Download Gene Regulation and Pathological Studies Using Mouse models

Gene Regulation and
Pathological Development
Studies Using Mouse models
Yaowu Zheng
Transgenic Research Center
[email protected]
(0431)85098583
Materials and Lecturers
• Oct. 9: Introduction to Gene regulation, Zheng
• Oct. 16: Gene expression and development, Zheng
• Oct. 23: Gene expression and coagulation system, Zheng
• Oct. 30: Gene expression and immune system, Professor Zhang
• Nov. 6: Gene expression and cancer, Zheng
• Nov. 13: Gene expression and vascular system, Professor Zhang
• Nov. 20: Gene expression and obesity, Dr. Xiaodan Lu
• Nov. 27: Gene expression and cardiovascular disease, Zheng
• Dec. 4: Gene expression and reproduction, Yan Ji
• Dec. 11: Transgenic technology, Zheng
Lecturers:
• Yan Ji: Ph.D student in Zheng lab.
• Xiaodan Lu: Postdoctoral fellow in Jilin University, former Ph.D graduate student from
Zheng’s lab
• Professor Zhang: Assistant professor, Ph.D graduate from Nanking University
• Yaowu Zheng: Professor in Transgenic Research Center, NENU
Gene Regulation
Introduction to gene regulation
Contents
•
•
•
•
•
•
•
•
Basic gene regulation
Higher level regulation
Prokaryotic regulation
Eukaryotic regulation
Regulation and development
Gene regulation and disease
Application of gene regulation
Gene regulation studies using transgenic mice
CENTRAL DOGMA
• Genetic information always goes
from DNA to RNA to protein
Updated Central Dogma
Biological sequence information
Primary structure in living organisms
• 3 linear biopolymers:
– Polydeoxyribonucleotide= DNA
– Polyribonucleotide= RNA
– Polypeptide=Proteins
•
3 classes of information transfer suggested by the dogma:
– General case
• Replication: DNA → DNA,
• Transcription: DNA → RNA,
• Translation: RNA → protein (general)
– Special case
• Reverse transcription: RNA → DNA,
• Viral: RNA → RNA,
• Non-natural: DNA → protein
– Unknown case
• protein → DNA,
• protein → RNA,
• protein → protein? prions
Regulation at DNA levels
Double helix Structure
Prokaryotic Gene Regulation
• Combination of a promoter and a gene or genes is called an OPERON
• Operon is a cluster of genes encoding related enzymes that are
regulated together and performed one related processes
• Operon consists of
– A promoter site where RNA polyerase binds and begins transcribing
the message.
• Genes coding protein from the message (mRNA) is called
structural genes.
• One mRNA codes for more than one protein is called
polycistronic.
– An upstream region (a gene) that makes a protein called repressor.
The gene codes for repressor is called regulatory gene. The
repressor is like a transcription factor in eukaryotes.
• Active repressor sits a site between promoter and structural genes and
blocks transcription, This site is called the operator which works like an
enhancer or silencer in eukaryotes.
Types of Operons
• Inducible Operon
– e.g lac operon
• Repressible Operon
– e.g trp operon
Trp Operon
• E. coli uses several proteins encoded by a cluster of 5 genes
to manufacture the amino acid tryptophan
• All 5 genes are transcribed together as a unit called an
operon, which produces a single long piece of mRNA for all
the proteins
• RNA polymerase binds to a promoter located at the
beginning of the first gene and transcribe the genes in
sequence
• A trp repressor protein is made constantly (constitutively).
• When tryptophan is present in the cell, it binds to repressor
and activates it, so it can binds operator and tell operon to
stop making tryptophan.
• When tryptophan is absent, the trp repressor is inactive and
can not binds to operator, so trp operon is making all the
necessary proteins for tryptophan synthesis.
Trp Operon
Tryptophan Gene Regulation (Negative control)
Lac Operon
• The lac Operon
– Regulates lactose metabolism
– It turns on when lactose is present & glucose is absent.
– Lactose is a disaccharide, a combination of Galactose & Glucose
• To Ferment Lactose E. coli Must:
– Transport lactose across cell membrane
– Separate the two sugars. To do that it needs three enzymes, it makes them all at
once!
