Heatshock-regulated transcription invitro fromareconstituted chromatin template

Toinvestigate themechanisms oftranscrip- tional regulation ofDrosophila heat shock genes westudied the activity ofaheatshock promoter invitro after reconstitution into chromatin. Increasing theduration ofnucleosome assem- blyprogressively inactivated aplasmid template whenitwas transcribed withextracts ofeither unshocked orheat-shocked Drosophila embryos, despite induction ofthetranscriptional activator heatshock factor. Addition ofthegeneral transcrip- tionfactor 1HD(TFIID) before nucleosome assembly didnot significantly relieve nucleosomal inhibition, butTFIIDpoten- tiated thepromoter toberesponsive toactivation byheat shock factor intheheat shock transcription extract. Thepotentiation byTFIIDcould berelated tothenucleosome-free, hypersen- sitive state ofheat shock promoters previously observed invivo before heatshockinduction andmaybenecessitated bythe needtoexpedite activation ofheatshock genes inresponse to environmental stress. configuration withboundTFIIDandpoised RNA polymer- aseII.Uponheatshockinduction theadditional binding of HSFtotheHSEsjust upstream oftheTATAboxleads tothe transcription ofthehsp70gene, presumably byadirect or indirect activation oftheblocked polymerase. Thesebasic features ofhsp70 generegulation arealso applicable toother members ofthehspgenefamily (16, 17). We haveinitiated experiments aimedatelucidating the mechanisms underlying thetranscriptional activation ofheat shock promoters inchromatin bystudying theactivity ofthe hsp70promoter invitro after thereconstitution ofnucleo- somesonthetemplate. Wefind that forachromatin template tobeefficiently transcribed tworequirements havetobemet. (i)TFIIDhastobepresent attheonsetofnucleosome assembly (potentiation) and(ii) thetranscription extract must contain anactivated HSFduring subsequent transcription of thereconstituted template (activation). Ineukaryotes, heatstress leads toadecrease ingeneral transcriptional activity andtothespecific induction ofgenes coding forheatshockproteins (1,2).Thetranscriptional stimulation ofheatshock genesdepends onakeyactivator protein, termed heatshockfactor (HSF)(3,4).Uponheat shock HSFisconverted fromapreexisting, inactive formto anactive species that binds toconserved heat shock elements (HSEs) (5-7) present inmultiple copies upstream ofall heat shock genes. Weareinterested inthemechanisms underlying thetranscriptional activation ofheatshock genes andinthe maintenance oftheinert state undernonshock conditions. Forexample, theDrosophila hsp70 geneisinactive under normal conditions invivo, butinaninvitro transcription assay anakedhsp7o template canbetranscribed efficiently withextracts prepared fromunshocked cells (refs. 8and9; this report). Theinactivity ofthehsp7o promoter invivo may thus depend onthenegative influence ofchromatin structure. Previous nuclease digestion studies ofhsp7o genechro- matin inintact nuclei showthat under normal conditions the promoter elements arelocated within abroad region (300 basepairs (bp)) ofDNaseIhypersensitivity embedded in nucleosomal DNA (10,11).TheDNaseI-hypersensitive promoter appears devoid ofnucleosomes, asnohistones can becrosslinked totheDNA under conditions wherehistones canbedetected onthecoding portion ofthegene(12). Detailed analyses ofnonhistone protein binding within the hypersensitive promoter region haverevealed thebinding of aprotein, presumably thegeneral transcription factor IID (TFIID), totheTATAboxundernonshock Conditions (13). Inaddition, thepresence ofatranscriptionaly engaged, but blocked, polymerase ontheheatshockpromoter inthe absence ofheatshockhasbeendemonstrated (14, 15). The uninduced hsp7o promoter isthuscharacterized byanopen MATERIALSANDMETHODS