1. Investigating heat shock-induced DNA breaks in spermatocytes Brittany Owen Libuda Lab University of Oregon OURS Research Presentation 2016 August 11, 2016

2. Why are DSBs important? Double Strand DNA Breaks (DSBs) –Proper chromosome segregation –Generate genetic diversity If problems occur during the formation and/or repair: –Miscarriages –Infertility –Birth defects –Cancer

3. Meiosis is necessary for sexually reproducing organisms By Rdbickel - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=49599354

4. Double strand DNA breaks are required for proper chromosome segregation paired homologous chromosomes crossover DSBs noncrossover chiasma Illustration by D. Libuda

5. Double strand DNA breaks are required for proper chromosome segregation paired homologous chromosomes crossover DSBs noncrossover chiasma Illustration by D. Libuda How is DSB repair regulated?

6. Caenorhabditis elegans Microscopic nematode Transparent Spatial-temporal gradient along germline –Can see all stages of meiotic prophase I at once Illustration courtesy (KDS444) Wikipedia

7. C. elegans germ line DAPI (DNA) crossover mutant 12 univalents wild type 6 bivalents Meiotic prophase progression Image taken by D. Libuda Wildtype (WT) 6 bivalents

8. SPO-11 induces DSBs and RAD-51 localizes at these breaks SPO-11 RAD-51 Sister Chromatids DNA strands DSBs 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

9. SPO-11 induces DSBs and RAD-51 localizes at these breaks SPO-11 Sister Chromatids DNA strands DSBs 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

10. SPO-11 induces DSBs and RAD-51 localizes at these breaks SPO-11 RAD-51 Sister Chromatids DNA strands DSBs 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

11. RAD-51 Meiotic prophase progression RAD-51 DAPI RAD-51 foci in the WT hermaphrodite gonad Image taken by D. Libuda

12. Heat shock induces DSBs Can induce DSBs in spermatocytes of C. elegans Type of wormNo heat shockHeat shock Wildtype (oogenesis)Normal # RAD-51 foci Wildtype ♂ (spermatogenesis) Normal # RAD-51 fociIncreased # RAD-51 foci spo-11 mutant No RAD-51 foci spo-11 mutant ♂ No RAD-51 fociRAD-51 foci

13. Heat shock induces DSBs Can induce DSBs in spermatocytes of C. elegans Type of wormNo heat shockHeat shock Wildtype (oogenesis)Normal # RAD-51 foci Wildtype ♂ (spermatogenesis) Normal # RAD-51 fociIncreased # RAD-51 foci spo-11 mutant No RAD-51 foci spo-11 mutant ♂ No RAD-51 fociRAD-51 foci

14. Heat shock induces DSBs Can induce DSBs in spermatocytes of C. elegans Type of wormNo heat shockHeat shock Wildtype (oogenesis)Normal # RAD-51 foci Wildtype ♂ (spermatogenesis) Normal # RAD-51 fociIncreased # RAD-51 foci spo-11 mutant No RAD-51 foci spo-11 mutant ♂ No RAD-51 fociRAD-51 foci

15. Heat shock induces DSBs Can induce DSBs in spermatocytes of C. elegans Type of wormNo heat shockHeat shock Wildtype (oogenesis)Normal # RAD-51 foci Wildtype ♂ (spermatogenesis) Normal # RAD-51 fociIncreased # RAD-51 foci spo-11 mutant No RAD-51 foci spo-11 mutant ♂ No RAD-51 foci RAD-51 foci

16. - heat shock ♀ spo-11 ♂ 2 hrs post-heat shock spo-11 DAPI RAD-51 Heat shock-induced RAD-51 foci in male germlines Image taken by D. Libuda

17. Heat shock induces DSBs Can induce DSBs in spermatocytes of C. elegans Type of wormNo heat shockHeat shock Wildtype (oogenesis)Normal # RAD-51 foci Wildtype ♂ (spermatogenesis) Normal # RAD-51 fociIncreased # RAD-51 foci spo-11 mutant No RAD-51 foci spo-11 mutant ♂ No RAD-51 foci RAD-51 foci What controls this spermatocyte specific phenotype?

