MOLECULAR REGULATION

AND SIGNALING
JOVIE E. NICOLAS, LPT
Regulation Of Gene Expression Starts With The Structure And Organization
Of The DNA

DNA Loop Linker DNA

(approx. 147bp) (10-70bp)

Present only
in Actively
dividing cells
 Overview of Gene
Expression in Eukaryotes

Protect the RNA

from exo- and
endonucleases
nRNA or pre-mRNA

How about the expression of

rRNA? tRNA?
Direction of Transcription
Specific RNA Polymerase for mRNA
(Type I), tRNA (Type III), rNA (Type II) GENE TRANSCRIPTION

Developing RNA Initiation point of Transcription (5’-3’)

CIS-Acting Elements Proximal-Promoter Element
• Non-coding DNA sequences • For true level expression (cell/time specific expression)
• Regulates the initiation of transcription by • Site of transcription factor binding
• Approx 250bp long
RNA Polymerase II (RNP II)
Enhancers and Silencers
Core Promoter • Increase efficiency or slows-down or inhibit transcription
• For basal level expression (minimum expression) • Potential Locations
• Approx 35bp long • Immediately upstream of a promoter
• Site of transcription initiation. • Immediately after a promoter
• Determines where RNP II will bind and initiate transcription • Within a gene
TATA BOX (TATAAA) • Kilobases away from the gene
• Goldberg-hogness Box • Can regulate transcription from a distance; uses certain
• In eukaryotes, influence the binding of RNP II to the core proteins to bend the DNA.
promoter
TRANS-Acting Elements
• Proteins binding to DNA; helps and affects
transcription
• Also called TRANSCRIPTION FACTORS
• Facilitate RNP II Binding

Two Categories
• General Transcription Factors (GTFs)
• Required for all RNP II – mediated
transcription
• Connects RNP II with cis-regulatory factors
• Transcriptional Activators and Repressors Domains
• Influence the efficiency of the action of RNP II • DNA Binding domain
• Connects RNP II with enhancers or silencers • Binds to the cis-acting element
GTFs involved in Human RNP II Binding • Trans-activating domain
• Transcription Factor (TF) IIA, IIB, IID • Binds to another transcription factor to
communicate the effects of the cis-acting
element.
 Regulation of Gene Expression through METHYLATION

DNA Methylation represses transcription

• Transcription of a gene is repressed by the
methylation (addition of methyl) to the 5th
carbon of cytosine bases in the promoter region
of the gene, inhibiting the binding of
transcription factors.
• Reasons why different proteins are produced by
cells despite having the same set of DNA.
• DNA in euchromatin are usually lowly
methylated or unmethylated (active in
transcription), while those in heterochromatin
are highly methylated.
• Genes coding for cell-specific proteins are
found in euchromatin (Ex. Euchromatin CpG = Cytosine – phosphate – Guanine
genes in muscles are coding for muscle SAM-CH3 = S-Adenosyl methionine (derived from methionine); methyl
proteins. These genes maybe in donor.
heterochromatin in blood cells) SAH = S-adenosylhomocysteine; Product of demethylation of SAM-CH3
Regulation of Gene Expression through METHYLATION

X-chromosome Inactivation
• RNA X-inactive specific transcript (XIST)
RNA encoded by the XIST Gene (found in
the X chromosome) enables X chromosome
inactivation. The XIST RNA wraps around
the X chromosome inactivating the
chromosome.
Regulation of Gene Expression through METHYLATION

Genomic Imprinting
• Only a gene inherited from the father or
the mother is expressed, while other genes
were silenced.
• Approximately 40-60 human genes are
imprinted and their methylation patterns
are established during spermatogenesis
and oogenesis.
Regulation of Gene Pre-mRNA

Expression through SPLICING Spliceosomes

Excision and Splicing

• Introns are excised (removed) from the pre-
mRNA and exons are spliced (ligated together)
• determines the final amino acid/protein snRNP
product after the translation of the mRNA.
• Excision of introns are catalyzed by snRNA
spliceosomes
COMPONENT OF SPLICEOSOMES
• Small nuclear RNAs (snRNAs)
• Small nuclear ribonucleoproteins (snRNPs
or SNURPS)
• In mitochondrial and chloroplast RNA, introns
themselves act as ribozymes and undergo self
excision.
Regulation of Gene Expression through SPLICING
Alternative splicing
• Splicing of exons in different patterns.
• a single mRNA can give rise to different proteins depending on the exons that will be ligated during splicing.
• Provides a means for cells to produce different proteins from a single gene.