– 3 Genes Turned On & Off Together:
• Lac Z code for b-galactosidase
• LacY codes for permease that allows lactose to enter cell
• LacA code for enzyme that acetylates lactose
Lac Operon
•
•
•
•
•
•
•
•
•
Near the lac operon is another gene, called lacI that codes for the lac
repressor protein
The lac repressor gene is expressed “constitutively”, meaning that it is always
on.
Just upstream from the transcription start point in the lac operon are two
regions called the operator (o) and the promoter (p).
Operator is the DNA sequence that repressor binds.
The promoter is the site where RNA polymerase binds and starts transcription.
Operator and promoter are “cis” or associated to lac operon.
Lac repressor protein is “trans” to lac operon, since the repressor is diffusible
and can bind to other lac operator sequences.
In the presence of lactose, the repressor binds to lactose, changes
conformation and floats away from the operator. RNA polymerase can bind to
the promoter and transcribed z, y and a. Permease allow lactose to enter the
cell, b-galactosidase digests the lactose and transacetylase modify galactose.
When level of lactose drops, the released repressor binds to the operator
DNA again and blocks lac operon
Lac Operon
Prokaryotic Translational
Control
• Shine-Dalgarno sequence is generally located 8
basepairs upstream of the start codon AUG
• The six-base consensus sequence AGGAGG
helps to recruit the ribosomes to the mRNA to
initiate protein synthesis
• The complementary sequence (CCUCCU),
called the anti-Shine-Dalgarno sequence, is
located at the 3' end of the 16S rRNA in the 30S
ribosome subunit.
Eukaryotic Gene Regulation
• A multi-cell organism may contain millions of cells.
• Virtually every cell in your body contains 1 or 2
complete set of genes
• But they are not all turned on at all the time or in
every tissues
• Each cell or tissue in your body expresses only a
small subset of genes at any time
• During development, different cells express
different sets of genes in a precisely regulated
fashion
• Some genes are expressed only at certain
conditions
Different morphology,
same genome
Neuron and lymphocyte
Multilevel Gene Regulation in
Eukaryotes
• Features of Eukaryotic Genomes
• Gene structure of eukaryotes
• The types of regulation in eukaryotes
• Multi-level of gene expression and
regulation
Chromatin
Chromosome
Nucleosome:
Two of each H2A, H2B, H3
and H4 subunits in the core
Average 200 bp
140bp in core area
Genome Level Regulation
–
–
–
–
–
–
Epigenetic Modification
Developmental methylation and Acetylation
DNA folding and unfolding
X-chromosomal inactivation
Chromosomal remodeling
Replication
Unpredictable Gene Regulation
at DNA Levels
•
•
•
•
•
•
Gene Deletion
Gene Duplication
DNA Rearrangement
Gene Amplification
Chemical Modification
Environmental DNA
damage, like UV
DNA Replication
• In the Central Dogma, DNA replication occurs in order to
faithfully transmit genetic material to the progeny.
• Replication is carried out by a complex group of proteins
called the replisome
• Replisome consists of a helicase that unwinds the
superhelix as well as the double-stranded DNA helix
• DNA polymerase and its associated proteins insert new
nucleotides in a sequence specific manner, like copy
machine.
• This process typically takes place during S phase of the
cell cycle.
Epigenetic Modification
• Epigenetics is the study of heritable changes in
gene expression or cellular phenotype caused by
changes other than underlying DNA sequence
• Epigenetic changes are preserved when cells
divide.
• DNA methylation is important in the control of
gene transcription and chromatin structure.
• The epigenetic changes in eukaryotic biology is
active in the process of cellular differentiation
• Methylation of mRNA was demonstrated having a
critical role in human energy homeostasis in 2011
DNA Methylation
•
•
•
•
•
Variation in methylation states of DNA can alter gene expression levels significantly.
Methylation variation usually occurs through the action of DNA methylases.
When the change is heritable, it is considered epigenetic.
When the change in information status is not heritable, it would be a somatic epitype.
The effective information content has been changed by means of the actions of a protein
or proteins on DNA, but the primary DNA sequence is not altered.
