GENOMICS

6, 333-340

(1990)

cDNA Isolation, Expression Analysis, and Chromosomal Localization of Two Human Zinc Finger Genes LUIGI LANIA,’ EMILIO DONTI,* ANTONIO PANNUTI, ANNA PASCUCCI,GINA PENGUE,ISIDOROFELICIELLO, GIROLAMA LA MANTIA, LUISA LANFRANCONE,*AND PIER-GIUSEPPE PELICCI* Department

of Genetics, General and Molecular Biology, University of Naples, via Mezzocannone 8, 80124 Naples, Italy; *Institute of Medical Clinics I, University of Perugia, 06100 Perugia, Italy Received

July 14, 1989;

revised

Press,

Inc.

INTRODUCTION

Differential regulation of gene expression is achieved at least in part by the specific binding of trans-acting factors and relevant &-acting DNA sequences. The best characterized DNA-binding structure is the evolutionarily conserved helix-turn-helix motif (reviewed in Pabo and Sauer, 1984; Gehring, 1987). A second type of DNA-binding domain is the zinc finger sequence motif that has been identified in a variety of transcriptional regulatory proteins and is thought to be directly involved in DNA binding (Klug and Rhodes, 1987; Evans and Hollenberg, 1988). This DNA-binding structure was initially identified as nine tandemly repeated sequences of approximately 30 amino acid residues in the RNA polymerase III transcription factor TFIIIA (Miller et al., 1985; Brown et al., 1985). It has been postulated that two Cys and two His residues in each repeat bind a Zinc(B) ion and that the resulting metalloprotein structures are involved in the sequenceSequence data from this article have been deposited with EMBL/GenBank Data Libraries under Accession No. 504751. 1 To whom correspondence should be addressed.

9, 1989

specific binding to DNA. Similar motifs homologous to the fingers in TFIIIA have been subsequently identified in other genes, including Drosophila genes (Rosenberg et al., 1986), in yeast regulatory proteins (Hartshorne et al., 1986), in frog (Ruiz i Altaba et al., 1987), in mouse (Chowdhury et al., 1987; Chavrier et al., 1988), and in human (Kadonaga et al., 1987; Page et al., 1987). We (Pannuti et al., 1988) and others (Ruppert et al., 1988) have utilized a finger-motif-containing probe to isolate human genes encoding putative DNA-binding finger domains. Here we report the isolation, characterization, and chromosomal location of two members of this human gene family, ZNF7 and ZNF8. Transcriptional studies indicate that these two human finger genes are ubiquitously expressed in many human cell lines. Moreover, upon in vitro induced terminal differentiation of human HL-60 myeloid cell line, their expression is drastically reduced.

On the basis of sequence similarity in the repeated zinc finger domain, we have identilled and characterized two human cDNA clones (ZNF7 and ZNFS), both encoding proteins containing potential finger-like nucleic acid binding motifs. Northern blot analysis indicates that both genes are expressed as multiple transcripts and they are ubiquitously present in many human cell lines of different embryological derivation. Moreover, their expression is modulated during in vitro induced terminal differentiation of human myeloid cell line I-IL-60. By in situ hybridization experiments, we have localized the ZNF7 gene to chromosome 8 (region q24) and the ZNFS gene to the terminal band of the long arm of chromosome 20 (2Oq13). o 1990 Academic

October

and

MATERIALS

AND

METHODS

Library Screening and DNA Sequencing A hgtll human placenta cDNA library (Clontech Laboratories Inc.) was screened with a 503-bp Hi&IIBamHI DNA fragment from an HF.10 cDNA clone (Pannuti et al., 1988). The ZNF nomenclature for human zinc finger genes described in this work was suggested by the Human Gene Mapping Workshop Nomenclature Committee. Approximately 5 X lo5 phages were plated out and after the plaques were transferred to nitrocellulose filters, the phage DNA was denatured and immobilized according to standard procedures. Hybridization to the DNA probe was carried out at 65°C in 5X SSPE, 5X Denhardt’s solution, 0.2% SDS, and 100 pg/ml of denatured salmon sperm DNA for 18 h. The DNA probe was labeled with a multiprime labeling kit (Amersham) to a specific activity of 5 X 10’ cpm/pg. The filters were washed at 37°C in 4X SSPE, 0.2% SDS for 2 h and in 2X SSPE, 0.2% SDS for 1 h

the

333 All

Copyright 0 1990 rights of reproduction

OSeiS-7543/90 $3.00 by Academic Press, Inc. in my form reserved.

334

LANIA

at 37“C. The filters were dried and exposed to Kodak X-Omat films at -70°C. The positive hybridizing plaques were isolated and purified. Inserts from positive clones were subcloned in pGEM-3 plasmid (Promega Biotech) for restriction enzyme mapping, hybridization analysis, and DNA sequencing. DNA sequencing was performed on both strands, and all DNA sequences were determined independently by two different methods using the GemSeq K/RT system as recommended by Promega Biotech. Nucleotide and amino acid sequences were analyzed using the MicroGenie Sequence Analysis Program software (Beckman).

