Histones H3 and H4 are Components of Upstream Activation Factor Required for the High-Level Transcription of Yeast rDNA by RNA Polymerase I

of 5
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Information Report



Views: 11 | Pages: 5

Extension: PDF | Download: 0

Histones H3 and H4 are Components of Upstream Activation Factor Required for the High-Level Transcription of Yeast rDNA by RNA Polymerase I
   Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13458–13462, December 1997Biochemistry Histones H3 and H4 are components of upstream activation factorrequired for the high-level transcription of yeast rDNA by RNA polymerase I J OHN  K  EENER *, J ONATHAN  A. D ODD *, D OMINIQUE  L   ALO ,  AND  M  ASAYASU  N OMURA  † Department of Biological Chemistry, University of California, Irvine, CA 92697-1700 Contributed by Masayasu Nomura, October 16, 1997   ABSTRACT RNA polymerase I (Pol I) transcription inthe yeast  Saccharomyces cerevisiae  is greatly stimulated  in vivo and  in vitro  by the multiprotein complex, upstream activationfactor(UAF).UAFbindstightlytotheupstreamelementoftherDNA promoter, such that once bound ( in vitro ), UAF does notreadily exchange onto a competing template. Of the polypep-tides previously identified in purified UAF, three are encodedby genes required for Pol I transcription  in vivo :  RRN5 ,  RRN9 ,and  RRN10 . Two others, p30 and p18, have remained unchar-acterized. We report here that the N-terminal amino acidsequence, its mobility in gel electrophoresis, and the immu-noreactivity of p18 shows that it is histone H3. In addition,histone H4 was found in UAF, and myc-tagged histone H4could be used to affinity-purify UAF. Histones H2A and H2B werenotdetectableinUAF.TheseresultssuggestthathistonesH3 and H4 probably account for the strong binding of UAF toDNA and may offer a means by which general nuclearregulatory signals could be transmitted to Pol I. Of the three nuclear RNA polymerases in eukaryotes, RNA polymerase I (Pol I) is responsible for rRNA synthesis. In the yeast  Saccharomyces cerevisiae , rRNA synthesis is the onlyessential function of Pol I, because strains that were mutatedor deleted for one of the large subunits of Pol I, but whichexpress rRNA from the  GAL7   promoter by RNA polymeraseII (Pol II), are viable (1). By using this  GAL7-rDNA  construc-tion, other mutants could be isolated that were specificallydefective in Pol I transcription (  rrn  mutants) and hence weredependent on galactose-induced Pol II transcription of rDNA for growth (2).In addition to identifying genes for subunits of Pol I notshared with the other polymerases, studies on the  rrn  mutantsled to discovery of Pol I-specific transcription factors. Extractsfrom such  rrn  mutant strains were defective for specific  in vitro transcription of a rDNA template, but a protein fraction fromawild-typeextractcouldrestoreactivity,providinganassayforpurification of Pol I transcription factors. The wild-type  RRN  genes, which were obtained by genetic complementation of themutants, were tagged with the influenza virus hemagglutinin(HA1) epitope, enabling construction of strains expressingonly the epitope-tagged  RRN   genes, and greatly aiding inpurification of the factors from these strains (3–5).The rDNA promoter of yeast, like that of higher eukaryotes,consists of two elements: an essential core element of about 50bp including the start site of transcription, and an upstreamelement extending to about  150, which is not essential but isrequired for a higher level of transcription (refs. 4 and 6; seealso refs. 7 and 8). The core element supports a low level of transcription, for which core factor (a multisubunit complex consisting of Rrn6p, Rrn7p, and Rrn11p; refs. 3, 9, and 10),Rrn3p (5), and Pol I are required. In addition to these factors,upstream activation factor (UAF) and TATA box-bindingprotein, as well as the upstream element, are required for ahigh level of transcription from the yeast rDNA promoter (4,11). Purified UAF previously was shown to contain threegenetically defined subunits, Rrn5p, Rrn9p, and Rrn10p, andtwo uncharacterized subunits, p30 and p18 (4).DNA in the eukaryotic nucleus is organized as chromatin,consisting mostly of regularly repeating nucleosomes in