CpG islands
Genomic regions that contain a high
frequency of CG dinucleotides.
CpG islands particularly occur at or near the
transcription start site of housekeeping genes.
Housekeeping gene: A gene is required for basic functions the
sustenance of the cell.
Housekeeping genes are constitutively
expressed
Luxury gene:
A gene has specialized functions
expressed in large amounts in particular
cell types or “inducible”.
Gene regulation by Methylation
TF
RNA pol
Active
transcription
Unmethylated CpG island
TF
RNA pol
CH
CH
CH
3
3
3
Methylated CpG island
Repressed
transcription
Histone modification
 Methylation
 Acetylation
TF
Mechanisms of gene regulation
a)
b)
Histone acetylation directly attracts
transcription factors
Histone acetylation further attracts coactivator proteins
Eukaryotic Gene Regulation
• Key Concept:
– Eukaryotic genes are controlled in a group or
Individually at different levels
– Have many regulatory sequences
– Are much more complex than prokaryotic genes
Comparisons
• Eukaryotes
• Prokaryotes
– Genes controlled in
group by operons
– 27% of E. coli genes
are controlled by
operons
– Housekeeping genes
not controlled by
operons
– Simultaneous
transcription and
translation
– No operons, but they still need
to coordinate regulation
– Genes are fragmented into
exons and introns
– More kinds of control elements,
many levels of control
– Separated transcription and
translation location and control
– Epigenetic regulation and
Chromatin remodeling
– Tissue-specific, developmental
and inducible regulation
Basics of eukaryotic gene
regulation
•Specific transcription factors bind to
regulatory elements (enhancers and
silencers) and regulate transcription
•Regulatory elements may be tissue
specific and will activate their gene
only in one kind of tissue
•Regulatory elements may be
developmental and only expressed
at certain stages
•Regulatory elements may be
inducible and expressed only at
certain condition
•Sometimes the expression of a
gene requires the function of two or
more different regulatory elements
Eukaryotic Gene Regulation
•
•
•
•
•
Monocistronic: one mRNA, one protein
Basic promoter: TATA box, CAT box, GC-rich
Enhancers: Distance and orientation independent
Silencers: Distance and orientation independent
Transcription factors: Contain at least 2
domains:_Enhancer specific DNA binding and activation
(silencing) of RNA polymerase
• Intron and exons: Number and size variable
• 3 RNA polymerases in eukaryotes
– RNA pol I for ribosomal RNAs
– RNA pol II for mRNAs
– RNA pol III for small RNAs
Mode of Gene Regulation
•
•
•
•
Developmental: When
Tissue-specific: Where
Event-induced: What
Intensity level: How much
Question:
How does this happen?
What decide tissue-specificity?
What decide the developmental stages?
What decide the expression level?
Eukaryotic Transcription
•
•
•
•
•
Promoter strength
Tissue-specific enhancers and silencers
Transcription Factors
Post-transcriptional regulation
RNA stability, RNA degradation and half
life
Eukaryotic Promoters
Core promoter
• Determine transcriptional start site
• In prokaryote: -10 region
• In eukaryote: TATA-box
Proximal elements of promoter
• in prokaryote: -35 region
• In eukaryote: CAAT-box, GC-box
Enhancer
• A regulatory DNA sequence that greatly enhances the transcription of a gene.
They are mostly tissue specific
Silencer
• A DNA sequence that helps to reduce or shut off the expression of a nearby
gene. They are mostly tissue-specific
Terminator
• A DNA sequence downstream of a gene and recognized by RNA polymerase as
a signal to stop transcription.
