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What is the fate of the nucleosome duringtranscription? This longstanding question1

has been a major focus for researchers work-ing on eukaryotic transcription. Four recentstudies2–5 now describe the involvement oftwo protein factors in facilitating RNA polymerase to overcome the repressivenucleosome barrier during transcriptionalelongation and in restoring it after the polymerase passes through.

Many studies have attempted to address thequestion described above, but their observa-tions and conclusions vary, depending on theexperimental system and the polymeraseemployed. In eukaryotes, RNA polymerase II(Pol II) is responsible for transcribing protein-encoding genes and is most likely toconfront nucleosomes in this process. Forefficient transcript elongation on a chromatintemplate, Pol II requires several additionalfactors. This requirement suggests how thetranscription machinery manages the nucleo-some barrier during the elongation process.

Of the numerous elongation factors thathave been identified, several belong to or arehomologous to a class of yeast genes knownas ‘suppressor of Ty’ (SPT)6. As the namesuggests, these factors affect the activity ofthe Ty transposon, which, when insertedupstream of a promoter, tends to impairtranscription from that promoter. As such,most SPT gene products are somehowinvolved in transcription. The SPT genes aresubdivided into a TATA-binding protein(TBP) group and a histone group. The TBPgroup includes genes encoding TBP itselfand the subunits of the SAGA histone acetyl-transferase complex, whereas the histonegroup include genes encoding the histoneproteins themselves and others whose prod-ucts are involved in histone function. Thefour recent studies2–5 discussed here primar-ily examine the function of two histonegroup members: Spt16, a subunit of the

FACT complex (facilitates chromatin transcription), and Spt6.

Spt16 and the nucleosomeBelotserkovskaya et al.2 took a biochemicalapproach to determine that the FACT com-plex facilitates the elongation reaction ofPol II by displacing the histone H2A–H2Bdimer during the process; they also demon-strated that FACT reassembles the nucleo-some. First, in vitro binding data revealed thathuman Spt16 interacts with the H2A–H2Bhistone dimer and mononucleosomesthrough its acidic C-terminal region. Theauthors then used immobilized mononucleo-somes containing H3–H4 tetramers andH2A–H2B dimers with different fluorescentlabels to demonstrate that FACT could releasethe dimer from nucleosomes. Electrophoreticmobility gel shift analysis (EMSA) onmononucleosome templates transcribed inthe presence of FACT in vitro further showedthat the FACT complex can displace theH2A–H2B dimer from nucleosome templatesundergoing transcription. Finally, EMSA inconjunction with the fluorescent dimers andtetramers provided evidence that FACT couldalso facilitate the formation of nucleosomesfrom a mixture of DNA, H3–H4 tetramersand H2A–H2B dimers. This last observationimplicates Spt16 as a histone chaperone; thisactivity also depended on the acidic C termi-nus of Spt16. Thus, it appears that the FACTcomplex can remove H2A–H2B dimers tofacilitate Pol II in overcoming the nucleosomebarrier. After Pol II transcribes through theregion, FACT may facilitate the restoration ofthe nucleosome. Such a mechanism couldexplain an earlier observation suggesting thatthe exchange of H2A–H2B dimers in chro-matin was enhanced by transcription7.

In a related study, Mason and Struhl5 usedchromatin immunoprecipitation (ChIP) toexamine the relationship between FACT andPol II in yeast. They found that FACT exhibitsa similar pattern of progression through agene as Pol II. Their analysis indicates thatFACT begins its association with a geneimmediately after Pol II has initiated tran-scription. When the activity of Spt16 was

compromised by mutation, the occupancy ofTBP, THIIB and Pol II on several promotersdecreased, while the occupancy of Pol II at the3´ end of several other genes increased. Theauthors further demonstrated that thisincreased Pol II occupancy on the 3´ end ofsome genes is due to transcription initiationfrom cryptic promoters within the codingregion of those genes; they also observedmodest levels of TBP binding to a TATA boxin the coding region of one gene. Mason andStruhl5 suggested that general transcriptionfactors, such as TBP and TFIIB, are present inlimiting amount within the cell. Thus, it ispossible that the reduction of TBP and TFIIBat upstream promoters in the spt16 mutant islikely a result of a redistribution of these lim-iting factors to 3´ cryptic promoters in thecoding regions. Once there, they inappropri-ately establish Pol II transcription initiation.

Spt6 and chromatin maintenanceKaplan et al.3 examined the role of the yeastelongation factor Spt6 in regulating tran-scription. In addition to being an elongationfactor, Spt6 is also known to affect chromatinstructure and interact with histones8. In theirstudy, Kaplan et al.3 found that a spt6 mutantincreased the transcription of several genes ina microarray analysis. A different set ofexperiments revealed that the spt6 mutant ledto aberrant transcription initiation within thecoding regions of several genes, includingFLO8. This observation with FLO8 is similarto what Mason and Struhl5 observed withtheir spt16 mutant. Notably, in the spt6mutant cell, TBP was found to occupy a TATAbox upstream of a cryptic 3´ initiation site inFLO8, and disruption of this TATA box consensus sequence abolished expression ofthe short transcript originating from the 3´initiation site.

