Bio 445 Chromatin structure and Gene expression

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Biol/Chem 473
Schulze lecture 6 Introduction to chromatin
structure
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Control regions upstream of pair-rule genes are
complex
General principle regulatory regions of
eukaryotic genes are complex the promoter of any
given gene has to integrate a large amount of
combinatorial inputs that will define its
activity depending on the context of the cell in
which the gene resides. (Are bicoid levels high
or low? What about Kruppel? Etc. )
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Eukaryotic gene expression through the occluding
effects of chromatin
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What is chromatin?

• It is a solution to a eukaryotes packaging
  problem.

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Eukaryotes have a BIG packaging problem

• How do you fit approximately 2 meters (human
  diploid nucleus) into a space that averages maybe
  5 millionths of a meter wide?
• How do you replicate, repair and transcribe
  tightly packaged DNA?

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Solution Chromatin!

• Chromatin is DNA packaged with specialised
  proteins (and even some RNA!) that serve to
  control the degree to which DNA sequences are
  accessible for synthesis and transcription
• These proteins include specialized structural
  proteins and enzymes

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Chromatin packaging heirarchy
Level 1 nucleosome formation
Level 2 30 nm fiber
Level 3 Nuclear scaffolding
Level 4 Mitotic (metaphase) chromosome
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Level One Building blocks of chromatin
nucleosomes

• string on a bead for obvious reasons
• Linker can vary (8-114bp or more)
• Compaction ratio is approx 7 fold

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Nucleosomes are composed of histones

• 2/3 of chromatin mass is protein
• 95 of chromatin protein are histones
• H1, H2A, H2B, H3, H4

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Nucleosome structure
Nucleosome core particle octamer of histones
plus 146 bp DNA
Chromatosome octamer of histones plus 146 bp
DNA AND linker histone H1 (this term rarely used
now)
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Nucleosome core particle
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Level Two the 30nm fiber

• Requires Histone H1
• Compaction ratio approx 100 fold

Lehninger
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Level three nuclear scaffolding

• Not well understood
• Organization is not random involved sequence
  elements (red dots), more non-histone chromatin
  proteins and tethering to the nuclear envelope
  and matrix

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Genome contortions during the cell cycle
Time for replication, transcription
Time for cell division no gene expression
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Metaphase chromatin level 4 packaging fully
condensed
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Interphase chromatin levels 1-3 relatively
decondensed chromosomes

• Heterochromatin dark-staining, condensed (mostly
  simple-sequence DNA)
• Euchromatin light-staining, less condensed
  (complex sequence DNA e.g. genes)

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Summary chromatin

• DNA plus protein
• Enables extraordinary condensation and packaging
  of eukaryotic genomes
• Fundamental unit is the nucleosome
• Nucleosome consists of an octamer of histone
  proteins 2XH2A, 2X H2B, 2XH3 and 2XH4
• Between nucleosomes, a fifth histone, H1, acts as
  a linker (among other mysterious things)
• Gene expression in eukaryotes takes place in the
  context of highly packaged chromatin
• Regulation of gene expression by chromatin
  structure is epigenetic regulation

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Heterochromatin

• WHY?? WHY?? WHY??
• It is a pain to work with (lots of repetitive
  DNA)
• It KILLS gene expression (transcriptionally
  repressive)
• Its boring.

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Turning genes OFF may be more important that
turning genes ON

• Inverse dose response
• Delete a chunk of chromosome and background gene
  expression tends to go UP (suggesting most of the
  deleted genes are repressors)
• Differential gene expression in development
• Coming attraction!
• Genome surveillance
• Keep parasitic (middle repetitive) DNA from
  wreaking havoc in the genome

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Gene silencing
X
Genes
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Genome architecture chromatin domains
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Heterochromatin vs Euchromatin

• Stains darkly (highly condensed)
• Repetitive sequences
• Replicates later in the cell cycle
• Little or no recombination
• Transcriptionally repressive silences gene
  expression

• Stains lightly (decondensed)
• Single copy sequences (genes)
• Replicates early in the cell cycle
• Recombines
• Transcriptionally active permissive for gene
  expression

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How do eukaryotes replicate their linear DNA?
Primase makes primers for okazaki fragments as
well as first primer
DNA synthesis is continuous on the leading
strand, but discontinuous on the lagging strand
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Solution telomerase!
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Solution telomerase!
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Solution telomerase!
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Heterochromatin summary

• Localized to telomeres and regions flanking
  centromeres.
• Consists of repetitive sequences (mostly).
• Transcriptionally repressive.
• Study of heterochromatin revealed how chromatin
  affects gene expression.
• Many of the silencing mechanisms operating
  constitutively in heterochromatin are used by
  euchromatin as well to locally regulate gene
  expression.

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Why is there heterochromatin?

• Structural role?
• Homolog pairing at meiosis?
• graveyard for potentially parasitic elements?
• No reason just too much trouble to get rid of?

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It all started with flies.
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Drosophila gene nomenclature

• Early days of Drosophila research a gene was
  named after the phenotype that resulted when that
  gene was mutant. (Example the white gene results
  in loss of red pigmentation in the eye, so the
  eye is white)

• Now it a lot more complicated (and less
  consistent). Genes tend to be named after the
  products they encode. (Example DEAD box protein
  80 Dbp80)

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Position Effect Variegation (PEV) in Drosophila

• A euchromatic gene relocated next to or within
  heterochromatin will variegate (show variable
  silencing)
• The sequence of the gene has not changed, only
  its position
• Therefore this is an epigenetic (beyond
  genetic) effect

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The wild type white gene variegates because of
its position (PEV)
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Genetic screen for modifiers of PEV
Wallrath LL, Cur. Opin. Genet. Dev. 1998, 8147
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Chromatin associated proteins

• Su(var) 2-5 encodes Heterochromatin Protein 1
• a chromatin structural protein which recognizes
  methylated histone H3 and can also recognize
  and bind to itself
• Su(var) 3-9 encodes a histone methyl-transferase
• an enzyme which transfers a methyl group onto a
  specific lysine residue on Histone H3
• Su(var) 326 encodes a histone deacetylase
• an enzyme which removes acetyl groups from
  histones deacetylated histones are correlated
  with repressed chromatin
• these proteins (and others) are reading a HISTONE
  CODE

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The histone code hypothesis
would it make a good movie? Is Tom Hanks
available???
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So whats this histone code all about?

• Histones are subjected to a variety of post
  translational modifications (most often on the
  N-terminal tails)
• These modifications are generated by specific
  enzymes
• These modifications are recognized by proteins
  that can influence gene expression and other
  chromatin functions

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Histone modifications cont
From Khorasanizadeh, 2004.
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Protein domains in chromatin associated proteins
SMART (simple modular architectural research
tool) http//smart.embl-heidelberg.de/
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There are new histone modifications being
discovered