Chromatin Conformation Capture (3C) techniques Flashcards
What are the four levels of chromatin packaging?
Level 1= DNA+histones = nucleosomes
Increases DNA packaging 7-fold
≈10nm
Level 2= Nucleosomes pack themselves in fibers. Increases DNA packaging 6-fold
30nm
Level 3= Fibers pack themselves into loops and TADs and form chromatin. Increases DNA packaging 3-fold. 100-250nm
Level 4 = Represented by the mitotic chromosome. Means 1000-fold packaging.
700-1000nm
Explain the dimensions of nucleosomes
Nucleosomes are the fundamental structural unit of the chromatic and represent first level of packaging and consist of DNA wrapped around proteins called histones. Their role is in both chromatin organisation (structural role) but also a functional role in maintaining gene regulation.
Nucleosomes joined by lined DNA to form 10 nanometre structure, appearing as beads on a string.
Nucleosomes are tightly packed together in a 30 nanometre fibre.
When do chromatin loops occur?
Molecular mechanisms that meditate this organisation are not fully well known, however what is currently understood?
When stretches of genomic sequence that lie on the same chromosome are in close physical proximity to each other.
- Cohesin: protein ring that binds to DNA and facilitates loops.
- CTCFs: Binding factor thought to regulate 3D structure of chromatin. Binds together strands of DNA.
Chromatin loops provide favourable environment to processes such as what?
DNA replication, transcription, and repair.
What is chromatin?
Chromatinis a substance within a chromosome consisting of DNA and protein. The DNA carries the cell’s genetic instructions. The major proteins inchromatinare histones, which help package the DNA in a compact form that fits in the cell nucleus.
What are TADS?
Topologically Associated Domains
Highly conserved chromatin domains that shape functional chromosomal organization.
Function not fully understood, their disruption lead to diseases.
TAD result in compartments, genome divided into B and C, based on epigenetic markers, A compartment is enriched in actively transcribed chromatin and B inactive chromatin. Dynamic changes in compartment organisation and transcription support physiological state of cell and organisational development.
What are chromatin territories?
- Non-overlapping domains/regions of the nucleus occupied by uncondensed chromosomes. They are composed of TADs.
Chromosome conformation capture was pursued using FISH as one method.
What were the issues with this?
What is needed?
Low throughput
Low resolution
Probe sequence specificity
GWAS of chromosome topology
Chromosome conformation capture (3C)-based techniques reconstruct what?
What do they provide?
What do they use and show?
The spatial topology at regional (loops and TADs), chromosome and genome levels and provide information about long-range chromatin interactions at the molecular level in vivo.
Platform to investigate Genome-wide level information
High throughput data and show Higher resolution
What steps are shared by all chromosome conformation capture techniques
Crosslink chromatin
Digest cross linked chromatin
Ligation
Reverse cross linking
What did 3C assays find?
First 3c assay of yeast chromosome 3 found that it forms an contorted ring. Next this method was adapted for mammalians and concluded existence of chromatin loops.
Chromosome Conformation Capture (3C) is designed to detect what?
Ligation junctions by PCR followed by gel electrophoresis (semiquantitative or quantitative) (one-versus-one).
What are the strengths of Chromosome Conformation Capture (3C)?
- Confirmed existence of chromatin loops
- Validation method
What are the weaknesses of Chromosome Conformation Capture (3C)?
- Primers design required
- Only contacts between known DNA sequences
- Contact only in a limited range (not exceeding a few hundred kilobases)
Circular Conformation Chromosome Capture (4C) was originally applied to elucidate what?
It has also be used for what?
DNA contact maps of B-globin and RAD23a genes in fertile liver tissue.
Has also been used to identify chromosomal rearrangements and uncover some mechanisms of disease
Circular Conformation Chromosome Capture (4C) provides a detailed analysis of what?
What does this involve?
The DNA contacts (mostly in cis, but also in trans) made across the genome by a given locus of interest (one-versus-all).
Cross-linking and cell lysis 1st RE digestion 1st Ligation Reverse cross-linking 2nd digestion 2nd ligation and purification Short primers and long primers PCRs
Same steps in 3C here, however second digestion and ligation required here
After this small DNA circles are created from which one half is known but other is not because 4C assays are used to interrogate all the physical contacts that known regions has in genome.
E.G know one region, but want to know other regions that are contacting with this known region
PCR used
Primers can be assigned towards against region known, using these primers can amplify regions not known
- PCRs (6nt barcodes and adaptors attached) are sequenced in a single flow cell on Illumina MiSeq platform.
- The bioinformatics analysis can be carried by using the R program: fourSig
What are strengths and weaknesses of 4C?
Strengths:
- Chromosomal rearrangements and disease mechanisms
- Specific genomic sites
Weaknesses:
- Inefficient amplification of GC-rich fragments (bias)
- Primers design required
- PCR duplication not identifiable
Chromosome conformation capture carbon copy (5C) interrogate what?
Interrogate their interactions between many regions
In contrast to 3C and 4C has a higher throughput and lower bias and improvements have been demonstrated by each application in the study of human beta globin locus
Chromosome conformation capture carbon copy (5C) designed to simultaneously detect what?
Millions of interactions through the use of thousands of primers in a single assay (many-versus-many).
5C provided first evidence of the assistance of topologically associated domains TADs by analysis of X chromosome
5c forward and reverse primers used
What are the advantages and disadvantages of 5C?
Advantages: - Higher throughput
- Lower bias
Disadvantages:
- Still limited size of the interrogated genome
- Require known sequences
- PCR duplication not identifiable
What is Hi-C?
3C-based method that generates genome-wide contact maps (all-versus-all).
Developed using high throughput sequencing technologies
What are advantages and disadvantages of Hi-C?
Advantages:
- Does not depend on primers
- Few biases
Disadvantages:
- Reliable high resolution contact map requires high sequencing depth
- Elevate cost and computational resources
What are other types of Chromosome conformation capture methods?
Capture-C
Capture-Hi-C
UMI-4C
ChiA-PET
What is a GWAS?
Genome-Wide Association Study observational study of a genome-wide set of genetic variants in different individuals to see if any variant is associated with a trait
What is a disadvantage of GWAS and is thus required?
- GWAS cannot identify causal genes/variants
- Fine-mapping analysis seeks to determine the genetic variant/s responsible for complex traits.
Genome-wide association studies (GWAS) and fine-mapping analysis have identified 170 low penetrance prostate cancer (PCa) susceptibility variants.
True of false
True
To investigate chromatin interactions between risk SNPs in RAD51B and RAD23B and whole genome
What assay is needed and why?
4C assay needed as had some information however lacked other information in concern to the other aspects of the genome that affected SNPS
RAD51 protein family member involved in homologous recombination repair (HRR) pathway of double-stranded DNA breaks.
True or false
True