ET AL.

hardt’s, 0.066 M phosphate buffer, pH 6.5, and 200 pg/ ml salmon sperm DNA. Slides were covered with a coverslip and incubated in a humidified chamber at 42’C for 16 h. The slides were washed repeatedly in 2X SSC at 45°C and in 0.2X SSC at room temperature, coated with autoradiographic emulsion NTB-2 (Kodak, Rochester, NY) diluted 1:l in tap water, and exposed for 15 days at 4°C. After development, slides were stained with Wright-Giemsa solution and destained two or three times to induce G-bands (Chandler and Yunis, 1978). RESULTS

Cell Cultures The following human cell lines were used: HL-60 (acute promyelocytic leukemia); NTERA2/Dl (embryonal carcinoma) (Andrews et al., 1984), MOLT-4 (T-cell lymphoma), WI-38 (embryonic lung cells), HeLa (cervical carcinoma), H5 (EBV-infected B lymphocytes), and CLL220.1 (colon carcinoma). Cell lines were cultured as previously described. HL-60-induced terminal differentiation was performed as described (Pannuti et al., 1988). Briefly, HL-60 cells were induced to differentiate to granulocytes by the addition of 1.25% DMSO to the culture medium (RPM1 1640 with 10% fetal calf serum). RNA Isolation and Northern Analysis Total RNA was extracted from the various cell lines by the guanidine isothiocyanate method (Chirgwin et al., 1979). Aliquots of 15 pg were electrophoresed on 1% agarose-formaldehyde gels and transferred to nitrocellulose filters. The blots were hybridized with the appropriate finger fragment in buffer containing 50% formamide, 5X SSPE, 1X Denhardt’s, 0.1% SDS, and 100 pg/ml salmon sperm DNA. The final washing was at 60°C in 0.2X SSC and 0.2% SDS. The entire ZNF8 cDNA insert and the large EcoRI fragment of the cZNF7 were used (see Figs. 1 and 2). The filters were autoradiographed at -70°C with an intensifying screen for 1 week. In Situ Hybridization Probe hybridization to metaphase chromosomes was performed according to method of Gerhard et cd. (1981), slightly modified. Briefly, chromosome spreads were obtained from PHA-stimulated peripheral blood lymphocytes (PBL), pretreated with RNase, and denatured in 70% formamide/2X SSC for 2 min at 7O’C. The appropriate finger probes were labeled by nick-translation using [3H]dCTP. The probe was denatured by boiling for 3-4 min and used at a concentration of SO100 rig/ml in a hybridization buffer containing 50% formamide, 5% dextran sulfate, 2X SSC, 2~ Den-

Isolation and Nucleotide Sequences of Two Human Finger cDNA Clones On the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Kruppel finger region, we have recently isolated two members of the human zinc finger family (Pannuti et al., 1988). Southern blotting analysis of human genomic DNA, carried out at low stringency with a probe consisting of a 503-bp HindIII/BamHI fragment containing the finger region of the HF.10 cDNA, revealed the presence of several related DNA sequences (data not shown). We used the 503-bp HindIII/BamHI fragment from the finger region of the HF.10 cDNA clone to screen a Xgtll cDNA library derived from human placenta. Several cDNA clones containing multiple copies of the zinc finger motif were identified. Two cDNA clones (cZNF7 and cZNF8) containing the longest inserts were further analyzed. The complete nucleotide and predicted amino acid sequencesof ZNF7 and ZNF8 cDNA clones are shown in Fig. 1 and Fig. 2, respectively. Translation of the longest open reading frame in both cDNA clones yields multifingered proteins containing 15 and 7 finger repeats, respectively. The cZNF7 clone is 2356 nucleotides long and contains a long open reading frame that begins at position 51 and extends to an in-frame TAG termination codon at position 2290; a putative ATG initiation codon (Kozak, 1987) is located at nucleotide 232. The open reading frame starting at the ATG at position 232 would code for a polypeptide of 687 amino acids with a calculated relative molecular mass of 77,894. The ZNF7-encoded protein contains 15 finger domains that comprise nearly 70% of the protein, and the 15 fingers are grouped into three sets of 6, 7, and 2 finger repeats, respectively. The nucleotide and amino acid sequences of cZNF8 are shown in Fig. 2. The longest open reading frame starts at the beginning of the cDNA clone and terminates at position 1623 with the TAG termination codon. The termination codon is followed by a 3’ untranslated region of 300 nucleotides, which lacks a polyadenylation signal and poly(A) tail. Seven zinc

