protein and dna interaction

Protein and DNA interaction

• How to detect nucleosome position

Xuefeng Zhu

• Department of Medical Biochemistry and Cell Biology• 2009-11-17

Nucleosome and Chromatin

contents

• What is the nucleosome?

• How the people discover the nucleosome?

• micrococcal nuclease cleavage

• Nucleosome and transcription

• How to get nucleosome position?

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?

Levels of chromatin structure

What is the nucleosome?

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?

What is the nucleosome?

What is the nucleosome?In vitro reconsistution of nucleosome

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?

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?

Micrococcal nuclease

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

Nucleosome

micrococcal nuclease cleavage

Micrococcal nuclease

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

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

Who decides nucleosome position?

• Is nucleosome position random?

• Does it depend on the DNA sequence?

It majorly depends on DNA sequences, But not all.

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

Nucleosome position depends on coding region DNA sequnces and couples to transcription

What is function of nucleosome positioning?

Nucleosome and transcription

Nucleosome positioning is couple to transcription activity.

Coding region

Nucleosome and transcription

How to get nucleosome postion?

Example: Yeast

How to get nucleosome position?

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