Bi190 �Mating Type Interconversion

2011 Sternberg, Caltech

Ira Herskowitz

a

α a

a

α

α

a/α zygote

Ascus with 4 spores

haploid cells

-C -N

mating

sporulation

germination +C +N

Saccharomyces cerevisiae (budding yeast) Life Cycle

α- specific genes

a - specific genes

non - specific genes

α mating

a mating

Production of α- factor Agglutination Response to a-factor

Production of a- factor Agglutination Response to α- factor

Response to pheromones

a

α a

a

α

α

a/α zygote

Ascus with 4 spores

haploid cells

-C -N

mating

sporulation

germination +C +N

Saccharomyces cerevisiae (budding yeast) Life Cycle

a a α α

α cells (α-factor producing)

a a α α α α

a a a a α α α α

a/α a/α

a/α a/α

ho HO

Homothallism: single haploid spore gives rise to diploid cells that can undergo meoisis and sporulation

shmoos zygotes

The genetic elements controlling mating type interconversion were defined by natural variants.

In current nomenclature:

S. cerevisiae HMLα MATα HMRa ; HO

S. cerevisiae HMLα MATα HMRa ; ho

S. oviformis HMLa MATα HMRa ; HO

S. norbensis HMLα MATα HMRα ; HO

S. diastaticus HMLα MATα-inc HMRa ; HO

MATa HO MATα ho

a HO Dip(loidizer) a ho a-mater α HO Dip α ho α mater

Tetratype Ascus

HO is necessary for Diploidization

HMLα MATa HMRa ; HO HMLα MATα HMRα ; HO

HMLα MATa HMRα HO Dip HMLα MATa HMRa HO Dip HMLα MATα HMRα HO α HMLα MATα HMRa HO Dip

HMLα MATa HMRα HO Dip HMLα MATa HMRα HO Dip HMLα MATα HMRa HO Dip HMLα MATα HMRa HO Dip

HMRα is necessary for a to α

Dip α

HMLα MATα HMRa

W X Yα ZL W X Yα Z X Ya ZR E EI I

W ~700 bp Ya ~600 bp Yα ~750 bp ZL ~300 bp ZR ~250 bp

CEN III

200 kb 150 kb

HO

HO cuts at ~18 bp site α-inc is the site

Mating Type Interconversion: the molecular level

The event is non-reciprocal --the cassette is lost. Gene Conversion via double-strand break repair

Tests of the cassette model

In 10% of the 10-6 ho switches, There is a deletion, called a-lethal or a Hawthorne deletion:

this is a fusion of MAT to HMRa

HML MAT HMR

Healing:

HO HMLα matα1-5 HMRa Sterile

can switch to an α-mater

Wounding:

HO HMLα-66 MATa HMRa a-mater

switches from a to Ste to a to Ste

Tests of the cassette model

HMLα MATα HMRa

W X Yα ZL W X Yα Z X Ya ZR E EI I

W ~700 bp Ya ~600 bp Yα ~750 bp ZL ~300 bp ZR ~250 bp

CEN III

200 kb 150 kb

HO

HO cuts at ~18 bp site α-inc is the site

Mating Type Interconversion: the molecular level

The event is non-reciprocal --the cassette is lost. Gene Conversion via double-strand break repair

E, essential for silencing I, important for silencing

mutagenize:

ho HMLα matα1-5 HMRa Sterile

pick α-mater

sir1-1 (silent regulator)

analyze: ho HMLα matα1-5 HMRa ; sir1-1 α-mater

ho HMLa matα1-5 HMRa ; sir1-1 Sterile

So, it depends on HML!

Tests of the cassette model

sir1 sir2 = mar1 sir3 = ste8 = cmt sir4 = ste9

The SIR proteins silence the HML and HMR loci

Jasper Rine:

a sir1-1 a sir1-1 Spo+

Thus, HMLα can provide α function

HMLα MATα HMRa

W X Yα ZL W X Yα Z X Ya ZR E EI I

W ~700 bp Ya ~600 bp Yα ~750 bp ZL ~300 bp ZR ~250 bp

CEN III

200 kb 150 kb

HO

HO cuts at ~18 bp site α-inc is the site

Mating Type Interconversion: the molecular level

The event is non-reciprocal --the cassette is lost. Gene Conversion via double-strand break repair

E, essential for silencing I, important for silencing

a a α

α

α α

α

Pedigree analysis

mother daughter

mother daughter

a or α but not a/α cells switch Only mothers switch Switching in pairs Directionality: a to α α to a

Strathern, Hicks & Herskowitz (Cell 1979)

Shmoo (G1 arrest at START)

a a α

α

α α

α

Pedigree analysis

mother daughter

mother daughter

a or α but not a/α cells switch Only mothers switch Switching in pairs Nasmyth (1983)

HO is Off in a/α cells Off in daughters On in late G1

a a α

α

α α

α

Pedigree analysis

a a

α α

a a

α α

SWI+ SIN+

swi5 SIN3+

SWI5+ sin3

mother daughter

mother daughter

SWI5 is an activator but is present in both mothers and daughters SIN3 is a repressor that is antagonized by SWI5, so we need to find Genes that regulate SWI5

a a α

α

α α

α

Sil & Herskowitz: Screen for microcolonies in which mother and daughters switch (1/66,000)

a a a a

ASH1+

ash1-1

mother daughter

mother daughter

ASH1 is a transcriptional repressor only in daughter cell nuclei ASH1 protein is unstable and gene expressed only at M/G1transition ASH1 mRNA is localized preferentially to daughter cells.

HO expression in mother versus daughter cells

Nasmyth: SWI5-dependent activators of HO Expression: Screen for genes necessary for HO-lacZ expression but not For GAL1-URS1-HO-lacZ expression 222 swi5 mutants, 315 she mutants, 5 complemenation groups

The SHE proteins are necessary for ASH1 mRNA localization Transport out of mother cells

SHE1 (MYO4) encodes a non-muscle myosin

Figure 3. The Effect of the she Mutants on Particle Localization and Formation Yeast strains disrupted for each one of the five SHE genes were transformed with the ASH1 reporter RNA and the GFP-MS2 fusion protein and the resultant particles observed by epifluorescence after fixation. Bar, 5 mm. (A) Wild-type cells (K699). Localization of the particle and its formation is inhibited in (B) she5 deletion strain (K5205): 36% of cells with signal formed bright, single particles; approximately half of the particles were localized at the bud neck and 2% were localized in the bud. (C) she3 deletion strain (K5235): 6% formed bright, single particles and 0% were localized in the bud. (D) she1 deletion strain (K5209): 16% of cells with signal formed bright, single particles and 0% were localized in the bud. (E) she2 deletion strain (K5547): 0% formed bright, single particles and 0% were localized in the bud. (F) she4 deletion strain (K5560): 32% formed bright, single particles and 16% with signal were localized in the bud. Colocalization (yellow) of a functional myctagged she protein (red) with the particle (green). (G) She1myc. (H) She2myc. (I) She3myc. (J–I) She1myc with nonlocalized particle in nonbudding cell. (Half of the particles showed colocalization with She1myc.)

Molecular Cell, Vol. 2, 437–445, October, 1998

Directional switching:

there is a recombinational enhancer on the left arm of III that is activated in a but not α cells.

(see Haber Annual Review of Genetics)

CEN3 MAT HML HMR RE

Haber (Ann Rev Genetics 1998)