CCCB talk

1. Does calorie restriction rescue age-related changes in isoform expression? Kyle Satterstrom September 10, 2015

2. Historical thoughts on longevity Luigi Cornaro (d. 1566)

3. Calorie restriction • Mice with various percent reductions in calorie intake Weindruch et al., 1986, J. Nutr. 116:641

4. Adaptation to reduced calorie intake • Metabolic reprogramming • ↓Glycolysis • ↓Fatty acid storage • ↑ Fatty acid oxidation • ↑ Mitochondrial biogenesis • Stress response • ↑ Antioxidant defenses • ↑ DNA repair • Rescue of age-related changes in gene expression Fat oxidation Glucose metabolism Anderson and Weindruch, 2009; Huang et al., 2010

5. RNA-seq of calorie-restricted mice • High-throughput RNA sequencing to identify effects of CR on isoform-specific expression in liver

6. RNA-seq experimental overview Tissue RNA Extraction DNAse Treatment Library Construction Purity Assay Purity Assay High-Throughput Sequencing

7. Data processing overview Millions of short reads Alignment to genome Quantification of gene expression Calculation of read depth at each bp Analysis of specific start site usage, isoform usage

8. CR reverses age-related gene expression changes: Liver

9. CR reverses age-related gene expression changes: Liver

10. “Isoform” score • Quantifies difference in normalized read distribution between a pair of samples • i.e., one score for aged control vs aged CR liver • Equal to number of bases with significantly different normalized read distribution at 5’ end (400 bp) of transcript • Christodoulou et al., 2014

11. “Isoform score” results

12. SEC31a 5’ 3’ Transcription start site Alternative transcription start site  Clear change in relative start site usage with age, reversed by CR

13. Conclusions • CR reverses many age-related changes in gene expression • CR can also reverse age-related changes in isoform usage

14. Thank you Marcia Haigis Lydia Finley Gaëlle Laurent Karina Gonzalez Herrera Natalie German Daniel Santos Wen Yang Noga Ron-Harel SeungminJeong Elma Zaganjor SejalVyas Koji Nagasawa Jaewon Lee Alexandra Bause Martha Bulyk (BWH Genetics) Bill Swindell Jon Seidman (HMS Genetics) Danos Christodoulou Josh Gorham Steve DePalma Michael Parfenov David Mooney (SEAS) Daniel Needleman (SEAS) ZoltArany (BI, Penn) The Joslin Diabetes Center NIH Training Grant No. T32 DK007260

15. SIRT3 transcripts

16. SIRT3 transcripts

17. SIRT3 transcripts  8 bp insert used twice as often in liver as in heart (H/L = 0.48)

18. SIRT3 transcripts • 8 bp insert: Higher in liver, lower in heart • Inclusion causes M1, M2 to hit premature stop codon  Heart is more permissive of letting M1, M2 isoforms reach full length • M1, M2 isoforms thought to localize to mitochondria more efficiently than M3 isoform  SIRT3 in heart more efficient at reaching mitochondria?

19. Sir2 • Yeast Genetic mimic of CR, but deleted Sir2 Genetic mimic of CR Lin et al., 2000

20. Sirtuins • Seven Sir2 homologs in mammals • NAD+-dependent protein deacetylases • Important roles in aging, metabolism, disease Kim et al., 2006; Haigis and Sinclair, 2010

21. SIRT3 • Major mitochondrial deacetylase • Upregulated in mouse liver and other tissues in response to CR Lombard et al., 2007; Hirschey et al., 2010; Hallows et al., 2011; Jin et al., 2009

22. SIRT3 in metabolism Someya et al., 2010; Hirschey et al., 2010; Hallows et al., 2011; Finley et al., 2011; Shimazu et al., 2010; Cimen et al., 2010

23. SIRT3 in metabolism Someya et al., 2010; Hirschey et al., 2010; Hallows et al., 2011; Finley et al., 2011; Shimazu et al., 2010; Cimen et al., 2010

24. SIRT3 in stress response Tao et al., 2010; Chen et al., 2011; Someya et al., 2010

25. SIRT3 in calorie restriction • Prevents age-related hearing loss in mice • Due to upregulatedanitoxidant activity • SIRT3 KO mice on CR diet suffer hearing loss Someya et al., 2010; Sebastian and Mostoslavsky, 2010

26. What regulates SIRT3 expression?

27. What regulates SIRT3 expression?

28. Bioinformatic identification of transcription factors that may induce SIRT3 expression • Satterstrom et al., 2015

29. Datasets with induced SIRT3

30. DNA sequence analysis • Analyze 20 kb around genes’ transcription start sites to calculate enrichment of transcription factor binding motifs • Weight by evolutionary conservation of motif Warner et al., 2008

31. Enrichment results, neocortex dataset Significantly enriched in top 50 Significantly enriched in top 25 Significantly enriched in top 100 • Satterstrom et al., 2015

32. Nuclear respiratory factor 2 • Tetramer composed of two heterodimers β α http://www.cs.duke.edu/

33. Nuclear respiratory factor 2 • Like ERRα, co-activated by PGC-1α • Central to mitochondrial biogenesis, metabolism • Scarpulla, 2002; Baldelli, 2013

34. SIRT3-PSMD13 promoter Analysis of SIRT3 and co-expressed genes Analysis of SIRT3 promoter alone NRF-2 • Satterstrom et al., 2015

35. NRF-2 level affects SIRT3 expression Overexpression: Knockdown: • Satterstrom et al., 2015

36. NRF-2 level affects SIRT3 expression • Satterstrom et al., 2015

37. NRF-2 level affects SIRT3 expression • Satterstrom et al., 2015

38. Chromatin immunoprecipitation Degrade proteins, leaving only associated DNA Mock Crosslink DNA, proteins IP with anti-HA antibody qPCR for DNA regions of interest Fragment DNA +NRF-2α-HA Positive NRF-2 controls Negative NRF-2 controls • Satterstrom et al., 2015

39. Potential SIRT3 induction in CR

40. Potential SIRT3 induction in CR

42. Mitochondrial ribosome Interaction data (width): 293T cells Correlation data (color): Calorie-restricted mice, skeletal muscle

43. Mass spec data: T cells, with time post-activation