Lecture 4: Differential

Gene Expression
Mechanisms of differential gene expression
Gene expression can be regulated at different levels of the central dogma.

DNA Reduced or increased transcription

• Epigenetic mechanisms (chromatin methylation/acetylation,
DNA methylation)
• Transcription factors

RNA Alternative mRNA splicing

• Differential mRNA longevity
• Selective inhibition of translation
• microRNAs
• Control of mRNA localization
protein Post-translational modifications
Regulating transcription
• Transcription can be regulated by many different mechanisms. Two of the
major mechanisms in embryonic development are:
• Epigenetic modifications
• Control through transcription factors
Epigenetic modification
• Epigenetics: refers to the study of gene expression changes that do not involve changes to the
DNA sequence.
• Examples: histone acetylation or histone methylation of the tails of histones H3 and H4.
• Acetylation and methylation occur on lysine residues on the histone tails.
Histone acetylation – opening chromatin
• Acetylation: Histone acetyltransferases place acetyl groups on histone tails. Acetyl groups
neutralize the positive charge on the histone tails. This loosens DNA and opens the
chromatin. Deacetyltransferases remove acetyl groups.
Histone methylation – opening or closing
chromatin
• Methylation: histone methyltransferases
catalyze the transfer of methyl groups onto
histone tails. Demethylases remove histone
groups.
• Depending on which lysine and which
histone tail is methylated, histone
methylation can enhance or prevent
transcription.
• Example:
• H3K9me2/3, H3K27me3, and H4K20me3
combined with no acetylation are
associated with highly condensed (and
silenced) chromatin.
• H3K4me3 in combination with acetylation
is associated with open (and active)
chromatin.
DNA methylation at promoters

CpG island
5’ ACTGA CGCG ATCC CG TCGTACGCG A CG Gene X 3’

Promoter
DNA methylation
• There are two types of promoters based on CpG content:
• High CpG-content promoters (HCPs):
• Developmental control genes (genes that regulate synthesis of TFs and regulatory proteins
used in the construction of the organism)
• Default state of these promoters is ON
• These CpG islands are not usually methylated
• To be turned off: histone methylation
• Low CpG-content promoters (LCPs):
• Genes whose products characterize mature, differentiated cells (e.g. globins of red blood
cells, hormones of pancreatic cells)
• Default state of these promoters is OFF
• The Cs in these promoters are methylated and this methylation is critical in preventing
transcription
• To be turned on: DNA methylation at the CpG sites is removed
DNA methylation – high CpG promoters (HCPs)

 Default

 Addition of repressing
histone methyl marks to turn
gene off
DNA methylation – low CpG promoters (LCPs)

 Active

 Removal of DNA methylation

You do not want cell-specific
marks and addition of activating
genes to be turned on in all
histone methyl groups
cells. They must be off by
default and turned on when
necessary.

 Default
DNA methylation blocks transcription
• DNA methylation can block transcription
in two ways:
• Methylation of C can physically prevent TFs
from binding DNA

• Methylated C can recruit proteins that

facilitate chromatin modification
• E.g. MeCP2. MeCP2 recruits histone
deacetylases and histone methyltransferases
to create heterochromatin.
DNA methyltransferases
• MeCP2 also recruits a DNA methyltransferase called
Dnmt3 which catalyzes de novo methylation →
spreading

• DNA methylation patterns are heritable during cell

division (mitosis).
• To maintain DNA methylation during cell division, an
enzyme called Dnmt1 recognizes the methylated
cytosine on the parental strand of DNA and
methylates the daughter strand.
DNA methyltransferases
5’- CGAGCCGTAGCG -3’
3’- GCTCGGCATCGC -5’
DNA methyltransferases
5’- CGAGCCGTAGCG -3’
3’- GCTCGGCATCGC -5’

Dnmt1

5’- CGAGCCGTAGCG -3’

3’- GCTCGGCATCGC -5’
Transcription factors
• Regulate gene expression by:
• Recruiting histone modifying enzymes
• E.g. Pax7 recruits the histone methyltransferase Trithorax complex to methylate H3K4,
resulting in transcription activation
• Stabilizing RNA polymerase activity (+ basal transcription factors)
• E.g. MyoD stabilizes a basal TF called TFIIB, which supports RNA polymerase II at the
promoter
• Coordinating the timely expression of multiple genes
• E.g. Pax6 binds enhancers for multiple genes that are supposed to be expressed in the
lens cells to activate simultaneous expression of those lens genes

• Control of gene expression can be positive or negative.

