Control of Gene Expression I

Gene Expression
- In both prokaryotes and eukaryotes

Why is prokaryotes's gene expression important?

1. Most of the cells in our body are bacterial. (play a big role in normal function, and disease)
2. Many techniques that we use in molecular biology to understand cells are derived from the
bacteria system, because they are simple, and manipulated and introduced into Eukaryotic cells
to understand Eukaryotes cells.

All the cells in your body have the same genomic sequence, what specified the cells?
- Where and when specific gene would be expressed

Different Gene Expression:

Spatial - where (what organ)
Temporal - when

Spatial Temporal Regulation => Differentiation

Ex. zygote => neuron

Stem Cells
- can be embryonic or adult

Embryonic Stem Cells: zygotes => blastocyst

- Inside the blastocyst, there is a inner cell mass =>
housed embryonic stem cells
- Stem cells can differentiate into all the specialized tissues that make up human body

Adult Stem Cells: somatic stem cells

- Many tissue has an area, called niche
- Inside the niche housed the stem cells
Why do adults need stem cells? => to regenerate tissues
- Stem cells send the signal, and the old with the new
- Different organ has a different RATE
 Control of Gene Expression in Prokaryotes
● Form of transcriptional regulation in E.coli
● Only want to express Lac operon when lactose is in the environment. Otherwise, glucose is
preferentially metabolized.

Why do bacteria need gene expression?

- Depends on the present/absence of lactose
- If lactose present => need to be metabolized, the gene lac Z, Y, A are activated (ZYA are
involved in the metabolism of lactose.)
- Lactose absent => nothing need to be metabolized, the lac operon are bound to the
● E.coli want to regulate expression of the Lac operon so that these genes are only expressed when
lactose is present in the environment. It does this through transcriptional regulation. If glucose
is present, E. coli do not want to express the lac operon because glucose is the preferential source
of energy.

Lac operon: involve in metabolism lactose

Lactose - breakdown by beta-galactosidase enzyme into glucose and galactose to processed in the cell

Active vs. Non-active: transcription occur or not

Structure of the Lac operon:

- Promoter:
- Operator (within promoter): next to the promoter, involve and drive the transcription
- Lac genes: lac Z, lac Y, and lac A
lacZ and lacY are involved in Galactose metabolism, encoding beta-galactosidase and
permease
Lac Z gene: DNA sequence translated into beta-galactosidase (enzyme)

Lac Y gene: translate into permease - enzyme that sits on cell membrane that allow
lactose come in the channel (require energy from the proton H+ gradient)
 - as part of an operon, either all 3 genes are transcribed and expressed simultaneously or
none are.
- this is fine, because operons usually group together genes that are all involved in a related
metabolic pathway.

● Regulatory lacI gene: upsterm of lac operon. LacI encode the lac repressor, a protein which
binds to the operator of the lac operon to suppress expression
● Lac repressor: repress transcription, by binding to the operator, therefore RNA polymerase
cannot go down, transcription is blocked.

Control of Lac Operon

When NO Lactose Present
- When no lactose is present, E.coli doesn’t want to express the lac operon.
- So the Lac repressor protein, in its active form, binds to the operator and prevents RNA
polymerase from binding to the promoter to do transcription.
= no transcription.
- No expression of lac operon. Glucose metabolized.
- lacI gene coding for the repressor is always expressed (constitutive), but its product can be
regulated. (next)

How does the repressor release itself off the operon? (how the inducer got the signal)
- Lactose in the cell, get converted into allolactose
Allolactose: the inducer 诱导物
- Allolactose binds to the repressor, change the confirmation of the repressor, inactivate
the repressor, make it can no longer binds to the operator

When Lactose Present

- When lactose is present, lactose => allolactose
- Allolactose isomer binds to Lac repressor and inactivates it.
= RNA polymerase can bind to promoter, transcription is initiated
- When lactose is present, it is converted to another form called allolactose. This binds to the Lac
repressor and inactivates it, meaning the lac repressor can no longer bind to the operator.
- RNA polymerase can now bind to the promoter and start transcription.
 Notice: lacZ, lacY, and lacA are all transcribed as one mRNA strand, but translated as separate
proteins because each gene has its own start and stop codon.

(refer to Textbook)

Mutations Affecting Lac Operon

● Mutation in Repressor – what consequence will this have?

● Mutation in lacZ - what consequence will this have?

● Mutation in operator - what consequence will this have?

