1. Histone Modifications Seminar in Bioinformatics Saar Gershuni 2005

2. Background – DNA Packing The DNA is packed in various levels of condensation in the nucleous (*10,000) Form of condensation – biological role Chromosomes, Euchromatin, heterochromatin, DNA strand

4. The Beads on a String The Histones form the 11nm strand. Are octamer build H3,H4,2HA,2HB monomers 146-147 bp are wrapped around every histone core The histone tail sequences account for 28% of the total amino acid content of the core histones “chromatin fiber folding: requirement for the Histone H4 n-terminal tail “, Benedetta Dorigo†, Thomas Schalch†, Kerstin Bystricky†, ‡ and timothy J. Richmond, journal of molecular biology 327, 1, 14 march 2003† ‡ Tail of histone h4 taken from cow

5. The Histone Core Wrapped With DNA

6. Chemical Review Acetyl Methyl Phosphoryl Ubiquitin

7. Histone Modifications De/Acetylation Methylation Phosphorylation Ubiquitination ADP-Rybosilation Swi/Snf complex, which, in vitro, uses the energy of ATP hydrolysis to disrupt histone-DNA interactions

8. Histone Modifications Map

9. Histone Modifications - Role Transcription – Acetylation/Methylation DNA repair – H2A -Phosphorilation Mitosis – chromosomal arrengement Chromatin assembly – DNA replication

11. Yi Zhang et al. Genes Dev. 2001; 15: 2343-2360 Figure 1. Sites of post-translational modificationson the histone tails

12. Examples of Biological Role of Histone Modifications Acetylation –Lisine Transcription – loosening the strand Replication – the positioning of histones Gcn5 – h3k14

13. Acetylation mechanism

14. Examples of Biological Role of Histone Modifications Phosphorylation – serine, threonine (scienceweek) Chromosomal condensation – H1,H3 Rsk-2, an H3 kinase, Coffin Lowry syndrome Transcription regulation - drosophila sex chromosomes serine 10 H3 in concert with H4K16

15. Coffin Lowry Syndrome Coffin-Lowry syndrome is a rare genetic disorder characterized by mental retardation; abnormalities of the head and facial area; large, soft hands with short, thin (tapered) fingers; short stature; and/or various skeletal abnormalities. Characteristic facial features may include an underdeveloped upper jawbone, an abnormally prominent brow, downslanting eyelid folds, widely spaced eyes, large ears, and/or unusually thick eyebrows. Skeletal abnormalities may include abnormal front-to-back and side-to-side curvature of the spine and unusual prominence of the breastbone. Coffin-Lowry syndrome is caused by mutations in the RSK2 gene and is inherited as an X-linked dominant genetic trait. Males are usually more severely affected than females.

16. Examples of Biological Role of Histone Modifications Methylation – Arginine, Lisine –Less studied, enzymology – not known –Can be mono-, bi-, tri- methylated –Transcription regulation - CARM1, arginine- specific, histone h3-selective methyltransferase activity, coactivator, with p160 family

17. Yi Zhang et al. Genes Dev. 2001; 15: 2343-2360 Figure 2. Chemistry of arginine and lysine methylation

18. Histone Code Code - a system of signals or symbols for communication (webster meriam online) Requirements from a code: – Consistent – Combinatorial (Kurdistany & Grunstein, 2003)

19. Mapping Global Histone Acetylation Patterns to Gene Expression Siavash K. Kurdistani, Saeed Tavazoie, and Michael Grunstein

20. Introduction The mechanism by which histone de/acetylation affect transcription involve two pathways: –By altering the folding properties of the chromatin fiber –By providing binding surface for recruitment of other elements To date (6/2004) there is no evidence for consistent patterns of de/acetylation from gene to gene or for the combinatorial use of histone modification sites

21. The Experiment – Data Collection & Methods Chromatin was extracted from YDS2 exponentially growing Using chip and DNA microarrays levels of modification was determined 11 sites of acetylation were examined – H4k8,12,16 H3k9,14,18,23,27 H2ak7,h2bk11,16

22. The Experiment - Methods Two Microarrays: 6700 IGR, 6200< ORF 2-4 repetitions Normalization by the ration of total intensities. Coefficient of variation < 0.5 between replicate experiment were counted End up with 2206 IGR, 2403 ORF Data were again normalized over the 11 sites per histone

23. ChIP

24. Results – Raw Data

25. Results – Correlation With Gene Expression These results – though significant might be artificially low due to technicalities.

