Download - Epigenetic
Epigenetics
*Changes in gene expression or phenotype that don’t involve
changes to the DNA sequence()
*Its defined as heritable changes in gene activity and
expression that occur without alteration in DNA sequence
*Modern definition is non-sequence dependent inheritance.
*How do different adult stem cells know their fate?
*Myoblasts can only form muscle cells
*Keratinocytes only form skin cells
*Hematopoetic cells only become blood cells
*But all have identical DNA sequences.
*Identical Twins with
Different Hair Color
High
ecdysone
Low
ecdysone
Endocrine system mediating temperature-dependent
polyphenism: Bicyclus
*Mechanism of EpigeneticsChromatin Remodelling & Histone Modification
DNA Methylation
Non-coding RNA mediated pathway
Prion
Epigenetic code:
Consist of Both DNA methylation and histone modifications
Genetic code in each cell is the same
Epigenetic code is tissue and cell specific
*
Gene Expression
RNA Interference
Histone ModificationsDNA Methylation
*
*De/Acetylation
*Methylation
*Phosphorylation
*Ubiquitination
*ADP-Rybosilation
*Swi/Snf complex, which, in vitro, uses the energy of ATP hydrolysis to disrupt histone-DNA interactions
* Chromatin modifications with
impact on gene expression
*K9 histone H3 and H4 acetylation
*K9 H3 methylation
*K4 H3 methylation
*Ser 10 phosphorylation
*Ubiquitination
*Association of chromatin remodeling complexes
Acetylation/deacetylation of DNA
Condensation of DNA involves coiling around proteins called
histones. Acetylation is when acetyl groups (-COCH3) are
attached to lysines in the histone tails. This reduces
condensation and promotes transcription because the
transcription machinery has better access to the DNA.
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*Histone deacetylases (HDAC)s
Removal of acetyl groups by histone deacetylases (HDAC)s is generally associatedwith re-packing of chromatin and a lack of gene expression or gene silencing
Lysine acetylation is reversible and is controlled by the opposing actions of HATs and HDACs in vivo
Adcock IM, Ford P, Ito K and Barnes PJ. Respiratory Research 2007; 7 :21.
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Lysine residues in the tails of histone H3 and H4 may be acetylated forming bromodomains enabling the association of other co-activators such as
*TATA box binding protein (TBP),
*TBP-associated factors,
*chromatin modifying engines and
*RNA polymerase II.
Gene transcription occurs when the chromatin structure is opened up, with loosening of the tight nucleosomal structure allowing RNA polymerase II and basal transcription complexes to interact with DNA and initiate transcription.
MBD protein
Me Me Me
DNA
HMTMe Me Me
*Chromatine silencing
*Transcriptional
repressors such as
REST, Rb or by methyl-
CpG bindingproteins
induces deacetylation
of histone tails by
HDACs
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*
Chromatin Immunoprecipitation
Cross Link
Sonicate
Primary Ab & Protein G Beads
Ab Enriched DNA
Supe
after IgG
IP
INPUT
IP
Quantitate by
Real Time PCR
Identify proteins that interact with DNA
* Non-coding RNAs and their
epigenetic regulatory mechanisms
*It is widely accepted that ncRNAs (non-coding RNAs),
as opposed to protein-coding RNAs, represent the
majority of human transcripts; and the regulatory roles
of many of these ncRNAs have been elucidated over
the past decade. One important role so far recognized
for ncRNAs is their participation in the epigenetic
regulation of genes. Indeed, it is becoming increasingly
apparent that most epigenetic mechanisms of gene
expression are controlled by ncRNAs.
*RNA silencing and its
transmission
*
*siRNA-mediated suppression of transcription
associated with histone and DNA methylation of
mammalian cells that targets the promoter
region has been reported by several
independent laboratories
*DNA Methylation
*DNA methylation occurs at the Cytosine bases of DNA after
which the Cytosine dinucleotide is converted to 5-
methylcytosine by the activity of DNA methyltransferase
(DNMTs) enzymes
*Silencing:
Methylation of CpG sites within the promoters of
genes can lead to their silencing, a feature
found in a number of human cancers (eg.
silencing of tumor suppressor genes)
* Activation:
In contrast, the hypomethylation of CpG sites
has been associated with the over-expression of
oncogenes within cancer cells.
*
*technique used for the analysis of methylation at CpG island
in a significant sequence. In bisulphite sequencing, bisulphite
converts methylated Cytosines into methylation-dependent
SNPs. The advantage of bisulphite sequencing is that DNA
methylation patterns are detected in a single cell.
Study of DNA
methylation
**Bisulfite Conversion is the most widely used technique for
studying DNA methylation
*Converts non-methylated cytosines to uracil
* Methylated Cytosine remains a C.
* After PCR amplification all Uracils are converted Thymine.
* Comparing the sequences of the native DNA to the
bisulfite treated DNA will show you the methylated bases.
*
Bisulfite conversion
PCR
*
Wide range of techniques used to study DNA post-bisulfite conversion
*Methylation Specific Restriction Enzymes
*Sanger Sequencing or Pyrosequencing
*Microarrays
*PCR Techniques
Bisulfite Specific PCR (BSP)
COBRA – Combined Bisulfite Restriction Analysis
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