15 (3D genome organization)

chromosome during cell phases

chromosomes are condensed only during M phase, cells spend most of their time in interphase, where chromosomes are uncondensed chromatin fibers, (M phase (mitosis + cytokinesis) , g1, S phase G2

Interphase chromosomes vs Metaphase chromosomes

Noodle in bowl vs alphabet

Interphase chromosomes: The cell performs its biological function (transcription of genes and mRNAs is translated into proteins )

Metaphase chromosomes, cell divisions (no transcription, no translation)

How are chromosomes organized in the nucleus

nuclear scale ( chromosomal territories)

Chromosomal scale (open and closed)

Mega base scale (domain of interacting chromatin)

Nested hierarchy of genome organization

(1) chromosome territories (2) Nuclear compartment (3) TADS (chromosomal domains) (4) Chromatin loops

Chromosome territories

each chromosome occupies its own space in the interphase nucleus

In the past, electron microscopy studies incorrectly concluded that chromosomes do not have defined territories

Chromosomes Territories ( Thomas Cremer’s Uv irradiation experiment)

Cremer thought electron microcopy studies could be wrong and wanted to experiment to test whether chromosomes are organized in territories

Cremer example

chromosomes intermingled: UV damages short stretches of many chromosomes

chromosomal territorial: UV damages larger stretches of fewer chromosomes

Cremer UV irradiation experiment

After irradiating the DNA, radioactive nucleotides would be incorporated into the chromosomes when the damage was repaired.

They could then visualize the location of the damage by imaging the chromosomes

DNA damage was concentrated on 2 and part of 1, providing 1st experimental support for chromosome territories

Chromosome territories

Chromosome painting allows imaging of chromosome territories

(1) Separate chromosomes by size (flow cytometry)

(2) Digestion into small fragments

(3) FISH each fragment set in a different color

Allows imaging of chromosome territories found they were like separate

The genome-wide study of these features was made possible by development of HI-C

nuclear compartments, TADs, chromatin loops

Hi-C: chromosome conformation capture

A chromosome can be folded or looped during interphase, such that 2 points that are far apart on linear chromosomes can be near each other in the nucleus

CTCF

Proteins known as architectural proteins help facilitate chromosome folding

Hi-C protocol

Covalent bonds fix DNA proteins in place , Create a chimeric molecule containing sequence from 2 diff places on the chromosome, enrich for chimeric molecules using beads that bind to biotin

HI-C data analysis

1. Align PE reads separately to the reference genome

2. Divide genomes into bins

3. Record the interaction between bins 1 and 5 in the matrix

4. A single Hi-C library will contain millions of chimeric molecules

5. All of the interactions are added to the contact matrix

6. Matrix cells are colored according to their values

7. The most common interactions are those between neighboring bins

8. Long distance interactions that are seen as dark color points away from matrix diagonal

Nuclear compartments

Domains of interactions differ in how much they interact with other domains

Creates a checkerboard pattern across the contact matrix

Each chromosome is organized into 2 diff compartments in the nucleus

(a) contains actively transcribed genes

(b) Compartments contain heterochromatic regions

Topological domains (TAD)

within compartments, chromosomes are organized into domains of interacting chromatin

Genetic roles with CTCF

genes within the same tads tend to similar expression patterns and epigenetic states

Insulator roles in maintaining TAD boundaries

TAD calculation

using directionality index

Position A had upstream left interactions but few downstream interactions which results in negative directionality index

TAD boundaries occurred when highly negative DI transitions to highly positive

fish and TADS

genes within the same tad should be closer to each other in 3d space compared to genes in neighboring TADS

Used the HOx gene clusters to test this

chromatin loops

occur between enhancer and promoters

Loops tend to occur between enhancers and genes that are within the same TAD

Loops can visualized as dark spots Hi-C contact map

Are organizational features important for genome function? (chromosome territories)

territories: chromosome territories are non-random and evolutionarily conserved, altered in disease,

Specific functions of chromosome territories remain unknown

Are organizational features important for genome function? (chromosome compartments)

Chromosomal compartments enforce spatial segregation of active and repressed regions of the genome

Compartments are reorganized during development and in response to environmental stress

Changes in transcription accompany compartment reorganization

Are organizational features important for genome function? (TADs and chromatin)

TADs limit interactions to specific chromosome domains to ensure that enhancers contact the correct gene

Chromosomal rearrangements can disrupt TADS and lead to improper enhancer-gene contacts