Tài liệu Báo cáo khóa học: Trichostatin A reduces hormone-induced transcription of the MMTV promoter

Trichostatin A reduces hormone-induced transcription of the MMTV

promoter and has pleiotropic effects on its chromatin structure

Carolina A˚ strand1,*, Tomas Klenka1,*, O¨ rjan Wrange1 and Sergey Belikov1,2

1

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden; 2

D.I.Ivanovsky

Institute of Virology, Moscow, Russia

The deacetylase inhibitor trichostatin A (TSA) has long

been used to study the relationship between gene transcrip￾tion and the acetylation status of chromatin. We have

used Xenopus laevis oocytes to study the effects of TSA on

glucocorticoid receptor (GR)-dependent transcription and

we have related these effects to changes in the chromatin

structure of a reporter mouse mammary tumor virus

(MMTV) promoter. We show that TSA induces a low level

of constitutive transcription. This correlates with a change of

acetylation pattern and a more open chromatin structure

over the MMTV chromatin, and with specific acetylation

and remodeling events in the promoter region. Specifically, a

repositioning of initially randomly positioned nucleosomes

along the distal MMTV long terminal repeat is seen. This

nucleosome rearrangement is similar to the translational

nucleosome positioning that occurs upon hormone activa￾tion. We also note a reduced hormone response in the

presence of TSA. TSA effects have for a long time been

associated with transcriptional activation and chromatin

opening through inhibition of the deacetylation of histones.

However, our results and those of others show that TSA￾induced changes in expression and chromatin structure can

be quite different in different promoter contexts and, thus,

the effects of TSA are more complex than previously

believed.

Keywords: MMTV promoter; chromatin structure; tran￾scription; Xenopus oocytes; TSA.

The role of the nucleosome as the fundamental unit of DNA

packaging has long been accepted, but its purely structural

role has been challenged by an increasing body of experi￾mental data [1]. Recent evidence suggests that the organ￾ization of promoters into nucleosome arrays provides

an additional mechanism of gene regulation [2]. In this

study, we have used a promoter from the 5¢-long terminal

repeat (LTR) region of the mouse mammary tumor virus

(MMTV) to correlate chromatin structure and gene activity.

The MMTV-LTR contains potential regulatory elements

which mediate transcription in the presence of glucocorti￾coid ligands and in the presence of androgen, progesterone

and their respective nuclear receptors (Fig. 1A) [3]. Six

translationally positioned nucleosomes (A–F) cover this

region [4], one of which, nucleosome B, covers the DNA

segment around position )60 to )240. This segment

contains four glucocorticoid response elements (GREs)

[4–6]. This whole DNA segment shows increased hyper￾sensitivity to DNase I upon binding of glucocorticoid

receptor (GR) homodimers [4,7,8].

We have used the Xenopus oocyte system to reconstitute

chromatin in vivo using single stranded DNA containing

the MMTV promoter as a template. Single-stranded DNA

reconstitutes chromatin more effectively than double-stran￾ded DNA as the second-strand synthesis is coupled to

chromatin assembly, and thus, seems to mimic the replica￾tion coupled chromatin assembly occurring during S phase

of the cell cycle [9].

While the ordered helical domains in the globular body

of the core histones provide a structure for DNA to wrap

around [10], the N-terminal histone tails have been shown to

protrude through and around the DNA helix in a far less

ordered manner [11]. They harbor positively charged lysine

residues at conserved positions. These lysine residues have

been shown to act as targets for post-translational modifi￾cation [12]. Deletion of H3 and H4 N-terminal tails is a

lethal event in yeast that significantly alters gene regulation,

nucleosome assembly and spacing [13]. It is believed that

reversible modifications of charged residues can alter

chromatin structure by causing changes in the overall

charge of the N-terminal tails, and hence their interactions

with the negatively charged sugar–phosphate DNA back￾bone, or with negatively charged regions located on adjacent

nucleosomes [11]. An alternative view is that the various

chemical modifications of specific amino acids in histones

act as a code by serving as binding sites for various effector

complexes. These complexes can modify the chromatin

structure and hence the expression of a gene [14].

The relationship between the histone acetylation status

of chromatin and transcription has been studied in many

systems using a variety of promoter constructs and native

Correspondence to S. Belikov, Department of Cell and Molecular

Biology, Medical Nobel Institute, Box 285 Karolinska Institute,

SE-171 77 Stockholm, Sweden. Fax: + 46 8 31 35 29,

Tel.: + 46 8 52 48 73, E-mail: [email protected]

Abbreviations: ChIP, chromatin immunoprecipitation; DMS,

dimethylsulphate methylation; GR, glucocorticoid receptor; GRE,

glucocorticoid response element; HAT, histone acetyltransferase;

HDAC, histone deacetylase; LTR, long terminal repeat; MNase,

micrococcal nuclease; MPE, methidiumpropyl-EDTA–Fe(II); NaBu,

sodium butyrate; TSA, trichostatin A; TA, triamcinolone acetonide.

*Note: Both these authors contributed equally to this work.

(Received 26 August 2003, revised 26 January 2004,

accepted 30 January 2004)

Eur.J.Biochem. 271, 1153–1162 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04019.x