Eukaryotic control of gene expression is similar to bacterial control but more complicated

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Eukaryotic Gene Regulation

• Eukaryotic control of gene expression is similar
  to bacterial control but more complicated
• Still involves activators and repressors and
  their associated binding sites, but there are
  many more and the interactions are more complex
• Also, regulation can take place at more levels
  due to the separation of the genome from the
  cytoplasm and the increased number of processing
  steps

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Eukaryotic Gene Regulation
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Eukaryotic Gene Regulation

• Three common regulatory DNA sequences
• Core promoter
• Right next to TSS
• Usually a TATA box some other binding sites
• Binds RNA polymerase II and associated TFs
• Proximal elements
• Just upstream (within 200 nt)
• Highly varied
• Enhancers and Silencers
• Can range from within the gene itself to 200
  100,000 nt away from promoter

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Eukaryotic Gene Regulation

• Three common regulatory DNA sequences
• All are referred to as cis-acting regulatory
  sequences
• Cis-acting impact genes on the same chromosome
• Proteins that interact with those regulatory
  sequence are trans-acting

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Eukaryotic Gene Regulation

• Three common regulatory DNA sequences
• Enhancers and Silencers contain sequences that
  are bound by regulatory proteins
• They act from a distance via DNA loops and
  protein intermediaries

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Eukaryotic Gene Regulation

• Enhancers and Silencers acting from a distance do
  so via DNA loops and protein intermediaries
• They contain sequences that are bound by
  regulatory proteins
• Sonic hedgehog (SHH) is a gene that directs limb
  formation in mammals
• Its expression is regulated by an enhancer
  sequence that is 1Mb away from the gene

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Eukaryotic Gene Regulation

• Different regulatory sequences can direct the
  same genes to be expressed in different ways
  under different circumstances or in different
  tissues
• SHH is expressed in both brain and limb
  development but under different circumstances and
  at different times
• Tissue-specific enhancers will be bound by
  tissue-specific TFs to modulate these differences

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Eukaryotic Gene Regulation

• Different regulatory sequences can direct the
  same genes to be expressed in different ways
  under different circumstances or in different
  tissues
• Locus control regions are specialized enhancers
  that regulate multiple genes in a coordinated
  fashion
• Multiple globin genes produce globins with
  slightly different oxygen affinities, which are
  expressed at different times during development

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Eukaryotic Gene Regulation

• Are mutations good or bad?
• Lactose tolerance
• Most adults in non-European populations are
  lactose-intolerant
• Normal in mammals Lactase gene is switched
  off after weaning
• Some human populations have high prevalence of
  lactase persistance in adults
• High prevalence is associated with cultures that
  began herding cattle 4-6 thousand years ago
• In these cultures, lactose tolerance confers an
  advantage
• Mechanism/mutation
• In European populations, the difference between
  persistence and non-persistence results from the
  difference in a single nucleotide located 13,910
  bases upstream of the lactase gene. T lactase
  persistence, C lactase non-persistence
• Another SNP -14,000 bp upstream of the lactase
  gene is associated with lactase persistence in
  some African populations.
• Gerbault et al. 2011 Phil. Trans R. Soc. B 366
  863-877

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Eukaryotic Gene Regulation

• Insulators Cis-acting sequences located between
  enhancers and the promoters of genes that need to
  be protected from their action
• Ensure that only the target gene is regulated by
  the enhancer
• Encourage loops or are bound by proteins that
  prevent interaction of the enhancer with the
  wrong promoter

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Eukaryotic Gene Regulation

• Regulation via chromatin remodeling
• Recall that chromatin can be either loosely
  compacted (euchromatin ) or densely compacted
  (heterochromatin)
• Euchromatin transcriptionally active
• Regions can switch back and forth depending on
  the needs of a cell

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Eukaryotic Gene Regulation

• Epigenetic control
• Some proteins tag histones and DNA by adding or
  removing methyl, acetyl and phosphoryl groups
• These tags alter (remodel) chromatin
• Epigenetic modifications
• Alter chromatin structure
• Are transmissible during cell division
• Are reversible
• Are directly associated with gene transcription
• DO NOT alter the DNA sequence

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Eukaryotic Gene Regulation

• If DNA is packed into chromatin, how do
  activators, repressors, etc. access the binding
  sites?
• 1. Some sites are just accessible in the linker
  DNA that extends between nucleosomes
• 2. Chromatin remodeling enzymes can move histones
  around
• 3. Chromatin modifiers can add or remove acetyl
  or methyl groups to alter packing
• Generally,
• Adding acetyl groups ? increased transcription
• Removing acetyl groups adding methyl groups ?
  silencing

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Eukaryotic Gene Regulation

• Open chromatin vs. closed chromatin
• Open
• loose association b/t DNA and histone
• DNA accessible to TFs
• Transcriptionally active
• Closed
• DNA bound tightly to histones
• DNA inaccessible
• Transcriptionally inert
• How do we tell which regions are which?

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Eukaryotic Gene Regulation

• How do we tell which regions are open or closed?
• DNase I is an enzyme that cuts naked DNA
• Only cuts in regions that are not bound by
  histones ? open
• DNase I hypersensitive sites are common in
  regions of transcribed genes, promoters, etc.

