Gene, 113 (1992) 269-274 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

269

GENE 06425

Cloning and characterization of the human gene encoding pll: structural similarity to other members of the S-IO0 gene family (Recombinant DNA; annexins; exon/intron structure; CpG island; Ca 2 ÷ ions; EF-hand)

Thomas Harder, Eckhard Kube and Volker Gerke Department of Biochemistry, Max-Planck-lnstitute for Biophysical Chemistry, D-3400 GOttingen(Germany) TeL (49-551)201486 Received by H. van Ormondt: 18 September 1991 Revised/Accepted: 9 December/16 December 1991 Received at publishers: 7 February 1992

SUMMARY The human gene (CLP11) encoding pl 1, the cellular ligand of the tyrosine kinase substrate, annexin II (AnxII), has been isolated from a human genomic library. Restriction mapping and sequencing reveals that CLPI 1 covers a stretch of approx. 11 kb in the human genome. The structure of CLPII resembles that of the other genes encoding S-100 proteins which have been characterized so far: the transcribed region is divided by two introns, one in the 5'-nontranslated portion and the second in the protein-coding region. Interestingly, in CLPII, as well as all other S-100 genes, the second intron separates the codons for two corresponding amino acids, which reside in the sequence connecting the two helix-loop-helix (EF-hand) motifs. The 5'-nontranscribed region, which most likely represents the CLPI 1 promoter, is characterized by a high G+C content and probably is part of a CpG-island. Several putative binding sites for transcription factors can be identified in the 5'nontranscribed region of CLPII. Among them, the [3DRE element, which was first described in the/~-globin promoter, is most notable, since it is also present in the promoter of ANXIL It could be responsible for the simultaneous induction of CLPII and ANXII expression during certain cell differentiation processes, e.g., the nerve growth factor-induced differentiation of the pheochromocytoma cell line, PC I2.

INTRODUCTION The response to elevation of cytoplasmic Ca 2+ levels following extra- or intracellular stimuli is mediated by pro-

Correspondence to: Dr. V. Gerke, Department of Biochemistry, MaxPlanck-lnstitute for BiophysicalChemistry,D-3400GOttingen(Germany) Tel. (49-551)201477; Fax (49-551)201578. Abbreviations: aa, amino acid(s); Anx, annexin; ANX, human gene encoding Anx; [JDRE, /l-globin direct repeat eleraent; bp, base pair(s); eDNA, DNA complementaryto mRNA; CLPIi, human gene encoding pll; 50xDenhardt's, 5% Ficoli/5% polyvinylpyrrolidone/5% bovine serum albumin;EF-hand, see INTRODUCTION;HTF, Hpall tinyfragments; kb, kilobase(s) or 1000bp; NGF, nerve growth factor; nt, nucleotide(s); pl 1, cellularligand of annexin II; SDS, sodium dodecyl sulfate; src, oncogene encoded by Rous sarcoma virus; SSC, 0.15 M NaCi/ 0.015 M Na3.citrate pH 7.6; SSPE, 0.18 M NaCl/0.01 M Na2HPO4pH 7.4/1 mM EDTA; TBE, 0.09 M Tris.borate pH 8.0/2 mM EDTA; tsp, transcription start point(s).

teins which are capable of binding Ca 2 ÷ ions. A particulax class of these proteins is characterized by the so-called EF-hand, a helix-loop-helix motif involved in coordinating the Ca 2 + ion (for review see Kretsinger et al., 1991). Within the EF-hand superfamily a distinct set of proteins is grouped in the so-called S-100 protein family, whose members share a high degree of sequence similarity with S-100a and S-100b, Ca 2 + -binding proteins originally isolated from cerebral fluid (Moore, 1965; Isobe et al., 1977). All S-100 proteins are characterized by two consecutive EF-hands (one in the N-terminal and one in the C-terminal half of the molecule), which are connected via a stretch of aa of a less defined secondary structure (for review on S-100 protein~ see Hilt and Kligrnan, 1991). Protein p l l is a member of the S-100 family but has several unique features. It has suffered crucial deletions and aa substitutions in both EF-hand motifs, which are thought to render both Ca 2 + -binding sites inactive (for review see

