Analysis of Gene Expression

Question 1

What is differential gene expression?

Answer 1

Different cells expressing different genes

Development of a single cell into a complex organism depends on formation of different cell types
Genes are expressed at different levels

Question 2

What is differential gene expression caused by?

Answer 2

Regulatory proteins

It is caused by changes in expression of an unchanging set of genes
Cells make selective use of their genes – they turn expression on or off depending on cues
This is controlled at many stages - most common control point is transcription - by regulatory proteins

Production of more RNA = more active protein

Question 3

What do regulatory proteins do?

Answer 3

Genes have long control regions often >10,000 bp
These bind regulatory proteins - enhancers/repressors
The proteins bind to short stretches of DNA
They work synergistically to amplify transcription and therefore expression

Different cells have different regulatory proteins
These affect RNA polymerase binding and transcription

Question 4

How can we measure gene expression?

Answer 4

We can look at abundance of mRNA or protein levels

We do this in more than one cell type to compare the genome

Question 5

What are some methods of detection of expression looking at abundance of mRNA?

Answer 5

Northern blot analysis
Quantitative PCR
Microarrays
RNA sequencing
In situ hybridization

They all rely on nucleic acid hybridisation (driven by Watson-Crick base pairings)
We know the sequence of the gene so we can design a complementary probe

Question 6

Detection of expression looking at abundance of mRNA: describe northern blot analysis?

Answer 6

Harvest cell types
RNAs separated by SDS-PAGE
RNAs are transferred to a filter
A labelled probe hybridises to the mRNA
The probe is detected and quantified

This is a very direct method - can be quantative
The level of darkness of a band indicates more material that it could bind to - therefore more expression
This is quantified by a machine measuring the density of material there

Question 7

Detection of expression looking at abundance of mRNA: describe quantitative PCR?

Answer 7

Harvest RNA from different cell types
Reverse transcription of mRNA into cDNA
Quantify PCR amplification with either fluorescent primers or dye that binds dsDNA
Rate of product appearance relates to concentration of mRNA

Benefits: quantitative, rapid and can detect several targets in one tube

Question 8

Detection of expression looking at abundance of mRNA: describe micro arrays?

Answer 8

DNA oligo representing 1000s of genes are immobilised on chip
Cell mRNA copied to cDNA using reverse transcriptase and then labelled with red or green dye
cDNAs hybridised washed and scanned
Red = expression in A
Green = expression in B
Yellow = both A and B

Can detect and quantify thousands of transcripts simultaneously

Question 9

Detection of expression looking at abundance of mRNA: describe RNA sequencing?

Answer 9

Determine abundance of all RNAs in a cell
Harvest total RNA
Select/amplify mRNAs
Perform RNA sequencing
Compare expression levels of all genes

We can look at every single gene expressed in an organism - but not cheap

Question 10

Detection of expression looking at abundance of mRNA: describe In situ hybridisation?

Answer 10

Very different method:
Tissue is prepared by fixing and permeabilization
Addition of labelled DNA or RNA probe (fluorescently tagged)
Probe detection by microscopy

Benefits
Can simultaneously show abundance of transcript expression in all tissues of an organism
No need to separate out all tissues of an organism – they can remain in situ
Reveals information of both mRNA abundance and location

Question 11

What are some methods of detection of expression looking at abundance of protein?

Answer 11

2D gels - MS

Specific antibodies

Question 12

Detection of expression looking at abundance of proteins: describe 2D gels followed by mass spectrometry?

Answer 12

Isolate specific cell types
Lyse cells - release proteins
Separate proteins on 2D protein gel 
They move to their isoelectric point (pH where their charge becomes 0) 
The gel had a fixed pH gradient
Then separate on another gel via size

Stain the separated proteins - Coomassie blue dye
They form a pattern of spots
We can compare spot patterns - possible to identify differences in protein expression
Identify by mass spectrometry - orbitrap

Question 13

Detection of expression looking at abundance of proteins: describe specific antiboidies?

Answer 13

Isolate specific cell types
Lyse cells - release proteins
Separate proteins on a protein gel
Transfer proteins to a membrane
Probe membrane with an antibody
Detect and quantify the antibody

Question 14

What can we use for protein expression detection is there is no easy marker?

Answer 14

Reporter gene

If we can’t detect the expression we could add a reporter gene
This will produce reporter mRNA and therefore a reporter protein
Common regulator genes - GFP, B-galactosidase, B-glucuronidase and luciferase
Where reporter protein is detected, the gene is being expressed

Question 15

How is can we find how gene expression regulated?