To understand the structural changes thatunderlie these observations, Kaplan et al.3

assayed the effects of the spt6 mutant on chro-matin sensitivity to micrococcal nuclease(MNase) digestion. They showed that, in thespt6 mutant cells, the transcribed FLO8 locuswas hypersensitive to MNase, while theuntranscribed GAL1 gene did not display

The authors are at the Stowers Institute forMedical Research, 1000 East 50th Street, KansasCity, Missouri 64110 USA. Correspondenceshould be addressed to M.J.C. (MJC@Stowers-Institute.org)

Repairing nucleosomes during transcriptionMichael J Carrozza, Thomas Kusch & Jerry L Workman

Recent studies suggest that the Spt16 protein of FACT shuttles H2A–H2B dimers off and on nucleosomes during transcriptionelongation. By restoring nucleosomes after passage of RNA polymerase II, Spt16 and Spt6 prevent transcription from crypticpromoters in coding regions that would otherwise be expressed in the absence of histones.

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hypersensitivity. Moreover, a mutation in thegene encoding the largest subunit of Pol II inthe spt6 mutant cells suppressed the FLO8chromatin hypersensitivity. Finally, thishypersensitivity was shown to correlate withthe loss of histone H4 from the nucleosomeson FLO8. Because hypersensitivity at theFLO8 locus depends on active transcription,these observations led the authors to hypoth-esize that Spt6 functions to restore chromatinstructure of a gene following the passage ofPol II. Thus, Spt6 facilitates maintenance ofchromatin structure on the transcribed geneto silence cryptic promoters that could other-wise be expressed as a result of the disruptionof chromatin structure by the elongation

machinery. The loss of histone H4 on FLO8 inthe spt6 mutant cells suggests that the H3–H4tetramer is also sensitive to displacement dur-ing transcription and likely requires restora-tion after passage of Pol II. Transcription-dependent exchange of H3–H4 tetramers canalso explain the fact that a histone H3 variantaccumulates on transcribed genes outside ofS-phase, when the majority of nucleosomeassembly occurs9.

Last but not least, Saunders et al.4 useDrosophila polytene chromosomes to bringtogether the actions of FACT and Spt6. Theyshowed that FACT colocalizes with the tran-scribing Pol II on the heat-inducible hsp70gene following heat shock. Time course stud-

ies using immunofluorescence along withChIPs for Pol II, FACT and Spt6 on the hsp70gene revealed that these factors progressthrough the coding region with overlappinglocalization after heat shock. They alsodemonstrated that another elongation factorSpt5 has the same localization patterns andkinetics of progression as Pol II, FACT andSpt6 on the induced hsp70 coding region. Theresults of Saunders et al.4 and of anotherrecent study10 suggests that Pol II, FACT, Spt6,and Spt5 progress along a gene in the form ofa loosely associated elongation complex.While it is not known how these proteinsmight associate with Pol II, a recent study hasshown that FACT can be recruited to genes bythe promoter-binding GAGA factor11.

An emerging modelIt now appears that both FACT and Spt6 actto prevent the expression of cryptic promot-ers as they become accessible in the wake ofthe transcribing Pol II. The colocalization andshared kinetics of progression through thehsp70 gene by Pol II, FACT, and Spt6 suggestthat these factors are working in a concertedfashion during transcriptional elongation.From these studies a model begins to emergefor how FACT and Spt6 function in transcrip-tional elongation (Fig. 1). In this model, theseproteins participate in both the partial orcomplete disassembly of nucleosomes aheadof the polymerase and then in the rapidreassembly of nucleosomes behind the poly-merase necessary to silence cryptic TATA ele-ments in the coding region. In the absence ofeither Spt6 or FACT, these cryptic TATAsequences are not silenced and, when used,give rise to inaccurate shorter transcripts ini-tiated internal to the gene.

1. van Holde, K.E., Lohr, D.E. & Robert, C. J. Biol.Chem. 267, 2837–2840 (1992).

2. Belotserkovskaya, R. et al. Science 301, 1090–1093(2003).

3. Kaplan, C.D., Laprade, L. & Winston, F. Science 301,1096–1099 (2003).

4. Saunders A. et al. Science 301, 1094–1096 (2003).5. Mason, P. B. & Struhl, K. Mol. Cell. Biol. 23, in the

press (2003).6. Winston, F. In Transcriptional Regulation (eds.

McKnight, S.L. & Yamamoto, K.R.) 1271–1293 (ColdSpring Harbor Laboratory Press, Cold Spring Harbor,NY, 1992).

7. Jackson, V. Biochemistry 29, 719–731 (1990).8. Bortvin, A. & Winston, F. Science 272, 1473–1476

(1996).9. Ahmad, K. & Henikoff, S. Mol. Cell. 9, 1191–1200

(2002).10. Lindstrom, D.L. et al. Mol. Cell. Biol. 23, 1368–1378

(2003).11. Shimojima, T. et al. Genes Dev. 17, 1605–1616

(2003).

880 VOLUME 10 NUMBER 11 NOVEMBER 2003 NATURE STRUCTURAL BIOLOGY

Figure 1 Spt6 and FACT regulate histone mobilization during elongation to regulate transcription. (a), FACT and Spt6 associate with RNA polymerase II on a elongating transcript. FACT mediates theremoval of histone H2A–H2B heterodimers (purple) downstream of RNA Pol II to facilitate transcriptionthrough chromatin templates. In regions that have been transcribed, FACT assists the redeposition ofH2A–H2B heterodimers, while Spt6 is required for reassembly of histone H3–H4 tetramers (blue). Therestoration of the chromatin organization in transcribed regions prevents the occupation of cryptic TATAsites (dotted yellow lines) in coding regions by TBP. (b), When Spt6 and FACT activities arecompromised, TBP can occupy cryptic TATA sites within the coding region and recruit Pol II, whichultimately leads to the generation of shortened transcripts.©

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