Estradiol-resistant mutants • Cre-EBD78 itself • Genes involved in localization of the EBD • Multidrug resistance • Logarithmically growing haploid cells produced resistant mutations at a rate of 1.4*10 -6 per cell division • In Diploids , 1.4*10 -8 mutations per cell division. 1
In a rapidly growing culture of yeast, ages of individual cells will distribute geometrically, with : 1/2 of the population virgins, 1/4 one-division old mothers , 1/8 two-division-old mothers , 1/16 original mother 2 M. Kaeberlein et al. Cell Dev. Biol. 2008
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In search of ageing regulating genes in Baker's yeast Dr. Haim Cohens’ lab. Thesis presentation October 2011 Shaylee Bar 4
What is ageing ? • For many years, ageing research was not popular among molecular biologists • ageing was believed to be a passive , entropic process of deterioration that occurred in a haphazard way. • We know now, however, that the ageing process, like many other biological processes, is subject to regulation by classical signaling pathways and transcription factors . 5
What is ageing ? molecular cellular organ damage 6
Why do we age? Kirkwood et al. Nature 2000 7
Why do we age? Ageing theories • The Mutation Accumulation Theory Suggested by Medawar 8 Kirkwood et al. Nature 2000
Why do we age? Ageing theories • The Theory Antagonistic Pleiotropy Suggested by Williams Kirkwood et al. Nature 2000 9
Why do we age? Ageing theories • The Disposable Soma Theory Suggested by Kirkwood and Holliday Kirkwood et al. Nature 2000 10
Ageing & Environment 11
Ageing & Environment – DR (Dietary Restriction) Dr. Clive McCay 12
Ageing & DR 13 Fontana et al. Science 2010
Ageing & DR Forkheads Forkheads Forkheads Forkheads 14 Kenyon et al. Nature 2010
Sirtuins & Ageing Sir2 - protein deacetylase SIR2 sir2 Δ sir2 Δ + OE SIR2 WT OE M. Kaeberlein et al. Cell Dev. Biol.2008 M. Kaeberlein et al. Genes Dev. 1999 Guarente L Genes Dev.2000 15
Insulin/IGF-1 signaling 16 Kenyon et al. Nature 2010 S. Broughton et al. Biochem.2009
Meet the Forkheads FOX proteins (Forkhead box proteins) first discovered more than 10 • years ago in Drosophila and are characterized by a shared 100 amino acid DNA- binding motif, termed “forkhead” domain The forkhead transcription factors have been shown to play diverse • roles in development, metabolism, immunology, cancer, and cell-cycle control Forkhead gene number: 39 in Homo • 20 in Drosophila 15 in Caenorhabditis 4 in Saccharomyces 17 Carlsson et al. Developmental Biology 2002
Forkheads in yeast • S.cerevisiae has 4 Forkhead proteins: 1. FHL1 - Pre-rRNA processing 2. HCM1 - S phase transition 3. FKH1 - G2-M transition 4. FKH2 - • Role in mediating Replicative Life Span in yeasts: Unknown 18
Ageing yeasts 19
Research goals Checking whether S.cerevisiae‟s forkhead proteins have a role in mediating RLS. 20
Results 21
RLS for forkheads‟ KO strains – old protocol 22
RLS for fkh1 KO - updated protocol 23
RLS for fkh2 KO - updated protocol 24
RLS for hcm1 KO - updated protocol 25
RLS for all 3 KO Strain WT fkh1 Δ fkh2 Δ hcm1 Δ Median lifespan 28 26 24 17 26
RLS of forkhead KO strains old vs. updated Updated protocol Old protocol OLD protocol UPDATED protocol strain WT fkh1 Δ fkh2 Δ hcm1 Δ WT fkh1 Δ fkh2 Δ hcm1 Δ 20.5 26 26.5 15.5 Median 28 26 24 17 37% 0% -6% 9% Change 27
Viability test -28 % 28
OLD vs. UPDATED - RLS protocol graveyard 1cm 4cm 29
RLS for forkheads‟ O/E strains • Synthesis of O/E strains of the proteins below : • FKH1 OE • FKH2 OE • HCM1 OE • FHL1 OE 30
Experimental procedure pGEM-T Vector Pac1 Asc1 HCM1 Restriction with Asc1+Pac1 M4366 Restriction with Not1 hisG hisG URA3 HCM1 HO-L HO-R Transformation To 31 BY4741=MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 Yeast To the HO locus
FKH1 Over-expression 32
FKH2 Over-expression 33
HCM1 Over-expression 34
FHL1 Over-expression • 3700bp • No luck with cloning 35
RLS for FKH1 over-expression 36
RLS for FKH2 over-expression 37
RLS for HCM1 over-expression 38
RLS for all 3 O/E Strain WT FKH1 OE FKH2 OE HCM1 OE Median lifespan 30 29 25 27 39
