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Zfp60, a mouse zinc finger gene expressed transiently during in vitro muscle differentiation
1 EBS 17085
FEBS Letters 387 (1996) 117 121
Zfp60, a mouse zinc finger gene expressed transiently during in vitro muscle differentiation Marie Pere#, Giovanni Rompato ~, Nicoletta CorbP, Laura De Gregorio b, Tommaso A. Dragani b, Claudio PassanantP ,e'* Istituto Pasteur-Fondazione Cenci BolognettL Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Universit3 di Roma La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy hDivision of Experimental Oneology, Istituto Nazionale Tumori, Via G. Veneziani 1, 20133 Milan, Italy "Istituto di Tecnologie Biomediche, CNR, Via Morgagni 30IE, 00161 Rome, Italy
Received 22 March 1996
~bstract The complete cDNA coding sequence of the zinc linger gene Zfp60 is reported. The predicted amino acid sequence of the Zfp60 protein has been found to contain 19 zinc finger motives clustered at the C-terminus. At its N-terminus, Zfp60 ~hares with other members of the zinc finger gene family two additional conserved amino acid modules named Kruppel ~.ssociated Boxes (KRAB). The expression patterns of Zfp60, MyoD and MHC mRNAs have been followed during in vitro myogenic differentiation of C2 cells. We show that the bacterial produced Zfp60 protein binds DNA only in presence of zinc ions. Zfp60 locus has been mapped in chromosome 7, where other Zfp loci are localised. Key words: cDNA cloning; Zinc finger protein; 'VIyogenic differentiation; Temporal expression; ) N A binding; Chromosome mapping
t. Introduction The formation of mature muscle fibers requires the coordin,tted expression of many different genes coding for the myo~ube-specific proteins. These functions are governed by a set .)f regulatory genes [1]. Myogenic cell lines and myoblast 0rimary cultures can mimic many of the steps characteristic ) f myogenic differentiation. The possibility to follow and to •~tudy the in vitro transition of myoblast cells to myotube !~rovides a useful system to study gene regulation during myo!genesis. In the last few years, several muscle regulatory genes !lave been identified and characterized such as many members •)f the MyoD gene family. This family of regulatory genes ~MyoD, myogenin, Myf5 and MRF4) share the same evoluionarily conserved domains, a basic region involved in DNA ~inding to the specific target sequence (E box), and a helix o o p helix (HLH) implicated in homodimerization and/or leterodimerization with other regulatory protein [2]. The badc idea of this work is the isolation and characterization of )ther regulatory genes, involved in myogenesis, coding for ~roteins with the 'zinc finger' motif as a DNA binding donain. A zinc finger motif was first identified in the Xenopus aevis RNA polymerase III transcription factor TFIIIA [3,4]. qince then, several zinc finger genes involved in gene regulaion have been identified in a variety of animals and plants 5,6].
~Corresponding author. Fax: (39) (6) 446 2891. E-mail: [email protected]
In a previous work we reported on the characterization of three zinc finger genes, isolated from an adult mouse skeletal muscle cDNA library [7]. In this work we describe one of those genes, termed Mfg3 and presently renamed Zfp60 according to the international convention. We determine the complete cDNA coding sequence and the predicted amino acid sequence of the ZFP60 protein. This protein is characterized by a cluster of 19 zinc finger motifs at the C-terminus and by the two Kruppel associated boxes (KRAB) A and B at the N-terminus. These modules have been shown to define a subfamily of multifinger proteins [8,9] and to be associated with transcriptional repression activity [10 13]. The expression patterns of Zfp60, MyoD and myosin heavy chain (MHC) mRNAs have been followed and compared during myoblast C2 cell line differentiation. We also show that the bacterial expressed Zfp60 protein is able to bind DNA only in presence of zinc ions. Using restriction length polymorphism (RFLP) we assigned the Zfp60 locus to the murine chromosome 7, at about 10 cM from the centromere, where a cluster of Zfp loci maps. This region shows homology with human chromosome 19q13.
2. Materials and methods 2.1. RNA extraction and Northern blotting Total RNA was extracted from cell lines using the method described by Chomczynski and Sacchi [14]. Total RNA (10 ~tg) was fractionated by electrophoresis on a 1.2% agarose-formaldehyde gel according to Maniatis and co-workers [15]. The gels were blotted on nylon filters (Hybond N, Amersham) and hybridized according to the manufacturer's instructions. DNA probes were labelled by [c~-32p]dATPincorporation using Megaprime Probe Kit (Amersham). 2.2. cDNA library screening A k gtll cDNA library, prepared from RNA of mouse F9 teratocarcinoma cell line differentiated using retinoic acid (0.1 p_M) was screened using as a probe the previously isolated cDNA fragment [7], spanning from nt 1447 to nt 1990 (see Fig. 1). 2.3. Cloning the cDNA 5' region In order to isolate the 5' region of Zfp60 mRNA we used the reverse PCR technique first described by Uematsu [16], and modified as previously described [17]. Briefly, three oligonucleotides were synthesized in the known extreme of the 5' end of Zfp60 cDNA: antisense oligonucleotide (A) from nt 551 to nt 573, sense oligonucleotide (B) from nt 524 to nt 547, antisense oligonucleotide (C) from nt 330 to nt 353. For cDNA synthesis, 1 ~tg of total RNA prepared from mouse F9 teratocarcinoma cell line induced to differentiate with retinoic acid [18] was used. RNA was primed using 10 ng of oligonucleotide A. First strand and second strand cDNA synthesis was performed using the Promega RiboClone cDNA System kit according to the manufacturer's instructions. The double strand cDNA in a total volume of 7 lal of TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA)
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GGGACGGTGCTGTGTGTGCGCGCGCGTCTCTGCGTACGCACC~AGCTATTTGCATTAT CACCGTGCTTGTCGCTTTCTCTCGCGGCCACCAGCAGAGGACCAAGCTTCACTGAGGAGA GGGGAGGAAG CCTGAAGAAGAATG G C C A A C T C T A G T T C T C A A C A C A T G G T C T G T G G G T C A M A N 8 S S Q H M V C ~ ~ G T G A C A T T T A G G G A T G T G G CTG TTG A CTTCT C T C A G G A G G A G T G G G C C T G C C ~ A T G C T V T F R D V A V D F S 0 E E W A C L D ~ A C T C A G A A G G T C T T A T A C A G GAA C A T A A T G T T G G A G A C C T A C A G C A A C C , ~ T C G C A G T G T Q K V L Y R N I M L E T Y S N L V A V GTGGGACGT~CAT~CAAGCCAGATCTGATCGTC~A CTAGAG C C G G A G A ~ G A G C C C V G R C I P K P D L r v L L E P E K E p TGGATC~TGAAGAAAGAAACAGG CAGGCCGAG C C A A G G ~ " I ~ A G A C A G G T ~ A A K A V K K ~ T G R P S 0 G ~, E T G F E GC~A~TAGATCT CCAAAGAATCATGTTTATAAT~GTTACC CAAACAAACC.ATA A E N R S P K N H V ¥ N K K L P K Q T I CAGCAATTAAGTAAAACTTCGGACG T C CAGGG CGTCAGTGTTAGTAA~,,GCC CTGGCTAT Q Q L S K T S D V Q G V S V S N G P G Y AGTGTAATCAAGGAGCCACAGAATTATCAAGAAGGAGA~CTAATCGA~TATTACTAAC S V I K E P Q N ¥ Q E G D A N R N I T N AAGAAAGAAATGTCTACTTACACTAGCAAGACTCTTGCTCACAATAAAGAA~CCGTAT K K E M S T ¥ T S K T L A H N K E K P Y A~TGTAAAGAC~GGGAAGTG CT~TTGTAAGTCAAACCTTCATCAGCATGAGAGT K ~ K D C G K C F G C K S N r. H Q H E S ATTCACACTGGAGAGAAG C C C T A T G A A T G T A A G G A C T G T G G G A A G A C C T T C A G A C T C C C T