Naturally Extended CT · AG Repeats Increase H-DNA Structures and Promoter Activity in the Smooth Muscle Myosin Light Chain Kinase Gene

Abstract

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.

ACKNOWLEDGMENTS

We thank Jennifer Cook and Guo-Fu Hu, Harvard Medical School, for advice on the details of the ChIP protocols and for generously providing repeat constructs, respectively, and Randal C. Jaffe, UIC, for generously providing the pGL3 vectors. We also thank our colleague Wilma Hofmann for critically reviewing the manuscript.

This work was supported in part by a grant from the National Science Foundation (0517468) to P.D.L. Y.-J.H. was supported by a postdoctoral fellowship from the American Heart Association (0525686Z).

We have no competing financial interests.