• PolyA signals (AATAAA) tell where to cut and add poly A sequences
Transcription factors
•
•
Trans-acting factors
Contain at least 2 domains
– DNA binding domain
– Transcription activation domain
 Acidic domains,
 Glutamine-rich domains
 Proline-rich domains
•
•
They are proteins that bind to the cis-acting elements to control gene
expression
Control gene expression in several ways:
–
–
–
–
–
may be expressed in a specific tissue, “tissue-specific”
may be expressed at specific time in development, “Developmental”
They may be induced by certain conditions, “inducible”
may be required for protein modification
may be activated by ligand binding, e.g. hormone receptors
HTH (helix-turn-helix)
α-helix (N-terminus)----DNA-specific
α-helix (C-terminus)----non-specific
Leu Zipper
Three Zinc Finger Motifs
forming the recognition site
Regulation of Transcription
Homeodomain Protein in Drosophila utilizing helix-turn-helix motif
to regulate developing patterns
Synergistic Regulation
INTRONS AND EXONS
• Eukaryotic DNA differs from prokaryotic DNA in that
the coding sequences are interspersed with
noncoding sequences called introns
• Sequences retain in mature mRNA are called EXONS.
A mature mRNA contains 5’-UT, coding region and
3’UT.
• Introns are spliced out, cap and polyA are added
before it matures and transported to cytoplasm, where
it is ready for translation
• Introns can be very large and numerous, so some
genes are much bigger than the final processed
mRNA
• Yeast has 4% genes with introns, Mammals have most
genes with introns.
Transcriptional Regulation
• Three RNA polymerases responsible for all the transcription
• Specific transcription factors bind to these regulatory
elements and regulate transcription
• Regulatory elements may be tissue specific and will activate
their gene only in one kind of tissue
• Regulatory elements may be developmental and only
expressed at certain stages
• Regulatory elements may be inducible and expressed only at
certain condition
• Sometimes the expression of a gene requires the function of
two or more different regulatory elements
• In eukaryotic cells the primary transcript (pre-mRNA) must be
processed further in order to ensure translation.
• This normally includes a 5' cap, a poly-A tail and splicing.
• Alternative splicing can contributes to the diversity of proteins
any single mRNA can produce.
Regulation of Eucaryotic Genes
Transcription complex
Insulator Elements (boundary elements) help to
coordinate the regulation
Post-Transcriptional
Regulation
•
•
•
•
•
•
•
•
•
Gene Regulation through mRNA Processing
Exon shuffling
Alternative splicing
RNA editing
RNA stability
RNA degradation
RNA half life
mRNA Transport Control
RNA Interference (RNAi)
–
–
miRNA
siRNA
Post transcriptional Regulation
•
RNA Splicing
– Introns are removed during this process, GT-AG junction
– Exons can be spliced together in different ways to generate variants and perform
different functions
– Alternate splicing is common in insects and vertebrates
•
RNA editing
– Nucleoside modifications such as cytidine (C) to uridine (U) and adenosine (A) to
inosine (I) deaminations, as well as non-templated nucleotide additions, deletions and
insertions.
– RNA editing in mRNAs effectively alters the amino acid sequence of the encoded
protein so that it differs from that predicted by the genomic DNA sequence.
– Such changes have been observed in tRNA, rRNA, mRNA and microRNA molecules
of eukaryotes but not prokaryotes
•
•
•
•
•
•
Capping: Adding two G in 3’-5’ direction, important for ribosomal binding.
Polyadenylation: Adding long stretches of As, related to mRNA stability.
Nuclear exporting, mRNA transport control
RNA stability
RNA degradation, RNA half life
RNA Interference (RNAi)
– miRNA
– siRNA
RNA Silencing
Reverse transcription
• Reverse transcription is the transfer of
information from RNA to DNA.
• Important for RNA virus propagation
• Occur in the case of retroviruses, such as
HIV, Involves chromosomal integration
• Occurs in eukaryotes in the case of
retrotransposons and telomere synthesis.
Transposons
•
•
•
•
•
A transposable element (TE) is a DNA sequence that can change its relative position (self-transpose) within the
genome of a single cell
The mechanism of transposition can be either "copy and paste" or "cut and paste"
Transposition can create phenotypically significant mutations and alter the cell's genome size.
Barbara McClintock's discovery of these jumping genes early in her career earned her a Nobel prize in 1983
Class I (retrotransposons):
– They copy themselves from DNA to RNA by transcription, from RNA back to DNA by reverse transcription.
– The DNA copy is then inserted into the genome in a new position.
– Reverse transcription is catalyzed by a reverse transcriptase often coded by itself, behave very similarly to
retroviruses, such as HIV.
