Histone deacetylation directs DNA methylation in survivin gene silencing

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Abstract

DNA methylation and histone acetylation are major epigenetic modifications in gene silencing. In our previous research, we found that the methylated oligonucleotide (SurKex) complementary to a region of promoter of survivin could induce DNA methylation in a site-specific manner leading to survivin silencing. Here, we further studied the role of histone acetylation in survivin silencing and the relationship between histone acetylation and DNA methylation.

First we observed the levels of histone H4 and H4K16 acetylation that were decreased after SurKex treatment by using the chromatin immunoprecipitation (ChIP) assay. Next, we investigated the roles of histone acetylation and DNA methylation in survivin silencing after blockade of histone deacetylation with Trichostatin A (TSA). We assessed survivin mRNA expression by RT-PCR, measured survivin promoter methylation by bisulfite sequencing and examined the level of histone acetylation by the ChIP assay. The results showed that histone deacetylation blocked by TSA reversed the effects of SurKex on inhibiting the expression of survivin mRNA, inducing a site-specific methylation on survivin promoter and decreasing the level of histone acetylation. Finally, we examined the role of histone acetylation in the expression of DNA methyltransferase 1 (DNMT1) mRNA. The results showed that histone deacetylation blocked by TSA reversed the increasing effect of histone deacetylation on the expression of survivin mRNA. This study suggests that histone deacetylation guides SurKex-induced DNA methylation in survivin silencing possibly through increasing the expression of DNMT1 mRNA.

Research highlights

Histone acetylation modification is the upriver event in survivin silencing. ► Histone deacetylation directs SurKex-induced DNA methylation in survivin silencing. ► Histone deacetylation guides DNA methylation possibly through increasing the expression of DNMT1 mRNA.

Introduction

Epigenetic information includes direct modifications on DNA or chromatin histones. DNA methylation and histone acetylation are two main reversible epigenetic modifications that may affect the structure and function of chromatin [1]. DNA methylation normally occurs on cytosine at CpG dinucleotides across the human genome and is associated with gene silencing. For example, DNA methylation is involved in X chromosome inactivation in females, DNA imprinting, and events involved in monoallelic gene expression. It also contributes to genome stability by preventing translocations of repetitive and transposable sequences, and likely plays a dynamic role in development [2], [3].

Histone hyperacetylation is generally associated with chromatin decondensation, which increases the accessibility of DNA to binding proteins as well as transcriptional activity, whereas histone hypoacetylation contributes to chromatin condensation and transcriptional repression [4], [5]. The N-terminal tails of the core histones are essential for condensation of chromatin and are involved in the genesis of chromatin structural states. Of the histone tails, the histone H4 tails have a prominent role in compacting the 30 nm fiber. An in vitro study demonstrated that K16 acetylation of H4 prevented the formation of the 30 nm chromatin fiber [6], [7]. Acetylation of histone H4K16 and loss or reduction of linker histones result in a decondensed chromatin fiber [8].

It is well established that both DNA methylation and histone modifications are strongly implicated in the process of gene silencing. However, there is no consensus on which epigenetic mechanism initiates and steers this communication. There is a heated debate about which event would be a dominant event in gene silencing. Some experiments supported that DNA methylation guide histone modifications [9], [10], [11], [12], however, other studies show contradictory evidence. Ou et al. [13] demonstrated that increase in histone acetylation by Trichostatin A (TSA) was associated with a significant decrease in global methylation. Januchowski et al. [14] confirmed that histone deacetylation affected DNA methylation by down regulation of DNA methyltransferase 1 (DNMT1) in Jurkat T cells. Smallwood et al. [15] considered histone methylation as initiated epigenetic modifications in survivin silencing. They found that the methylation of histone H3K9 created a binding platform for HP1, allowing DNMT1 to interact with HP1, resulting in increased DNA methylation on DNA and chromatin templates. Wu et al. [16] found that blockade of histone deacetylation by a depsipeptide actively silenced genes such as p16, SALL3 and GATA4 in different human cancer cell lines through decreasing methylation of both CpG and H3K9 on their promoters. These experiments support the theory that histone modifications guide DNA methylation.

SurKex is a methylated oligonucleotide complementary to a region of human survivin promoter (sequence from 2766 to 2787, genbank accession no. NC_000017), designed according to the method from Yao et al. [17]. In our previous study, we demonstrated that SurKex inhibited not only survivin expression in vitro, but also the growth of NSCLC tumors in nude mice as well. Moreover we confirmed that SurKex led to site- and sequence-specific methylation of the survivin promoter by 50% at 5 μM [18]. In this study, we attempted to reveal the relationship between DNA methylation and histone acetylation in SurKex-induced survivin by using the histone deacetylase inhibitor TSA.

Section snippets

Materials

SurKex sequences were designed as follows: AmCGGGTCCCGmCG ATTCAAATCT, which is complementary to a region of the human survivin promoter (sequence from 2766 to 2787, genbank accession no. NC_000017). Random is a control cognate of the SurKex oligonucleotide, consisting of a methylated 22-mer oligonucleotide with a random sequence (ATGCTmCGGAACCTTTmCGCAGGA), which is not related to survivin, but it contains the same number of m5C residues as SurKex. TSA (Trichostatin A), a histone deacetylase

Effects of SurKex on histone acetylation

Histone acetylation is tightly related to gene expression. Histone hyperacetylation is generally associated with chromatin decondensation, allowing DNA to be accessible to binding proteins and thus increased transcriptional activity, whereas histone hypoacetylation contributes to chromatin condensation and transcriptional repression. In order to determine the changes of histone acetylation of the survivin promoter after SurKex treatment, the ChIP assay was used. Compared with lipofectamin 2000

Discussion

Methylation of the cytosine in CpG dinucleotides has emerged as an important epigenetic modification that regulates gene transcription [19]. In general, promoter DNA methylation is correlated with gene repression. When a gene is hypermethylated, especially in its promoter region, transcription is usually diminished [20]. In view of this, oncogenes could be silenced by inducing methylation of their promoter regions.

Yao et al. [17] established a novel method, by which expression of a given gene

Acknowledgments

This work was supported by Grants (03JC14049 and 034319209) from Shanghai Science and Technology Administration and a Grant (2009CB521900 PI: Dr. Ding-Feng Su) from National Basic Research “973” Program, Ministry of Science and Technology, China.

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