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 Louisiana Biomedical Research Network

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 Louisiana Biomedical Research Network

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)? Louisiana Biomedical Research Network

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 Louisiana Biomedical Research Network

5. Objective • Utilize the C. elegans system to address requirement of a direct DNA-binding mechanism for NR4A Louisiana Biomedical Research Network

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? Louisiana Biomedical Research Network

7. Experimental approach and results: the spermatheca model organ distal constriction spermatheca- uterine valve sac gonad arm uterus Sheath Spermatheca Anterior Posterior Louisiana Biomedical Research Network

8. L C H V Y R K Y A G D I P luc R A R 3X 5’-AAAGGTCA-3’ D R S S NBRE Reporter N T R C C C C T K V Q E Zn N Zn Y G A G A C C C C LDADKM KGFFKRTVQKNSKYT RYQKCLEVGM NHR-6 can transactivate from the NR4A response element (NBRE) in mammalian cell culture Louisiana Biomedical Research Network

9. NHR-6 binds the NBRE in vitro GST-NHR-6 - - - + Cold Competitor (50X) Louisiana Biomedical Research Network

10. Expression and function of a C-terminal tagged NHR-6::GFP 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-6::GFP 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 Louisiana Biomedical Research Network

11. Expression and function of a C-terminal tagged NHR-6::GFP fusion GFP Expressing * F G H I R +/+ -/- +/- Transgenic lines (nhr-6(-)/nhr-6(-); Ex) * Transgenic R line does not express NHR-6::GFP Louisiana Biomedical Research Network

12. NHR-6::GFP 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 C H 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 Y G A G A C C C C LDADKM KGFFKRTVQKNSKYT RYQKCLEVGM

14. A functional NHR-6 DNA-binding is necessary Line Z: DBD mutated NHR-6::GFP is expressed and nuclear localized Line G: Wild-type NHR-6::GFP 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-6::GFP 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