The 5′ sequence of human Factor XII gene contains transcription regulatory elements typical of liver specific, estrogen-modulated genes

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Biochimica et Biophysica Acta, 1172 (1993) 197-199 Elsevier Science Publishers B.V.

BBAEXP9~56

Short Sequence-Paper

The 5' sequence of human Factor XII gene contains transcription regulatory elements typical of liver specific, estrogen-modulated genes F. Citarella, S. Misiti, A. Felici, A. Aiuti, C. La Porta and A. Fantoni Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Universith di Roma 'La Sapienza ', Roma (Italy) (Received 13 October 1992)

Key words: Factor XII; Liver; Estrogen; Fibrinolysis; Promoter

The human Factor XII gene codes for a serine proteinase synthesized in liver that activates both the coagulation and the fibrinolytic cascades. The nucleotide sequence analysis of a HincII-HincII 3129 bp fragment was performed showing that the FXII promoter region contains neither CAAT and TATA regulatory elements, nor GC islands, but revealing the presence of two tandemly repeated sequences in opposite orientation, two LFoA1 elements typical of the liver specific genes and one estrogen responsive element, that substantiates the observation of Factor XII gene modulation by estrogens.

Human coagulation Factor XII (FXII) is a serine proteinase zymogen secreted by the liver and present in plasma ( 2 5 - 3 0 / z g / m l ) . Factor XII gene is localized on chromosome 5 [1], at chromosome map position 5q33-qter [2]. The gene measures about 12 kb and is organized in 14 exons and 13 introns [3]. The complete nucleotide structure of the coding sequence was reported by Cool et al. [4] and completed by Tripodi et al. [5]. The primary structure of FXII has revealed [6] that FXII has a structural organization similar to that of other serine proteinases involved in blood coagulation and in fibrinolysis. The function of FXII is not clear and indeed, FXII deficiency is not associated with bleeding disorders. However, active FXII may have an important role in the pathophysiology of inflammation [7] as it is involved in kinin formation, complement activation and fibrinolysis. Recent evidence indicates that FXII concentration in plasma is enhanced by estrogens [8]. Information relevant to FXII role in human physiology may be obtained either from the study of properly engineered, recombinant FXII proteins [9] or else,

Correspondence to: F. Citarella, Dipartimento di Biopatologia Umana, Sezione di Biologia Cellulare, Viale Regina Elena 324, 00161 Roma, Italy. The sequence data reported in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession number X67330.

from the regulatory pattern of FXII gene transcription. On this purpose we have isolated a 3174 bp fragment of human genomic DNA at the 5' of the FXII transcription unit and we have analyzed its nucleotide sequence. A human genomic library in pCOS 2EMBL cosmid vectors was screened with a 280 bp fragment at the 5' of the FXII cDNA, starting with the coding position 1 [5]. From HincII digests of a positive cosmid a 3.17 kb fragment was subcloned in pBluescript (clone 5' FXII 3174) and a restriction map of the region was derived. This fragment (3.174 kb) was subdivided by PvulI digestion into four pieces of which two of 1810 and 1290 bp were subcloned in pBlueScript (clone 5' FXII1810 and 5' FXII-1290). Clones FXII 3174, 5'FXII1810 and 5' FXII-1290 were used to derive the nucleotide sequence of the FXII 5' region by the dideoxy chain-termination method (see Fig. 1). At least 30% of the sequence analysis was performed on both strands. The sequenced region is comprehensive of 3077 bp 5' to first transcription initiation site, and 97 bp of the transcription unit. As it does not contain any valid open reading frame, control elements encountered in this region may be relevant to FXII gene regulation. By SI mapping and primer extension experiments [3] five sites of transcription initiation were identified at positions +1, +14, +22, +27, +33, where transcription initiation ranges from 49 to 17 bp prior to AUG. This indicates that FXII m R N A has a very short non translated 5' flanking region.

