Part 3 How Genomes Function

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Part 3How Genomes Function

• Transcription, translation and their regulation

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Chapter 10Accessing the Genome

• Chromosome package status, the accessibility

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10.1 Inside the nucleus

• Not all parts of the genome are readily
  accessible to the DNA-binding proteins that are
  responsible for its expression.
• Histones and other packaging proteins are not
  simply inert structures around which the DNA is
  wound

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10.1.1 The internal architecture of the
eukaryotic nucleus

• The inside of the nucleus is just as complex as
  the cytoplasm of the cell, the only difference
  being that, in contrast to the cytoplasm, the
  functional compartments within the nucleus are
  not individually enclosed by membranes, and so
  are not visible when the cell is observed using
  conventional light or electron microscopy
  techniques.
• The nucleus has a highly ordered internal
  structure.

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Each chromosome has its own territory within the
nucleus

• Remain stationary throughout the cell cycle

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Something New

• We describe Hi-C, a method that probes the
  three-dimensional architecture of whole genomes
  by coupling proximity-based ligation with
  massively parallel sequencing. We constructed
  spatial proximity maps of the human genome with
  Hi-C at a resolution of 1 megabase. These maps
  confirm the presence of chromosome territories
  and the spatial proximity of small, gene-rich
  chromosomes. We identified an additional level of
  genome organization that is characterized by the
  spatial segregation of open and closed chromatin
  to form two genome-wide compartments. At the
  megabase scale, the chromatin conformation is
  consistent with a fractal globule, a knot-free,
  polymer conformation that enables maximally dense
  packing while preserving the ability to easily
  fold and unfold any genomic locus. The fractal
  globule is distinct from the more commonly used
  globular equilibrium model. Our results
  demonstrate the power of Hi-C to map the dynamic
  conformations of whole genomes.

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Something New

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Something New
Lieberman-Aiden et al 2009. Comprehensive
mapping of long-range interactions reveals
folding principles of the human genome. Science
326289-293.
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Something New
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10.1.2 Chromatin domains

• Chromatin structure is hierarchic, ranging from
  the two lowest levels of DNA packaging the
  nucleosome and the 30 nm chromatin fiber to the
  metaphase chromosomes.
• Constitutive heterochromatin Centromeric and
  telomeric DNA as well as certain regions of some
  other chromosomes (human Y).
• Facultative heterochromatin
• Euchromatin less compact, accessible

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• Structural domains and functional domains
• Some structural domains contain genes that are
  not functionally related, and the boundaries of
  some structural domains lie within genes.

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Functional domains are defined by insulators

• Insulator 1-2kb in length, first discovered in
  Drosophila and have now been identified in a
  range of eukaryotes.

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Loop formation at insulator sites

• Krivega M, Dean A Insulators Organize Chromatin
  Emerging Rules of the Game. Mol Cell 2011,
  441-2.
• Wood Ashley M, Van Bortle K, Ramos E, Takenaka N,
  Rohrbaugh M, Jones Brian C, Jones Keith C, Corces
  Victor G Regulation of Chromatin Organization
  and Inducible Gene Expression by a Drosophila
  Insulator. Mol Cell 2011, 4429-38.

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Some functional domains contain locus control
regions
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10.2 Chromatin modifications and genome expression
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10.2.1 Chemical modification of histones

• Acetylation (???) of histones, the most best
  studied type in various modifications, influences
  many nuclear activities including genome
  expression.

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The histones in heterochromatin are generally
unacetylated whereas those in functional domains
are acetylated.
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• Histones deacetylation represses active regions
  of the genome
• Other types of histone modifications methylation
  (repress or activate, depends on, irreversible?,
  so long term), phosphorylation, ubiquitination,
  etc.
• Histone code
• Cause and effect?

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10.2.2 The influence of nucleosome remodeling on
genome expression
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10.3 DNA modification and genome expression

• Making chemical changes to the DNA, semipermanent
  silencing.
• The modified state is inherited in cell division.

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10.3.1 Genome silencing by DNA methylation
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• Cytosine methylation is relatively rare in lower
  eukaryotes, but in vertebrates up to 10 of the
  total cytosine bases in a genome are methylated,
  and in plants, the figure can be as high as 30.