18. My Projects Looking at the role of SWSN-1 in induced DSBs –Creating a swsn-1 mutant in a spo-11 mutant background Do transposons cause heat shock-induced DSBs? –Mutation repair assay

19. SWSN-1  Chromatin remodeling protein

20. SWSN-1  Chromatin remodeling protein  SWSN-1 is specific to the male germline

21. SWSN-1  Chromatin remodeling protein  SWSN-1 is specific to the male germline  Important/required for embryonic and larval development

22. SWSN-1  Chromatin remodeling protein  SWSN-1 is specific to the male germline  Important/required for embryonic and larval development  Temperature sensitive phenotypes

23. swsn-1 Mutant Phenotypes swsn-1(os22) @ 15 °C – Nearly WT Mingxue Cui et al. Genetics 2004;167:1177-1185 http://www.cgc.cbs.umn.edu/strain.php?id=8841

24. swsn-1 Mutant Phenotypes swsn-1(os22) @ 15 °C – Nearly WT @ 25 °C – Growth arrest at larval stage Mingxue Cui et al. Genetics 2004;167:1177-1185 http://www.cgc.cbs.umn.edu/strain.php?id=8841 Does SWSN-1 have a role in DSBs?

25. Strain Construction to obtain spo-11;swsn-1 double mutant Crossed spo-11(me44) ♂ to swsn-1(os22) Screened progeny for spo-11 and swsn-1 mutations Selfed specific progeny and allowed them to reproduce

26. Strain Construction: Screening for spo-11(me44) total platesDAPI 6 bivalentsDAPI 12 univalentstotal screened 147541670 12 univalents 6 bivalents Images taken by D. Libuda WT spo-11 mutant

27. Strain Construction: Screening for swsn-1(os22) Selfed and grew @ 25 °C WT and spo-11 mutant swsn-1 and spo-11;swsn-1 mutant 10 had some plates like this 6 had all plates like this Got the double mutant!!

28. Next Steps:  Run immunofluorescence experiment on spo-11;swsn-1 mutant males and look at RAD-51 foci

29. My Projects Looking at the role of SWSN-1 in induced DSBs –Creating a swsn-1 mutant in a spo-11 mutant background Do transposons cause heat shock-induced DSBs? –Mutation repair assay

30. Transposons are mobile units of DNA Transposon DNA Strand DSB

31. Transposons are mobile units of DNA Transposon DNA Strand “hops” out DSB

32. Transposons are mobile units of DNA Transposon DNA Strand “hops” out DSB

33. Does heat shock excise transposons? Using unc-22(st136) homozygous mutant unc-22(st136)TC1 DNA Heat shock Genotype Phenotype uncoordinated

34. Does heat shock excise transposons? Using unc-22(st136) homozygous mutant unc-22(st136)TC1 DNA Heat shock unc-22(st136) TC1 DSB Induced excision Genotype Phenotype uncoordinated

35. Does heat shock excise transposons? Using unc-22(st136) homozygous mutant unc-22(st136)TC1 DNA Heat shock unc-22(st136) TC1 DSB unc-22 (+) Repaired gene Induced excision Genotype Phenotype TC1 unc-22(st136) Allele from oocyte uncoordinated ?

36. Does heat shock excise transposons? Phenotype of heterozygous ( ) worms = ??? N2 (WT) ♂ X unc-22(st136) Conclusion: unc-22(st136)/+ are WT moving

37. Does heat shock excise transposons? unc-22(st136) 34 °C (for 60, 90, or 120 minutes) Step 1 – heat shock Petri dish

38. Does heat shock excise transposons? unc-22(st136) 34 °C (for 60, 90, or 120 minutes) Step 1 – heat shock Petri dish Step 2 – self worms and store for 3-4 days @ 20 °C

39. Does heat shock excise transposons? unc-22(st136) 34 °C (for 60, 90, or 120 minutes) Step 1 – heat shock Petri dish Step 2 – self worms and store for 3-4 days @ 20 °C Step 3 – observe phenotype of F1 progeny (Unc or WT moving) Results: No WT moving worms in any of the groups

40. Next Steps:  Run heat shock and immunofluorescence experiments on a WT strain with many TC1 (a transposon) copies

41. Acknowledgments Libuda Lab at U of O –Diana Libuda –Jackie Helm –Marissa Glover –Kaycee Schoellhorn –Cordell Clark –Austin Harvey –Nicole Kurhanewicz NICHD Summer Research Program at the University of Oregon (NIH-R25HD070817) Peter O’Day Marilyn Drennan