The Genetic Core

of Development:
Differential Gene Expression

LEE KUI SOON

 Developmental Genetics

The discipline that examines how the

genotypes is transformed into the
phenotype and the major paradigm of
development genetics is differential gene
expression from the same nuclear
repertoire

LEE KUI SOON

 Question--THINK

How different genes from the

sam egenetic repertoire are
activated and repressed at
particular times and places to
cuase cells to become different
form one another?
Regulation of gene expression

1. Differential gene transcription

2. Selective nuclear RNA processing
3. Selective mRNA translation
4. Differential protein modification

LEE KUI SOON

 Differential gene transcription
Two fundamental differences between
eukaryotic genes from most prokaryotic genes:

• The eukaryotic genes are contained within a

complex of DNA and protein called
chromatin

• The eukaryotic genes are not co-linear with

their peptide products
LEE KUI SOON
GENERAL CHARACTERISTIC OF A EUKARYOTIC GENE

1.Promoter
2.Transcription initiation site (cap sequence)
3.Translation initiation site (ATG)
4.5’ untranslated region ( 5’ UTR or leader sequence)
5.Exon and Intron
6.Translation termination codon (TAA)
7.3’ untranslated region (3’ UTR
8.Poly A tail

LEE KUI SOON

LEE KUI SOON
 Promoters and Enhancer
Promoter
-Are sites where RNA polymerase binds to the DNA
to initiate transcription

Enhancers
- Is a DNA sequence that can activate the utilization
of a promoter, controlling the efficiency and rate of
the transcription from that particular promoter

LEE KUI SOON

The generic elements regulating cell type-
specific transcription can be identified by
fusing reporter genes to the enhancer
regions of the genes found in particular cell
types. (A) the enhancer regions of the
muscle-specific protein Myf-5 is fused to a
β-galactosidase reporter gene and is
incorporated into t a mouse embryos.
When stained for β-galactosidase activity
(arrowhead), the mouse embryos shows
that the reporter gene is expressed in the
eyes muscle, forelimb, neck muscles and
segmented myotomes. (B) the green
flourescent (GFP) is fused to a lens
crystallin enhancer in Xenopus tropicalis.
The result is the placement of green
fluorescent protein in the tadlpole lens
 6 general emphasis for differential gene expression

1. Most gene require enhances for their transcription

2. Enhancers are the major determinant of differential transcription
in space (cell type) and time
3. Enhancer could have multiple signals to determine whether a
given is transcribed. A given gene can have several enhancer sites
linked to it and each enhancer can be bound by more than one
transcription factor
4. The interaction between the proteins bound to the enhancer sites
and the transcription initiation complex assembled at the
promoter is thought to regulate transcription
5. Enhancers are modular
6. A gene can have several enhancer elements, each elements, each
turning it on in a different set of cells

LEE KUI SOON

 Transcription Factors
•Are proteins that bind to enhancer or promoter region and
interact to activate or repress the transcription of a
particular gene

•Most transcription factors can bind to specific DNA

sequences

• Have tree major domains:

1. DNA-binding domain
2. Trans-activating domain
3. Protein-protein interacting domain
Eg: MITF and PAX6
LEE KUI SOON
 CASCADE OF TRANSCRIPTION FACTORS
How do the transcription factors themselves get to be
expressed in a tissue-specific manner?
- Activated by other transcription factors
- Self-activated/regulated

LEE KUI SOON

 Methylation Pattern and
the control of Transcription

How does a pattern of transcription become stable?

How can a lens cell continue to remain in a lens cell
and not activate muscle-specific genes?
How can cells undergo rounds of mitosis and still
maintain their differentiated characteristics?

Answer: DNA methylation

LEE KUI SOON

- The promoters of inactive genes become methylated at
certain cytosine residues and the resulting methylcytosine
stabilizes nucleosomes and prevents transcription factors
from binding

*Cytosine methylation appear to be a major mechanism of

transcriptional in vertebrates but not in invertebrate (most
of them) LEE KUI SOON
Methylation Pattern and the control of Transcription

In vertebrate, the presence of methylated cytosine in the

promoter of a gene correlates with the repression of
transcription from that gene and proven experimentally.

-adding transgenes to cells and giving them different

patterns of methylation, the promoter regions or enhancer
of a gene correlates extremely well the repression of gene
transcription

How is methylation involved in repressing genes?

Hypothesis: DNA methylation stabilizes nucleosomes

LEE KUI SOON
 Genetic Imprinting
Epigenetic phenomenon that causes a subset of
mammalian genes to be expressed from one of the two
parental chromosomes

P M P M P M

LEE KUI SOON

Biallelically expressed Maternally expressed Paternally expressed
 Genomic imprinting
The ability to mark a gene as coming from either from the
father or the mother is called genomic imprinting
- Adds addition information to the inherited genome,
information that may regulated spatial and temporal gene
activity

In certain mutation mouse and human, a severe or lethal

condition arises if the mutant gene is derived from one
parent, but that same mutant gene has no deleterious
effects if inherited from other parent.

