procaryotic chromatin is packaged somewhat but not nearly as much as eucaryotic chromatin
Eukaryotic chromatin = DNA (1 part) + proteins (2 parts)
- chromatins 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 10Ám (microns) in diameter.
What happens to chromatin structure during transcription and replication?
Enzymes need access to DNA, which is never naked, so changes are necessary in chromatin structure for these processes to take place.
Levels of Organization the Packing Ratio
Nucleosomes around 30 nm fiber
Attachment of 30 nm fiber to matrix/core
1000X (interphase nucleus, which is when transcription and replication occur)
10000X (mitotic chromosome is even more condensed so that it can be packaged during cell division)
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
To Analyze the Nucleosome (FIGURE 19.17)
- 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
Spacing of Nucleosomes
- 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
400 bp = dinucleosome
600 bp = tirnucleosome etc
How is long is the linker and how long is the DNA around the histone core?
digest mononucleosome with MN for longer
- 165 bp
- 146 bp tightly bound to histones and this number is constant and universal
Whats in a nucleosome?
Nucleosome = core particle + linker DNA
- 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.
DNAs 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.
To determine if DNA at all points in wrapping is equivalent
- 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.
Whats the role of H1?
- 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
Is there a consensus sequence for attachment to the matrix? Whats the sequence? What are the proteins that make up 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
MARs = SARs (scaffold attachment regions)
start with mitotic chromosomes, extract out histones
use restriction enzyme (RE) DNAase to find scaffold attachment regions on chromosome scaffold
thought that same proteins are involved in SAR and MAR attachments and that the matrix may be a loose scaffold
The Histone Octamer
- 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
How does transcription machinery deal with chromatin structures? How does RNA polymerase deal with nucleosomes? How do DNA-binding proteins (tx factors) interact with DNA?
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?
FIGURE 19.29 and 19.30
How can we tell if nucleosomes are in specific positions?
- were interested in the specific red sequence (lets say its 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 its 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.
Two Types of Nucleosome Positioning
- 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.
- 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
isolate chromatin from specific cell type (specific because of gene expression particular to cell type)
lets 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
continue experiment with plasmidcontaining b
-globin gene in a test tube and only see hypersensitivity if RBC protein extract is added before or at the same time as histones. Histones, if added first, can block the binding of proteins from RBC extract. DNA-binding proteins can influence nucleosome position.
- often have hypersensitive sites in promoter region
- cleavage sites neednt 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
- but not with b
-globin from muscle è
- gene OFF 10% degraded
- gene ON 50% degraded
- transcribed genes are more open, making transcription unit more susceptible