Unconvinced by the RT-PCR results, you perform a qRT-PCR reaction using primers for Gene X and GAPDH again. You get the
following results. The Ct for GAPDH is the same for both Wt and mutant cells (the value is not important in this case). The Ct for Gene X is 24 for the Wt sample, and is 25 for the mutant sample. What can you conclude based on these results?
A. Gene X transcription is about 2-fold reduced in the
mutant compared to Wt
B. Gene X transcription is slightly reduced in the
mutant compared to Wt
C. You cannot conclude anything because you have
know way to know if each sample had the same amount of total RNA
A.)
Global gene expression in eukaryotes versus bacteria
In bacteria, most genes are expressed (unless specifically repressed).
In eukaryotes, most genes are silent until activated.
Most repressors turn genes OFF (rather than preventing them from being turned ON)
In most cells in multicellular eukaryotes, only about 20% of the genes are expressed (this number is difficult to calculate).
What’s the biggest difference between bacterial and eukaryotic DNA?
chromatin
General (basal) transcription factors
common to most promoters, assemble at promoter, recruit RNA pol (e.g. TFIID, TBP, TAFs, etc.)
aka. transcriptional machinery
Transcription activators, DNA-binding activators
Bind to other sites (e,g. enhancers), more specific to specific genes, and not as universal as general TFs
aka. transcriptional factors
HMG (high mobility group) Proteins
bends DNA to facilitate looping
HMG is a:
B. )
Mediator is a
D. )
Insulators
Insulators block enhancers from regulating transcription across from the insulator site
Insulator sequences
about 50 nucleotides, contain a conserved CCCTC sequence
CTCF – CTC-binding factor
protein with 11 zinc fingers
Binds to insulators
______ structure is the first barrier to transcription
Chromatin
Heterochromatin
region of chromatin associated with gene silencing
Euchromatin
a more accessible form of chromatin to the transcription machinery. Sensitive to DNaseI digestion.
Chromatin remodeling
changing chromatin structure to make it more or less accessible
Chromatin remodelling is controlled by at least three mechanisms:
1) Nucleosome repositioning on the DNA
2) Histone variants
3) Histone modification
Nucleosome repositioning on the DNA
Chromatin remodeling complexes shift the position of nucleosomes so that the promoter is no longer is wrapped around a nucleosome.
Histone variants
Histone variants can be inserted to substitute for the main histone subunits, altering DNA binding ability
Histone modification
Histones are modified by acetylation or methylation, altering DNA accessibility.
Acetylation
almost always associated with increasing accessibility and activating transcription
• Specific lysines on histone tails are acetylated (e.g. H3K9ac)
mostly associated with silencing transcription, but in some cases activation. Usually tri-methylated.
Example: H3K9me3
Nucleosome repositioning
DNA is wrapped tightly around nucleosomes
In order to allow access to those sequences to transcription machinery, the nucleosomes must be shifted
SWI/SNF – a chromatin remodeling complex that can reposition nucleosomes to free up the promoter
SWI/SNF is recruited to specific regions by transcription activators, thus the transcription activators must bind to their DNA sequences first
How to turn on a gene (7 steps)
1. Transcription factors specific to a gene bind to regulatory sites near or far from the promoter of that gene
2. HATs are recruited to acetylate histone tails
3. Chromatin remodeling complexes (SWI/SNF) are also recruited and reposition the nucleosomes to expose the promoter
4. The chromatin structure around the gene (particularly the promoter) goes from a closed configuration to an open configuration
5. Transcription factors recruit mediator, and architectural regulators bend the DNA into loops, collectively bringing these complexes in close proximity to the promoters
6. All of this begins to recruit the transcription machinery (aka general transcription factors and RNA pol) to assemble on the promoter
7. Transcription initiation occurs
General (basal) transcription factors are:
C. )
Which term is least associated with positive regulation of transcription?
C. )
Which factor is least likely to have a positive effect on transcriptional
activation?
D. )
Turning off a gene
Unlike bacteria (think lac repressor), eukaryotic repressors generally only turn off transcription after it’s already started.
