1. Protein and DNA interaction How to detect nucleosome position Xuefeng Zhu Department of Medical Biochemistry and Cell Biology 2009-11-17

2. Nucleosome and Chromatin

3. contents What is the nucleosome? How the people discover the nucleosome? micrococcal nuclease cleavage Nucleosome and transcription How to get nucleosome position?

4. DNA structure within the cell DNA is associated with proteins in cells, both prokaryotes and eukaryotes, even viruses. DNA and its associated proteins is called a chromosome What is the nucleosome?

5. Levels of chromatin structure What is the nucleosome?

6. Nucleosome Nucleosome is the basic block (lowest level) of chromatin organization. Most of genomic DNA is confined in nucleosomes (one nucleosome per 146 base pairs on average). Nucleosome core particle consist of histone octamer (two molecules each of H2A, H2B, H3 and H4 histone proteins) and super-coiled double- stranded DNA 125-166 base pairs long. What is the nucleosome?

8. In vitro reconsistution of nucleosome

9. Chromatin structure; oligomers of the histones. Kornberg RD, Thomas JO. Science. 1974 May 24;184(139):865-8. No abstract available. Chromatin structure: a repeating unit of histones and DNA. Kornberg RD. Science. 1974 May 24;184(139):868-71. No abstract available. The nucleosome, was described by Roger Kornberg in 1974. 1975,Cambridge 2006,Standford How the people discover the nucleosome?

10. First, partial digestion of chromatin with micrococcal nuclease (an enzyme that degrades DNA) was found to yield DNA fragments approximately 200 base pairs long. In contrast, a similar digestion of naked DNA (not associated with proteins) yielded a continuous smear of randomly sized fragments. These results suggested that the binding of proteins to DNA in chromatin protects regions of the DNA from nuclease digestion, so that the enzyme can attack DNA only at sites separated by approximately 200 base pairs. Consistent with this notion, electron microscopy revealed that chromatin fibers have a beaded appearance, with the beads spaced at intervals of approximately 200 base pairs. Thus, both the nuclease digestion and the electron microscopic studies suggested that chromatin is composed of repeating 200-base-pair units, which were called nucleosomes. Two types of experiments led to Kornberg's proposal of the nucleosome model. How the people discover the nucleosome?

11. Micrococcal nuclease Micrococcal nuclease is an endonuclease that cleaves DNA; in chromatin, DNA is cleaved preferentially between nucleosomes. Nucleosome micrococcal nuclease cleavage Micrococcal nuclease

12. Microccocal nuclease initially cleaves between nucleosomes. Mononucleosomes typically have ~200 bp DNA. End-trimming reduces the length of DNA first to ~165 bp, and then generates core particles with 146 bp. Microccocal nuclease initially cleaves between nucleosomes micrococcal nuclease cleavage

13. Micrococcal nuclease digests chromatin in nuclei into a multimeric series of DNA bands that can be separated by gel electrophoresis. Micrococcal nuclease digestion pattern micrococcal nuclease cleavage

14. Who decides nucleosome position? Is nucleosome position random? Does it depend on the DNA sequence? It majorly depends on DNA sequences, But not all.

15. How does DNA sequence contribute to nucleosome positioning? significant periodicities of dinucleotides and that DNA bending or flexibility helps in determining nucleosome position. Backbone inward Backbone out ~60% of predicted high occupancy sites confirmed in vivo. AA/TT/TA Dinucleotides model

16. Nucleosome position depends on coding region DNA sequnces and couples to transcription What is function of nucleosome positioning? Nucleosome and transcription

17. Nucleosome positioning is couple to transcription activity. Coding region Nucleosome and transcription

18. How to get nucleosome postion? Example: Yeast How to get nucleosome position?

19. Experimental work Bioinformatic Analysis Raw data from tiling array Affymetrix tiling array software Normlization & calculation P Value & signal Export Visualize in Integrated Genome Browser How to get nucleosome position? Yeast culture