Eukaryotic chromatin = DNA (1 part) + proteins (2 parts)

• chromatin’s proteins are divided into two types: histones and nonhistones (such as transcription factors

The human genome is about 2 yards long but the average size of a nucleus is 10µm (microns) in diameter.

Enzymes need access to DNA, which is never naked, so changes are necessary in chromatin structure for these processes to take place.

Nucleosome — 67 nm DNA around 11 nm histone core (beads on a string), the fundamental unit of organization, the 6X comes from 67/11

EXPERIMENTS FROM THE TEXTBOOK

• break open the cells
• isolate the nuclei
• put nucleus in hypotonic media (low salt)
• nucleus is ruptured and chromatin spills out

beads = histone core

fiber = linker DNA

• lyse nuclei in hypotonic media
• mild digestion by micrococcal nuclease, which cuts both strands of the DNA helix for a short time and with little enzyme for incomplete digestion (not a restriction enzyme because it only has little sequence specificity)
• centrifuge mixture on sucrose gradient in excess of 50000 RPM (low density/low sucrose at top of gradient and high sucrose/high density at bottom). The particles will move according to shape and size (histones and DNA are still complexed together)
• fractionate according to gradient
• extract DNA so that DNA goes into solution and get rid of the protein
• run agarose gel with each lane corresponding to a fraction in order to separate DNA according to size
• micrococcal nuclease (MN) seems to cut in 200 bp intervals and the DNA of each lane/fraction is of a specific length that is a multiple of 200 bps
• linker region DNA is more accessible to MN cutting and the linker region is every 200 bps so nucleosomes are spaced every 200 bps
• 200 bp = mononucleosome

• digest mononucleosome with MN for longer
• 200
• 165 bp
• 146 bp — tightly bound to histones and this number is constant and universal

• histones
• 2 molecules of each core histone è H2A, H2B, H3, H4
• 1 molecule of H1
• 200 bp of DNA
• mononucleosome = core particle + linker
• core particle = core histones + 146 bp

200 bp DNA = 67 nm (6X packing)

DNA wraps almost twice around when its 67 nm of DNA is wound around 11 nm core.

DNA’s two turns, each diameter, take up almost all the 6nm height of the nucleosome

Two regions that are 80 bp apart on linear DNA are close to together on the nucleosome.

DNA helix with 10.5 bp per turn is wrapped around nucleosome. Is the DNA at every point in this wrapping equivalent in structure? Nope.

• use DNAase I, which cuts only one strand
• isolate and denature DNA

• on a flat glass surface, DNAase I cuts every 10.5 bps
• cutting periodicity = structural periodicity = 10.5 bp/turn
• once a turn the DNA is more accessible and so is cut there

• same experiment with core particle instead of glass surface and DNAase I cuts every 10.7 bp in some regions and every 10 bp at other regions
• high resolution mapping indicates that there is variation in cutting periodicity along the DNA that is wrapped around the core particle
• location of helix on core changes the structure slightly
• some regions are more stressed than others

TAKE HOME MESSAGE: THIS HAS IMPLICATIONS FOR DNA BINDING PROTEINS.

• binds linker DNA
• required for 30 nm fiber, somehow involved in organizing nucleosomes

• extraction under high salt conditions yields 30 nm fiber in presence, but not in absence of H1

• guess at 30 nm structure è helix of nucleosomes with a diameter of 30 nm (what the polymerases deal with)
• It is thought that H1 binds at enter and exit of helix at each nucleosome and interacts with other H1s at the helix center, holding all this together at the 40X level of packing
• 30 nm fiber thought to be organized into loops, with some regions attached to the matrix

• isolate chromatin
• extract (remove) histones
• treat with complete digestion by a combination of restriction enzymes. (No histones on the loops so restriction enzymes can cut) but the regions bound and protected by the matrix will not be cut
• extract DNA away from protein and sequence these matrix associated regions (MARs)
• no consensus sequence
• very A-T rich
• occur in many cases near 5’ regulatory region of transcription units ( not all regulatory regions are near attachment sites)
• DNA may be organized into transcription unit(s) loops

• crystallization tells us how the histones fit together
• H2A-H2B dimmer on either side
• All four histone proteins have histone fold structure

• Histone fold = helix + loop + helix + loop + helix ( + loop + helix for two of the core histones)
• A LOT OF DNA BINDING PROTEINS HAVE SIMILAR HELIX-LOOP-HELIX

• N terminal tails stick out from the core and are subject to post translational modifications
• Modifications of amino acid in tail can lead to changes in affinity of histone core with DNA by making the core less positive and bind the negatively charged DNA less tightly
• Examples: acetylation (of lysine), methylation, phosphorylation of serine of histone during mitosis to increase condensing)
• IMPORTANT FOR TRANSCRIPTION

• Look in 10 different cells to see if nucleosomes on our specific gene are arranged in the same or random places. Does the promoter always lie in the linker region?

• we’re interested in the specific red sequence (let’s say it’s a promoter)
• take nuclei from many cells and treat with MN to get mononucleosomes
• digest with restriction enzyme that will cut just adjacent to our sequence and look to see if RE site is in same place relative to linker region
• run agarose gel and get all fragments between 0-200 bp. Use radioactive P-32 in a probe we hybridize to our red sequence, melting the probe and incubating with the gel in order to locate our piece of DNA
• if this yields (and it does!) a unique, distinct band it’s because RE site is fixed and MN site is fixed from the structure
• if we observed instead a continuous smear on the gel it would mean that the MN site is at random distances from the RE site (this is not what we find!)
• In many cases, position of histone octamers is NOT random. THIS HAS IMPORTANT IMPLICATIONS FOR TRANSCRIPTION.
• This is called nucleosome positioning or nucleosome phasing.

• linear placement of DNA relative to histone
• changing location of histone alters which turns are in the linker region

• exposure of DNA helix on nucleosome surface
• changing location of turns by rotating affects the accessibility of the double helixbecause of the exposure or lack of exposure due to protection by the nucleosome surface

Changes in positioning are associated with transcription.

Experiment:

• URA3 gene in yeast has an inducible promoter and we can turn gene expression on and off and look at the two positioning cases
• Nucleosome positioning can change depending on the "transcriptional state" of the gene
• when the URA3 gene isturned off, nucleosomes have particular positions but when gene expression is turned on the nucleosome positions are randomized, return to ladder positions when turned off again
• ladder è smear è ladder

• isolate chromatin from specific cell type (specific because of gene expression particular to cell type)
• let’s use RBCs and b -globin gene ON (-300 to —50 region)
• digest with DNAase I incompletely
• certain sites are 100X more sensitive to cleavage
• use gel and hybridize probe

• often have hypersensitive sites in promoter region
• cleavage sites needn’t be continuous
• probably not bound to nucleosomes as need to be bound by transcription factors, which will later protect them

• occurs all along transcription unit, not just promoter
• if we look at sensitivity of b -globin isolated from RBCs we get digestion è sensitivity
• but not with b -globin from muscle è no sensitivity
• gene OFF 10% degraded
• gene ON 50% degraded
• transcribed genes are more open, making transcription unit more susceptible