The antithetical regulation of the cardiac MHC genes (1 and 2) is highly coordinated in response to altered thyroid state, diabetes, pressure overload, and during development, yet the mechanism underlying this regulation is poorly understood. In previous studies, we discovered a naturally occurring antisense 2 RNA transcript that starts in the middle of the intergenic spacer (IGS) between the 2 and 1 genes and extends upstream to the 2MHC gene promoter region, fully overlapping the 2 gene. This antisense transcription originating from the IGS was previously proposed to coordinate cardiac MHC gene expression in normal rodent hearts and in response to altered thyroid state diabetes, and pressure overload. Recently, more comprehensive analyses of intergenic RNA via strand specific RT-PCR revealed the existence of intergenic RNA in the sense direction that is transcribed toward the 1MHC basic promoter region and continues through the 1MHC gene (see figure 1). These results (and preliminary results on promoter reporter assays) strongly support the novel concept that the IGS is transcriptionally active in both directions in rodent heart. Transcription of the lower strand, which proceeds upstream toward the 2MHC gene, produces antisense RNA: a process that may interfere with 2 gene transcription. Transcription of the upper strand, which starts from ~2kb upstream from the 1MHC gene TSS and proceeds through the 1 promoter to within the 1 gene: a process that may enhance 1 gene transcription. Thus, we hypothesize that the intergenic bidirectional transcription controls the coordinated antithetical regulation of adjacent 1 and 2 MHC genes. The goal of this proposed research is to examine the in vivo regulation of the bidirectional intergenic transcription in the context of altering the expression of the two adjacent genes on the cardiac MHC gene locus via an epigenetic mechanism which involves DNA methylation, chromatin remodeling and histone modification. For comparative purposes and as part of our approach to understanding the gene regulation on this locus, studies will also involve the atria and slow skeletal muscle. These tissues are unique in that the former expresses predominantly 1, while the latter expresses only traces of 1, with 2 being predominant. Consequently, this research will explore a new area of gene regulation and investigate an intriguing regulatory mechanism involving non-coding intergenic RNA, and epigenetic regulation of the cardiac MHC gene locus via bidirectional intergenic transcription.