GCGCGGCGGCGGACCTCGGGTTGCCCTC

28

GGTCCGAGTGATCCCTGGTCGCTTCCTTAGCCCTCCCGCCTTCGGCATTGGGGTCCCCGCGTCCCCCGGGCCTCCAGGCGGGAAAGCGCGGGGGCTTTGCGGGGC ***

133

CTTGAGCGCCTGGTGTGGGAGGTGGTCGAGCCCAGCCACCCTCCCCCGCGGCGGCGCGAGGTCTCTCGGCCAGAACACGTGGATGCCCACCCACCACTGAGCCTC

238 343

ATGGAGGTGGTAACATTTGGCGATGTGGCTGTGCACTTCTCTCGGGAGGAGTGGCAGTGTCTGGACCCTGGCCAGAGGGCCCTCTACAGGGAAGTGATGCTGGAG 1 MetGluValValThrPheGlyAspValAlaValHisPheSerArgGluGluTrpGlnCysLeuAspProGlyGlnArgAlaLeuTyrArgGluValMe~LeuGlu AACCACAGCAGTGTGGCTGGACTAGCAGGATTCCTGGTTTTCAAGCCTGAGCTGATCTCTCGGCTGGAGCAGGGAGAAGAGCCATGGGTCCTCGACCTGCAGGGA 36 AsnHisSerSerValAlaGlyLeuAlaGlyPheLeuValPheLysProGluLeuIleSerArgLeuGluGlnGlyGluGluProTrpValLeuAspLeuGlnGly

448

GCAGAGGGGACAGAGGCACCAAGGACCTCCAAGACAGATTCTACGATTAGGACTGAAAATGAGCAGGCCTGTGAGGACATGGACATCCTAAAATCAGAATCCTAT 71 AlaGluGlyThrGluAlaProArgThrSerLysThrAspSerThrIleArgThrGluAsnGluGlnAlaCysGluAspMe~AsplleLeuLysSerGluSerTyr

553

GGGACAGTGGTCAGAATCTCCCCACAGGACTTTCCTCAGAATCCTGGCTTTGGAGACGTTTCTGATTCTGAGGTCTGGTTAGACAGTCATCTGGGCAGTCCCGGG GlyThrValValArgIleSerProGlnAspPheProGlnAsnProGlyPheGlyAspValSerAspSerGluValTrpLeuAspSerHisLeuGlySerProGly

658

106

CTGAAAGTGACAGGCTTTACCTTCCAAAATAACTGTTTGAATGAGGAGACTGTGGTTCCCAAGACCTTCACCAAGGACGCACCCCAGGGATGTAAGGAGCTGGGA LeuLysValThrGlyPheThrPheGlnAsnAsnCysLeuAsnGluGluThrValValProLysThrPheThrLysAspAlaProGlnGlyCysLysGluLeuGly

763

141

AGCAGCGGCCTGGATTGTCAGCCTCTTGAAAGTCAGGGAGAGAGTGCGGAAGGGATGTCCCAGAGATGCGAGGAGTGTGGCAAAGGCATCAGAGCCACTTCAGAT SerSerGlyLeuAspCysGlnProLeuGluSerGlnGlyGluSerAlaGluGlyMe~SerGlnArgCysGluGluCysGlyLysGlyIleArgAlaThrSerAsp

868

176

ATCGCTCTGCATTGGGAAATTAATACACAGAAAATTAGCAGATGTCAAGAATGCCAAAAAAAGTTATCTGACTGCTTGCAGGGGAAACATACAAATAACTGCCAT lleAlaLeuHisTrpGlulleAsnThrGlnLyslleSerArgCysGlnGluCysGlnLysLysLeuSerAspCysLeuGlnGlyLysHisThrAsnAsnCysHis (1) GGAGAGAAGCCGTACGAATGTGCAGAGTGTGGGAAAGTCTTCAGGCTCTGCTCGCAGCTTAATCAGCATCAGAGAATCCACACGGGAGAGAAACCCTTTAAATGC GlyGluLysProTyrG~uCysAlaGluCysGlyLysValPheArgLeuCysSerGlnLeuAsnGlnHisGlnArglleHisThrGlyGluLysproPheLys~

973

211

246

1078

(2)

(31 . I

316

ACTGAGTGTGGAAAAGCCTTCCGCCTGAGCTCAAAACTTATTCAGCATCAAAGAATCCACACTGGGGAGAAGCCCTACAGATGTGAGGAATGTGGAAAAGCTTTT ThrGluCysGlyLysAlaPheArgLeuSerSerLysLeuIleGlnHisGlnArgIleHisThrGlyGluLysProTyrArgCysGluGluCysGlyLysAlaPhe (4) GGTCAGAGCTCAAGCCTCATCCACCATCAGAGAATCCACACAGGAGAGAGGCCCTATGGTTGTCGTGAGTGTGGGAAAGCCTTCAGCCAGCAGTCGCAGCTGGTT GlyGlnSerSerSerLeuIleHisHisGlnArgIleHisThrGlyGluArgProTyrGlyCysArgGluCysGlyLysAlaPheSerGlnGlnSerGlnLeuVal

351

(5) AGACACCAGAGAACTCACACTGGGGAGAGGCCCTACCCTTGCAAGGAGTGTGGGAAGGCCTTCAGCCAGAGCTCCACCCTAGCCCAGCATCAAAGGATGCATACT ArgHisGlnArgThrHisThrGlyGluArgProTyrProCysLysGluCysGlyLysAlaPheSerGlnSerSerThrLeuAlaGlnHisGlnArgMe~HisThr

