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

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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Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5433-5441.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5433-5441

Author(s):

B Y Ahn ◽

P D Gershon ◽

E V Jones ◽

B Moss

Keyword(s):

Rna Polymerase ◽

Vaccinia Virus ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Viral Rna ◽

In Vitro Translation ◽

Virus Protein ◽

Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

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The FACT chromatin modulator: genetic and structure/function relationships

Biochemistry and Cell Biology ◽

10.1139/o04-050 ◽

2004 ◽

Vol 82 (4) ◽

pp. 419-427 ◽

Cited By ~ 23

Author(s):

Richard A Singer ◽

Gerald C Johnston

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Unfolding ◽

Elongation Factor ◽

Yeast Genetics ◽

Genetic Studies ◽

Hmg Box ◽

Transcription Elongation Factor ◽

C Terminus ◽

Chromatin Configuration

The chromatin configuration of DNA inhibits access by enzymes such as RNA polymerase II. This inhibition is alleviated by FACT, a conserved transcription elongation factor that has been found to reconfigure nucleosomes to allow transit along the DNA by RNA polymerase II, thus facilitating transcription. FACT also reorganizes nucleosomes after the passage of RNA polymerase II, as indicated by the effects of certain FACT mutations. The larger of the two subunits of FACT is Spt16/Cdc68, while the smaller is termed SSRP1 (vertebrates) or Pob3 (budding yeast). The HMG-box domain at the C terminus of SSRP1 is absent from Pob3; the function of this domain for yeast FACT is supplied by the small HMG-box protein Nhp6. In yeast, this "detachable" HMG domain is a general chromatin component, unlike FACT, which is found only in transcribed regions and associated with RNA polymerase II. The several domains of the larger FACT subunit are also likely to have different functions. Genetic studies suggest that FACT mediates nucleosome reorganization along several pathways, and reinforce the notion that protein unfolding and (or) refolding is involved in FACT activity for transcription.Key words: nucleosomes, transcription, FACT, yeast, genetics.

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A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification

Blood ◽

10.1182/blood-2007-05-090514 ◽

2007 ◽

Vol 110 (13) ◽

pp. 4445-4454 ◽

Cited By ~ 268

Author(s):

Dorothee Mueller ◽

Christian Bach ◽

Deniz Zeisig ◽

Maria-Paz Garcia-Cuellar ◽

Sara Monroe ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Chromatin Modification ◽

Histone H3 ◽

Elongation Factor ◽

Polycomb Group ◽

Fusion Partner ◽

Transcriptional Elongation ◽

Associated Proteins ◽

Group Members

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79–specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

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Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.2970-2983.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 2970-2983 ◽

Cited By ~ 141

Author(s):

Dmitri Ivanov ◽

Youn Tae Kwak ◽

Jun Guo ◽

Richard B. Gaynor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Transcriptional Elongation ◽

Tat Protein ◽

C Terminus ◽

Heptad Repeats ◽

And Function ◽

Hiv 1

ABSTRACT SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.

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Faculty Opinions recommendation of Histone H2A monoubiquitination represses transcription by inhibiting RNA polymerase II transcriptional elongation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1100226.556399 ◽

2008 ◽

Author(s):

Cheng-Ming Chiang

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Elongation ◽

Histone H2a

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Interaction between P-TEFb and the C-Terminal Domain of RNA Polymerase II Activates Transcriptional Elongation from Sites Upstream or Downstream of Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.321-331.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 321-331 ◽

Cited By ~ 76

Author(s):

Ran Taube ◽

Xin Lin ◽

Dan Irwin ◽

Koh Fujinaga ◽

B. Matija Peterlin

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Target Genes ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Cyclin T1 ◽

Positive Transcription Elongation Factor ◽

Terminal Domain ◽

Activation Of Transcription

ABSTRACT Transcriptional elongation by RNA polymerase II (RNAPII) is regulated by the positive transcription elongation factor b (P-TEFb). P-TEFb is composed of Cdk9 and C-type cyclin T1 (CycT1), CycT2a, CycT2b, or CycK. The role of the C-terminal region of CycT1 and CycT2 remains unknown. In this report, we demonstrate that these sequences are essential for the activation of transcription by P-TEFb via DNA, i.e., when CycT1 is tethered upstream or downstream of promoters and coding sequences. A histidine-rich stretch, which is conserved between CycT1 and CycT2 in this region, bound the C-terminal domain of RNAPII. This binding was required for the subsequent expression of full-length transcripts from target genes. Thus, P-TEFb could mediate effects of enhancers on the elongation of transcription.