Answer 15

Identify the gene regulatory sequences

Identify gene regulatory proteins

Question 16

Give an example of a gene we can identify the gene regulatory sequence and the gene regulatory proteins?

Answer 16

Even skipped gene (Eve)
Eve is essential for development of Drosophila
It helps define formation of the segmented body plan
Acts very early in the organization of the embryo
Eve expression occurs in 7 discrete stripes

Stretches for 20 kb; >7 kb upstream and >13 kb downstream
5 regulatory sequence modules control expression in 7 stripes
Stripe modules exert control of Eve expression by interacting with over 20 regulatory proteins
The 480 nt stope 2 module - binds 4 regulatory proteins: hunchback+, bicoid+, Kruppel- and Giant-

To determine this - Eve ORF was substituted for a reporter ORF to see where the gene was expressed

Question 17

How do we identify the gene regulatory sequences

Answer 17

Make deletions through: restriction enzyme digestion

Site directed mutagenesis
Gene synthesis

Question 18

Describe identification of gene regulatory sequences through restriction enzyme digestion?

Answer 18

Sequence entire regulatory region
Generate restriction map
Remove sequences by double restriction enzyme digests
Introduce the altered genes into Drosophilia eggs

Removal of BstEII - BssHII fragment abolished stripe 2 expression
These 480 nts contain all signals needed for stripe 2 expression
Delete all other sequences from the gene regulatory regions

Question 19

Describe identification of gene regulatory sequences through site directed mutagenesis and gene synthesis?

Answer 19

Site directed mutagenesis:
Can make precise nucleotide changes to define required sequences:
Precisely shorten region
Remove internal sequences
Make single/multiple nt changes

Gene synthesis:
You can make any sequence you want but it is very expensive

Using these methods alone or in combination allows the regulatory sequences to be defined

Question 20

How do we identify the strip 2 (eve) gene regulatory proteins?

Answer 20

Two methods:
Electrophoretic mobility-shift analysis
Affinity chromatography

Question 21

Describe identification of gene regulatory proteins through electrophoretic mobility-shift analysis?

Answer 21

1. dsDNA fragment (20-35 bp) containing a protein binding site is prepared (end-labelled)
2. A DNA probe is incubated with a protein fraction - protein-DNA complexes form
   A non-specific competitor is also added to eliminate non-specific interactions
3. Run in gel electrophoresis - under native conditions to free the bound probe
4. The gel is dried and position of the probe is detected using X-ray film
   The probe + cell fraction won’t move as far as the probe alone on the gel

Antibody can also be used to find out the presence of a protein
Due to the extra mass of the antibody (slower) this produces a super shift on the gel
If no antibodies are available, use MS analysis

It is very simple and highly sensitive

Question 22

Describe identification of gene regulatory proteins through affinity chromatography?

Answer 22

1. Make a DNA fragment of the regulatory region
2. Attach to a solid matrix - eg agarose
3. Add cell lysate to column -> regulatory proteins bind the immobilized DNA
   This will be washed and eluted with salt
4. Analyse by MS ID protein

Question 23

How can we determine where regulatory proteins bind exactly?

Answer 23

2 main methods:
DNA footprint analysis
Chromatin immunoprecipitation (ChIP) analysis

Question 24

Describe identification of location of gene regulatory proteins through DNA footprint analysis?

Answer 24

This allows us to identify nucleotides that are in contact with a DNA binding protein

Purify the binding protein
Incubate the DNA with the binding protein - forms a ‘hot’ regulatory region
The binding protein is in excess of the probe
Add DNase I - this binds to the minor groove and produces random nicks (only cuts once on a strand)
As this produces random cutes this can result in gaps/bands varying in intensity
The binding protein will protect its own binding site
Wash away the binding protein
Look at the sizes of the remaining DNA fragments

The products are separated in electrophoresis to reveal the ‘footprint’
There is a gap in the sizes of the labelled DNA fragments
The gap is where the binding protein binds
We can generate binding curves and equilibrium constants from this data

Question 25

Describe identification of location of gene regulatory proteins through Chromatin immuno-precipitation (ChIP)?

