The Spt4, Spt5, and Spt6 proteins are conserved eukaryotic transcription-elongation factors. Recent studies have provided the first evidence that they are generally required in multicellular eukaryotes, including during development and for viral gene expression.Molecular and Cellular Biolog

Winston, Fred Marshall

Genome Biology

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 Control of Eukaryotic Transcription Elongation  (Article begins on next page)The Harvard community has made this article openly available.Please share how this access benefits you. Your story matters.Citation Winston, Fred. 2001. Control of eukaryotic transcriptionelongation. Genome Biology 2(2): reviews1006.1-reviews1006.3.Published Version http://genomebiology.com/2001/2/2/reviews/1006.1Accessed February 18, 2015 10:46:12 PM ESTCitable Link http://nrs.harvard.edu/urn-3:HUL.InstRepos:4506287Terms of Use This article was downloaded from Harvard University's DASHrepository, and is made available under the terms and conditionsapplicable to Other Posted Material, as set forth athttp://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAAhttp://genomebiology.com/2001/2/2/reviews/1006.1commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchMinireviewControl of eukaryotic transcription elongationFred WinstonAddress: Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. E-mail: winston@rascal.med.harvard.eduAbstractThe Spt4, Spt5, and Spt6 proteins are conserved eukaryotic transcription-elongation factors. Recentstudies have provided the first evidence that they are generally required in multicellular eukaryotes,including during development and for viral gene expression. Published: 31 January 2001Genome Biology 2001, 2(2):reviews1006.1–1006.3The electronic version of this article is the complete one and can befound online at http://genomebiology.com/2001/2/2/reviews/1006© BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914)It has been clear for over 20 years that the control of tran-scription elongation is a prominent mechanism of gene regu-lation  in  prokaryotes.  In  eukaryotes,  although  this  level  ofregulation was clearly demonstrated in 1988 [1], it is mainlyover the past five years that transcription elongation has blos-somed into a broadly active area of investigation [2,3]. Forexample, several recent studies of the human immunodefi-ciency virus (HIV) Tat protein, a transcriptional activator thatstimulates transcription past a pause site, have helped us tounderstand factors required for elongation [4,5]. Three transcription-elongation factors that have emerged asplaying critical roles are the Spt4, Spt5, and Spt6 proteins.These three proteins, conserved among eukaryotes, were ini-tially discovered by genetic studies in Saccharomyces cere-visiae  [6].  Further  studies  in  S.  cerevisiae  suggested  thatSpt4, Spt5, and Spt6 are essential for transcription via themodulation of chromatin structure [7,8]. Spt4 and Spt5 aretightly associated in a complex, while the physical associa-tion of the Spt4-Spt5 complex with Spt6 appears to be con-siderably weaker [9,10]. Evidence over the past two yearshas  demonstrated  that  Spt4,  Spt5,  and  Spt6  play  roles  intranscription elongation in both yeast and humans [9,10],including a role in activation by Tat [11]. The breadth of therequirement for these factors in multicellular eukaryotes wasnot known, however. Now, four papers [12-15] have illumi-nated the roles of these factors in vivo. These new studies,taken  together  with  earlier  work,  portray  Spt4,  Spt5,  andSpt6 as general transcription-elongation factors, controllingtranscription  both  positively  and  negatively  in  importantregulatory and developmental roles. Spt4, Spt5, and Spt6 have characteristics ofgeneral transcription-elongation factorsTwo recent studies [12,13] of Spt5 and Spt6 have providedthe first view of their roles in vivo in a multicellular eukary-ote, in this case Drosophila melanogaster. In both studies,specific antisera were used to determine the localization ofSpt5  and  Spt6  on  polytene  chromosomes.  