270 EXPERIMENTAL AND DISCUSSION

Gerke, 1991). In all tissues and cells studies so far, pl 1 is found in a heterotetrameric complex with another Ca: + binding protein, AnxII (Erikson et al., 1984; Gerke and Weber 1985). AnxII, a major cellular substrate of the tyrosine kinase encoded by the src-oncogene, belongs to the Anx family of Ca-" +-dependent phospholipid- and membrane-binding proteins (for review see Crompton et al., 1988; Klee, 1988). At least some of its biochemical properties, e.g., the aggregation and fusion of phospholipid vesicles (Drust and Creutz, 1988), are modulated by p l l induced complex formation, which involves the binding of one p l 1 dimer to two Anxll monomers (for review see Gerke, 1989). Several genes of the S-100 family have been isolated and characterized recently and have been shown to have a very similar structure (see legend of Fig. 4 for references). The two EF-hands are encoded by different exons (exons 2 and 3), while the first exon comprises 30-100 nt of nontranslated sequence at the very 5' end (for review see Heizmann and Hunziker, 1991). In order to address the question whether the C L P I I gene has a similar structure we isolated generate D N A clones for CLP11 and mapped the intron positions. In addition, we report the 5'-nontranscribed sequence, which most likely represents the CLPI 1 promoter and contains putative binding sites for transcription factors. Since CLPI 1 and ANXII are likely to be co-regulated at the transcriptional level, these 5'-nontranscribed sequences of the C L P I I gene are also discussed in~the light ofthe recently published ANXII promoter sequence (Spano et al., 1990).

H

B

S

S H E B E SH

BESH

(a) Isolation and characterization of A-phage clones covering the entire human C L P l l gene A human genomic library in A-Fix II (Stratagene, La Jo!la, CA) was screened with an EcoRI-HindIII fragment (nt 1-586) covering the 5'-nontranslated, the proteincoding and most of the 3'-nontranslated region of the human C L P l l e D N A (Kube et al., 1991). Four independent A-phage clones were identified and characterized by restriction mapping and partial sequence analysis. The results are summarized in Figs. I and 2. They show that the human CLPI 1 gene spans approx, l 1 kb and contains two introns (Fig. 1). Intron I (approx. 8 kb) is located in the 5'-nontranslated region, whereas intron II (2.6 kb) resides in the protein-coding region and separates the first and the second EF-hand motif (Fig. 2). Exert I contains 90 nt of nontranslated sequence, whereas the remaining 21 nt of the 5'-nontranslated region are on exert If. In addition, exon II harbours the ATG start codon and the sequence coding for aa 1-43. The rest of the coding region and the complete 3'-nontranslated region including the canonical poly(A) signal are found on exon Ill. The 5' and 3' boundaries of the introns fit well to the consensus for splice sites (Mount, 1982). In order to exclude the occurrence of additional introns the exonic sequences were sequenced completely. Exons II and Ill proved to be identical and colinear to the human e D N A clone (Kube et al., 1991), indicating that no additional intron is present in the protein-coding region. However, the sequence of exert I showed several differ-

