genomics - 3d genome

GWAS

genome-wife association studies

-identify disease or trait associated variants

-majority of GWAS hits are located in non-coding regions

-majority of GWAS hits are located in cis-regulatory elements

how do con-coding mutations contribute to disease?

-gain of TF binding sites

-loss of TF binding sites

-enhancer hijacking

how are target genes for distal enhancers found?

-nearest genes by genomic distance

-correlation based on gene expression and enhancer activities

-3d space proximity

-CRISPR/Cas9 to delete enhancers and observe their effect on gene expression

Imaging technologies to study genome folding

-bright-field

-fluorescence

-EM

-fluorescence in situ hybrization (FISh)

genomics technologies to study genome folding

-DamID

-ChIA-PET

-GAM

-Chromosome conformation capture-derived

FISH

fluorescence in situ hybridization

-cytogenetic technique

-uses fluorescent molecules to “paint” (regions of interest on) chromosomes in cells often in metaphase or interphase

-aids in analysis of chromosome structure, structural aberrations, ploidy determination, etc

FISH advantages

-rapid and sensitive

-lots of cells can be analyzed

-no cell culture needed

FISH disadvantages

-low throughput

-limited number of commercial probes available

-needs specialized camera and image capture system

STORM

stochastic optical reconstruction microscopy

a super-resolution microscopy technique that generates high-resolution images by precisely locating individual fluorescent molecules that are randomly activated one at a time, allowing for the reconstruction of a detailed image with significantly better resolution than conventional light microscopy

can be used w multiplexed super-resolution FISH

Capturing chromosome conformation

-cross-linking

-digestion

-ligation

-detection by PCR

-3C, 4C, 5C, Hi-C

3C

-ligation

-formation of 3C library

-secondary digestion

-form 3c-qpcr library

genomic scale: ~250 kilobases

“one to one”

advantages: very high dynamic range, highly quantitative, easy data analysis

limits: very low-throughput, limited to few viewpoints in a selected region

4C

-ligation

-3c library

-secondary digestion

-circularization

-inverse PCR

“one to all”

-4c library: sequencing

genomic scale: complete genome

advantages: good sensitivity at large separation distances

limitations: genome-wide contact map limited to a unique viewpoint

5C

-ligation

-3C library

-oligonucleotides hybridization, ligation

-pcr

-5c library sequencing

“many to many”

genomic scale: few megabases

advantages: good dynamic range, complete contact map (all possible viewpoints) of a specific locus

limits: contact map obtained is limited to a selected region

Hi-C

For genome-wide analysis of higher order chromatin structure

-biotinylation

-ligation

-sonication

-purification on biotin-strepavidin beads

-ligation of adapters and pcr

-hi c library sequencing

“all to all”

genomic scale: complete genome

advantages: very high throughput, complete contact map

limits: poor dynamic range, complex data processing

in situ Hi-C

-in situ hi-c maps dna-dna contacts ocurring in intact nuclei, by proximity ligation

-reaches kilobase resolution

micro-C

nucleosome resolution Hi-C

-crosslink

-mnase digestion

HiChIP/PLAC-seq

-combines ChIP and in situ hi C

-restriction enzyme cutting and biotinylation

-proximity ligation

-sonication and immunoprecipitation

-dna purification and biotin enrichment

-pair end sequencing

subcompartments in Hi-C maps

the long range contact pattern of a locus indicates its nuclear neighborhood

ordinary domains in Hi-C maps

squares of enhanced contact frequency along the diagonal indicate the presence of small domains of condensed chromatin

loop domains in Hi-C maps

-peaks in the contact map indicate the presence of loops. these loops tend to lie at domain boundaries and bind CTCF in a convergent orientation

distribution of contact frequencies

vary cell to cell

chromatin organization is dynamic, changes throughout cell cycle

“C” technologies

pros: high throughput, high resolution

cons: low ligation (detection) efficiency, indirect cross-linking via nuclear structure, not readily applicable to low cell number

imaging technologies

pros: high detection efficiency, direct visualization of proximity, readily applicable to single cells

cons: low throughput, limited resolution

loop extrusion model

CTCF regulated cohesin’s loop extrusion activity by changing direction and inducing loop shrinkage

-uses CTCF motif

one sided extrusion

Rabl-like chromosome architecture

Hi-C defines species specific chromosome architectures

Tail-Tail, Centromere-Centromere, C-T axis

passively specified post mitosis

territorial chromosome architecture

hi-C defines species-specific chromosome architectures

condensin II defined territories

condensin II KO → rabl-like config after mitosis

4D Nucleome project

aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions

a) mapping

b) model building

c) functional validation

3D genome key take aways

-Hi-C analysis reveals that the mammalian genome is spatially compartmentalized, and consists of mb sized topological domains (TADs)

-TADs are stable across cell types and largely preserved during evolution, suggesting that they are a basic property of the chromosome architecture

-partitioning of the genome into TADs would naturally restrict the enhancers to selective promoters

-long range looping interactions between enhancers and promoters correlate w higher transcriptional responsiveness of promoters

-cell specific enhancer/promoter interactions are formed in each cell type, some time prior to activation of the genes, and are not significantly altered by transient signaling induction

-pre-existing lineage specific chromatin looping interactions between enhancers and promoters predict transcriptional responses to extracellular signaling, suggesting chromatin conformation is another layer of transcriptional control

-Hi-C types of assays interrogate population average