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FEBS Letters 409 (1997) 74-78 FEBS 18631

Human Supt5h , a putative modulator of chromatin structure, is reversibly phosphorylated in mitosis

Axel A. Stachora1, Ramon Enriquez Schäfer, Matthias Pohlmeier, Gernot Maier, Herwig Ponstingl* Deutsches Krebsforschungszentrum, Division for Molecular Biology of Mitosis, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

Received 18 March 1997

that a main function of the complex is carried out by the Abstract The Saccharomyces cerevisiae Spt4p, Spt5p and Spt6p are involved in transcriptional repression by modulat- SPT5 and SPT6 products. Recently, SUPT4H and ing the structure of chromatin. From HeLa cells we have purified SUPT6H, the human homologues of the SPT4 and SPT6 a human homologue of Spt5p, SuptShp, and show here that the , have been cloned [11-13]. We report here the purifica- protein is reversibly phosphorylated in mitosis. The cloned cDNA tion of Supt5hp, a human homologue of yeast Spt5p, its mi- predicts a protein of 1087 residues with 31% identity to yeast totic phosphorylation, and the cloning and sequencing of the Spt5p. It includes an acidic N-terminus, a putative nuclear SUPT5H cDNA. localization signal and a C-terminal region containing two different repeated motifs. One of them, with the consensus 2. Materials and methods sequence P-T/S-P-S-P-Q/A-S/G-Y, is similar to the C-terminal domain in the largest subunit of RNA polymerase II. 2.1. Generation of antibodies and purification of SuptShp © 1997 Federation of European Biochemical Societies. HeLa cells were cultivated as described before [14]. For cell cycle studies, cells were synchronized in S-phase by a double thymidine Key words: Transcriptional regulation; Chromatin structure; block [15] and the subsequent arrest in mitosis was achieved by a Cell cycle; C-terminal domain; Phosphorylation nocodazole block [16]. Monoclonal antibody 8001 was one of a set raised against proteins from mitotic HeLa cell lysates. It was charac- terized as IgMK and used to monitor the purification of Supt5hp. This was then used to generate polyclonal antibody AS40 by immunizing chicken and preparing antibodies from the eggs. 1. Introduction Initially, monoclonal antibody 8001 was used to monitor Supt5hp isolation. In subsequent routine procedures the more sensitive anti- The structure of chromatin is modified by proteins that body AS40 was used. Fractions containing Supt5hp were identified in immunoblots stained with either of the two antibodies, and the inten- interact with histones and promote either a relaxed state of sity of the stain was determined densitometrically. All purification chromatin, suitable for transcription, or a more compact and steps were performed at 4°C. Frozen HeLa cells (4X1010, 100 g wet transcriptionally inactive state [1,2]. Factors, like the large Snf/ weight) were thawed, resuspended in 200 ml of buffer A (20 mM bis- Swi complex, facilitate the initiation of transcription by inter- Tris-propane-HCl (Sigma), pH 6.9, containing 20% glycerol, 1 mM action with nucleosomes and the RNA polymerase II complex dithiothreitol and protease inhibitors, swollen on ice for 30 min and homogenized by 35 strokes in a S-type Dounce homogenizer (Braun, [3].In Saccharomyces cerevisiae a deficiency in the activating Melsungen). Insoluble material was removed by centrifugation (30 Swi/Snf complex is suppressed by mutations in the SPT4, min at 70000Xg). The pellet was resuspended in 100 ml of buffer SPT5 and SPT6 genes [4—6], indicating that the wild-type A containing 50 mM NaCl, and again centrifuged for 30 min at proteins are involved in transcriptional repression. Mutations 70 000Xg. The two supernatants were pooled, solid ammonium sul- in these genes were also shown to alter transcription of a fate was added with continuous stirring up to 30% saturation. The suspension was further stirred for 1 h at 4°C. After spinning down subset of histone genes in S. cerevisiae [7]. Since double mu- precipitated protein (30 min at 70 000 X g), the pellet was dissolved in tant combinations of spt4, spt5 and spt6 are generally lethal in 25 ml of buffer A containing 100 mM NaCl and applied to an haploid strains, the corresponding wild-type proteins may DMAE-Fractogel 650 S column (1.2x10 cm, Merck) equilibrated form a heterotrimeric complex. The co-immunoprecipitation with buffer A containing 100 mM NaCl. Bound protein was eluted by applying a linear gradient of 250 mM-1 M NaCl at a flow rate of of Spt5p and Spt6p also indicates a physical interaction of 1.2 ml/min. The fractions containing Supt5hp were diluted 10-fold in these proteins [8]. The chromatin remodelling-mechanism of buffer B (50 mM Mes-NaOH, pH 6.9, containing 20% glycerol and the postulated Spt protein complex may in part be explained 1 mM dithiothreitol) and applied to an S03-Fractogel 650S column the finding that Spt6p interacts directly with the globular do- (0.5X5 cm, Merck) equilibrated with buffer B containing 140 mM main of histone H3 and that it functions as a nucleosome NaCl. Bound protein was eluted by applying a linear gradient of 140 mM-2 M NaCl at a flow rate of 0.4 ml/min. The fractions con- assembly factor which assists histones in binding to DNA [9]. taining Supt5hp were diluted 10-fold in buffer A containing 200 mM The SPT4 gene encodes a small protein of 102 amino acid NaCl, and applied to a MonoQ column (0.5X5 cm, Pharmacia) equi- librated with the same buffer. Bound protein was eluted by applying a residues, whereas Spt5p (1063 residues) and Spt6p (1726 res- linear gradient of 200^100 mM NaCl at a flow rate of 0.4 ml/min. idues) are considerably larger proteins with highly acidic N- After electrophoresis, SDS gels were stained with Coomassie blue terminal regions. Null mutants of SPT4 are viable [10], while R250 and scanned in a laser densitometer, with bovine serum albumin the deletion of SPT5 or SPT6 has a lethal effect, indicating as standard.