Splicing Isoforms = Proteins derived from the same gene

Regulation of Gene Expression through SPLICING
mRNA capping and polyadenylation
• mRNA capping is the addition of the 5’-methylguanosine cap to the ligated exons and functions in the stability
and protection of the 5’-end of the mRNA from nucleases. Cap is added even before splicing.
• mRNA polyadenylation is the addition of the poly-A tail at the 3’ end of the ligated exons. The poly-A tail is a
series of adenine nucleotide residues (approx. 250 Adenine residues) being added at the 3’ end of the mRNA
(at a conserved sequence containing AAUAAA) and is essential stabilization, and for the protection of the
mRNA from exonucleases
Regulation of Gene Expression through Post-translational
Modifications

Translation
Regulation of Gene Expression through Post-translational
Modifications

Protein Modification

Proteome = total number

of proteins that can be
produced by an organism
Regulation of Gene Expression through Post-translational
Modifications

Protein Modification Kinase

add phosphate
w/o breaking a
PHOSPHORYLATIION
bond
• plays critical roles in the regulation
of many cellular processes,
including cell cycle, growth,
apoptosis and signal transduction
pathways.
• Activates inactivated proteins
Ex. Addition of phosphate to
different enzymes in Glycogenolysis,
the removal of glucose from Phosphorylase
glycogen breaks bond to
add phosphate
Regulation of Gene Expression through Post-translational
Modifications
Protein Modification

GLYCOSYLATION
• Addition of carbohydrate
• Production of glycoprotein
• encompasses a diverse selection of sugar-
moiety additions to proteins that ranges
from simple monosaccharide
modifications of nuclear transcription
factors to highly complex branched
polysaccharide changes of cell surface
receptors.
Ex. Carbohydrates in the form of aspargine-
linked (N-linked) or serine/threonine-linked
(O-linked) oligosaccharides are major
N-linked glycosylation in asparagine
structural components of many cell surface
and secreted proteins.
Cell Signaling
Communication between cells
Essential for development and organ formation
 the cell is producing the chemical signal
affects itself.

Juxtacrine Direct Signaling

involves communication between cells that
are in direct contact with each other
through gap junctions

Uses Local mediators (Paracrine factors or

Growth and Differentiation Factors, GDFs)
the signal is short range

Uses HORMONES
Through the bloodstream, since the
signal is long range
Endocrine Hormones
Amino Acid Derivatives
Comes from a single amino acid
Produced in the cytosol and ribosomes
• Norepinephrine
• Epinephrine
• Thyroxine
Peptides
Comes from multiple amino acids
Produced in ribosomes and RER
• Antidiuretic hormone (ADH) or Vasopressin
Proteins
Produced through gene expression
• Insulin
• Glucagon
Steroids
Produced in the SER
• Androgens
• Estrogens
• Corticosteroids
Corticosteroid
Paracrine Signaling Factors (GDFs)
Fibroblast Growth Factors (FGF)
• Around 2 dozens identified
• Produce protein isoforms by altering splicing
• Activate Tyrosine Receptor Kinases called Fibroblast Growth Factor Receptors (FGFRs)
• Important for angiogenesis, axon growth, and mesoderm differentiation
WNT (“Wingless/Integrated”)
• About 15 already identified
• Receptors are members of the Frizzled family of proteins
• Involved in regulating limb patterning, midbrain development, somite and urogenital differentiation.
Hedgehog
• 3 hedgehog genes: Indian, Desert, and sonic hedgehog
• Binds to the receptor Patched, which in turn promotes a pathway to inhibit the inhibitor of a transducer.
• Involved in limb patterning, neural tube induction and patterning, somite differentiation, gut regionalization
etc.
Transforming Growth Factor-ß
• 30 identified
• Involved in extracellular matrix formation, epithelial branching of the lungs, kidneys, and salivary glands.
Induces bone formation, regulating cell division, apoptosis, and cell migration.
Receptors

