The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episomebased eNOS promoters, we examined the methylation status of 5-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.

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Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long-range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence-structure-fitness relationships in other common motifs.