198 The region at the 5' of the FXII gene seems to be particularly rich in Alu sequences (see Fig. 2). In addition to two Alu elements described [3] within the first intron of FXII transcription unit, we have found in the region - 1 0 9 4 / - 3 5 6 two tandemly repeated, closely spaced and highly homologous Alu sequences. Two additional Alu sequences, both in opposite orien-

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tation to the first two, are present at positions - 2 7 7 4 and - 2126. TATA and CAAT promoter sequences are not present in close proximity to transcription initiation sites, which would inserts FXII gene in the TATA less group of eukaryotic genes. FXII promoter contains at position - 6 2 3 , - 8 8 5 , and - 9 8 2 three G G C G G G regula-

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0 0 0 1 - AACAGC'i~TC ACGTCAATGA ~ A A A C C

100 I

AGTTAC~IT~" GAAACCAGTG GTTGGAAAGG TGCCCCTCCT CTCCTGCCCT CCTCTTTC'I~ TG'I"TTC%-"F~

0 1 0 1 - CCAAGAAGTC TTCCACAGCC TGCCTCTCTT CCAGCAGAAT TAATAAATTG CTACACTCAG T C T ' I ~ A ~

CCATAAA~TA CTTTGTATAA CGTTAGGACT

0 2 0 1 - TTGATAATCG TAAAGTGACA GAAGTCACTC AAATTGGTTT ATAACTGAAA GTGACAGAAG TCCACTCAAA TTGG'FXTx'AT TGGCTCATAT AAC'I~AGAAG 0 3 0 1 - TCTA'I-L-I-~-~-~- "FL'FI'F~'F~G AGATACAGTC TCAGTCTGCT TCCCAGACTG GAGTGCAGTG GCACAATCAC GGC'I~ACAGC A'~TI';'GGACC

TCTGGGCTCA

0 4 0 1 - AGCAATCCTC CTGCCTCAGA CTCACAAGTA GCCGGAACTA CAGGCACGCG CCACCATGCC TGGCTAATGT TAAA'~'FI.Ti'~" TGGAGAGATG GGATCTCACT 0501- A ~ C T A

CATCAGTCTG AACTCC'I~GC TCAAGTGATC CTCAAGTGAT CCCCAGCCTC CCAGAGTGTT GGCTAATTAC AGGCGTGAGG CACTGTGCCT

0 6 0 1 - AGCCATAACT GACCAAGTCT TAAGGATACT TCTAG'~TFrA GGATGTAGGC TCTCAAAGTC ATGAGAAATC AATCCATCAG TACTAGTTCT CAAATGTCGT 0 7 0 1 - GTAACAAATC ACCCTAAC'I~ GTGGC'I~AAA GCAACAACAT "~/'AATGATGA 'FI'TATCACAG TTTC'I~A'I'GG TCAG'FFF~'GC C'FI'GAGGTCT TTCATAAGAT 0801-