whichDNA is wrapped around an octameric structure of corehistones (for reviews, see refs. 12 and 13). The core histones,H2A, H2B, H3, and H4, are small (15–18 kDa) and very basicproteins,andarepresentintwocopiesofeachpernucleosome.Their N-terminal tails contain multiple lysine residues, whichare acetylated or deacetylated in various combinations toregulate nucleosome assembly or interactions with other pro-teins, including transcription factors. Nucleosome structuresgenerally prevent access of RNA polymerase and proteinfactors to promoters and are used to maintain genes in inactivestates; some special mechanisms appear to be used to remodelor disrupt the nucleosome structures, allowing initiation of transcription (for reviews, see refs. 14–17). Thus, histones, thecomponents of the nucleosome that bind DNA tightly, gener-ally are considered to be repressive in gene expression. In thispaper, we report our finding that histones H3 and H4 arecomponents of UAF, a transcription activator used for yeastrDNA transcription by Pol I. MATERIALS AND METHODS Strains and Plasmids.  Strains and plasmids used are listedin Table 1. Strain NOY847, in which  RRN5  is tagged withhexa-histidine [(His) 6 ] and a triple HA1 [(HA1) 3 ]-epitope atthe C terminus [‘‘  RRN5-(HA1) 3 -(His) 6 ’’], and  HHF2  (encodinghistone H4) is  myc -tagged, was constructed as follows: First,NOY844, carrying (His) 6 -tagged and (HA1) 3 -tagged  RRN5  ona  HIS3 -marked CEN plasmid, was crossed with MX1–4C (agift from M. M. Smith, University of Virginia; ref. 18), in whichboth chromosomal loci encoding histones H3 and H4 aredeleted and are complemented by a  URA3 -marked CENplasmid carrying one of the loci,  HHT1-HHF1 . The resultingdiploid was His  Ura  and Leu  . The diploid was sporulated,and the tetrads were dissected. Haploid segregants that wereHis  [  RRN5-(HA1) 3 -(His) 6 ], Ura  (  HHT1-HHF1 ) and Leu  (chromosomal disruption of   RRN5 ) were screened for sensi-tivity to 5-fluoroorotic acid (5-FOA), indicating that the  URA3 plasmid carrying  HHT1-HHF1  was essential for viability be-cause both chromosomal deletions of the histone H3, H4 loci The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked ‘‘  advertisement ’’ inaccordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424  97  9413458-5$2.00  0PNAS is available online at http:   www.pnas.org.  Abbreviations: UAF, upstream activation factor; Pol I, RNA poly-merase I; HA1, influenza virus hemagglutinin; TCA, trichloroaceticacid.*J.K. and J.A.D. contributed equally to this work. † To whom reprint requests should be addressed. e-mail: mnomura@uci.edu. 13458   were present. The resulting strain, NOY846, was transformed with pNOY436, a  TRP1 -marked CEN plasmid carrying  HHT2 and  myc -tagged  HHF2  (see Table 1 for construction details).NOY847 was a transformant selected for 5-FOA resistance,indicating loss of the  URA3  (pMS329) plasmid carrying wild-type  HHT1  and  HHF1 . Thus all of the histone H4 in NOY847is  myc -tagged. PurificationofUAF. PurificationofUAFusuallywascarriedout by using strain NOY798 according to the scheme shown inFig. 1  A . For preparation of extracts, cells were grown in yeastextract  peptone  glucose medium (1) to OD 600  1.0 to 1.2. Cells were collected by centrifugation, washed in 200 mM Tris  HCl,pH 8.0  0.3 M (NH 4 ) 2 SO 4  10 mM MgCl 2  10% glycerol  0.1%Tween-20  1.0 mM phenylmethylsulfonyl fluoride, centrifugedagain, and stored at  70°C. Frozen cells were thawed in 6 mlof breakage buffer per gram of cell paste [buffer as above but0.4 M (NH 4 ) 2 SO 4  10 mM imidazole], disrupted in a Frenchpressure cell at 20,000 psi, and centrifuged at 100,000   g   for1 hr. The supernatant was added to NiSO 4 -charged chelatingSepharose (Pharmacia) and mixed by rotation at 4°C for 1 hr. After low-speed centrifugation (1,000   g  , 5 min) the nickel-resin was poured into a column and washed with 1 vol of 20mM Tris  acetate, pH 8.0  400 mM KCl  10 mM Mg-acetate  20% glycerol  0.1% Tween-20. (His) 6 -tagged UAF was eluted with the above buffer containing 250 mM imidazole. Forlarge-scale purification, the elution was done directly onto aheparin-Sepharose cartridge (5 ml, Pharmacia). The heparincartridge was detached from the nickel-resin column andeluted