Tài liệu Báo cáo khoa học: Effect of deletion of the DNase I hypersensitive sites on the

Effect of deletion of the DNase I hypersensitive sites on

the transcription of chicken Ig-b gene and on the

maintenance of active chromatin state in the Ig-b locus

Hiroki Matsudo1

, Kyoichi Osano1

, Hiroshi Arakawa2 and Masao Ono1

1 Department of Life Science, and Frontier Project ‘Life’s Adaptation Strategies to Environmental Changes’, Rikkyo University,

College of Science, Toshima-ku, Tokyo, Japan

2 GSF, Institute for Molecular Radiobiology, Neuherberg-Munich, Germany

In vertebrate cells, chromatin of active or potentially

active genes and flanking regions are characterized by

(a) sensitivity to DNase I [1–6]; (b) the presence of cell

type-specific DNase I hypersensitive sites (DHSs) [7,8];

and (c) core histone modifications such as acetylation

and methylation specific for active chromatin [9–11].

These characteristics have been reported specifically

for loci such as b-globin [1,3,12,13], and Ig-b ⁄ growth

hormone (GH) [14–16]. Thus, it is possible to differen￾tiate between active, or potentially active, and inactive

chromatin states by comparing differences in chroma￾tin sensitivity to nuclease and histone modifications.

However, the mechanism by which the chromatin

structure is modified in order to initiate cell type-speci￾fic gene expression as well as the maintenance of this

active state in differentiated cells remains to be elucida￾ted [17–19]. Along with membrane immunoglobulin

and Ig-a ⁄ mb1, Ig-b is a component of the antigen

receptor complex and belongs to the immunoglobulin

superfamily [20,21]. The Ig-b gene is expressed early in

B cell development [22–24]. The mechanism of B cell￾specific expression of mouse and human Ig-b genes has

been studied mainly in the proximal promoter region,

and several cis-elements and transacting factors have

Keywords

chicken Ig-b gene; DNase I hypersensitive

sites; DT40; histone acetylation;

transcription

Correspondence

M. Ono, Department of Life Science,

College of Science, Rikkyo University,

Toshima-ku, Tokyo 171-8501, Japan

Fax ⁄ Tel: +81 339852387

E-mail: [email protected]

(Received 11 September 2004, revised 3

November 2004, accepted 15 November

2004)

doi:10.1111/j.1742-4658.2004.04482.x

The role of DNase I hypersensitive sites (DHSs) in transcription of the B

cell-specific Ig-b gene and in maintenance of active chromatin state in the

Ig-b locus were examined. A total of 10 DHSs were divided into four

regions, and each region was deleted separately in chicken B lymphocyte￾derived DT40 cells. Deletion of three DHSs located between the Ig-b pro￾moter and its upstream Na channel gene, resulted in the absence of Ig-b

mRNA. Three regions except the region in the Na channel gene were

involved in the transcription of Ig-b gene. The enhancing activity of DHSs

as determined by transient transfection assays did not always correlate with

the effect of DHS deletion on the expression level of Ig-b mRNA. In each

deletion, cells contained the same DHSs as observed in the predeletion

cells, indicating that deleted DHSs did not participate in the maintenance

of DT40-specific DHSs. Enhanced acetylation of H3 and H4 histones at

the Ig-b promoter and at DT40-specific DHSs was observed in cells in

which DHSs between the Na channel gene and Ig-b promoter were deleted;

therefore, these DHSs are prerequisite for transcription of the Ig-b gene

but not required for the maintenance of active chromatin state in the Ig-b

locus. Thus, epigenetic factors required for the maintenance of the active

chromatin state are suggested to reside in other regions than those deleted

in this study.

Abbreviations

ChIP, chromatin immunoprecipitation; DHS, DNase I hypersensitive site; GH, growth hormone; LCR, locus control region; R-PCR, real-time

polymerase chain reaction.

422 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS