Proc. Nall. Acad. Sci. USAVol. 89, pp. 11678-11682, December 1992Biochemistry

Transcription factor IIA is inactivated during terminaldifferentiation of avian erythroid cellsJ. BUNGERT, R. WALDSCHMIDT, I. KOBER, AND K. H. SEIFART*Institut fMr Molekularbiologie und Tumorforschung, Lahnstrasse 3, 3550 Marburg, Federal Republic of Germany

Communicated by E. Peter Geiduschek, August 13, 1992 (receivedfor review June 24, 1992)

ABSTRACT Avian histone H5 and aA-glObin genes aretranscribed much more efficently in whole cell extracts derivedfrom immature polychromatic erythrocytes than in extractsfrom mature duck erythrocytes. We found that these differ-ential activities are detectable only if assayed with promoterscontaining a functional TATA box. The addition of eitherhighly purifed human or recombinant yeast transcriptionfactor IIA (TFHA) to extracts from mature erythrocytes re-sulted in a ficant increase in transcription from TATA-containing promoters, whereas transcription from TATA-lesspromoters remained unaffected. Moreover, the activity ofTFIIA was found to be reduced in extracts from matureerythrocytes. These data support the proposition that Inacti-vation ofTFHA may contribute to a general repression of geneactivity in avian erythrocytes, and only those genes withalternative mechanisms of Iitiation complex formation con-tinue to be expressed in these cells. In the case of the histone H5gene, such an alternative mechanism could be mediated va theinteraction between duck erythrocyte upsrem stimulatingfactor and TYI.

(16). A TFIIA-like activity was also shown to be involved inpolymerase III-directed, TFIID-dependent transcription ofmammalian U6 genes (17).Very recently, we demonstrated that the efficiency of

transcription of a TATA-less H5 construct was reduced butthat TFIID was nonetheless required (7). In this particularcase, the interaction of TFIID with the TATA-less H5promoter was shown to be mediated by duck erythrocyte TFupstream stimulating factor (eUSF), which binds specificallyto a sequence element in the vicinity of the initiation site.Hence, eUSF may play an alternative role during the forma-tion of initiation complexes on the H5 promoter.

In this report we analyzed the role of TFIIA duringdifferential expression of the H5 gene of the duck. Bycomparatively analyzing TEIIA activities in whole cell ex-tracts (WCE) derived either from mature (e) or from imma-ture (pe) duck erythrocytes, we could show that the activityofTFIIA is drastically reduced when erythroid cells enter thefinal stages of differentiation.

Histone H5 is specifically expressed in nucleated avian,amphibian, fish, and reptile erythroid cells. During differen-tiation it replaces the linker histone H1 and as a consequencethe chromatin becomes more compact. Its transcription isregulated by 5' promoter elements as well as by a 3' enhancer(1, 2). Like the j3A-globin gene, it continues to be expressedeven in very late stages of differentiating erythrocytes (3) andis therefore an interesting model system for studying mech-anisms by which certain genes can still be expressed indifferentiated cells while transcription of nearly all othergenes is blocked.

Multiple protein components beside RNA polymerase IIare required to initiate basal transcription from protein-coding genes. Among these components are transcriptionfactor IID (TFIID) as the TATA box binding protein [TBP;plus associated factors (TAFs) (4)], TFIIB, TFIIE, andTFIIF (5, 6). In the absence of the TATA box, additionalfactors may be required to mediate the incorporation ofTFILD into functional transcription complexes (4, 7). Se-quence-specific regulatory TFs, acting as activators or re-pressors, can interact either directly or via coactivators(adaptors) with the complex anchored at the start site (8-12).TFIIA was shown to interact directly with TFIID and to

facilitate the binding of TEIID to the TATA box (6). Corteset al. (13) used this feature of TFIIA and purified its activityover a TFIID affinity column. They demonstrated that theactivity ofTEITA from HeLa cells can be separated into threepolypeptides of 34, 19, and 14 kDa, which had previouslybeen described as components ofTFIIA by different authors(14, 15). Genes encoding two subunits of the yeast homologofTFIIA have now been isolated, and it was shown that thesepolypeptides of32 and 13.5 kDa display the function ofTEIIA