386

GGGGAGAAAGCTCAAATTCTAAAAGCCTCAGACAGTCCAAGCCTTGTTGCACATCAGAGAATTCACGCTGTAGAGAAACCATTTAAGTGTGATGAGTGTGGGAAA GlyGluLysAlaGlnIleLeuLysAlaSerAspSerProSerLeuValAlaHisGlnArgIleHisAlaValGluLysProPheLysCysAspGluCysGlyLys

421

171 *, I GCTTTTAGGTGGATCTCTCGCCTGAGTCAGCATCAGCTGATTCACACTGGAGAGAAGCCTTATAAATGCAACAAGTGTACAAAAGCCTTTGGTTGTAGTTCACGG ~laPheArgTrpIleSerArgLeuSerGlnHisGlnLeulleHisThrGlyGluLysProTyrLysCysAsnLysCysThrLysAlaPheGlyCysSerSerArg

281

1183

1288

1393

(6) 1498

1603

(8)

491

CTTATTCGCCATCAGAGAACTCACACTGGAGAAAAACCATTTAAATGTGATGAGTGTGGCAAAGGCTTTGTTCAGGGCTCACACCTTATTCAGCATCAGCGAATC LeuIleArgHisGlnArgThrHisThrGlyGluLysProPheLysCysAspGluCysGlyLysGlyPheValGlnGlySerHisLeuIleGlnHisGlnArgIle (9) CACACTGGAGAGAAACCCTATGTGTGTAATGACTGTGGAAAAGCCTTCAGTCAGAGTTCCAGCCTTATTTACCATCAGAGAATCCATAAAGGAGAGAAGCCCTAC ~ThrGlyG1uLysProTyrValCysAsnAspCysGlyLysAlaPheSerG1nSerSerSerLeuI1eTyrHisG1nArgI1eHisLysGlyG1uLysProTyr

1918

526

(10) GAATGCCTCCAATGCGGAAAAGCCTTCAGTATGAGCACACAGCTTACAATACATCAAAGGGTTCACACTGGAGAGAGGCCCTATAAATGTAATGAATGTGGGAAA GluCysLeuGlnCysGlyLysAlaPheSerMetSerThrGlnLeuThrIleHisGlnArgValHisThrGlyGluArgProTyrLysCysAsnGluCysGlyLys

2023

561

(11) (12) GCCTTCAGTCAAAACTCAACCCTTTTCCAACACCAGATAATTCATGCAGGGGTGAAGCCCTATGAGTGCAGTGAGTGTGGAAAAGCCTTCAGCCGGAGCTCATAT AlaPheSerGlnAsnSerThrLeuPheGlnHisGlnIleIleHisAlaGlyValLysProTyrGluCysSerGluCysGlyLysAlaPheSerArgSerSerTyr

2128

596

(13) CTTATTGAACACCAGAGAATACACACTAGGGCCCAGTGGTTTTACGAATATGGGAATGCCCTGGAAGGGTCCACCTTTGTGAGCCGTAAAAAGGTTAATACTATA LeuIleGluHisGlnArgIleHisThrArgAlaGlnTrpPheTyrGluTyrGlyAsnAlaLeuGluGlySerThrPheValSerArgLysLysValAsnThrIle

456

631

666

AAGAAACTGCATCAGTGTGAAGACTGTGAGAAGATATTTAGGTGGCGTTCACACCTAATTATACACCAGAGAATTCACACCGGGGAGAAGCCTTATAAATGCAAT LysLysLeuHisGln~ysG1uAspCysGluLysIlePheArgTrpArgSe~HisLeuIleIleHisGlnArgIleHisThrGlyGluLysProTyrLysCysAsn (14) GACTGTGGCAAAGCTTTTAATCGTAGCTCAAGGCTTACCCAGCATCAAAAAATTCACATGGGATAGACCACTTACATATAAATGTGTATATATGTG~CCT AspCysGlyLysAlaPheAsnArgSerSerArgLeuThrGlnHisGlnLysIleHisMe~Gly***

1708

1813

2233 (15) 2338

ATAGCCTTAACTTAAAAAAAAAAAA

FIG. 1. Nucleotide and deduced amino acid sequences of ZNFP cDNA. The DNA sequence is numbered on the right. The 15 finger *** indicates an in-frame termination codon. The AATAAA polyadenylation signal is underlined. At domains are underlined and numbered. the bottom is a schematic representation of the ZNF7 protein showing the relative positions of the finger domains.

336

LANIA

ET

AL.

