HUMAN BETA-MYOSIN HEAVY-CHAIN MESSENGER-RNA PREVALENCE IS INVERSELY RELATED TO THE DEGREE OF METHYLATION OF REGULATORY ELEMENTS

Objective: Methylation of cytosine in CG dinucleotides within regulato ry elements is believed to silence gene expression. These dinucleotide s occur in certain important regulatory elements in the promoter regio n of the human p-myosin heavy chain (P-MHC) gene. We therefore investi gated whether methylation of these elements correlates with P-MHC gene transcription in human 'expressing' (right atrial) and 'non-expressin g' (peripheral blood leucocytes) cells. Methods: We employed 2 techniq ues to assess promoter methylation: (i) analysis of the susceptibility to digestion of a particular CCGG restriction site in the promoter re gion when genomic DNA is cleaved with the restriction endonucleases Ms pI (methylation-insensitive) and HpaII (methylation-sensitive), and (i i) the bisulphite-PCR method to examine in detail the methylation patt erns of 3 important regulatory elements that contain CG dinucleotides. beta-MHC mRNA exprsssion in right atrium and leucocytes was assessed using reverse-transcription-PCR with specific primers that do not dete ct alpha-MHC cDNA. Results: The digestion pattern observed with MspI o r HpaII indicated that the CCGG site was almost completely methylated in leucocytes, but relatively unmethylated in atrial myocardium from t he same patients. When methylation was examined with the bisulphite-PC R method we found a reciprocal relationship between the level of beta- MHC mRNA expression in leucocytes and atrial myocardium and the degree of methylation of CG dinucleotides in the 5' regulatory elements of t he gene. Conclusions: Tissue-specific methylation of the human beta-MH C gene promoter may play a role in determining the pattern of expressi on of this gene. Furthermore, alteration of the level of methylation m ay underlie the changes in transcription of this gene that occur, for example, when atrial or ventricular myocardium hypertrophies. (C) 1998 Elsevier Science B.V. All rights reserved.