with a 0.4 M to 1 M KCl gradient [in 20 mM Tris  HCl,pH 8.0  10 mM MgCl 2  20% glycerol  0.1% Tween-20  0.2 mMEDTA   0.5 mM DTT]. UAF was eluted at about 0.7 M KCl.Peak fractions were identified by immunoreactivity with12CA5 anti-HA1 mAb (3). For anti-HA1 affinity chromatog-raphy, the anti-HA1 antibody was precipitated from ascitesfluid with 55% (NH 4 ) 2 SO 4  (wt   vol), purified by gradientelution from Q Sepharose and crosslinked to NHS-activatedSepharose (Pharmacia). To pooled UAF-containing peakfractions from the heparin Sepharose column, protease inhib-itors (see ref. 4) were added followed by mixing with anti-HA1beads. The mixture was rotated in the cold for 2 hr. Beads then were washed, and UAF was eluted with HA1 peptide onto aheparin cartridge as described previously (4). The heparincartridge was eluted by a KCl gradient as above and gave onlya single protein peak. The peak fractions contained essentiallypure UAF as determined by SDS  PAGE followed by silverstaining. Purified UAF was trichloroacetic acid (TCA)-precipitated, applied to SDS  PAGE, transferred to a (poly) vinylidene difluoride membrane, and stained by Coomassieblue. The p18 band was excised and sequenced by the Mac-romolecular Structure Facility at Michigan State University.UAF also was prepared from a strain in which histone H4 was myc-tagged (NOY847) as described above through thenickel column step. UAF was applied to anti-HA1 beads and washed as described above, then batch eluted in 10 mMTris  HCl, pH 8.0  0.4 M KCl  20% glycerol  0.05% Tween  0.5mM DTT  protease inhibitors  0.2 mM phenylmethylsulfonylfluoride  2 mg/ml HA1 peptide. Anti-myc affinity resin wasprepared by first purifying 9E10 mAb from ascites fluid(Babco, Richmond, CA) and then crosslinking it to activatedSepharose as described above for anti-HA1 antibody. Controlresin was prepared in parallel without the anti-myc antibody.The purified UAF was applied to anti-myc or control resin, washed in 10 mM Tris  HCl, pH 8.0  0.1 M KCl  20% glycerol  0.5% Tween  0.5 mM DTT, and batch-eluted twice in the samebuffer as used for anti-HA1 elution above, with myc peptideinstead of HA1 peptide, and the eluates were combined. F IG . 1. Purification of UAF. (  A ) Scheme for purification of UAF.(  B ) A silver-stained gel of a peak fraction from the final column. Thepolypeptide components of UAF previously identified (4) are indi-cated. Aprotinin added as carrier for TCA precipitation appears as aheavy band at the bottom of the gel. A band just above this heavy bandmay represent histone H4 (see  Discussion ). Separation was in a10–15% SDS  pHast gel.Table 1. Yeast strains and plasmids usedDesignation DescriptionStrainsNOY577  MAT    ade2 ade3 leu2 ura3 trp1 his can1 rrn5  :: TRP1 ,pNOY103 ( URA3, GAL7  -35S rDNA) (4)NOY798 NOY577 carrying pNOY402 [  LEU2, RRN5-(HA1) 3 -(  His ) 6 ] instead of pNOY103NOY700  MAT  a  ade2-1 ura3-1 his3-11 trp1-1 leu2-3, 112 can1-100 rrn5  ::  LEU2 , pNOY103; same as NOY699(4) except for mating typeNOY844 NOY700 carrying pNOY434 [  HIS3, RRN5-(HA1 ) 3 -(  His ) 6 ] instead of pNOY103MX1-4C  MAT    ura3-52 leu2-3, 112 trp1 his3    (hht1-hhf1)  (hht2-hhf2 ), pMS329 ( CEN4 URA3 SUP11 HHT1-HHF1 ) (18)NOY846  MAT    ura3 his3 trp1 leu2-3,112 rrn5  ::  LEU2  (hht1-hhf1)   (hht2-hhf2 ), pNOY434[  HIS3 RRN5-(HA1) 3 -(  His ) 6 ], pMS329 ( URA3 SUP11 HHT1-HHF1 )NOY847 NOY846 carrying pNOY436 [ TRP1 HHT2, myc-HHF2 ) instead of pMS329PlasmidspNOY103 High-copy number plasmid carrying  GAL7  -35SrDNA,  ADE3, URA3,  2  , (2)pNOY330  LEU2, CEN6, RRN5-(HA1) 3  (4)pNOY402  LEU2, CEN6, RRN5-(HA1) 3 -(  His ) 6  derivative of pNOY330 in which an oligonucleotide encoding Histag (GSSHHHHHHSSG) is inserted immediatelyafter the third HA1 repeatpRS313 Yeast-  E. coli  shuttle vector (19);  HIS3, CEN6, ARS4 pNOY434  HIS3, CEN6, ARS4, RRN5 -(  HA1 ) 3 -(  His ) 6 , the 1.7-kb Sal I  Sac I fragment from pNOY402 carrying theHis-tagged, HA1-tagged  RRN5  gene cloned betweenthe  Xho I and  Sac I sites of pRS313pRS314 Yeast-  E. coli  shuttle vector (19);  TRP1, CEN6, ARS4 pCC66 Plasmid clone carrying the  HHT2-HHF2  locus (a giftfrom F. Winston, Harvard; ref. 20)pNOY435 A 2.1-kb  Sac I   Eco RI fragment carrying  HHT2  and  HHF2  was amplified by PCR and cloned between the Sac I and  Eco RI sites of pRS314pNOY436  TRP1, CEN6, ARS4, HHT2, myc-HHF2 ;oligonucleotide encoding a myc tag sequence(SEQKLISEEDL) inserted between the first andsecond codons of   HHF2  in pNOY435 by site-directedmutagenesis Biochemistry: Keener  et al. Proc. Natl. Acad. Sci. USA 94 (1997)  13459  Pol I Transcription Assay.  