MATERIALS AND METHODSDNA Templates and Fragments. DNA templates or frag-

ments containing sequences of the H5 gene were derivedfrom plasmid pUCH5 [H5 sequences from -1000 to +1500(18)]. Deletion mutants of the H5 gene were generated frompUCH5 by exonuclease III digestions (exonuclease III kit;Promega) and defined as pH5-116 (containing H5 sequencesfrom -116 to +278), pH5-35 (H5 sequences -35 to +278) andpH5ATATA (a TATA-less mutant with H5 sequences from-9 to +278). Plasmid pSW3 contains homologous aA-globingene sequences from -1265 to +399 (19). TheDNA carryingthe mouse U6 gene, pUmU60.34, contains U6 sequences from-150 to +190 (20). The DNA probe harboring the H5 TATAbox used in binding studies is a 77-base-pair (bp) EcoRI/HindIII restriction fragment [H5 sequences from -35 to -10cloned into the HinclI site of pUC18 (7)].For competition experiments, the following oligonucleo-

tides were used: (i) a 22-bp oligonucleotide containing theUSF binding site ofthe adenovirus type 2 major late promoter(Ad2MLP), (ii) the 77-bp oligonucleotide described aboveharboring the H5 TATA box.

Protein Fractions. WCEfrom HeLa cells (hWCE) oreWCEorpeWCE were prepared by the method ofManley et al. (21)with slight modifications described by Weingart et al. (19). Toobtain immature erythrocytes, adult female ducks were madeanemic by intramuscular injection of phenylhydrazine on 5successive days at a concentration of 15 mg per kg of bodyweight. The induced hemolytic anemia leads to a prematurerelease of reticulocytes, erythroblasts, and other immaturestages, amounting to at least 80%1o of the erythroid cells and

Abbreviations: TF, transcription factor; TBP, TATA binding pro-tein; hWCE, peWCE, and eWCE, whole cell extracts from HeLacells, from polychromatic immature, and mature duck erythrocytes;USF, upstream stimulating factor; Ad2MLP, adenovirus type 2major late promoter.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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collectively designated as polychromatic erythrocytes (pe)(22). The cell extracts contained 10-15 mg of protein per ml.eUSF and TFIID from duck erythrocytes were partiallypurified as described (7, 19) and contained 0.2 (eUSF) or 0.3(TFIID) mg of protein per ml, respectively. The purificationof eUSF involved a heat treatment at 700C, and crosscontamination with TFIIA and TFIID could hence be ex-cluded (17, 23). Human recombinant TBP was expressed andpurified as described (17). The final protein concentrationwas 0.2 mg/ml. TEIA was partially purified from HeLa cellextracts or from duck erythrocytes as described by Wald-schmidt and Seifart (17), with an additional step of affinitychromatography over a TBP column according to the pro-cedure described by Usuda et al. (15). The protein was elutedfrom the TBP column at 1 M KC1 and had a final concentra-tion of25 ,&g ofprotein per ml. Subunits ofrecombinant yeastTFIIA (expression vectors pJR7 encoding TOAI and pJR16encoding TOAII; kindly provided by Steven Hahn and JeffRanish, Seattle) were expressed in Escherichia coli (BL21)and prepared as described by Ranish et al. (16). The finalprotein fraction contained 0.12 mg/ml, >80% of which wascomposed of subunits TOAI and TOAII as estimated bySDS/PAGE.

Electrophoretic Mobility-Shift Analyses. The electropho-retic mobility-shift analyses were performed exactly as de-scribed by Bungert et al. (7). Approximately 2 x 104 cpm wasused per assay; incubation was for 45 min at 30TC. Incompetition experiments, the protein fractions were prein-cubated with appropriate oligonucleotides or plasmid DNAfor 15 min at 30°C prior to the addition of labeled fragments.

In Vtro Transcription Assays. Transcription of templatescontaining H5 or aA-globin gene sequences was assayed byprimer extension as described (18). Transcription of themouse U6 gene was performed exactly as described byWaldschmidt et al. (20).

