Transcript Epigenetic regulation of lymphocyte development and function

Epigenetic regulation of lymphocyte
development and function
Hodaka Fujii, M.D., Ph.D.
Combined Program on Microbiology and Immunology
Research Institute for Microbial Diseases
April 14, 2015
Today’s topics
1. What is epigenetics?
2. Known mechanisms of epigenetic regulation
3. Methods to analyze epigenetic regulation
4. Future direction
1. What is epigenetics?
What is epigenetics?
Epigenetics is the study of heritable changes in
gene expression or cellular phenotype caused by
mechanisms other than changes in the underlying
DNA sequence – hence the name epi- (Greek: επίover, above, outer) -genetics. It refers to
functionally relevant modifications to the genome
that do not involve a change in the nucleotide
sequence.
Central issue in the post-genome era:
Molecular mechanisms of epigenetic regulation
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Leads to discovery of
potential novel
biological phenomena
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Lineage commitment
Cell differentiation
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Genome Imprinting
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RNA
X chromosome inactivation
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2. Known mechanisms of
epigenetic regulation
Known mechanisms of epigenetic regulation
・ DNA modification, eg. methylation
・ Modification / replacement of histones
・ DNA-binding proteins
・ Intra / inter-chromosomal interaction
・ Binding of RNA
DNA modification: methylation of cytosine 1
DNA modification: methylation of cytosine 2
DNA modification: methylation of cytosine 3
hydroxymethylation
Histone modification 1
Histone modification 2
Histone code hypothesis
Histone replacement 1
Histone replacement 2
DNA-binding proteins
Inter- & intra-chromosome interaction 1
Inter- & intra-chromosome interaction 2
olfactory receptors
nuclear receptor target genes
CD4 & CD8
Non-coding RNA in epigenetic regulation
3. Methods to analyze epigenetic
regulation
Conventional methods for epigenetics research I
Genetic approaches 1
1. Forward genetics
Mutagenize (mutagen, P-element, etc.)
↓
Isolate mutants
↓
Identify causative genes
Bacteria
Yeasts
Plants
Nematodes
Fruit flies
Zebra fish
Mouse
Human
mutant
WT
Cannot find redundant genes
Takes long time to elucidate molecular mechanisms
Conventional methods for epigenetics research I
Genetic approaches 2
2. Reverse genetics
Knockout genes
encoding
modification
enzymes of DNA or
histones or other
candidate molecules
Not non-biased screening
Global but not local
Conventional methods for epigenetics research II
Identification of gene regulatory regions 1
DNase hypersensitivity sites
Conventional methods for epigenetics research II
Identification of gene regulatory regions 2
Formaldehyde-assisted isolation of regulatory elements (FAIRE)
Conventional methods for epigenetics research II
Identification of gene regulatory regions 2
Promoter / enhancer analysis
Reporter assay
Not physiological
How to find regulatory regions?
Conventional methods for epigenetics research III
Analysis of specific genomic regions 1
1. Protein analysis：Identification of chromatin-binding proteins
(i) Identification of chromatin-binding proteins
DNA affinity purification, Yeast one-hybrid, IP, etc.
non-physiological
conditions
Conventional methods for epigenetics research III
Analysis of specific genomic regions 2
1. Protein analysis：Identification of chromatin-binding proteins
(i) Identification of chromatin-binding proteins
DNA affinity purification, Yeast one-hybrid, IP, etc.
non-physiological
conditions
Conventional methods for epigenetics research III
Analysis of specific genomic regions 3
1. Protein analysis：Identification of chromatin-binding proteins
(ii) Chromatin Immunoprecipitation
(ChIP)
Histone modification, detection of DNAbinding proteins
not non-bias screening
Conventional methods for epigenetics research III
Analysis of specific genomic regions 4
1. Protein analysis：Identification of chromatin-binding proteins
(iii) Imaging: localization of proteins
2. DNA analysis：Detection of DNA modification and
intra/interchromosomal interaction
(i) Fluorescence in situ Hybridization (FISH)
3. RNA analysis：Identification of chromatin-bound RNA
(i) FISH
not non-bias screening
low resolution
Conventional methods for epigenetics research IV
Epigenomics
Genome-wide mapping of epigenetic marks
1. DNA modifications
2. Histone modifications
3. ChIP analysis of DNA-binding proteins / Histones
4. 3C (see below)
5. RNA
Descriptive
Methods to directly identify
molecular interaction in the
genome
Why are methods to directly identify molecular
interaction in the genome necessary?
