A functional NR4A nuclear receptor DNA-binding domain is required for organ morphogenesis in C. elegans

1
A 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

2
Project 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

3
Project 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)?

4
Significance

• 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

5
Objective

• Utilize the C. elegans system to address
  requirement of a direct DNA-binding mechanism for
  NR4A

6
Experimental 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?

7
Experimental approach and results the
spermatheca model organ
Anterior
Posterior
8
NHR-6 can transactivate from the NR4A response
element (NBRE) in mammalian cell culture
9
NHR-6 binds the NBRE in vitro
GST-NHR-6
- - -
Cold Competitor (50X)
10
Expression 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
11
Expression 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
12
NHR-6GFP is nuclear localized during development
13
Genetic 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
14
A 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
15
Conclusions

• 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

16
Future 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)

17
Project 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
18
Acknowledgements

• 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