RESULTSAvian Erythroid Genes Are Differentlally Transcribed by

WCE Derived from Erythrocytes of Various Stages of CellularDifferentiation. We found (Fig. 1) that peWCE transcribedthe homologous aA-globin (lanes 1-4) and histone H5 genes(lanes 5-8) much more actively than comparable extractsderived from mature erythrocytes (eWCE). Appropriate con-trols (lanes 4 and 8) show that with the amount ofWCE used(5 ,Ju), endogenous RNA did not interfere with the assay.Mixing experiments and analyses of conceivable degradingactivities (proteases, RNases, or DNases) showed that the

*!A-giobin..-: -_

histone H5

j~

DNA

__p pe WCE

e WCE

2 3 4 5 6 8

FIG. 1. aA-Globin and histone H5 genes are differentially tran-scribed in peWCE or eWCE. Transcription experiments were per-formed with 5 ul of peWCE or eWCE. Increasing amounts oftemplate DNA (pSW3 containing the aA-globin gene: lane 1,0.5 jtg;lane 2, 1 ug; lane 3, 2 pLg; lane 4, no DNA; pH5-116 carrying histoneH5 gene sequences: lane 5, 0.5 j.g; lane 6, 1 ,ug; lane 7, 2 ,ug; lane 8,no DNA) were added before transcription reactions were started.aA-Globin or H5 mRNA synthesis was assayed by primer extension.

differential transcription activity was not due to trivial arte-facts (data not shown).As will become evident from results in Figs. 3 and 4, the

differential transcription activity in the extracts was observedonly with templates containing the TATA box, thus focusingthe attention on factors interacting with this sequence. SinceTFIID was shown not to be the limiting component for invitro transcription in mature duck erythrocytes (unpublisheddata), we attempted to analyze the activity of TFIIA invarious stages of terminally differentiated erythroid cells.The Effect ofTFUIA on Bindin T the 115 Promoter

Strngy Depends on thePe of the TATA Box. In initialexperiments, we analyzed the effect ofpurified human TFIIAon the binding of duck erythrocyte TFIID to the TATA boxofthe H5 gene. As shown in Fig. 2A, neitherTFIIA (1 M KClfraction of the TBP affinity column) nor duck erythrocyteTFIID alone formed a detectable complex with the H5 TATAbox (lanes 2 and 3). However, in the presence ofboth TEIAand duck erythrocyte TFIID, a stable comlx appegred(lane 4). Formation of this complex was inhibited by prein-cubation of the protein fractions with either an oligonucleo-tide (lanes 6 and 7) or plasmid DNA (lanes 8 and 9) harboringthe H5 TATA sequence but not by preincubation with anoligonucleotide containing the USF-binding site of theAd2MLP (lane 5) or plasmid DNA containing H5 genesequences from -9 to +278, lacking the TATA box(pH5ATATA; lanes 10 and 11). These results support theconclusion that binding occurs to the TATA sequence. Insome competition experiments, additional protein-DNAcomplexes were observed (for example, lane 6); these mayrepresent residual binding of TFIID alone to the fragmentunder conditions where the TFIIA-TFIID complex partiallydissociates.We have shown previously that a TF isolated from duck

erythrocytes and resembling USF (eUSF) is also able tostabilize the binding of TFIID to the H5 TATA box and tomediate the interaction of TFIID with a TATA-less H5promoter (7). To compare the effects ofTFIIA and eUSF onTFIID binding to the H5 TATA box, electrohoretic mobil-ity-shift experiments were performed with human recombi-nant TBP in combination with either TFIIA or eUSF (Fig.2B). None of the protein fractions alone bound to the DNAfragment (anes 2, 6, and 7) under the conditions described.When bothTBPand eUSF were incubatedjointly with the H5TATA box, a clear protein-DNA complex appeared (lane 3).In this case, complex formation could specifically be inhib-ited by competition with plasmid DNA lacking the H5 TATAbox but containing a USF-binding site at around position +40[pH5ATATA (7)], thus serving as a competitor for eUSFprotein.The simultaneous incubation of TBP and TFIUA with the

