Home LiteratureArticle Details
PMID: 19742326 Published · epublish English Journal Article Research Support, N.I.H., Extramural

Repression of RNA polymerase II elongation in vivo is critically dependent on the C-terminus of Spt5.

PloS one ·Vol. 4 ·No. 9 ·2009-09-09 ·Pages e6918

Chen H, Contreras X, Yamaguchi Y, Handa H, Peterlin BM, Guo S

Abstract

The stalling of RNA polymerase II (RNAPII) at the promoters of many genes, including developmental regulators, stress-responsive genes, and HIVLTR, suggests transcription elongation as a critical regulatory step in addition to initiation. Spt5, the large subunit of the DRB sensitivity-inducing factor (DSIF), represses or activates RNAPII elongation in vitro. How RNAPII elongation is repressed in vivo is not well understood. Here we report that CTR1 and CTR2CT, the two repeat-containing regions constituting the C-terminus of Spt5, play a redundant role in repressing RNAPII elongation in vivo. First, mis-expression of Spt5 lacking CTR1 or CTR2CT is inconsequential, but mis-expression of Spt5 lacking the entire C-terminus (termed NSpt5) dominantly impairs embryogenesis in zebrafish. Second, NSpt5 de-represses the transcription of hsp70-4 in zebrafish embryos and HIVLTR in cultured human cells, which are repressed at the RNAPII elongation step under non-inducible conditions. Third, NSpt5 directly associates with hsp70-4 chromatin in vivo and increases the occupancy of RNAPII, positive transcription elongation factor b (P-TEFb), histone H3 Lys 4 trimethylation (H3K4Me3), and surprisingly, the negative elongation factor A (NELF-A) at the locus, indicating a direct action of NSpt5 on the elongation repressed locus. Together, these results reveal a dominant activity of NSpt5 to de-repress RNAPII elongation, and suggest that the C-terminus of Spt5 is critical for repressing RNAPII elongation in vivo.

