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

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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Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

Molecular and Cellular Biology ◽

10.1128/mcb.00857-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4641-4651 ◽

Cited By ~ 40

Author(s):

Junjiang Fu ◽

Ho-Geun Yoon ◽

Jun Qin ◽

Jiemin Wong

Keyword(s):

Rna Polymerase Ii ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Elongation Complex ◽

Complex Activity ◽

Interacting Protein ◽

Pol Ii ◽

Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

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The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency

Genetics Research International ◽

10.4061/2011/726901 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Kyle A. Nilson ◽

David H. Price

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Health Concern ◽

Pol Ii ◽

Positive Transcription Elongation Factor ◽

Elongation Control ◽

Hiv 1 ◽

Latent Phase

HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR) is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II), leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.

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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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Cross-talk of GATA-1 and P-TEFb in megakaryocyte differentiation

Blood ◽

10.1182/blood-2008-03-145722 ◽

2008 ◽

Vol 112 (13) ◽

pp. 4884-4894 ◽

Cited By ~ 30

Author(s):

Kamaleldin E. Elagib ◽

Ivailo S. Mihaylov ◽

Lorrie L. Delehanty ◽

Grant C. Bullock ◽

Kevin D. Ouma ◽

...

Keyword(s):

Down Syndrome ◽

Rna Polymerase Ii ◽

Cross Talk ◽

Elongation Factor ◽

Cardiomyocyte Hypertrophy ◽

Physical Interaction ◽

Transcriptional Elongation ◽

Developmental Abnormalities ◽

Megakaryocytic Differentiation ◽

Cyclin T1

Abstract The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclin-dependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy.

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Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.777-788.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 777-788 ◽

Cited By ~ 39

Author(s):

Yi Pei ◽

Hongyan Du ◽

Juliet Singer ◽

Courtney St. Amour ◽

Selena Granitto ◽

...

Keyword(s):

Fission Yeast ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Growth Defect ◽

Transcriptional Elongation ◽

Cyclin Dependent Kinase ◽

Elongation Complex ◽

Pol Ii

ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.

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HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia

Nature Communications ◽

10.1038/s41467-021-27944-8 ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Yongkang Yang ◽

Haiquan Lu ◽

Chelsey Chen ◽

Yajing Lyu ◽

Robert N. Cole ◽

...

Keyword(s):

Breast Cancer ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Transcription ◽

Target Genes ◽

Large Subunit ◽

Hypoxia Inducible Factor ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Pol Ii

AbstractHypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30–60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

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PTEN modulates gene transcription by redistributing genome-wide RNA polymerase II occupancy

Human Molecular Genetics ◽

10.1093/hmg/ddz112 ◽

2019 ◽

Vol 28 (17) ◽

pp. 2826-2834 ◽

Cited By ~ 3

Author(s):

Ata Abbas ◽

Roshan Padmanabhan ◽

Todd Romigh ◽

Charis Eng

Keyword(s):

Gene Regulation ◽

Transcriptional Regulation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Control Of Gene Expression ◽

Pol Ii ◽

Genome Wide ◽

Pol Ii Pausing

Abstract Control of gene expression is one of the most complex yet continuous physiological processes impacting cellular homeostasis. RNA polymerase II (Pol II) transcription is tightly regulated at promoter-proximal regions by intricate dynamic processes including Pol II pausing, release into elongation and premature termination. Pol II pausing is a phenomenon where Pol II complex pauses within 30–60 nucleotides after initiating the transcription. Negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) contribute in the establishment of Pol II pausing, and positive transcription elongation factor b releases (P-TEFb) paused complex after phosphorylating DSIF that leads to dissociation of NELF. Pol II pausing is observed in most expressed genes across the metazoan. The precise role of Pol II pausing is not well understood; however, it’s required for integration of signals for gene regulation. In the present study, we investigated the role of phosphatase and tensin homolog (PTEN) in genome-wide transcriptional regulation using PTEN overexpression and PTEN knock-down models. Here we identify that PTEN alters the expression of hundreds of genes, and its restoration establishes genome-wide Pol II promoter-proximal pausing in PTEN null cells. Furthermore, PTEN re-distributes Pol II occupancy across the genome and possibly impacts Pol II pause duration, release and elongation rate in order to enable precise gene regulation at the genome-wide scale. Our observations demonstrate an imperative role of PTEN in global transcriptional regulation that will provide a new direction to understand PTEN-associated pathologies and its management.

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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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Subnuclear Localization and Cajal Body Targeting of Transcription Elongation Factor TFIIS in Amphibian Oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e02-09-0601 ◽

2003 ◽

Vol 14 (3) ◽

pp. 1255-1267 ◽

Cited By ~ 9

Author(s):

Abigail J. Smith ◽

Yan Ling ◽

Garry T. Morgan

Keyword(s):

Rna Polymerase Ii ◽

Transcription Elongation ◽

Elongation Factor ◽

Cajal Body ◽

Cajal Bodies ◽

Site Specific ◽

Pol Ii ◽

Subnuclear Localization ◽

Transcription Elongation Factor ◽

Multiple Regions

We have examined the localization and targeting of the RNA polymerase II (pol II) transcription elongation factor TFIIS in amphibian oocyte nuclei by immunofluorescence. Using a novel antibody against Xenopus TFIIS the major sites of immunostaining were found to be Cajal bodies, nuclear organelles that also contain pol II. Small granular structures attached to lampbrush chromosomes were also specifically stained but the transcriptionally active loops were not. Similar localization patterns were found for the newly synthesizedmyc-tagged TFIIS produced after injection of synthetic transcripts into the cytoplasm. The basis of the rapid and preferential targeting of TFIIS to Cajal bodies was investigated by examining the effects of deletion and site-specific mutations. Multiple regions of TFIIS contributed to efficient targeting including the domain required for its binding to pol II. The localization of TFIIS in Cajal bodies, and in particular the apparent involvement of pol II binding in achieving it, offer further support for a model in which Cajal bodies function in the preassembly of the transcriptional machinery. Although our findings are therefore consistent with TFIIS playing a role in early events of the transcription cycle, they also suggest that this elongation factor is not generally required during transcription in oocytes.

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The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes

Scientific Reports ◽

10.1038/s41598-020-80650-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marina Yu. Mazina ◽

Elena V. Kovalenko ◽

Nadezhda E. Vorobyeva

Keyword(s):

Rna Polymerase Ii ◽

Target Genes ◽

Elongation Factor ◽

Transcriptional Response ◽

Pol Ii ◽

A Subunit ◽

Active Transcription ◽

Pol Ii Pausing ◽

Negative Elongation Factor ◽

Regulatory Sites

AbstractFor many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). The complete NELF complex is formed at the 20E-dependent promoters and participates in both their induced transcriptional response and maintenance of the uninduced state to keep them ready for the forthcoming transcription. NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, we show the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex.

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