2.2. Amino acid sequences of peptides and cloning of SUPT5H "Corresponding author. Telefax: (49) 6221-423460. Ten micrograms of the purified protein was cleaved in 0.1 M E-mail : [email protected] NH4HCO3 and 1 M urea with N-Asp protease for 2 h at 25°C. The reaction was stopped by addition of 15% (v/v) TFA. Another xThe first two authors contributed equally. aliquot of 10 jig was cleaved in 75% formic acid with CNBr for 20

0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved. P//S0014-5793(97)00486-9 A.A. Stachora et al.lFEBS Letters 409 (1997) 74-78 75 h at 25°C. The resulting peptides were separated by reversed phase HPLC (C18) in 0.1% TFA, 5-72% acetonitrile and sequenced with a gas-phase sequencer (Model 470A, Applied Biosystems). The Tblastn program (part of the HUSAR program package, DKFZ) was used to search the databases for nucleotide sequences corresponding to the peptide sequences obtained. Total RNA was obtained from logarithmically growing HeLa cells using the RNA-Clean kit (AGS, Heidelberg, Germany). Reverse tran- scriptase-mediated PCR was performed on total RNA using the SuperScript reverse transcriptase (Life Technologies). Clone 322 was amplified by RT-PCR using as forward and reverse primers: 5'-gac c- ga ccc ccc aag aaa ccc-3' and 5'-ggg ate ctt gac tec ctg gga-3', re- spectively. PCR products were cloned directly into the pCRII vector (Invitrogen). HeLa and testis libraries were plated on E. coli Y1090- with ap- proximately 5X 104 plaque-forming units per 150 mm plate, and were lifted on Hybond N+ membranes (Amersham). Fifty nanograms of cDNA clones were labelled (DECAprimell kit, Ambion) with 50 uCi Fig. 1. Purification of Supt5hp from HeLa cells. A: 12.5% SDS- [oc-32P]dCTP and hybridized to the membranes. Positive clones were PAGE of protein fractions stained with Coomassie brilliant blue. B : selected and DNA was eluted (35 mM Tris/HCl, pH 7.5, 10 mM 10% SDS-PAGE: Total soluble HeLa protein, immunostained with MgSC-4, 0.1 M NaCl, 0.01% gelatin). Phage DNA was replated and monoclonal antibody 8001 (lane 10), or immunostained using the further rounds of screening were performed. Phage DNA of single polyclonal antibody AS40 (lane 11), or pre-immune immunoglobulin plaques was purified and cut with EcoRl in the presence of 10 mM (lane 12). spermidine. The inserts obtained were purified as before, eluted and cloned into the EcoKI sites of pCRII. DNA sequencing was per- formed with an ALFexpress (Pharmacia) DNA sequencer and with 155 kDa protein in immunoblots (Fig. 1A, lane 9), and both an ABI sequencer Model 373A (Applied Biosystems). stained the nuclei of interphase HeLa cells. By comparative 5'-RACE was performed on testis cDNA (Marathon-Ready kit, immunoblotting using AS40, we estimated the amount of the Clontech) with the primer: 5'-etc tag aat gtc etc tgc tec ate etc c- protein to be 20000 copies/HeLa cell; representing ~0.0025% ca ctg gtc ctc-3'. Twenty-six cycles were performed with 68°C for of the total cellular protein (data not shown). 1 min, 72°C for 2 min and 92°C for 1 min. Two microliters of the 50 ul reaction mixture were removed and used as a template for further reamplification under the same conditions. The PCR products 3.2. Cloning of SUPT5H were analyzed on agarose gels and cloned into pCRII vectors. EST We cleaved aliquots of the antigenic protein by N-Asp pro- clones from the IMAGE Consortium (LLNL) [17] were obtained from tease and CNBr. The resulting fragments were separated by Research Genetics (Huntsville, AL). HPLC and 15 of them were sequenced. A data base search using the Tblastn program yielded one cDNA clone (T18964) 3. Results which, in its translated open reading frame, contained the se- quence of one of the peptide fragments (residues 131-152 in 3.1. Purification of Supt5hp from HeLa cells Fig. 2). Based on this sequence, primers were designed and In a study to identify proteins involved in cell cycle regula- used to amplify a fragment of 342 bp (clone 322) from HeLa tion, we had generated a set of monoclonal antibodies raised cDNA. A A,gtl0 HeLa library was screened with this fragment against proteins from mitotic HeLa cells. One of them, anti- yielding a clone with a 792 bp insert, designated clone 13. body 8001, in immunofluorescence studies stained a nuclear Furthermore, another peptide fragment (residues 796-810 in epitope in interphase HeLa cells (data not shown) and de- Fig. 2) was found in the translated sequences of a number of tected a protein with an apparent mass of approximately 155 expressed sequence tags in the GenBank database (R59954, kDa in immunoblots (Fig. IB, lane 10). Using this antibody to R73139, R14898 and T80145). Clone R14898 represents a se- monitor enrichment, the protein we later identified as Supt5hp quence of 1517 bp including a poly(A) tail and a polyadenyla- was purified from lysates of logarithmically growing HeLa tion signal. With pooled DNA from clones 13 and R14898 a cells by fractionation as described in Section 2 and enriched JigtlO testis library was screened, yielding two overlapping 32500-fold to apparent homogeneity (Fig. 1A and Table 1). In clones of 972 bp and 1430 bp, respectively. The sequence order to confirm the results obtained with monoclonal anti- was extended by 5'-RACE for a further 320 bp. A new data- body 8001, the purified protein was used to raise polyclonal base search with the complete sequence identified two addi- antibodies in a chicken. Antibody preparations collected from tional EST clones (R18289 and W73325) of 2391 bp and the eggs of this chicken were designated AS40. Both antibodies 3276 bp, respectively, which were sequenced and confirmed displayed an identical distribution pattern of a 155 kDa pro- the sequence already established. The sequence data are corro- tein in the various protein fractions, both detected the purified borated by the microsequencing data of the Supt5hp peptides.

Table 1 Purification of Supt5h from HeLa cells Fraction Total protein (mg) Supt5hp (|ig) Purification factor Recovery (%) Crude lysate 7920 200 100 Soluble protein 3872 160 1.3 80 30% Ammonium sulfate precipitate 184 150 26.7 75 DMAE-Fractogel 4.2 120 943 60 SCVFractogel 0.37 94 8300 47 MonoQ 0.06 60 32500 30 Crude lysate was obtained from 100 g of packed HeLa cells (4X 1010 cells). Supt5h was quantitated by the staining of gels with Coomassie blue and of immunoblots with AS40 antibodies to Supt5h. 76 A.A. Stachora et allFEBS Letters 409 (1997) 74-78

