA histone acetylase complex. Transcriptional repression is bypassed by direct recruitment of TBP and several TBP-associated factors, but not by natural activation domains or direct recruitment of polymerase II holoenzyme components. These results suggest that the domain of localized histone deacetylation generated by recruitment of Rpd3 mediates repression by inhibiting recruitment of chromatin-modifying activities and TBP.

scholarly journals Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein

2005 ◽  
Vol 25 (13) ◽  
pp. 5552-5566 ◽  
Author(s):  
Fabien Guidez ◽  
Louise Howell ◽  
Mark Isalan ◽  
Marek Cebrat ◽  
Rhoda M. Alani ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Histone Deacetylase ◽  
Zinc Finger ◽  
Transcriptional Repression ◽  
Histone Acetyltransferase ◽  
Transcriptional Repressor ◽  
Promyelocytic Leukemia ◽  
Promyelocytic Leukemia Zinc Finger

ABSTRACT Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. 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 coactivator protein in transcriptional repression.

scholarly journals A large protein complex containing the yeast Sin3p and Rpd3p transcriptional regulators.

1997 ◽  
Vol 17 (8) ◽  
pp. 4852-4858 ◽  
Author(s):  
M M Kasten ◽  
S Dorland ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Gel Filtration ◽  
Transcriptional Regulators ◽  
Dna Binding Proteins ◽  
Multiprotein Complex ◽  
Large Protein ◽  
Genetic Studies ◽  
Large Protein Complex

The SIN3 gene is required for the transcriptional repression of diverse genes in Saccharomyces cerevisiae. Sin3p does not bind directly to DNA but is thought to be targeted to promoters by interacting with sequence-specific DNA-binding proteins. We show here that Sin3p is present in a large multiprotein complex with an apparent molecular mass, estimated by gel filtration chromatography, of greater than 2 million Da. Genetic studies have shown that the yeast RPD3 gene has a function similar to that of SIN3 in transcriptional regulation, as SIN3 and RPD3 negatively regulate the same set of genes. The SIN3 and RPD3 genes are conserved from yeasts to mammals, and recent work suggests that RPD3 may encode a histone deacetylase. We show that Rpd3p is present in the Sin3p complex and that an rpd3 mutation eliminates SIN3-dependent repression. Thus, Sin3p may function as a bridge to recruit the Rpd3p histone deacetylase to specific promoters.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814 ◽  
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

scholarly journals The Rpd3-Sin3 Histone Deacetylase Regulates Replication Timing and Enables Intra-S Origin Control in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (11) ◽  
pp. 4769-4780 ◽  
Author(s):  
Jennifer G. Aparicio ◽  
Christopher J. Viggiani ◽  
Daniel G. Gibson ◽  
Oscar M. Aparicio
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Replication Timing ◽  
Histone Deacetylation ◽  
Origin Firing ◽  
Replication Checkpoint ◽  
Genome Transcription ◽  
Late Origin

ABSTRACT The replication of eukaryotic genomes follows a temporally staged program, in which late origin firing often occurs within domains of altered chromatin structure(s) and silenced genes. Histone deacetylation functions in gene silencing in some late-replicating regions, prompting an investigation of the role of histone deacetylation in replication timing control in Saccharomyces cerevisiae. Deletion of the histone deacetylase Rpd3 or its interacting partner Sin3 caused early activation of late origins at internal chromosomal loci but did not alter the initiation timing of early origins or a late-firing, telomere-proximal origin. By delaying initiation relative to the earliest origins, Rpd3 enables regulation of late origins by the intra-S replication checkpoint. RPD3 deletion suppresses the slow S phase of clb5Δ cells by enabling late origins to fire earlier, suggesting that Rpd3 modulates the initiation timing of many origins throughout the genome. Examination of factors such as Ume6 that function together with Rpd3 in transcriptional repression indicates that Rpd3 regulates origin initiation timing independently of its role in transcriptional repression. This supports growing evidence that for much of the S. cerevisiae genome transcription and replication timing are not linked.

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

Methylated DNA-binding proteins and chromatin structure

1993 ◽  
pp. 377-389
Author(s):  
R. Meehan ◽  
P. Jeppesen ◽  
J. Lewis ◽  
A. Bird
Keyword(s):  
Dna Binding ◽  
Chromatin Structure ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Methylated Dna

scholarly journals Residues in the Swi5 Zinc Finger Protein That Mediate Cooperative DNA Binding with the Pho2 Homeodomain Protein

1998 ◽  
Vol 18 (11) ◽  
pp. 6436-6446 ◽  
Author(s):  
Leena T. Bhoite ◽  
David J. Stillman
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Binding Proteins ◽  
Zinc Finger Protein ◽  
Interaction Analysis ◽  
Dna Binding Proteins ◽  
Two Hybrid

ABSTRACT The Swi5 zinc finger and the Pho2 homeodomain DNA-binding proteins bind cooperatively to the HO promoter.Pho2 (also known as Bas2 or Grf10) activates transcription of diverse genes, acting with multiple distinct DNA-binding proteins. We have performed a genetic screen to identify amino acid residues in Swi5 that are required for synergistic transcriptional activation of a reporter construct in vivo. Nine unique amino acid substitutions within a 24-amino-acid region of Swi5, upstream of the DNA-binding domain, reduce expression of promoters that require both Swi5 and Pho2 for activation. In vitro DNA binding experiments show that the mutant Swi5 proteins bind DNA normally, but some mutant Swi5 proteins (resulting from SWI5* mutations) show reduced cooperative DNA binding with Pho2. In vivo experiments show that these SWI5* mutations sharply reduce expression of promoters that require both SWI5 and PHO2, while expression of promoters that require SWI5 but arePHO2 independent is largely unaffected. This suggests that these SWI5* mutations do not affect the ability of Swi5 to bind DNA or activate transcription but specifically affect the region of Swi5 required for interaction with Pho2. Two-hybrid experiments show that amino acids 471 to 513 of Swi5 are necessary and sufficient for interaction with Pho2 and that the SWI5* point mutations cause a severe reduction in this two-hybrid interaction. Analysis of promoter activation by these mutants suggests that this small region of Swi5 has at least two distinct functions, conferring specificity for activation of the HO promoter and for interaction with Pho2.

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