H

exon I

HE

E

exon 11

I

SH HE

ESB

E

,

exon I11

I

i

~.- pll A

i I kb

l

~- pll B

I

I I

~.- pll C

I

~.- pll D

I

Fig, I. Restrictionmap of the human CLP! I gen¢. B, BamHl; E, EcoRl', H, Hindllk S, Sacl. Black bars indicate the relative location of the three exons, the portion of the gene contained in the individual ~,clones ()~-plI A,B,C,D) is given below. A ).-Fix II library made from generate DNA of the human lung fibroblast cell line W134 was obtained from Stratagene (La Jolla, CA, USA). 10~' clones (approx. five times the haman genome equivalent) were screened by hybridizationof the phage DNA immobilized on Hybond N Filters (Amersham) with an EcoRI-HindIII fragment covering nt 1-586 of the human CLPil eDNA (Kubo et al., 1991), Prehybridization and hybridization were carried out at 65~C in 5 x SSPE/0.2% polyvinylpyrrolidone/0.2% Ficoli/0,i% SDS/0. ! mg/ml of denatured salmon-sperm DNA at 65:C. 10-20 ng of hybridization probe were radiolabelled to approx, 109cpm/pg DNA using a random-primerlabelling kit (Pharmacia, Piscataway, NJ) and [~t-32P]dCTP. The final high-stringencywash was performed in 0.5 x SSC/0.1% SDS at 65:C. Posifve clones were identified after autoradiographyfor 12-36 h on Kodak X-OMAT film exposed at -70°C with an intensifyingscreen. DNA from positive phage clones was prepared according to routine procedures (Sambrook et al., 1989). Appropriate subfragments of the human DNA inserts were obtained in preparative restriction digests and subcloned into pBluescriptIIKS+ (Stratagene, La Jolla, CA) for further restriction mapping and sequence analysis.

271 -370

5'~ccggt

t acct ct ggt t ct gcgccacgt

-314

gat t t gt acact t t ct aaaaccaaacccgagaggaagggcaggct

-254

t aaat at t cgagagcaggaccgt

-194

~ggcgggJcgagcgctctgclgaggcgggltccgggagcgagggcaJgggcgtggJgccgcgcgc

-134

ccggggt

-74

t gccgagcgcccgccaggct

-14

eDNA | G ..... a c g t a c t aaggaag'GCGCACAGCCCGCCGCGCTCGCCTCTCCGCCCCGCGTCCACGTCGC

cgggggagt

i

....

gccccacccgcaggacggccgggt

t tct act gaagagaagt

cagggt gggat gccc

t t acaagaacgct

cgggggcaggaagagggggaggagacagggct cct ccccgt

cccgcaccgcct

t ct t t

ct gt ct g

gggggagcgccc

ccct ct acccacccgccgc

I

C

+47

CCACGTCGCCCAGCTCGCCCAGCGTCCGCCGCGCCTCGGCCAA~g t g a g c t c c c a g t

+!07

gccc

Intron I: 8,0 kb I I

tacttlacatcct

t cg

_ M t taata~GCTTCAACGGACCACACCAAA,~TG

|

P S Q M E H A M'E T M M F T F H K F A G CCATCTCAAATGGAACACGCCA TGGAAACCATGATGT TTACATTTCACAAATTCGCTGGG

21

D K G Y L T K E D L R V L M E K E F P G GATAAAGGCTACTTAACAAAGGAGGACCTGAGAGTACTCATGGAAAAGGAGTTCCCTGGA

41

F L E . , TTTTTGGA~gtaagtgt

tggaaaagct

Intron I1:2,6 kh tg i ittgttctt

N tt t t ttttcaglAAT

45

Q K D P L A V D K I M K D L D Q C R D G CAAAAAGACCCTCTGGCTGTGGACAAAATAATGAAGGACCTGGACCAGTGTAGAGATGGC

65

K V G F Q S F F S L I A G L T I A C N D AAAGTGGGCTTCCAGAGCTTCTTTTCCCTAATTGCGGGCCTCACCATTGCATGCAATGAC

85

Y F V V H M K Q K G K K * TATTTTGTAGTACACATGAAGCAGAAGGGAAAGAAGTAGGCAGAAATGAGCAGTTCGCT

CCTCCCTGATAAGAGTTGTCCAAAGGGTCGCTTAAGGAATCTGCCCCACAGCTTCCCCCC ATAGAAGGATTTCATGAGCAGATCAGGACACTTAGCAAATGTAAAAATAAAATCTAACTC TCATTTGACAAGCAGAGAAAGAAAAGTTAAATACCAGATAAGCTTTTGATTTTTGTATTG

eDNA

TT TGCATCCCCTTGCCCTCAATAAATAAAGTTCTT TTTTAGTTCC=a a a t t t g a g a c a g a a tgtttgttcttccctcagaaat

tcttgt

tcccccaagaggcagcttagccc

3'