Both  studiesdemonstrated that Spt5 and Spt6 colocalize to hundreds ofsites  on  polytene  chromosomes  and  that  this  localizationstrikingly coincides with that of actively transcribing RNApolymerase II (Pol II), Pol IIo (Figure 1). This correlationbetween  the  Spt  proteins  and  active  transcription  wasstrengthened  by  the  demonstration  that  the  induction  ofheat shock (hsp) gene transcription causes massive recruit-ment of both Spt5 and Spt6 to hsp loci. These results, then,show  that  Spt5  and  Spt6  are  associated  with  virtually  allactive transcription, strongly suggesting a general positiverole for these factors. Evidence  for  a  negative  role,  although  less  general,  camefrom  the  colocalization  between  Spt5,  Spt6  and  the  non-transcribing form of Pol II, Pol IIa, at a small subset of poly-tene positions [12]. Most of these positions correspond tothe Salivary gland secreted (Sgs) loci, and the colocalizationresults  suggest  that  the  Sgs genes  are  regulated  by  Spt5,Spt6, and perhaps by a transcriptional pause, similar to hspgenes [12]. In addition, Spt5, but not Spt6, is present at theuninduced  hsp70 locus,  where  Pol IIa  is  paused  approxi-mately 25 nucleotides from the initiation site [12,13]. As wellas suggesting a negative role for Spt5, this result indicatesthat Spt5 and Spt6 do not play identical roles in vivo. While2 Genome Biology Vol 2 No 2 Winstonthe other member of this triad of proteins, Spt4, was notdirectly examined in either of these studies [12,13], its strongphysical association with Spt5 suggests that Spt4 is likely tobe similarly localized. These results, suggesting negative aswell as positive control by the Spt proteins in vivo, are con-sistent with earlier in vitro results [10,16].In addition to the genome-wide view provided by the poly-tene  chromosome  studies,  higher-resolution  analysis  hasprovided additional insights about Spt5 and Spt6 [13]. Chro-matin immunoprecipitation analysis revealed that Spt5 andSpt6 are bound within the open reading frames of heat shock(hsp) genes after heat shock induction, providing strong evi-dence for a direct role in transcription elongation. These twopapers  [12,13],  then,  provided  strong  evidence  for  generalroles  for  Spt4,  Spt5,  and  Spt6  in  elongation. In  futurestudies,  genetic  analysis  of  Spt4,  Spt5,  and  Spt6  inD. melanogaster should  provide  important  insights  intoboth their positive and negative roles in vivo. A role for Spt5 in neuronal development inzebrafishAn intriguing result emerged from recent studies of foggy, azebrafish mutant identified in a screen for mutations thatcause neuronal development defects [14]. The foggy muta-tion  causes  a  previously  unidentified  pattern  of  neuronaldefect: a reduction of dopamine-containing neurons and acorresponding increase in serotonin-containing neurons inthe hypothalamus. Positional cloning and sequence analysisrevealed  that  foggy encodes  the  zebrafish  Spt5  homolog.Thus,  a  mutation  that  impairs  a  transcription-elongationfactor  causes  a  novel  neuronal  developmental  defect.  Bio-chemical analysis of the mutant Foggy/Spt5 protein demon-strated a defect in its ability to negatively control elongationin vitro, suggesting that the foggy mutation allows increasedexpression of particular genes in vivo, thereby causing thisspecific neuronal defect [14]. Similarly, a developmental rolefor Spt6 had been previously suggested on the basis of thedevelopmental  defects  of  a  Caenorhabditis  elegans spt6(emb-5) mutant during gastrulation [17].Studies of the foggy mutant raise the issue of the roles ofgeneral  factors  in  developmental  control.  Is  the  role  ofFoggy/Spt5 in zebrafish dedicated to specific aspects of neu-ronal  development,  or  does  the  foggy mutant  phenotypereflect a particular type of defect in a general factor? On thebasis  of  other  studies  of  Spt5,  the  latter  possibility  seemsmore likely. The foggy mutation also causes cardiovasculardefects  [18],  indicating  that  this  mutation  is  pleiotropic,albeit  still  highly  specific  in  the  neuronal  defects  that  itcauses. How might a mutation that alters a general elonga-tion factor cause specific neuronal defects? One possibility isthat  particular  genes  have  a  stronger  requirement  forFoggy/Spt5 than do others: a mutation that mildly reducesFoggy/Spt5  activity  might  affect  a  small  subset  of  genes,causing  specific  mutant  phenotypes.  