RESUMEN
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 Ca(2+) 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.
Asunto(s)
Proteína de Unión a CREB/genética , Metilación de ADN , Homocisteína/farmacología , Proteínas de la Membrana/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , Islas de CpG , Regulación de la Expresión Génica , Humanos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero , Factores de Transcripción/genética , TransfecciónRESUMEN
BACKGROUND: Elevated plasma homocysteine concentrations can influence genomic and gene-specific DNA methylation in peripheral blood cells. The aim of this study was to investigate in patients with alcohol dependence, who show chronically elevated homocysteine levels, whether DNA methylation pattern within the HERP (homocysteine-induced endoplasmic reticulum protein) promoter region and expression of HERP mRNA is altered. METHODS: The HERP mRNA expression level was measured by quantitative PCR in the blood of 66 male alcoholic patients and 55 nondrinking healthy controls. Epigenetic genomic DNA methylation status and HERP promoter methylation were measured with a nonradioactive elongation assay. RESULTS: We observed a significant increase (7.6%) in the HERP promoter DNA methylation in patients with alcohol dependence (t test, t = -2.45, p < 0.02) when compared with healthy controls (80.4%, SD 14.5), which was significantly associated with their elevated homocysteine levels (multiple linear regression, p < 0.007). Furthermore, we found a significantly lower HERP mRNA expression in patients with alcohol dependence (t test, -7.61 DeltaCT; SD 1.87, p < 0.001) when compared with healthy controls (-6.04 DeltaCT; SD 2.41). The lowered HERP mRNA expression in alcoholic patients was best explained by the hypermethylation of the regulatory HERP gene promoter (regression analysis, p = 0.004). CONCLUSIONS: To our knowledge, this is the first study evaluating HERP mRNA expression and its specific gene promoter methylation in alcoholic patients. As hypermethylation of DNA is an important epigenetic factor in the down-regulation of gene expression, and as HERP has been considered to play an essential role within the intracellular defense system, these findings may be useful in the understanding and treatment of different disease conditions associated with alcohol dependence.