Lipid-based
 Extracellular Domain
Transmembrane Domain

Cytoplasmic domain
Key Endocrine and Paracrine Signaling
pathways for Development
 SONIC HEDGEHOG PROTEIN - Master Morphogen,
Sonic Hedgehog: Master produced in the notochord and limb bud, secreted
Gene for Embryogenesis molecule that would establish concentration gradients and
instruct cells in how to become different tissues and organs.

Upon binding of SHH to

Ptc, Ptc activity is
eliminated, the inhibition
of Smo is removed, and
Smo Ptc
Smo is activated to,
ultimately, upregulate
activity of the GLI family
(1 to 3) of transcription
factors that control
expression of target
genes.
 FRIZZLED Signal
Planar Cell Polarity:
Convergent Extension
Pathway
Regulates the process of convergent
extension whereby a tissue becomes longer Diego
and narrower. Convergent extension Prickle
requires changes in cell shape together with Activates
cell movement and intercalation with other Dishevelled
cells Activates

Kinases

Activates

c-Jun N-terminal kinases

-Binds to transcription factors
to upregulate expression of
genes involved in
cytoskeletal changes
Juxtacrine Signaling
Does not involve diffusible factors

Instead, there are three (3) juxtacrine signaling:

• A protein on one cell surface interacts with a receptor on an adjacent cell in a
process analogous to paracrine signaling
• Ligands in the extracellular matrix secreted by one cell interact with their
receptors on neighboring cells
• There is direct transmission of signals from one cell to another by gap junctions
Juxtacrine Signaling
1. A protein on one cell surface
interacts with a receptor on an
adjacent cell in a process
analogous to paracrine signaling
Juxtacrine Signaling
2. Ligands in the extracellular matrix
secreted by one cell interact with
their receptors on neighboring
cells

Integrins anchor cell into the

extracellular matrix. The binding of
integrins to the enzyme
Spermidine/spermine acetyl
transferase inhibits the activity of
spermidine and spermine by adding an
acetyl (CH3OR) group. The acetylation
of Spermidine and spermine inhibits
them from inhibiting the activity of the
Kir 4.2 potassium channel. The activity
of the potassium channel modulates
cell migration
Juxtacrine Signaling
3. There is direct transmission of
signals from one cell to another by
gap junctions
Key Juxtacrine Signaling pathways for
Development
The Notch Pathway
the cleaved portion of the protein
enters the nucleus directly and binds
to a DNA-binding protein that normally
represses transcription of Notch target
genes. Binding of
Notch removes
the inhibitory
activity of the
repressor and
permits
Notch signaling is involved in cell activation of
proliferation, apoptosis, and epithelial to downstream
mesenchymal transitions. It is especially genes
important in neuronal differentiation, blood
vessel formation and specification
(angiogenesis), somite segmentation,
pancreatic B-cell development, B- and T-cell
differentiation in the immune system,
development of inner ear hair cells, and
septation of the outflow tract of the heart.
Induction and Organ Formation
Communication between cells
Essential for development and organ formation
Organizers, which comprise groups of cells with the ability to instruct adjacent cells into specific
states, represent a key principle in developmental biology.

The concept was first introduced by Spemann and Mangold, who showed that there is a cellular
population in the new embryo that elicits the development of a secondary axis from adjacent cells.
Similar experiments in chicken and rabbit embryos.
Spemann organizer:
A multicellular structure situated above the developing
blastopore lip in the gastrula stage amphibian embryo that
when transplanted to a different but specific area of the Grafted
embryo (the opposite pole) is able to: cells

(1) induce an ectopic neural plate;

(2) act as the source of cells for the prechordal plate and the
notochord; and

(3) promote convergence and extension movements in host

cells (Gerhart, 2001; Harland and Gerhart, 1997).