Consistent  with  thisidea, reduced levels of Pol II in S. cerevisiae cause specificmutant  phenotypes  [19].  A  second  possibility  is  thatFoggy/Spt5 interacts with several gene-specific transcriptionfactors and the foggy mutation impairs only an interactionbetween Spt5 and a neuronal-specific transcription factor.This idea is supported by the demonstration of allele-specificphenotypes among different  spt5 mutants in  S. cerevisiae[9]. Allele-specific developmental defects have been identi-fied  for  mutants  of  general  factors,  including  Pol II  (forexamples, see [20,21]). It would be of great interest to iden-tify additional foggy alleles and study their effect on neu-ronal  development.  The  identification  of  proteins  thatinteract  with  Foggy/Spt5  might  identify  neuron-specifictranscription-elongation factors. Human cytomegalovirus and Spt6The  fourth  recent  study  [15]  concerns  the  humancytomegalovirus (CMV) activator pUL69. This regulator isrequired for transcription of several CMV genes [22] and isalso  responsible  for  arresting  CMV-infected  cells  in  G1phase of the cell cycle [23]. To learn more about the func-tion  of  pUL69,  a  two-hybrid  protein-protein  interactionscreen was performed to identify pUL69-interacting pro-teins, and human Spt6 was identified. Additional experi-ments  provide  compelling  evidence  that  pUL69  isphysically associated with Spt6 in CMV-infected cells. Thebiological relevance of this interaction was established bythe  complete  correlation  between  pUL69  mutations  thatimpair interaction with Spt6 and those that impair pUL69-dependent transcriptional activation. How might an interaction with Spt6 help explain transcrip-tional activation by pUL69? One model comes from experi-ments  that  addressed  the  interaction  of  human  Spt6  withhistones. Previous studies of yeast Spt6 had demonstratedFigure 1Colocalization of (a) Spt5 and (b) Spt6 with Pol IIoser2(Pol IIo phosphorylated on Ser2) on Drosophila polytenechromosomes. Spt5 and Spt6 are red, Pol IIoser2 is green andcolocalization is shown as yellow. Polytene positions 74EFand 75B are indicated. Adapted with permission from [12].commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchhttp://genomebiology.com/2001/2/2/reviews/1006.3an Spt-histone interaction in vitro [8]. In the current study[15], the authors have demonstrated a human Spt6-histoneH3 interaction and show that this interaction is antagonizedby pUL69. These results suggest that activation by pUL69occurs by overcoming the repression of transcription causedby an Spt6-histone interaction. Of interest with respect tothe models discussed above for the effects of Foggy/Spt5 inthe zebrafish, this work demonstrates that Spt6 specificallyinteracts with a particular transcriptional activator. Earlierwork  suggested  that  Spt6  also  interacts  with  the  estrogenreceptor [24]. Spt6 may, therefore, interact with many gene-specific transcription factors. The pUL69 studies also raiseinteresting and related issues. First, does the pUL69-Spt6interaction serve to control transcription initiation or elon-gation? Second, what directs the association of Spt6 with theCMV genome after infection? Third, does Spt6 play a role inall  pUL69-dependent  transcription?  Finally,  do  Spt4  andSpt5 also play a role in pUL69 activation? The answers tothese questions will undoubtedly teach us more about Spt4,Spt5, and Spt6.The recent studies discussed in this article portray Spt4, Spt5,and Spt6 as general elongation factors that are likely to havemany interactions with other factors in vivo, including Pol II,histones,  and  gene-specific  transcription  factors.  