Modulation of SIR3 Activity and Effects on Aging in Annelids

KO of Sir3 in C. Elegans

Will Gibson and Marta Milan
20.109(S08)
Pink T/R

Begin to define your research proposal by making a wiki page to collect your ideas and resources (you can do this on one page with your partner or split the effort and each turn in an individual page). Keep in mind that your presentation to the class will ultimately need:

• a brief project overview
• sufficient background information for everyone to understand your proposal
• a statement of the research problem and goals
• project details and methods
• predicted outcomes if everything goes according to plan and if nothing does
• needed resources to complete the work

For now, focus on coming up with a research problem and giving a little background about it. Print your user page(s) for next time, making sure it defines your topic, your idea and two or more references you've collected and summarized. Keep in mind that you're not committed to this idea - if you come up with something that you like better later on, that's fine.

Research

Research the effect of SIR3 on aging of C. Elegans. Much work has been done on SIR2 and the SIR genes in yeast, we intend to extend insight into higher organisms.

Background

• Increased activity of SIR2 has been shown to increase lifespan of various organisms. Restrictions in caloric intake activate SIR2 and studies positively linking caloric intake to increased lifespan have been done in species ranging from yeast to apes. SIR3 has been shown to inhibit activity of SIR2 and SIR4. In this study we propose to modulate SIR3 activity and establish its effect SIR2 and aging.

References

1. A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family
   • http://www.pnas.org/cgi/reprint/97/12/6658?ck=nck
   • Yeast SIR2 has a NAD+ dependent histone deacetylase activity.
   • The NPT1 gene encodes an enzyme which catalyzes the synthesis of NAD+.
   • Null mutants of this gene were created with highly decreased amounts of NAD+. They display characteristics similar to SIR2 null mutants.
   • Mutants in which all 5 SIR2 -like genes have been deleted display normal histone acetylation levels. Researchers concluded that the SIR2 family of proteins monitors cellular energy and redox states.
2. Identification of a Novel Allele of SIR3 Defective in the Maintenance, but Not the Establishment, of Silencing in Saccharomyces cerevisiae
   • http://www.genetics.org/cgi/content/abstract/155/2/523
   • Previously, knockout Sir2 mice have been sterile, and we worried about knockout Sir3 (which affects telomere silencing) C. Elegans having the same problem.
   • In this paper, they found a way to mutate Sir3 without affecting mating.
   • The specific allel, Sir3-P898Rp, was shown to be defective in maintenance, but not the establishment of silencing.
   • This should lead us in the correct path to creating a non-sterile KO.
3. Long-Range Communication Between the Silencers of HMR
   • http://mcb.asm.org/cgi/reprint/28/6/1924
   • Gives insight on silencers and how they affect Sir3

Ideas

1. Knockout SIR3 in C. Elegans with targeted homologous recombination.
2. Measure SIR2 Activity
3. Modulate Translation of SIR3 via RNA interference. Induce plasmids containing complementary sequence to SIR3.
4. Controls: In case of knockout, C. Elegans with intact SIR3,induced with plasmid backbone. In case of RNA interference, induce plasmids containing nonsense sequence.

Websites

• Caloric Restriction and Lifespan in Yeast: http://web.mit.edu/newsoffice/2000/yeast-0927.html
• The SIR3 Gene: http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=retrieve&dopt=full_report&list_uids=851163
• RNA Interference: http://en.wikipedia.org/wiki/RNA_interference
• RNA Interference with siRNA's: http://en.wikipedia.org/wiki/SiRNA
• Guarante Lab: http://web.mit.edu/biology/guarente/about.html
• Knockout Mice: http://en.wikipedia.org/wiki/Knockout_mouse