1. 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

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

3. Chromatin

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

5. Fredrik Fagerström Billai, BEA The Chromatin Subunit model

6. Fredrik Fagerström Billai, BEA The Nucleosome Crystal Structure

7. 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.

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

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

10. Fredrik Fagerström Billai, BEA

11. Eukaryotic mRNAs are synthesized by the Pol II complex

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

13. Fredrik Fagerström Billai, BEA Chromatin Immunoprecipitation

14. Fredrik Fagerström Billai, BEA Chip- General outline

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

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

17. Fredrik Fagerström Billai, BEA Real Time PCR quantification

18. Fredrik Fagerström Billai, BEA Real Time PCR quantification

19. Fredrik Fagerström Billai, BEA Chip on Chip Outline

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

21. 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

22. Fredrik Fagerström Billai, BEA Affymetrix genechip family

23. Fredrik Fagerström Billai, BEA Tiling Arrays

24. Fredrik Fagerström Billai, BEA Boyer et al. 2005 Tiling arrays provide high resolution for identifying bound fragments Overlapping 25-mer fragments

25. Fredrik Fagerström Billai, BEA Quantile Normalization

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

27. Fredrik Fagerström Billai, BEA Integrated Genome Browser, IGB

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

29. Fredrik Fagerström Billai, BEA Chip profiles