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Eukaryotic Gene Regulation

• How are the nucleosomes moved around to
  expose/hide binding sites?
• Chromatin remodelers
• Reposition or eject histones via multiple
  mechanisms and multiple enzyme complexes

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Eukaryotic Gene Regulation

• Chromatin remodelers
• Imitation switch (ISWI) complex
• Can measure spaces between nucleosomes
• Arranges nucleosomes into regular spaced pattern
  that serves to close chromatin

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Eukaryotic Gene Regulation

• Chromatin remodelers
• SWR1 complex
• Replaces H2A with H2A.Z variant histone
• Interactions with other histone proteins are
  disrupted
• Makes the histone octamer easy to displace

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Eukaryotic Gene Regulation

• Chromatin remodelers
• Switch/Sucrose non-fermenting (SWI/SNF) complex
• Described in yeast
• Slides or ejects histones to open chromatin
• Consists of multiple proteins that vary by
  species

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Eukaryotic Gene Regulation

• Chromatin modifiers
• Dont remove histones or move them
• Instead, they chemically alter them by adding or
  removing chemical groups
• Alter the strength of the DNA-histone
  interactions, leading to open or closed promoters
• Most common chemical modifications
• Acetyl and methyl groups
• Chromatin writers, erasers, readers

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Eukaryotic Gene Regulation

• Histone acetyltransferases (HATs)
• Add acetyl groups (writers)
• Addition of acetyl groups neutralizes positive
  charge on histone tails, relaxes histone-DNA
  interaction
• Recruited by activators
• Histone deacetylases (HDACs)
• Remove acetyl groups (erasers)
• Recruited by repressors

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Eukaryotic Gene Regulation

• Histone acetyltransferases (HATs)
• Add acetyl groups (writers)
• Addition of acetyl groups neutralizes positive
  charge on histone tails, relaxes histone-DNA
  interaction
• Recruited by activators
• Histone deacetylases (HDACs)
• Remove acetyl groups (erasers)
• Recruited by repressors

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Eukaryotic Gene Regulation

• Histone methyltransferases (HMTs)
• Add methyl groups (writers)
• Addition of methyl groups can lead to either open
  or closed chromatin depending on which amino
  acids are methylated and how many methyl groups
  are transferred
• Histone demethylases
• Remove methyl groups (erasers)

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Eukaryotic Gene Regulation

• Imprinting
• DNA methylation in mammalian cells
• Methylated DNA bound by MeCP2
• MeCP2 recruits histone deacetylases and
  methylases
• Compacted chromatin, genes turned off

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Eukaryotic Gene Regulation

• Gene silencing
• Imprinting selective expression of one parental
  allele
• Neighboring genes, Igf2 and H19, are on and off
  depending on parental source
• What is involved in this regulation?
• Downstream enhancer
• CTCF regulatory protein
• ICR imprinting control region

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Eukaryotic Gene Regulation

• Gene silencing
• Activators bound to enhancer could potentially
  activate both genes
• Maternal chromosome is unmethylated in this
  region
• Lack of methylation allows binding of CTCF to ICR
• CTCF blocks activation of Igf2
• allows activation of H19
• Paternal chromosome is methylated in this region
• Methylation blocks binding of ICR
• blocks activation of H19 via MeCP2

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Eukaryotic Gene Regulation

• Gene silencing
• Beckwith-Wiedemann syndrome (BWS)
• 1/15,000 births
• Increased risk of cancer (Wilms tumor)
• Hemihypertrophy

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Eukaryotic Gene Regulation

• RNA-mediated control
• Small RNAs have been found to be key components
  of gene regulation
• Discovered when researchers wanted to induce a
  particular color petal in petunias by injecting
  transcripts that would encode the pigment
• Instead, they found that all pigment production
  stopped
• Referred to as RNAi (RNA interference)
• Can act transcriptionally or post-transcription
• The basic idea
• Short RNAs complementary to the target gene
  direct proteins to that gene or to the transcript
  to eliminate production of the gene product
• The small RNA/protein complex either
• A. enters the nucleus to shut down transcription
  of the gene or,
• B. targets the transcripts of the gene for
  destruction or,
• C. prevents translation of the transcript.

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Eukaryotic Gene Regulation

• RNA-mediated control
• Source of the small RNAs?
• Various hairpin forming transcripts in the genome
  ? microRNAs (miRNA)
• Externally supplied dsRNA ? small interfering
  RNAs (siRNA)
• These source RNAs are usually 100-200 bp
• Two different but overlapping pathways
• Both pathways involve
• Processing of the original RNA
• Formation of a RISC (RNA-Induced Silencing
  Complex)
• Discard one strand of the RNA
• Targeting and silencing

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Eukaryotic Gene Regulation

• RNA-mediated control
• siRNA pathway
• Processing of the original RNA with Dicer
• Formation of a RISC (RNA-induced silencing
  complex)
• Formation of single strand
• Targeting and silencing
• https//www.youtube.com/watch?vEtFHIT2mcsM

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Eukaryotic Gene Regulation

• RNA-mediated control
• miRNA pathway
• Processing of the original RNA with Dicer and
  Drosha
• Formation of a RISC (RNA-induced silencing
  complex)
• Formation of single strand
• Targeting and silencing
• https//www.youtube.com/watch?vcK-OGB1_ELE
• Do NOT click - https//www.youtube.com/watch?vVfz
  C-P3dhzs

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Eukaryotic Gene Regulation

• RNA-mediated control

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Eukaryotic Gene Regulation

• RNA-mediated control
• Thought to have evolved to protect against
  viruses and TEs
• VERY useful as an experimental tool
• RNAi vs knockouts