MeSH Terms
Animals Chromatin/chemistry Chromosomal Proteins, Non-Histone/chemistry,physiology Gene Expression Regulation, Enzymologic HSP70 Heat-Shock Proteins/chemistry HeLa Cells Heat-Shock Proteins/chemistry Histones/chemistry Humans Models, Biological Promoter Regions, Genetic Protein Structure, Tertiary RNA Polymerase II/antagonists & inhibitors Transcription, Genetic Transcriptional Elongation Factors/chemistry,physiology Zebrafish
Chemicals
Chromatin Chromosomal Proteins, Non-Histone HSP70 Heat-Shock Proteins Heat-Shock Proteins Histones Transcriptional Elongation Factors SPT5 transcriptional elongation factor RNA Polymerase II
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Chen Hui
Department of Biopharmaceutical Sciences, and Programs in Biological Sciences and Human Genetics, University of California San Francisco, San Francisco, California, United States of America.
Contreras Xavier
Yamaguchi Yuki
Handa Hiroshi
Peterlin B Matija
Guo Su
References (55)
55 references, click to expand
  1. A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS.
    Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15036-41 PMID: 16214896
  2. A regulator of transcriptional elongation controls vertebrate neuronal development.
    Nature. 2000 Nov 16;408(6810):366-9 PMID: 11099044
  3. A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation.
    PLoS Biol. 2005 Feb;3(2):e76 PMID: 15719065
  4. The elongation factors Pandora/Spt6 and Foggy/Spt5 promote transcription in the zebrafish embryo.
    Development. 2002 Apr;129(7):1623-32 PMID: 11923199
  5. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product.
    Nature. 1987 Dec 3-9;330(6147):489-93 PMID: 2825027
  6. Controlling the elongation phase of transcription with P-TEFb.
    Mol Cell. 2006 Aug 4;23(3):297-305 PMID: 16885020
  7. Positive transcription elongation factor B phosphorylates hSPT5 and RNA polymerase II carboxyl-terminal domain independently of cyclin-dependent kinase-activating kinase.
    J Biol Chem. 2001 Apr 13;276(15):12317-23 PMID: 11145967
  8. Zebrafish foggy/spt 5 is required for migration of facial branchiomotor neurons but not for their survival.
    Dev Dyn. 2005 Nov;234(3):651-8 PMID: 16193504
  9. Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster.
    Genes Dev. 2000 Oct 15;14(20):2623-34 PMID: 11040216
  10. Role of the human homolog of the yeast transcription factor SPT5 in HIV-1 Tat-activation.
    J Mol Biol. 1998 Mar 27;277(2):179-97 PMID: 9514752
  11. Npl3 is an antagonist of mRNA 3' end formation by RNA polymerase II.
    EMBO J. 2005 Jun 15;24(12):2150-60 PMID: 15902270
  12. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation.
    Cell. 1999 Apr 2;97(1):41-51 PMID: 10199401
  13. Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5.
    Retrovirology. 2004 Dec 27;1:46 PMID: 15620346
  14. Locus-specific requirements for Spt5 in transcriptional activation and repression in Drosophila.
    Curr Biol. 2004 Sep 21;14(18):1680-4 PMID: 15380072
  15. NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila.
    Mol Cell Biol. 2008 May;28(10):3290-300 PMID: 18332113
  16. The peptidyl-prolyl isomerase Pin1 interacts with hSpt5 phosphorylated by Cdk9.
    J Mol Biol. 2001 Sep 28;312(4):675-85 PMID: 11575923
  17. Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 protein kinase: Spt5 phosphorylation, autophosphorylation, and mutational analysis.
    J Biol Chem. 2003 Oct 31;278(44):43346-56 PMID: 12904290
  18. Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae.
    Mol Gen Genet. 1993 Mar;237(3):449-59 PMID: 8483459
  19. Laser-induced gene expression in specific cells of transgenic zebrafish.
    Development. 2000 May;127(9):1953-60 PMID: 10751183
  20. Transcription elongation factor hSPT5 stimulates mRNA capping.
    Genes Dev. 1999 Jul 15;13(14):1774-9 PMID: 10421630
  21. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.
    Nature. 2007 Aug 2;448(7153):553-60 PMID: 17603471
  22. Promoter melting and TFIID complexes on Drosophila genes in vivo.
    Genes Dev. 1992 Nov;6(11):2190-200 PMID: 1427079
  23. CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo.
    Genes Dev. 2002 Aug 15;16(16):2135-46 PMID: 12183367
  24. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.
    Mol Cell Biol. 1986 Nov;6(11):3984-9 PMID: 3099167
  25. Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro.
    EMBO J. 1998 Dec 15;17(24):7395-403 PMID: 9857195
  26. Structure and function of the human transcription elongation factor DSIF.