UAF activity was assayed byusing a reconstituted  in vitro  transcription system that will bedescribed in detail elsewhere. Briefly, the reaction mixturecontained linear template DNA that carried up to   210 bprelative to the rDNA transcription start site and produced arunoff transcript of about 550 bases, recombinant TATA box-binding protein, and Rrn3p purified from  Escherichia coli ,purified Pol I, and partially purified core factor that was freefrom UAF. Transcripts were labeled with   -[ 32 P]GTP andquantified by PhosphorImager.  Analysis of Histones.  To analyze which core histones copu-rified with UAF, nickel affinity-purified UAF (as above) waschromatographed on a Superdex 200 sizing column in 20 mMTris  HCl, pH 8.0  0.4 M KCl  20% glycerol  0.1% Tween-20  0.2 mM EDTA   0.5 mM DTT, and UAF-containing fractions were identified immunologically by using anti-HA1 antibody.The peak fractions were pooled, applied to heparin Sepharose,and step-eluted batch-wise in the same buffer but containing1 M KCl. After TCA precipitation, the UAF-containingmaterial was subjected to 12% SDS  PAGE and Westernblotted. Blots were incubated overnight with rabbit polyclonalantibodies made against purified yeast core histones H2A andH2B (a gift of A. Carmen and M. Grunstein, University of California, Los Angeles), diluted 1:2,500 and 1:1,000, respec-tively, or with rabbit serum directed against the unacetylatedand acetylated amino terminal tails of histone H3 (a gift of S.Y. Roth, University of Texas, M.D. Anderson Cancer Center,Houston), each diluted 1:500. Secondary goat antibody against whole rabbit IgG conjugated to alkaline phosphatase (SigmaNo. A8025) was diluted 1:4,000.Mouse mAb against an epitope common to all core histones(Boehringer),butwhichdetectedonlyH3andH4inourhands, was diluted to 1   g  ml for overnight incubation. Secondarygoat anti-mouse (against whole IgG) antibody (Sigma No. A3688) was diluted 1:4,000. Yeast histones for use as standards were prepared as described (21). RESULTS Identification of Histones H3 and H4 as Subunits of UAF. We wanted to purify enough UAF to obtain amino acidsequences from the p30 and p18 subunits, which had not beenidentified genetically. We first constructed a yeast strain(NOY798) in which  RRN5  is replaced with  RRN5  tagged withhexa-histidine as well as with a triple HA1 epitope. Extracts were made from this strain, and UAF was purified accordingto the scheme described in Fig. 1  A , which consists of a nickelcolumn affinity step and an immunoaffinity purification thatused anti-HA1 antibodies combined with two heparin Sepha-rose chromatographic steps. Fig. 1  B  shows one of the finalheparin agarose fractions after analytical SDS  PAGE andsilver staining. The subunit composition appeared to be thesame as previously described, including the three geneticallydefined subunits Rrn9p, Rrn5p, and Rrn10p, and also p30 andp18. By using preparative SDS  PAGE, which enabled betterseparation of p18 from Rrn10p than that shown in Fig. 1  B , p18 was isolated, and its N-terminal amino acid sequence wasdetermined by a conventional Edman degradation. The 20-aaresidues identified were the same as those of yeast histone H3.(The p30 band also was analyzed, but we have not succeededin obtaining sequence information for this protein.)The identification of p18 as histone H3 raised an obviousquestion, namely, if other histones, especially histone H4, arealso present in UAF, because histone H3 usually is complexed with H4  in vivo . To examine this question, we first analyzed thepurified UAF preparation shown in Fig. 1  B  in a higherpercentage gel for improved separation in the low molecular weight range, and carried out Western immunoblot analysis byusing a mAb directed against all four mammalian core his-tones. In our hands the antibody reacted most strongly withhistones H3 and H4 in the preparations of yeast core histonesusedforstandards(Fig.2  A ).Asexpected,thep18(histoneH3)band was detectable in UAF. In addition, a band correspond-ing to histone H4 was evident in the purified UAF (Fig. 2  A ).