 The carboxyl termini are thought to be the trans-
activating domains

Transcription factors
• TFs contain 3 main domains:
• DNA binding domain
• Trans-activation domain
• Protein-protein interaction domain
• A domain is a region of the
protein’s polypeptide chain that
forms independently from the rest.
They are the structural and
functional units of proteins.
• MITF transcription factor →
Pioneer transcription factors
• When transcription factors bind enhancers
and loop towards promoters, they can open
the chromatin near the transcription start
site, allowing pol II to bind.
• But how do transcription factors bind
enhancers if the enhancers are covered by
nucleosomes?
• Pioneer transcription factors
• They are the first to begin the process of
making a locus available for transcription
because they can bind and open
heterochromatin.

Mayran and Drouin. 2018. Journal of Biological Chemistry. Pioneer transcription factors shape the epigenetic landscape.
 Gene Regulatory Network (GRN)
• Pioneer TFs begin the process of differentiation; they are not sufficient to complete the process.
• Different cell types are created through gene regulatory networks.
Mechanisms of differential gene expression
Gene expression can be regulated at different levels of the central dogma.

Reduced or increased transcription

• Epigenetic mechanisms (chromatin methylation/acetylation,
DNA DNA methylation)
• Transcription factors

RNA • Alternative mRNA splicing

• Differential mRNA longevity
• Selective inhibition of translation
• microRNAs
• Control of mRNA localization
protein • Post-translational modifications
Mechanism of differential gene
expression: pre-mRNA processing
• To be translated, once transcribed
a pre-mRNA molecule must be:
translation
• Spliced in the nucleus to remove
introns
• Translocated from the nucleus to the
cytoplasm
• Alternative splicing: a process that
allows for a single gene to form
more than one protein translation
 Alternative splicing
allows different
proteins to be made
from the same gene.
Mechanism of differential gene
expression: pre-mRNA processing

• All introns have a 5’ splice site and a 3’ splice site.

• These splice sites are recognized by a multi-subunit protein/RNA
complex called the spliceosome.
Mechanism of differential gene
expression: pre-mRNA processing

The spliceosome cuts out the

sequence in between the intron
between the “GU” and the
“AG”.
 Mechanism of differential gene
expression: pre-mRNA processing

cis-regulatory sequences in the pre-mRNA:

ISS – intronic splicing silencer
Alternative splicing regulatory proteins: ISE – intronic splicing enhancer
hnRNP - bind splicing silencers to ESE – exonic splicing enhancer
inhibit splicing ESS – exonic splicing silencer
SR proteins – bind splicing
enhancers to promote splicing
Kornblihtt et al. 2013
Mechanism of differential gene
expression: pre-mRNA processing
• Cell type-specific proteins can promote or inhibit splicing at specific introns and exons, leading to
alternative splicing.
Dscam: a gene that can produce 38,016
different proteins by alternative splicing
• Dscam contains 115 exons.
• Each neuron expresses a different isoform. When two dendrites from the same Dscam-expressing
neuron come in contact, they repel each other.
• Ensures that dendrites from the same neuron don’t form synapses on each other.
Mechanism of differential gene
expression: mRNA translation
• Once the mRNA has been efficiently processed (spliced, capped,
polyadenylated) it will be exported for translation in the cytoplasm.
• The rate of translation is another mechanism by which cells can control gene
expression.
• mRNA stability
• mRNA accessibility (translatability)
mRNA stability – half-life
• The longer an mRNA is stable in the cytoplasm, the more protein will be translated from it.
• Half-life: the time required for degradation of 50% of the existing RNA molecules
• Half-life is measured by using a molecule called Actinomycin D which stops transcription. You then measure
how long it takes for all the mRNA molecules of a given gene to be degraded.

Sequences in the 3’UTR recruit

either destabilizing or stabilizing
RNA-binding proteins (RBP) that
dictate the half-life of the mRNA.
mRNA translatability - stored oocyte
mRNAs
• Eggs/oocytes are often filled with mRNA. This is Oocyte
called maternal mRNA.

• This maternal mRNA in the oocyte is kept

translationally inactive until fertilization.

• Example of translation inhibition in the early embryo:

• The oocyte of the tobacco hornworm moth produces
some uncapped mRNAs that cannot be translated. A
methyltransferase adds the 5’ cap upon fertilization,
and mRNA translation can proceed.

It is the maternal mRNA that provides instructions for

early developmental events after fertilization has
occurred.
Stored maternal
mRNAs
• In zebrafish futile mutants, the female
and male pronuclei do not fuse.
• Despite not having a functional zygotic
genome, early cleavage processes
progress perfectly well due to the
presence of maternal mRNAs that can
dictate cell divisions.
 mRNA stability and translatability –
microRNAs miRNA

