Beruflich Dokumente
Kultur Dokumente
Corey Henderson
Kyle Fowler
Introduction to Epigenetics
http://en.wikipedia.org/wiki/Epigenetics
The study of heritable changes in gene expression that occur without a change in DNA sequence.
Regulation differentiation From Structure not sequence
Heritable
Types
H1 H2A H2b H3 H4
Species Conservation
Eukaryotes Archea
Nucleosome Formation
Chromatin
Modifiable Tails
Methylation Acetylation
http://en.wikipedia.org/wiki/Ima ge:NucleosomeKG.jpg
Chromatin
DNA + Protien Enables extraordinary condensation and packaging of eukaryotic genomes Fundamental unit: NUCLEOSOME Gene expression in Eukaryotes takes place with highly packaged chromatin Regulation of gene expression by chromatin structure is epigenetic regulation
Heterochromatin
Highly condensed DNA
Lots of repeats
MIP
Methylation induced premeiotically
RNA
Sequence-specific degradation
RNAi
AKA
Cosuppression (plants) Quelling (Neurospora)
Epigenetics
Chromatin: An Overview
http://www.sciencemuseum.org.uk/on-line/lifecycle/133.asp
http://www.blackwellpublishing.com/11thhour/book5/about/images/c5t2.gif
Methylation
Phosphorylation Ubiquitination
http://www.umanitoba.ca/institutes/manitoba_institute_cell_biology/MICB/davie_jim_2.htm
http://www.benbest.com/health/cancer.html
Code regulates accessibility of DNA and transcription of genes Language by which information about chromatin and underlying genes is conveyed to other protein complexes Combinatorial (Jenuwein and Allis, 2001)
In this study: H3-K4 dimethylation, H3-K4 trimethylation, H3-Ac, H4-Ac, and H3-K79 dimethylation
6.
1.
Proteins not bound to DNA not crosslinked even at high concentrations Mild conditions can be used to reverse cross-links Effective in vivo or in vitro (Solomon and Varshavsky, 1985)
DNA
DNA
http://publish.uwo.ca/~jkiernan/formglut.htm
2. Fragmentation of Chromatin Sonication to break apart nucleosomes Believed to fragment DNA at random 500-700 bp Exposure of antibody-binding epitopes
http://openlearn.open.ac.uk/file.php/2645/formats/print.htm
ARTme2KQTARKSC IAQDFme2KTDLRF
http://www.creationsbydawn.net/pi/tutorials/rabbit.jpg
http://www.imgenex.com/images/antibody_image.jpg
http://www.abcam.com/index.html?datasheet=21623
Fluorescent labeling- Cy5 (red) and Cy3 (green)-labeled nucleotides incorporated in PCR
ChIP-on-Chip
5. Cohybridization of chromatin-bound DNA and nonimmunoprecipitated DNA sample (labeled with different fluorescent marker) to microarray 6. Ratio of fluorescent signals measure of enrichment due to ChIP
Mass Spectrometry
MS can be used to identify and quantify histone post-translational modifications (PTMs) on a proteomic scale If DNA sequence known, deviation from expected mass PTMs Identification of previously unknown modifications
Mass Spectrometry
Methodology
Histone Code
Post-translational covalent modifications to the histone tails
Methodology
Chromatin Immunoprecipitation (ChIP)
Isolation of DNA bound to chromatin Requires specific antibody or epitope tag to pull down protein (or modification) of interest Looked at relationship between genes and specific modifications (acetylation, methylation, phosphorylation, etc.)
Methodology
Schulze, S.
Methodology
Chromatin Immunoprecipitation (ChIP) ChIP on Chip (ChIPs!)
ChIP coupled with array Genomic approach to ChIP
Methodology
Methods in Epigenetics
Chromatin Immunoprecipitation (ChIP) ChIP on Chip ChIPs signal analysis
Bound
Input
Methods in Epigenetics
Transcriptional correlation beyond the gene level? Chromosome- & Genome-wide analysis
Entirety of H3-K4 methylation across chromosome 2L in Drosophila Related these data to cDNA array
www.cartoonstock.com
Dosage Compensation
Xist: X inactive-specific transcript (non coding RNA) Xist is expressed from both X chromosomes but only spreads and initiates silencing on ONE X
Schulze,S
Schulze, S
Questions?