TGCAAGCCAA

ATGTCAGCGA

GGGCTGCAGG

GGCTGCATGT

GTGTCCTCAC

AACACAGTGG

CTGGCTTCCT

CCAGAGTGAG

CAACCTAAGA

GACCGAGGCA

0901-

GAAACTGCAA

TGTC'FFF~'AT G A C C T G G G C T

TGGAAGTCCA

ACACCCTCAC

TTCTGCCATA

'FFX'~,TFF~.T~" T T T G A G A C A G

TCCTGTCGCC

CAGGTCGGAG

1001-

TGCAATGGCA

C A A ~ T

TCCACTTCCT

GGTTCAAGCG

A~CTGC

CTCAGCCTCA

CGGTAGCTGG

GATTACAGGC

ACACGCCACT

1101-

ACGCCCGGCT

AA'x-FITz<~TA 'FFFI"I~ATAG ATGATC, GC,G T T T G G C C A A C A

CGTI'GGCCAG

GCCGGTCq~'G

AACTCCTGAC

CTAGAGTGAT

CTGCCTGCCT

1201-

TGGCATCCCA

AAGTGCTGGG

A'FI'ACAGGTG T G A G C C A C C A

CACCCAGCCA

CTTCTGTCAT

ATTCTGATGG

ACACACAGAA

CAGCCCTAAC

TCAATGTGGG

1301- A A G A C A C C A T

ACAAGGGCAT

GAATCCTAGG

TTGCTGAGGA

TCAATGGGGG

CTGTCTTTGA

GGCTGTCTAC

CACAGCATCT

TTCCATCCCT

TCCTGCCCTG

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~

'FFFI'CCTATG TGTAC, C,C T T C

ATTCTCAAAC

AGGCCCTCCC

TAGAGAGTGA

CAAAGGTGAT

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~AGTAAC

CCCAGTGCAA

CACTACAACC

C'~'FF~'FI~CT T T C C C A A A A G

CATCAATGTG

TTCAGACCCA

CATGCTCTGT

'FI'CTGGCAAA A G T C C C A A G C

TAGCAC'FI'TA A T T G G C C T A A

ATTGTGTATA

TGCTTATCTC

1601- T G A A C C A A T C

ACTGTGGA'FI ~ A G A G A T G T C A

TGCTCTGATT

GACCAGACCT

AGGCCACATC

TCTAGCCCTA

GCTCTGAGGG

TAGAGTTGGC

AGCACTAGAG

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CCCATGGAAG

AAGTAAGAGA

GGAGTCGTTG

CTAAAGGAAA

AATCAAAGTG

TCATTACCGA

ACCAGGACAG

ATGCTGGGCA

GCACATGTGC

ACCCCGTC'FI ~

1801- C T T C T C A T G T

TCCAGCTGCA

CATCTTAGTG

CCCCTTC.GTT

TAGCAC'FFFI" C T C A T T A A A T

CATTTGCTTT

CTTGCCTCAC

TTCCTGTC, G T

TGGTAGAATG

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CTAAGATGGC

CCCAAGATCT

CTACCCCTGG

TG~ACA

CCTCCCAGTT

ATTCTGTCAA

ACATGAATGT

AGATGCTTCT

GTGAAAGAAT

TTGCACATGT

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AATTTAAGTC

CAAATTGTTT

GACCTTAAAA

TAAGGAGAAT

GGCAGGGCCA

~ATGGTGG

CTCATACCTG

TAACCCAGCA

C ~ A G G

CCAAGGCGGG

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CAGATCACGA

GGTCAGGAGA

TCGAGACCAT

CCTGC, C'~AAC A C A G T G A A A C

CCCATCTCTA

CTAAAAATAC

AAAAAAATTA

GCTC, G G C G T G

GTGGCGGGTG

2201-

CCTGTATCCC

AGCTACCCAG

GAGGCTGAGG

CAGGAGAATG

GCGTGAACCC

GGGAGGCGTA

GC~F?GCAGTG

AGCCAAGATC

GTGCCACTGC

ACTCCAGCCT

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GGGTGACAGA

C,C C A G A C T C T

GTCTCAAAAA

AAAAAAAAAA

AAAAAAAGGA

GAATGGCTTT

GGTGGGCCTG

ACCTAGTCAG

GTCAGTTCTT

AAAAGGCGAC

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ACATGGCCCG

GTGCAGTGGC

TCAGGCCTGT

AATCCCAGCA

CTTTGGGAGG

CCGAGGCGGG

TGGATCACGA

GGTCAGGAGA

TCGAGACCAT

C ~ A A C

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ATGGTGAAAC

CCCGTCTCTA

CTAAAAAGAC

AAAAAA'I~I~AG C T G G G C G T G G

TGGTGGGCTC

CTOTAGTCCC

AGCTACTCGG

GAGGCTGAGG

CAGGAGAATG

TCCAGCCTC, G C,C G A C A G A G C

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GCGTGAACCC

GGAGGCGGAG

CTTC,C A G T G A

GCGGAGTTGC

C,C C A C T G C A C

GAGACTCCGT

CTCAAAAAAA

AAAAAAAAAA

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AAAAAAAAAA

AGAAAATTAA

AAGTGGGTAT

TGTTGTAAGA

TGCTGAGT'I~ ~ A T G G T A G T T T

GTTACATGAC

AATAGAAAAT

GAACACACTT

CACAGTGGAC

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TCCAAGATCC

CCATGATCTT

TGATCTCCTT

AACCTCCTGA

TCTCCACAGG

ACCCAGAGCA

TAAGAATGTC

CCTTCTTCTG

CTTCCAGTCC

CACTATCTAG

2901-

AAAAGAGAGG

AGGAGCCCAG

CTCTTCAT'FI" C A C C C C C A C C

CACAAACTCC

CAACT'FI'CCG G C C C T C A A G G

GGTGACCAAG

GAAGTTGCTC

C A ~ T T

3001-

TCCACAAACA

GCCTG~CC

CACCAGG~

CTTGACCAAT

CTCTATTTCC

GCCAGTCCTA

TTGATCTGGA

CTCCTGGATA

CCAACGGACG

GACGCCATG

3 I0 i- GC,C A G C T G G A

AGGAGGGCAG

AAGACCTTTG

Fig. h Nucleotide sequence of 3.129 kb 5' flanking region of the human FXII gene.