1 agtcaggcgtcgtgcgaacagcagctggtaccgaaggcggaggtggagcccgagggggaaccagcggggaaactgaggctcggggtggagcgcaggattg 100 01 tgqgacgcgccaaggctgctgtctttcccagcagcagcggaag ATG TCG GÂC AGC GAG GAC AGC AAC TTT TCC GAG GAG GAG GÄC 185 iggggg gy """^^^MSOsF^OSNFSlSgl) 14 AGC GAG CGC AGC AGT GAC GGC GCC GAS GTA (3AC GAR CAS CGG CGG AGT GCA GCG GGC AGT GAS AAA GAR S E R S S O G AEVDSERRSAAGSEKB GAA GAG CCT GAS GAC GAA GAS GAG GAG GAA GAG GAG GAG GAR TJ .-. ■ -. ■' .-■. ■ ■■ ■-.■ :.-./■ -■ ' ■•■. »■' 335 EgPSDÏKKEEÏSESY L . : . .. 64 CGA CCC CCC AAG AAA CCC CGC CAT GGA GGC TTC ATT CTG GAC G? - '- - --' - "' - ■- - - -*• 410 RPPKKPRHGGFILDE ' - '89 GAC CAG TGG GAG GAÏ GGA GCA GAG GAC ATT CTA GAG AAA GAA GAG ATT GAS GCC TCC AAT ATC GAÏ AAT GTT GTC 485 »QWEDGABDILEKEglKASNlDNVV 114 CTG GAT GAA GAÎ CGT TCT GGG GCT CGC CGC CTG CAA AAC CTC TGG AGG GAC CAG CGA GAA GAA GAA CTG GGC GAG 560 L D , E D R 5 _G A R R L_Q___N L W R D 0 R E _ E E L G E 139 TAT TAC ATG AAG AAA TAC GCC AAG TCA TCT GTG GGA GAG ACG GTG TAT GGA GGA TCT GAT GAG CTC TCA GAC GAC 635 Y Y M K K Y A K S _._S_ V, G__ E TVYGGSDELSDD 164 636 ATC ACC CAG CAG CAG CTG CTC 710 165 I T Q Q Q L L 189 711 CGG GCC ACG GCC ATT TCC TTG ATG CGC AAG TTC ATT GCC TAC CAG TTC ACA GAC ACG CCC CTG CAG ATC AAG TCA 190 RATAISLMRKFIAYQFTDTPLQIKS GTA GTG GCA CCA GAG CAT GTG AAG GGC TAC ATC TAC GTG GAG GCC TAC AAG CAG ACC CAC GTG AAG CAG GCC ATT \\\ VVAPEHVKGYIYVEAYKQTHVKQAI GAA GGG GTG GGC AAC CTG CGG CTT GGC TAC ?GG AAC CAG CAG ATG GTG CCC ATC AAG GAG ATG ACA GAC GTG CTC JNQQMVPIKEMTDVL

1011 ATT GCT CAG GTG GAC TAC GTG GAG CCC AGC CAG AAC ACC ATC TCC CTG AAG ATG ATC CCA CGC ATC GAC TAC GAT 1085 290 I- A Q __y___ _.D X. Y E P S_ Q N __T ISLKMIPRID .._Y.__.._D_ 314 1086 CGC ATC AAG GCC CGC ATG AGC TTG AAA GAC TGG TTT GCC AAA AGG AAG AAG TTT AAG CGG CCT CCA CAG AGG CTG P P Q R L TTT GAT GCT GAG AAG ATC AGG TCC CTG GGG GGT GAT GTT GCC TCT GAT GGT GAC TTC CTC ATC TTT GAG GGG AAC 5 FDAEKIRSLGGDVASDGDFLIFEGN III CGT TAC AGC CGG AAG GGC TTT CTG TTC AAG AGC TTC GCC ATG TCT GCT GTG ATC ACG GAG GGT GTG AAG CCA ACA K K M V I V K CTC TCT GAG CTG GAA AAG TTT GAG GAC CAG CCA GAG GGC ATT GAC CTG GAG GTG GTG ACT GAG AGC ACA GGG AAG ELEKFEDQPEGIDLEVVTESTGK L S E L E GAG CGG GAG CAC AAC 7C CAA CCT GGG GAC AAC GTG GAG GTC TGT GAG GGT GAG CTC ATC AAC CTG CAG GGC AAG E R E H N QPGDNVEVCEGSLINLQGK \\t° ATC CTC AGC GTG GAT GGC AAC AAG ATC ACC ATC ATG CCC AAG CAT GAG GAC CTC AAG GAC ATG TTG GAG TTC CCA V N K I I M K H D K D M GCC CAG GAA CTT AGA AAA TAC TTC AAG ATG GGG GAC CAC GTG AAG GTG ATT GCT GGC CGA TTC GAG GGC GAC ACA A. Q E L „R... K Y FKMGDHVKVrAGRFEGDT GGC CTC ATT GTG CGG GTG GAG GAG AAT TTC GTT ATC CTG TTC TCT GAC CTC ACC ATG CAT GAG CTG AAG GTG CTC

