Chromatin Structure and Its Effects on Transcription

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Chromatin Structureand Its Effects
onTranscription

• Du tian peng

Nov 5
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The Content of this Chapter

• 1 Histones and Nucleosome
• 2 DNase Hypersensitivity
• 3 Histone Acetylation
• 4 Histone Deacetylation
• 5 Chromatin Immunoprecipitation ( ChIP )
• 6 Chromatin Remodeling

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1.1 Histones

• Kinds H1, H2A, H2B, H3 and H4
• Extremely abundant the mass of histones in
  eukaryotic nuclei is equal to the mass of DNA
• Basic at least 20 of their amino acids are
  arginine or lysine , have a pronounced positive
  charge at neutral pH and can be extracted from
  cells with strong acids.

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1.2 Nucleosomes

• Nucleosomeshave a core of histones, around which
  the DNA winds
• Core histones a tetramer ( H3-H4 )2 and two
  dimers H2A-H2B
• DNA wrapped outside 146bp

The DNA winds almost twice around the
core,condensing the length of the DNA by a
factor of 6 - 7
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• The core histone tails binds to DNA and
  associates more closely with DNA,stabilizing the
  nuclosomes, and thereby inhibiting transcription.

• In addition, the very basic region of the
  N-terminal tail of histone H4 interacts with an
  acidic pocket in the H2A-H2B dimer of the
  adjoining nucleosome.So the cross-linking of
  nuclosomes can repress transtription.

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The Folding Order of Chromatin

• The nucleosome fiber is further folded into a
  30-nm, or condensed, fiber. One candidate for the
  structure of this fiber is a solenoid. Another is
  a variable zigzag ribbon. Histone H1 appears to
  play a part in formation of the 30-nm fiber, but
  its exact role is unclear

• Higher Order Chromatin Folding(unknown)

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1.3 The Effects of Histones on 5SrRNA Gene
Transcription

• Tow families
• oocyte 5S rRNA genes, 98, transcription only
  occurs in oocytes
• somatic 5S rRNA genes, about 400 genes, are
  transcribed in both oocytes and somatic cells

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• The apparent reason for this difference is that
  the somatic genes form more stable complexes with
  transcription factors. The transcription factors
  seem to keep the somatic genes active by
  preventing nucleosomes from forming a stable
  complex. Stable complex requires the
  participation of histone H1. Once the complex
  forms, transcription factors are excluded and the
  gene is repressed.

Fig1
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1.4 The Effect of Histones on Transcription of
Class ? Genes

• A detailed study to distinguish between the
  effects of the core histones on transcription by
  RNA polymerase ? in vitro.
• Core histones a mild repression, about 4-fold of
  genetic activity.
• histone H1 the repression became much more
  profound 25- to 100-fold.
• Transcription factors had no effect on this
  repression.
• This repression could be blocked by activators

Fig2
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First, (1) the nucleosomes could slow the
progress of all RNA polymerases by about 75, but
not stop any of them. (2) 75 of the polymerases
could be blocked entirely by nucleosomes, but 25
of the promoters might have been left free of
nucleosomes and thus could remain available to
RNA polymerase. A control experiment showed that
the remaining 25 transcription could be
eliminated by cutting the chromatin with a
restriction enzyme that cleaves just downstream
of the transcription start site. The fact that
this site was available indicated that it was
nucleosome-free. Thus, Second, hypothesis 2
is the right one.
Two possible explanations

• .

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2 DNase Hypersensitivity

• Sign of a nucleosome-free DNA region to DNase
• Active genes tend to have DNase-hypersensitive
  control regions. At least part of this
  hypersensitivity is due to the absence of
  nucleosomes.

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3 Histone Acetylation

• Acetylation occurs on the amino groups on lysine
  side chains and correlates with gene activity.
• Hypothesis there are enzymes in nuclei acetylate
  and deacetylate histones and thereby influence
  gene activity.
• These enzymes are present in low quantities in
  cells.
• Finally, in 1996, researchers succeeded in
  identifying and purifing a histone
  acetyltransferase ( HAT ), an enzyme that
  transfers acetyl groups from a donor ( acetyl-CoA
  ) to core histones.

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Experiment

• Extracts from macronuclei of tetrabymena(ciliated
  protozoan)
• SDS-gel electrophoresis (containing histones)
• Detect HAT activity by soaking the gel in a
  solution of acetyl-CoA labeled in its acetyl
  group with 3H
• Detect labeled histones first wash away the
  unreacted acetyl-CoA,then subjected the gel to
  fluorography

Fig3
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4 Histone Deacetylation

• The deacetylation of core histones allows the
  basic tails of the histones to bind strongly to
  DNA and to histones in neighboring nucleosomes,
  stabilizing and cross-linking the nucleosomes,
  and thereby inhibiting transcription

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The apparent mechanism of core histone
deacetylation would be a transcription repressing
event. (Model)
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How do we know a physical association exists
among transcription factors(Mad1), corepressors
(SIN3A) , and histone deacetylases(HDAC2)?
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Example of protein that can either activators or
repressors,depending on other molecules bound to
them
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5 Chromatin Immunoprecipitation ( ChIP )
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6 Chromatin Remodeling

• At least four classes of proteins participate in
  this chromatin remodeling, and all require ATP
  for activity.
• SWI/SNF family, ISWI family, NuRD family, and
  INO80 family.
• All four classes of proteins mobilizes
  nucleosomes and allows nucleosomes to move by
  sliding or by other mechanisms. making the DNA
  more accessible

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6.1 Remodeling in the Human IFN- ß Gene

• This process involves the following events the
  transcription factors recruit HATs, the SWI/SNF
  complex, and the general transcription factors.

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Thank you!
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Fig3
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Fig2
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Fig1