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[~Nincll-~ull

t il

~

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LF-A1

+50

~incll

ERE ~-A1 ATG

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Fig. 2. Sequencing strategy and regulatory elements of the human FXII promoter region. Numbers indicate the nucleotide functions from the transcription initiation site. LFA1, ERE, ALU and ATG are positions of elements reported in text. Hincll and P u v l I are restriction sites indicated in text.

tory elements which are recognized by Spl factors, as typical of TATA less housekeeping genes, constitutively expressed in all cells. Though, these elements seem to be too remote from each other to be able to exert functions typical of Spl factors. For the absence of TATA and CAAT regulatory elements and for the presence of multiple initiating sites, FXII gene is very similar to coagulation factors VIII and IX, to protein C and prothrombin. One putative control element in common with a human coagulation factor, the Factor IX silencer [10] (CCTCTCCTG) is present at position -3011. Other putative control elements recognized in the FXII promoter region seem to indicate that FXII gene is liver specific and estrogen regulated. Indeed, at position + 30 and -2693 has been identified the consensus sequence TGGACC recognized by the liver specific transcription factor LF-AI [11]. The sequence AGGGCAGCTTGACC, present at position - 4 4 / 31, is homologous except for one nucleotide to the

palindrome AGGTCANNNTGACC reported as ERE, estrogen responsive element [12]; in addition the sequence AGGTCA, homologous to the first hemisite of ERE, is found at positions -1314, - 9 6 8 and -608. This study was supported in part by the Consiglio Nazionale deIle Ricerche, progetto finalizzato Biotecnologia e Biostrutturazione e Progetto finalizzato Ingegneria Genetica, by the Ministero dell'Universit~ e della Ricerca Scientifica, by the Fondazione Istituto Pasteur-Fondazione Cenci Bolognetti and by the Ministero della SanitY, progetto AIDS. References 1 Citarella, F., Tripodi, M., Fantoni, A., Bernardi, F., Patracchini, P., Romeo, G. and Rocchi, M. (1988) Human Genet. 80, 397-398. 2 Royle, N.J., Nigli, M., Cool, D., MacGillivray, R.T. and Hamerton, J.L. (1988) Somat. Cell. Mol. Genet. 12, 217-221. 3 Cool, D.E. and MacGillivray, R.T.A. (1987) J. Biol. Chem. 262, 13662-13673. 4 Cool, D.E., Edgell, C.J.S., Louie, G.V., Zoller, M.J., Brayer, G.D. and MacGilliwray, R.T.A. (1985)J. Biol. Chem. 260, 13666-13676. 5 Tripodi, M., Citarella, F., Guida, S., Galeffi, P., Fantoni, A. and Cortese, R. 11986) Nucleic Acids Res. 14, 3146. 6 McMullen, B.A. and Fujikawa, K. 119831 J. Biol. Chem. 260, 5328-5341. 7 Colman, R.W. (1984) J. Clin. Invest. 73, 1249-1253. 8 Gordon, E.M., Johnson, T.R., Ramos, L.P. and Schmeider-Sapiro, K.T. 11991)J. Lab. Clin. Meal. 117~ 353-358. 9 Citarella, F., Aiuti, A., La Porta, C., Russo, G., Pietropaolo, C., Rinaldi, M. and Fantoni, A. (1992) Eur. J. Biochem. 208, 23-31/. 10 Salier, J.P., Hirosawa, S. and Kurachi, K. (1990) J. Biol. Chem. 265, 7062-7068. 11 Ramji, D.P., Tadros, M.N., Harden, E.M. and Cortese, R. (1991) Nucleic Acids Res. 19, 1139-1146. 12 Evans, R.M. (1988) Science 240, 889-895.