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

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4884-4894 ◽  
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.

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

2002 ◽  
Vol 22 (1) ◽  
pp. 321-331 ◽  
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.

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

2003 ◽  
Vol 14 (4) ◽  
pp. 1517-1528 ◽  
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.

scholarly journals MAQ1 and 7SK RNA Interact with CDK9/Cyclin T Complexes in a Transcription-Dependent Manner

2003 ◽  
Vol 23 (14) ◽  
pp. 4859-4869 ◽  
Author(s):  
Annemieke A. Michels ◽  
Van Trung Nguyen ◽  
Alessandro Fraldi ◽  
Valérie Labas ◽  
Mia Edwards ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Specific Interaction ◽  
Elongation Factor ◽  
Virus Genome ◽  
Transcriptional Elongation ◽  
Dependent Manner ◽  
Small Nuclear Rna ◽  
Cyclin T1 ◽  
Terminal Domain ◽  
Immunodeficiency Virus

ABSTRACT Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
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.

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

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.

scholarly journals The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ursula Schulze-Gahmen ◽  
Heather Upton ◽  
Andrew Birnberg ◽  
Katherine Bao ◽  
Seemay Chou ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Regulatory Proteins ◽  
Tat Protein ◽  
Elongation Complex ◽  
Cyclin T1 ◽  
Subunit Interface ◽  
Hiv Tat ◽  
Gene Transcripts ◽  
Hiv 1

Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS.

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

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4445-4454 ◽  
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.

scholarly journals Ubiquitylation of Cdk9 by Skp2 Facilitates Optimal Tat Transactivation

2005 ◽  
Vol 79 (17) ◽  
pp. 11135-11141 ◽  
Author(s):  
Matjaz Barboric ◽  
Fan Zhang ◽  
Mojca Besenicar ◽  
Ana Plemenitas ◽  
B. Matija Peterlin
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Primary Mouse ◽  
Immunodeficiency Virus ◽  
F Box Protein ◽  
Hiv 1

ABSTRACT By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that cyclin-dependent kinase 9 (Cdk9), the catalytic subunit of P-TEFb, is ubiquitylated in vivo. This ubiquitylation depended on the Skp1/Cul1/F-box protein E3 ubiquitin ligase Skp2. Likewise, Tat required Skp2 since its transactivation of the HIV-1 long terminal repeat decreased in primary mouse embryonic fibroblasts, which lacked Skp2. The ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and transactivation response element. Thus, our findings underscore the requirement of ubiquitylation for the coactivator function in regulating HIV-1 transcriptional elongation.

scholarly journals The Recruitment of theSaccharomyces cerevisiaePaf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

2013 ◽  
Vol 33 (16) ◽  
pp. 3259-3273 ◽  
Author(s):  
Manasi K. Mayekar ◽  
Richard G. Gardner ◽  
Karen M. Arndt
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Physical Interaction ◽  
Repeat Domain ◽  
Necessary And Sufficient ◽  
Active Genes ◽  
Interacting Domain

Transcription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR.In vitroexperiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

scholarly journals Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

2009 ◽  
Vol 106 (17) ◽  
pp. 6956-6961 ◽  
Author(s):  
Karen Zhou ◽  
Wei Hung William Kuo ◽  
Jeffrey Fillingham ◽  
Jack F. Greenblatt
Keyword(s):  
Histone Modification ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Consensus Sequence ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Histone H2b ◽  
Paf Complex

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

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