I H] T G E K P Y E ~ K D C G K T F R L P C A G A T G C T T T C A A G A C A T C A G A A G T C T C A C A G T G A T G AGAGAC ~-t-FFrGAGTGTAATATA Q M L S R H Q K S H] S D E R P F E [C N I TGTGGA~GTCT~ CATCTTC CTAC C C T G C ~ C A G T A C C A T A A A A A T A T T C A T A C A G G T C G K S F H I". p T L L O Y H K N I H] T G T T A A A A C C A T T T G A A T G T G A G G A G T G TGGG A A G T C C T T C A A G T C C T T C A A C C G C A T C T C T L K P F E [C E E C G K S F K S F N R I S ACC~-'~'x'~'I"I'CAGCATAGGACTATTCATGCAGGTATGAAACCGTATAAATGTAATGTGTGT T L F Q H R T I H] A G M K P Y K [C N V e GGGAAAGCCTTTAATCGTCGCTCAAACCTCC~CAACATCAGAAAATTCATTCTGAGGAC G K A F N R R S N L L Q H Q K I H] S E D AGAC~-FFFI"CATTGTAAGGTGTGTGGAAAGGCCTTCACTGTGCTGGCTCAGCTTACTCGG R P F H [C K V C G K A F T V L A Q L T R CAeGAGAACATTCACACTGAAGACAAATCGTTTGAATGTAAGCAGTGTGGTAAGATATTT H E N I H] T E D K S F E [C K Q C G K I F AGTAATGGCTCTTACCTCTTACGACACTACGATACTCACACCAATGAGA~CCCT'~AG S N G S Y L L R H ¥ D T H] T N E K P F E TGTAATATATGTGGCAAGGCTTTTAGGCTTCATCTATACCTTTCTGAGCATCAGAAAACT [C N I C G K A F R L H L ¥ L S E H Q K T CACACCGATGAGAAGCCTTTCAAGTGTAAGCTGTGTGAGTCAGCCTTCAGACGTAAGTAC H] T D E K P F K [e K L C E S A F R R K Y CAGCTCAGTGAACATCAGAGAATTCACACTGATGGTAAACCCTATCAGTGCAAGGAT~T Q L S E H Q R I H] T D G K P Y Q [C K D C TGGGAAT~CT~CGTCGACGATCAAATT~ATCGAACATCAGAGTATTCACACTGGAAAG W E F F R R R S N F I E H Q S I H] T G K AAACCC~GAGTGTAAGGACTGTGGGAAGGTCT~AGACTA~TATACATC~TTCGA K P F E [C K D C G K V. F R L N I H L I R C A T C A G A G A T T T C A T A G T G ATGAGAAG C C CTTTG A A T G T A A A G A A T G T G G A A A G G ~-FFFI" . ~ R r a] s D E K p ~ ~ ( c ~ ~ c ~ x ^ ~
M. Perez et al./FEBS Letters 387 (1996) 11~121
60 120 180 13 240 33 300 $3 360 78 420 83 480 113 540 133 600 1S3 660 I73 720 193 780 213 840 233 900 253 960 273 1020 283 1080 213 1140 333 1200 3S3 1260 373 1320 383 1380 413 1440 433 1500 4S3 1560 473 1628
C A T T T ~ C CAG CCAG C T T A A T A A C C A T A A ~ C C A G T C A T A C A G G TCAG ACA C ~-LTL-t'GAA 1 6 8 0 H F S S Q L N N H K T S H] T G Q T P F E 513 TGTAAAGAATGTGGGAAGTCGTTTAAGCGTGTCTCCAGCCTTGTGGAACATAGGATTATT 1740 [C K E C G K S F K R V S S L V E H R I I 533 CACAGTGGTGTGAAACCATATAAATGTAATG CGTGTGGAAGAGCCTTCAATCGTCGCTCA 1800 H]S G V K P ¥ K [C N A C G R A F N R R S 553 AACCTCATGC~CATGAGA~TTCATTCTGATGAGAGACCCT~GAGTGTAAGGACTGT 1860 N L M Q H E K I H l S D E R P F E [C K D C $73 G G A ~ G G C C T T C A C T G T T C T G G CTCAGCTCA CACGG CAC C A G A C G A T T CATAATC, G A A A G 1 9 2 0 G K A F T V L A Q L T R H Q T I H] N G K $93 AAATCATA~%2AATGTGAGCAGTGTG GG TCAG C CTTCAGA CT C CCGTAC CAA CT C A C T C A A 1 9 8 0 K S Y E [C E Q C G S A F R L P ¥ Q L T Q 613 C A T C A G A G A A ~ F C A T T A T G A T G TGAA G C CATTT C A G T G C A A G G A A T G T G G A A G G G ~'FFFL" 2 0 4 0 H Q R I H] y D V K P F Q [c K E C G R A F 633 G T T C G T A G T A C A G G CCTTAG A A T T C A T G A A A G G A T C C A C A C ~ T G A G A A G C CCTTTCAG 2100 V R S T G L R I H E R I H]T G E K P F Q 683 TGTAAGGAGTGTGGAGAAG CCTTTCAGTATCATTACCAATTTCTTGGACACTTTAGAATT 2160 [C K E C G E A F Q ¥ H Y Q F L G H F R I 673 CATACTGGC~AACCCTTATGAATGTAGTGAATGTGGGAAGTACTTTACTTATGGTCGA 2220 H] T G K N P Y E [C S E C G K Y F T Y G R 693 GACCTTAAAGTACATCAGAGTATTCACAATCT~AGA~CCGTAAGAATGTGGG AAGA C C 2 2 8 0 D L K V H Q S I H] N L E K P * 707 TTCAG~rGA~TCAAAGGTGGTTTGACATGCTAAACTTAATATTATGAAGAAGAAGCCT 2340 TACAATGCTTATAAGGAATGTGGAAAGCCTTCAGTAATTCTAAGTAGGTGTATATCAGAG 2400 AATTCATACTGGTCTGAAGCACTGTTGATATGAAAATTGTGAGAAGTTTAGATTAAGTT C 2460 AGCCTTCAGTGTACACTCAAGATTCATATTGGCTGAACCCATCACTGTAAGATGT~ 2520 GACCCG C R A C ~ G C T C A G G C C T T C A G CATA~GTATCCATGCTGTCGTGAAACACTA 2580 TGCAGC, G G T G T G T A A A C A T T T A T A G A A T T C A T T C T G A A A A C T C A T G A A G G A G T C C A T A T A 2640 GGTCTGAAACCATAAGGATGTGAAAAAAATTTTATTGTTAAATGGTGAGCTTATACAG CA 2 7 0 0 TGAGAAAAGGAAGACGGATAAGACTTTATGGAAATTTTACTGAACATCAAAGAAGTCATT 2760 AACAAACTGGGACTGAAAGGAGTCTGGGAAGAAC"~-~"~AATCTATCATAGGATTTAACCAA 2820 CATCTGAGTGTTCATATAAAAAAAATCCTTAGAGAATGAGAGTGGGAATTCTGTTCACAG 2880 AAAATACTGT~GAAGGACAGAAAGAGATATTGTAGCC~CATGCTC~AAGTTAC~TC 2940 A~vxTx'AATACTACTGTTAATTGCATAAGCTTAATTCACCAAACACAATTCAGCTCACTT 3000 AGGAAGAACAGTTATTGAGACTATTATTATTCGAGGCACCT~CeACCAATTACATCCCA 3060 TGCCTCCTGTGCTATTACCTGGATGTTTGCTTTCTTTCTGCATAGTTCATGGTGAGCACG 3120 GTAe~ATGTGTGGAAATCGGGGTCATAATGGATAGAGAATCCTTGAGAGTATTT 3180 GGTATGT~TGAAGTAGCTTCTACAGTGTTGGTGCTCTCAGGGGCGTTACTGTAACC, G 3240 ATATA~GACAAGTTGTGTCGTAGATAGT~AGA~GCCTCATGCAGCCCTCCAGTAT 3300 TAAATTCTAAATATTTTCTTCTGGGTAAAATCTAATAGATGAACAGGTACATGAAAGCAT 3360 ~-FI-L'ATTCTCAATCCGTTTCTTTAGCTTTCGTGCAGAGATGTCTATAAACTAGCTGTTGG 3420 TGAATAAGTAC-L-L-I-~AAACGGGGATAAACACATTTAACTTTCAAAAGCTAAAATCCTGTA 3480 ATTTATGTTCTCTGCCCAAACATTAAGATAAAATGCATTTGTGACTCAGCAGGACAGTGC 3540 AGTCCCCTCCATCTCATGCTGCTGAGACCATTTTATGTTGACTAGAATTTGA-~-~-L-L.AGAT 3600 GGAGGAGATTCTTGAGATTTACCCACCTGTGATCTGTGAGCCAGCGGTCAGATC~CTG 3668 CTATTTTA~-L"~"L~GCTTTTCTTAGACTGCCTGCCTGTGCAAACTTTCCCCTTCCACTTAA 3720 CTGCTTACCC~CTTCTGTTA~C'~CTGC'~GGCAAATGCTCCCCCTGTAGCTGCT 3780 GAGCAAGATATCAGAATGTGGTTCTTG.xTx-F~.AATAGCCCCTTGCTGTAAATGCTTGTGA 3840 GTGTATATGGATCCCGAATA~TGAGTGTAGTCCCAGCCAACTGGAATItI~GACq-iqCAG 3800 TTGGCTAAAAAAAAACGGAATTC 3823
483
Fig. 1. Nucleotide sequence of Zfp60 cDNA and its deduced amino acid sequence. Nucleotides are numbered on the right, amino acid sequence is numbered in bold characters. The amino terminal KRAB boxes A and B are underlined and double underlined respectively. Individual zinc finger domains are shown between square brackets. The polyadenylation signals are in bold characters; the 'ATTTA' sequences, potentially involved in mRNA selective degradation, are underlined. The Zfp60 nucleotide sequence has been deposited at GenBank, with accession number U48721.
was circularized according to Uematsu [16]. The amplification of the circularized double strand cDNA was accomplished using the Zfp60 oligonucleotides B and C and the GeneAmp PCR Core Reagents Kit (Perkin Elmer Cetus). The amplification cycles consisted of denaturation for 30 s at 94°C, annealing for 30 s at 66°C, and extension for 1 min at 72°C. 10 ~1 of the PCR products were analyzed by 1.6% agarose gel electrophoresis and transferred to Amersham Hybond N nylon membrane. Specific products for Zfp60 sequences were detected by hybridization using [7-32P]ATP end labelled oligonucleotide A as a probe. The PCR products were then directly subcloned using the TA Cloning Kit (Invitrogen). 2.4. DNA sequencing DNA fragments of cDNA clones were subcloned either in pCR vector (Invitrogen) or pBluescript (Stratagene) and both strands were sequenced using the Sequenase kit according to the manufacturer's instructions (United States Biochemical). 2.5. Cell cultures The mouse myoblast C2 cell line [19] clone 7 was obtained from Dr. M. Buckingham (Institut Pasteur, Paris, France). Cells were maintained as undifferentiated myoblasts in Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal calf serum (FCS) in a 6.5% carbon dioxide atmosphere. To induce cell differentiation, myoblasts were grown to confluence and shifted to DMEM supplemented with 2% FCS. 2.6. Expression o f recombinant fusion protein The entire coding region of Zfp60 cDNA was cloned in pQE-30 vector (Qiagen). Zfp60 bacterial fusion protein was expressed and purified according to the manufacturer's instructions.