Discussion • Why didn‟t fkh 1 Δ , fkh2 Δ & hcm1 Δ have the same lifespan increase like WT in the updated protocol? • Hcm1 Δ viability test + 35% decreased in life span tetO7-HCM1 = stress resistance 40
Sum-up • Unlike their roles in developed organisms (flies, nematodes, mice), the yeast forkheads transcription factors do not effects yeasts BUT replicative life span • 2X expression (mRNA level) is enough to induce potential benefits ? • 2 X in mRNA level doesn‟t always means 2X protein levels? • What about Fhl1? • End of part 1 Questions? 41
RLS measuring by “MEP” assay The Mother Enrichment Program 42
Measuring RLS • The current „„gold standard‟‟ for isolating aged mother cells is by micromanipulation • 40 yeasts + 3 strains on each plate + Every 90 min. = 3-4 strains in 3 weeks 43
And then came Gottschling.. • Using a faster method to analyze RLS of tens of strains without using the micromanipulation method. • Novel genetic method that creates selection against daughter cell replication, while allowing mother cells to achieve a normal RLS. 44
A Cre-lox mediated selection against daughter cells • MEP strain was created using the following way: 1. Loxing of UBC9 and CDC20: Both are required for the degradation of mitotic cyclins and other targets vital to cell cycle progression 45
A Cre-lox mediated selection against daughter cells 2. Expression of the Cre recombinase restricted by a daughter- specific promoter derived from SCW11 Daughter specific promoter: Conditional ability: controlled by Ace2 TF, which is Cre is fused to an EBD domain (estradiol- asymmetrically distributed to binding domain). EBD sequesters the fusion daughter cell nuclei prior to protein in the cytoplasm until estradiol is cytokinesis. introduced . 46
A Cre-lox mediated selection against daughter cells 3. Illustration of the expected localization of Cre-EBD78 (shading) in response to estradiol. PSCW11-cre-EBD78 expression is restricted to daughter cells. • In the absence of estradiol the fusion protein is sequestered in the cytoplasm. • Upon ligand binding, the fusion protein is translocated into the nucleus where it • can act on loxP target sites. 47
Research goals Perform a genetic screen for genes that effect RLS by using the MEP assay 48
MEP assay - protocol MEP 30 for strain 2 x 10 3 cells/ml 3 hr 30 O/N 25 ml recovering Estradiol ..… 0 hrs 5 hrs 10 hrs 30 hrs 120 hrs 49
MEP assay - protocol 50% of cells are daughters, thus immediately arrested Normal RLS aging behavior Daughters constituted approximately half of the initial inoculum. These daughter cells are immediately arrested by estradiol, leaving only a cohort of mother cells in the viable population. 50
Results (2) 51
Assay Calibration – Estradiol effects Diploid , Haploid and Control strains grown for 16 hours in YPD with or without estradiol to verify the effect of estradiol on life span 52
CR & MEP 53
CR & MEP- take 2 54
Discussion Micromanipulation MEP RLS protocol • Why didn‟t I get life span extension by caloric restriction? Why didn‟t Gottschlings‟ lab check the effects of CR on lifespan? • All the genes of interest must be manipulated on the MEP diploid strain • RLS as a function of time - “As long as the cell division rates remains constant within a cohort of mother cells, viability in liquid culture will be directly proportional to RLS..” (Gottschling et al. Genetics.2009) • It is not possible to do the assay on more than tens strains at a time. 55
Discussion • MEP as a method to purify aged population As an alternative to micromanipulation, methods were developed to • isolate aged cells from liquid cultures. due to the exponential growth of progeny cells, these populations are technically limited to 7 – 12 generations before nutrient depletion interferes with replicative aging 56
Sum-up • A good method to measure RLS and replace the micromanipulation is yet to be found • Possible potential to the MEP as a method to purify aged population 57
Dr. Haim Cohen‟s lab Dr. Batia Lerrer Dr. Yariv Kanfi Dr. Gilad Gibor Dr. Yahav Dikstein Moran Finkel Noam Maoz Orshay Gabay Maya Greenbloom Sivan Elchanati Liat Nachum Asaf Gertler Shaul Bert Reuven Gil Shoshana Naiman Shai Lubel Daniel Marton Yael Buchnik 58
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