Elsevier

Experimental Cell Research

Volume 312, Issue 20, 10 December 2006, Pages 4049-4055
Experimental Cell Research

Research Article
Homocysteine regulates expression of Herp by DNA methylation involving the AARE and CREB binding sites

https://doi.org/10.1016/j.yexcr.2006.09.004Get rights and content

Abstract

Herp (homocysteine-induced endoplasmic reticulum protein) is an ER-resident membrane protein, which has a ubiquitin-like domain at its N-terminus. Expression of Herp protein is up-regulated in response to ER stress, including homocysteine. Herp stabilizes neuronal Ca2+ homeostasis and is involved in improving the balance of the folding capacity and protein loading in the ER. In patients with alcoholism, we observed a significant decrease in Herp mRNA expression, and an increase of Herp promoter DNA methylation, which was associated with elevated homocysteine levels. Therefore, we studied the mechanism of Herp CpG islands regulation by luciferase assays and mRNA analysis in neuronal SH-SY5Y (human neuroblastoma cell line) and HEK 293T (human embryonic kidney 293T) cells. Acute homocysteine treatment caused transient demethylation of the Herp promoter and an increase in Herp mRNA level. Global DNA methylation was increased over the following 48 h period. We identified the transcription factor binding site AARE (amino acid response element) by mutational analysis involved in Herp induction in SH-SY5Y cells, and the more significant role of the CREB binding site (cyclic AMP response element-binding protein) compared to AARE in HEK 293T cells. Stimulation with SAM (S-adenosyl methionine) and homocysteine led to an increase in Herp promoter methylation, which correlated to an acute decrease in luciferase expression in SAM, but not in homocysteine stimulated cells. Complete methylation of the CpG islands resulted in suppressed gene expression.

Introduction

The endoplasmic reticulum (ER) is an intracellular target of homocysteine toxicity [1]. ER stress is a condition in which misfolded proteins accumulate in the ER lumen. Homocysteine enhances the expression of both ER chaperone proteins (e.g. grp78, grp94, gadd153) [2] [3] [4] and the homocysteine-induced unfolded non-chaperone Herp (homocysteine-induced ER protein) in cells [5]. This response results in the inhibition of translation which in turn prevents further accumulation of unfolded proteins; but also increases the expression of proteins involved in polypeptide folding, known as the unfolded protein response (UPR), and results in the destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system. Herp is dually regulated by both the shared (PERK/eIF-2alpha dependent) and the ER stress-specific branches (Ire1/XBP-1 and ATF6 dependent) during UPR activation [6].

Herp is an endoplasmic reticulum (ER)-resident membrane protein, which has a ubiquitin-like domain at its N-terminus. It is also present in neurons in the developing and adult brain. The decrease in Herp protein levels following a lethal ER stress occurs prior to mitochondrial dysfunction and cell death. A role for Herp in regulating neuronal Ca2+ signaling has been suggested [7]. Ca2+ overload inside the cell damages mitochondria and causes neurotoxicity.

Recently, it has been shown that global DNA methylation status in peripheral mononuclear cells correlates with plasma homocysteine concentrations in healthy individuals [8] [9]. Most recently, it was observed that elevated plasma homocysteine levels are associated with an increase of global (genomic) and gene-specific DNA methylation in patients with alcoholism [10] [11].

In a preliminary study, we observed a significant increase in gene-specific DNA methylation within the Herp promoter in patients with alcoholism which significantly correlated with their elevated homocysteine levels [12]. Herp mRNA expression was significantly lowered in these patients, which was correlated to hypermethylation status of regulatory Herp gene promoter CpG islands.

As Herp has been linked to the intracellular defense system, the aim of this study was to further investigate Herp regulation by homocysteine because knowledge of the exact mechanisms may be useful in the understanding and treatment of alcohol associated disease conditions.

Section snippets

Oligonucleotides

Herpmeth-F: 5′-GGTCTGCTAGGATACCCCACTCC-3′;

Herpmeth-R: 5′-GCCTTTTATAGAAGCCCCGCGCC-3′.

HerpmRNA-F: 5′-CACCGCGACTTGGAGCTGAGTGG-3′,

HerpmRNA-R: 5′-TCTGTGGATTCAGCCACCTTGG-3′;

Beta-Actin-F: 5′-CTGGAACGGTGAAGGTGACA-3′,

Beta-Actin-R: 5′-AAGGGACTTCCTGTAACAATGCA-3′.

GL-1: 5′-ATGTATGTTATGGTACTGTAAC-3′;

GL-2: 5′-TTCTTTATGTTTTTGGCGTCTTC-3′.

Herp-AARE-mod-F: 5′-AGACGCGGCGGGTTTCATCAGCCCGTGC-3′,

Herp-AARE-mod-R: 5′-CGGGCTGATGAAACCCGCCGCGTCTGCG-3′,

Herp-CREB-mod-F: 5′-GCGCCTCTGGGTTACGCAGACGCGGCGG-3′,

Herp-CREB-mod-R:

Results

In a preliminary study in patients with alcoholism, we observed a significant decrease in Herp mRNA expression and an increase of Herp promoter DNA methylation, which was associated with elevated homocysteine levels [12]. Therefore, we went on to study the mechanism of Herp CpG islands regulation in neuronal SH-SY5Y and HEK 293T cells. The region containing CpG islands within the promoter was identified by database analysis using the UCSC genome browser (http://genome.ucsc.edu/).

Discussion

By “monitoring” Herp promoter methylation during stimulation with homocysteine in untransfected SH-SY5Y cells, we were able to demonstrate that demethylation induced by homocysteine was accompanied by a distinct increase in Herp mRNA expression after 4 h. After 8 h, expression was reduced again, and the promoter remethylated. At the same time, we found that homocysteine leads to a general increase in total DNA methylation, which blocked gene expression and may itself be involved in setting up a

Acknowledgments

We thank PD Dr. Andreas Henkel, Dr. Hans Klafki and Kathryn Watson for critical review of the manuscript.

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These authors contributed equally to this work.

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