Fig. 7. Twist dimerizes with Da and represses muscle transcription in vitro. (A)
Mobility shift analysis using rho CATATG (Ip et al.,
1992) (lanes 2, 3, 4) and
CACCTG E boxes (lanes 5, 6, 7) performed with Twist (lanes 2, 5), Da
(lanes 4, 7), and Twist + Da (lanes 3, 6). Equal amounts of protein are used
in each lane. Heterodimer shifts are marked with an asterisk. The Da homodimer
shows binding to class A sites (CACCTG), whereas the Twist homodimer
shows a marked affinity for a class B site (CATATG) (Ohsako et al.,
1994). Cotranslation of both
proteins and incubation with class A or B DNA sites results in formation of an
intermediate shift between the Da homodimer and the Twist homodimer, which
corresponds to the Twist/Da heterodimer (*). The apparent
Kds for Twi/Da and Da/Da on the rho E box are
1.6x10-6 and 3.2x10-6, respectively. (B) Da
represses Twist activation of the 175 bp Mef2 enhancer in SL2 cells.
SL2 cells were transfected with indicated amounts of actin-lacZ
plasmid, the 175 bp Mef2 enhancer-luciferase reporter plasmid,
equimolar amounts of either Twist, Da, Twi+Da, or Twist-Da linked dimer
expression vectors and carrier DNA. The values are expressed relative to
controls (see Methods). Error bars represent the standard error of means of
triplicated experiments. Transfection of Twist alone results in reporter gene
activation. The same molar amount of transfected Da results in little
activation. Cotransfection of equimolar amounts of Twist and Da results in
reduction of reporter gene activity. Transfection of Twist-Da linked dimers
led to little reporter gene activity. (C) Schematic representation of the
tethered Twist-Da protein. DNA segments encoding Twist and Da are joined in
frame via a flexible linker. The boxes denote the basic helix-loop-helix
domain. Linker sequence is given in the single letter amino acid code. Numbers
below the line represent the nucleotide sequence of twist and
da cDNAs (Caudy et al., 1988; Cronmiller and Cline, 1988). (D)
Mobility shift analysis with the tethered Twi-Da heterodimer. In vitro
translated products are assayed for binding to class A E box.
Twist-Daughterless linked dimers (lane 2 and 4) have the same DNA binding
specificity as Twist/Da unlinked heterodimers (*, lane 1).