GACTTTACCCAGGAGGAATGGGGGCAGCTGCACCCTACCCAGAGGATCCTCTACCGTGACGTGATGCTGGAGACCTTTGGCTACCTGCTCTCCATAGGT AspPheThrGlnGluGluTrpGlyGlnLeuAspProThrGlnArgIleLeuTyrArgAspValMetLeuGluThrPheGlyTyrLeuLeuSerlleGly

99

CCTGAGCTTCCGAAGCCTGAAGTCATCTCCCAGCTGGAGCAAGGGACCGAGCTATGGGTGGCTGAGAGAGGAACCACCCAGGGCTGCCATCCAGCCTGGGAGCCT 35 ProGluLeuProLysProGluVallleSerGlnLeuGluGlnGlyThrGluLeuTrpValAlaGluArgGlyThrThrGlnGlyCysHisProAlaTrpGluPro

204

CGATCTGAAAGCCAAGCATCACGCAAGGAAGAGGGCCTGCCTGAAGAGGAGCCATCCCATGTCACGGGAAGGGAAGGATTCCCGACAGATGCTCCTTATCCCACC 66 ArgSerGluSerGlnAlaSerArqLysGluGluGlyLeuProGluGluGluProSerHisValThrGlyArgGluGlyPheProThrAspAlaProTyrProThr

309

ACGTTAGGGAAAGACAGGGAGTGTCAGAGCCAGAGTCTGGCACTCAAGGAGCAGAATAACTTGAAGCAGTTGGAATTTGGCCTCAAGGAAGCACCAGTTCAAGAT 97 ThrLeuGlyLysAspArgGluCysGlnSerGlnSerLeuAlaLeuLysGluGlnAsnAsnLeuLysGlnLeuGluPheGlyLeuLysGluAlaProValGlnAsp

414

CAAGCCTACAAAACTCTCAGACTCAGGGAAAACTGCGTCCTGAGTTCAAGCCCAAATCCATTCCCAGAGATCTCTAGAGGGGAGTATTTGTATACTTACGACTCA GlnGlyTyrLysThrLeuArgLeuArgGluAsnCysValLeuSerSerSerProAsnProPheProGluIleSerArgGlyGluTyrLeuTyrThrlyrAspSe~

519

132

CAGATTACAGACTCAGAACATAACTCCAGCTTAGTCAGTCAGCAGACAGGCTCCCCAGGAAAACAGCCCGGTGAAAACAGTGACTGTCACAGAGATTCCAGTCAG GlnIleThrAspSerGluHisAsnSerSerLeuValSerGlnGlnThrGlySerProGlyLysGlnProGlyGluAsnSerAspCysHisArgAspSerSerGln

624

167

GCCATTCCAATTACGGAACTCACAAAAAGCCAGGTGCAGGACAAACCCTACAAATGTACTGACTGTGGGAAGTCGTTTAACCATAACGCACACCTCACCGTGCAC AlaIleProIleThrGluLeuThrLysSerGlnValGlnAspLysProTyrLysCysThrAspCysGlyLyrSerPheAsnHisAsnAlaHisLeuThrValHis (1) AAGAGGATTCATACGGGAGAAAGACCTTATATGTGCAAGGAGTGTGGGAAAGCCTTCAGCCAGAACTCCTCCCTCGTCCAGCATGAGCGCATCCACACTGGAGAC LysArglleHisThrGlyGluArgProTyrMetCysLysGluCysGlyLysAlaPheSerGlnAsnSerSerLeuValGlnHisGluArgIleHisThrGlyAsp

729

202

237

834

(2)

342

AAGCCCTACAAGTGTGCCGAATGTGGGAAGTCTTTCTGCCATAGTACACACCTTACCGTCCATCGGAGGATTCACACTGGGGAGAAGCCCTATGAGTGTCAGGAC LysProTyrLysCysAlaGluCysGlyLysSerPheCysHisSerThrHisLeuThrValHisArgArgIleHisThrGlyGluLysProTyrGluCysGlnAsp (3) (4) TGTGGGAGGGCCTTCAACCAGAACTCCTCCCTGGGGCGGCACAAGAGGACACACACTGGGGAGAAGCCATACACCTGCAGTGTGTGTGGGAAATCCTTCTCTCGG CysGlyArgAlaPheAsnG1nAsnSerSerLeuGlyAr~HisLysArgThrHisThrGlyGluLysProTyrThrCysSerValCysGl~LysSerPheSerArq (5) ACCACTTGCCTTTTCCTGCACCTGAGAACTCACACCGAGGAGAGGCCCTACGAGTGTAACCACTGCGGGAAGGGCTTCAGGCACAGCTCATCCCTGGCCCAGCAC ThrThrCysLeuPheLeuHisLeuArgThrHisThrGluGluArgProTyrGluCysAsnHisCysGlyLysGlyPheArgHysSerSerSerLeuAlaGlnHis

1254

377

CAGCGGAAGCACGCGGGGGAGAAGCCCTTTGAGTGCCGCCAGAGGCTGATCTTTGAGCAGACGCCAGCTCTCACAAAGCATGAATGGACAGAAGCCCTGGGCTGT GlnArgLysHisAlaGlyGluLysProPheGluCysArgGlnArgLeuIlePheGluGlnThrProAlaLeuThrLysHisGluTrpThrGluAlaLeuGlyCys

