Chromatin Immunoprecipitation and the Chip on Chip technique. Fredrik Fagerström Billai B E A- Core Facility for Bioinformatics and Expression Analysis.

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Chromatin Immunoprecipitation and the Chip on Chip technique. Fredrik Fagerström Billai B E A- Core Facility for Bioinformatics and Expression Analysis. Department of Biosciences & Nutrition Karolinska Institutet

Agenda -General introduction to Chromatin and Transcriptional and Epigenetic control. -Chromatin Imunnoprecipitation, tiling arrays and the ”Chip on Chip” technique Fredrik Fagerström Billai, BEA

Chromatin

Fredrik Fagerström Billai, BEA A = 30nm fiber of an interphase chromosome B = Nucleosomes along a strand of DNA Images of Chromatin

Fredrik Fagerström Billai, BEA The Chromatin Subunit model

Fredrik Fagerström Billai, BEA The Nucleosome Crystal Structure

Fredrik Fagerström Billai, BEA Histones are modified at Specific Amino Acids Histone Code hypothesis - Modifications of the Histone tails act as marks that can be read by other proteins to control the expression or replication of chromosomal regions. The coding in the histones may be heritable.

Fredrik Fagerström Billai, BEA Specific modifications are associated with specific functions.

Transcriptional gene silencing Chromatin compaction Genome stability X chromosome inactivation (females) Fredrik Fagerström Billai, BEA Methylation of DNA

Fredrik Fagerström Billai, BEA

Eukaryotic mRNAs are synthesized by the Pol II complex

Fredrik Fagerström Billai, BEA Gene regulation in the eukaryotes Wasserman and Sandelin (2004)

Fredrik Fagerström Billai, BEA Chromatin Immunoprecipitation

Fredrik Fagerström Billai, BEA Chip- General outline

Fredrik Fagerström Billai, BEA Chip analysis of ERα recruitment to estrogen responsive gene promoters. Ray et al. 2006

Chip Quantification Fredrik Fagerström Billai, BEA Calella et al. 2007

Fredrik Fagerström Billai, BEA Real Time PCR quantification

Fredrik Fagerström Billai, BEA Real Time PCR quantification

Fredrik Fagerström Billai, BEA Chip on Chip Outline

Fredrik Fagerström Billai, BEA Genome Wide Chip Solutions Affymetrix Tiling ArraysClassic Two Color ArraysIllumina Deep Sequencing

Fredrik Fagerström Billai, BEA Millions of copies of a specific oligonucleotide probe Single stranded, labeled ‘target’ Oligonucleotide ‘probe’ * * * * * 1.28cm GeneChip Probe Array 1.28cm Affymetrix array architecture

Fredrik Fagerström Billai, BEA Affymetrix genechip family

Fredrik Fagerström Billai, BEA Tiling Arrays

Fredrik Fagerström Billai, BEA Boyer et al Tiling arrays provide high resolution for identifying bound fragments Overlapping 25-mer fragments

Fredrik Fagerström Billai, BEA Quantile Normalization

Fredrik Fagerström Billai, BEA Identifying bound genomic fragments by Intensity Percentile Ranking.

Fredrik Fagerström Billai, BEA Integrated Genome Browser, IGB

Fredrik Fagerström Billai, BEA Genome-wide profiling of PRC1Polycomb chromatin binding in Drosophila melanogaster.

Fredrik Fagerström Billai, BEA Chip profiles

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