Mcb 317 genetics and genomics
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MCB 317 Genetics and Genomics. MCB 317 Topic 10, part 6 A 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 ?.

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Mcb 317 genetics and genomics

MCB 317Genetics and Genomics

MCB 317 Topic 10, part 6

A Story of Transcription


Mcb 317 genetics and genomics

What is order of action in vivo?


Mcb 317 genetics and genomics

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?


Mcb 317 genetics and genomics

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


Mcb 317 genetics and genomics

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.

C

D

B

A

An antibody to A, B, C, OR D will ppt this

segment of DNA

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


Mcb 317 genetics and genomics

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

D

C

C

D

C

B

B

A

A

A

Wild-type

ChIP signal from:

A, B, C, D

Gene B deleted

ChIP signal from:

A only

Gene D deleted

ChIP signal from:

A, B and C


Mcb 317 genetics and genomics

What is order of action in vivo?


An imaginary yeast gene

An Imaginary Yeast Gene

t = 0 min

UAS

Pr

YFG1 ORF

Act

t = 5 min

UAS

Pr

YFG1 ORF

Act

TBP

t = 10 min

UAS

Pr

YFG1 ORF


Mcb 317 genetics and genomics

UAS

Pr

YFG1 ORF

Primer

Set 2

Primer

Set 1

PCR on Total Purified Genomic DNA (not ChIP):

Primer

Set 1

Primer

Set 2

Set 1 +

Set 2


Strain 1 activator is epitope tagged

Act

Act

Strain 1 = Activator is Epitope Tagged

t = 0 min

UAS

Pr

YFG1 ORF

t = 5 min

UAS

Pr

YFG1 ORF

TBP

t = 10 min

UAS

Pr

YFG1 ORF


Strain 1 activator is epitope tagged1

Act

Act

Strain 1 = Activator is Epitope Tagged

t = 0 min

UAS

Pr

YFG1 ORF

Set1

t = 5 min

UAS

Pr

YFG1 ORF

Set2

TBP

t = 10 min

0

5

10

UAS

Pr

YFG1 ORF

Time (min)

Primer Set 1 = UAS; Primer Set 2 = Pr

Both Sets of Primers are in each PCR rxn


Strain 2 tbp is epitope tagged

Strain 2 = TBP is Epitope Tagged

t = 0 min

UAS

Pr

YFG1 ORF

Act

Set1

t = 5 min

UAS

Pr

YFG1 ORF

Set2

Act

TBP

t = 10 min

0

5

10

UAS

Pr

YFG1 ORF

Time (min)

Primer Set 1 = UAS; Primer Set 2 = Pr

Both Sets of Primers are in each PCR rxn


Strain 3 mediator is epitope tagged what would you conclude

Strain 3 = Mediator is Epitope TaggedWhat Would You Conclude?

Set1

Set2

5

10

15

C

0

Time (min)

Primer Set 1 = UAS; Primer Set 2 = Pr

Both Sets of Primers are in each PCR rxn

C = Control = Pruified Genomic DNA (no ChIP)


Combine data from 3 strains model

Combine Data from 3 Strains -> Model

t = 0 min

UAS

Pr

YFG1 ORF

Act

t = 5 min

UAS

Pr

YFG1 ORF

Act

TBP

t = 10 min

UAS

Pr

YFG1 ORF

Mediator

Act

TBP

t = 15 min

UAS

Pr

YFG1 ORF


Order of events action at the gal1 promoter

Order of events/action at the GAL1 promoter


Components

Components

UAS

Pr

GAL1 ORF

Also Gal4 activator protein


Binding in vitro

Binding in vitro

Gal4

TBP

UAS

Pr

GAL1 ORF

Gal4 activator protein


Mcb 317 genetics and genomics

UAS

UAS

Pr

Pr

GAL1 ORF

GAL1 ORF

Strategy:GAL1 OFF in Glucose -> ON in GalactoseGrow in Glucose -> shift to Galactose -> ChIP each component at various time points to determine when they bind control region

Glucose

Galactose

ChIP at 1 min, 2 min, 3 min, etc…..


Gal1 chip

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

ChIP Resolution Limited by Fragment Size

75 bp

UAS

Pr

GAL1 ORF

PCR

Shear DNA

500-1000 bp Fragments

U

P

U

P

U

P

U

P

U

P

U

P


Conclusions from chip of gal1 control region

Conclusions from ChIP of GAL1 Control Region

Resolution could not distinguish binding at UAS vs. Promoter

1. Gal4 bound constitutively

2. Gal4 recruits SAGA and Mediator independently

3.SAGA does not recruit mediator

4.Recruitment of mediator is not sufficient to recruit the basal factors

5.Mediator bound before RNAPII


Model of recruitment at gal1

Model of Recruitment at Gal1

Saga

RNAPII and Basal Factors

Gal4

Mediator

Dashed arrows = not addressed by this experiment


Surprises at gal1

“Surprises” at Gal1

  • Gal4 bound constitutively

  • Mediator binds independently of RNAPII


Order of events action at the ho promoter

Order of events/action at the HO promoter


Ho txn is cell cycle regulated off in m on in g 1

URS1

URS2

Pr

HO ORF

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

Both URS1 and URS2 are required for txn of HO

RNAPII

Basal Factors

Mediator

Swi/Snf chromatin remodeling complex

SAGA (co-activator)