D. If allolactose cannot bind to the repressor, then the

repressor will remain in its active form and always block
transcription. Therefore, the lac genes would never be
expressed efficiently, even in the presence of lactose.
 Control of Gene Expression in Eukaryotes

Transcriptional Regulation => determines which gene are transcribed

Regulate transcription happen or not
- Regulation of transcription initiation
- Chromatin remodeling to make genes accessible for transcription

Post-transcriptional Regulation => determine types and availability of

mRNA to ribosomes
Regulate translation occur or not
Regulate the mRNA (pre-mRNA)
- RNA editing
- Variations in pre-mRNA processing

Translational Regulation => determines rate at which proteins are made

Regulate the translation in different mechanism
- Variations in rate of initiation of protein synthesis

Post-translational Regulation => determines availability of finished proteins

regulate the amount of protein
- Variations in rate of protein breakdown

Spatial and Temporal Gene Expression

1. Every cell nucleus contains the complete genome established in the fertilized egg. The DNA of
all differentiated cells are “identical”.
DNA of all differentiated cells are identical
2. Only a small percentage of the genome is expressed in each cell, and the transcripts are specific
for that cell type.
Not all gene expressed in each cells
3. The unused genes in differentiated cells are not destroyed or mutated, and they retain the
potential for being expressed.

Cells retain the potential for being expressed!

Spatial and Temporal Regulation

● All stem cells have the exact same genome (excluding spontaneous mutations)
● Spatial and temporal regulation of gene expression determine what cell type a stem cell can give
rise to.
● Tissue-specific transcription factors play a big role.

Spatial regulation = where (in what cell/part of the body)

Temporal regulation = when (at what time/under what conditions)
At what stages can we control gene expression?
- Transcription, post-transcription, translation, post-translation
- It is most important at the transcriptional level because why waste energy transcribing and
translation only to inhibit a protein’s function later down the line?

● Transcription is controlled by transcription factors at its regulatory sites (promoter, proximal

and distal regulatory sites).
● When and where transcription occurs determines what types of proteins are present in a
certain cell, which is ultimately what dictates a cell type.
● If you want a precise control of genes, then you need a precise control on transcription factors
themselves.
● We have both tissue-specific transcription factors and tissue-specific proteins.

Regulating Transcription Initiation

Regulatory Proteins:
- Positive Regulator: tissue-specific regulatory proteins =>regulate this genes only can express
in specific tissue (upstream)
- Negative Regulator: the gene are not expressed are bind by negative regulator (downstream)

Promoter: the gene control region

Transcription Factors: binds (?) to TATA box

Recall mRNA Splicing
pre-mRNA => mRNA

- 3’ splicing site: NCAG

How Miss Splicing Leads to Diseases:
● Mutations at both splice sites can lead to abnormal variance.
● Mutations in the middle in introns (nothing to do with splice sites) can also lead to disease states.
Normal:

Abnormal:
 Mutation in Intron can Lead to β-thalassemia
β-thalassemia – Hereditary Blood Disorder (Abnormal Hemoglobin = Anemia)

Two Key Points:

1. G → A mutation in intron creates new 3’ splice site
2. Expression of an early stop codon causes premature termination of translation

● β-thalassemia is a hereditary blood disorder that results in the formation of an abnormal

hemoglobin.
Hemoglobin: the protein in red blood cells that carries oxygen.

Below is a sequence from a small part of the β-globin gene (normal and β-thalassemia).
Normal: β-globin gene

Mutated: β-thalassemia => form abnormal hemoglobin; cannot carrier O2 through the body

Splicing Site: NCAGG

The G → A point mutation in the gene leads to the formation of an abnormal hemoglobin.
Describe how this point mutation leads to aberrant (abnormal) formation of the β-globin
protein.

Stop codon: TAG

 RNA Editing
Background:
● Apolipoprotein B (Apo. B) is a protein that plays an important role in the transport of
cholesterol in the body.
○ Apo B-100 is produced in the liver
○ Apo B-48 (a truncated version of Apo B-100) is produced in the small intestine.
● APOBEC-1 is an RNA editing enzyme that converts cytosine (C) to Uracil (U) via a process
called deamination.
● The open reading frame is edited which leads to premature termination of translation producing a
smaller version (or variant) of the protein called Apo-B48

1. Apo B-100 and Apo B-48 are proteins transcribed from the SAME gene.
● Apo B-100: produced in the liver; gets transcribed fully and its mRNA is then translated
into protein and this protein functions in the liver.
● Apo B-48: produced in the small intestine; also gets fully transcribed but AFTER
transcription and splicing of pre-mRNA, the mature mRNA gets "edited" so that the
protein derived from this edited mRNA is shorter.
APOBEC-1: enzyme that converts C to a U in the middle of the mature transcript
through the process of deamination. This creates a new stop codon (UAA instead of
CAA). Translation stops at this place (roughly half way through the ORF) and creates
Apo B-48 that is only synthesized in the small intestine.
* Transcription and translation of APOBEC-1 only occurs in small intestine and not in
the liver

2. Post-transcriptional regulation because the regulation (ie, the editing) is occurring AFTER
transcription has been completed

3. Transcription and translation of APOBEC-1 only occurs in the small intestine and not in the liver
(ie, tissue-specific expression). Transcription factors for APOBEC-1 do not bind to the
 APOBEC-1 promoter in the liver, only in small intestine. Therefore, the mRNA of Apo B-100 is
only edited in the small intestine leading to tissue-specific expression of Apo B-48.