2. 7. Gel electrophoresis and South- Western blotting Purified protein obtained in bacteria (0.5 ~tg per lane) was separated on 9% SDS-polyacrylamide gel, blotted to nitrocellulose paper (Hybond C, Amersham) as described by Harlow and Lane [20]. South-Western blotting was performed according to Sukegawa and Blobel [21]. In particular two identical filter blots (A and B) were incubated for 12 h in renaturation buffer A (50 mM Tris-HC1 pH 7.5, 100 mM KC1, 1% Triton X-100, 10% glycerol, and 0.1 mM ZnC12) and in renaturing buffer B (50 mM Tris-HC1 pH 7.5, 100 mM KC1, 1% Triton X-100, 10% glycerol, 10 mM EDTA, and 2 mM DTT) respectively. Total mouse genomic DNA was sonicated to a final size of about 1 kb, labelled with [a-32P]dATP and random hexanucleotide primers. 5 × 104 cpm per ml were added and incubated for 3 h to both filters. The filters were washed five times, for 10 s, in the respective buffers A and B, and autoradiographed. All incubations were performed at 4°C. In order to normalize the amount of protein loaded in each lane, the same filters were then incubated with Zfp60 mouse polyclonal antibody. Proteins were then visualized using an alkaline phosphatase-conjugated goat anti-mouse. 2.8. Genetic mapping of Zfp60 Details on the interspecific test cross population of 106 male (C3H/ He)< M. spretus)× C57BL/6J, designed HSB, have already been reported [22]. The marker locus Ligl was identified by segregation analysis of the three M. spretus polymorphic bands, detected by the hybridization of HSB Taql-digested genomic DNAs with the Ligl cDNA [23]. A genetic linkage map was constructed by multipoint analysis of the data, with the MAPMARKER/EXP program [24,25]. Genetic distances were computed by use of Haldane's mapping function. Linkage between markers was considered significant if the LOD score was >3. Zfp60 locus was mapped by restriction length polymorphism (RFPL) analysis of a 813-bp fragment from 3'-UTR
119
M. Perez et al./FEBS Letters 387 (1996) 117 121
region of the murine Zfp60 gene. The fragment was obtained by PCR amplification of 75 ng of genomic DNA. 5 pmol of specific primers: from nt 3042 to nt 3072 (5'-GCCACCAATTACATCCCATGC('TCCTGTGC-3') and from nt 3856 to nt 3827 (5'-GGGATCCATATACACTCACAAGCATTTAC-3') were designed from Zfp60 eDNA. Samples were subjected to 40 PCR cycles consisting of dena~aration at 95°C for 20 s, annealing at 52°C for 30 s and extension at ~2°C for 30 s. PCR samples were digested using the restriction enzyme ;[intl. We detected a RFLP between HinfI-digested 3'-UTR fraglents of C3H/He (H) and C57BL/6J (B) mouse strains and M. spretus ~) strain. A HinfI site, present in H and B strains but absent in the S train, gave rise to a RFLP. We analyzed the segregation of this band ] the HBS population. The segregation showed the expected 1:1 ,attern.
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~.1. Structural organization To isolate the Zfp60 complete c D N A we screened a ~, : D N A library prepared from the mouse F9 teratocarcinoma ;ell line differentiated using retinoic acid [18]. We used as a ~robe the previously isolated c D N A fragment, corresponding o a portion of the zinc finger coding region [7]. Several Zfp60 ~ositive ~, c D N A clones were isolated. The longest c D N A :lone comprised both the 3' untranslated region and the com>lete zinc finger coding portion of the Zfp60 m R N A , but it ~¢as missing the 5' region of the transcript. To isolate this :>ortion of the Zfp60 m R N A , we used the inverse circular ~CR technique [16,17]. The longest Zfp60 c D N A sequence )btained using different cloning strategies was 3923 bp long G e n B a n k accession n u m b e r U48721). An A U G was identiied at nt 142-144, located in an appropriate context for an nitiation codon [26]. This A U G was preceded by an in-frame
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Fig. 2, Northern blot analysis of total RNA isolated from the mouse myoblast C2 cell line on different days during differentiation. On day 2 cells are shifted to the differentiation medium. The same filter was hybridized with the probes indicated on the left. Panel A: Zfp60 cDNA, panel B: MyoD1 cDNA, panel C: myosin heavy chain cDNA, and panel D: glyceraldehyde-3-phosphate-dehydrogenase cDNA.
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B Fig. 3. South-Western blot analysis of a bacterially expressed and purified Zfp60 fusion protein. The proteins were separated on a 9% SDS-polyacrylamide gel and blotted to nitrocellulose filter. Blots were probed with [0t-z2P]dATP labelled mouse genomic DNA either in the presence of zinc ions (lane 1) or in the presence of EDTA and DTT (lane 2). Panel A: The fusion protein Zfp60 is marked by an arrow. Positions of the molecular mass standard proteins are shown. Panel B: The same filters were incubated with a Zfp60 mouse polyclonal antibody in order to normalize the amount of protein loaded in each lane. Proteins were visualized using an alkaline phosphatase-conjugated goat anti-mouse. stop codon: nt 133-135. As shown in Fig. 1, the A U G codon opens an uninterrupted reading frame encoding a 707 aa long polypeptide. At the C-terminus, 19 zinc finger motifs are clustered, classifying Zfp60 as a multifingered protein. The Nterminal portion of the protein contains two conserved peptide modules termed KRAB, box A and box B, which have been found to occur in several members of the zinc finger multigene family [8,9]. A 1660 nt long untranslated region is present at the 3' end of Zfp60 m R N A . In this region several ' A U U U A ' sequences are present (see Fig. 1), frequently associated with transcripts having an elevated turn-over [27]. Two canonical polyadenylation consensus sequences are arranged in tandem at nt 3857-3862 and nt 3886-3891, a poly A tail is present at the end of the c D N A . 3.2. Expression analysis We have previously reported that Zfp60 gene expression is strongly induced upon differentiation both in the F9 teratocarcinoma cell line and in the C2 myogenic cell line [7]. Here we show a detailed analysis of the expression pattern of Zfp60 in total R N A prepared at different times following the onset of differentiation of the myogenic C2 cell line. As shown in Fig. 2A a specific Zfp60 hybridization signal is present at low level in C2 cycling cells on days 1 and 2, it peaks on day 3,
M. Perez et al./FEBS Letters 387 (1996) 117-121
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Fig. 4. Haplotype data of completely typed HSB mice. Panel A: Each column represents the type of chromosome identified in the progeny. The number of progeny exhibiting each type of chromosome is listed at the bottom. The open squares represent the M. spretus allele; the black squares stand for the C3H/HeJ allele. Panel B: The partial genetic linkage map of the mouse chromosome 7 showing the location of the Zfp60 gene. The recombination frequencies are shown on the left and are calculated by multipoint analysis of recombinants over the number of animals typed for each pair of loci, and expressed as genetic distances in cM (computed using Haldane's function). Distance of the Ligl marker from the centromere is also indicated. concomitantly with the differentiation commitment, and then it slowly declines from day 4 on. Fig. 2B shows the expression pattern of MyoD, one of the muscle-specific determining/ transcription factors [1,2]; its hybridization signal remains nearly constant during differentiation progression. In fact, in the C2 cell line, the MyoD gene product is present in an inactive form in cycling conditions. In order to follow the differentiation progression the MHC gene transcript has been visualized (Fig. 2C). It appears on day 4 and then remains constant during the terminal differentiation. The amount of R N A loaded in each lane has been normalized using as a probe G A P D H cDNA (Fig. 2D). These results suggest that Zfp60 gene has a refined pattern of expression correlated to the onset of differentiation, but not to the maintenance of this stage. 3.3. DNA binding activity A fusion protein coding for the complete Zfp60 was expressed and purified in E. coll. Purified protein was separated by SDS-PAGE, blotted to nitrocellulose, and then probed with sonicated and [c~-32P]dATP labelled mouse genomic
DNA. The probing was performed either in solutions containing ZnC12 or in solutions containing EDTA and DTT [21]. As shown in Fig. 3 the mouse D N A binds Zfp60 protein in the presence of zinc ions, whereas no D N A binding was observed in the presence of EDTA and DTT. These data demonstrate that Zfp60 protein is capable of binding D N A in a zinc-dependent fashion. 3.4. Genetic mapping o f Zfp60 R F L P analysis was used to identify restriction polymorphisms in a PCR amplified fragment (813 bp) from the 3'-UTR region of the murine Zfp60 gene. The fragment was obtained by PCR amplification of mouse genomic D N A using specific primers of Zfp60 cDNA (see Section 2). We detected a R F L P between the HinfI-digested 3'-UTR fragment of C3H/He (H) and C57BL/6J (B) mouse strains and M. spretus (S) strain. A HinfI site, present in strains H and B, but absent in the S strain gave rise to a RFLP. Its segregation was analyzed in the HBS population showing the expected 1:1 pattern (Fig. 4A). Therefore, we could assign the Zfp60 locus to the murine chromosome 7, at about 10 cM from the centromere (Fig. 4B). Other zinc finger coding genes have been mapped in this chromosome region, such as Zfp29, Zfp30, Zfp36, Zfp56 [28-30] and Zfp59 (our unpublished data). This region shows homology with human chromosome 19q13, where the human ZNF36 gene maps, the homologue of the murine Zfp36 [30].