DNA fragment led to formation of a stable complex (Fig. 2B,lane 8). In contrast to the complex seen for eUSF/T'BP (lane3) the interaction ofTFIIA/TBP with the H5 TATA box wasnot blocked by competition with plasmidDNA containing theTATA-less H5 construct (lanes 9 and 10). These differencesclearly indicate that there are alternative mechanisms tostabilize TEIID binding to the H5 promoter.. One of thesedepends on the TFIIA-mediated, direct interaction ofTFIIDwith the TATA box, while the other uses interactions be-tween TFIID and eUSF (anchored to its appropriate bindingsite), thereby directing TFIID to the initiation site, of the H5promoter.

Activity of TFUA Decreases During the Transiton fomImmature to Mature Erythrocytes. Fig. 3A summarizes ex-periments in which different H5 templates were transcribedin extracts derived either from HeLa cells (hWCE) or fromimmature polychromatic (peWCE) or mature (eWCE) duckerythrocytes. As a functional control, we simultaneouslyanalyzed transcription of the U6 gene by RNA polymerase

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PlSI IATATTA AIA

IIA e 1[)

Ir'peet It:

.)N A

Pr it eir,

pii

4A 1.

1 2 3 4 5 6 7 8 9 10 1 1

B Ir 5 H5,.AJA A1 competitor0.4 0.8 jig

TOP 1SJFXTBP 1SF IIA IIA/1 BP------l1r- l- r--Or

Proteir

Of'D 10 5 5/5 pi

1 3 4 5 (6 8 9 10

FIG. 2. Effect of TFIIA on binding of duck erythrocyte TFIDdepends on the presence of the H5 TATA box. (A) Electrophoreticmobility shift showing that TFIIA stabilizes the binding ofTFPIIDfromduck erythrocytes (e ID) toaDNAfragment containingthe H5TATAbox. The 77-bp DNA probe used in the binding studies (see also Fig.2B) contains H5 gene sequences from -35 to -9. TFIIA was purifiedfrom HeLa cells by chromatography over a TBP affinity column (1 MKCI fraction; 25 pug of protein per ml). The TFIID fraction was derivedfrom mature duck erythrocytes (0.3 mg of protein per ml). Fractionscontaining TFIIA and TFlD were incubated with the DNA probealone orin combination, as appropriately indicated, for45 min at30C.In competition experiments, protein fractions were preincubated witholigonucleotides or plasmid DNA (lane 5, 0.4 ,ug of USF oligonucle-

otide; lanes 6and 7,0.4 or0.8 ,LgofTATA oligonucleotide; lanes 8 and9, 0.4 or 0.8 jig of pH5-35; lanes 10 and 11, 0.4 or 0.8 pg ofpHSATATA) for 15 min at 300C before addition of labeled fragment.(B) Electrophoretic mobility-shift assay comparng the effect ofTFIIAand eUSF on binding of human recombinant TBP to the H5 TATAbox. Binding conditions were the same as in A. Protein componentsTFIIA, eUSF, and TBP were added as indicated.

III, which essentially depends on both TFIID (20) and TFIIA(17) for efficient transcription.The results obtained in hWCE show that both H5 templates

containing the TATA box (pH5-116, pH5-35) as well as theU6 gene were efficiently transcribed. Transcription of theTATA-less H5 template (pH5ATATA) was significantly re-duced but correctly initiated as described (7).

Transcription in peWCE leads to efficient transcription,showing that all components required for productive expres-

sion of the H5 gene must be present and active in immaturecells. It should be emphasized that there is a marked differ-ence in transcription from TATA-containing versus TATA-less H5 templates as was also observed in HeLa cell extracts.

