mcb 317 genetics and genomics

MCB 317Genetics and Genomics

MCB 317 Topic 10, part 6A Story of Transcription

What is order of action in vivo?

How do we get at what’s going on in vivo?

Chromatin Immunoprecipitation(ChIP)

Does a specific protein of interest bind to a specific site on a chromosome in vivo?

UNLESS STATED OTHERWISE, WE ONLY LOOK AT PCR PRODUCTS FROM THE PPT

A

B

C D

Because formaldehyde crosslinks protein-DNA and protein-protein, each of the different proteins, A, B, C and D, will “ChIP” to the DNA that is bound by A.

An antibody to A, B, C, OR D will ppt thissegment of DNA

Also, can use epitope tagged versions of a gene rather than raise antibodies to every protein you want to ChIP

A

CD

Performing ChIP on mutant strains can give insight into the arrangement of proteins in the complex relative to DNA

A

B

C D

A

B

C

Wild-typeChIP signal from:A, B, C, D

Gene B deletedChIP signal from:A only

Gene D deletedChIP signal from:A, B and C

What is order of action in vivo?

An Imaginary Yeast Gene

UAS Pr YFG1 ORF

Act TBP

UAS Pr YFG1 ORF

Act

UAS Pr YFG1 ORFt = 0 min

t = 5 min

t = 10 min

UAS Pr YFG1 ORF

PrimerSet 1

PrimerSet 2

PCR on Total Purified Genomic DNA (not ChIP):

PrimerSet 1

PrimerSet 2

Set 1 +Set 2

Strain 1 = Activator is Epitope Tagged

UAS Pr YFG1 ORF

TBP

UAS Pr YFG1 ORF

Act


t = 5 min

t = 10 minAct

Strain 1 = Activator is Epitope Tagged

UAS Pr YFG1 ORF

TBP

UAS Pr YFG1 ORF

Act


t = 5 min

t = 10 minAct

Time (min)0 5 10

Set1

Set2

Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn

Strain 2 = TBP is Epitope Tagged

UAS Pr YFG1 ORF

TBP

UAS Pr YFG1 ORF

Act


t = 5 min

t = 10 minAct

Time (min)0 5 10

Set1

Set2

Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn

Strain 3 = Mediator is Epitope TaggedWhat Would You Conclude?

Time (min)0 5 10

Set1

Set2

Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxnC = Control = Pruified Genomic DNA (no ChIP)

15 C

Combine Data from 3 Strains -> Model

UAS Pr YFG1 ORF

TBP

UAS Pr YFG1 ORF

Act


t = 5 min

t = 10 minAct

UAS Pr YFG1 ORF

TBPt = 15 min

Act

Mediator

Order of events/action at the GAL1 promoter

Components

Also Gal4 activator protein

UAS Pr GAL1 ORF

Binding in vitro

Gal4 activator protein

UAS Pr GAL1 ORF

Gal4 TBP

Strategy:GAL1 OFF in Glucose -> ON in Galactose

Grow in Glucose -> shift to Galactose -> ChIP each component at various time points to determine when they bind control region

UAS Pr GAL1 ORF

UAS Pr GAL1 ORF

Glucose

Galactose

ChIP at 1 min, 2 min, 3 min, etc…..

GAL1 ChIP

UAS Pr GAL1 ORF

PCR

Perform ChIP for each component at each time point. NOTE: Each Component = different strain

Primer does not distinguish binding at UAS from binding at the Promoter

ChIP Resolution Limited by Fragment Size

UAS Pr GAL1 ORF

PCR

75 bp

Shear DNA 500-1000 bp Fragments

PU

PU

PU

PU

PU

PU

Conclusions from ChIP of GAL1 Control Region

Resolution could not distinguish binding at UAS vs. Promoter

1. Gal4 bound constitutively2. Gal4 recruits SAGA and Mediator independently3. SAGA does not recruit mediator4. Recruitment of mediator is not sufficient to recruit the

basal factors5. Mediator bound before RNAPII

Model of Recruitment at Gal1

Gal4

Saga

Mediator

RNAPII and Basal Factors

Dashed arrows = not addressed by this experiment

“Surprises” at Gal1

1. Gal4 bound constitutively2. Mediator binds independently of RNAPII

Order of events/action at the HO promoter

HO txn is cell cycle regulated: OFF in M -> ON in G1

URS2 Pr HO ORFURS1

Both URS1 and URS2 are required for txn of HO

RNAPIIBasal FactorsMediatorSwi/Snf chromatin remodeling complexSAGA (co-activator)SBF activatorSwi5 activator