(The amount of histone H4 relative to H3 in the purified UAFpreparation analyzed in this way appears to be significantly lessthan that found in the standards. The question of stoichiom-etry of H4 relative to H3 needs further study.) Silver stainingof the purified UAF preparation in a higher-percentage gelalso visualized a band corresponding to histone H4 (Fig. 2  B ). A separate immunoblot analysis confirmed that Rrn10p mi-grated between histones H3 and H4 with a mobility similar tothat of histone H2A (data not shown).To verify that histone H4 was part of the UAF complex, weconstructed a strain (NOY847) in which histone H4 wasmyc-tagged in addition to the tagging of   RRN5  with (His) 6  and(HA1) 3 . From this strain, UAF was purified by nickel andanti-HA1 affinity chromatography but without the heparinsteps used in the method described in Fig. 1  A . When this UAFpreparation was analyzed by SDS  PAGE and silver staining,the Rrn5p, Rrn9p, p30, and histone H3 bands were present.The histone H4 band was not present, but instead there was aband of increased molecular weight corresponding to myc-H4(Fig. 2  B , lane myc-H4 UAF). Immunoblotting that usedanti-myc antibody confirmed that this band corresponded tomyc-tagged histone H4 (data not shown). The Rrn10p band inmyc-H4 UAF is obscured by the myc-tagged H4 band. F IG . 2. The presence of histone H4 in purified UAF. (  A ) Westernimmunoblot analysis of yeast core histone standards and the purifiedUAF preparation shown in Fig. 1. Separation was in 12% SDS  PAGE.The blot was probed with mAb against all four mammalian corehistones, but which reacts mostly with yeast histones H3 and H4. (  B )Silver-stained gel of yeast core histone standards, the UAF shown inFig. 1, and UAF purified from a strain (NOY847) in which histone H4is myc-tagged. The latter preparation was obtained without heparinSepharose steps and shows several contaminating protein bands. Thelanesofstandardsare2-foldserialdilutions.Thecorehistonesinorderof decreasing molecular weight are H3, H2B (the darkest band), H2A (faint), H3* (a fragment of H3, see ref. 21), and H4. Separation wasin a 8–25% SDS  pHast gel. The heavy lowest band in the UAF laneis aprotinin (carrier), and Rrn10p appears as a diffuse band betweenH3 and H4. 13460 Biochemistry: Keener  et al. Proc. Natl. Acad. Sci. USA 94 (1997)  The above-described UAF preparation that contained myc-tagged histone H4 was applied to affinity resin with anti-mycantibody crosslinked to it, or to control resin. After washing,proteins bound via the myc-epitope were eluted with myc-peptide, and their UAF activities were assayed by  in vitro transcription. This  in vitro  system has been reconstituted frompurified components. Without UAF, a low basal level of activity was observed, whereas addition of UAF stimulatedtranscription (Fig. 3). The activities of the myc-peptide eluatesindicate that the eluate from the anti-myc resin contained asignificant amount of UAF relative to the eluate from thecontrol resin (Fig. 3). Thus myc-tagged histone H4 is a stablybound component of UAF.The association of histone H4 with UAF also was demon-strated by carrying out sizing column chromatography. Theextract prepared from the strain carrying the myc-tagged H4(NOY847) was subjected to nickel chelate chromatographyand then applied to a Superdex 200 sizing column. Fractions were analyzed for UAF activity in rDNA transcription, and forthe presence of Rrn5p (by using anti-HA1 antibodies), histoneH3 (by using a mixture of antibodies against an unacetylatedH3 peptide and an acetylated H3 peptide), and histone H4 (byusing anti-myc antibodies) by SDS  PAGE followed by West-ern blot analysis. The peak position of histone H4 coincided with the peak positions of UAF activity, Rrn5p, and histoneH3. No significant trailing of H4 relative to H3 or Rrn5p wasobserved,indicatingthattheassociationofH4withotherUAFcomponents is strong (data not shown). From the position of size markers, the size of the UAF complex in this experiment was estimated to be roughly 250 kDa, which is the same, withinerror, as that previously published for a highly purified UAFpreparation (4). From all of these experiments, we concludethat both histones H3 and H4 are tightly associated with otherUAF components and UAF activity. The Absence of Histones H2A and H2B in UAF.  