SBF activator

Swi5 activator


Proteins that bind ho control region in vitro

Proteins that bind HO control region in vitro:

TBP et al

Swi5

SBF

URS1

URS2

Pr

HO ORF

RNAPII

Basal Factors

Mediator

Swi/Snf = chromatin remodeling complex

SAGA = co-activator, histone acetylase

SBF = activator

Swi5 = activator


Mcb 317 genetics and genomics

URS1

URS2

Pr

HO ORF

Set 1

Set 2

Set 3

S1

S3

S2

1

2

3

4

Primer Set 1 = S1 = URS1

Primer Set 2 = S2 = URS2

Primer Set 3 = S3 = Pr

PCR = Genomic DNA (not ChIP)

Lane 1 = Set 1 only

Lane 2 = Set 2 only

Lane 3 = Set 3 only

Lane 4 = Set 1 + 2 + 3

“Multiplex PCR”

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


Mcb 317 genetics and genomics

URS1

URS2

Pr

HO ORF

Set 1

Set 2

Set 3

Swi5-tag

S1

S3

S2

1

2

3

4

5

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


Mcb 317 genetics and genomics

URS1

URS2

Pr

HO ORF

Set 1

Set 2

Set 3

Mediator-tag

Swi/Snf-tag

Swi5-tag

S1

S3

S2

1

1

1

2

3

4

5

2

3

4

5

2

3

4

5

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

Lane 5 = 8 min

Conclusions???


Model derived from data on previous slide

Model Derived From Data on Previous Slide

t = 0 min

URS1

URS2

Pr

HO ORF

Swi5

t = 2 min

URS1

URS2

Pr

HO ORF

Swi/Snf

Mediator

Swi5

t = 4 min

URS1

URS2

Pr

HO ORF

Swi/Snf

Mediator

t = 6 and

8 min

URS1

URS2

Pr

HO ORF


Is swi snf chromatin remodeling complex required to recruit mediator or is swi5 sufficient

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

t = 0 min

URS1

URS2

Pr

HO ORF

Swi5

t = 2 min

URS1

URS2

Pr

HO ORF

Swi/Snf

Mediator

Swi5

t = 4 min

URS1

URS2

Pr

HO ORF

Swi/Snf

Mediator

t = 6 and

8 min

URS1

URS2

Pr

HO ORF


Swi2 no functional swi snf chromatin remodeling complex

URS1

URS2

Pr

HO ORF

Set 1

Set 2

Set 3

Swi2 = No functional Swi/Snf chromatin remodeling complex

Mediator-tag

Swi/Snf-tag

Swi5-tag

S1

S3

S2

1

1

1

2

3

4

5

2

3

4

5

2

3

4

5

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


Swi2 no swi snf chromatin remodelling complex

Swi2 = No Swi/Snf chromatin remodelling complex

t = 0 min

URS1

URS2

Pr

HO ORF

Swi5

t = 2 min

URS1

URS2

Pr

HO ORF

Swi5

t = 4 min

URS1

URS2

Pr

HO ORF

t = 6 and

8 min

URS1

URS2

Pr

HO ORF


One model derived from data on wt and snf2 strains

One Model Derived From Data on WT and snf2 strains

t = 0 min

URS1

URS2

Pr

HO ORF

Swi5

t = 2 min

URS1

URS2

Pr

HO ORF

Mediator

Swi/Snf

Swi5

t = 4 min

URS1

URS2

Pr

HO ORF

Mediator

Swi/Snf

t = 6 and

8 min

URS1

URS2

Pr

HO ORF


Mcb 317 genetics and genomics

Order of initial events at HO in vivo


Mcb 317 genetics and genomics

Order of initial events at HO in vivo

What evidence might lead you to draw this arrow?


Mcb 317 genetics and genomics

In vivo order of

events leading

to txn of the HO gene


Surprises at ho

“Surprises” at HO

  • Swi5 binds transiently

  • Mediator at URS1, URS2 and Promoter

  • Swi/Snf and mediator stay bound at URS1 after Swi5 is no longer bound-- how?

  • Swi/Snf and Saga arrive at URS2 before SBF -- how?

  • SBF recruited to URS2 by Saga (activator recruited by a co-activator)


Mcb 317 genetics and genomics

  • Swi5 binds transiently

  • Mediator at URS1, URS2 and Promoter

  • Swi/Snf and mediator stay bound at URS1 after Swi5 is no longer bound- how?

  • Swi/Snf and Saga arrive at URS2 before SBF- how?

  • SBF recruited to URS2 by Saga (activator recruited by a co-activator)


Mcb 317 genetics and genomics

PIR1, CLN2 and HO puzzles


Mcb 317 genetics and genomics

Lodish 11-32


Mcb 317 genetics and genomics

URS = Upstream Repressor Sequence

Not Regulatory Seq as in HO

UAS = enhancer

URS = silencer

Lodish 11-32


Writers and readers of the histone code

“Writers” and “Readers”of the Histone Code


Chip of histones antibodies against different modified forms of the histone tails

ChIP of HistonesAntibodies against different modified forms of the Histone Tails


Mcb 317 genetics and genomics

Use antibodies that are specific for histone modifications


Mcb 317 genetics and genomics

Lodish 11-33

Puzzle?

Why does ChIP using Abs to the histone tails work?

What do the histone tails do?


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