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