26. Results - Clustering

27. Results – coexpression of Clusters Problem: what is the expression level of randomly selected cluster of genes? The study also checked expression levels at different stress conditions (255) – correlation was found in 6 IGR ’ s and 13 ORF ’ s

28. Results – Clusters Biological Relevance Annotations for all the genes in a cluster were taken from MIPS, GO, MDS 12/53 IGR’s, 13/68 ORF’s – with significant results Motif search using AlignACE algorithm found 102 of 29/53 IGR’s, 110 of 34/68 ORF’s

29. Does the Modifications Constitute a Code? The authors believe that the answer is no because: The total number of modifications does not contain more information than the sum of individual modification. Problem: it has been shown to be combinatorial – bdf1 in vitro preference for tetra acetylated H4.

30. Problems Cutting the chromatin fiber was done using sonicator bath thus creating various size of fiber – with various number of nucleosome – problem with measuring acetylation levels. Microarrays are 1kb in length can contain up to 5 nucleosomes.

31. Problems Normalization – step 1 – average number of 1 through all the genome. Step 2 – normalizing groups of 11 lysines in each and every locus (  =0, var=1) All the problems relates with k means algorithm, AlignACE, and gene expression data

32. Genomic Maps and Comparative Analysis of Histone Modifications in Human and Mouse Bradley E. Bernstein, Michael Kamal, Kerstin Lindblad-Toh, Stefan Bekiranov, Dione K. Bailey, Dana J. Huebert, Scott McMahon, Elinor K. Karlsson, Edward J. Kulbokas III, Thomas R. Gingeras, Stuart L. Schreiber, and Eric S. Lander

33. The Experiment Large scale study of histone modifications (methylation, acetylation) patterns in human and mouse cells Methods: ChIP, RTPCR – for validating the data, tiling oligonucleotide arrays – 35 bp intervalss Focus: chromosomes 21,22, (H3K4 di/trimethylation and H3K9,14 acetylation) and cytokine cluster, IL4 Receptor, and Hox clusters (H3K4 dimethylation)

34. Results – Raw Data 90%< correlation between methylated H3K4, and acetylated H3K9,14 Di/Trimethyl – gene start

35. Conservation of Modification pettern Between Human and Mouse For this purpose the IL4R methylation analysis were preformed 55% conservation in human, 68% conservation in mouse, (*7 than random) with no correlation with sequence conservation

36. Hox Clusters A group of linked regulatory homeobox genes that are involved in patterning the animal body axis during development. Homeobox genes are defined as those that contain an 180-base-pair sequence that encodes a DNA-binding helix–lturn–helix motif (a homeodomain). (Nature) The remaining orthologous regions between human and mouse

37. Methylation Patterns in Hox Clusters Completely unique. Contain huge methylated regions encompass multiple genes Evolutionary conserved (human-mouse) Methylation correlates with expression both in ORF’s and IGR’s unlike IL4R

38. Problems The method of creating the lysate is still sonication. No relation with genes functionality, cell cycle phase – can change nucleosome concentration

39. What Is It Good for? Genome wide understanding of chromatin role (structure, functionality( in biology The use of all the methods we learned Improve our understanding regarding various mechanisms and processes within the nucleous Develop new bioinformatic and biological methods for research (advanced ChIP technics, combination with tiling microarrays, and data analyzing tools – normalizations, intergrated tools)

40. Where Do We Go Next? Characterization of lysine 56 of histone H3 as an acetylation site in Saccharomyces cerevisiae. Ozdemir A, Spicuglia S, Lasonder E, Vermeulen M, Campsteijn C, Stunnenberg HG, Logie C. Epigenomic mapping in Arabidopsis using tiling microarrays. Martienssen RA, Doerge RW, Colot V. The epigenetic breakdown of cancer cells: from DNA methylation to histone modifications. Ballestar E, Esteller M.

41. Questions?