Becausehistones H3 and H4 had been shown to be components of UAF, it was important to determine if the other core histonespresent in nucleosomes, H2A and H2B, also might be associ-ated with UAF. For this experiment, UAF was purified fromstrain NOY798 expressing untagged, wild-type histones byusingnickelchelatechromatographyfollowedbySuperdex200sizing column chromatography in the same way as describedabove. The UAF peak fractions from the sizing column werepooled and then concentrated by binding to heparin Sepharosefollowed by step elution with high salt. The purified UAF wasanalyzedbySDS  PAGEfollowedbyimmunoblot.Serial2-folddilutions of yeast core histones, in which H3, H2B, H2A, andH4 were of about equal intensity after Coomassie staining,served as standards. The blot was probed first with a mixtureof two antibodies directed against yeast histones H2A andH2B. Histone H2A was detected with the greatest sensitivityamong the standards; a band was visible in the 0.06-  l lane(Fig. 4). By contrast neither histones H2A nor H2B weredetected in the UAF sample (Fig. 4). Next, the same blot wasprobed with antibodies directed against histone H3. The H3band was evident in the UAF samples and in the lanes with thelargest amounts of standards (Fig. 4). From the results, weestimated that histone H2A, if present, was below 0.4% of thelevel of H3 in UAF (see the legend to Fig. 4). Histone H2B inthe standard preparation used in Fig. 2  B  was stained moststrongly by silver. In contrast, no corresponding band wasobserved in the purified UAF preparation (Fig. 2  B , laneUAF). By comparing UAF with the standard as was done forH2A, we estimated that we would have been able to detecthistone H2B in an amount that was 10% of H3 in UAF. Weconclude that the other core histones are not components of UAF. DISCUSSION We have presented evidence that the p18 component of the yeast Pol I activator complex, UAF, is histone H3. TheN-terminal amino acid sequence, its mobility in SDS  PAGE,and the immunoreactivity of p18 are the same as histone H3.We then looked for and found histone H4 in UAF. The bandin UAF that comigrated with histone H4 showed similarimmunoreactivity as well. When histone H4 was tagged withthe myc epitope, the band shifted as expected from theincreaseinmolecularweight,andUAFactivitycouldbeboundto and then eluted from anti-myc affinity resin. [We attributeour failure to previously note the presence of histone H4 inpure UAF to diffuse bands in the low molecular weight regionof our SDS  PAGE gels and to interference by artifacts visu-alized by silver staining or by a peptide used as carrier. Indeed,a band corresponding to histone H4 may be visible in figure 6  a of the previous paper (4), and a likely band is evident justabove the aprotinin band in Fig. 1 of this paper.] The othercore histones, H2A and H2B, were not found in UAF; ourdetection limit was, for H2A, less than 1% of the abundanceof H3, and for H2B less than 10%. Thus, it is unlikely that the F IG . 3. Transcription stimulatory activity of UAF preparationsobtained from the myc-tagged histone H4 strain (NOY847) with and without a myc-affinity purification step. The UAF preparation (Load)containing myc-tagged histone H4 (shown in Fig. 2  B ) was incubated with control beads or with beads carrying anti-myc antibodycrosslinked to them.After repeated washing,proteinswereeluted withmyc peptide. Samples of the indicated amounts were added to 20-  lreactions of a yeast Pol I  in vitro  transcription system that lacked UAF.Portions of a gel displaying the resulting transcripts are shown. Therelative activities were corrected for activity in the absence of addedUAF.F IG . 