4. Discussion The differentiation processes require the involvement of a cascade of signals and regulatory molecules in order to govern the expression of specific structural genes. The search for regulatory genes appears to be fundamental for the understanding of these sophisticated processes. Here we report on Zfp60, a mouse zinc finger coding gene, which is transiently expressed during in vitro muscle differentiation. To date, only one other member of the multifinger gene family, named ZT3, has been reported to be expressed during skeletal muscle differentiation [31]. The Zfp60 protein has a cluster of 19 zinc fingers and belongs to the KRAB multifinger gene family. The data presented here clearly demonstrate that the bacterially expressed Zfp60 protein binds D N A and that the binding is dependent on the presence of zinc ions. The KRAB domain, located in the N-terminal portion of Zfp60, has been shown to be a potent transcriptional repressor element [10-13]. Its predicted c~-helical structure has been proposed to mediate protein-protein interaction. This hypothesis has been recently supported by the report of co-immunoprecipitation of a protein likely interacting with the KRAB domain [32]. In the present study we follow the expression of Zfp60 during the differentiation of the myogenic C2 cell line. A specific hybridization signal is present at a very low level in C2 cycling cells and increases when the cells are switched to the differentiation medium. Its maximum expression coincides with the massive entry of cells in GO phase and then declines concomitantly with myoblast cells fusion. This transcription pattern has been confirmed in experiments performed on mouse embryonic primary myogenic cells (data not shown). Moreover, the presence in the 3' untranslated region of Zfp60 m R N A of several destabilizing ' A U U U A ' sequences is coherent with the presence of Zfp60 transcript in a very narrow window during myogenic
M. Perez et aI.IFEBS Letters 387 (1996) 11~121
differentiation. This peculiar expression pattern suggests that Zfp60 protein is transiently required during myogenic differeatiation. Zfp60 m R N A peaks concomitantly with the cell e_,atry to the myogenic terminal differentiation program. Duri~g this process myogenic cells undergo profound structural a ad functional changes, including cell growth arrest, cell ~lignment and fusion to form multinucleate myotubes. 7fp60 with its large number of zinc fingers and the K R A B i~gulatory element could effect multiple contacts with D N A nd play a role in the change of chromatin structure, which :companies the transition of a cycling myoblast to mature Hultinucleate myotubes. The fact that several ZFPs have been iadicated to participate in the formation of chromatin struc1 1re [17,21,33 35] supports this hypothesis. Moreover, a gene 1 1at shares a high degree of homology with Zfp60, namely ~;fp59, has been shown to be tightly associated with nuclear ,~affold structure during murine male germ line differentiation [L7]. In particular it is interesting to note that the Zfp60 and 'Zfp59 genes have been assigned to the murine chromosome 7, ia a region where a cluster of other Zfp loci maps. Taken all 1 9gether these data suggest that Zfp60, as well as Zfp59, may I elong to a cluster of structurally and functionally correlated ~enes. cknowledgements." We are grateful to Prof. Paolo Amati for contin-
ous support and critical discussion. We thank Mr. N. Falcone for 1,-chnical assistance. This work was supported by TELETHON 95 I 9rogetto A51, C.P.) and by Associazione Italiana Ricerca sul Cancro, ~,~ilan (to Prof. Paolo Amati). References [1] Ontell, M., Ontell, M.P. and Buckingham, M. (1995) Microsc. Res. Tech. 30, 354-365. [2] Ludolph, D.C. and Konieczny, S.F. (1995) FASEB J. 9, 15951604. [3] Brown, R.S., Sander, C. and Argos, P. (1985) FEBS Lett. 186, 271-274. [4] Miller, J., McLachlan, A.D. and Klug, A. (1985) EMBO J. 4, 1609 1614.. [5] EI-Baradi, T. and Pieler, T. (1991) Mech. Dev. 35, 155-169. [6] Berg, J.M. (1993) Curr. Opin. Struct. Biol. 3, 11-16. [7] Passananti, C., Felsani, A., Caruso, M. and Amati, P. (1989) Proc. Natl. Acad. Sci. USA 86, 9417 9421. [8] Bellefroid, E.J., Poncelet, D.A., Lecocq, P.J., Revelant, O. and Martial, J.A. (1991) Proc. Natl. Acad. Sci. USA 88, 3608 3612. [9] Rosati, M., Marino, M., Franz6, A., Tramontano, A. and Grimaldi, G. (1991) Nucleic Acids Res. 19, 5661-5667.
121 [10] Margolin, J.F., Friedman, J.R., Meyer, W.K.-H., Vissing, H., Thiesen, H.-J. and Rauscher III, F.J. (1994) Proc. Natl. Acad. Sci. USA 91, 45094513. [11] Witzgall, R., O'Leary, E., Leaf, A., E)naldi, D. and Bonventre, J.V. (1994) Proc. Natl. Acad. Sci. USA 91, 45144518. [12] Pengue, G., Calabr6, V., Cannada Bartoli, P., Pagliuca, A. and Lania, L. (1994) Nucleic Acids Res. 22, 2908-2914. [13] Vissing, H , Meyer, W.K., Aagaard, L., Tommerup, N. and Thiesen, H.J. (1995) FEBS Lett. 369, 153 157. [14] Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 56159. [15] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboatory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [16] Uematsu, Y. (1991) Immunogenetics 34, 174-178. [17] Passananti, C., Corbi, N., Paggi, M.G., Russo, M.A., Perez, M., Cotelli, F., Stefanini, M. and Amati, P. (1995) Cell Growth Diff. 6, 1037 1044. [18] Silver, L.M., Martin, G.R. and Strickland, S. (Eds.) (1983) Teratocarcinoma Stem Cells, Cold Spring Harbor Conference on Cell Proliferation, Vol. 10. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [19] Yaffe, D. and Saxel, O. (1977) Nature 270, 725 727. [20] Harlow, E. and Lane, D. (1988) Antibody. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [21] Sukegawa, J. and Blobel, G. (1993) Cell 72, 29-32. [22] Dragani, T.A. and Pierotti, M.A. (1994) Genomics 23, 118-124. [23] Gariboldi, M., Montecucco, A., Columbano, A., Ledda-Columbano, G.M., Savini, E., Manenti, G., Pierotti, M.A. and Dragani, T.A. (1995) Mol. Carcinogen. 14, 71-74. [24] Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E. and Newburg, L. (1987) Genomics 1, 174-181. [25] Lincoln, S.E., Daly, M. and Lander, E.S. (1992) Whitehead Institute Technical Report. [26] Kozak, M. (1987) Nucleic Acids Res. 15, 8125 8148. [27] Brawerman, G. (1990) Trends Biotechnol. 8, 171 174. [28] Denny, P. and Ashworth, A. (1995) Mamm. Genome 6, 683. [29] Denny, P. and Ashworth, A. (1994) Mamm. Genome 5, 643-645. [30] Brilliant, M.H., Williams, R.W., Conti, C.J., Angel, J.M., Oakey, R.J. and Holdener, B.C. (1994) Mamm. Genome 5, 104-123. [31] Polimeni, M., Giorgi, S., De Gregorio, L., Dragani, T.A., Molinaro, M., Cossu, G. and Bouche, M. (1996) Mech. Dev. 54, 107 117. [32] Deuschle, U., Meyer, W.K.-H. and Thiesen, H.-J. (1995) Mol. Cell. Biol. 15, 1907-1914. [33] Noselli, S., Payre, F. and Vincent, A. (1992) Mol. Cell. Biol. 12, 724-733. [34] Cleard, F., Matsarskaia, M. and Spierer, P. (1995) Nucleic Acids Res. 23, 796-802. [35] Yokoyama, N., Hayashi, N., Seki, T., Pant6, N., Ohba, T., Nishii, K., Kuma, K., Hayashida, T., Miyata, T., Aebi, U., Fukui, M. and Nishimoto, T. (1995) Nature 376, 184-188.
