Title: A functional NR4A nuclear receptor DNA-binding domain is required for organ morphogenesis in C. elegans
1A functional NR4A nuclear receptor DNA-binding
domain is required for organ morphogenesis in C.
elegans
- PI Chris R. Gissendanner, ULM
- Mentor Brian Rowan, Tulane University School of
Medicine
2Project Goals
- Utilize an organogenesis system in the model
organism Caenorhabditis elegans to dissect the
cellular activities of the NR4A nuclear receptor
transcription factor
- Background
- NR4A has critical functions in regulating cell
proliferation and cell differentiation in a wide
variety of developmental and disease contexts - The NR4A ortholog in C. elegans, NHR-6, is
required for cell proliferation and cell
differentiation during organ formation,
indicating NR4A functions are conserved in C.
elegans
3Project Goals Using the C. elegans model system
to dissect the cellular activities of NR4A
- Key questions this project is addressing
- 1) What signaling pathways modulate NHR-6
activity? - 2) What are the downstream target genes of NHR-6?
- 3) What are the biochemical mechanisms of NHR-6
activity (at the protein level)?
4Significance
- NR4A pathways regulating cell proliferation, cell
differentiation, and cell survival decisions have
not yet been elucidated - It has not been determined if NR4A functions
through a DNA-binding mechanism in vivo in the
regulation of cell proliferation and cell
differentiation
5Objective
- Utilize the C. elegans system to address
requirement of a direct DNA-binding mechanism for
NR4A
6Experimental Approach
- 1) Can NHR-6 bind to, and activate transcription
from, the canonical NR4A binding site? - 2) Is the ability to bind DNA critical for NHR-6
function in vivo?
7Experimental approach and results the
spermatheca model organ
Anterior
Posterior
8NHR-6 can transactivate from the NR4A response
element (NBRE) in mammalian cell culture
9NHR-6 binds the NBRE in vitro
GST-NHR-6
- - -
Cold Competitor (50X)
10Expression and function of a C-terminal tagged
NHR-6GFP fusion
9283 bps
GFP
1 2 3 4 5
6 7 8 9 10
11 12 13
nhr-6 C-terminal tagged GFP construct
Genetic rescue assay -nematode transgenics
generated with 1 ug/ml nhr-6GFP and 100 ug/ml
transformation marker -determine if a GFP tagged
NHR-6 rescues the nhr-6 mutant phenotype -assess
GFP localization within the cell -if GFP tagged
NHR-6 is functional, use as an in vivo assay for
structure/function experiments. GFP allows
monitoring of protein expression and localization
11Expression and function of a C-terminal tagged
NHR-6GFP fusion
GFP Expressing
F G H I R /
-/- /-
Transgenic lines (nhr-6(-)/nhr-6(-) Ex)
Transgenic R line does not express NHR-6GFP
12NHR-6GFP is nuclear localized during development
13Genetic rescue with a DNA-binding domain mutation
9283 bps
GFP
1 2 3 4 5
6 7 8 9 10
11 12 13
L
H
C
V
Y
R
K
Y
A
G
D I P
R
A
R
D
R
S
S
N
T
R
C
C
C
C
T
K
V
Q
E
Zn
N
Zn
G
Y
A
G
A
C
C
C
C
RYQKCLEVGM
LDADKM
KGFFKRTVQKNSKYT
14A functional NHR-6 DNA-binding is necessary
Line Z DBD mutated NHR-6GFP is expressed and
nuclear localized
Line G Wild-type NHR-6GFP
Y Y Z Z W M / -/- /- (/-)
(-/-) (/-) (-/-) (-/-) (-/-)
nhr-6 genetic background
15Conclusions
- NHR-6 can bind the canonical NR4A binding site in
vitro and in cell culture - A C-terminal GFP tagged NHR-6 is fully functional
and localized to the nucleus - A functional NHR-6 DNA-binding domain is required
for the organ morphogenesis
16Future Directions (NHR-6 structure/function and
target gene identification)
- Use genetic rescue assay system to determine
other functional regions of the protein in vivo - Use NHR-6GFP for ChIP-chip experiments to
identify NHR-6 binding sites in vivo (recently
initiated INBRE project)
17Project Progress (2009 to date)
- Students Trained
- M.S. Students
- Melissa Heard-defended July, 2009
- Anna Kleshayeva
- Mayur Fagwani
- Undergraduate Students (stipend funded by INBRE)
- Elodie Burlet, Chris Wilson, Taylor Barnes,
Daniel Ellis - Presentations 4 regional and 1 international
- Manuscripts (INBRE)
- Heard, M., Morehead, B., Hoener, M., Nguyen,
T., Maina, C., Williams, C., Rowan, B.,
Gissendanner, C.R. (2009). A functional NR4A
nuclear receptor DNA-binding domain is required
for organ morphogenesis in C. elegans, to be
submitted to Development, August, 2009. - Manuscripts (non-INBRE)
- Parihar, M., R.L. Minton, S. Flowers, A.
Kleshayeva, J. Paille, C.R. Gissendanner,
Identification and larval expression of EcR and
RXR/Usp homologs in the nematode Pristionchus
pacificus, submitted to Gen. Comp. Endo. - Grants
- NIH NICHD R15 funded July 20, 2009
graduate student undergraduate
18Acknowledgements
- Melissa Heard
- Dr. Brian Rowan
- Dr. Sushma Krishnamurthy, Dr. Eric Pani, Dr. Ann
Findley (ULM) - Dr. Victor Hsia (ULM)
- Marius Hoener and Tri Q. Nguyen GFP construct
- Claude Maina (New England Biolabs) assistance
with gel shifts