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Review
. 2015 Sep;79(3):347-72.
doi: 10.1128/MMBR.00006-15.

An Overview of Genome Organization and How We Got There: from FISH to Hi-C

James Fraser  1 , Iain Williamson  2 , Wendy A Bickmore  3 , Josée Dostie  4
Affiliations
Review

An Overview of Genome Organization and How We Got There: from FISH to Hi-C

James Fraser et al. Microbiol Mol Biol Rev. 2015 Sep.

Abstract

In humans, nearly two meters of genomic material must be folded to fit inside each micrometer-scale cell nucleus while remaining accessible for gene transcription, DNA replication, and DNA repair. This fact highlights the need for mechanisms governing genome organization during any activity and to maintain the physical organization of chromosomes at all times. Insight into the functions and three-dimensional structures of genomes comes mostly from the application of visual techniques such as fluorescence in situ hybridization (FISH) and molecular approaches including chromosome conformation capture (3C) technologies. Recent developments in both types of approaches now offer the possibility of exploring the folded state of an entire genome and maybe even the identification of how complex molecular machines govern its shape. In this review, we present key methodologies used to study genome organization and discuss what they reveal about chromosome conformation as it relates to transcription regulation across genomic scales in mammals.

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Figures

FIG 1
FIG 1
Human genome organization in a three-dimensional nucleus. (A) Chromosome territories observed during interphase. Nuclear pore complexes are shown perforating the nuclear envelope. The nucleolus is shown in white. The nuclear lamina is represented as a filamentous mesh inside the double nuclear membrane. (B) Example of a normal female karyotype as would be observed by SKY (322) of mitotic cells.
FIG 2
FIG 2
Inferring chromatin organization. The original 3C method is outlined from top to bottom on the left. Formaldehyde cross-linking captures interactions between DNA segments (blue and green lines) mediated by protein complexes (colored shapes). The chromatin is next digested with a restriction enzyme, and the free DNA ends are joined by proximity ligation before reverse cross-linking and purification. The genome-wide ChIA-PET and Hi-C techniques are related to 3C, and key steps are shown from left to right. The Y-shaped molecule represents antibodies. Biotinylated nucleotides are shown as red dots. Streptavidin beads are shown in brown. The genome-scale 4C and 5C methods indicated at the bottom require the production of 3C libraries, and specific key steps are outlined from left to right. Green arrows represent PCR primers specific to the bait region. 5C primers used during the ligation-mediated amplification step are illustrated with green and blue lines, where the light and dark gray moieties represent universal primer sequences.
FIG 3
FIG 3
Chromatin organization across genomic scales. The chromatin fiber from one chromosome is unraveled to illustrate four different organization levels described previously in the text. Chromatin conformations are presented from low (top) to high (bottom) resolutions. The chromatin fiber and corresponding chromosome territory are shown in pink. A and B compartments (multimegabase scale) are shown separately to highlight their inherently distinct nature, although there is no evidence that their conformations differ at the level of TADs (megabase scale). Three examples of chromatin looping (submegabase scale) are shown: (i) enhancer-promoter, (ii) enhancer-silencer, and (iii) insulator-insulator. E, enhancer; P, promoter; S, silencer; I, insulator.
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