CCC CGG GAC CTG CAG CT TGC TCA GAG ACA GCA TCA GGT GTG GAT GTT GGG GGC CAG CAT GAA TGG GGC GAG CTG 0 CSETASGVDVG. G ._Q _H E_ W G E L _ P R D L Q L = lit 1761 GTG CAG CTG GAT CCC CAG ACT ;TG GGT GTC ATC GTG CGA CTA GAA CGG GAG ACC TTC CAG GTG CTG AAC ATG TAC 540 V _ÇL ..L D P Q T 'GVIVRLERETFQVLNMY ïlV 1836 GGG AAG GTG GTG ACT GTC AGA CAT CAG GCT GTG ACC CGG AAG AAG GAC AAC CGC TTT GCT GTG GCC TTG GAC TCA 565 GKVVTVRHQAVTRKKDNR F A V A = L _ D S 1911 GAG CAG AAC AAC ATC CAT GTG AAA GAC ATC GTT AAG GTC ATT GAT GGC CCC CAC TCA GGC CGA GAA GGG GAG ATT 1985 614 590 EQNNIHVKDIVKVIDGPHSGREGEI 1986 CGC CAH T CTC TTC CGA AGC TTC GCC TTC CTA CAT TGC AAG AAA CTG GTG GAG AAC GGG GGC ATG TTT GTC TGC AAG ACC CGC CAC CTG GTG CTG GCT JGG GGC TCA AAG CCC CGT GAT GTG ACC AAC TTC ACC GTG GGT GGC TTT GCG CCT T R H L V L A ÏGSKPRDVTNFTVGGFAP ATG AGT CCC CGG ATC AGC AGC :CC ATG CAC CCC AGT GCT GGA GGT CAG CGT GGC GGC TTT GGT AGC CCA GGT GGC M S P R I S S >MHPSAGGQRGGFGSPGG GGC AGT GGT GGC ATG AGC AGG 5GC CGG GGC CGG AGG GAC AAC GAA CTC ATC GGC CAG ACC GTG CGC ATC TCC CAG 5 G S G G M S R ^RGRRDNELIGQTVRISQ ?ÏS GGG CCC TAC AAA GGC TAC ATC ÎGT GTG GTG AAA GAT GCC ACA GAG TCC ACG GCC CGT GTG GAG CTG CAC TCC ACC K I jVVKDATESTARVELHST TGC CAG ACC ATC TCT GTG GAC :GT CAG CGG CTC ACC ACG GTG GGC TCA CGG CGC CCG GGC GGC ATG ACC TCG ACC C Q T I S V D ^ _ _Q _R L T_ TV G S R R P G G M T S T TAT GGG AGG ACG CCC ATG TAT GGC TCC CAG ACG CCC ATG TAT GGC TCT GGC TCC CGA ACA CCC ATG TAC GGC TCA 2510 YGRTPMYGSQTPMYGS GSRTPMYGS 789 CAG ACA CCC CTC CAG GAT GGT AGC CGC ACC CCA CAC TAC GGC TCA CAG ACG CCC CTG CAT GAT GGC AGC CGC ACT Q T P L Q D G S R T P H Y G S 0 T P L H D G S R T 2586 CCT GCC CAG AGT GGG GCC TGG GAC CCC AAC AAC CCC AAC ACG CCG TCA CGG GCT GAG GAA GAA TAT GAG TAT GCT 815 PAQSGAWDPNNPNTPSRAEEEYEYA TTC GAT GAT GAG CCC ACC CCG ÏCC CCG CAG GCC TAT GGG GGA ACC CCC ÄA.T CCC CftA ACA CCS GGC TAC CCA GAC F D-.