Naturally extended CT · AG repeats increase H-DNA structures and promoter activity in the smooth muscle myosin light chain kinase gene

Simple sequence repeats (SSRs), or microsatellites, are genetic loci where one to six bases are tandemly repeated for various numbers of times. They are ubiquitous in prokaryotes and eukaryotes, and they are present even in the smallest bacterial genomes (13). SSRs are inherently unstable, and one of their interesting characteristics is their mutability. SSRs mutate by inserting or deleting a few repeats, and the mutation rates generally increase in concert with the length of repeat tracts (48). Because of their high mutability, SSRs play a significant role in genome evolution by creating and maintaining quantitative genetic variation (16). Moreover, mutations occurring at SSRs are associated with neurodegenerative diseases (fragile X syndrome, Huntington's disease, and myotonic dystrophy) (32) and some human cancers, such as hereditary nonpolyposis colorectal carcinoma (50). (CT)n · (AG)n dinucleotide sequences are among the most abundant dinucleotide repeats in the mammalian genome (43). These sequences are unique in that they form noncanonical DNA secondary structures at promoter regions of many genes. Along with the trinucleotide (GAA)n · (TTC)n or (CTT)n · (AAG)n repeats, dinucleotide (CT)n · (AG)n sequences can adopt a non-B DNA structure, an intramolecular triplex or H-DNA. These structures form at regions containing homopurine-homopyrimidine sequences, where part of the tract can dissociate into single strands using the energy provided by supercoiling. One of the single strands can then swivel its backbone parallel to the duplex, forming a three-stranded helix and leaving its complementary strand unpaired (14, 45). Naturally occurring repeat sequences capable of adopting H-DNA structures are abundant in mammalian genomes and are found as frequently as 1 in every 50,000 bp in the human genome (37). They are mutagenic in mammalian cells (46) and are preferentially located at promoter regions of many genes, including proto-oncogenes such as human c-myc (8, 18), mouse Ki-ras (29), and human c-Src (33). Because of this preferential distribution, (CT)n · (AG)n repeats have been implicated in the formation of H-DNA structures and the regulation of gene expression. Indeed, (CT)n · (AG)n dinucleotide sequences function as a positive transcription element to stimulate gene expression (23), and deleting these sequences reduced promoter activity of heat shock genes in Drosophila melanogaster (35). Moreover, the trans-acting GAGA factor has been shown to bind dinucleotide repeats and to stimulate promoter activities of heat shock genes in Drosophila melanogaster by establishing nucleosome-free DNase-hypersensitive sites (19, 20). Nucleosome structure and positioning are also critical in promoter and enhancer regions because nucleosomes limit accessibility to regulatory factors (21). The packaging of promoter DNA into nucleosomes has been thought to be a major obstacle to transcription. However, many recent reports have shown that chromatin structure is dynamic (1) and a vital component of transcription (42). The dynamic state of chromatin is regulated partly by posttranslational modifications of histones. Residues with histone tails are subject to diverse posttranslational modifications, including phosphorylation, acetylation, and methylation, which together govern the higher-order structure of chromatin and gene expression. Of the modifications, histone acetylation/deacetylation is highly dynamic (11). Acetylation of conserved lysine residues in histone tails stimulates gene expression by neutralizing positive charges, resulting in unstable histone-histone and histone-DNA interactions that otherwise limit the binding of transcription factors to DNA. While it is well established that acetylation of the core histone plays a fundamental role in transcriptional regulation, there is almost no literature on how histone modifications interact with DNA structure, especially non-B DNA structures like H-DNA. Therefore, we studied the promoter of the smooth muscle MLCK (smMLCK) gene. smMLCK regulates smooth muscle contraction by phosphorylating myosin light chains (5). Myosin light chain phosphorylation also regulates cell motility (49) and a host of other cellular responses, including cell spreading and cytokinesis (15). smMLCK is an important regulatory enzyme in the vasculature, and we recently showed that it plays a central role in hypertension. The expression of smMLCK is developmentally regulated and correlates with the onset of hypertension in spontaneously hypertensive rats (SHR), and inhibiting smMLCK expression or activity prevents the development of hypertension (12). Importantly, the smMLCK promoter in normotensive Wistar-Kyoto (WKY) rats contains a dinucleotide repeat motif consisting of 22 (CT) · (AG) repeats. SHR, in contrast, have six additional repeats that increase promoter activity and responsiveness to serum response factor (12), an important regulator of vascular smooth muscle differentiation (28). Because they only differ by these six (CT) · (AG) repeats, the promoters from SHR and WKY rats provide a unique opportunity to determine the effects of nucleotide repeats on gene structure and promoter activity and to relate those results to an important disease. We report below that the CT · AG repeats in both promoters form H-DNA structures without affecting their ability to be packed into nucleosomes and that the extended repeats in the SHR promoter expand the H-DNA structure and increase histone modifications. This is, to the best of our knowledge, the first report on the role of H-DNA in regulating gene expression in a vascular disorder.