Chromatin Structure and Its Effects on Transcription

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Chromatin Structureand Its Effects
onTranscription

• Jian Zhang (??)

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The Content of Chapter 13

• Histones
• Nucleosomes
• Chromatin Structure and Gene Activity

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13.1 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.
• Homogeneous as show in Table 13.1

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The reason for this variety

• gene reiteration repreated times ( 10-20 times
  in the mouse, and about 100 times in Drosophila )
• posttranslational modification acetylation on
• N-terminal amino groups and on lysinee-amino
• groups, lysinee-amino methylation and
• phosphorylation, serine and threonine O-
• phosphorylation, lysine and histidine N-
• phosphorylation.

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13.2 Nucleosomes

• Definition structures which are the first order
  of folding in eukaryotic chromatin.
• Core histones a tetramer ( H3-H4 )2 and two
  dimers H2A-H2B
• DNA wrapped outside 146bp

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The Folding Order of Chromatin

• Nucleosomes
• Nucleosome Filament
• The 30-nm Fiber
• Higher Order Chromatin Folding
• But,it has not been possible to crystallize any
  component of chromatin larger than the nucleosome
  core.

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13.3 Chromatin Structure and Gene Activity

• The Effects of Histones on 5SrRNA Gene
  Transcription
• The Effect of Histones on Transcription of Class
  ? Genes
• Nucleosome Positioning
• Histone Acetylation
• Histone Deacetylation
• Chromatin Remodeling
• Heterochromatin and Silening
• Nucleosomes and Transcription Elongation

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13.3.1The Effects of Histones on 5SrRNA Gene
Transcription

• Xenopus laevis 5S rRNA of frog
• Transcribed by RNA polymerase?, with the help of
  TF?A, TF?B, and TF?C
• Haploid number 20,000
• 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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What causes the oocyte genes to be active in
oocytes but inactive in somatic cells?

• An attractive interpretation
• Somatic cells contain transcription factors that
  are able to form stable preinitiation complexes
  with the somatic 5S rRNA genes, but are less
  successful in forming these comlexes with the
  oocyte 5S genes.
• So , nucleosomes form on the oocyte genes,
  including their promoters, and histone H1
  cross-links these nucleosomes in an ordered array
  and keeps them repressed.But, the transcription
  factors engaged on the somatic genes prevent
  nucleosomes from forming, or at least from
  forming an ordered, cross-linked structure.
  Therefore, these genes remain active.

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13.3.2 The Effect of Histones on Transcription of
Class ? Genes

• A detailed study to distinguish between the
  effects of the core histones and of histone H1 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

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• Two possible explanations
• First, the nucleosomes could slow the
  progress of all RNA polymerases by about 75, but
  not stop any of them.
• Second, 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,
  hypothesis 2 is the right one.
• Summary The core histones (H2A, H2B, H3, and H4
  ) assemble nucleosome cores on naked DNA.
  Transcription of reconstituted chromatin with an
  average of one nucleosome core per 200 bp of DNA
  exhibits about 75 repression relative to naked
  DNA. The remaining 25 is due to promoter sites
  not covered by nucleosome cores.

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13.3.3 Nucleosome Positioning

• Nucleosome-Free Zones
• DNase Hypersensitivity is another 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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13.3.4 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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Experiment1 Activity gel assay

• 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

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Summary

• Histone acetylation occurs in both the cytoplasm
  and nucleus.
• Cytoplasmic acetylation is carried out by a HAT
  B and prepares histones for incorporation into
  nucleosomes. The acetyl groups are later removed
  in the nucleus.
• Nuclear acetylation of core histone N-terminal
  tails is catalyzed by a HAT A and correlates
  with transcription activation. A variety of
  coactivators have HAT A activity, which may allow
  them to loosen the association of nucleosomes
  with a genes control region. Acetylation of core
  histone tails also attracts bromodomain proteins
  such as TAF?250 ,which are essential for
  transcription

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13.3.6 Chromatin Remodeling

• At least four classes of proteins SWI/SNF
  family, ISWI family, NuRD family, and INO80
  family. All require ATP for activity.
• All four classes of proteins alter the structure
  of nucleosome cores to make the DNA more
  accessible, not only to transcription factors,
  but also to nucleases.

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Remodeling in the Yeast HO Gene

• Researchers studied protein association with the
  HO gene of yeast, which plays a key role in
  switching the mating type.
• The expression of HO depends on a series of
  protein factors that appear at different phases
  of the cell cycle.

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Experiment2 Chromatin Immunoprecipitation ( ChIP
)

• Fuse DNA fragments encoding short regions of a
  protein(Myc) to the ends of genes encoding the
  proteins known to associate with the HO gene
• Make cell extracts and immunoprecipitate the
  DNA-protein complexes with antibodies against the
  Myc epitopes
• Verify the HO gene by PCR(with HO-specific
  primers)
• As show in Figure 13.32

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Summary

• Nucleosome remodeling agents such as SWI/SNF and
  ISWI are required for activation ( and repression
  ) of some genes. These proteins disrupt the core
  histones in nucleosomes, and may also move
  nucleosomes. Such remodeling, combined with core
  histone acetylation and binding of other
  proteins, can create nucleosome-free enhancers
  that can bind readily to transcription
  activators. Remodeling can also help move
  nucleosomes into position to repress
  transcription.

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13.3.7 Heterochromatin and Silening

• Euchromatin is relatively extended and open and
  at least potentially active.
• Heterochromatin is very condensed and its DNA is
  inaccessible. In higher eukaryotes it even
  appears as clumps when viewed microscopically (
  as shown in Figure 13.36 ).
• Heterochromatin is found at the telomeres and
  the centromeres of chromosomes.

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Histone Methylation

• Methylation can have either an activating or a
  repressing effect.
• Certain proteins involved in forming
  heterochromatin have conserved regions called
  chromodomains.
• One such protein is a histone methyl transferase
  ( HMTase ) whose human form is known as SUV39H
  HMTase.
• Another is a histone methyl transferase-associated
  protein called HP1.
• Targets for methylation lysine 9 and lysine 4 of
  histone H3

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13.3.8 Nucleosomes and Transcription Elongation

• How does RNA polymerase deal with the
  nucleosomes that lie within the transcribed
  region of a gene?
• As shown in Figure 13.42

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• When RNA polymerase moves through a single
  nucleosome, it can displace the nucleosome core
  to a new location behind the advancing polymerase
  on the same DNA. This may be accomplished by a
  spooling mechanism in which upstream DNA spools
  around core histones as downstream DNA unspools,
  with the RNA polymerase transcribing through a
  loop between these spooling and unspooling DNAs.
• Helpers in this process SWI/SNF HMG14 FACT
  and Spt4, 5, and 6.

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Thanks
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histoneH1

• Reconstitute chromatin with DNA containing two
  enhancer-promoter constructs
• ( 1 ) pG5E4, five GAL4-bingding sites coupled to
  the adenovirus E4 minimal promoter
• ( 2 ) pSV-Kr, six GC boxes from the SV40 early
  promoter coupled to the Drosophila Kruppel
  minimal promoter
• Not only add the core histones, but histone H1 in
  various
• quantities, from 0 to 1.5 molecules per core
  nucleosome
• Then transcribed the reconstituted chromatin in
  vitro.

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