Class II (DNA transposons):
• Cut-and-paste transposition mechanism, No RNA
intermediate.
• Catalyzed by various types of transposase enzymes.
• Transposases bind non-specifically to any target
site or to specific sequence targets.
• The transposase makes a staggered cut at the
target site producing sticky ends, cuts out the DNA
transposon and ligates it into the target site.
• A DNA polymerase fills in gaps from the sticky ends
and DNA ligase closes backbone resulting in target
site duplication.
• The duplications at the target site can result in gene
duplication, which plays an important role in evolution
Function of transposable
elements
•
•
•
•
From the immediate point of
view, transposons have no
necessary function in the cell called, junk DNA"; or "selfish
DNA", as transposons
propagate on behalf of the
cellular resources.
On a wider scale, the motility of
the retrotransposable elements
can be important for genome
plasticity.
Occasional insertion into genes
can disrupt the gene function
and cause an inherited disease
(Fig. 3C).
LTR and LINE elements can
also change gene expression, if
inserted near a gene, as LTRs
and LINE 5´UTR have strong
promoter activity in both
directions (Fig. 3F).
Eukaryotic Translation
•
•
•
•
•
•
•
•
•
•
Ribosome bind to cap site and scan down. When meet the first good ATG (with
Kozak sequences), translation starts.
mRNA is read by the ribosome as triplet codons, usually beginning with an AUG,
or initiator methionine codon downstream of the ribosome binding site.
Complexes of initiation factors and elongation factors bring aminoacylated
transfer RNAs (tRNAs) into the ribosome-mRNA complex, matching the codon in
the mRNA to the anti-codon on the tRNA, thereby adding the correct amino acid
in the sequence encoding the gene.
As the amino acids are linked into the growing peptide chain, they begin folding
into the correct conformation.
Translation ends with a UAA, UGA, or UAG stop codon.
The nascent polypeptide chain is then released from the ribosome as a mature
protein.
One mRNA can only be translated once, one mRNA, one protein. Occationally
(viral) ribosome can start translation from the middle of a mRNA when it contains
a IRES (internal ribosomal entry sequence) sequence.
In some cases the new polypeptide chain requires additional processing to make
a mature protein (post-translational modification).
The correct folding process is quite complex and may require other proteins,
called chaperone proteins.
Occasionally, proteins themselves can be further spliced; when this happens, the
inside "discarded" section is known as an intein.
Translational Regulation
• Translation efficiency:
– Ribosomal binding, Kozak sequence
– Blocking mRNA Attachment to Ribosomes
– Elongation
• Coordinated translation, folding,
processing, modification, translocation and
secretion
• Protein stability, N-terminal rule
• Protein degradation, ubiquitylation
Translational
and Post-translational Regulation
•
•
•
•
•
Translation Control
Blocking mRNA Attachment to Ribosomes
Regulation of Protein Processing
Protein Modification
Phosphorylation
–
–
–
–
–
–
Ser/Thr type
Tyr type
Reversible
Integrated signals from different pathways effectively
The same kind kinase or phosphatase is multiple-substrates.
Modified different amino acids, have different influences
Chaperone functions: Hsp70
The Fate of Proteins after translation
Post-translational Modification
•
Addition of functional groups
–
–
–
•
•
•
•
Addition of other proteins or peptides, e.g.,dimerization
Changing the chemical nature of amino acids
Structural changes, amino acid aditing
Protease processing
–
–
–
•
•
•
•
•
Ser/Thr type, Tyr type
Reversible, Integrated signals from different pathways
Sumoylation: Small Ubiquitin-like Modifier (SUMO) proteins
–
–
•
Activation of proteases, e.g., coagulation factors
Structure maturation, e.g., Insulin
Signal peptide removal during translocation and secretion
Glycosylation: Epitope recognition
Acetylation: Histone modification
Methylation: Histone modification
Ubiquitylation: Direct protein to degradation
phosphorylation
–
–
•
addition of cofactors for enhanced enzymatic activity
modifications of translation factors
addition of smaller chemical groups
A family of small proteins that are covalently attached to and detached from other proteins in cells to
modify their function.
Involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation,
apoptosis, protein stability, response to stress, and progression through the cell cycle
Lipidation: Addition of hydrophobic groups for membrane localization
–
–
–
myristoylation
palmitoylation
isoprenylation or prenylation
Protein Degradation
• The half-lives of proteins within cells vary widely, from minutes to
several days, and differential rates of protein degradation are an
important aspect of cell regulation.
• Many rapidly degraded proteins function as regulatory molecules,
such as transcription factors.
• The rapid turnover of these proteins is necessary to allow their
levels to change quickly in response to external stimuli.
• Other proteins are rapidly degraded in response to specific signals,
providing another mechanism for the regulation of intracellular
enzyme activity.
• In addition, faulty or damaged proteins are recognized and rapidly
degraded within cells, thereby eliminating the consequences of
mistakes made during protein synthesis.
• In eukaryotic cells, two major pathways—the ubiquitin-proteasome
pathway and lysosomal proteolysis—mediate protein degradation.
The Ubiquitin-Proteasome
Pathway
•
•
•
•
•
•
The major pathway of selective protein
degradation in eukaryotic cells uses
ubiquitin as a marker that targets cytosolic
and nuclear proteins for rapid proteolysis.
Ubiquitin is a 76-amino-acid polypeptide
that is highly conserved in all eukaryotes
(yeasts, animals, and plants).
Proteins are marked for degradation by
the attachment of ubiquitin to the amino
group of the side chain of a lysine residue.
Additional ubiquitins are then added to
form a multiubiquitin chain and recognized
and degraded by a large, multisubunit
protease complex, called the proteasome.
Ubiquitin is released in the process, so it
can be reused in another cycle.
It is noteworthy that both the attachment
of ubiquitin and the degradation of
marked proteins require energy in the
form of ATP.
N-end rule
•
•
•
•
•
The N-end rule is a rule related to ubiquitination, discovered by
Alexander Varshavsky in 1986.
The rule states that the N-terminal amino acid of a protein
determines its half-life.
The rule applies to both eukaryotic and prokaryotic organisms,
but with different strength.
However, only rough estimations of protein half-life can be
deduced from this 'rule', as N-terminal amino acid modification
can lead to variability and anomalies, whilst amino acid impact
can also change from organism to organism.
Other degradation signals, known as degrons, can also be found
in sequence.
N-terminal residues in Yeast
Met, Gly, Ala, Ser, Thr, Val, Pro - > 20 hrs
(stabilising)
Ile, Glu - approx. 30 min (stabilising)
Tyr, Gln - approx. 10 min (destabilisiing)
Leu, Phe, Asp, Lys - approx. 3 min
(destabilising)
Arg - approx. 2 min (destabilising)
"N"-terminal residues approximate half-life of
proteins in mammalian
systems
Val -> 100h
Met, Gly -> 30h
Pro - > 20h
Ile -> 20h
Thr -> 7.2h
Leu -> 5.5h
Ala -> 4.4h
His -> 3.5h
Trp -> 2.8h
Tyr -> 2.8h
Ser -> 1.9h
Asn -> 1.4h
Lys -> 1.3h
Cys -> 1.2h
Asp -> 1.1h
Phe -> 1.1h
Glu -> 1.0h
Arg -> 1.0h
Gln -> 0.8h
Lysosomal Proteolysis
•
•
•
•
•
•
•
•
The other major pathway of protein degradation in
eukaryotic cells involves the uptake of proteins by
lysosomes.
Lysosomes are membrane-enclosed organelles that
contain an array of digestive enzymes, including
several proteases.
They have several roles in cell metabolism, including
the digestion of extracellular proteins taken up by
endocytosis as well as the gradual turnover of
cytoplasmic organelles and cytosolic proteins.
The containment of proteases and other digestive
enzymes within lysosomes prevents uncontrolled
degradation of the contents of the cell.
Therefore, in order to be degraded by lysosomal
proteolysis, cellular proteins must first be taken up by
lysosomes.
