Post on 28-Dec-2015
The Genome is Organized in Chromatin
Nucleosome Breathing, Opening, and Gaping
A model for gaining access to core DNA
Nucleosome movement catalyzed by nucleosome remodeling complexes alter nucleosome phasing
250 bp
+ SWI/SNF
Position 1 Position 2Chromatin remodeler
1 2
Native PAGE
ATP
Nucleosome Sliding
Nucleosome Sliding using Micrococcal nuclease digestion
MNase
MNase
core
Linker
T
D
M
MNase
0.5kb
1kb
Silent vs. active chromatin show different micrococcal nuclease digestion patterns
MNaseEuchromatin
heterochromatinMore mobile nucleosome
Strongly positioned nucleosome
Genome Analysis of Nucleosome Spacing
linker
Nucleosome
ISWI is a Nucleosome Spacing Factor
• The implication is that ISWI chromatin remodelers can organize chromatin into a more repressive configuration, OR a more active one
• Since yeast has two ISWI ATPases with different effects on chromatin in vitro, they may have evolved contrasting activities
EcoRVBsp1286I Bsp1286I
Time
In vivo nucleosome mobility assay
Restriction enzyme accessibility
0 1.5’ 3’ 9’ 27’ 80’
0’
t1
Nucleosome Positioning (Phasing)Positioning may affect which regions of DNA are in the linker and which face of DNA is exposed on the nucleosome surface
Translationally Positioned Nucleosomes
Displacement of the DNA by 10 bp changes the sequences that are in the more exposed linker regions but does not alter which face of the DNA is protected by the histone surface and which is exposed to the exterior regions
DNAse I digestion and chromatin remodeling
Rotational Positioning describes the ‘Exposure’ of DNA on thesurface of the Nucleosome and Determines its Interactions with Proteins and other Factors.
Rotational Positioning
Nuclease resistant
•Any movement that differs from the helical repeat (~10.2 bp/turn) displaces DNA with reference to the histoneSurface
•A translational movement of half a helical turn (e.g., 5 bp) will alter thesurface exposure of a DNA sequence
•Nucleotides on the inside next to the histone octamer are more protected against nucleases than nucleotideson the outside.
Nucleosome movement during nucleosome remodeling
alter nucleosome phasing
• Histones can be modified by– Acetylation (Ac)– Ubiquitination (Ub)– Methylation (Me)– Phosphorylation (P)– Sumoylation (Su)
Unwrapping of chromatin can be facilitated two phenomenon:
• Changes in DNA methylation within promoters, CpG islands, and genic/intergenic regions.
• Modification in histones or histone variants.
Histone Modification MapHistone Modification Map
Map Target Protein DNA Binding Sites and Methylated DNA Regions Using Robust Protocols
Chromatin immunoprecipitation-on-chip (ChIPchip)is a powerful tool to map target protein DNA binding sites across entire genomes or within biologically important regions such as promoters.
This method is used to map chromatin structure and DNA binding sites of transcription factors andpolymerases.
Methylated DNA regions are accurately mapped using a combination of affinity-based enrichment, such as Methylated DNA Immunoprecipitation(MeDIP) or the Methylated CpG Island Recovery Assay (MIRA), followed by microarray analysis.
CpG islands are genomic regions that contain dense clusters of CG dinucleotides that are oftenassociated with gene promoters
Chromatin Immunoprecipitation-on-chip(ChIP-chip)
DNA Methylation (MeDIP-chip)
– A high throughput technology that allows detection of thousands of genes simultaneously
– Principle: base-pairing hybridization
Base-pairing– DNA: A-T and G-C– RNA: A-U and G-C
– Much rely on computer aids
– Central platform for functional genomics
Microarray
Types of DNA microarrays and their uses
• What is measured depends on the chip design and the laboratory protocol:– Expression
• Measure mRNA expression levels (usually polyadenylatedmRNA)
– Resequencing• Detect changes in genomic regions of interest
– Tiling• Tiles probes over an entire genome for various applications
(novel transcripts, ChIP, epigenetic modifications)
– SNP• Detect which known SNPs are in the tested DNA
Affymetrix Microarray Gene ChipsTwo types of microarray chips
cDNA chips: Probe cDNA (500~5,000 bases long) is immobilized
oligo chip: Oligonucleotide (20~80-mer oligos) is synthesized either in situ
(on-chip) or by conventional synthesis followed by on-chip immobilization
Clustering of entire yeast genome
The total genome activity of transcription factors and epigenetic modifications are far more complex than previously predicted.
• It is no longer sufficient to limit regulatory studies to promoter regionsor defined genomic loci. • To better understand the regulation of transcription, an unbiased, whole-genome approach is needed to reveals the full regulatory network activity of transcription factors and epigenetic modifications.
§ Mapping regions of transcription
§ Transcription factor binding sites
§ Sites of DNA methylation
§ Chromosomal origins of replication
§ RNA binding protein sites
§ LOH/ Chromosome copy numbers
Affymetrix Tiling ArraysAffymetrix Tiling Arrays
Universal Array for multiple applications
Surrogate StrategyMost expression arrays to date
Annotation StrategyExon arraysSplice variants
Tiling strategyUnbiased look at the genome
What is Tiling Array?What is Tiling Array?
Known Exons Unknown transcript
25-mer probes spaced every 35bp across an entire genome
High-Resolution Profiling of Histone Methylations in the Human Genome
Histone Methylation near Transcription Start Sites
Histone Methylation Patterns of Active and Inactive Genes
ChIP on chip
Cancer biology
Developmental biology
Immunology and infectious
disease
Plant research stress biology,
transgenic research
Application of Epigenetic control using Chip on chip