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Nucleic Acids Research, 1992, Vol. 20, No. 19 5105-5113
© 1992

MOLECULAR BIOLOGY

Different E-box regulatory sequences are functionally distinct when placed within the context of the troponin I enhancer

Katherine E. Yutzey+ and Stephen F. Konieczny*

Department of Biology Sciences, Purdue University West Lafayette, IN 47907, USA

* To whom correspondence should be addressed

Received June 29, 1992. Revised September 9, 1992. Accepted September 9, 1992.

Basic hellx-loop-hellx (bHLH) regulatory proteins are known to bind to a single DNA consensus sequence referred to as an E-box. The E-box is present in the regulatory elements of many developmentally controlled genes, including most muscle-specific genes such as troponin I (Tnl). Although the E-box consensus is minimally defined as CANNTG, the adjacent nucleotides of functional E-boxes are variable for genes regulated by the bHLH proteins. In order to examine how E-box regulatory regions containing different internal and flanking nucleotides function when placed within the context of a single regulatory element, the E-box region (14 bp) present within the Tnl enhancer was substituted with the corresponding E-box sequences derived from the muscle-specific M-creatine kinase (MCK) and cardiac {alpha}-actin regulatory elements as well as from the immunoglobulin {chi} (Ig{chi}) enhancer. Within the Tnl enhancer, the E-box sequence derived from cardiac {alpha}-actln was inactive whereas the corresponding sequence from the MCK right E-box efficiently restored wild-type enhancer activity in muscle cells. Intermediate levels of gene activity were observed for Tnl enhancers containing E-boxes derived from the MCK left E-box site or from the lg{chi}E2 E-box. DNA binding studies of MyoD: E12 protein complexes with each substituted Tnl enhancer confirmed that DNA binding activity in vitro mimics the relative strength of the enhancers in vivo. These studies demonstrate that the specific nucleotide composition of individual E-boxes, which are contained within the regulatory elements of most if not all muscle-specific genes, contributes to the complex regulatory mechanisms governing bHLH-medlated gene expression.

+ Present address: Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021, USA


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