FEBS Letters 387 (1996) 117 121
Zfp60, a mouse zinc finger gene expressed transiently during in vitro muscle differentiation Marie Pere#, Giovanni Rompato ~, Nicoletta CorbP, Laura De Gregorio b, Tommaso A. Dragani b, Claudio PassanantP ,e'* Istituto Pasteur-Fondazione Cenci BolognettL Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Universit3 di Roma La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy hDivision of Experimental Oneology, Istituto Nazionale Tumori, Via G. Veneziani 1, 20133 Milan, Italy "Istituto di Tecnologie Biomediche, CNR, Via Morgagni 30IE, 00161 Rome, Italy
Received 22 March 1996
~bstract The complete cDNA coding sequence of the zinc linger gene Zfp60 is reported. The predicted amino acid sequence of the Zfp60 protein has been found to contain 19 zinc finger motives clustered at the C-terminus. At its N-terminus, Zfp60 ~hares with other members of the zinc finger gene family two additional conserved amino acid modules named Kruppel ~.ssociated Boxes (KRAB). The expression patterns of Zfp60, MyoD and MHC mRNAs have been followed during in vitro myogenic differentiation of C2 cells. We show that the bacterial produced Zfp60 protein binds DNA only in presence of zinc ions. Zfp60 locus has been mapped in chromosome 7, where other Zfp loci are localised. Key words: cDNA cloning; Zinc finger protein; 'VIyogenic differentiation; Temporal expression; ) N A binding; Chromosome mapping
t. Introduction The formation of mature muscle fibers requires the coordin,tted expression of many different genes coding for the myo~ube-specific proteins. These functions are governed by a set .)f regulatory genes [1]. Myogenic cell lines and myoblast 0rimary cultures can mimic many of the steps characteristic ) f myogenic differentiation. The possibility to follow and to •~tudy the in vitro transition of myoblast cells to myotube !~rovides a useful system to study gene regulation during myo!genesis. In the last few years, several muscle regulatory genes !lave been identified and characterized such as many members •)f the MyoD gene family. This family of regulatory genes ~MyoD, myogenin, Myf5 and MRF4) share the same evoluionarily conserved domains, a basic region involved in DNA ~inding to the specific target sequence (E box), and a helix o o p helix (HLH) implicated in homodimerization and/or leterodimerization with other regulatory protein [2]. The badc idea of this work is the isolation and characterization of )ther regulatory genes, involved in myogenesis, coding for ~roteins with the 'zinc finger' motif as a DNA binding donain. A zinc finger motif was first identified in the Xenopus aevis RNA polymerase III transcription factor TFIIIA [3,4]. qince then, several zinc finger genes involved in gene regulaion have been identified in a variety of animals and plants 5,6].
~Corresponding author. Fax: (39) (6) 446 2891. E-mail: [email protected]
In a previous work we reported on the characterization of three zinc finger genes, isolated from an adult mouse skeletal muscle cDNA library [7]. In this work we describe one of those genes, termed Mfg3 and presently renamed Zfp60 according to the international convention. We determine the complete cDNA coding sequence and the predicted amino acid sequence of the ZFP60 protein. This protein is characterized by a cluster of 19 zinc finger motifs at the C-terminus and by the two Kruppel associated boxes (KRAB) A and B at the N-terminus. These modules have been shown to define a subfamily of multifinger proteins [8,9] and to be associated with transcriptional repression activity [10 13]. The expression patterns of Zfp60, MyoD and myosin heavy chain (MHC) mRNAs have been followed and compared during myoblast C2 cell line differentiation. We also show that the bacterial expressed Zfp60 protein is able to bind DNA only in presence of zinc ions. Using restriction length polymorphism (RFLP) we assigned the Zfp60 locus to the murine chromosome 7, at about 10 cM from the centromere, where a cluster of Zfp loci maps. This region shows homology with human chromosome 19q13.
2. Materials and methods 2.1. RNA extraction and Northern blotting Total RNA was extracted from cell lines using the method described by Chomczynski and Sacchi [14]. Total RNA (10 ~tg) was fractionated by electrophoresis on a 1.2% agarose-formaldehyde gel according to Maniatis and co-workers [15]. The gels were blotted on nylon filters (Hybond N, Amersham) and hybridized according to the manufacturer's instructions. DNA probes were labelled by [c~-32p]dATPincorporation using Megaprime Probe Kit (Amersham). 2.2. cDNA library screening A k gtll cDNA library, prepared from RNA of mouse F9 teratocarcinoma cell line differentiated using retinoic acid (0.1 p_M) was screened using as a probe the previously isolated cDNA fragment [7], spanning from nt 1447 to nt 1990 (see Fig. 1). 2.3. Cloning the cDNA 5' region In order to isolate the 5' region of Zfp60 mRNA we used the reverse PCR technique first described by Uematsu [16], and modified as previously described [17]. Briefly, three oligonucleotides were synthesized in the known extreme of the 5' end of Zfp60 cDNA: antisense oligonucleotide (A) from nt 551 to nt 573, sense oligonucleotide (B) from nt 524 to nt 547, antisense oligonucleotide (C) from nt 330 to nt 353. For cDNA synthesis, 1 ~tg of total RNA prepared from mouse F9 teratocarcinoma cell line induced to differentiate with retinoic acid [18] was used. RNA was primed using 10 ng of oligonucleotide A. First strand and second strand cDNA synthesis was performed using the Promega RiboClone cDNA System kit according to the manufacturer's instructions. The double strand cDNA in a total volume of 7 lal of TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA)
~1014-5793196/$12.00 © 1996 Federation of European Biochemical Societies. All rights reserved. DIIS001 4 - 5 7 9 3 ( 9 6 ) 0 0 4 7 4 - 7
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GGGACGGTGCTGTGTGTGCGCGCGCGTCTCTGCGTACGCACC~AGCTATTTGCATTAT CACCGTGCTTGTCGCTTTCTCTCGCGGCCACCAGCAGAGGACCAAGCTTCACTGAGGAGA GGGGAGGAAG CCTGAAGAAGAATG G C C A A C T C T A G T T C T C A A C A C A T G G T C T G T G G G T C A M A N 8 S S Q H M V C ~ ~ G T G A C A T T T A G G G A T G T G G CTG TTG A CTTCT C T C A G G A G G A G T G G G C C T G C C ~ A T G C T V T F R D V A V D F S 0 E E W A C L D ~ A C T C A G A A G G T C T T A T A C A G GAA C A T A A T G T T G G A G A C C T A C A G C A A C C , ~ T C G C A G T G T Q K V L Y R N I M L E T Y S N L V A V GTGGGACGT~CAT~CAAGCCAGATCTGATCGTC~A CTAGAG C C G G A G A ~ G A G C C C V G R C I P K P D L r v L L E P E K E p TGGATC~TGAAGAAAGAAACAGG CAGGCCGAG C C A A G G ~ " I ~ A G A C A G G T ~ A A K A V K K ~ T G R P S 0 G ~, E T G F E GC~A~TAGATCT CCAAAGAATCATGTTTATAAT~GTTACC CAAACAAACC.ATA A E N R S P K N H V ¥ N K K L P K Q T I CAGCAATTAAGTAAAACTTCGGACG T C CAGGG CGTCAGTGTTAGTAA~,,GCC CTGGCTAT Q Q L S K T S D V Q G V S V S N G P G Y AGTGTAATCAAGGAGCCACAGAATTATCAAGAAGGAGA~CTAATCGA~TATTACTAAC S V I K E P Q N ¥ Q E G D A N R N I T N AAGAAAGAAATGTCTACTTACACTAGCAAGACTCTTGCTCACAATAAAGAA~CCGTAT K K E M S T ¥ T S K T L A H N K E K P Y A~TGTAAAGAC~GGGAAGTG CT~TTGTAAGTCAAACCTTCATCAGCATGAGAGT K ~ K D C G K C F G C K S N r. H Q H E S ATTCACACTGGAGAGAAG C C C T A T G A A T G T A A G G A C T G T G G G A A G A C C T T C A G A C T C C C T I H] T G