Fig. 2. Partial sequence of the human CLPI 1 gene. The coding region, cxon/intron boundaries as well as 5'- and 3'-flanking regions are shown. Appropriate restriction fragments containing exon sequences were identified by hybridization with the human CLPI 1 cDNA, and cloned into pBiuescriptll or M 13mp 19. Sequence analysis of these subfragments was carried out following the dideoxy chain-termination method (Sanger et al., 1977) using a T7 sequencing kit (Pharmacia). Capital letters show cxonic, small letters indicate intronic or 5' and 3' flanking sequences of the CLPI i gone. Boxes in the 5'-flanking region of the gene indicate the consensus for Sp ! binding. The sequences homologous to the [JDRE element in the/~-globin promoter are underlined. Splice sites are indicated by downward lines. Within the protein-coding ~cquen~e the ATG start codon is underlined and the stop codon marked by an asterisk. The aa numbers are bold. The canonical polyadenylation signal in the 3'.~iontranslated region is underlined. The 9 nt, which are deleted from the 5'-nontranslated region of our eDNA clone flanked by two base exchanges, are indicated by dots. The tsp, which was determined by primer-extension analysis, coincides with the 5' end of our cDNA (not shown). The downward arrow at position + 30 marks an additional strong stop in the primer extension reaction. Primer extension was performed with poly(A) + RNA from HeLa cells (3 pg per reaction) and a primer complementary to nt 91-102 of the CLPi i eDNA. The annealing (at 50'C in 0.1 M Tris.HC! pH 8.3/' 140 mM KCI/0.1 mM EDTA) and the polymerisation reaction (at 55 ~C) with avian myeloblastosis virus reverse transcriptase (Amersham, Atling:.a= Hcigbl.s, 11..I were carried out as described (Ausubel et al., 1987). The 5'nontranscribed sequence of the CLP! ! gent is listed in the GenBank database under the accession No. M77483.

ences when compared to our initial e D N A clone. Most mismatches occurred at the very 5' end of the transcribed region. They were eliminated after re-isolation of the original c D N A clone. Sequence analysis of this isolate reveals that the sequence of the very 5' end of our initial c D N A (Kube et al., 1991) was erroneous. The sequence of the new isolate now proved to be almost identical to that of exon I in our genomic clones indicating that the genomic sequence is indeed correct. However, there remains a deletion of 9 bp flanked by two base exchanges 60 nt upstream from the ATG start codon in our CLPI1 c D N A clone.

Most likely, these differences are due to artifacts that had occurred during the e D N A cloning procedure. The human CLP11 gene is considerably larger ( > 11 kb) than those for the other members of the S-IO0 gene family ( < 6 kb). Nevertheless, all gene structures are remarkably similar with the two introns residing in the 5'-nontranslated region and in the middle of the protein-coding part. Most notably, in CLPII, as well as in all other S-lO0 genes known so far, the second intron separates exactly the codons for two aa, which are located at a correspoqding position in the region connecting the two EF-ha':d motifs

272 aa Immnll 1PSQMEHAMETMMFTFHKFA---GDKGYLTKEDLRVLMEKEFPGFLE NQKDPL IGSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLD AQKDAD $100 a 1SELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLE EIKEQE $100 6 1ARPLEEALDVIVSTFHKYSGKEGDKFKLNKTELKELLTRELPSFLF KRTDEA p9 Ka 1LTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIR KK . . . . MRP8 1 TCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLK KENKNEK MRP14 1 ACPLDQAIGLLVAIFHKYSGREGDKHTLSKKELKELIQKEL-TIGS KLQD-A 2A9 1 SAKKSPEEMKSIFQKYAAKEGDPNQLSKEELKLLIQSEFPSLLK A S S - - CaBP9k Ioll