Answer 25

Allows in vivo identification of DNA sequences associated with proteins (DNA is in native chromatin state) Treat in vivo cells with formaldehyde or UV to cross-link - creating a temporary physical bond between DNA-protein and chromatin-protein complexes Chromatin fragmentation - cleave DNA into 300 bp fragments by sonication, restriction endonucleases or micrococcal nuclease Immunoprecipitation - of the protein of interest and its associated chromatin fragments - using a specific antibody This antibody needs to be compatible - even after cross-linking, which could adversely affect it DNA capture/isolation - antibody-chromatin complexes are collected using beads containing proteins or secondary antibodies A series of wash steps reduces non-specific interactions Isolate - heat (remove cross-links), digest antibody/chromatin proteins with protease K and purify the DNA with affinity chromatography Compare fragment sequences - deduce binding protein binding site Do this through an qPCR and then alignment

Question 26

What can we conclude about Eve after these identification experiments?

Answer 26

This allows us to understand how Eve expression is regulated in terms of inducers and repressors Activators bound = repressors can't bind = Eve expressed Repressors bound = activators can't bind = Eve not expressed This is found in strip 2 for Eve as neither repressor is activated/bound

Question 27

How do we determine gene function?

Answer 27

``` We produce mutants where gene function has been perturbed Two approaches: Forward genetics (Classical genetics) First - look for a phenotype Second - look for a genetic change Slow laborious costly ``` Reverse genetics Second - look for a phenotype Rapid and precise If a gene can be linked to a phenotype then this can reveal clues about its function

Question 28

Describe reverse genetics?

Answer 28

This can specifically perturb gene expression in two ways: Genetic - associated with irreversible changes to the genome E.g. nt/gene deletion, promotor deletion, splicing mutant or poly(A) mutant This perturbs expression - some might not even produce mRNA Epigenetic - No changes to the genome (changes elsewhere) E.g. Regulatory proteins or RNA interference

Question 29

What were some older strategies that were used for genetic/epigenetic changes in organisms?

Answer 29

They used to rely on introduction of altered genes and homologous recombination to swap genetic material There are drawbacks in mutational efficiency and specificity Transposons - jump into the area you want to change (could go to the wrong place) Insert copies into a fused egg (single cell - pronucleus) Recombination can be unpredictable If diploid - must replace both WT genes Efficiency is low and cost is very high

Question 30

Describe RNA interference molecules - used in epigenetic changes?

Answer 30

Native pathway of RNA degradation and defence found in many higher organisms - plants, animals and fungi RNAi (RNA interference) - 20-30 nt noncoding RNAs, with associated proteins, can control the expression of genetic information Controls vital processes e.g. Cell growth, tissue differentiation, cardiovascular disease, neurological disorders and many types of cancer Some believe we owe are sentience (capacity to feel, perceive, or experience subjectively) to small RNAs, as increased miRNAs in a genome seems to correlate to the complexity of an organism 3 related pathways that share the same central complex: siRNA (small interfering RNAs) miRNA (micro RNAs) piRNA (Peewee interacting RNAs)

Question 31

What is involved in the formation of RNAi?

Answer 31

Involves the generation of double stranded RNAs that can target existing mRNAs in a cell and block their expression All 3 pathways start with dsRNA and involve a complex of RNA and proteins called the RNA-induced silencing complex (RISC) RISC silences a target mRNA via degradation and/or translational repression It induces non-endonucleolytic translational repression before or after initiation, followed deadenylation and degradation

Question 32

Describe the formation of miRNA?

Answer 32

Transcribed as ssRNA from host cell genome - pri-miRNA They fold into dsRNAs, greater than 1000 nts long Processed by cellular machinery to short dsRNAs - Microprocessor Microprocessor contains Drosha - an RNA III family enzyme and can perform endolytic cleavage Dicer + miRNA + dsRNA binding protein (dsRBP) then recruit argonaute to form the RISC loading complex Guide RNA selected, passenger RNA ejected = dsRNA is separated Guide RNA + argonaute = RISC Guide RNA binds target mRNA at the 3' end by base pairing – RISC mostly blocks translation

Question 33

Describe the formation of siRNA?

Answer 33

Source of dsRNAs is exogenous, often viral dsRNAs are perfectly complementary Processed by cellular machinery to short 21-25 nt dsRNAs - only ‘Dicer’ Dicer + miRNA + dsRNA binding protein (dsRBP) then recruit argonaute to form the RISC loading complex Guide RNA selected, passenger RNA ejected = dsRNA is separated Guide RNA + argonaute = RISC Guide RNA binds target mRNA by base pairing – RISC mostly cleaves it Argonaute has nuclease activity