-D- EPTPSPQAYG _ G ..f I- K g 0. f P G Y S S II? 2736 CCC ÏCG ÏCC CCA CAG GTC AAC CCA CAA TAC AAC CCG CAG ACG CCA GGG ACG CCG GCC ATG $A AAC ACA GAC CAG 865 P S S P Q V N__ P _Û Y N _P_ _Q_ JT __JL_ G .T P. A. M . Y N T D__ Q _ 2811 TTC TCT CCC TAT GCT GCC CCC ÏCC CCA CAA GGT ÏCC ^C CAG CCC AGC CCC AGC CGC CAG AGC ÏAC CAC CAG GTG 890 F S P Y A A ..gL^...&..,....g .._Q,_.._...,S^,.JL^JL:-- Q ■■* S P S P O S Y H _ Q V__ Iff 2 88 6 GCG CCA AGC CCA G€Â GSC TAC CAG AAT ACC CAC TCC CCA GCC MQ TAC CAC CCÏ ACA CCG TCG CCC ATG GCC TA? 915 AP3PAGVQ NTSSPASYSPÏPSPMAY 2961 CAG GCT AGC CCC AGC CCG AGC CCC GTT GGC TAC AGT CCT ATG ACA CCT GGA GCT CCC TCC CCT GGT GGC TAC AAC 940 QASPSPSPVGÏSPMTPGAPSPGGYN 3036 CCA CAC ACG CCA GGC TCA GGC ATC GAG CAG AAC TCC AGC GAC TGG GTA ACC ACT GAC ATT CAG GTG AAG GTG CGG 965 PHTPGSGIEQNSSDWVTTDIQVKVR 3111 GAC ACC TAC CTG GAT ACA CAG GTG GTG GGA CAG ACA GGT GTC ATC CGC AGT GTC ACG GGG GGC ATG TGC TCT GTG 3185 990 DTYLDTQVVGQTGVIRSVTGGMCSV 1014 318 6 TAC CTG AAG GAC AGT GAG AAG GTT GTC AGC ATT TCC AGT GAG CAC CTG GAG CCT ATC ACC CCC ACC AAG AAC AAC 1015 YLKDSEKVVS ISSEHLEPITPTKNN 3261 AAG GTG AAA GTG ATC CTG GGC GAG GAT CGG GAA GCC ACG GGC GTC CTA CTG AGC ATT GAT GGT GAG GAT GGC ATT 1040 KVKVILGEDREATGVLLSIDGEDGI 3336 GTC CGT ATG GAC CTT GAT GAG CAG CTC AAG ATC CTC AAC CTC CGC TTC CTG GGG AAG CTC CTG GAA GCC tqaaqcag 1065 V R M DLDEQLKILNL __ R__. F_ _L_. G. K L L E A 3413 gcagggccggtggacttcgtcggatgaagagtgatcctccttccttccctggcccttggctgtgacacaagatcctcctgcagggctaggcggattgttc 3512 3513 tggatttccttttgtttttccttttagttttccatcttttccctccctggtgctcattggaatctgagtagagtctgggggagggtccccaccttcctgt 3612 3613 acctcctccccacagcttgcttttgttgtaccgtctttcaataaaaagaagctgtttggtctaaaaaaaaaaaaaaaaaaaaaaaa 3698