4. FALSE. The mRNA of the Apo B-48 protein is the SAME size as the mRNA of Apo B-100. It
has been edited such that there is a stop codon producing a shorter protein (not a shorter mRNA).

Common Mistake on test:

Students often answer that the mRNA of ApoB-48 is shorter than mRNA of Apo-100. This is not
true. In both cases, the mRNA is the SAME length and the sequence is almost identical (except
CAA in ApoB-100 and UAA in ApoB-48). Post-transcriptional regulation
The protein sequence is the same except ApoB-48 is almost half the size.
Post-transcriptional regulation => produce shorter protein not shorter mRNA
 Control of Gene Expression II - Stem Cells

Telomerase and Hayflick Limit

Telomere: repeat sequence at the end of chromosome to protect the gene

We lose Telomere length everytime replicate, until reach the critical limit that would affect the
gene, no longer replicative => Senescence Stage (=> cells can still function, but no longer replicate)
Hayflick Limit: the critical limit point; stop proliferation

Telomerase: enzyme that maintain, restore the length

Telomerase active cells: cancer cells; germ cells (egg and sperm); stem cells (embryonic and adult);
early embryogenesis

Stem Cells
Stem Cells Express Telomerase.
● Stem cells: cells that have the potential to differentiate into different cell types in the body
during embryogenesis and adult life.
* Not All stem cells can differentiate into any type of cells
● Potency: The potential to differentiate into specialized cell types and be able to give rise to any
mature cell type.
Potency of the stem cell specifies the differentiation potential 分化潜力
i.e., the potential to differentiate into different cell types.

Have different levels of potency:

High Potency => Low Potency

Testing for Potency

Unknown sample → extract the stem cells → label with fluorescent marker → inject into
inner cell mass of blastocyst to form a chimera → implant blastocyst into pseudopregnant
mouse → scan offspring for fluorescent marker
- If marker is in a wide range of tissue, sample is pluripotent b/c it was able to develop
into different tissues
- If marker is restricted to 1 type of tissue, sample is unipotent bc it was unable to
develop into different tissues

4 Types of Stem Cells

1. Embryonic Stem Cells 胚胎干细胞
- Ethical issue

2. Adult/Somatic Stem Cells 体细胞干细胞

3. Induced Pluripotent Stem Cells (iPS Cells) 诱导多功能干细胞

- derive form somatic cells (converted somatic cells to pluripotent stem cells)

4. Umbilical Cord Stem Cells 脐带干细胞

Sources of Different Types of Stem Cells (Potency)

 ● Totipotent stem cells can differentiate into embryonic and extraembryonic cell types creating a
complete, viable organism.
○ The only totipotent cell is the zygote.
○ Extraembryonic: make umbilical cord

● Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly
all cells of the human body.
○ Don’t have extraembryonic cells, cannot create entire viable organism

● Multipotent stem cells can differentiate into a number of cells, but only those of a closely
related family of cells.
○ For example, the bone marrow contains multipotent stem cells that give rise to all the
cells of the blood but not to other types of cells.
○ Bone marrow: make blood cells

● Unipotent stem cells can produce only one cell type but have the property of self- renewal
○ Distinguishes them from non-stem cells (eg, skin stem cells).
Unipotent stem cells vs. Somatic cells
● Unipotent stem cells: give rise to cells that won’t reach cell senescence. They
self-renew.
● Somatic cells: not maintaining telomere length, eventually reach their Hayflick
limit and must enter irreversible cell cycle arrest. Cannot self-renew

Embryonic Stem Cells

- ICM - Inner Cell Mass contain pluripotent stem cells

Adult Stem Cells

Adult stem cells: cells of variable potency that can self-renew

Adult stem cells stored in various parts of the body (stem cell niche). Organ maintenance (cell turnover)
and repair
How Stem Cells Divide