E K P Y E ~ K D C G K T F R L P C A G A T G C T T T C A A G A C A T C A G A A G T C T C A C A G T G A T G AGAGAC ~-t-FFrGAGTGTAATATA Q M L S R H Q K S H] S D E R P F E [C N I TGTGGA~GTCT~ CATCTTC CTAC C C T G C ~ C A G T A C C A T A A A A A T A T T C A T A C A G G T C G K S F H I". p T L L O Y H K N I H] T G T T A A A A C C A T T T G A A T G T G A G G A G T G TGGG A A G T C C T T C A A G T C C T T C A A C C G C A T C T C T L K P F E [C E E C G K S F K S F N R I S ACC~-'~'x'~'I"I'CAGCATAGGACTATTCATGCAGGTATGAAACCGTATAAATGTAATGTGTGT T L F Q H R T I H] A G M K P Y K [C N V e GGGAAAGCCTTTAATCGTCGCTCAAACCTCC~CAACATCAGAAAATTCATTCTGAGGAC G K A F N R R S N L L Q H Q K I H] S E D AGAC~-FFFI"CATTGTAAGGTGTGTGGAAAGGCCTTCACTGTGCTGGCTCAGCTTACTCGG R P F H [C K V C G K A F T V L A Q L T R CAeGAGAACATTCACACTGAAGACAAATCGTTTGAATGTAAGCAGTGTGGTAAGATATTT H E N I H] T E D K S F E [C K Q C G K I F AGTAATGGCTCTTACCTCTTACGACACTACGATACTCACACCAATGAGA~CCCT'~AG S N G S Y L L R H ¥ D T H] T N E K P F E TGTAATATATGTGGCAAGGCTTTTAGGCTTCATCTATACCTTTCTGAGCATCAGAAAACT [C N I C G K A F R L H L ¥ L S E H Q K T CACACCGATGAGAAGCCTTTCAAGTGTAAGCTGTGTGAGTCAGCCTTCAGACGTAAGTAC H] T D E K P F K [e K L C E S A F R R K Y CAGCTCAGTGAACATCAGAGAATTCACACTGATGGTAAACCCTATCAGTGCAAGGAT~T Q L S E H Q R I H] T D G K P Y Q [C K D C TGGGAAT~CT~CGTCGACGATCAAATT~ATCGAACATCAGAGTATTCACACTGGAAAG W E F F R R R S N F I E H Q S I H] T G K AAACCC~GAGTGTAAGGACTGTGGGAAGGTCT~AGACTA~TATACATC~TTCGA K P F E [C K D C G K V. F R L N I H L I R C A T C A G A G A T T T C A T A G T G ATGAGAAG C C CTTTG A A T G T A A A G A A T G T G G A A A G G ~-FFFI" . ~ R r a] s D E K p ~ ~ ( c ~ ~ c ~ x ^ ~
M. Perez et al./FEBS Letters 387 (1996) 11~121
60 120 180 13 240 33 300 $3 360 78 420 83 480 113 540 133 600 1S3 660 I73 720 193 780 213 840 233 900 253 960 273 1020 283 1080 213 1140 333 1200 3S3 1260 373 1320 383 1380 413 1440 433 1500 4S3 1560 473 1628
C A T T T ~ C CAG CCAG C T T A A T A A C C A T A A ~ C C A G T C A T A C A G G TCAG ACA C ~-LTL-t'GAA 1 6 8 0 H F S S Q L N N H K T S H] T G Q T P F E 513 TGTAAAGAATGTGGGAAGTCGTTTAAGCGTGTCTCCAGCCTTGTGGAACATAGGATTATT 1740 [C K E C G K S F K R V S S L V E H R I I 533 CACAGTGGTGTGAAACCATATAAATGTAATG CGTGTGGAAGAGCCTTCAATCGTCGCTCA 1800 H]S G V K P ¥ K [C N A C G R A F N R R S 553 AACCTCATGC~CATGAGA~TTCATTCTGATGAGAGACCCT~GAGTGTAAGGACTGT 1860 N L M Q H E K I H l S D E R P F E [C K D C $73 G G A ~ G G C C T T C A C T G T T C T G G CTCAGCTCA CACGG CAC C A G A C G A T T CATAATC, G A A A G 1 9 2 0 G K A F T V L A Q L T R H Q T I H] N G K $93 AAATCATA~%2AATGTGAGCAGTGTG GG TCAG C CTTCAGA CT C CCGTAC CAA CT C A C T C A A 1 9 8 0 K S Y E [C E Q C G S A F R L P ¥ Q L T Q 613 C A T C A G A G A A ~ F C A T T A T G A T G TGAA G C CATTT C A G T G C A A G G A A T G T G G A A G G G ~'FFFL" 2 0 4 0 H Q R I H] y D V K P F Q [c K E C G R A F 633 G T T C G T A G T A C A G G CCTTAG A A T T C A T G A A A G G A T C C A C A C ~ T G A G A A G C CCTTTCAG 2100 V R S T G L R I H E R I H]T G E K P F Q 683 TGTAAGGAGTGTGGAGAAG CCTTTCAGTATCATTACCAATTTCTTGGACACTTTAGAATT 2160 [C K E C G E A F Q ¥ H Y Q F L G H F R I 673 CATACTGGC~AACCCTTATGAATGTAGTGAATGTGGGAAGTACTTTACTTATGGTCGA 2220 H] T G K N P Y E [C S E C G K Y F T Y G R 693 GACCTTAAAGTACATCAGAGTATTCACAATCT~AGA~CCGTAAGAATGTGGG AAGA C C 2 2 8 0 D L K V H Q S I H] N L E K P * 707 TTCAG~rGA~TCAAAGGTGGTTTGACATGCTAAACTTAATATTATGAAGAAGAAGCCT 2340 TACAATGCTTATAAGGAATGTGGAAAGCCTTCAGTAATTCTAAGTAGGTGTATATCAGAG 2400 AATTCATACTGGTCTGAAGCACTGTTGATATGAAAATTGTGAGAAGTTTAGATTAAGTT C 2460 AGCCTTCAGTGTACACTCAAGATTCATATTGGCTGAACCCATCACTGTAAGATGT~ 2520 GACCCG C R A C ~ G C T C A G G C C T T C A G CATA~GTATCCATGCTGTCGTGAAACACTA 2580 TGCAGC, G G T G T G T A A A C A T T T A T A G A A T T C A T T C T G A A A A C T C A T G A A G G A G T C C A T A T A 2640 GGTCTGAAACCATAAGGATGTGAAAAAAATTTTATTGTTAAATGGTGAGCTTATACAG CA 2 7 0 0 TGAGAAAAGGAAGACGGATAAGACTTTATGGAAATTTTACTGAACATCAAAGAAGTCATT 2760 AACAAACTGGGACTGAAAGGAGTCTGGGAAGAAC"~-~"~AATCTATCATAGGATTTAACCAA 2820 CATCTGAGTGTTCATATAAAAAAAATCCTTAGAGAATGAGAGTGGGAATTCTGTTCACAG 2880 AAAATACTGT~GAAGGACAGAAAGAGATATTGTAGCC~CATGCTC~AAGTTAC~TC 2940 A~vxTx'AATACTACTGTTAATTGCATAAGCTTAATTCACCAAACACAATTCAGCTCACTT 3000 AGGAAGAACAGTTATTGAGACTATTATTATTCGAGGCACCT~CeACCAATTACATCCCA 3060 TGCCTCCTGTGCTATTACCTGGATGTTTGCTTTCTTTCTGCATAGTTCATGGTGAGCACG 3120 GTAe~ATGTGTGGAAATCGGGGTCATAATGGATAGAGAATCCTTGAGAGTATTT 3180 GGTATGT~TGAAGTAGCTTCTACAGTGTTGGTGCTCTCAGGGGCGTTACTGTAACC, G 3240 ATATA~GACAAGTTGTGTCGTAGATAGT~AGA~GCCTCATGCAGCCCTCCAGTAT 3300 TAAATTCTAAATATTTTCTTCTGGGTAAAATCTAATAGATGAACAGGTACATGAAAGCAT 3360 ~-FI-L'ATTCTCAATCCGTTTCTTTAGCTTTCGTGCAGAGATGTCTATAAACTAGCTGTTGG 3420 TGAATAAGTAC-L-L-I-~AAACGGGGATAAACACATTTAACTTTCAAAAGCTAAAATCCTGTA 3480 ATTTATGTTCTCTGCCCAAACATTAAGATAAAATGCATTTGTGACTCAGCAGGACAGTGC 3540 AGTCCCCTCCATCTCATGCTGCTGAGACCATTTTATGTTGACTAGAATTTGA-~-~-L-L.AGAT 3600 GGAGGAGATTCTTGAGATTTACCCACCTGTGATCTGTGAGCCAGCGGTCAGATC~CTG 3668 CTATTTTA~-L"~"L~GCTTTTCTTAGACTGCCTGCCTGTGCAAACTTTCCCCTTCCACTTAA 3720 CTGCTTACCC~CTTCTGTTA~C'~CTGC'~GGCAAATGCTCCCCCTGTAGCTGCT 3780 GAGCAAGATATCAGAATGTGGTTCTTG.xTx-F~.AATAGCCCCTTGCTGTAAATGCTTGTGA 3840 GTGTATATGGATCCCGAATA~TGAGTGTAGTCCCAGCCAACTGGAATItI~GACq-iqCAG 3800 TTGGCTAAAAAAAAACGGAATTC 3823
483
Fig. 1. Nucleotide sequence of Zfp60 cDNA and its deduced amino acid sequence. Nucleotides are numbered on the right, amino acid sequence is numbered in bold characters. The amino terminal KRAB boxes A and B are underlined and double underlined respectively. Individual zinc finger domains are shown between square brackets. The polyadenylation signals are in bold characters; the 'ATTTA' sequences, potentially involved in mRNA selective degradation, are underlined. The Zfp60 nucleotide sequence has been deposited at GenBank, with accession number U48721.
was circularized according to Uematsu [16]. The amplification of the circularized double strand cDNA was accomplished using the Zfp60 oligonucleotides B and C and the GeneAmp PCR Core Reagents Kit (Perkin Elmer Cetus). The amplification cycles consisted of denaturation for 30 s at 94°C, annealing for 30 s at 66°C, and extension for 1 min at 72°C. 10 ~1 of the PCR products were analyzed by 1.6% agarose gel electrophoresis and transferred to Amersham Hybond N nylon membrane. Specific products for Zfp60 sequences were detected by hybridization using [7-32P]ATP end labelled oligonucleotide A as a probe. The PCR products were then directly subcloned using the TA Cloning Kit (Invitrogen). 2.4. DNA sequencing DNA fragments of cDNA clones were subcloned either in pCR vector (Invitrogen) or pBluescript (Stratagene) and both strands were sequenced using the Sequenase kit according to the manufacturer's instructions (United States Biochemical). 2.5. Cell cultures The mouse myoblast C2 cell line [19] clone 7 was obtained from Dr. M. Buckingham (Institut Pasteur, Paris, France). Cells were maintained as undifferentiated myoblasts in Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal calf serum (FCS) in a 6.5% carbon dioxide atmosphere. To induce cell differentiation, myoblasts were grown to confluence and shifted to DMEM supplemented with 2% FCS. 2.6. Expression o f recombinant fusion protein The entire coding region of Zfp60 cDNA was cloned in pQE-30 vector (Qiagen). Zfp60 bacterial fusion protein was expressed and purified according to the manufacturer's instructions.