Fig. 2. cDNA and derived amino acid sequence of SUPT5H. The 5'- and 3'-UTRs are printed in lower case, the polyadenylation signal is highlighted in bold. Amino acid sequences confirmed by microsequencing of purified Supt5hp are underlined. Acidic residues of the N-terminal region are boxed, as is the putative NLS signal. The CTRl and CTR2 repeats are boxed and marked differently. A.A. Stachora et al.lFEBS Letters 409 (1997) 74-78 11

3.3. SUPT5H cDNA and predicted structure of the encoded CTR1 CTR2 protein position sequence position sequence The SUPT5H cDNA has a length of 3699 bp, with an open 754 G R G 844 pHpF" P p Q reading frame of 3261 bp, predicting a protein of 1087 amino 760 G M T s T 854 LïL p Q TP[S]|Y| acid residues (Fig. 2). The 3'-UTR of 270 bp contains a poly- 766 G R T P M Y 863 [P] D p Q 772 G [si Q T p M Y 881 T p A M |Y adenylation signal (AATAAA), preceding a poly(A) tail by 17 781 G R R T p M Y 896 P S p V G S y 788 G S 0 T p T, Q 904 [Pj[Sj P s p Q S Y bp. A 5'-UTR region of 143 bp precedes a start ATG codon 796 G S R T p H Y] 916 P s p A G ï which is in agreement with other functional initiation sequen- 803 G S Q T p L H 924 '1' H p y 811 _G_ S R T p A Q 932 T s p 1*1 A Y ces [18]. There is 31% overall amino acid identity between 943 H S 0 s p M Y V 21 Supt5hp and its yeast homologue, with the domain 1-816 e s R T p X Y P » P S P Q S Y displaying a stronger identity of 34%, and the C-terminal Q S Ä, G 271 residues showing a weaker one of 25%> (Fig. 3). Supt5hp, RNAp jlymeras eil (g P T s p s * similar to its yeast counterpart, has a very acidic N-terminal domain of 118 amino acid residues 62 of which are either Asp Fig. 4. Structure of the C-terminal repeats. The C-terminal repeat domains (CTR) 1 and 2 are listed and the consensus motifs are giv- or Glu (Fig. 4). Residues 77-81 contain an incomplete (L-D- en. On the left side the number of the first residue of each repeat is E-A-D) DEAD-box motive (normally implicated in ATP displayed. Below these repeats the consensus sequence of the CTR binding) which, however, lacks the characteristic additional of RNA-polymerase II is listed, which shows a similar pattern. conserved domains normally flanking such a motif. A putative nuclear localization signal KRKKFKR (residues 328-334), Pro and Tyr, contains two different C-terminal repeat motifs may be responsible for the observed nuclear localization of (Fig. 4), unrelated to the motif S-T/A-W-G-G-A/Q, present in Supt5hp. A C-terminal domain of 280 residues, rich in Ser, yeast Spt5p. The first motif (CTR1) with the consensus se- quence G-S-R/Q-T-P-X-Y is repeated 9 times and the second,