pll $I00 a 8100 P p9 Ka MRP8 MRP14 2A9 CaBPgK

5~VDKIMKDLDQCRDGKVGFQSFFSLIAGLTIACNDYF-VVHMKQKGKK s~VDKVMKELDEDGDGEVDFQEYVVLVAALTVACNNFFWENS S2VVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE S~FQKVMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGCPDKEPRKK 4~ADVWFKELDINTDGAVNFQEFLILVIKMGVAAHKKSHEESHKE STVIEHIMEDLDTNAOKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTP 51EIARLMEDLDRNKDQEVNFQEYVTFLGALALIYNEALKG 48TLDNLFKELDKNGDGEVSYEEFEVFFKKLSQ

Fig. 3. Alignment of the aa sequences of S-100 proteins with known gene structure. The vertical line marks the position of intron II in the genes coding for hum:m pli (this report), bovine S-100~ (Isobe and Okayama, 1981; Morii et al., 1991), human S-100fl (AIIore et al., 1990), rat p9Ka (Barraclough et al., 1987), human MRPI4 and MRP8 (Lagasse and Cleft, 1988), human 2A9 (Calcyclin; Ferrari et al., 1987), and rat CaBPgK (Perret et al., 1988). Several other names have been given to the individual S-100 proteins which are not included in the figure, pl I is also known as 42C and calpactin I light chain; MRP8 as CF antigene, p8, and calgranulin A; MRPI4 as p14 and calgranulin B. 2A9, calcyclin and PRA as well as 18A2, pEL68, p9Ka and 42A are synonyms or species homologs (for review and nomenclature see Hilt and Kligman, 1991; Heizmann and Hunziker, 1991). Putative Ca-"÷-binding loops of the EF-hand motifs are marked by a horizontal line. Dashes indicate gaps introduced for better alignment of the aa sequences.

(Fig. 3). Thus, each EF-hand motif is encoded by one exon. The similarities in gene structure and sequence support the hypothesis that all members of the S-100 protein family have a common ancestor, which contained a single EFhand encoded by one exon. Via gene duplication and mutation the whole family could have arisen from this putative primordial gene. Although p l I has lost the ability to bind Ca-"+, the protein sequence and the gene structure described here clearly place pll within the S-i00 family. Most probably its loss of Ca 2÷-binding is a secondary effect due to mutations within the EF-hand loops (Gerke and Weber, 1985). Consequently, the conformation of p l 1 might be frozen in a permanently active state, which allows the binding to its cellular target, Anxll, even in the absence of Ca-"+.

(b) CLPll is a single-copy gene in humans The gene encoding Anxll, the cellular iigand of pll, exists in several copies within the human genome (Huebner et al., 1988; Spano et at., 1990; T.H. and V.G., unpublished observations). Only one of the loci detected contain,~ the structural ANXII gene, the others represent nontranscribed pseudogenes. To address the question whether the situation is similar for CLPI 1, a human genomic Southern blot was hybridized with the EcoRI-HindIII fragment covering nt 1-586 of the human pl I eDNA. The hybridization pattern (Fig. 4)can be explained on the basis of the restriction map of the cloned CLPI l gene shown in Fig. 1. Fragments containing exons II and III yield a strong hybridization signal, e.g., the 1.3-kb and 2.6-kb HindIII fragments which harbour exons II and Ill, respectively. The signal resulting from exon I is

visible only in the BamHl-digested DNA (indicated by an asterisk). Since exon I is rather short and contains some mismatches to the cDNA probe (see above) the signal is relatively weak. All bands decorated in the Southern blot can be accounted for by the restriction map shown in Fig. 1. Thus the 2 clones described here cover the single-copy locus of the human CLPI 1 gene. Based on a Southern blot analysis it has been suggested that the gene encoding murine pl I is also a single-copy gene in mice (Saris et al., 1987).