Question 34

What are the key differences between formation of miRNA and siRNA?

Answer 34

1. Initial source and structure is different miRNAs are transcribed from a genome, that form partially double stranded RNAs siRNAs are exogenous, and usually perfectly complementary dsRNAs 2. The way the guide RNA binds the target is different miRNAs bind target mRNAs with partial complementarity, contains mismatches and extended terminal loops siRNAs bind target mRNAs with perfect complementarity for base pairings 3. The activity of argonaute is different miRNA - argonaute binds mRNA and blocks its translation – rarely cleavage siRNA - argonaute binds mRNA and most often causes its degradation

Question 35

How is RNAi used in the lab and beyond?

Answer 35

RNAi is used for gene silencing (knock out gene expression) in order to determine gene expression To target a single gene To target multiple genes As a therapy for human disease

Question 36

How does RNAi form dsRNA?

Answer 36

Synthetic RNAi triggers are generally perfectly base-paired dsRNAs or short hairpin RNAs Transfecting a plasmid into the cell expressing a short hairpin RNA (shRNA) The hairpin loop is formed so it is complementary The plasmid will express a shRNA with the sequence of the target gene - that will fold into a dsRNA, before being incorporated into RISC

Question 37

What some roles of RNAi regarding virus entry?

Answer 37

Low throughput example: Transfect siRNA specific for ARCN1 gene into cells - this is important for flu virus entry (uses COPI vesicles) Infect with eGFP-influenza virus Look for phenotype by microscopy High throughput example: Transfect siRNAs specific for ALL 19K human genes into cells in 20x 96-well plates Infect plates with eGFP-influenza virus Look for loss of eGFP signal by screening all wells Identified 12 out of 19,000 cellular genes needed for virus entry

Question 38

How is RNAi used in the clinic?

Answer 38

Patisiran – an siRNA packaged into a lipid nanoparticle FDA approved in 2018 Treatment of a human disease - hereditary transthyretin (hATTR) amyloidosis siRNA delivery by injection every 3 weeks Targets the 3’ UTR of the TTR gene Blocks mutant TTR production by 70% High efficacy in controlling hATTR

Question 39

Give a summary of RNAi?

Answer 39

RNA interference is an epigenetic mechanism of gene expression perturbation that effects mRNA translation efficiency RNAi relies on generation of double stranded RNAs, with one strand – the guide RNA – being complementary to a target mRNA Incorporation of the guide RNA into RISC leads to perturbation of gene expression from the target mRNA

Question 40

What is the CRISPR-Cas9 system?

Answer 40

CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats They are repeat sequences, next to short sequences A system for editing and regulating genomes CRISPR are transcribed into RNA - crRNA - which is bound by Cas proteins These complexes bind to dsDNA - at a complemenary genomic sequence, next to a protospacer, and cleave the DNA Here DNA may be inserted by: nonhomologous end-joining or homology directed repair tracrRNA is also needed to identify the crRNA to Cas9

Question 41

What is the basis of the CRISPR mechanism?

Answer 41

Many prokaryotes have a genetic locus where short DNA fragments of past invaders are integrated - this DNA sequences is known as ‘protospacers’ Sequences are stored between the short palindromic repeats (SPR) of the CRISPR locus These fragments are called spacers and serve as vaccinations to combat future infections These RNAs transcribed from these sequences, associate with Cas proteins (CRISPR associated proteins) eg. Cas9 Cas proteins process these RNAs into crRNAs (CRISPR RNAs) The crRNA joins with an RNA called tracrRNA (transactivating CRISPR RNA) to form a small guide RNA (sgRNA) Cas + sgRNA seek out identical protospacer sequences to direct their destruction by nuclease attack (cleave it)

Question 42

What are some critical features of Cas and the crRNA?

Answer 42

The sgRNA allows Cas9 to identify the protospacer target DNA through base-pairing Cas9 cuts at a specific sequence next to the protospacer called the protospacer adjacent motif (PAM) – the PAM for Cas9 is NGG Cas9 cuts both strands of the protospacer target to generate a double stranded break - 3bp on the 3' site upstream of the PAM This double stranded break is the key to gene editing

Question 43

What is the end mechanism of CRISPR?

Answer 43

Editing relies on using homologous recombination to repair the double strand break 1. Cas9/sgRNA causes a ds break by the PAM sequence 2. Supply a ‘repair template’ with the desired sequence 3. Homologous recombination occurs to replace host DNA with repair template 4. Genes can be knocked out by eg. adding stop codons