1. AIS (Androgen Insensitivity Syndrome) and the androgen receptor (AR) Sam Trammell

2. AIS and AR outline What is Androgen Insensitivity Syndrome? How is the androgen receptor linked to AIS? What accounts for the variability in the syndrome? Are there AR homologs in other species? What genes are interacting with AR? What compounds interact with the receptor?

3. Androgen Insensitivity Syndrome (AIS) Androgen=testosterone Usually, modified to dihydrotestosterone in the cytoplasm Insensitive=“never calls back” Put the two together and you get the idea

4. Alias: Testicular Feminization Prevalence: 1 in 20,000 births Most prevalent form of intersexism XY karyotype Possibly feminized features Ambiguous or undeveloped genitalia Take it away Androgen Insensitivity Syndrome (AIS) Two individuals with AIS AISSG-USA Support Group Retrieved from: http://www.indiana.edu/~ais/ html/ais_facts.html

5. Hermaphrodite ≠ Intersex Not necessarily associated with homosexuality Androgen Insensitivity Syndrome (AIS)

6. Mild AIS (MAIS), Partial AIS (PAIS), Complete AIS (CAIS) All images from: Geneva Foundation for Medical Education and Research Retrieved from: http://www.gfmer.ch/selected_images_v2/d etail_list.php?cat1=15&cat2=124&cat3=34 6&cat4=2&stype=n Androgen Insensitivity Syndrome (AIS)

7. Popular Press Article Accomplished international runner Stripped of her medals Why? I can’t walk! Well, you have legs. Taken from “Punishing Difference” http://www.expressbuzz.com/edition/story.aspx?Title=Punishing+difference &artid=kunBaJO7yWk=&SectionID=f4OberbKin4=&MainSectionID=fyV9T2j Ia4A=&SectionName=cxWvYpmNp4fBHAeKn3LcnQ==&SEO=gender,%20 sexualities,%20santhi%20soundararajan Santhi Soundararajan

8. Androgen Receptor First linked to AIS in mice Homolog found in humans Those with CAIS found to have a mutation in AR

9. AR Motifs Using MOTIF Sex-determining Y gene product region Expected! Others were unrelated or insignificant to AIS

10. Protein Domains Gene encodes 920 amino acid protein Using SMART 3 Domains Steroid or nuclear hormone receptor (NR) Zinc FingerLigand binding

11. Protein Domains Androgen receptor N-terminus, DNA binding, ligand binding Zinc finger DNA binding, dimerizes upon activation Ligand Binds ligand, for us DHT (dihydrotestosterone)

12. Protein Domain Function Androgen receptor responds to androgen in cytoplasm Conformational change increases affinity for ligand in ligand domain Protein binds DNA to initiate transcription of other genes Accounts for phenotypic variance?

13. Homology IdentitiesExpect Chimpanzee0.736E-50 Rat0.840 Mouse0.840 Dog0.870 Cattle0.792E-171 Zebra fish0.672E-161 Chicken0.580

14. Homology X-axis: Human AR Y-axis: Predicted similar gene to human AR in chimpanzees

15. Homology X-axis: Human AR Y-axis: Predicted similar gene to human AR in cattle

16. Homolog in C. elegans Using Wormbase nhr-69 nuclear hormone receptor-69 Does not determine sex, but mutations in it can lead to both maternal and offspring infertility –Acts to develop germline Would not use as a model organism for AIS

17. Phylogeny Aligned with T-Coffee

18. Cellular Component: Cytoplasm Nucleus Gene Ontology (using GO Consortium) Biological processes: Cell growth Prostate gland development Cell to cell signaling Signal transduction Sex determination Molecular processes: Androgen receptor activity Transcription activity DNA binding

19. Protein Interactions Using String

20. Protein Interactions UBE2I CCND1 HTATIP BRCA1 BAG1 NCOA1 NCOA2 RNF14 TGFB1I1 NCOA4 Those highlighted are protooncogenes.

21. Drugs interacting with AR Flutamide Nilutamide Bicalutamide Finasteride Oxandrolone Testosterone Nandrolone Fluoxymesterone Dromostanolone Cyproterone

22. Conclusions Mutations in androgen receptor cause AIS –Severity depends on mutation location in domain and mutation type (nonsynonymous or synonymous or nonsense) Androgen receptor accounts for many trait differences in males and females –Determines gonad type –Puberty –Regulates body and pubic hair growth Androgen receptor is highly conserved in mammals and higher organisms but does not have a direct homolog in more distantly related organisms –Should not be used a to determine speciation events, example, chimpanzee and cattle Regulates cell division –Target for anti-cancer drugs

23. Future Directions Search for more interacting genes –RNAi screen –Chemical genetics Does the androgen receptor play an important role in upregulating genes and/or silencing genes during puberty? –Microarrays! Current microarray studies focus on differential gene expression on the body

24. Questions?

25. Thank you A special thank you to Ahna Skop for all of her help and guidance.

26. References 1. Galani, A., Kitsiou-Tzeli, S., Sofokleous, C., Kanavakis, E., Kalpini-Mavrou, A. (2008). Androgen insensitivity syndrome: clinical features and molecular defects. HORMONES 7(3). Retrieved from: http://hormones.gr/preview.php?c_id=227http://hormones.gr/preview.php?c_id=227 2.Wisniewski, A., Migeon, C., Meyer-Bahlburg, H., Gearhart, J., Berkovitz, G., Brown, T., Money, J. (2000). Complete Androgen Insensitivity Syndrome: Long-Term Medical, Surgical, and Psychosexual Outcome. Clinical Endocrinology and Metabolism 85(8). Retrieved from: http://jcem.endojournals.org/cgi/content/abstract/85/8/2664 http://jcem.endojournals.org/cgi/content/abstract/85/8/2664 3.Hines, M., Ahmed, S., Hughes, I. (2003). Psychological Outcomes and Gender-Related Development in Complete Androgen Insensitivity Syndrome. Archives of Sexual Behaviors 32(2). doi: 10.1023/A:102249210697410.1023/A:1022492106974 4.Diamond, M., Watson, L. A. (2004). Androgen insensitivity and Klinefelter's syndrome: sex and gender considerations. Child Adolesc Psychiatric Clin N. Arm. of North America 13. Retrieved from: http://www.hawaii.edu/PCSS/online_artcls/intersex/AndrogenInsensitivity.htmhttp://www.hawaii.edu/PCSS/online_artcls/intersex/AndrogenInsensitivity.htm 5.Lubahn, D., Brown, T., Simental, J., Higgs, H., Migeon, C., Wilson, E., French, F. (1989). Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity syndrome. Proc. Natl. Acad. Sci 86. Retrieved from: http://www.jstor.org/stable/35117?seq=1http://www.jstor.org/stable/35117?seq=1 6.Holterhus, P., Hiort, O., Demeter, J., Brown, P., Brooks, J. (2003). Differential gene-expression patterns in genital fibroblasts of normal males and 46,XY females with androgen insensitivity syndrome: evidence for early programming involving the androgen receptor. Genome Biology 4(R37). Doi: 10.1186/gb-2003-4-6-r37