1359

447

GACCCACCTTTGAGTCAAGATGAGAGGACTCACCGAAGCGACAGACCCTTCAAATGTAATCAGTGTGGGAAGTGTTTCATTCAGAGCTCTCACCTCATCCGGCAC AspProProLeuSerGlnAspGluArgThrHisArgSerAspArgProPheLysCysAsnGlnCysGlyLysCysPheIleGlnSerSerHisLeuIleArgHis (7) CAGATAACTCACACCAGAGAGGAGCAGCCCCATGGGCGAAGCCGGCGGCGTGAACAATCCTCGAGCAGGAACTCACACCTGGTTCAGCATCAACACCCGAACTCC GlnIleThrHisThrArgGluGluGlnProHisGlyArgSerArgArgArgGluGlnSerSerSerArgAsnSerHisLeuValGlnHisGlnHisproAsnS~r AGAAAGAGCTCTGCAGGAGGACGAAAGGCAGGGCAGCCGGAAAGCAGAGCCCTGGCTTTGTTTGACATCCAAAAAATCATGC~GAGAAAAACCCTGTGCACGTT ArgLysSerSerAlaGlyGlyArgLysAlaGlyGlnproGluSerArgAlaLeuAlaLeuPheAsplleGlnLysIleMetGlnGluLysAsnProValHisVal

I569

482

ATTGGGGTGGAAGAGCCTT~TGTGGGTGCTTCCATGTTATTTGACATCAGAGAATCCACATAGGAGAGAAACTTTGCTGATGACTTTTAACCACAAGTAAAAAAT IleGlyValGluG~uProSerValGlyAlaSerMetLeuPheAspfleArgGluSerThr***

1674

537

GTGGTAAGTCCACATAGTGTACTCATGGAAGGAGGGGCTGGGGGTAGAAATGTCATGGGTGACTTCTGACTTTCTAAGGAAATGATGCTTCCCAAGCACCCGAGG

1779

TTGGTTGGTCCCAAATCTATCAAACTCAGTGCCCTCTTTAGCGACATATTTTGTGACATTCCTTCCATTACACCACAGTGAGTTCACAGGTAATATAACCTACCC

I884

272

307

939

1044

1149

(‘5)

412

1464

ACCTGTGTAATGTCGAAAAAAATCAATATGCGGCCCCTATTTGTAAAGGATCATTAAAAT

FIG. 2. termination the bottom,

DNA and amino acid sequences of ZNFB cDNA. The DNA sequence is numbered on the right. *** indicates the in-frame codon. The seven finger domains are underlined and numbered. In the schematic representation of the ZNF8 protein shown at the dotted line that extends past the cDNA clone indicates the portion of the amino terminus lacking in ZNFS.

finger domains can be identified in the ZNFB-encoded protein; however, six of them are clustered between amino acid positions 220 and 380, and the seventh finger domain is located at position 430. Therefore, the last two finger domains are not separated by the classical H/C-link consensus sequence (Schuh et al., 1986).

A comparison of the finger motifs present in both ZNF7- and ZNFB-encoded proteins indicated that each unit conforms almost exactly to the consensus sequence CX2CX3FXSLX2HX3H (Fig. 3) diagnostic of DNAbinding zinc fingers. Furthermore, in both proteins the fingers are connected by “H/C links” (TGEKPF/YX,

HUMAN 1

CQE CAE CTE CEE CRE CKE CDE C N‘K CCE CND

CLQ

ZINC

10

Y CQKKL CGKVF CGKAF CGKAF

CGKAF CGKAF

CGKAF CTKAF

CGKGF CGKAF CGKAF CGKAF CGKAF CEKIF

SQQSQ

G

NNC

ii:: IQ IH VR

SQSST RWISR GCSSR VQGSH

AQ SQ

siisss

IY TI

IR

IQ

337

GENES

20

r SDCLQ RLCSQ RLSSK GQSSS

FINGER

28

ORI ORI QRI QRT QRM QLI QRT QRI QRI QRV QII QRI ORI QKI

H H H H H H H H H H H H H H H

GEKPYE TGEKPFK TGEKPYR TGERPYG TGERPYP TGEKAQI TGEKPYK TGEKPFK TGEKPYV KGEKPYE TGERPYK AGVKPYE TRAQWFY TGEKPYK MG

Qri

H

tGekpyx

Consensus

KRI ERI RR1 KRT LRT

H H H H H H H

TGERPYM TGDKPYK TGEKPYE TGEKPYT TEERPYE AGEKPFE TREEQPH

ZNF8

ZNF7

CGKAF

SMSTQ SQNST SRSSY RWRSH NRSSR

Cxe

CgKaF

xxxsx

CTD CKE CAE

NHNAH SQNSS CHSTH

CNQ

CGKSF CGKAF CGKSF CGRAF CGKSF CGKGF CGKCF

cxx

CGkxF

H

tgekP:x

Consensus

ZNF8

cxx

CxxxF

H

TGEKPYx

Consensus

Kruppel

cxx

CgKrf

H

TGExxFx

Consensus

Spl

CNE CSE CED CND

CQD csv CNH

FQ IE I I

TQ

TV

VQ

NQNSS

TV GR

SRTTC RHSSS IQSSH

1; IR

QRK QIT

ZNF7

FIG. 3. Amino acid comparison of ZNF7, ZNF8, Spl, and Krnppel zinc finger motifs. The sequences are given in one-letter code and are aligned tc show the repeated units. Amino acids that are strictly conserved in the finger motifs are boxed. For each gene, a consensus for the repeated motif is displayed: uppercase letters correspond to strictly conserved amino acids, and lowercase letters to amino acids conserved >60%; x indicates no conservation.

