Nucleic Acids Research, Vol 25, Issue 15 3026-3033, Copyright © 1997 by Oxford University Press
D Porte, P Oertel-Buchheit, M John, M Granger-Schnarr and M Schnarr
The mammalian Fos and Fos-related proteins are unable to form homodimers
and to bind DNA in the absence of a second protein, like c- Jun for
example. In order to study the implications of hydrophobic point mutations
in the c-Fox leucine zipper on DNA binding of the entire c-Fos protein, we
have constructed and purified a set of Fos mutant proteins harboring one or
several isoleucine or leucine residues in the five Fos zipper a positions.
We show that a single point mutation in the hydrophobic interface of the
c-Fos leucine zipper enables the c-Fos mutant protein to bind specifically
to an oligonucleotide duplex harboring the TRE consensus sequence
TGA(C/G)TCA. This point mutation (Thr196-->Ile) is situated in the a
position of the second heptade (a2) of the Fos zipper. The introduction of
additional isoleucine residues in the other a positions progressively
increases the DNA binding affinity of these homodimerizing Fos zipper
variants. Heterodimerization of these c-Fos variants with c-Jun reveals a
complex behavior, in that the DNA binding affinity of these heterodimers
does not simply increase with the number of isoleucine side chains in
position a. For example, a c-Fos variant harboring a wild-type Thr in
position a1 aad Ile in the four other a positions (c-Fos4I) interacts more
tightly with c-Jun than a variant harboring Ile in all five a positions
(c-Fos5I). The same holds true for the corresponding leucine variants,
suggesting that the wild-type a1 residue of the Fox zipper (Thr162) is
thermodynamically relevant for Fos-Jun heterodimer formations and DNA
binding. The c-Fos4I variant forms heterodimers with c-Jun slightly better
than the wild-type zipper protein, suggesting that the driving force for
Fos-Jun heterodimerization is not the simple fact that the Fos protein is
unable to form homodimers. These c-Fos variants were further tested for
their transactivation properties in F9 and NIH3T3 cells. At low expression
levels the most efficiently homodimerizing variant (c-Fos5I) activates
transcription in F9 cells about 6-fold. However part of this activation may
be due to the formation of heterodimers with a member of the Jun family
(like JunD for example), since a wild type c-Fos expression vector confers
a 3-fold activation under these conditions. In the case of the
homodimerizing c-Fos variants however, this activation is abrogated at
higher expression levels due to a strong inhibition of basal transcription
activity.
ARTICLES
DNA binding and transactivation properties of Fos variants with homodimerization capacity
Institut de Biologie Moleculaire et Cellulaire, UPR 9002 du CNRS, F- 67084 Strasbourg Cedex, France.
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