Final Report for Regulation of Embryonic Development in Higher Plants Page: 2 of 6
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John J. Harada Final Report
DE-FG02-03ER15392, Regulation of Embryonic Development in Higher Plants Page 2
subunits do not.
We analyzed CBF complexes containing LEC1 versus NLH3 subunits to understand the
basis for their different functions. We first showed that both types of HAP3 subunits form CBF
complexes in vitro and in yeast. This result suggested that LEC1 and NLH3 subunits are able to
interact with the other CBF subunits, HAP2 and HAP5, to form complexes.
We compared the DNA binding specificity of CBF complexes containing LEC1 and
NLH3 subunits and showed that both the LEC1 CBF and the NLH3 CBF bind DNA sequences
in vitro that correspond to the consensus CBF binding sites in mammals. We compared DNA
binding interactions of the two complexes by measuring the equilibrium dissociation constants
for DNA binding to the complexes. Similar Kd values for LEC1 CBF and the NLH3 CBFs were
measured using two different CCAAT-containing DNA fragment. These experiments suggest
that the LEC 1 CBF and NLH3 CBF do not exhibit significant differences in the specificity or
relative affinity of binding with the CCAAT DNA motif in vitro.
We next explored interactions between the LEC1 CBF and NLH3 CBFs with DNA by
measuring their rate constants for dissociation (koff) in off-time electrophoretic mobility shift
assays experiments. We found that the CCAAT DNA probe was competed much more rapidly
from LEC1-CBF than NLH3-CBF. The calculated koff indicated that, although the DNA binding
affinities of the LEC1 and NLH3 CBFs are similar, the half-life of the LEC1 CBF-DNA complex
is significantly shorter than that of the NLH3-CBF complex.
We analyzed DNA conformational change induced by LEC1 CBF and NLH3 CBF
complexes. We showed previously that the LEC1 B domain contains an Asp residue at position
28 that is essential for LEC1 function and that differs from a conserved Lys residue that is
present in the B domains of NLH3 subunits. This Lys residue lies in the L1 loop of the histone-
fold motif that has been implicated to make contact with DNA and potentially mediate bending
of the DNA. Given differences in the off rates for LEC1 CBF and NLH3 CBFs, we performed
circular permutation assays to determine if the two complexes bend DNA differently. The
experimental data showed that both the LEC1 CBF and NLH3 CBF caused DNA bending,
although no appreciable difference in the bending angle was detected. These results suggest that
LEC1 CBF and NLH3 CBF do not bend DNA to significantly different extents.
One key to resolving questions about functional differences between the LEC1-CBF and
NLH3-CBF complexes is to compare DNA sequences bound by the two complexes in planta.
We conducted chromatin immunoprecipitation coupled with DNA tilling array hybridization
(ChIP-chip) experiments to define all of the LEC 1 binding sites in the Arabidopsis genome. We
used transgenic plants containing LEC1 tagged with the FLAG peptide and fused with the
steroid binding domain of glucocorticoid receptor (35S:FLAG-LEC1-GR) or 35S:FLAG-NLH3.
ChIP experiments were done on 35S:FLAG-NLH3 seedlings grown for eight days and on eight-
day 35S:FLAG-LEC1-GR seedlings treated with dexamethsone, an inducer of LEC1 activity, for
4 h. DNA immunoprecipitated with anti-FLAG antibodies or with non-specific anti-GST
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Harada, John J. Final Report for Regulation of Embryonic Development in Higher Plants, report, October 22, 2013; United States. (https://digital.library.unt.edu/ark:/67531/metadc841520/m1/2/: accessed April 24, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.