3’UTR

miRNAs:
• Small RNA molecules
• Bind complementary sites in the
3’ UTR of mRNAs (not 100% Blocking Triggering
complementary – bulges) translation mRNA
• > 1000 miRNA sequences in initiation degradation
humans, and over 50% of
genes contain binding sites for
miRNAs in their 3’ UTR
microRNAs – lin-4
• lin-4 was the first microRNA discovered. Discovered in C.
elegans.
• lin-4 regulates a gene called lin-14. lin-14 has 7 binding
sites for the lin-4 microRNA.
• The LIN-14 protein is a transcription factor that is only
needed for the first phase of larval development, the mRNA
needs to be quickly degraded after to stop production of
the protein to allow the worm to proceed to the next larval
stage.
• lin-4 is responsible for this degradation.
microRNA biogenesis No need to memorize this.
The maternal-to-zygotic transition
• Maternal-to-zygotic switch:
• For the first few cell divisions after
fertilization, all of the proteins produced in
the embryo come from the translation of
maternal mRNAs.
• Most genes in the zygote genome are not
being transcribed.
microRNAs and the maternal-to-zygotic
transition
• Maternal-to-zygotic switch:
• At a specific time in embryogenesis
(different for every organism), the maternal
mRNAs are degraded, and the zygotic
genome becomes activated.
• Many mechanisms work together to
rapidly degrade maternal mRNAs,
including miRNAs.
microRNAs and the maternal-to-zygotic
transition
• How are maternal mRNAs
degraded?
• In zebrafish, the microRNA
miR430 is responsible. It targets
about 40% of the maternal mRNA
in the zebrafish embryo.
Upto here in quiz 1
Tools – in situ hybridization

What it is:
• A way to visualize RNA
in a cell or embryo.

When to use it:

• You want to know
where or when a
specific mRNA is
expressed in a cell or
embryo.
Tools – ChIP-sequencing (chromatin
immunoprecipitation)
What it is:
• A way to determine the DNA-binding site of
specific proteins

When to use it:

• You want to know which enhancers or silencers
a specific transcription factor is binding at a
given moment during development.
• You want to know which genes are covered in
specific histone modifications (e.g. which genes
contain the silencing H3K9me3 mark on day 12
of development)
Tools – RNA-sequencing
What it is:
• A way to identify the full repertoire of mRNAs expressed
in an embryo, tissue, or a single cell at a given moment.
• Like taking a snapshot of all mRNAs present in the
cytoplasm at a given timepoint.

When to use it:

• You want to ask questions like “which mRNAs are
expressed on day 4 of development and how does this
compare to the mRNAs expressed on day 9?”
• You want to know how much of a specific mRNA
molecule is present at a given time.
Tools – reverse genetics
• Is gene X necessary for tail development?
• Remove gene X from embryo → observe development.
• What is the function of gene Y?
• Remove gene Y from cell/embryo → observe the cell/embryo

• Many tools to delete or mutate genes.

• Mutating a gene to uncover its function – reverse genetics
Tools – reverse genetics: RNAi
• The microRNA pathway can be hijacked by researchers
to knock down genes of interest.
• Small double-stranded RNA molecules called siRNAs
(small-interfering RNAs) are introduced into cells and bind
complementary mRNA targets and lead to their
degradation.
• This is not permanent as the siRNAs become diluted after
several cell divisions, and not 100% of the target mRNA is
degraded. Therefore, it’s called a gene knock down.
Tools – reverse genetics:
CRISPR/Cas9
• CRISPR is a naturally occurring anti-viral system in
bacteria.
• When bacteria become exposed to a virus, they integrate a
small fragment of that virus into their own genome.
• When they encounter this virus again, the small DNA
fragment is transcribed into a guide RNA (gRNA).
• The gRNA is bound by an endonuclease called Cas9.
• When the gRNA binds the viral DNA (through sequence
complementarity), Cas9 cleaves the viral DNA generating a
double-strand break, disabling the virus.
Tools – reverse genetics:
CRISPR/Cas9
• The double strand break can be repaired by one of two
pathways: non-homologous end-joining (NHEJ) and homology
directed repair (HDR).
• NHEJ is an imperfect repair mechanism – often introduces
INDELS into DNA.
• HDR uses a repair template to swap in a fragment of DNA
based on homologous sequences between the repair template
and the sequences surrounding the double-stranded break.
You are a PhD student studying the regulation of a gene called “Florinex-1” (fake gene name) in
Drosophila embryos. Your supervisor wants you to determine:
a) When and where during embryogenesis it is expressed.
b) Which enhancer(s) regulate(s) expression of Florinex-1. There are a number of regions of DNA you
suspect may be acting as enhancers: region A, region B, region C.
c) This experiment will be performed after you have determined which enhancer activates Florinex-1.
If the transcription factor “PEX-1” (that your fellow PhD student is studying) is involved in the
activation of an enhancer that regulates the expression of Florinex-1.

Describe experiments that will allow you to determine the answers to the above questions.
You’re studying the expression of gene Y in zebrafish embryos. You know that
protein Y is highly expressed in the developing brain at 60 hours post-
fertilization for 2 hours and then expression rapidly drops. By 63 hours post-
fertilization there is no protein Y in the brain. Explain four different mechanisms
that could have led to a rapid decrease in protein Y expression.