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How are contact between the mouse β-globin and activated promotor mediated?
Through multiple component complexes
Incl. GATA 1 and Lbd 1
Lbd 1 recruited by GATA 1
Promotes LCR looping
What are drawbacks of 3C?(4)
Req's previous knowledge of interacting sequences
Need sequence-specific primers
Need naked DNA primer controls
Describe 5C and what 5C stands for. Name 2 advantages.
Chromosome conformation capture carbon copy
High throughput version of 3C
Still need prior knowledge of interacting sequences for primer design
Still ligate designed primers
Don't need naked DNA
How does 5C overcome the 3C problem of varying PCR efficiency?
Add an additional primer annealing and ligation step
Allows for PCR w/ the same 3' and 5' primers
What happens after the ligation step in 5C?
Get a 3C library
Add 2nd set of primers w/ your 2 regions of interest
Primers have the same 3' and 5' ends so you can use universal primers T7 and T3
No P on the 5' end, so only the middle can be ligated - ensures right orientation
Can now multiplex w/ Taqman probe
How did Dotsie and Dekker use 5C?
β-globin LCR as model and
human K562 cell line (Leukemia) that expresses fetal
γ-globin genes (not adult
Used 78 5C primers to analyse 845 possible 5C ligation products
If the primer touches, get a ligation product
No touch = no product
What is a drawback of 5C?
Still have to know prior knowledge of gene interactions/predicted interactions
What are the main advantages of Hi-C?
Doesn't require prior knowledge of the interaction
Don't need to design primers - just use T7 and T3
Entire human genome already sequenced, just need a computer to locate your results in the genome
Produces a matrix so you can view the entire genome at once
Describe the Hi-C process.
Same as 3C up to RE
Get staggered overhang, add biotin marker, cut w/ another RE to get smaller fragments
Ligate under v. dilute conditions
Lose HindIII site, create Nhe I site
Purify and shear DNA, ill down biotin w/ streptavidin beads; sequence w/ paired ends
What is the purpose of biotinylation in the Hi-C technique?
Specifically isolates ligated fragments so you only pull out the junctions
Allows for the construction of a matrix
Darker red = higher frequency
White = no ligation
What were the results of the 5C paper?
Validated that genomes of K562 and GM06690 cells organized into 2 distinct compartments
Both types found in both cell lines
BUT - many loci that were open in one were closed in the other and vice versa
What was found in a 3C study?
FTO was implicated in a 3C study as determinant of obesity/diabetes in humans
Specifically mutations and SNPs w/in intron of FTO gene
Deletion of FTO = skinnier mice but smaller everywhere (not just fat)
HS sites in FTO introns found in both expressive and non-expressive tissues
What 4C study revealed more about FTO?
Showed FTO intron associated w/ IRX3 located ~ 500Kb downstream on the same chromosome
Seen in mice, zebrafish and humans
Describe the FTO and IRX3 mechanism.
Enhancer element in the first intron of FTO associates w/ the IRX3 promotor and regulates its expression
IRX3 encodes transcription factors involved in multiple development processes
153 human brain samples analyzed showed obesity-linked SNPs associated w/ IRX3 but not FTO expression
What did genetic deletion of IRX3 in mice result in?
30% weight reduction
Higher base metabolism rates
Less body fat
What are 3 important areas of regulation of DNA replication in eukaryotes?
Location of origins of replication (i.e. where assemble pre-RC)
Selection of specific origins to activate (many created, not all are used)
Timing of activation of DNA replication during the cell cycle (i.e. when to fire the selected origins)
Origins of replication in prokaryotes
In E. coli, there's only one oriC/genome
Origins of replication in eukaryotes
Don't have specific DNA sequences for origins
Except for in budding yeast
Classical methods of identifying origins (esp. for yeast - S. cerevisae) (2)
Functional assay/extra-chromosomal plasmid maintenance assay
Describe the functional assay.