It was noted by C. Stern that Spemann had initially referred

to ‘a piece of embryonic tissue that creates an “organization
field” of a certain [axial] orientation and extent, in the
indifferent material in which it is normally located or to
which it is transplanted’, and that ‘this concept embodies
both induction and patterning: the grafted cells change the
fate of the responding tissue, and also generate a coherent
(“organized”) set of structures’ (Stern, 2001).
Induction and Competence
Induction—one cell or group of cells changes the behavior (fate, differentiation, shape, mitotic
activity, etc.) of another cell or group of cells. In each such interaction, one cell type or tissue is the
inducer that produces a signal, and one is the responder to that signal.

Competence—The capacity to respond to a signal from an inducer. Essentially means competent

cells have receptors and all necessary second messengers necessary to respond appropriately to the
signal
• Receptor must be present
• Signal transduction pathway must be present and active
• may need to remove an inhibitor to respond
• cell fate may already be determined (gene expression profile “locked in”)
• Often a temporal component—responding tissue may only be competent during a
• specific “developmental window”; earlier or later and the responding tissue is not
competent.
Two main types of interactions
Instructive – occurs where the responding cell has a choice of fates and will follow
one developmental pathway following induction, and an alternative pathway in
absence of the inductive signals.

Example: in the early frog embryo, ectoderm will form the neural plate in
presence of inductive signals from the notochord, but epidermis in the absence
of induction. 

Example: notochord induces neural tube cells to form floor plate if notochord
is removed --> no floor plate differentiation. If notochord is transplanted to
lateral position-->lateral neural tube cells differentiate as floor plate cells

• Sonic hedgehog (Shh) is expressed in notochord can induce floor plate differentiation with
SHH (Sonic hedgehog protein)
Two main types of interactions
Permissive – Occurs where the responding cell is already committed to a certain
fate, and requires the inducing signal to proceed in the developmental pathway.
Cells contain the information but require specific environment to express fate. –
Signals allow a response –Signals do not designate a specific response –ECM allows
differentiation

• Here, the responding tissue contains all the potentials that are to be expressed,
and needs only an environment that allows the expression of these traits,

Example: many tissues need a solid substrate containing fibronectin or laminin in

order to develop. The fibronectin or laminin does not alter the type of cell that is to
be produced, but only enables what has been determined to be expressed.
Epithelial Mesenchymal
Interactions
Many inductive interactions occur between
epithelial and mesenchymal cells. Epithelial cells
are joined together in tubes or sheets, whereas
mesenchymal cells
are fibroblastic in appearance and dispersed in
extracellular matrices. Although an initial signal
by the inducer to the responder initiates the
inductive event, crosstalk between the two
tissues or cell types is essential for
differentiation to continue (Fig. 1.5, arrows).

All organs consist of an epithelium and an

associated mesenchyme, so epithelial-
mesenchymal interactions are among the most
important phenomena in nature
Regional Specificity of
Induction
Skin is composed of two main tissues: an outer
epidermis, an epithelial tissue derived from
ectoderm, and a dermis, a mesenchymal tissue
derived from mesoderm. The chick epidermis
signals the underlying dermal cells to form
condensations (probably by secreting Sonic
hedgehog and TGF-β2 proteins), and the
condensed dermal mesenchyme responds by
secreting factors that cause the epidermis to
form regionally specific cutaneous structures.
These structures can be the broad feathers of When cells from different regions of the dermis
the wing, the narrow feathers of the thigh, or (mesenchyme) are recombined with the epidermis
the scales and claws of the feet. (epithelium) in the chick, the type of cutaneous structure
made by the epidermal epithelium is determined by the
original source of the mesenchyme
 Genetic Specificity of
Induction
the mesenchyme may instruct the epithelium as
to what sets of genes to activate, the
responding epithelium can comply with these
instructions only so far as its genome permits
(factors may include packaging of DNA and
other regulatory levels).

Since the ectodermal cells of each donor have unique Reciprocal transplantation between the presumptive oral
genomes, the structures of the donor developed and not of ectoderm regions of salamander and frog gastrulae leads to
the recipient newts with tadpole suckers and tadpoles with newt
balancers.