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

Microbiology and Molecular Biology Reviews

Fraser J, Williamson I, Bickmore WA, Dostie J. An Overview of Genome Organization and How We Got There: from FISH to Hi-C. Microbiol Mol Biol Rev. 2015 Sep;79(3):347-72. doi: 10.1128/MMBR.00006-15. PMID: 26223848; PMCID: PMC4517094.

Genome Organization

Interactions between chromatin and the nuclear lamina occur through Lamin Associated Domains (LADs) which can be cell-type specific. Chromatin near the lamina generally has low GC content and are gene poor. Similarly Nucleolus Associated Domains also have GC- and gene-poor chromatin regions as does chromatin localized in the nucleolus.

Within the nucleus the genome is broadly divided into chromatin territories. Chromosomes fold into territories and individual chromosomes fold into active or open A compartments and closed or silent B compartments. Within compartments, chromatin is organized into topologically associated domains (TADs) which may be generally conserved between cell types and across species. Sub-TADs topology vary with tissue.

Chromosome territories (CTs)

Chromosomal interactions tend to be more within than between them. Homologous chromosomes are far from each other in diploid cells and haplotypes don’t interact frequently. Examining DNA with FISH shows that chromosome form individual domains. There is a cell-specific ‘intermingling’ between these CTs, but how much is unknown.

Gene-rich chromosomes tend to be nearer the nuclear center and small gene-rich chromosomes like 19 interact more with each other than with other small, but gene-poor, chromosomes, which are found more at the periphery.

The importance of chromosome position and interactions are not fully understood. The position of CTs can be cell-type specific but can vary a lot between cells. Gene regulation in there cells may be controlled by the position of genes as genes deep in a CT are harder to activate.

Chromosome compartments

PCA of Hi-C data shows contact frequencies grouping into open A compartment or closed B compartments. A compartments have higher GC content, more gene enrichment, transcription activity, DNAse I hypersensitivity and active (H3K36me3) and poised (H3K27me3) chromatin histone modifications. B compartments have H3K9 silencing marks. B compartment position is highly correlated with late replication timing and LADs. The distribution of CTs and compartments along chromosomes is stable across cell-types, but can vary. However, within-compartment contacts are weak and they may be a transient feature.

TADs

TADs are blocks of chromatin that interact more with each other than with nearby regions. They show up as sharp contact frequency changes between regions in 5C and Hi-C data. Modifying TAD boundary sequences can cause partial domain fusion and affect nearby gene expression.

TADs are thought to partition genomes into distinct globular units that can remain spatially distant even if they are adjacent along chromosomes. Most long-range gene regulation by enhancers is constrained within TADs.

Sub-TADs

TADs can be divided into Sub-TADs. Large TADs found previously could be divided into smaller domains that were stable between cell lines and whose boundaries had enriched CTCF binding and activating histone marks. At this level, chromatin organization is tissue-specific.

Looping

Chromatin can have long range interactions through looping which promotes or prevents contact between promoters and regulatory elements like enhancers. > cellular enhancers have been found genome-wide and are largely responsible for > the tissue-specific expression of genes enhancers can activate transcription > in either orientation, whereas promoters cannot. Enhancers can also drive > transcription from a position upstream of, downstream of, or within target > genes.

Long-range promoter interactions were often not blocked by CTCF and cohesin binding sites and that enhancers physically interact with the nearest gene in only 7% of cases. Interactions between promoters and sequences were most frequent 120 kb upstream of transcription start sites and were asymmetric, suggesting a directionality of chromatin looping.

Cell-type specific elements

• LADs: those with high GC content and correlate with tissue-specific gene expression

• TADs

• The position of CTs and compartments along the chromosome

Key regulators of genome architecture

CTCF and gene regulation

CTCF is a highly conserved protein that can bind to DNA insulator sequences which suppress transcription. It can bind to more than 30,000 sites and frequently bound sites have genes with tissue-specific promoters. It may also be involved in LAD formation. CTCF can form long-range DNA contacts and spatially organize chromatin as part of its insulator function.

Cohesin

Cohesin is a protein complex involved in sister chromatid cohesis, chromosome segregation and DNA repair as well as transcription regulation. > Cohesin colocalizes extensively with CTCF throughout the genome. In fact, many > of the original CTCF-mediated looping contacts were later found to require > cohesin.

By physically segregating chromatin regions into topological domains, CTCF and cohesin might define functional microenvironments for regulatory elements and target genes where contacts are more easily nucleated while preventing chromatin states from spreading and limiting contacts with the rest of the genome. The partitioning of the genome into TADs correlates with enhancer-promoter units, clusters of coregulated promoters and enhancers. Cohesin may organize chromatin in such a way as to prevent interactions between particular TADs and isolate gene expression states from one another

Assays

Chromatin conformation capture (3C)

3C can examine genomic organization in short regions as high resolution as interactions are measured individually. 3C cannot easily distinguish between haplotypes and reads are averaged between chromosomes, along with any abnormalities. Data are averaged chromatin interaction patterns and does not have interaction stability or strength information or cell-specific information. Variability from cell cycle stage can affect chromatin conformation.

Chromatin conformation capture-on-chip (4C)

4C improves on the throughput and resolution of 3C with microarrays and sequencing. However, it only captures interactions between one restriction fragment and the rest of the genome and its data cannot be used to predict domain or chromosome-level conformation.

Chromosome conformation capture carbon copy (5C)

5C can detect up to millions of 3C ligation junctions between many restriction fragment pairs simultaneously, as long as the regions are covered by the primer library.

HI-C

Hi-C is also called genome-wide chromosome conformation capture and can study the spatial organization of an entire genome. Hi-C complexity is very high and the deep sequencing required is expensive. 4C and 5C are better for submegabase-scale analyses.