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Cardiovascular Research 1998 38(3):736-743; doi:10.1016/S0008-6363(98)00058-3
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Human β-myosin heavy chain mRNA prevalence is inversely related to the degree of methylation of regulatory elements

C.P. Clifford and D.J.R. Nunez*

Section on Clinical Pharmacology, Division of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK

* Corresponding author. Tel.: +44 (181) 383 3219; Fax: +44 (181) 383 2066; E-mail: d.nunez@rpms.ac.uk

Objective: Methylation of cytosine in CG dinucleotides within regulatory elements is believed to silence gene expression. These dinucleotides occur in certain important regulatory elements in the promoter region of the human β-myosin heavy chain (β-MHC) gene. We therefore investigated whether methylation of these elements correlates with β-MHC gene transcription in human ‘expressing’ (right atrial) and ‘non-expressing' (peripheral blood leucocytes) cells. Methods: We employed 2 techniques to assess promoter methylation: (i) analysis of the susceptibility to digestion of a particular CCGG restriction site in the promoter region when genomic DNA is cleaved with the restriction endonucleases MspI (methylation-insensitive) and HpaII (methylation-sensitive), and (ii) the bisulphite-PCR method to examine in detail the methylation patterns of 3 important regulatory elements that contain CG dinucleotides. β-MHC mRNA expression in right atrium and leucocytes was assessed using reverse-transcription-PCR with specific primers that do not detect {alpha}-MHC cDNA. Results: The digestion pattern observed with MspI or HpaII indicated that the CCGG site was almost completely methylated in leucocytes, but relatively unmethylated in atrial myocardium from the same patients. When methylation was examined with the bisulphite-PCR method we found a reciprocal relationship between the level of β-MHC mRNA expression in leucocytes and atrial myocardium and the degree of methylation of CG dinucleotides in the 5' regulatory elements of the gene. Conclusions: Tissue-specific methylation of the human β-MHC gene promoter may play a role in determining the pattern of expression of this gene. Furthermore, alteration of the level of methylation may underlie the changes in transcription of this gene that occur, for example, when atrial or ventricular myocardium hypertrophies.

KEYWORDS Human; Myosin; DNA methylation; Hypertrophic cardiomyopathy; Bisulphite; Gene expression; Heart; RT-PCR


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