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Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5433 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5433-5441 ◽

Cited By ~ 62

Author(s):

B Y Ahn ◽

P D Gershon ◽

E V Jones ◽

B Moss

Keyword(s):

Rna Polymerase ◽

Vaccinia Virus ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Viral Rna ◽

In Vitro Translation ◽

Virus Protein ◽

Transcriptional Elongation

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Runx1 Binds Positive Transcription Elongation Factor b and Represses Transcriptional Elongation by RNA Polymerase II: Possible Mechanism of CD4 Silencing

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.10675-10683.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 10675-10683 ◽

Cited By ~ 34

Author(s):

Huimin Jiang ◽

Fan Zhang ◽

Takeshi Kurosu ◽

B. Matija Peterlin

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Immunoprecipitation ◽

T Cell Development ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Positive Transcription Elongation Factor ◽

Transcription Elongation Factor ◽

Inhibitory Domain

ABSTRACT Runx1 binds the silencer and represses CD4 transcription in immature thymocytes. In this study, we found that Runx1 inhibits P-TEFb, which contains CycT1, CycT2, or CycK and Cdk9 and stimulates transcriptional elongation by RNA polymerase II (RNAPII) in eukaryotic cells. Indeed, its inhibitory domain, spanning positions 371 to 411, not only bound CycT1 but was required for silencing CD4 transcription in vivo. Our chromatin immunoprecipitation assays revealed that Runx1 inhibits the elongation but not initiation of transcription and that RNAPII is engaged at the CD4 promoter but is unable to elongate in CD4− CD8+ thymoma cells. These results suggest that active repression by Runx1 occurs by blocking the elongation by RNAPII, which may contribute to CD4 silencing during T-cell development.

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ELL and EAF1 are Cajal Body Components That Are Disrupted in MLL-ELL Leukemia

Molecular Biology of the Cell ◽

10.1091/mbc.e02-07-0394 ◽

2003 ◽

Vol 14 (4) ◽

pp. 1517-1528 ◽

Cited By ~ 28

Author(s):

Paul E. Polak ◽

Federico Simone ◽

Joseph J. Kaberlein ◽

Roger T. Luo ◽

Michael J. Thirman

Keyword(s):

Rna Polymerase Ii ◽

Elongation Factor ◽

Cajal Body ◽

Leukemia Cells ◽

Physical Interaction ◽

Transcriptional Elongation ◽

Direct Role ◽

Pol Ii ◽

Active Transcription

The (11;19)(q23;p13.1) translocation in acute leukemia results in the formation of a chimeric MLL-ELL fusion protein. ELL is an RNA Polymerase II (Pol II) transcriptional elongation factor that interacts with the recently identified EAF1 protein. Here, we show that ELL and EAF1 are components of Cajal bodies (CBs). Although ELL and EAF1 colocalize with p80 coilin, the signature protein of CBs, ELL and EAF1 do not exhibit a direct physical interaction with p80 coilin. Treatment of cells with actinomycin D, DRB, or α-amanitin, specific inhibitors of Pol II, disperses ELL and EAF1 from CBs, indicating that localization of ELL and EAF1 in CBs is dependent on active transcription by Pol II. The concentration of ELL and EAF1 in CBs links the transcriptional elongation activity of ELL to the RNA processing functions previously identified in CBs. Strikingly, CBs are disrupted in MLL-ELL leukemia. EAF1 and p80 coilin are delocalized from CBs in murine MLL-ELL leukemia cells and in HeLa cells transiently transfected with MLL-ELL. Nuclear and cytoplasmic fractionation revealed diminished expression of p80 coilin and EAF1 in the nuclei of MLL-ELL leukemia cells. These studies are the first demonstration of a direct role of CB components in leukemogenesis.

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