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Molecular and Cellular Biology, December 1998, p. 7243-7258, Vol. 18, No. 12
The Heart Institute for Children, Hope
Children's Hospital, Oak Lawn, Illinois 60453, and Department of
Physiology and Biophysics, The University of Illinois, Chicago,
Illinois 606121;
Department of Medicine,
The University of Chicago, Chicago, Illinois
606372; and
New England Baptist Bone
Joint Institute, Beth Israel Deaconess Medical Center, Harvard
Institute of Medicine, Boston, Massachusetts
021153
Received 30 June 1998/Returned for modification 18 August
1998/Accepted 10 September 1998
The expression of the
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Tissue-Restricted Expression of the Cardiac
-Myosin Heavy
Chain Gene Is Controlled by a Downstream Repressor Element
Containing a Palindrome of Two Ets-Binding Sites
-myosin heavy chain (MHC) gene is
restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in -MHC gene
regulation have been identified; however, the mechanism restricting the
expression of this gene to cardiac myocytes has yet to be elucidated.
In this study, we have identified by using sequential deletion mutants
of the rat cardiac -MHC gene a 30-bp purine-rich negative regulatory
(PNR) element located in the first intronic region that appeared to be
essential for the tissue-specific expression of the -MHC gene.
Removal of this element alone elevated (20- to 30-fold) the expression
of the -MHC gene in cardiac myocyte cultures and in heart muscle
directly injected with plasmid DNA. Surprisingly, this deletion also
allowed a significant expression of the -MHC gene in HeLa and other
nonmuscle cells, where it is normally inactive. The PNR element
required upstream sequences of the -MHC gene for negative gene
regulation. By DNase I footprint analysis of the PNR element, a
palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG)
was identified. Furthermore, by analyses of site-specific
base-pair mutation, mobility gel shift competition, and UV
cross-linking, two different Ets-like proteins from cardiac and HeLa
cell nuclear extracts were found to bind to the PNR motif. Moreover,
the activity of the PNR-binding factor was found to be increased two-
to threefold in adult rat hearts subjected to pressure overload
hypertrophy, where the -MHC gene is usually suppressed. These data
demonstrate that the PNR element plays a dual role, both downregulating
the expression of the -MHC gene in cardiac myocytes and silencing
the muscle gene activity in nonmuscle cells. Similar palindromic
Ets-binding motifs are found conserved in the -MHC genes from
different species and in other cardiac myocyte-restricted genes. These
results are the first to reveal a role of the Ets class of proteins in
controlling the tissue-specific expression of a cardiac muscle gene.
*
Corresponding author. Mailing address: Department of
Medicine, MC 5105, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Phone: (773) 702-3546. Fax: (773) 702-9764. E-mail:
mgupta{at}medicine.bsd.uchicago.edu.
Molecular and Cellular Biology, December 1998, p. 7243-7258, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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