Tài liệu Báo cáo khoa học: Characterization of human deoxyribonuclease I gene (DNASE1) promoters

Characterization of human deoxyribonuclease I gene

(DNASE1) promoters reveals the utilization of two

transcription-starting exons and the involvement of Sp1

in its transcriptional regulation

Yoshihiko Kominato1

, Misuzu Ueki2

, Reiko Iida3

, Yasuyuki Kawai4

, Tamiko Nakajima1

, Chikako

Makita1

, Masako Itoi1

, Yutaka Tajima1

, Koichiro Kishi1 and Toshihiro Yasuda2

1 Department of Legal Medicine and Medical Genetics, Gunma University, Japan

2 Division of Medical Genetics and Biochemistry, University of Fukui, Japan

3 Division of Legal Medicine, University of Fukui, Japan

4 Third Division of Internal Medicine, University of Fukui, Japan

Deoxyribonuclease I (DNase I, EC 3.1.21.1) is an

enzyme that preferentially attacks double-stranded

DNA by Ca2+- and Mg2+ ⁄Mn2+-dependent endo￾nucleolytic cleavage to produce oligonucleotides with

5¢-phosphoryl- and 3¢-hydroxy termini [1,2]. DNase I

is considered to play a major role in the digestion of

dietary DNA, because in vertebrates it is secreted by

exocrine ⁄ endocrine glands such as the pancreas and

parotid gland into the alimentary tract [3–5]. However,

the presence of the enzyme in mammalian tissues other

than the digestive organs [6–8] suggested that it might

have other function(s) in vivo; endogenous DNase I

has been regarded as a candidate endonuclease respon￾sible for internucleosomal DNA degradation during

apoptosis [9]. Furthermore, Napirei et al. have shown

that extracellular (serum) DNase I participates in the

chromatin breakdown of necrotic cells, achieved by

its diffusion from the extracellular fluid into the

Keywords

alternative splicing; deoxyribonuclease I;

genes; promoter; Sp1

Correspondence

T. Yasuda, Division of Medical Genetics and

Biochemistry, Faculty of Medical Sciences,

University of Fukui, Eiheiji, Fukui 910-1193,

Japan

Fax: +81 776 61 8149

Tel: +81 776 61 8287

E-mail: [email protected]

Database

The nucleotide sequences reported here

have been submitted to the GenBank/

EMBL/DDBJ Data Bank with accession

numbers AB188151 and AB188152

(Received 22 March 2006, revised 8 May

2006, accepted 15 May 2006)

doi:10.1111/j.1742-4658.2006.05320.x

Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown

to be altered by physiological and ⁄ or pathological processes. However, no

information is available on the regulation of DNase I gene (DNASE1)

expression in vivo or in vitro. We first mapped the transcription start sites

of DNASE1 in human pancreas and in the DNase I-producing human pan￾creatic cancer cell line QGP-1, and revealed a novel site
12 kb upstream

of exon 1, which was previously believed to be the single transcription￾starting exon. This initiation site marks an alternative starting exon,

designated 1a. Exons 1 and 1a were used simultaneously as transcription￾starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and

chromatin immunoprecipitation analysis with QGP-1 cells showed the pro￾moter region of exon 1a in which the Sp1 transcription factor is specifically

involved in promoter activity. This is the first to be identified as a tran￾scription factor responsible for gene expression of vertebrate DNase I

genes. Furthermore, RT-PCR analysis indicated alternative splicing of

human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two tran￾scripts among eight alternative splicing products identified can be transla￾ted to produce intact DNase I protein. These results suggest that human

DNASE1 expression is regulated through the use of alternative promoter

and alternative splicing.

Abbreviations

AMI, acute myocardial infarction; ChIP, chromatin immunoprecipitation; DNase I, deoxyribonuclease I; DNASE1, DNase I gene; EMSA,

electrophoretic mobility shift assay; PCI, percutaneous coronary intervention; SRED, single radial enzyme diffusion.

3094 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS