Faculty Opinions recommendation of Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II.

ABSTRACT Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.

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Methylation of Histone H3 by Set2 in Saccharomyces cerevisiae Is Linked to Transcriptional Elongation by RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.23.12.4207-4218.2003 ◽

2003 ◽

Vol 23 (12) ◽

pp. 4207-4218 ◽

Cited By ~ 454

Author(s):

Nevan J. Krogan ◽

Minkyu Kim ◽

Amy Tong ◽

Ashkan Golshani ◽

Gerard Cagney ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Histone H3 ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Histone H2a ◽

Ww Domain ◽

Growth Defects ◽

C Terminus ◽

Active Transcription

ABSTRACT Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less β-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.

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Pdx-1 Links Histone H3-Lys-4 Methylation to RNA Polymerase II Elongation during Activation of Insulin Transcription

Journal of Biological Chemistry ◽

10.1074/jbc.m505741200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36244-36253 ◽

Cited By ~ 74

Author(s):

Joshua Francis ◽

Swarup K. Chakrabarti ◽

James C. Garmey ◽

Raghavendra G. Mirmira

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Structure ◽

Small Interfering Rna ◽

Histone H3 ◽

Insulin Gene ◽

Transcriptional Elongation ◽

Pol Ii ◽

Insulin Promoter ◽

Interfering Rna

Expression of the insulin gene is nearly exclusive to the β cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in βTC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.

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Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.3.737 ◽

1996 ◽

Vol 142 (3) ◽

pp. 737-747 ◽

Cited By ~ 7

Author(s):

Jacques Archambault ◽

David B Jansma ◽

James D Friesen

Keyword(s):

Saccharomyces Cerevisiae ◽

Steady State ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Reporter Gene ◽

Growth Medium ◽

Temperature Sensitivity ◽

Slow Growth ◽

Yeast Saccharomyces Cerevisiae ◽

Genes Encoding

Abstract In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

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Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)50101-0 ◽

1992 ◽

Vol 267 (32) ◽

pp. 23376-23382 ◽

Cited By ~ 3

Author(s):

M.H. Sayre ◽

H Tschochner ◽

R.D. Kornberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Initiation Factors

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The rye Mutants Identify a Role for Ssn/Srb Proteins of the RNA Polymerase II Holoenzyme During Stationary Phase Entry in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.17 ◽

2001 ◽

Vol 157 (1) ◽

pp. 17-26 ◽

Cited By ~ 3

Author(s):

Ya-Wen Chang ◽

Susie C Howard ◽

Yelena V Budovskaya ◽

Jasper Rine ◽

Paul K Herman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Growth ◽

Rna Polymerase ◽

Stationary Phase ◽

Yeast Cell ◽

Rna Polymerase Ii ◽

Transcriptional Response ◽

Nutrient Deprivation ◽

Ras Signaling ◽

Rna Polymerase Ii Holoenzyme

Abstract Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

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Genetic interactions between an RNA polymerase II phosphatase and centromeric elements in Saccharomyces cerevisiae

Molecular Genetics and Genomics ◽

10.1007/s00438-004-1009-5 ◽

2004 ◽

Vol 271 (5) ◽

pp. 603-615 ◽

Cited By ~ 3

Author(s):

E. Pierstorff ◽

C. M. Kane

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genetic Interactions

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The Essential WD Repeat Protein Swd2 Has Dual Functions in RNA Polymerase II Transcription Termination and Lysine 4 Methylation of Histone H3

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2932-2943.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2932-2943 ◽

Cited By ~ 60

Author(s):

Hailing Cheng ◽

Xiaoyuan He ◽

Claire Moore

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Modification ◽

Histone H3 ◽

Temperature Sensitive ◽

Repeat Protein ◽

Tightly Coupled ◽

Cleavage And Polyadenylation ◽

Wd Repeat

ABSTRACT Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3′ end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled.

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The Ras/PKA Signaling Pathway May Control RNA Polymerase II Elongation via the Spt4p/Spt5p Complex in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/165.3.1059 ◽

2003 ◽

Vol 165 (3) ◽

pp. 1059-1070

Author(s):

Susie C Howard ◽

Arelis Hester ◽

Paul K Herman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Growth ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Signaling Pathway ◽

Elongation Factor ◽

Ras Signaling ◽

Elongation Step ◽

Rna Polymerase Ii Transcription

Abstract The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription.

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Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0591 ◽

2014 ◽

Vol 25 (12) ◽

pp. 1916-1924 ◽

Cited By ~ 3

Author(s):

David Öling ◽

Rehan Masoom ◽

Kristian Kvint

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Life Span ◽

Rna Polymerase Ii ◽

Ribosomal Dna ◽

Genetic Evidence ◽

Wild Type ◽

Replicative Life Span ◽

Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

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