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
Eukaryotic Gene Regulation
Eukaryotic Gene Regulation
• Three common regulatory DNA sequences
• Core promoter
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Right next to TSS
Usually a TATA box + some other binding sites
Binds RNA polymerase II and associated TFs
• Proximal elements
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Just upstream (within ~200 nt)
Highly varied
• Enhancers and Silencers
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Can range from within the gene itself to ~200 – 100,000 nt away from promoter
Eukaryotic Gene Regulation
• Three common regulatory DNA sequences
• All are referred to as cis-acting regulatory sequences
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Cis-acting – impact genes on the same chromosome
• Proteins that interact with those regulatory sequence are trans-acting
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
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
• It’s expression is regulated by an enhancer sequence that is ~1Mb
away from the gene
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
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
Eukaryotic Gene Regulation
• Are mutations good or bad?
• Lactose tolerance
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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
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
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
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
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Alter chromatin structure
Are transmissible during cell division
Are reversible
Are directly associated with gene transcription
DO NOT alter the DNA sequence
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
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Generally,
Adding acetyl groups  increased transcription
Removing acetyl groups + adding methyl groups  silencing
Eukaryotic Gene Regulation
• Open chromatin vs. closed chromatin
• Open
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loose association b/t DNA and histone
DNA accessible to TFs
Transcriptionally active
• Closed
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DNA bound tightly to histones
DNA inaccessible
Transcriptionally inert
• How do we tell which regions are which?
Eukaryotic Gene Regulation
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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.
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
Eukaryotic Gene Regulation
• Chromatin remodelers
• Imitation switch (ISWI) complex
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Can ‘measure’ spaces between nucleosomes
Arranges nucleosomes into regular spaced pattern that serves to close
chromatin
Eukaryotic Gene Regulation
• Chromatin remodelers
• SWR1 complex
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Replaces H2A with H2A.Z variant histone
Interactions with other histone proteins are disrupted
Makes the histone octamer easy to displace
Eukaryotic Gene Regulation
• Chromatin remodelers
• Switch/Sucrose non-fermenting (SWI/SNF) complex
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Described in yeast
Slides or ejects histones to open chromatin
Consists of multiple proteins that vary by species
Eukaryotic Gene Regulation
• Chromatin modifiers
• Don’t 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
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Acetyl and methyl groups
• Chromatin writers, erasers, readers
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
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
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)
Eukaryotic Gene Regulation
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Imprinting
DNA methylation in mammalian cells
Methylated DNA bound by MeCP2
MeCP2 recruits histone deacetylases and methylases
Compacted chromatin, genes turned off
Eukaryotic Gene Regulation
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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
Eukaryotic Gene Regulation
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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
Eukaryotic Gene Regulation
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Gene silencing
Beckwith-Wiedemann syndrome (BWS)
~1/15,000 births
Increased risk of cancer (Wilms’ tumor)
Hemihypertrophy
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
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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.
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
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Processing of the original RNA
Formation of a RISC (RNA-Induced Silencing Complex)
Discard one strand of the RNA
Targeting and silencing
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
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https://www.youtube.com/watch?v=EtFHIT2mcsM
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
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https://www.youtube.com/watch?v=cK-OGB1_ELE
Do NOT click - https://www.youtube.com/watch?v=VfzCP3dhzs
Eukaryotic Gene Regulation
• RNA-mediated control
Eukaryotic Gene Regulation
• RNA-mediated control
• Thought to have evolved to protect
against viruses and TEs
• VERY useful as an experimental tool
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RNAi vs knockouts