The 4D Genome and Human Disease

This represents the idea that DNA exists in a much more COMPLEX state than we usually think about. It is very dynamic and can change in _______ and time.

Chromosome Arrangement
- Chromosomes are not ____________ distributed in the nucleus
- This implies that there are 2 types of interactions happening:
1. __________ interactions: communication between different chromosomes
2. __________ interactions: communication within the same chromosome (what we'll be looking at)

This allows the maternal and paternal alleles in the H19/IGF2 locus to have two different 3D structures

Germ Layers

1. _________ (inner layer): lung cells, thyroid, digestive

2. _________ (middle layer): cardiac, skeletal, smooth muscle and red blood cells

3. __________(outer layer): skin and pigment cells, neurons

CTCF and Genome Expression
- CTCF is a methylation-sensitive insulator protein
-- it regulates many imprinted gene loci, but also has a more global role in the ______ dimensional orginization of the genome

- its role at any given binding site depends on its context (which other proteins is it partnering with?)
- CTCF can cause chromatin looping so that distal sites interact (an enhancer or silencer with a promoter)
- How could we map these DNA-DNA interactions?

______________ Conformation Capture (3C)
- This is a technique for mapping DNA-DNA interactions; also known as "3C"
- If I was interested in the interactions between a promoter and enhancer one DNA sequence to another (one to one), I would use this assay

From 3C to 4C, 5C, Hi-C and Beyond

3C provides "one-to-one" mapping

4C provides "___-to-many"

5C provides "_____ to ______"

Hi-C provides "all-to-all"

Hi-C Data Output
- Heatmaps produced indicate interactions (pyramids): the brighter the signal, the more physcal interactions were measured by Hi-C
- The seperation of these pyramids into different domains is ____
- The insulator protein is enriched at these boundaries and fits its function as a boundary protein

Hi-C allows for the identification of chromatin sub-domains that share 3D space
- These operationally defined units are called topically associated domains (TADs)
- TADs that have been pushed out towards the edge of the nucleus are known as ____, laminar associated domains

There are two types of TADs

1. _ compartent TADs: correlate with early replication; enriched for proteins and histone modifications; involved with active transcription

2. _ compartment TADs: correlate with late replication; enriched for epigenetic modifications; involved with gene SILENCING

LADs are a subtype of _ compartment TADs
- The DNA is associated with the nuclear lamina

Importance of TADs/LADs and Genome Organization
- Genome organization is controlled temporally (through development) and spatially (between tissues)
- Keeping active genes together (A TADs) facilitates ___ polymerase reloading
- Keeping enhancers in one domain prevents them from causing random transcription; i.e. it helps achieve specificity of interaction

Implications for Disease
- Most disease associated genetic changes do not affect the protein-coding region, but instead change the levels of gene expression

- Distinct human limb malformations are caused by DNA deletions, inversions or duplications altering the structure of three ___s at the WNT6/IHH/EPHA4/PAX3 locus
- This locus is important for limb development
- Scientists studied 3 familial limb defects in multiple families:
1. Brachydactyly (_____ digits)
2. F-syndrome (syndactyly) = digits are fused
3. Polysyndactyly (polydactyly + syndactyly) = extra digits that may be fused
- All of these diseases are caused by chromatin organization defects at ___ locus

Limb Malformations

- What do all of these differfent diseases have in common?
- Their mutations cross ___ boundaries!

- The scientists hypothesized that this causes ectopic interactions between regulatory elements and genes in different ___s
(There are regulatory elements, like enhancers that are talking to promoters that shouldn't be doing that, leading to inappropriate gene expression)
- When the mutations were introduced in mice, CTCF couldn't seperate TAD domains anymore, causing miscommunications