    J Biol Chem. 1999 Mar 19;274(12):8085-92 PMID: 10075709
  27. In vivo evidence that defects in the transcriptional elongation factors RPB2, TFIIS, and SPT5 enhance upstream poly(A) site utilization.
    Mol Cell Biol. 2003 Nov;23(21):7887-901 PMID: 14560031
  28. SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.
    Mol Cell Biol. 1991 Jun;11(6):3009-19 PMID: 1840633
  29. Heat shock gene expression and function during zebrafish embryogenesis.
    Semin Cell Dev Biol. 2003 Oct;14(5):267-74 PMID: 14986856
  30. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element.
    Mol Cell Biol. 2004 Jan;24(2):787-95 PMID: 14701750
  31. NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila.
    Genes Dev. 2003 Jun 1;17(11):1402-14 PMID: 12782658
  32. Stages of embryonic development of the zebrafish.
    Dev Dyn. 1995 Jul;203(3):253-310 PMID: 8589427
  33. Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation.
    Mol Cell Biol. 2002 Jul;22(13):4622-37 PMID: 12052871
  34. A chromatin landmark and transcription initiation at most promoters in human cells.
    Cell. 2007 Jul 13;130(1):77-88 PMID: 17632057
  35. Breaking barriers to transcription elongation.
    Nat Rev Mol Cell Biol. 2006 Aug;7(8):557-67 PMID: 16936696
  36. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.
    Nat Genet. 2007 Dec;39(12):1512-6 PMID: 17994019
  37. RNA polymerase is poised for activation across the genome.
    Nat Genet. 2007 Dec;39(12):1507-11 PMID: 17994021
  38. AIRE recruits P-TEFb for transcriptional elongation of target genes in medullary thymic epithelial cells.
    Mol Cell Biol. 2007 Dec;27(24):8815-23 PMID: 17938200
  39. Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors.
    Cell. 2004 Jan 23;116(2):247-57 PMID: 14744435
  40. Elongation by RNA polymerase II: the short and long of it.
    Genes Dev. 2004 Oct 15;18(20):2437-68 PMID: 15489290
  41. Properties of RNA polymerase II elongation complexes before and after the P-TEFb-mediated transition into productive elongation.
    J Biol Chem. 2007 Jul 27;282(30):21901-12 PMID: 17548348
  42. Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins.
    Mol Cell Biol. 2003 Feb;23(4):1368-78 PMID: 12556496
  43. DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs.
    Genes Dev. 1998 Feb 1;12(3):343-56 PMID: 9450929
  44. Interactions between fission yeast mRNA capping enzymes and elongation factor Spt5.
    J Biol Chem. 2002 May 31;277(22):19639-48 PMID: 11893740
  45. High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation.
    Genes Dev. 2000 Oct 15;14(20):2635-49 PMID: 11040217
  46. Exploring functional relationships between components of the gene expression machinery.
    Nat Struct Mol Biol. 2005 Feb;12(2):175-82 PMID: 15702072
  47. A highly purified RNA polymerase II elongation control system.
    J Biol Chem. 2001 Nov 9;276(45):42601-9 PMID: 11553615
  48. NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation.
    EMBO J. 2006 Jan 11;25(1):139-49 PMID: 16319923
  49. P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation.
    Mol Cell. 2006 Jan 20;21(2):227-37 PMID: 16427012
  50. Domains in the SPT5 protein that modulate its transcriptional regulatory properties.
    Mol Cell Biol. 2000 May;20(9):2970-83 PMID: 10757782
  51. Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.
    PLoS One. 2008;3(11):e3621 PMID: 18978947
  52. Development of noradrenergic neurons in the zebrafish hindbrain requires BMP, FGF8, and the homeodomain protein soulless/Phox2a.
    Neuron. 1999 Nov;24(3):555-66 PMID: 10595509
  53. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote.
    Genes Dev. 2004 Jun 1;18(11):1263-71 PMID: 15175259
  54. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly.
    Genes Dev. 2008 Jul 15;22(14):1921-33 PMID: 18628398
  55. Transcription elongation and human disease.
    Annu Rev Biochem. 1999;68:301-19 PMID: 10872452
Article Info
Journal
PloS one
Abbr.
PLoS One
ISSN
1932-6203
DOI
10.1371/journal.pone.0006918
Published
2009-09-09
Epub
2009-00-09
Pages
e6918
Language
English
Region
United States
NLM ID
101285081
PMCID
PMC2735033
Subset
IM
Grants
NINDS NIH HHS · NS042626 · United States
NIGMS NIH HHS · P50 GM082250 · United States
NIAID NIH HHS · AI49104 · United States
NIGMS NIH HHS · P50 GM082250-02 · United States
NIAID NIH HHS · R01 AI049104 · United States
NINDS NIH HHS · R01 NS042626 · United States
External Links
PubMed source DOI: 10.1371/journal.pone.0006918 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2