Estradiol-resistant mutants Cre-EBD78 itself Genes involved in - - PowerPoint PPT Presentation

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Estradiol-resistant mutants Cre-EBD78 itself Genes involved in - - PowerPoint PPT Presentation

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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

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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.

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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

  • M. Kaeberlein et al. Cell Dev. Biol. 2008

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In search of ageing regulating genes in Baker's yeast

Thesis presentation October 2011 Shaylee Bar

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  • Dr. Haim Cohens’ lab.
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  • For many years, ageing research was not

popular among molecular biologists

  • ageing was believed to be a passive,

entropic process of deterioration that

  • ccurred 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.

What is ageing ?

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SLIDE 6

What is ageing ?

molecular cellular

  • rgan damage

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Why do we age?

Kirkwood et al. Nature 2000

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  • The Mutation Accumulation Theory

Suggested by Medawar

Why do we age? Ageing theories

Kirkwood et al. Nature 2000

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  • The Theory

Suggested by Williams

Why do we age? Ageing theories

Antagonistic Pleiotropy

Kirkwood et al. Nature 2000

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  • The Disposable Soma Theory

Suggested by Kirkwood and Holliday

Why do we age? Ageing theories

Kirkwood et al. Nature 2000

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Ageing & Environment

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Ageing & Environment – DR

(Dietary Restriction)

  • Dr. Clive McCay

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Fontana et al. Science 2010

Ageing & DR

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Ageing & DR

Kenyon et al. Nature 2010

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Forkheads Forkheads Forkheads Forkheads

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Sir2 - protein deacetylase

Sirtuins & Ageing

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  • M. Kaeberlein et al. Genes Dev. 1999

sir2Δ

sir2Δ+ SIR2 OE SIR2 OE

WT Guarente L Genes Dev.2000

  • M. Kaeberlein et al. Cell Dev. Biol.2008
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SLIDE 16
  • S. Broughton et al. Biochem.2009

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Insulin/IGF-1 signaling

Kenyon et al. Nature 2010

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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

Carlsson et al. Developmental Biology 2002

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Forkheads in yeast

  • S.cerevisiae has 4 Forkhead proteins:
  • 1. FHL1 - Pre-rRNA processing
  • 2. HCM1 -
  • 3. FKH1 -
  • 4. FKH2 -
  • Role in mediating Replicative Life Span in

yeasts: Unknown

G2-M transition S phase transition

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Ageing yeasts

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Research goals

Checking whether S.cerevisiae‟s forkhead proteins have a role in mediating RLS.

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Results

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RLS for forkheads‟ KO strains –

  • ld protocol

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RLS for fkh1 KO - updated protocol

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RLS for fkh2 KO - updated protocol

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RLS for hcm1 KO - updated protocol

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RLS for all 3 KO

hcm1Δ fkh2Δ fkh1Δ WT Strain 17 24 26 28 Median lifespan

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RLS of forkhead KO strains

  • ld vs. updated

Old protocol Updated protocol

UPDATED protocol OLD protocol

hcm1Δ fkh2Δ fkh1Δ WT hcm1Δ fkh2Δ fkh1Δ WT strain

17 24 26 28 15.5 26.5 26 20.5

Median

9%

  • 6%

0% 37%

Change 27

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Viability test

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  • 28%
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OLD vs. UPDATED - RLS protocol

1cm 4cm

graveyard

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  • Synthesis of O/E strains of the

proteins below :

  • FKH1 OE
  • FKH2 OE
  • HCM1 OE
  • FHL1 OE

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RLS for forkheads‟ O/E strains

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Asc1

HCM1

Pac1

pGEM-T Vector

Restriction with Asc1+Pac1 Restriction with Not1

HCM1 hisG URA3 hisG

HO-R HO-L Transformation To Yeast To the HO locus

M4366

BY4741=MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

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Experimental procedure

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FKH1 Over-expression

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FKH2 Over-expression

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HCM1 Over-expression

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  • 3700bp
  • No luck with cloning

FHL1 Over-expression

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RLS for FKH1 over-expression

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RLS for FKH2 over-expression

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RLS for HCM1 over-expression

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RLS for all 3 O/E

HCM1 OE FKH2 OE FKH1 OE WT Strain 27 25 29 30 Median lifespan

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Discussion

  • Why didn‟t fkh1Δ, fkh2 Δ & hcm1 Δ have the

same lifespan increase like WT in the updated protocol?

  • Hcm1Δ viability test + 35% decreased in life

span

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tetO7-HCM1 = stress resistance

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Sum-up

  • Unlike their roles in developed organisms (flies,

nematodes, mice), the yeast forkheads transcription factors do not effects yeasts replicative life span

  • 2X expression (mRNA level) is enough to induce

potential benefits ?

  • 2X in mRNA level doesn‟t always means 2X

protein levels?

  • What about Fhl1?
  • End of part 1

Questions?

BUT

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RLS measuring by “MEP” assay The Mother Enrichment Program

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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

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And then came Gottschling..

  • Using a faster method to analyze RLS of tens
  • f 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.

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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

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  • 2. Expression of the Cre recombinase

restricted by a daughter-specific promoter derived from SCW11

Daughter specific promoter: controlled by Ace2 TF, which is asymmetrically distributed to daughter cell nuclei prior to cytokinesis. Conditional ability: Cre is fused to an EBD domain (estradiol- binding domain). EBD sequesters the fusion protein in the cytoplasm until estradiol is introduced .

A Cre-lox mediated selection against daughter cells

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  • 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.

  • 3. Illustration of the expected localization of Cre-EBD78

(shading) in response to estradiol.

A Cre-lox mediated selection against daughter cells

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Research goals

Perform a genetic screen for genes that effect RLS by using the MEP assay

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MEP strain 30 O/N 30 for 3 hr recovering cells/ml

3

10 x 2 ml 25

Estradiol

0 hrs 5 hrs 10 hrs 30 hrs 120 hrs

..…

MEP assay - protocol

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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

  • nly a cohort of mother cells in the viable population.

MEP assay - protocol

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Results (2)

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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

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CR & MEP

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CR & MEP- take 2

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Discussion

  • 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.

Micromanipulation

MEP RLS protocol

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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

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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

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  • 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

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