Prader-willi and Angelman syndrome explain well this

phenomenon LEE KUI SOON
LEE KUI SOON
 Prader-Willi and Angelman Syndrome

Prader-willi Syndrome
-Associated with mild mental retardation, obesity, small
gonads and short statue

Angelman syndrome
-Associated with sever mental retardation, seizures, lack of
speech and inappropriate laughter

LEE KUI SOON

 Differential RNA processing
• There is no guarantee that after the synthesis of
the RNA transcription, a function protein will be
produced. To become an active protein, the RNA
must
i. Processed into a mRNA by introns removal
ii. Translocated from the nucleus to the cytoplasm
iii.Translated by the protein-synthesizing
apparatus
iv.Posttranslational modification to become
active
Control of early development by nuclear RNA

• mRNA was not the primary transcripts from

the genes but rather nuclear RNA (nRNA or
hnRNA)
• nRNA is usually many times longer than
mRNA due to presence of intron
• Different cells types could be transcribing
the same type of nuclear RNA in different
types of cells
Control of early development by nuclear RNA
 Creating families of protein through
differential nRNA splicing
• nRNA consists of exons and separated by introns
• By splicing different sets of exons, different cells can make
different types of mRNA and hence different types of
proteins
• Alternative nRNA splicing is based on determining which
sequences can be spliced out as introns
• Splicing of nRNA is mediated through a complex called a
spliceosome, made up of small nuclear RNA (snRNA) and
proteins, that assembles at a splice site.
• Differential RNA processing has be found to control the
alternative forms of expression of genes encodings over
100 proteins.
 Posttranslational Gene Regulation
• Proteins are often sequestered in certain regions such
as membranes, lysosomes, nuclei or mitochondria
• Some newly synthesized proteins are inactive without
the cleaving away of certain inhibitory sections (insulin)
• Some proteins must be “addressed” to their specific
intracellular destinations in order to function
• some proteins are not active unless they bind an ion
such as calcium, or are modified by the covalent
addition of a phosphate or acetate group
• Some proteins need to assemble with other proteins to
form a functional unit.
 Summary

1. Differential gene expression from genetically identical

nuclei creates different cell types. Differential gene
expression can occur at the levels of gene transcription,
nuclear RNA processing, mRNA translation, and protein
modification.
2. Genes are usually repressed. Activation of a gene often
means inhibiting its repressor. This leads to thinking in
double and triple negatives: Activation is often the
inhibition of the inhibitor; repression is the inhibition of
the inhibitor of the inhibitor.
3. Eukaryotic genes contain promoter sequences to which
RNA polymerase can bind to initiate transcription. The
eukaryotic RNA polymerases are bound by a series of
proteins called basal transcription factors.
4. Eukaryotic genes expressed in specific cell types
contain enhancer sequences that regulate their
transcription in time and space.
5. Specific transcription factors can recognize specific
sequences of DNA in the promoter and enhancer
regions. They activate or repress transcription from
the genes to which they have bound.
6. Enhancers work in a combinatorial fashion. The
binding of several transcription factors can act to
promote or inhibit transcription from a certain
promoter. In some cases transcription is activated only
if both factor A and factor B are present, while in other
cases, transcription is activated if either factor
A or factor B is present.
7. A gene encoding a transcription factor can keep itself
activated if the transcription factor it encodes also
activates its own promoter. Thus, a transcription factor
gene can have one set of enhancer sites to initiate its
activation and a second set of enhancer sites (that bind
the encoded transcription factor) to maintain its
activation.
8. Often, the same transcription factors that are used during
the differentiation of a particular cell type are also used
to activate the genes for that cell type's specific products.
For instance, Pax6 is needed both for the differentiation
of the lens and for the transcription of the lens crystallin
genes, and Mitf is needed for pigment cell differentiation
and for the transcription of the genes whose products
catalyze the synthesis of melanin.
9. Enhancers can act as silencers to suppress the
transcription of a gene in inappropriate cell types.
10.Locus control regions may function by making
relatively large portions of a chromosome accessible
to transcription factors.
11.Transcription factors act in different ways to regulate
RNA synthesis. Some transcription factors stabilize
RNA polymerase binding to the DNA, some disrupt
nucleosomes, and some increase the efficiency of
transcription.
12.Transcription correlates with a lack of methylation on
the promoter and enhancer regions of genes.
Methylation differences can account for examples of
genomic imprinting, wherein a gene transmitted
through the sperm is expressed differently from the
same gene transmitted through the egg.
13.Differential RNA selection can allow certain transcripts
to enter the cytoplasm while preventing other
transcripts from leaving the nucleus.
14.Differential RNA splicing can create a family of related
proteins by causing different regions of the nRNA to
be read as exons and introns. What is an exon in one
set of circumstances may be an intron in another.
15.Some messages are translated only at certain times.
The oocyte, in particular, uses translational regulation
to set aside certain messages that it transcribes during
egg development but uses only after the egg is
fertilized. This activation is often accomplished either
by the removal of inhibitory proteins or by the
polyadenylation of the message.