Transcript on-chip
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 Nucleosome Sliding 250 bp + Position 1 1 SWI/SNF Chromatin remodeler 2 Native PAGE ATP Position 2 Nucleosome Sliding using Micrococcal nuclease digestion MNase core MNase Linker MNase 1kb 0.5kb T D M Silent vs. active chromatin show different micrococcal nuclease digestion patterns Euchromatin heterochromatin MNase More mobile nucleosome Strongly positioned nucleosome Genome Analysis of Nucleosome Spacing Nucleosome linker 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 Restriction enzyme accessibility Bsp1286I EcoRV Bsp1286I 0’ t1 Time 0 In vivo nucleosome mobility assay 1.5’ 3’ 9’ 27’ 80’ 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 Rotational Positioning describes the ‘Exposure’ of DNA on the surface of the Nucleosome and Determines its Interactions with Proteins and other Factors. •Any movement that differs from the helical repeat (~10.2 bp/turn) displaces DNA with reference to the histone Surface •A translational movement of half a helical turn (e.g., 5 bp) will alter the surface exposure of a DNA sequence •Nucleotides on the inside next to the histone octamer are more protected against nucleases than nucleotides on the outside. Nuclease resistant Nucleosome movement during nucleosome remodeling alter nucleosome phasing 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. • Histones can be modified by – Acetylation (Ac) – Ubiquitination (Ub) – Methylation (Me) – Phosphorylation (P) – Sumoylation (Su) Histone 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 often associated with gene promoters Chromatin Immunoprecipitation-on-chip (ChIP-chip) DNA Methylation (MeDIP-chip) Microarray – 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 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 polyadenylated mRNA) – 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 Chips Two 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 regions or defined genomic loci. • To better understand the regulation of transcription, an unbiased, wholegenome approach is needed to reveals the full regulatory network activity of transcription factors and epigenetic modifications. Affymetrix Tiling Arrays § Mapping regions of transcription § Transcription factor binding sites § Sites of DNA methylation § Chromosomal origins of replication § RNA binding protein sites § LOH/ Chromosome copy numbers Universal Array for multiple applications What is Tiling Array? Unknown transcript Known Exons Surrogate Strategy Most expression arrays to date Annotation Strategy Exon arrays Splice variants 25-mer probes spaced every 35bp across an entire genome Tiling strategy Unbiased look at the 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 Application of Epigenetic control using Chip on chip Immunology and infectious disease Cancer biology ChIP on chip Developmental biology Plant research stress biology, transgenic research