Program of the 2001 International Worm Meeting - Sternberg Lab ...

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191. Functional Analysis of C.
elegans Nuclear Hormone Receptors
Marc R. Van Gilst, Keith R.
Yamamoto
Cellular and Molecular Pharmacology,
University of California-San Francisco
Nuclear hormone receptors (NHRs) comprise a
large family of metazoan transcription factors
that participate in numerous developmental and
metabolic processes. NHRs target their
transcriptional regulation to specific genes
through interaction with DNA sequences called
hormone response elements (HREs) and the
type of transcriptional regulation carried out by
an NHR at these genes is often determined by
interaction with a small hydrophobic ligand
and/or various protein cofactors. Sensitivity to
regulation by these modulators allows NHRs to
be quite useful regulators of development, as
their activity can be specifically modified by
environmental signals to orchestrate distinct
transcriptional effects at different genes and in
various cell types. Additionally, these responses
can be modified by regulatory factors in a
target-gene-specific fashion over the course of
development.
In order to understand the utility of NHRs in
metazoan development, we are investigating
NHRs in C. elegans. Our goals are to understand
how NHRs are required for worm development
and to identify the C. elegans factors that serve
as determinants of NHR activity. Initially, we
plan: (i) to isolate C. elegans NHRs and
characterize their physiological roles, (ii) to
identify DNA binding sites and target genes for
individual NHRs, (iii) to characterize C. elegans
proteins that function as NHR cofactors and (iv)
to purify small molecules that function as NHR
ligands. Thus far we have cloned over 20 NHRs
from C. elegans and have characterized their
RNA interference phenotypes by bacterial
feeding and/or by injection. Using yeast
transcriptional reporter assays we have
identified the DNA binding sites of many of
these NHRs and have quantified their ligand
independent transcriptional activities. Our
results suggest that C. elegans NHRs function
similarly to mammalian NHRs in regards to
DNA binding site recognition and
transcriptional activation. We have found that,
like mammalian NHRs, C. elegans receptors
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recognize their DNA binding site using a
conserved amino acid stretch in the zinc-finger
DNA binding domain called a P-box.
Additionally, some C. elegans NHRs also utilize
an additional sequence located at the C-terminal
end of the DNA binding domain called the CTE
to recognize their DNA binding site. Our results
show that C. elegans NHRs with conserved
P-boxes recognize DNA binding sites that are
identical to their mammalian homologs. Many
C. elegans NHRs, however, possess novel P-box
elements that mediate the recognition of novel
DNA binding sites. We are currently using this
DNA binding site information to carry out
computational searches for regulatory elements
that support regulation of target genes by NHRs
in vivo. While some C. elegans NHRs have
strong activity in yeast in the absence of ligand,
we have found that others have little activity,
suggesting the these receptors likely require
interaction with a ligand and/or cofactors in
order to affect transcription. Thus we have
extended our yeast system to identify ligands
and proteins that serve as regulators of C.
elegans NHR activity. Indeed, we have found
that the mammalian NHR coactivator GRIP can
also serve as a functional coactivator of the C.
elegans receptor CHR3 (nhr-23). Mutations that
interrupt the predicted coactivator interface in
CHR3 block the ability of GRIP to coactivate,
arguing that the basic NHR coactivator binding
interface is conserved in C. elegans. We are
currently conducting yeast screens using a C.
elegans protein expression library to identify
such C. elegans coactivators.