Repressors/co-repressors kick off activators and inhibit RNA pol, turning off transcription
Histone deacetylation by HDACs leads to a closed chromatin configuration, sealing the closed state
DNA methylation and gene expression
Cytosines of CG pairs (also called CpG) can be methylated
DNA Methylation at the promoter is almosT always associated with the repression of gene expression and closed chromatin
1) Methylated DNA could inhibit transcriptional machinery from binding to the promoter
2) Methylated DNA could recruit HDACs and other complexes associated with closed chromatin configurations
siRNA
short interfering RNA. About 21-27 nucleotides long. Sequence is the reverse complement of transcript and will hybridize with it to form double-stranded RNA.
Anti-sense RNA
an RNA whose sequence is the reverse complement of another RNA (sense RNA). Anti-sense and sense RNA will hybridize to form double-stranded RNA
microRNA (miRNA)
RNAs produced naturally by cells to regulate gene expression. After processing, they’re around 20-22 nucleotides
The gene whose mRNA is bound to by siRNA becomes ____
silenced
Who discovered miRNA and what experiment led to the discovery?
Craig Mello and Andrew Fire
hypothesized that adding RNA that was complementary to an mRNA (i.e. anti-sense RNA) would make the transcript double stranded and block expression. They added anti-sense RNA to mex-3 in nematodes, then measured mex-3 transcripts by staining (the dark color). They also added double stranded RNA (dsRNA) that matched mex-3 as well.
Two ways miRNAs silence genes
1) Perfect or near perfect
complementarity
• Forms dsRNA structure and the mRNA is rapidly degraded
2) Partial complementarity
• RISC complex remains bound to miRNA and mRNA, and translation is physically
blocked. Eventually the mRNA is degraded
How do miRNAs recognize their targets?
Usually 8mers in the 3’ UTRs of mRNAs (but not always)
1 miRNA can recognize many, many target genes
~5000 human genes are targeted by at least 1 miRNA
Each gene can have binding sites for multiple miRNAs
miRNAs likely have more subtle regulatory effects on an individual gene, but can impact a lot of genes at once
There are estimated to be around 800 miRNA genes e.g. miR-142, miR-181a
miRNA vs. siRNA
miRNA: encoded by the genome
siRNA: exagernously added (viruses, researchers)
shRNA
short-hairpin RNA. Created by researchers. When placed into a DNA plasmid, once transcribed will form a short-hairpin, which will be processed by the miRNA machinery. siRNAs are typically created as single-stranded or double-stranded RNA, whereas shRNA starts out as a DNA sequence.
what is CRISPR/Cas9
A way to introduce mutations to specific sequences (i.e. gene targeting)
CRISPR/Cas9 Components
gRNA – guide RNA. Sometimes called sgRNA (single guide RNA).
A short sequence that contains:
1) Sequences of the gene you want to target
2) Sequences to interact with Cas9 proteins
Cas9 – a protein complex with DNA endonuclease activity
Targeting construct – A fragment of DNA (i.e. not a circular plasmid). Contains the sequences you want to insert into the genome. The ends of the targeting construct have sequences that match the gene
you want to target (“homology arms")
Guide RNA + Cas9 will scan the genome and cut the DNA when the sequence matches the gRNA
How does CRISPR/Cas9 work? (Step 1 & Step 2 a., 2b.)
1) Guide RNA + Cas9 will scan the genome and cut the DNA where the sequence matches the gRNA
2) During repair, two things can happen:
a) If there’s no targeting construct or other template for repair, then it will join the severed ends by nonhomologous end joining (NHEJ) which often shortens the DNA and usually inactivates the gene
b) If there’s a targeting construct or other template, the repair machinery will use that sequence to guide repair (homology directed repair), inserting the construct sequences into the gene
You perform CRISPR/Cas9 gene targeting to insert a LacZ gene into
Gene X in mice. You’re worried your construct may have also inserted
elsewhere in the genome, so you sequence the genome of your mice.
Which sequence will you focus on in your sequence file to further examine
if an off-target integration has occurred?
C. )
How will you determine if you successfully inserted LacZ into Gene X?
A. Use Xgal to measure β-gal activity in cells known to express Gene X.
B. Use a Northern blot and probe for LacZ and Gene X and see if they
have the same expression level.
C. Perform an RT-PCR reaction with primers against lacZ and Gene X in
cells known to express Gene X.
D. Perform a qRT-PCR reaction with primers against lacZ and Gene X in
cells known to express Gene X.
E. Any of those would work.
