Chromatin 99 Part I.

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Chromatin Structure

• Overview
• The structure of a promoter
• Levels of Chromatin Structure
• Levels of Regulation
• Role of the Nucleosome in gene regulation
• Role of histone modifying enzymes

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Chromatin Structure fulfills two key functions

• Packing
• Regulation of Gene Expression

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Packing

• DNA is incredibly long (and fragile) it can be
  up to 1.6 meters for the largest human
  chromosome. And yet it is packed within a nucleus
  of diameter only a few tens of microns.
• As a result the DNA must be extensively packed
  but at the same time it must be extensively
  available.
• We understand quite a lot about how it is
  packaged, but not so much about how it can be
  read.

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Details of chromatin packing

• Based on a fundamental unit called the
  nucleosome.
• An octamer of histones compacts146 bp of DNA (of
  length 14.6x34A) into a flat disk of thickness
  ca. 110A ie. 510A--gt 110A (About
  5x packing).
• Under the influence of H1 histone the extended
  nucleosome array collapses into a solenoid.
  (about 30X packing)
• Large solenoid loops are then anchored within the
  nuclear matrix. (Up to 1500x packing)

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Nucleosome Structure
This shows a single nucleosome with protein on
the inside.
And here is a polynucleosome with some DNA
between individual nucleosomes.
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How do we know this is the structure of the
nucleosome?
1. Of course we now have an X-Ray analysis. 2.
But the structure was pretty well
understood before the crystallography
studies. This was based upon 1.
Histone-Histone interactions. 2. Neutron
scattering. 3. DNase I footprinting. 4.
Micrococcal nuclease digestions. 5. Electron
Microscopic observations.
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Histone-Histone Interactions

• Histones are very basic proteins. This helps
  with the interaction with DNA. They are quite
  small. They were long known to aggregate at
  neutral PHs.
• Isenberg showed Histones H3 and H4 interact
  specifically, likewise H2A and H2B.
• Kornberg found that an octamer of two each of the
  above histones was stable. He then went on to
  argue that the octamer was the basic unit in
  vivo.
• The fifth histone H1 does not form complexes with
  the other histones

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DNase I footprinting of Nuclesomes.
1. Prepare nucleosomes and digest with DNaseI
for footprinting. 2. The results were
unexpected. 3. Need to be aware that DNase I
cuts single strands within a
duplex. The results are
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One does not get a footprint but rather a set of
bands spaced 10 bp apart!
This reflects cutting DNA which is on the outside
of a surface (the histones within).
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End-on and side views of models based on
nucleosome X-ray analysis (Nature, 1997, 389,
251-258).
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Histone-DNA interactions in one turn of the
nucleosome.
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Histone modifications.

• Added post translationally.
• Acetylation.
• Phosphorylation.
• Methylation.
• Ubiquitination.
• Wide range of stability of modifications.

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Sites of Histone Tail Modification in the
nucleosome.
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Micrococcal Nuclease, in contrast to DNase I,
cuts both strands at the same time
So this enzyme cannot cut DNA in a
nucleosome...but it can cut between nuclesomes.
Obtain a partial digest.
After digestion remove Histones.
DNA Products
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Electron Microscopy revealed nucleosomes!
(this work was done in Tennessee by Don and Ada
Olins).
They lysed nuclei, fixed with formalin at pH
9.0 and looked at the product.
But at lower pH!
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This led to the idea of the solenoid structure.
Solenoid requires H1 to form, H1 is fairly
extended and links nucleosomes, interacting
with spacer DNA.
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Finally, large loops of solenoid are anchored
into the nuclear matrix (protein).
Matrix
Solenoid
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Still alot to learn about chromatin structure.
So...we know alot about chromatin structure, and
how information is packaged within the
nucleus...but this just makes the problems of
finding the information harder. If we focused
upon a string of nucleosomes, then since some of
the DNA is on the outside, then this is not
insuperable. But with a solenoid, much of the DNA
is internal. Possibilities include some sort of
local extension for scanning purposes. Or
perhaps some way of marking important points in
the chromatin. Maybe a protein which says
important tissue-specific gene here, or,
ubiquitous gene nearby!. But how does the
marker protein know where to go?
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Role of Chromatin Structure in Gene Regulation.

• Chromatin structure of coding sequences of gene.
• Chromatin structure of the promoter.
• Role of the nucleosome in blocking access.
• Can nucleosomes be moved?
• Chromosome position effects.

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Chromatin Structure of a gene in the process of
being transcribed.

• Originally thought that transcriptionally active
  genes were nucleosome free.
• This is now known to be false.
• Probably due to using ribosomal genes as model,
  as well as artifactual proteolysis.
• Sound biochemical and microscopic evidence for
  active templates being nucleosomal.
• Ergo conclude that there is some mechanism for
  the polymerases to negotiate nucleosomes.

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The Nucleosome can block access to the PIC.
Experiments by Roeder showed that the nucleosome
can block TBP, factors and pol II.
Add nucleosome first Then add TFIID, factors and
pol II
Add factors and pol II first, then histones


II
II
TATA
Transcription!
No transcription!
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Chromatin Structure and Gene Expression
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Activation of the PHO 5 Gene by low phosphate
levels
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Chromatin Structure of HS 26 Gene.
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Structure of Human Interferon Beta Promoter.
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MMTV is an Inducible Gene in Mammals
-32
-210
-430
-600
-810
F
E
D
C
B
A
-200
NF1
HRE
Oct 1

• All these factors can bind naked DNA, but not all
  at once.
• NF1 and Oct1 cannot bind phased nucleosome alone.
• Two HRE sites become occupied on nucleosome
  surface.
• This exposes the next HRE site and binding
  occurs.
• This permits the binding of BOTH NF1 and Oct1.

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Binding depends on the nature of the MMTV
promoter substrate
Factors cannot all bind at the same time to DNA
substrate
Factors can all bind to DNA substrate when is it
is in nucleosomal conformation.
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An Example of Repression by Histones...Yeast
Telomeres.
A gene inserted into the telomere region becomes
repressed, depending on how far it is from the
end of the csome.
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Organization of Chromatin Structure in a Higher
eucaryote.
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Organization of Chromatin Structure in a Higher
eucaryote.
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Organization of Chromatin Structure in a Higher
eucaryote.
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Position Matters

• Chromosomes have both active and inactive genes.
• Variegated Position Effects.
• Activation from proximal and distal
  promoter.also repression.
• Enhancers (and repression).usually far from
  promoter.
• Locus Control Regions, silencers and insulators.

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Long Distance Regulation

• Transgenic constructs often dont transcribe.
• In differentiated cells, some promoters wont
  bind ubiquitous factors.
• British-Dutch thalessemia.
• Role of upstream hypersensitive sites.

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Locus Control Region (LCR).

• Is a DNA binding site far from the promoter.
• Usually binds both tissue specific and ubiquitous
  factors.
• Renders the promoter active regardless of its
  position in the chromatin.
• Works by opening up the nucleosome structure of
  the promoter, so factors can bind.

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How does an LCR work?
LCR
Acetylase
Pol II
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Sites of Histone Tail Modification in the
nucleosome.
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How do we find out where nucleosomes are excluded?
DNase I Hypersensitive Site Mapping.
Isolate nuclei
Naked DNA
Use very small amount of DNase I so that only ca.
one cut per gene is made, This cuts only naked
DNA, not nucleosomal DNA. Deproteinize Chromatin.
RI
RI
Probe
Cut with RI, run gel and blot.