Proteins that bind HO control region in vitro:

URS2 Pr HO ORFURS1

Swi5 SBF TBP et al

RNAPIIBasal FactorsMediatorSwi/Snf = chromatin remodeling complexSAGA = co-activator, histone acetylaseSBF = activatorSwi5 = activator

Primer Set 1 = S1 = URS1Primer Set 2 = S2 = URS2Primer Set 3 = S3 = Pr

URS2 Pr HO ORFURS1

Set 1 Set 2 Set 3

S1

S3

S2

1 2 3 4

PCR = Genomic DNA (not ChIP)Lane 1 = Set 1 onlyLane 2 = Set 2 onlyLane 3 = Set 3 onlyLane 4 = Set 1 + 2 + 3

“Multiplex PCR”

Primer sets can resolve URS1, URS2 and Pr in ChIP analysis

URS2 Pr HO ORFURS1

Set 1 Set 2 Set 3

Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min

S1

S3

S2

1 2 3 4

Swi5-tag

5

URS2 Pr HO ORFURS1

Set 1 Set 2 Set 3

Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min;Lane 5 = 8 min

S1

S3

S2

1 2 3 4

Swi5-tag Mediator-tag Swi/Snf-tag

5 1 2 3 4 5 1 2 3 4 5

Conclusions???

Model Derived From Data on Previous Slide

HO ORFPrURS2URS1t = 0 min



HO ORFPrURS2URS1t = 6 and 8 min

Swi5M

edia

tor

Swi/

Snf

Swi5

MediatorSwi/Snf





Swi5

Med

iato

r

Swi/

Snf

Swi5

MediatorSwi/Snf

Is Swi/Snf Chromatin remodeling complex required to recruit Mediator or is Swi5 sufficient?

URS2 Pr HO ORFURS1

Set 1 Set 2 Set 3

Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min

S1

S3

S2

1 2 3 4

Swi5-tag Mediator-tag Swi/Snf-tag

5 1 2 3 4 5 1 2 3 4 5

Swi2 = No functional Swi/Snf chromatin remodeling complex

Swi2 = No Swi/Snf chromatin remodelling complex





Swi5

Swi5

One Model Derived From Data on WT and snf2 strains





Swi5

Mediator

Swi/Snf

Swi5

Mediator

Swi/Snf

Order of initial events at HO in vivo

Order of initial events at HO in vivo

What evidence might lead you to draw this arrow?

In vivo order ofevents leadingto txn of the HO gene

“Surprises” at HO

1. Swi5 binds transiently2. Mediator at URS1, URS2 and Promoter3. Swi/Snf and mediator stay bound at URS1 after Swi5 is

no longer bound-- how?4. Swi/Snf and Saga arrive at URS2 before SBF -- how?5. SBF recruited to URS2 by Saga (activator recruited by a

co-activator)

1. Swi5 binds transiently2. Mediator at URS1, URS2 and Promoter3. Swi/Snf and mediator stay bound at URS1 after Swi5 is no longer bound- how?4. Swi/Snf and Saga arrive at URS2 before SBF- how?5. SBF recruited to URS2 by Saga (activator recruited by a co-activator)

PIR1, CLN2 and HO puzzles

Lodish 11-32

Lodish 11-32

URS = Upstream Repressor SequenceNot Regulatory Seq as in HO

UAS = enhancerURS = silencer

“Writers” and “Readers” of the Histone Code

ChIP of Histones

Antibodies against different modified forms of the Histone Tails

Use antibodies that are specific for histone modifications

Lodish 11-33

Puzzle?

Why does ChIP using Abs to the histone tails work?

What do the histone tails do?

mcb 317 genetics and genomics

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