1 MSDSEDSNFS EEEDSERSS 19 less conserved motif (CTR2), with the consensus sequence P- III III :h h: T/S-P-S-P-Q/A-S/G-Y is repeated 10 times. 1 MSDNSDTNVSMQDHDQQFADPWVPQSTDTKDENTSDKDTVDSGNVTTTE 50 20 DGEEAEV 26 3.4. Cell cycle-dependent phosphorylation of the Supt5hp 51 STERAESTSNIPPLDGEEKEAKSEPQQPEDNAETAATEQVSSSNGPATDD 100 To monitor the amount of Supt5hp throughout the cell 27 DEERRSAAGSEKEEEPEDEEE 47 cycle, we performed immunostaining with AS40 of lysates 101 AQATLNTDSSEANEIVKKEEGSDERKRPRÉEDTKNSDGDTKDEGDNKDED 150 from synchronized HeLa cells. We found the amount to re- 48 EEEEEEYDEEEEEEDDDRPPKKPRHG-GFILD-EADVTjDEYEDEDQWEDG 95 main constant during the cell cycle, like that of actin, while 151 DDEDDDDDDDDEDDDDÈAPTKRRRQÉRNRFLDIEAÉVSDD-EDED--EDE 197 the cyclin B level showed its characteristic fluctuation (Fig. 96 AEDILEKEEIEASNIDNWLDEDRS--GARRLQNLWR--DO REEE 13 6 5A). During mitosis, Supt5hp appeared as a more slowly mi- 198 EDSELVREGFITHGDDE DDEASAPGARRDDRLHRQLDQDLNKTSEED 244 grating form in SDS-polyacrylamide gels than in interphase. This effect is likely to result from protein phosphorylation, 137 LGEYYMKKYAKSSVGETVYGGSDELSDDÏTQQQLLPGVKDPNLWTV|: :|::|| : :: |: : T MM : :|K 18|:3 245 AQRLAKELRERYGRSSSKCjYRAAAQDGY VPQRFLLPSVDTATIWGVR 291 since it can be reverted upon treatment with calf intestinal 184 CKIGEERATAISLKRKFIAYQ--FTDTPLQIKSWAPEHVKGYIYVEAYK 231 phosphatase (Fig. 5B). 292 CRPGKEKELIRKLLKKKFNLDRAMGKKKLKILsiFQRBNYTGRIYiEAPK 341 232 OTHVKQAIEGVGNLRLGYWNQQMVPIKEMTDVLKWK--EVANLKPKSIW 279 4. Discussion 342 QSviEKFCNGVPDiYis--QKLLipVQELPLLLKPNKSDDVA'LEEGSYV 388 280 RLKRGIYKDDIAQVDYVEPSQNTISLKMIPRIDYDR1XA-RMSLKDWFAK 328 Genetic evidence in the yeast S. cerevisiae suggests that 389 RIKRGIYKGDLAMVDQISENNLEVMLKIVPRLDYGKFDEIDPTTQQRKSR 438 Spt4p, Spt5p and Spt6p modify chromatin structure and 329 RKKF-KRPPQRLFDAEKIRSLGGD—VASDGDFLIFEGNRYSRKGFLFKS 375 thereby change transcriptional regulation of a large number 439 RPTFAHRÀPPQLFNPTMALRLDQANLYKRDDRHFTYKNEDYI-DGYLYKS 487 of genes in vivo. We have purified Supt5hp, a human homo- 376 FAMSAVITEGVKPTLSELEKFEDQPEGIDLEWTESTGKEREHN--FOPG 423 logue of Spt5p and cloned its cDNA. The open reading frame 488 FRiQHVETKNiQPTVEELARFGSKEGÀVDLTSVSQSIKKAQAAKVTFQPG 537 of SUPT5H predicts a protein of 1087 amino acid residues, a 424 DNVEVCEGELINEQGKILSVDGNKITIMPKHEDLKDMLEFPAQELRKYFK 473 calculated molecular mass of 121 kDa and a pi of 4.8, com- 538 DRIEVLBGEQRGSKGIOTRTTKDIATI--KLNGFTTPLEFPISTLRKIFE 585 pared to the yeast Spt5p protein with 1063 residues, a calcu- 474 MGDHVKVIAGRFEGDTGLIVRVEENFVILFSDLTMHELKVLPRDLQLCSE 523 lated mass of 116 kDa and a p/ of 5.0. Both proteins share 586 PGDHVTVINGEHQGDAGLVLMVEQGQVTFMSTQTSREVTITANNLSKSID 635 31% sequence identity. The apparent