4. Western immunoblot analysis of histone H2A. The indi-cated amounts of yeast core histone standards or the purified UAFpreparation shown in Fig. 1 were separated by 12% SDS  PAGE,blotted, and probed first for histones H2A (the strongest signal in thestandards) and H2B, and subsequently for histone H3. (In the exper-iment shown here, UAF was concentrated by binding to heparinSepharosefollowed by elutionand TCA precipitation,whereashistonestandards were directly analyzed. In separate experiments, yeaststandards were subjected to the same treatments, that is, binding toheparin Sepharose followed by elution and TCA precipitation, andthen analyzed. No significant change in relative amounts of histones was observed.) It can be seen that the immunoreactivity of the histoneH3 band in 6   l of UAF is greater than or equal to that of 4   l of standard, whereas the histone H2A band in 0.06   l of standard (64times less than 4   l) is greater than that in the 24-  l sample of UAF(4 times greater than 6  l). Thus the ratio of histone H3 to H2A in the24-  l sample is at least 256:1 (4    64), that is, histone H2A can beestimated to be present at less than 0.4% of the level of H3 in UAF. Biochemistry: Keener  et al. Proc. Natl. Acad. Sci. USA 94 (1997)  13461  presence of histones H3 and H4 in our UAF preparations issimply because of contamination by nucleosomes.Histones H3 and H4 are tightly bound components of UAF.They were retained with UAF through a variety of purificationsteps, including mAb affinity chromatography directed againstthe triple HA1 tag on the Rrn5p subunit, nickel chelatechromatography directed against the hexa-histidine tag onRrn5p, gradient elution from heparin Sepharose and from thecation exchanger MonoS, and molecular sizing chromatogra-phy. Thus, the interactions of histones H3 and H4 with theother UAF components appear to be distinguished from therecently described interactions between transcriptional repres-sors, such as Tup1 (21) or silencing proteins Sir3 and Sir4 (22),and the H3-H4 components of nucleosomes through theN-terminal tails of these histones. Instead, UAF containinghistones H3 and H4 is reminiscent of chromatin assemblycomplex (CAC). In CAC, a histone H3-H4 tetramer was stablybound to chromatin assembly factor (CAF-1) so that it wasretained with CAF-1 through an antibody affinity purificationstep and a glycerol gradient (23). However, CAC functions ininitiating assembly of nucleosomes by depositing the H3-H4tetramer onto newly replicated DNA and is not known to haveany function related to transcription. By contrast, UAF is atranscriptional activator and is not known to have any functionin nucleosome assembly or rearrangement.UAF previously was shown to bind and commit a rRNA template to transcription  in vitro . That is, UAF binding to thetemplate was necessary for subsequent binding of TATA box-binding protein and core factor into a stable preinitiationcomplex. Once bound, UAF is not appreciably exchanged ontoa second competing template, in the presence or absence of other factors (4). Thus UAF appears to bind strongly to DNA.The genetically identified subunits of UAF, Rrn5p, Rrn9p, andRrn10p, all have isolectric points of less than 7. Therefore,histonesH3andH4probablycontributesubstantiallytoUAF’sapparent high affinity for its binding site, and perhaps one (ormore) of the other subunits is responsible for specificity of binding. In view of the observed tight  in vitro  binding of UAFto the upstream element of the promoter, there is a goodpossibility that a UAF-rDNA promoter complex might beformed at the time of DNA replication  in vivo , and that theUAF-rDNA promoter complex, with or without other tran-scription factors, might remain present regardless of whetheror not rDNA transcription is actively taking place. Thus, arDNA repeat with bound UAF might correspond to a rDNA repeat proposed by Sogo and coworkers (24, 25) that is‘‘activatable’’ but is distinct from a totally inactive repeat witha typical nucleosome beads structure. If this is, in fact, the case,regulation of rDNA transcription might take place withoutaltering the number of such activatable repeats, for example,by causing modification of components of UAF bound to thepromoter, histones H3 and H4, in particular (or componentsinvolved in subsequent steps that would lead to transcriptioninitiation).ItisknownthathistonesH3andH4areposttranscriptionallymodified by acetylation of lysine residues in their amino-terminal tails (for recent reviews, see refs. 26 and 27). Al-though we have not yet determined their acetylation state inUAF, histones H3 and H4 would seem to offer a simple