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Molecular and Cellular Biology, January 2000, p. 261-272, Vol. 20, No. 1
Center for Blood Research and Department of
Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received 23 July 1999/Returned for modification 1 September
1999/Accepted 23 September 1999
Basic helix-loop-helix (bHLH) proteins perform a wide variety of
biological functions. Most bHLH proteins recognize the consensus DNA
sequence CAN NTG (the E-box consensus sequence
is underlined) but acquire further functional specificity by preferring
distinct internal and flanking bases. In addition, induction of
myogenesis by MyoD-related bHLH proteins depends on myogenic basic
region (BR) and BR-HLH junction residues that are not essential for
binding to a muscle-specific site, implying that their BRs may be
involved in other critical interactions. We have investigated whether
the myogenic residues influence DNA sequence recognition and how MyoD, Twist, and their E2A partner proteins prefer distinct CAN
NTG sites. In MyoD, the myogenic BR residues establish
specificity for particular CAN NTG sites
indirectly, by influencing the conformation through which the BR helix
binds DNA. An analysis of DNA binding by BR and junction mutants
suggests that an appropriate BR-DNA conformation is necessary but not
sufficient for myogenesis, supporting the model that additional
interactions with this region are important. The sequence specificities
of E2A and Twist proteins require the corresponding BR residues. In
addition, mechanisms that position the BR allow E2A to prefer distinct
half-sites as a heterodimer with MyoD or Twist, indicating that the E2A
BR can be directed toward different targets by dimerization with
different partners. Our findings indicate that E2A and its partner bHLH
proteins bind to CAN NTG sites by adopting
particular preferred BR-DNA conformations, from which they derive
differences in sequence recognition that can be important for
functional specificity.
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Establishment of Distinct MyoD, E2A, and Twist DNA
Binding Specificities by Different Basic Region-DNA
Conformations
*
Corresponding author. Mailing address: Center for Blood
Research and Department of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 278-3150. Fax: (617) 278-3131. E-mail: blackwell{at}cbr.med.harvard.edu.
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