Yeast has naturally occurring plasmids that can autonomously replicate once per cell cycle
(e.g. 2 μm plasmid)
Each has own origin of replication
Can replace origin w/ different genomic sequence - test whether plasmid can replicate once/cycle
Identify many ARSs within the yeast genome
Once ID'd can validate w/ 2D electro.
What is an ARS?
Autonomously replicating sequence
Found in yeast
Describe 2D electrophoresis
1st dimension: separate by size
Looks like normal gel
2nd dimension: sep. by shape
Cut out the gel, flip and run again
Use a southern blot to detect replication forks/bubbles
Use info to map ARS location(s)
What are the 5 images you can get after 2D electrophoresis?
Simple Y - full arch
Bubble - half arch
Double Y - straight angle
Bubble → Y - disconnected arch
→ double Y - imperfect arch
What are the 3 steps needed to prepare a sample for 2D? What do you end up with?
Synch yeast cells so get a culture of all newly replicating 2μm plasmids for analysis
Collect all cells in S-phase to catch replication fork/bubble
Harvest for DNA analysis
End up w/ 2 isoforms (A & B) of the
2μm plasmid due to flipping of an internal sequence
Why do you digest with 2 different REs?
Do the same analysis process but cut differently to see if there are different bubbles
Describe origins of replication in S. cerevisiae.
At least 300 origins
Origins are 100-150 bp long
3 domains: A, B, C
A: conserved 11bp sequence called the ARS consensus sequence (ACS)
B and C: auxiliary domains that support efficient initiation
What is Abf1?
ABS binding factor 1
Functions as a transcriptional activator protein
What are B1 and B2?
Bind to origin
Bubble can only start when helicases come on
What is the relationship b/w yeast origins and nucleosome positioning?
Most ORC binding sites in yeast are nucleosome-free regions (NFR) flank d by phased nucleosomes
Non-ORC binding sites: only modifications associated w/ NFRs
Lack phased nucleosomes
Not all NFRs promote ORC binding
Subset allow ORC to position nucleosomes to allow sufficient space for mcm loading
Origins of replication, in general
ORC binding sites
AT rich sequences (weakest link)
DNA unwinding region have DUEs (DNA unwinding elements)
Auxiliary motifs as binding sites
Why don't classical methods of identifying and studying origins work on mammals?
If your genome sequence is long enough, you'll find an area capable of replication
Need to be able to see origins that function in vivo
Could behave differently in vitro
2 ways mammalian replication can form:
Confined replication zones
Initiation occurs at high frequency
Extended replication zones
Multiple, infrequently used initiation sites
Where are origins typically located?
Around elements of transcriptional regulation
Can distal elements influence origin activity? Provide an example.
Yes. Human β-globin gene origin is dependent upon upstream LCR.
How do you find mammalian origins of replication? (2 ways)
1. Genome-wide analysis of origin-binding proteins (ChIP microarray/sequencing)
2. ID'ing sequences corresponding to replication bubbles
2A. Identifying by isolating small nascent leading strands
2B. Bubble trap method
How come where the ORC binds doesn't necessarily mean you'll find an origin in mammals?
Many places where the ORC can bind on the genome, but few actually fire
Why does ChIP work so well for identifying yeast ORCs?
Found ORC binding that correlates w/ NFRs
How does mapping SNSs work? Name the steps.
Harvest total genomic DNA from synchronized cells (use cells from S-phase)
Sucrose gradient to isolate fragments 500-1500 bp (have to be larger than Okazakis)
Key: avoid random shearing
Will look like Oka otherwise, giving a false read
For comparison, isolate DNA from cells synched at G1 = total genomic sequence
What is the purpose of using a sucrose gradient
Separates DNA based on size
How do you spot the genomic fragments on a microarray based on colour and origin efficiency?
Pale orange: inefficient origin
Dark orange: moderately efficient origin
Red: very efficient origin
Green: non-origin region
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