The closely linked (863 bp), divergently arranged mouse myotonic dystrophy kinase binding protein (Mkbp)/HspB2 and small heat shock protein (shsp)/alphaB-crystallin genes have different patterns of tissue-specific expression. We showed previously that an intergenic enhancing region (-436/-257 relative to alphaB-crystallin transcription start site) selectively activates the alphaB-crystallin promoter in an orientation-dependent manner (Swamynathan, S.K. and J. Piatigorsky 2002. J. Biol. Chem. 277:49700-6). Here we show that cis-elements alphaBE1 (-420/-396) and alphaBE3 (-320/-300) functionally interact with glucocorticoid receptor (GR) and Sp1, respectively, both in vitro and in vivo. alphaBE1:GR regulates both the HspB2 and alphaB-crystallin promoters, while alphaBE3:Sp1 selectively regulates the alphaB-crystallin promoter, as judged by mutagenesis and co-transfection tests. Enhancer blocking assays indicate that the -836/-622 fragment can act as a negative regulator in transfection tests, raising the possibility that it contributes to the differential expression of the proximal HspB2 promoter and distal alphaB-crystallin promoter. Finally, experiments utilizing transiently transfected cells and transgenic mice show that two conserved E-box elements (-726/-721 and -702/-697) bind nuclear proteins and differentially regulate the HspB2 and alphaB-crystallin promoters in a tissue-specific manner. Taken together, our results indicate that the linked, differentially expressed HspB2 and alphaB-crystallin genes have evolved shared and promoter-preferred cis-control elements within the intergenic sequence. The context-dependency of cis-elements provides multiple opportunities for evolutionary novelty by small sequence changes.