Interestingly, different results were obtained in transcrip-tion reactions conducted in eWCE. In agreement with resultsshown in Fig. 1, significant reduction of transcription fromthe TATA-containing H5 templates was observed (lanes 1and 2) and transcription of the U6 gene was abolished (lane4). The efficiency of transcription in extracts from matureerythrocytes from either TATA-containing or TATA-less H5templates was repeatedly observed to be comparable. Theseresults strongly suggest that a component, present and activein polychromatic erythrocytes and re4quii for efficienttranscription from TATA-containing promoters, is inactiveor not present in mature erythrocytes. Since the efficiency oftranscription of the TATA-less H5 template in the differentextracts remained approximately similar (lane 3) it is. clearthat this component acts only in the presence ofthe H5 TATAbox.From the results presented in Figs. 2 and 3A., TFIIA was

considered to be a likely candidate involved in the observeddifferential transcription. To further strengthen this assump-tion, transcription from different H5 templates and the U6gene was performed in cell extracts from mature erythrocytesto which increasing amounts of highly purified human TFIIA(1 M KCl fraction of the TBP affinity column) was added. Asshown in Fig. 3B, transcription from the TATA-containiH5 promoters (pH5-116, lanes 1-4; pH5-35, lanes 5-8) couldbe strongly stimulated by addition of TFIIA. In contrast,addition of TFIIA had no effect on transcription from theTATA-less H5 promoter (pH5ATATA, lanes 9-12). Expres-sion of the U6 gene was virtually absent in extracts frommature erythrocytes (lane 13) but could be stimulated todetectable levels by the addition of TFIIA (lanes 14 and 15).The reduction of U6 transcription observed with higheramounts of TFIIA (lane 16) is due to overtitration.To eliminate the possibility that contaminant(s) in the

TFIIA-containing protein fraction are responsible for thestimulatory activity, we expressed yeast clones. encodingTFIIA (kindly provided by Steven Hahn and Jeff Ranish).The yeast protein, although immunologically unrelated, waspreviously shown to be biochemically active in a mammaliantranscription system (16). As shown in Fig. 4, yeast TFIIAhas only a weak stimulatory effect on transcription of the H5gene (pH5-35) in peWCE (lanes 1-4). In contrast, a markedstimulation of transcription was observed when eWCE weresupplemented with increasing amounts of yeast TFIIA (lanes5-8). Most importantly, this stimulation resulted in an extentof transcription comparable to that observed with peWCE.Again, no stimulatory effect was observed in the absence ofthe TATA box (pH5ATATA, lanes 9-12).These results strongly support the conclusion that TFIIA

is involved in the differential transcription activities of bothextracts. To prove this point, the activities of T1FIIA inprotein fractions from various extracts were assayed by theirability to support the binding of human recombinant TBP tothe H5 TATA box. For this purpose, the fractions frompolychromatic or from mature duck erythrocytes, eluted with0.3 M KCI from the DEAE-Sephacel column, were comparedin relation to highly purified HeLa TFIIA (1 M KC1 fractionfrom the TBP affinity column) serving as a control. As shownin Fig. 5, none of the individual components (TBP or TFIIA)alone formed a stable complex on the H5 TATA box (lanes1-4). However, a clear protein-DNA complex appearedwhen TBP was incubated with TFIIA from HeLa cells (lane5) or TFIIA from immature polychromatic duck erythrocytes(lanes 6-8). With the highest amount of the TFIIA fractionfrom mature erythrocytes (10 Ap; lane 11), formation of a veryfaint complex could be observed, indicating that TFIIA may

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H

L1) Lf) L

ImxII) w.

D

_ - h WCEzo~~~ B

w - pe WCEH5-116

0 2.5 5 10

e WCEH5-35 H5ATATA

0 2.5 5 10 0 2.5 5 10U6

0 2.5 5 16

0*2*3

12 3 4

- e WCE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

FIG. 3. Activity of TETIA varies during differentiation of duck erythroid cells. (A) Transcription of H5 templates containing (pH5-116,pH5-35) or lacking the TATA sequence (pH5ATATA) as well as the U6 gene were conducted in hWCE, peWCE or eWCE as indicated.Transcription of plasmids pH5-116, pH5-35, and pH5ATATA was performed with 1 p&g ofDNA and 5 Al ofWCE (hWCE, peWCE, and eWCE).H5 RNA synthesis was assayed by primer extension; U6 RNA synthesis was performed by runoff transcription with 2 jig ofDNA (PUmU6o.34)and 5 pl (hWCE/peWCE/eWCE) of WCE as described (20). (B) Effect of purified human TFIIA on transcription ofH5 and U6 gene templatesin eWCE. Transcription of individual templates was performed with eWCE under exactly the same conditions as described in A. Increasingamounts of HeLa TEIIA (1 M KCI fraction of the TBP affinity column) were added to the reaction mixtures as indicated.