S => stem cells; can go back into the niche and stay
P => progenitor; commit to be differentiate, cannot go back into the niche, have to go down the
differentiate path
2 types of progenitor cells:
● Multipotent progenitor (Upon differentiation from a stem cell, progenitors are
multipotent)
● Committed progenitor (more differentiated and committed than the multipotent
progenitor)
- Progenitor cells are stem cells that have committed to differentiation, cannot go back to the niche
- Upon differentiation from a stem cell, progenitors are multipotent
- After further differentiation, they are committed progenitors -> means they are more differentiated and committed than the multipotent
progenitor

Hibernating:
- stem cells sit in G0.
- when sense the tissue needed to bre regenerate, the stem cell awake and divide
- Send a stem cell differentiate into that specific cell type
Stem cells can divide in 3 ways:
● Symmetric self-renewal: 1 Stem cell => 2 Stem cells
○ Maintains count, the extra cell can stay or go
● Asymmetric: 1 Stem cell => 1 Stem cell + 1 Progenitor
○ The stem cell back into niche, cannot go down, need to maintains count; while the
progenitor is committed to differentiation
● Symmetric differentiation: 1 Stem cell => 2 Progenitor
○ Loses count, so neighbor stem cell has to undergo Symmetric self-renewal to maintain
count
 Role of Stem Cells in the Body
● Regenerate adult tissue, maintain tissues in the body
● Examples:
○ Epidermal stem cells => epidermal stem cells are unipotent cells that
replenish epithelial skin cells; differentiate and migrate to the
epithelial layer
○ Intestinal villi stem cells => Intestinal villi stem cells stay in a
quiescent stage (G0) in the crypt of the villi until sense neighboring
cells release transcription factors Wnt, EGF and Notch that tell the cell to enter G1
and begin migration

● Transcription factors are key for determining differentiation; TFs tell the stem cell to differentiate
as they can turn on/off entire sets of genes, very powerful
○ Wnt, EGF, Notch
○ Stem cells require these factors to enter G1

Induced Pluripotent Stem Cells (iPS Cells)

● iPS are fibroblasts (obtained easily and non-invasively) that have
been reprogrammed by adding the 4 Yamanaka factors.
Yamanaka factors: transcription factors (TF) that tell the fibroblast
to revert to a stem cell.
● Very powerful as they can recreate organ systems in vitro without having to get embryonic
stem cells → better to study organ systems that extracting a primary sample from a patient.
More predictable behavior and easier to culture.
○ Macular degeneration (1st clinical trial, but develop into ey tumor)
○ Risk of cancer
○ Burn victims
■ iPS skin not morphologically identical to original skin
○ Heart attack patients
○ Pharmacogenetics
● can even make a viable organism => Mice made from induced stem cell

● Shinya Yamanaka - induced the first iPS cells in 2006

● Normal somatic adult cells were induced to become pluripotent stem cells, by inducing 4 4
Yamanaka factors
Somatic adult cells => pluripotent stem cells (Reprogram)
Adult=> Embryotes
“The program is there”
- iPS cells: not embryonic stem cells, but behave like embryonic stem cells

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia

● CRISPR-Cas9: the technique used to edit a genome

● use gene editing to treat sickle cells disease and β-thalassemia
○ β-thalassemia: blood disorder due to mutation of intronic region lead to , missen splicing
lead hemoglobin cannot efficiently carry oxygen
○ Sickle cells: missense mutation; exonic
● Fetal Hemoglobin (HbF): during embryonic
development
● Fetal Hemoglobin (HbF) =BCL11A=> Adult
Hemoglobin (HbA) (figure on the right)
● HbF has a higher affinity for oxygen than adult HbA;
because the mom’s oxygen come from lower partial
pressure
● Since HbA has a problem in both of the disease
○ The mutation is on the β

=> Solution: turn on the HbF; its program is still

there (* all genes maintain the potential to be
expressed)
Treatment: CTX001 Drug
● Hematopoietic stem cells (HSPCs) and progenitor cells from healthy adult donors
○ But are from adult, don’t express HbF
● Edit using CRISPR-Cas9
○ Targeting red blood cells => specific to erythroid enhancer region (since don’t
wanna target BCL11A in other molecules)
○ Mutate the erythroid enhancer region of BCL11A, so that BCL11A cannot
produced
BCL11A: transcription factor that repressed Fetal Hb; inhibit Gamma-globin
gene
KLF-1: transcription factors bind to the erythroid enhancer region, control the
transcription of BCL11A
○ KLF cannot bind to the region anymore => BCL11A is not transcript
○ Gamma-globin gene is depressed => Turn on Fetal Hb
● Grow and put in patient bone marrow
● Patient stem cells can send out progenitors, differentiate into RBC with HbF
● Stem cells self renew
○ If supply RBC, cannot last very long
● Always have supply of stem cells with HbF