3Ctechnology: Identifying and connecting functional elements - PowerPoint PPT Presentation

Title: 3Ctechnology: Identifying and connecting functional elements


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3C-technologyIdentifying and connecting
functional elements

• Dekker Lab
• University of Massachusetts
• Program in Gene Function and Expression
• Department of Biochemistry and Molecular
  Pharmacology

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RationaleFunctional elements physically
interact with others
Gene-enhancer interaction
Packing the genome inside chromosomes
Interactions between unknown elements
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Chromosome Conformation Capture
(3C)Identification of elements by detection of
connections
Dekker et al. Science 2002 Dekker TiBS 2003
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Identification of transcription regulatory
elements
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3C

• Detection of connections between elements
• Identification of new elements
• Connecting known elements
• Connections suggest functional relationships
  between elements

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3C technology development

• Developed fully validated protocol
• Obtained proof-of-principle
• In progress increasing throughput
• No large-scale data yet
• I will present several proof-of-principles

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Identification of new elements that interact
with a known regulatory element
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The Beta-globin locus (ENm009)
?
LCR
e
g2
g1
b
d
Identify elements that interact with the
LCR K562 cells express the beta-globin
locus GM06990 cells no globin expression
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LCR interactions in the beta-globin locus
g2
g1
b
d
LCR
e
Globin OFF
Globin ON
10
Focus analysis on three looping interactions
g2
g1
b
d
LCR
e
Globin OFF
Globin ON
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Correlation between 3C interaction and
transcription
Most expressed globin gene
Globin OFF
Globin ON
EST found in erythroid cells
Expressed globin pseudogene
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Correlations with histone modifications
g2
g1
b
d
LCR
e
Globin OFF
Globin ON
Globin ON
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Correlations with histone modifications
g2
g1
b
d
LCR
e
Globin OFF
Globin ON
Globin ON
Correlation -
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Correlations with Dnase Hypersensitive sites
g2
g1
b
d
LCR
e
Globin OFF
Globin ON
Globin ON
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Correlations with Dnase Hypersensitive sites
g2
g1
b
d
LCR
e
Globin OFF
Globin ON
Globin ON
Correlation - ?
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Beta-globin summary
LCR interactors
g2
g1
b
d
LCR
e
Correlation with Transcription His.
Modifications Dnase HS




-


?
-
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Identification of new elements that regulate
known genes
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Identification of elements that regulate CFTR
expression (ENm001)
?
?
CFTR
Identify elements that interact with the promoter
of CFTR HT-29 CFTR expression K562 cells no
CFTR expression
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3C interactions of the promoter of CFTR
CFTR OFF
CFTR ON
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3C interactions of the promoter of CFTR
CFTR OFF
CFTR ON
Transcription-dependent interaction. Enhancer?
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Correlations with Dnase Hypersensitive sites
CFTR OFF
CFTR ON
CFTR OFF
CFTR ON
DnaseI HS correlates with CFTR expression
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A closer look
CFTR OFF
CFTR ON
CFTR OFF
CFTR ON
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Summary
g2
g1
b
d
LCR
e
Beta-globin
LCR-interactors are expressed some, but not
all, contain modified histones some, but not
all, contain HSs
CFTR
CFTR-interactor contains two HSs, one is
correlated with expression some evidence for
conservation