routeby which general regulatory signals could be transmitted viaUAF to control Pol I transcription, regardless of the questionof reversibility of UAF binding to DNA.It is known that the histone H3-H4 tetramer interacts withDNA and forms a structure similar to a nucleosome (28, 29),presumably by using positively charged residues on the H3-H4tetramersurfaceliningthepathoftheDNAasanalyzedforthenucleosome(30,31).IfhistonesH3andH4arepresentinUAFas a tetramer, and if UAF might wrap DNA like a nucleosome,ashasbeenproposedforTFIID(32),arequestionsthatremainto be addressed in future studies. We thank Drs. M. M. Smith (University of Virginia), M. Grunstein(University of California-Los Angeles), F. Winston (Harvard Univer-sity), C. D. Allis (University of Rochester), and S. Y. Roth (Universityof Texas, M. D. Anderson Cancer Center) for kindly providing us with yeast strains, plasmids, and antibodies, as described in this paper.PlasmidpNOY402andstrainNOY798wereconstructedbyD.A.Keysin this laboratory. We also thank Drs. M. Waterman and S. Arfin forcritical reading of the manuscript, and D. Semanko for assistance in itspreparation. This work was supported by U.S. Public Health GrantR37GM35949 from the National Institutes of Health.1. Nogi, Y., Yano, R. & Nomura, M. (1991)  Proc. Natl. Acad. Sci.USA  88,  3962–3966.2. Nogi, Y., Vu, L. & Nomura, M. (1991)  Proc. Natl. Acad. Sci. USA 88,  7026–7030.3. Keys, D. A., Vu, L., Steffan, J. S., Dodd, J. A., Yamamoto, R. T.,Nogi, Y. & Nomura, M. (1994)  Genes Dev.  8,  2349–2362.4. Keys, D. A., Lee, B.-S., Dodd, J. A., Nguyen, T. T., Vu, L.,Fantino, E., Burson, L. M., Nogi, Y. & Nomura, M. (1996)  Genes Dev.  10,  887–903.5. Yamamoto, R. T., Nogi, Y., Dodd, J. A. & Nomura, M. (1996)  EMBO J.  15,  533–561.6. Choe, S. Y., Schultz, M. C. & Reeder, R. H. (1992)  Nucleic Acids Res.  20,  279–285.7. Musters, W., Knol, J., Maas, P., Dekker, A. F., van Heerikhuizen,H. & Planta, R. J. (1989)  Nucleic Acids Res.  17,  9661–9678.8. Kulkens, T., Riggs, D. L., Heck, J. D., Planta, R. J. & Nomura,M. (1991)  Nucleic Acids Res.  19,  5363–5370.9. Lalo, D., Steffan, J. S., Dodd, J. A. & Nomura, M. (1996)  J. Biol.Chem.  271,  21062–21067.10. Lin, C. W., Moorefield, B., Payne, J., Aprikian, P., Mitomo, K. &Reeder, R. H. (1996)  Mol. Cell Biol.  16,  6436–6443.11. Steffan, J. S., Keys, D. A., Dodd, J. A. & Nomura, M. (1996) Genes Dev.  10,  2551–2563.12. van Holde, K. E. (1988)  Chromatin  (Springer, New York).13. Wolffe, A. (1992)  Chromatin: Structure and Function  (Academic,London).14. Paranjape, S. M., Kamakaka, R. T. & Kadonaga, J. T. (1994)  Annu. Rev. Biochem.  63,  265–297.15. Felsenfeld, G. (1992)  Nature (London)  355,  219–224.16. Felsenfeld, G. (1996)  Cell  86,  13–19.17. Pazin, M. J. & Kadonaga, J. T. (1997)  Cell  88,  737–740.18. Morgan, B. A., Mittman, B. A. & Smith, M. M. (1991)  Mol. Cell Biol.  11,  4111–4120.19. Sikorski, R. S. & Heiter, P. (1989)  Genetics  122,  19–27.20. Clark-Adams, C. D., Norris, D., Osley, M. A., Frassler, J. S. &Winston, F. (1988)  Genes Dev.  2,  150–159.21. Edmondson,D.G.,Smith,M.M.&Roth,S.Y.(1996) GenesDev. 10,  1247–1259.22. Hecht, A., Laroche, T., Strahl-Bolsinger, S., Gasser, S. M. &Grunstein, M. (1995)  Cell  80,  583–592.23. Verreault, A., Kaufman, P. D., Kobayashi, R. & Stillman, B.(1996)  Cell  87,  95–104.24. Conconi, A., Widmer, R. M., Keller, T. & Sogo, J. M. (1989)  Cell 57,  753–761.25. Dammann, R., Lucchini, R., Koller, T. & Sogo, J. M. (1993)  Nucleic Acids Res.  21,  2331–2338.26. Pazin, M. J. & Kadonaga, J. T. (1997)  Cell  89,  325–328.27. Roth, S. Y. & Allis, C. D. (1996)  Cell  87,  5–8.28. Camerini-Otero,R.D.,Sollner-Webb,B.&Felsenfeld,G.(1976) Cell  8,  333–347.29. Hayes, J. J., Clark, D. J. & Wolffe, A. P. (1991)  Proc. Natl. Acad.Sci. USA  88,  6829–6833.30. Arents, G. & Moudrianakis, E. N. (1993)  Proc. Natl. Acad. Sci.USA  90,  10489–10493.31. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. &Richmond, T. J. (1997)  Nature (London)  389,  251–260.32. Hoffmann, A., Chiang, C.-M., Oelgeschlager, T., Xie, X., Burley,S. K., Nakatani, Y. & Roeder, R. G. (1996)  Nature (London)  380, 356–359. 13462 Biochemistry: Keener  et al. Proc. Natl. Acad. Sci. USA 94 (1997)
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!