One pathway for this uptake of cellular proteins,
autophagy, involves the formation of vesicles
(autophagosomes) in which small areas of cytoplasm
or cytoplasmic organelles are enclosed in membranes
derived from the endoplasmic reticulum.
These vesicles then fuse with lysosomes, and the
degradative lysosomal enzymes digest their contents.
The uptake of proteins into autophagosomes appears
to be nonselective, so it results in the eventual slow
degradation of long-lived cytoplasmic proteins.
Inteins
• An intein is a segment of a protein able to excise itself from the
chain of amino acids as they emerge from the ribosome and rejoin
the remaining portions with a peptide bond.
• This is a case of a protein affecting its own primary sequence
encoded originally by the DNA of a gene.
• Additionally, most inteins contain a Homing endonuclease or HEG
domain which is capable of finding a copy of the parent gene not
containing the intein nucleotide sequence.
• On contact with the intein free copy the HEG domain initiates the
DNA double-stranded break repair mechanism.
• This process causes the intein sequence to be copied from the
original source gene to the intein free gene.
• This is an example of protein directly editing DNA sequence, as well
as increasing the sequences heritable propagation.
Prions
•
•
•
•
•
•
•
Reported in 1982 by Stanley B. Prusiner
Prions are infectious agent composed of
protein in a misfolded form
Proteins that propagate themselves by
making conformational changes in other
molecules of the same type of protein.
This change affects behavior of the
protein.
In fungi this change happens from one
generation to the next, i.e. Protein →
Protein.
A transfer of information through protein,
prion interactions leave the sequence of
the protein unchanged.
Causing many diseases in mammals
– ability to catalytically convert native
protein to an infectious state
– capable of inducing a phenotypic
change without a modification of the
genome
Microscopic "holes" are
characteristic in prionaffected tissue sections,
causing the tissue to develop
a "spongy" architecture.
How to study gene functions
• High throughput sequencing has uncovered thousands of
genome and predicted huge numbers of genes.
• Biologic functions of many are still unknown.
• Systematic study of gene functions are needed.
• Gene level: Gene inactivation (Gene knockout)
– Global
– Tissue-specific
• Transgenic Studies: over expression
• Anti-sense RNA knock down (siRNA)
• Protein level: Mutant protein, dominant-negative
– Site-directed mutagenesis: amino acid change and function
– Biochemical studies
• In vitro (cellular level) and In vivo (animal) studies
Application of Gene Regulation
• Gene Therapy
– To correct mutations
– To provide proper amount of functional
proteins
– To curb the bad gene expression
• Transgenics
– To facilitate research, animal models of
diseases
– To improve live stocks for better nutrition,
better health, better economic values.
Identification of Genetic Association of Disease by
Human Genome Sequencing
•
•
•
•
•
•
•
•
Chromosomal studies
SNPs
Non-coding regions
Non-translated regions
Non-coding RNAs
Small RNAs
SiRNAs
Auto-immune diseases
Aspect of Gene regulation
• Systematic: Conserved and Species-specific
programming
• Genetic: Each individual predetermined
• Epigenetic: Predetermined and developmentally
determined
– There are several layers of regulation of gene expression.
– One way that genes are regulated is through the remodeling of
chromatin
• Chromatin Remodeling
–
–
–
–
DNase I hypersensitive site
DNA methylation
Histone Methylation
Histone acetylation
• Environmental: Unpredictable
Homeostasis
• Genetic determinants try to maintain the balanced
development internally
• At the same time try to adapt environment changes
externally
• When the balances is lost, a pathologic development
comes
– Genetic disease
– Acquired disease
Environment Causes of Cancer:
 Viruses, bacteria infection
 Smoking
 Unhealthy Food
 Radiation
 Chemicals, pollution
 Depression
Summary
• Gene regulation happens every where
• Genomic level_developmental
• Gene level
• Transcriptional, post-transcriptional
• Translational, post-translational
• Protein activation and deactivation
• Degradation and Half life
• Gene out of control causes disease
• One gene, one disease (protein disease, Hemoglobin)
• One gene, many diseases (regulatory factor disease, VEGF)
• Many genes, one disease (pathway disease)
• Direct cause, easy to identify
• In direct effect, hard to cure