not be absent in mature erythrocytes but that its activity iseither reduced or inhibited in these cells. To exclude thepossibility that TFIIA from mature erythrocytes has a dif-ferent chromatographic behavior due to conceivable modifi-cations, the other column fractions obtained from DEAE-Sephacel were assayed and found to be devoid of TFIIAactivity (data not shown).

DISCUSSIONDuring terminal differentiation of avian erythrocytes, geneexpression is sequentially shut down. The histone H5 gene isturned off last and still remains active while most other genesare repressed. In an attempt to investigate the underlyingmechanisms, we analyzed WCE from various stages ofdifferentiating duck erythroid cells (Fig. 1). We found evi-dence supporting the proposition that TFIIA may be involvedduring the general repression ofmost genes in erythroid cellswhile alternative mechanisms of initiation complex formationcould transiently maintain transcription from the H5 pro-moter. This promoter is characterized by an extremely highG+C content (24), conceivably limiting direct interaction ofTFIID with the TATA box and requiring the activity ofauxiliary factors. In electrophoretic mobility-shift assays, thesimultaneous presence ofTFIIA was required to stabilize thebinding of TFIID (Fig. 2A). This observation proved to be auseful tool to assay for the auxiliary activity of TFIIA.We have demonstrated previously that eUSF mediates the

interaction of TFIID with the H5 promoter even in theabsence of its TATA box (7). Here we show that TFIIA is

unable to stabilize the binding ofTFID to the TATA-less H5promoter (Fig. 2). These results indicate that the effect ofTEIIA on preinitiation complex formation on the H5 genedepends on the presence of a functional TATA box, and theysuggest that alternative mechanisms exist for incorporationof TFIID into transcription complexes. These data are inagreement with those of Roy et al. (25) showing that TFII-I,which binds to an initiator element of the Ad2MLP, cansubstitute for TFIIA to direct TFIID to the Ad2MLP. It ispossible that the potential function of eUSF to mediateincorporation of TFIID into initiation complexes on the H5promoter could reflect a mechanism that allows HS genetranscription to progress in cells in which most of the othergenes are repressed.To test this assumption, we attempted to analyze the

activities of TFIIA in peWCE or eWCE. The results in Figs.1 and 3A show that peWCE express the H5 gene-in thepresence of its TATA box-much more efficiently thaneWCE, hence reflecting the situation in vivo. However,transcription from the TATA-less H5 gene construct wasfound to be comparable in hWCE, peWCE, or eWCE,

\\ e x I[)f ,H> :!a

I |! (

peWCE

H5-35

Om 4 a

eWCE

H5-35 H5 TATA DNA

- yllA

go_.1 2 3 4 5 6 7 8 9 10 1 1 2

FIG. 4. Complementation of eWCE with recombinant yeastTFIIA (yIIA) results in transcription efficiencies comparable to thoseobserved in peWCE. Transcription of individual templates (pH5-35;pH5ATATA) in peWCE or eWCE was performed under the sameconditions as described in Fig. 3A. Increasing amounts of recombi-nant yeast TFIIA were added to the reaction mixtures (lanes 1, 5, and9, 0 1; lanes 2, 6, and 10, 2.5,ul; lanes 3, 7, and 11, 5 pl; lanes 4, 8,and 12, 10 Al).

FIG. 5. Activity ofTFIIA, assayed by stabilization ofthe bindingof recombinant human TBP, is decreased in mature duck erythro-cytes. Electrophoretic mobility-shift assay was performed as de-scribed in Materials and Methods and Fig. 2A. TFIIA was purifiedeither from HeLa cells (h IIA; 1 M KCl fraction of TBP affinitycolumn) or from polychromatic (pe IIA) or mature (e IIA) duckerythrocytes (0.3 M KCI fraction ofthe DEAE-Sephacel column; 0.2mg/ml). Protein fractions were added either alone or in pairwisecombination as indicated.