(c) The 5'-nontranscribed region of the CLPI! gene Primer extension analysis on polyfA) + RNA from HeLa cells reveals that a start site for transcription coincides with the 5' end of CLPII eDNA (Fig. 2). However, an additional strong stop in the extension reaction is visible at position +30 (indicated by an arrow in Fig. 2). This stop could either indicate an alternative tsp or could be due to secondary structure of the G+C-rich CLP11 mRNA in this particular region. Thus, the 370 nt of 5'-nontranscribed sequence shown in Fig. 2 most likely cover at least a part of the promoter. The 5'-nontranscribed region of the CLPII gene has a high G+C content (66%) with the dinucleotide CG occurring frequently. This nt preference is typical of HTF islands (Bird, 1986), which are often found in the vicinity of the 5' ends of mammalian genes, especially of those which are not strictly tissue-specific. Thus, CLP11 might be regarded as a so-called housekeeping gene (Bird, 1986). Detailed analysis of the 5'-nontranscribed sequence c ~ the CLPll gene reveals several consensus sequences for the binding of transcription factors. In particular, three GC boxes which are putative Spl-binding sites (Briggs et at.,

273

4 kb 23 9.4 6.5

---

4.4

-

induced upon differentiation of erythroid cells, but is also found in genes expressed in other differentiated cells like muscle or lymphoid cells (Stuve and Meyers, 1990). Thus flDRE might be responsible for the induction of CLPII and ANXII expression during certain cell differentiation processes, e.g. upon the N G F - i n d u c e d differentiation of the p h e o c h r o m o c y t o m a cell line PC12 ( M a s i a k o w s k i and Shooter, 1988; Schlaepfer and Haigler, 1990).

ACKNOWLEDGEMENTS 2.3

2.0---"

1.3

W e thank Dr. Kiaus W e b e r for support and stimulating discussions. This study w a s supported in part by a grant from the Bundesministerium fur Forschung und Technologic ( B M F T ) .

1.0 REFERENCES

0.88 - Fig. 4. Genomic Southern blot of humat~ DNA probed with the human CLPII eDNA. 10 Fg of DNA from the human myeloblastic cell line HL60 were digested to completion with different restriction enzymes. The DNA fragments were separated on an 0.87~, agarose gel in I/2xTBE and transferred to a GeneScreenPlus membrane (Dupont) according to the manufacturer's instructions. Digestion was carried out with: BamHl (lane 1); Hindlll (lane 2); EcoRl (lane 3); Sacl (lane 4). The immobilized DNA was hybridized in 5 x SSC/50 mM Tris.HCI pH 7.5/1 × Denhardt's/50?o formamide/0.1 mg per ml of denatured salmon sperm DNA at 42: C with I x l0 s cpm/ml of the radiolabelled EcoRI-Hindlll (nt 1-586) fragment of the human CLPII eDNA (covering exons I and II and most ofexon III). The final wash was performed in 2x SSC/0.1'~o SDS as 65:C. The membrane was subsequently exposed for 24 h to a Kodak X-OMAT film at -70 ° C using an intensifying screen. 1986) are present at nt - 194, - 175 and - 151, respectively (boxed sequences in Fig. 2). However, an a p p a r e n t T A T A box, a C A A T motif a n d possible binding sites for the transcription factor A P 1 can not be identified• A comparison of this region of the CLPI 1 gene with the ANXII promoter was performed to identify c o m m o n sequence motifs, which might mediate the co-regulation of CLP11 a n d ANXII transcription. Interestingly, a sequence motif which occurs twice in the 5 ' - n o n t r a n s c r i b e d region of the CLPII gene (5'-AGGGCAG(G)GC; underlined in Fig. 2) can also be identified in the ANXII promoter (nt - 2 1 6 to - 2 0 6 ; Spano et al., 1990)• The sequence resembles a motif described in the h u m a n S-10013 gene (Allore et al., 1990) and in the fl-globin genes of several species (Stuve and Meyers, 1991). This sequence element, termed "pDRE', seems to be involved in the maximum-level induction of the fl-globinencoding gene in murine erythroleukemia cells, which occurs during differentiation along the p a t h w a y towards erythrocytes, flDRE is not restricted to genes which are

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