2. 7. Gel electrophoresis and South- Western blotting Purified protein obtained in bacteria (0.5 ~tg per lane) was separated on 9% SDS-polyacrylamide gel, blotted to nitrocellulose paper (Hybond C, Amersham) as described by Harlow and Lane [20]. South-Western blotting was performed according to Sukegawa and Blobel [21]. In particular two identical filter blots (A and B) were incubated for 12 h in renaturation buffer A (50 mM Tris-HC1 pH 7.5, 100 mM KC1, 1% Triton X-100, 10% glycerol, and 0.1 mM ZnC12) and in renaturing buffer B (50 mM Tris-HC1 pH 7.5, 100 mM KC1, 1% Triton X-100, 10% glycerol, 10 mM EDTA, and 2 mM DTT) respectively. Total mouse genomic DNA was sonicated to a final size of about 1 kb, labelled with [a-32P]dATP and random hexanucleotide primers. 5 × 104 cpm per ml were added and incubated for 3 h to both filters. The filters were washed five times, for 10 s, in the respective buffers A and B, and autoradiographed. All incubations were performed at 4°C. In order to normalize the amount of protein loaded in each lane, the same filters were then incubated with Zfp60 mouse polyclonal antibody. Proteins were then visualized using an alkaline phosphatase-conjugated goat anti-mouse. 2.8. Genetic mapping of Zfp60 Details on the interspecific test cross population of 106 male (C3H/ He)< M. spretus)× C57BL/6J, designed HSB, have already been reported [22]. The marker locus Ligl was identified by segregation analysis of the three M. spretus polymorphic bands, detected by the hybridization of HSB Taql-digested genomic DNAs with the Ligl cDNA [23]. A genetic linkage map was constructed by multipoint analysis of the data, with the MAPMARKER/EXP program [24,25]. Genetic distances were computed by use of Haldane's mapping function. Linkage between markers was considered significant if the LOD score was >3. Zfp60 locus was mapped by restriction length polymorphism (RFPL) analysis of a 813-bp fragment from 3'-UTR
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M. Perez et al./FEBS Letters 387 (1996) 117 121
region of the murine Zfp60 gene. The fragment was obtained by PCR amplification of 75 ng of genomic DNA. 5 pmol of specific primers: from nt 3042 to nt 3072 (5'-GCCACCAATTACATCCCATGC('TCCTGTGC-3') and from nt 3856 to nt 3827 (5'-GGGATCCATATACACTCACAAGCATTTAC-3') were designed from Zfp60 eDNA. Samples were subjected to 40 PCR cycles consisting of dena~aration at 95°C for 20 s, annealing at 52°C for 30 s and extension at ~2°C for 30 s. PCR samples were digested using the restriction enzyme ;[intl. We detected a RFLP between HinfI-digested 3'-UTR fraglents of C3H/He (H) and C57BL/6J (B) mouse strains and M. spretus ~) strain. A HinfI site, present in H and B strains but absent in the S train, gave rise to a RFLP. We analyzed the segregation of this band ] the HBS population. The segregation showed the expected 1:1 ,attern.
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~.1. Structural organization To isolate the Zfp60 complete c D N A we screened a ~, : D N A library prepared from the mouse F9 teratocarcinoma ;ell line differentiated using retinoic acid [18]. We used as a ~robe the previously isolated c D N A fragment, corresponding o a portion of the zinc finger coding region [7]. Several Zfp60 ~ositive ~, c D N A clones were isolated. The longest c D N A :lone comprised both the 3' untranslated region and the com>lete zinc finger coding portion of the Zfp60 m R N A , but it ~¢as missing the 5' region of the transcript. To isolate this :>ortion of the Zfp60 m R N A , we used the inverse circular ~CR technique [16,17]. The longest Zfp60 c D N A sequence )btained using different cloning strategies was 3923 bp long G e n B a n k accession n u m b e r U48721). An A U G was identiied at nt 142-144, located in an appropriate context for an nitiation codon [26]. This A U G was preceded by an in-frame
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Fig. 2, Northern blot analysis of total RNA isolated from the mouse myoblast C2 cell line on different days during differentiation. On day 2 cells are shifted to the differentiation medium. The same filter was hybridized with the probes indicated on the left. Panel A: Zfp60 cDNA, panel B: MyoD1 cDNA, panel C: myosin heavy chain cDNA, and panel D: glyceraldehyde-3-phosphate-dehydrogenase cDNA.
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B Fig. 3. South-Western blot analysis of a bacterially expressed and purified Zfp60 fusion protein. The proteins were separated on a 9% SDS-polyacrylamide gel and blotted to nitrocellulose filter. Blots were probed with [0t-z2P]dATP labelled mouse genomic DNA either in the presence of zinc ions (lane 1) or in the presence of EDTA and DTT (lane 2). Panel A: The fusion protein Zfp60 is marked by an arrow. Positions of the molecular mass standard proteins are shown. Panel B: The same filters were incubated with a Zfp60 mouse polyclonal antibody in order to normalize the amount of protein loaded in each lane. Proteins were visualized using an alkaline phosphatase-conjugated goat anti-mouse. stop codon: nt 133-135. As shown in Fig. 1, the A U G codon opens an uninterrupted reading frame encoding a 707 aa long polypeptide. At the C-terminus, 19 zinc finger motifs are clustered, classifying Zfp60 as a multifingered protein. The Nterminal portion of the protein contains two conserved peptide modules termed KRAB, box A and box B, which have been found to occur in several members of the zinc finger multigene family [8,9]. A 1660 nt long untranslated region is present at the 3' end of Zfp60 m R N A . In this region several ' A U U U A ' sequences are present (see Fig. 1), frequently associated with transcripts having an elevated turn-over [27]. Two canonical polyadenylation consensus sequences are arranged in tandem at nt 3857-3862 and nt 3886-3891, a poly A tail is present at the end of the c D N A . 3.2. Expression analysis We have previously reported that Zfp60 gene expression is strongly induced upon differentiation both in the F9 teratocarcinoma cell line and in the C2 myogenic cell line [7]. Here we show a detailed analysis of the expression pattern of Zfp60 in total R N A prepared at different times following the onset of differentiation of the myogenic C2 cell line. As shown in Fig. 2A a specific Zfp60 hybridization signal is present at low level in C2 cycling cells on days 1 and 2, it peaks on day 3,
M. Perez et al./FEBS Letters 387 (1996) 117-121
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Fig. 4. Haplotype data of completely typed HSB mice. Panel A: Each column represents the type of chromosome identified in the progeny. The number of progeny exhibiting each type of chromosome is listed at the bottom. The open squares represent the M. spretus allele; the black squares stand for the C3H/HeJ allele. Panel B: The partial genetic linkage map of the mouse chromosome 7 showing the location of the Zfp60 gene. The recombination frequencies are shown on the left and are calculated by multipoint analysis of recombinants over the number of animals typed for each pair of loci, and expressed as genetic distances in cM (computed using Haldane's function). Distance of the Ligl marker from the centromere is also indicated. concomitantly with the differentiation commitment, and then it slowly declines from day 4 on. Fig. 2B shows the expression pattern of MyoD, one of the muscle-specific determining/ transcription factors [1,2]; its hybridization signal remains nearly constant during differentiation progression. In fact, in the C2 cell line, the MyoD gene product is present in an inactive form in cycling conditions. In order to follow the differentiation progression the MHC gene transcript has been visualized (Fig. 2C). It appears on day 4 and then remains constant during the terminal differentiation. The amount of R N A loaded in each lane has been normalized using as a probe G A P D H cDNA (Fig. 2D). These results suggest that Zfp60 gene has a refined pattern of expression correlated to the onset of differentiation, but not to the maintenance of this stage. 3.3. DNA binding activity A fusion protein coding for the complete Zfp60 was expressed and purified in E. coll. Purified protein was separated by SDS-PAGE, blotted to nitrocellulose, and then probed with sonicated and [c~-32P]dATP labelled mouse genomic
DNA. The probing was performed either in solutions containing ZnC12 or in solutions containing EDTA and DTT [21]. As shown in Fig. 3 the mouse D N A binds Zfp60 protein in the presence of zinc ions, whereas no D N A binding was observed in the presence of EDTA and DTT. These data demonstrate that Zfp60 protein is capable of binding D N A in a zinc-dependent fashion. 3.4. Genetic mapping o f Zfp60 R F L P analysis was used to identify restriction polymorphisms in a PCR amplified fragment (813 bp) from the 3'-UTR region of the murine Zfp60 gene. The fragment was obtained by PCR amplification of mouse genomic D N A using specific primers of Zfp60 cDNA (see Section 2). We detected a R F L P between the HinfI-digested 3'-UTR fragment of C3H/He (H) and C57BL/6J (B) mouse strains and M. spretus (S) strain. A HinfI site, present in strains H and B, but absent in the S strain gave rise to a RFLP. Its segregation was analyzed in the HBS population showing the expected 1:1 pattern (Fig. 4A). Therefore, we could assign the Zfp60 locus to the murine chromosome 7, at about 10 cM from the centromere (Fig. 4B). Other zinc finger coding genes have been mapped in this chromosome region, such as Zfp29, Zfp30, Zfp36, Zfp56 [28-30] and Zfp59 (our unpublished data). This region shows homology with human chromosome 19q13, where the human ZNF36 gene maps, the homologue of the murine Zfp36 [30].