Schuh et al., 1986) found in genes from mouse, Drosophila, and Xenopus that cross-hybridize to the Kruppel finger domains (Schuh et al., 1986; Chowdhury et al., 1987; Ruiz i Altaba et al., 1987). The sequence similarity among the ZNF7 fingers is 65-80% and that among the ZNF8 fingers is 60-75%, whereas the sequence similarity between any of the ZNF7 fingers and those present in ZNF8 is 50-60%. No sequence similarity was found outside the finger regions. Expression of Human Finger Genes in Various Cell Lines and during in Vitro Induced Differentiation To determine the expression and distribution of ZNF7 and ZNFS finger gene transcripts, a number of RNAs extracted from different established human cell lines were analyzed by Northern blotting; the result of this analysis is shown in Fig. 4. The ZNF7 probe detected two discrete transcripts of sizes 2.8 and 3.3 kb (Fig. 4A). Both transcripts were expressed at constant level in all the cell lines tested; however, the level of the 3.3-kb transcript was in each case three to fivefold lower than that of the 2.8-kb transcript. A similar pat-

tern of expression was also observed in poly(A)-selected RNA (data not shown). The ZNFS probe detected two transcripts 11 and 2.2 kb long. However, no expression of either ZNF8 transcript was detected in HeLa RNA, and only the 2.3-kb transcript was observed in NTERAS/Dl cells (Fig. 4B). In light of alternate processing of ZNF7 and ZNFS transcripts, we suggest that the cDNA clones reported in Figs. 1 and 2 represent the major messages from these loci. This suggestion is based on our isolation of additional cDNA clones; after restriction map analysis and partial DNA sequencing, the shorter cDNA clones were found to be identical to the ZNF7 and ZNF8 cDNA clones reported in Figs. 1 and 2 (data not shown). Next, we sought to determine the expression of ZNF7 and ZNF8 genes during in vitro induced terminal differentiation of HL-60 cells. HL-60 is a human acute leukemia cell line with phenotypic features of immature myeloid cells and is capable of undergoing terminal differentiation upon treatment with dimethyl sulfoxide (DMSO) (Collins et aZ., 1977). Figure 5 shows a Northern blot analysis of total RNA isolated from untreated and DMSO-treated HL-60 cells and hybridized to

338

LANIA 1

234567

ET

AL.

1

234567

FIG. 4. Northern blot analysis of RNA isolated from various human cell lines hybridized to ZNF7 (A) and ZNFB (B) cDNA probes. The RNA samples are HeLa (lane l), NTERAQ/Dl embryonal carcinoma (lane 2), HL-60 (lane 3), MOLT-4 (lane 4), WI-38 (lane 6), B lymphocytes (lane 6), and colon carcinoma (lane 7). Aliquots of 15 fig were electrophoresed on 1% agarose-formaldehyde gels and processed as described under Materials and Methods. Ribosomal RNAs (28 S and 18 S) are indicated by arrowheads.

ZNF7 and ZNFB probes. The relative amounts of the ZNF7 and ZNF8 transcripts decreased after DMSOinduced terminal differentiation. Rehybridization of the Northern blots to the GADPH probe (Piechaczyk et al., 1984) indicated that comparable amounts of total RNA were present in each lane. We have also monitored the expression of ZNF7 and ZNF8 finger genes during the in vitro induced differentiation of the human embryonal carcinoma cell line NTERAB/Dl (Andrews et aZ., 1984). No differences in the level of expression of either the ZNF7 or the ZNFS finger gene were found upon retinoic acid-induced differentiation of NTERA2/Dl cells (data not shown).

Chromosome Mapping of ZNF? and ZNF8 Genes by in Situ Hybridization Analysis DNA probes representative of the ZNF7 and ZNF8 genes were hybridized to human chromosome metaphases obtained from normal peripheral blood lymphocytes. Using the 1459-bp EcoRI fragment of ZNF7, of 146 metaphases analyzed, a total of 246 silver grains (1.68 grains/metaphase) were found. Thirty-nine grains (15.8% of the total) were located on chromosome 8 (P < O.OOl), 27 of which (69.2%) were present on band q24.1-qter (Fig. 6). Using the entire EcoRI insert of the ZNFS cDNA clone, a total of 62 metaphases were scored for the assignment of 110 labeled sites (1.77 grains/metaphase) to chromosome bands. Twenty-one (19% of the total) were observed on chromosome 20 (P < O.OOl), 17 of which (80.9%) were clustered on band 2Oq13 (Fig. 7). In both experiments no specific labeling was observed in any other chromosomal region. Moreover, these results were further confirmed using

GAPDHFIG. 6. Expression of ZNF7 (left) and ZNF8 (right) genes during in vitro induced differentiation of HL-60 myeloid cells. HL-60 cells were treated with DMSO for 5 days and RNAs were extracted from undifferentiated cells (lane l), at Day 1 (lane 2), Day 3 (lane 3), and Day 5 (lane 4) of culture in the presence of the inducing agent. The same blots were rehybridised to the GAPDH probe. RNA extraction and Northern blot analysis were performed as described in the text. In overexposed autoradiograms of total RNAs, some hyhridisation to both 28 S and 18 S RNA is detectable. Ribosomal RNAs (28 S and 18 S) are indicated by arrowheads.

a FIG. 6. Idiogram of chromosome 8 showing ZNF7 hybridization grains from 146 metaphases.

the distribution

of

HUMAN

FIG. 7. Idiogram of ZNFS hybridization

of chromosome 20 showing grains from 63 metaphases.