Futurestudies  will  need  to  address  the  distinctions  between  theirpositive and negative roles, the individual roles of Spt4, Spt5,and Spt6, and their possible regulation during development. AcknowledgementsWork from my laboratory is supported by grants from the National Insti-tutes of Health. References1. Rougvie AE, Lis JT: The RNA polymerase II molecule at the 5’end of the uninduced hsp70 gene of D. melanogaster is tran-scriptionally engaged. Cell 1988, 54:795-804.2. Conaway JW, Shilatifard A, Dvir A, Conaway RC: Control of elon-gation by RNA polymerase II. Trends Biochem Sci 2000, 25:375-380.3. Orphanides G, Reinberg D: RNA polymerase II elongationthrough chromatin. Nature 2000, 407:471-475.4. Price DH: P-TEFb, a cyclin-dependent kinase controllingelongation by RNA polymerase II. Mol Cell Biol 2000, 20:2629-2634.5. Jones KA: Taking a new TAK on tat transactivation. Genes Dev1997, 11:2593-2599.6. Winston F, Chaleff DT, Valent B, Fink GR: Mutations affectingTy-mediated expression of the HIS4 gene of Saccharomycescerevisiae. Genetics 1984, 107:179-197.7. Swanson MS, Winston F: SPT4, SPT5 and  SPT6 interactions:effects on transcription and viability in Saccharomyces cere-visiae. Genetics 1992, 132:325-336.8. Bortvin A, Winston F: Evidence that Spt6p controls chromatinstructure by a direct interaction with histones. Science 1996,272:1473-1476.9. Hartzog GA, Wada T, Handa H, Winston F: Evidence that Spt4,Spt5, and Spt6 control transcription elongation by RNApolymerase II in Saccharomyces cerevisiae. Genes Dev 1998,12:357-369.10. Wada T, Takagi T, Yamaguchi Y, Ferdous A, Imai T, Hirose S, Sugi-moto S, Yano K, Hartzog GA, Winston F, et al.: DSIF, a novel tran-scription elongation factor that regulates RNA polymeraseII processivity, is composed of human Spt4 and Spt5homologs. Genes Dev 1998, 12:343-356.11. Wu-Baer F, Lane WS, Gaynor RB: Role of the human homologof the yeast transcription factor SPT5 in HIV- 1 Tat-activa-tion. J Mol Biol 1998, 277:179-197.12. Kaplan CD, Morris JR, Wu Ct, Winston F: Spt5 and Spt6 areassociated with active transcription and have characteristicsof general elongation factors in D. melanogaster. Genes Dev2000, 14:2623-2634.13. Andrulis ED, Guzman E, Doring P, Werner J, Lis JT: High-resolu-tion localization of Drosophila Spt5 and Spt6 at heat shockgenes in vivo: roles in promoter proximal pausing and tran-scription elongation. Genes Dev 2000, 14:2635-2649.14. Guo S, Yamaguchi Y, Schilbach S, Wada T, Lee J, Goddard A, FrenchD, Handa H, Rosenthal A: A regulator of transcriptional elon-gation controls vertebrate neuronal development. Nature2000, 408:366-369.15. Winkler M, aus Dem Siepen T, Stamminger T: Functional interac-tion between pleiotropic transactivator pUL69 of humancytomegalovirus and the human homolog of yeast chro-matin regulatory protein SPT6. J Virol 2000, 74:8053-8064.16. Wada T, Takagi T, Yamaguchi Y, Watanabe D, Handa H: Evidencethat P-TEFb alleviates the negative effect of DSIF on RNApolymerase II-dependent transcription in vitro. EMBO J 1998,17:7395-7403.17. Nishiwaki K, Sano T, Miwa J: emb-5, a gene required for thecorrect timing of gut precursor cell division during gastrula-tion in Caenorhabditis elegans, encodes a protein similar tothe yeast nuclear protein SPT6. Mol Gen Genet 1993, 239:313-322.18. Guo S, Wilson SW, Cooke S, Chitnis AB, Driever W, Rosenthal A:Mutations in the zebrafish unmask shared regulatory path-ways controlling the development of catecholaminergicneurons. Dev Biol 1999, 208:473-487.19. Archambault J, Jansma DB, Friesen JD: Underproduction of thelargest subunit of RNA polymerase II causes temperaturesensitivity, slow growth, and inositol auxotrophy in Saccha-romyces cerevisiae. Genetics 1996, 142:737-747.20. Parkhurst SM, Ish-Horowicz D: wimp, a dominant maternal-effect mutation, reduces transcription of a specific subset ofsegmentation genes in Drosophila. Genes Dev 1991, 5:341-357.21. Mortin MA, Kaufman TC: Developmental effects of a tempera-ture-sensitive RNA polymerase II mutation in Drosophilamelanogaster. Dev Biol 1984, 103:343-354.22. Winkler M, Rice SA, Stamminger T: UL69 of humancytomegalovirus, an open reading frame with homology toICP27 of herpes simplex virus, encodes a transactivator ofgene expression. J Virol 1994, 68:3943-3954.23. Lu M, Shenk T: Human cytomegalovirus UL69 proteininduces cells to accumulate in G1 phase of the cell cycle. JVirol 1999, 73:676-683.24. Baniahmad C, Nawaz Z, Baniahmad A, Gleeson MA, Tsai MJ, O’Mal-ley BW: Enhancement of human estrogen receptor activityby SPT6: a potential coactivator. Mol Endocrinol 1995, 9:34-43.

Control of Eukaryotic Transcription Elongation

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Control of Eukaryotic Transcription Elongation

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