mass of the purified 524 TASGVWGGQHEWGELVQLDPQTVGVIOTLERETFQVLNMYGKWTVRHQ 573 protein as determined by SDS-PAGE is somewhat higher 636 TTAT SSEYALHDivELSAKNVACliQAGHDIFKviDETGKVSTITKG 682 than the calculated mass. This discrepancy is probably due 574 AVTRKKDNRFA'-VALDSEQNNIHVKDIVKVIDGPHSGREGEIRHLFRSI F 621 to the abundance of acidic residues present in Supt5hp, caus- 683 SILSKINT. :VSSVDANGNEIKIGD--TIVEKVGSRREGQVLYIQTQGQVI Q 730 ing a reduced electrophoretic mobility in SDS-PAGE. Similar 622 AFLHCKKLVENGGMFVCKTRHLVLAGGSKPRDVTNFTVGGFAPMSPRISS 671 differences between masses calculated from electrophoretic 731 IFWSKKivENAGVPVVNPSN-VEAVASKDNMLSN--KMDLSKMNPEIIS 777 mobilities and from the derived sequence have been described 672 PMHPSAGGQRGGFGSPGGGSGGMSRGRGRRTJNELIGQTVRISQGPYKGYI 721 for many other proteins [19-21]. I I ■ II : I h IM' : III : 778 KMGPPSS KTFQQP IQSRGGREVAL-GKTVR1RSAGYKGQL 816 A carboxyterminal domain of Supt5hp is rich in Ser, Pro 722 GWKDATESTARVELHSTCQ'TISVDRQRLTTVGSRR?GGM7STYGRTPMY 771 and Tyr and contains two C-terminal repeat motifs (CTR). 817 GivKDVHGDKATVELHSKNKHITIDKHKLTYYN--REGGEGITYD 859 Nine variants of the consensus sequence G-S-R/Q-T-P-X-Y Fig. 3. of Supt5hp (this work, upper lines) and (CTR1) are found and 10 copies of the type P-T/S-P-S-P-Q/ yeast Spt5p ([22], lower lines). The C-terminal regions of both se- A-S/G-Y (CTR2). The yeast Spt5p also contains a C-terminal quences, with a less significant homology, are omitted. Identities are repeat, yet with a different consensus motif (S-T/A-W-G-G-A/ marked '1', related residues are labeled ':' and deletions introduced to maximize the homology are indicated by '-'. Q). The importance of the repeat domain for the yeast protein 78 A.A. Stachora et al.lFEBS Letters 409 (1997) 74-78

Supt5hp binds ATP in photolabelling experiments (data not shown). This finding is particularly interesting, as proteins with a function implicated in the modulation of chromatin structure were shown to possess an ATPase activity [27,28]. We do not know yet whether the incomplete DEAD-box mo- tif present in Supt5h protein is involved in ATP-binding by Supt5hp. As yeast Spt4p and human Supt4hp are functional homo- logues [11], one may expect a similar functional conservation from yeast to human for Spt5p and Spt6p and their human homologues. Further functional studies will address this issue.

Acknowledgements: We are grateful to H. Krebber for help with cDNA screening, A. Hunziker for sequencing parts of the cDNA clones and J. Kretschmer for technical assistance. We also want to thank R. Deane for critically reading the manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to H.P.

References

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