A

DNA

pi IIA

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indicating that the activity, which is reduced in matureerythrocytes, depends on the presence of the H5 TATA box.When eWCE were supplemented with purified human or

recombinant yeast TFIIA, they exhibited transcription effi-ciencies on TATA-containing H5 promoters similar to thoseobserved in peWCE (Figs. 3B and 4). Addition of TEJID(either human recombinant protein or purified from matureduck erythrocytes) or eUSF to eWCE did not stimulatetranscription of the H5 gene (data not shown). These resultsstrongly support the conclusion that activity of TFIIA isdrastically reduced in mature erythrocytes, while the otherbasal transcription factors are apparently not rate-limiting. Atthis point in our investigation, however, we cannot differen-tiate whether the activity of TFIIA is reduced or inhibited inthese cells.

Protein components that can repress basal transcriptionfrom genes transcribed by RNA polymerase II were shown tointerfere with TFIIA function (26). These components, de-fined as NC1 and NC2, contact TFIID and inhibit interactionof TFIID with TFIIA (12). In contrast to these findings, aninhibitor that conceivably inactivates TFIIA in mature duckerythrocytes would have to associate with TFIIA sincetranscription in these extracts can be stimulated by additionof purified TFIIA but not by addition of TFIID. The assump-tion of specific inhibitory components for TFIIA offersseveral advantages for regulation of gene expression. Inac-tivation of TFIIA would repress general transcription whileretaining the possibility of directing TFIID to specific pro-moters by alternative protein-protein interactions [e.g., viaTFIl-I (25); Spl plus tethering factor (4); or eUSF (7)].

It should be pointed out that the assembly of an initiationcomplex can be inhibited by the association of a nucleosomenear the transcription start site. Workman et al. (27) haveshown that preincubation of template DNA with TRIID andUSF efficiently prevents the association of a nucleosome onthe Ad2MLP. The same could be true for TFIIA, known tostabilize the binding of TFIID to the TATA box and to act atan early step during initiation of transcription (28). In thisrespect, TFIIA or sequence-specific binding proteins likeeUSF may act as antirepressors by stabilizing the preinitia-tion complex and thereby preventing the association ofnucleosomes or linker histones (H1/H5) with essential pro-moter regions. The difference between true activation andantirepression has recently been discussed by Croston et al.(29).During the differentiation of erythroid cells, the chromatin

becomes more compact and most of the genes are repressed(30). Our data suggest that inactivation of TFIIA couldcontribute to a general repression of transcription of genesrequiring TFIIA for the assembly of stable initiation com-plexes. Under conditions where TFIIA is inactivated, stabletranscription complexes can only be assembled or remainstable on genes that exhibit alternative mechanisms (viainitiator elements) to stabilize the binding of TFIID. Thehistone H5 gene may represent such a case and it is inter-esting that transcription complexes isolated on the TATA-less promoter (stabilized by eUSF) are as stable as complexesisolated on the TATA-containing template (stabilized byTFIIA/eUSF; ref. 7).The alternative mechanism ofcomplex formation viaeUSF

would permit transcription of the H5 gene in erythroid cells,while most of the other genes are repressed due to inactiva-tion of TFIIA. Ultimately, repression of the H5 gene in thefinal stages of differentiation could then be explained by the

binding of chicken initiation binding repressor to the tran-scription initiation region of the H5 gene as is discussed byGomez-Cuadrado et a!. (31).

We are grateful to Frauke Seifart, Ulla Kopiniak, and ChristofAlbers for expert technical assistance. We also thank Drs. StevenHahn and Jeff Ranish (Seattle) for having made availble the clonesfor recombinant yeast TFIIA and Edgar Wingender (Braunschweig)and Wolfgang Meissner (IMT Marburg) for critically reading themanuscript. Financial support from the Deutsche Forschungsge-meinschaft as well as the Fonds der Chemischen Industrie is grate-fully acknowledged.

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