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Methyl-CpG-binding proteins are components of the
Sin3 and NuRD histone deacetylase complex.
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Methylation is involved in genomic imprinting and
X activation

• Imprinting genes tend to occur in clusters.
• Igf2, a growth factor gene.
• H19 gene
• Evolutionary conflicts between the males and
  females.
• David Haig, Professor of Biology in Harvard
  university

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X activation

• Some 20 genes escape the process and remain
  functional.
• X inactivation center (Xic),
• Xist, transcribed into 25 kb noncoding RNA.
• Replacement of histone H2A, one of the members of
  the core octamer of the nucleosome (Section
  2.2.1), with a special histone, macroH2A1
• Deacetylation of histone H4, as usually occurs in
  heterochromatin
• Hypermethylation of certain DNA sequences,
  although this appears to occur after the inactive
  state has been set up.

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Something Not New

• DNA wrapped in nucleosomes is sterically
  occluded, creating obstacles for proteins that
  must bind it. How proteins gain access to DNA
  buried inside nucleosomes is not known.
• Here we report measurements of the rates of
  spontaneous nucleosome conformational changes in
  which a stretch of DNA transiently unwraps off
  the histone surface, starting from one end of the
  nucleosome, and then rewraps. The rates are
  rapid. Nucleosomal DNA remains fully wrapped for
  only about 250 ms before spontaneously
  unwrapping unwrapped DNA rewraps within about
  1050 ms.

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Something Not New

• Spontaneous unwrapping of nucleosomal DNA allows
  any protein rapid access even to buried stretches
  of the DNA. Our results explain how remodeling
  factors can be recruited to particular
  nucleosomes on a biologically relevant timescale,
  and they imply that the major impediment to entry
  of RNA polymerase into a nucleosome is rewrapping
  of nucleosomal DNA, not unwrapping.
• From Li G, et al. (2005) Rapid spontaneous
  accessibility of nucleosomal DNA. Nat Struct Mol
  Biol 1246-53

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Something New

• Then how to transcribe a gene?
• The nucleosome behaves as a fluctuating barrier
  that locally increases pause density, slows pause
  recovery, and reduces the apparent pause-free
  velocity of Pol II. The polymerase, rather than
  actively separating DNA from histones, functions
  instead as a ratchet that rectifies nucleosomal
  fluctuations.
• Hodges C, et al (2009) Nucleosomal Fluctuations
  Govern the Transcription Dynamics of RNA
  Polymerase II. Science 325626-628
• Otterstrom JJ, M. van Oijen A (2009) Nudging
  Through a Nucleosome. Science 325547-548

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Something New

• Multiple transcribing Pol II complexes can
  efficiently overcome the high nucleosomal barrier
  and displace the entire histone octamer.
  DNA-bound histone hexamer left behind the first
  complex of transcribing enzyme is evicted by the
  next Pol II complex. Thus transcription by single
  Pol II complexes allows survival of the original
  H3/H4 histones, while multiple, closely spaced
  complexes of transcribing Pol II can induce
  displacement of all core histones along the gene.
  
• Kulaeva OI, et al. (2010) RNA polymerase
  complexes cooperate to relieve the nucleosomal
  barrier and evict histones. PNAS 10711325-11330

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Something New

• At a low Pol II density transcription is
  accompanied by transient displacement/exchange of
  H2A/H2B dimer(s) the nucleosome structure is
  recovered before arrival of the next Pol II
  complex. At a higher density Pol II complexes
  encounter hexasomes that are missing H2A/H2B
  dimer(s) therefore, all core histones are
  evicted and exchanged.

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Discussion and speculation

• Niu DK and Wang YF (1995). Why animals have
  tumours. Acta Biotheoretica 43 279-280. ?????

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Further Reading

• Buratowski, S., and D. Moazed. 2005. Gene
  regulation Expression and silencing coupled.
  Nature 4351174-1175.
• Haig, D. 2004 Genomic imprinting and kinship how
  good is the evidence? Annual Review of Genetics
  38 553-585.
• Prendergast et al. 2007. Chromatin structure and
  evolution in the human genome. BMC Evol. Biol. 7
  72.
• Lieberman-Aiden et al 2009. Comprehensive
  mapping of long-range interactions reveals
  folding principles of the human genome. Science
  326289-293.