4. Discussion The differentiation processes require the involvement of a cascade of signals and regulatory molecules in order to govern the expression of specific structural genes. The search for regulatory genes appears to be fundamental for the understanding of these sophisticated processes. Here we report on Zfp60, a mouse zinc finger coding gene, which is transiently expressed during in vitro muscle differentiation. To date, only one other member of the multifinger gene family, named ZT3, has been reported to be expressed during skeletal muscle differentiation [31]. The Zfp60 protein has a cluster of 19 zinc fingers and belongs to the KRAB multifinger gene family. The data presented here clearly demonstrate that the bacterially expressed Zfp60 protein binds D N A and that the binding is dependent on the presence of zinc ions. The KRAB domain, located in the N-terminal portion of Zfp60, has been shown to be a potent transcriptional repressor element [10-13]. Its predicted c~-helical structure has been proposed to mediate protein-protein interaction. This hypothesis has been recently supported by the report of co-immunoprecipitation of a protein likely interacting with the KRAB domain [32]. In the present study we follow the expression of Zfp60 during the differentiation of the myogenic C2 cell line. A specific hybridization signal is present at a very low level in C2 cycling cells and increases when the cells are switched to the differentiation medium. Its maximum expression coincides with the massive entry of cells in GO phase and then declines concomitantly with myoblast cells fusion. This transcription pattern has been confirmed in experiments performed on mouse embryonic primary myogenic cells (data not shown). Moreover, the presence in the 3' untranslated region of Zfp60 m R N A of several destabilizing ' A U U U A ' sequences is coherent with the presence of Zfp60 transcript in a very narrow window during myogenic
M. Perez et aI.IFEBS Letters 387 (1996) 11~121
differentiation. This peculiar expression pattern suggests that Zfp60 protein is transiently required during myogenic differeatiation. Zfp60 m R N A peaks concomitantly with the cell e_,atry to the myogenic terminal differentiation program. Duri~g this process myogenic cells undergo profound structural a ad functional changes, including cell growth arrest, cell ~lignment and fusion to form multinucleate myotubes. 7fp60 with its large number of zinc fingers and the K R A B i~gulatory element could effect multiple contacts with D N A nd play a role in the change of chromatin structure, which :companies the transition of a cycling myoblast to mature Hultinucleate myotubes. The fact that several ZFPs have been iadicated to participate in the formation of chromatin struc1 1re [17,21,33 35] supports this hypothesis. Moreover, a gene 1 1at shares a high degree of homology with Zfp60, namely ~;fp59, has been shown to be tightly associated with nuclear ,~affold structure during murine male germ line differentiation [L7]. In particular it is interesting to note that the Zfp60 and 'Zfp59 genes have been assigned to the murine chromosome 7, ia a region where a cluster of other Zfp loci maps. Taken all 1 9gether these data suggest that Zfp60, as well as Zfp59, may I elong to a cluster of structurally and functionally correlated ~enes. cknowledgements." We are grateful to Prof. Paolo Amati for contin-
ous support and critical discussion. We thank Mr. N. Falcone for 1,-chnical assistance. This work was supported by TELETHON 95 I 9rogetto A51, C.P.) and by Associazione Italiana Ricerca sul Cancro, ~,~ilan (to Prof. Paolo Amati). References [1] Ontell, M., Ontell, M.P. and Buckingham, M. (1995) Microsc. Res. Tech. 30, 354-365. [2] Ludolph, D.C. and Konieczny, S.F. (1995) FASEB J. 9, 15951604. [3] Brown, R.S., Sander, C. and Argos, P. (1985) FEBS Lett. 186, 271-274. [4] Miller, J., McLachlan, A.D. and Klug, A. (1985) EMBO J. 4, 1609 1614.. [5] EI-Baradi, T. and Pieler, T. (1991) Mech. Dev. 35, 155-169. [6] Berg, J.M. (1993) Curr. Opin. Struct. Biol. 3, 11-16. [7] Passananti, C., Felsani, A., Caruso, M. and Amati, P. (1989) Proc. Natl. Acad. Sci. USA 86, 9417 9421. [8] Bellefroid, E.J., Poncelet, D.A., Lecocq, P.J., Revelant, O. and Martial, J.A. (1991) Proc. Natl. Acad. Sci. USA 88, 3608 3612. [9] Rosati, M., Marino, M., Franz6, A., Tramontano, A. and Grimaldi, G. (1991) Nucleic Acids Res. 19, 5661-5667.
121 [10] Margolin, J.F., Friedman, J.R., Meyer, W.K.-H., Vissing, H., Thiesen, H.-J. and Rauscher III, F.J. (1994) Proc. Natl. Acad. Sci. USA 91, 45094513. [11] Witzgall, R., O'Leary, E., Leaf, A., E)naldi, D. and Bonventre, J.V. (1994) Proc. Natl. Acad. Sci. USA 91, 45144518. [12] Pengue, G., Calabr6, V., Cannada Bartoli, P., Pagliuca, A. and Lania, L. (1994) Nucleic Acids Res. 22, 2908-2914. [13] Vissing, H , Meyer, W.K., Aagaard, L., Tommerup, N. and Thiesen, H.J. (1995) FEBS Lett. 369, 153 157. [14] Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 56159. [15] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboatory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [16] Uematsu, Y. (1991) Immunogenetics 34, 174-178. [17] Passananti, C., Corbi, N., Paggi, M.G., Russo, M.A., Perez, M., Cotelli, F., Stefanini, M. and Amati, P. (1995) Cell Growth Diff. 6, 1037 1044. [18] Silver, L.M., Martin, G.R. and Strickland, S. (Eds.) (1983) Teratocarcinoma Stem Cells, Cold Spring Harbor Conference on Cell Proliferation, Vol. 10. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [19] Yaffe, D. and Saxel, O. (1977) Nature 270, 725 727. [20] Harlow, E. and Lane, D. (1988) Antibody. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [21] Sukegawa, J. and Blobel, G. (1993) Cell 72, 29-32. [22] Dragani, T.A. and Pierotti, M.A. (1994) Genomics 23, 118-124. [23] Gariboldi, M., Montecucco, A., Columbano, A., Ledda-Columbano, G.M., Savini, E., Manenti, G., Pierotti, M.A. and Dragani, T.A. (1995) Mol. Carcinogen. 14, 71-74. [24] Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E. and Newburg, L. (1987) Genomics 1, 174-181. [25] Lincoln, S.E., Daly, M. and Lander, E.S. (1992) Whitehead Institute Technical Report. [26] Kozak, M. (1987) Nucleic Acids Res. 15, 8125 8148. [27] Brawerman, G. (1990) Trends Biotechnol. 8, 171 174. [28] Denny, P. and Ashworth, A. (1995) Mamm. Genome 6, 683. [29] Denny, P. and Ashworth, A. (1994) Mamm. Genome 5, 643-645. [30] Brilliant, M.H., Williams, R.W., Conti, C.J., Angel, J.M., Oakey, R.J. and Holdener, B.C. (1994) Mamm. Genome 5, 104-123. [31] Polimeni, M., Giorgi, S., De Gregorio, L., Dragani, T.A., Molinaro, M., Cossu, G. and Bouche, M. (1996) Mech. Dev. 54, 107 117. [32] Deuschle, U., Meyer, W.K.-H. and Thiesen, H.-J. (1995) Mol. Cell. Biol. 15, 1907-1914. [33] Noselli, S., Payre, F. and Vincent, A. (1992) Mol. Cell. Biol. 12, 724-733. [34] Cleard, F., Matsarskaia, M. and Spierer, P. (1995) Nucleic Acids Res. 23, 796-802. [35] Yokoyama, N., Hayashi, N., Seki, T., Pant6, N., Ohba, T., Nishii, K., Kuma, K., Hayashida, T., Miyata, T., Aebi, U., Fukui, M. and Nishimoto, T. (1995) Nature 376, 184-188.