1. Conventional methods are time consuming.
eg. Analysis of regulation of gene expression
a. Identification of cis-regulatory elements: reporter assay
b. Identification of binding proteins: in vitro affinity purification, yeast
one hybrid
c. Verification of interaction in vivo: ChIP
d. Functional analysis: loss-of-function experiments
It takes more than ten years to make a story!
2. Frequently, conventional methods produce nonsense results.
Chromosome Conformation Capture (3C) and its derivatives
Dekker et al., Science (2002)
- Incomplete digestion causes amplification of adjacent regions, which
inhibits amplification of interacting genomic regions.
- Multiple enzymatic reactions in non-optimal conditions
- 3C-based methods generate some data, but do they really represent
interaction? Another indication of accessibility?
de Wit & de Laat, Genes & Dev., 2012
Non-biased analysis of molecules bound to specific
genomic regions of interest
PURIFY!
Biochemical and molecular biological analysis
Proteomics of isolated chromatin segments (PICh)
Déjardin et al. Cell 2009
LNA: locked nucleic acid
- Successful in
identification of
telomere-binding
proteins
- Telomere: multiple
copies
- Applicable to singlecopy or low-copy
genes?
insertional chromatin immunoprecipitation: iChIP
8 x LexA BE
160 bp
Direct detection of molecular
interaction in the genome
Hoshino & Fujii, J. Biosci. Bioeng., 2009
Fujita & Fujii, PLoS One, 2011
US patent: 8415098; Japan patent 5413924
iChIP by using homologous recombination
ZFN or TALEN
targeting vector
LexA-binding elements
Homologous
recombination
Neor
genomic DNA
genomic DNA
a target region
-Neor
(Cre-LoxP)
iChIP
Guidelines:
1. Evolutionarily NON-conserved regions
a target region
(e.g. promoter)
2. Several
hundred base apart from the
Random integration
Into genomic DNA
reporter
genomic
regions of interest
LexA-binding elements
iChIP by using random integration
genomic DNA
Selection marker
(e.g. Neor)
iChIP
Zinc finger nuclease (ZFN), TALEN & Cas9
ZFN
Cas9
TAL (trans activator-like)
engineered DNA-binding molecule-mediated
chromatin immunoprecipitation: enChIP
Fujita et al., Sci. Rep. (2013)
enChIP using the CRISPR system
Fujita & Fujii, BBRC (2013)
Advantages of locus-specific ChIP (iChIP & enChIP)
1. Non-biased method: does not need any prior knowledge
about targets.
2. Speed: takes much shorter time.
eg. p68 in an insulator complex
x 70 acceleration!!
conventional methods: 17 years
iChIP:
3 months
3. Generality: any molecules including DNA, RNA, and proteins
can be analyzed.
4. Robustness: calibration is easy.
5. Higher sensitivity:
x 103 - 104 more sensitive!!
PICh: telomere 103 - 104 copies / genome
iChIP:
1 copies / genome
Comparison of non-biased methods to analyze
specific genomic regions
Method
iChIP
enChIP
3C
PICh
DNA
analysis
Protein
analysis
RNA
analysis
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○ X X
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Not
reported
Not
reported
Include
enzyme
reactions
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(-)
(+)
(-)
Low copy #
genes
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?
Need
transgenic
Allelespecific
analysis
LexA BE
Insertion
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Yes
X
No
X
No
X
Yes
Contact
藤井穂高
Hodaka Fujii
感染症学免疫学融合プログラム推進室・准教授 (Principal
Investigator)
Associate Professor
Combined Program on Microbiology and Immunology
大阪大学 微生物病研究所
Research Institute for Microbial Diseases
[email protected]
http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/index.html
Tel/Fax: 06-6879-8358