ZINC

the distribution

ZNF7 and ZNF8 subclones that did not contain finger domains as probes (data not shown).

the

DISCUSSION

Taking advantage of the relatively conserved nature of zinc finger domains (Schuh et aZ., 1986), we have isolated and characterized two human finger genes by finger-motif-containing DNA probe hybridization. The finger motifs of both ZNF7- and ZNFB-encoded proteins contain the appropriate amino acids residues to fit the consensus sequence of the Cys/His finger (Klug and Rhodes, 1987; Evans and Hollenberg, 1988). The predicted amino acid sequences of ZNF7 and ZNFB cDNA clones indicate the presence of 15 and 7 finger repeats, respectively. The 15 fingers in ZNF7 are grouped into three sets of 6, 7, and 2 finger repeats, respectively. This organization resembles that of the Xenopus Xfin gene, in which the 37 fingers are grouped into six sets (Ruiz i Altaba et al., 1987). Seven zinc finger domains can be identified in the ZNF8 protein; however, six of them are clustered, with the last being a single finger domain. A recent study on a single-finger peptide indicates that a single zinc finger can exist as an independent structure sufficient for DNA binding (Parraga et al., 1988). Transcription studies indicate that the ZNF7 and ZNF8 genes are expressed as multiple transcripts in many human cell lines of different embryological derivation. Moreover, the expression of both genesis negatively regulated during in vitro induced terminal differentiation of HL-60 myeloid cells. The low levels of expression of ZNF7 and ZNF8 transcripts in terminally differentiated HL-60 cells may be due to expression of the genesin a small subpopulation of proliferating cells. Alternatively, the expression patterns of ZNF7 and ZNFS genes may be due to cell-type specificity. Current research is aimed at addressing this hypothesis. Although we have not yet performed experiments to test whether the ZNF7 and ZNF8-encoded proteins bind DNA, we believe that this is likely becausespecific DNA binding is a common feature of other multifingered proteins (Miller et al., 1985; Kadonaga et al., 1987). Moreover, because of the precedent of TFIIIA

FINGER

339

GENES

protein (Klug and Rodes, 1987), we cannot exclude the possibility that ZNF7 and ZNF8 proteins bind RNA and/or DNA. Using the in situ hybridization technique, we found that the ZNF7 finger gene is located on band q24 of the long arm of human chromosome 8. The same chromosomal region contains the c-myc oncogene locus and is implicated in the 8,14 translocation associated with Burkitt’s lymphoma (Dalla Favera et al., 1982). In addition, it is involved in microdeletions associated with the Langer-Giedion syndrome (tricho-rhino-phalangeal, TRP syndrome) (Bowen et al, 1985), in which 8q24.11-q24.13 appears to be the shortest overlapping region (Fryns and Van den Berghe, 1986). The ZNFS gene was localized on the terminal band of the long arm of human chromosome 20. Chromosome 20 is involved in a nonrandom karyotype rearrangement, i.e., deletion of its long arm (2Oq-), in chronic myeloproliferative disorders, most frequently in polycythemia vera (27% of the cases) (Heim and Mitelman, 1987). The 2Oq- marker chromosome is the result of an interstitial deletion; molecular-cytogenetic analysis demonstrated that the C-Q-Cgene, which normally maps to the most distal band of 2Oq, still remains on the rearranged chromosome in 2Oq- (Le Beau et al., 1985). We are currently investigating whether ZNF8 sequences are lost in the 2Oq- marker. In the last year the localizations of other human multifingered protein genes have been described: TDF to chromosome Y (Page et al., 1987), ZPX to Xp21.3 (Muller and Schempp, 1989), the GLI gene to chromosome 12q13.3-q14.1 (Arheden et al., 1989), EGR.l to 5q23-31 (Sukhatme et al., 1988), the human homolog of mouse Krox-20 to chromosome lOq21.1-22.1 (Chavrier et aZ., 1989), and ZFP3 to chromosome 17p1217pter (Ashworth et al., 1989). The chromosomal assignment of members of the finger gene family will be useful in establishing the evolutionary relatedness of this gene family and also in determining their possible functions. ACKNOWLEDGMENTS This work was paid for by grants from Progetto Finalizzato CNR “Oncologia” and MPI to L. L. and by the Italian Association for Cancer Research (AIRC) to L.L. and P-G.P.

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