Naturally extended CT . AG repeats increase H-DNA structures and promoter activity in the smooth muscle myosin light chain kinase gene.
Naturally occurring repeat sequences capable of adopting H-DNA structures are abundant in promoters of disease-related genes. In support of this, we found (CT)(22) . (AG)(22) repeats in the promoter of smooth muscle myosin light chain kinase (smMLCK), a key regulator of vascular smooth muscle function. We also found an insertion mutation that adds another six pairs of CT . AG repeats and increases smMLCK promoter activity in spontaneously hypertensive rats (SHR). Therefore, we used the smMLCK promoters from normotensive and hypertensive rats as a model system to determine how CT . AG repeats form H-DNA, an intramolecular triplex, and regulate promoter activity. High-resolution mapping with a chemical probe selective for H-DNA showed that the CT . AG repeats adopt H-DNA structures at a neutral pH. Importantly, the SHR promoter forms longer H-DNA structures than the promoter from normotensive rats. Reconstituting nucleosomes on the promoters, in vitro, showed no difference in nucleosome positioning between the two promoters. However, chromatin immunoprecipitation analyses revealed that histone acetylations are greater in the hypertensive promoter. Thus, our findings suggest that the extended CT . AG repeats in the SHR promoter increase H-DNA structures, histone modifications, and promoter activity of the smMLCK, perhaps contributing to vascular disorders in hypertension.
Han
Yoo-Jeong
YJ
Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott, Chicago, IL 60612, USA.
de Lanerolle
Primal
P
D000107:Acetylation; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D015342:DNA Probes / Q000235:genetics; D006657:Histones / Q000378:metabolism; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000235:genetics* / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D011918:Rats, Inbred SHR; D012680:Sensitivity and Specificity; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D015342:DNA Probes; D006657:Histones; D009707:Nucleosomes; C051802:triplex DNA; D004247:DNA; D009219:Myosin-Light-Chain Kinase; D015719:Single-Strand Specific DNA and RNA Endonucleases