Genome-wide chromosome conformation capture analyses, such as Hi-C, have provided us with snapshots of the large-scale spatial architecture of mammalian genomes, describing chromatin compartments, topologically associating domains (TADs), and CTCF– and cohesin-mediated chromatin loops in many cell types. However, the resolution of Hi-C-based protocols, limited by the size and uniformity of chromatin fragmentation, represents a barrier to those seeking to discern gene-level features of chromatin conformation and study the connection between genome organization and gene transcription.
Encouragingly, epigeneticists longing for “macro” insight into small-scale chromosomal architecture have a new tool at their disposal; specifically, two new studies have described a “micro” take on Hi-C and report on the huge potential of Micro-C as a means to provide nucleosome-resolution analysis of chromosomal architecture.
To avoid the problems related to the general heterogeneity of crosslinked chromatin fragments that have restricted Hi-C analysis at the small scale, researchers led by Oliver J. Rando (University of Massachusetts, USA) modified the standard Hi-C protocol by fragmenting chromatin to mononucleosomes using micrococcal nuclease. In this way, Micro-C significantly increases both fragment density and spacing uniformity and takes chromosome conformation capture down to the nucleosome level.
So, let’s hear more on the making of Micro-C and the potential of this hugely impressive technique by Krietenstein and colleagues through their studies in human embryonic stem cells (ESCs) and foreskin fibroblasts:
- Micro-C captures known features of chromosomal architecture with an improved signal-to-noise ratio and significantly improved resolution (~200 base pairs) when compared to previous Hi-C maps
- Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of previously unappreciated mammalian chromosome architectural features
- Mapping with nucleosomal precision enables the identification of ∼20,000 more loops in each cell type when compared to Hi-C
- Micro-C maps demonstrate that TADs represent collections of transient loops; furthermore, many newly identified loops bridge regulatory elements and reveal weak pause sites along cohesin extrusion tracks
- Micro-C also identifies nucleosome-depleted regions as boundaries between contact domains, suggesting that factors other than CTCF can regulate interactions between adjacent chromatin domains
Overall, Micro-C should represent a hugely valuable resource to those seeking to describe the architecture of our genome at the smallest of scales. Indeed, Micro-C has provided the highest-resolution maps of chromosome folding in human cells, which should permit new insight into multiple aspects of chromosomal organization.
Micro-C Moves Beyond its Making and Into Transcriptional Regulation
Moving beyond the making of Micro-C, researchers led by Robert Tjian and Xavier Darzacq (University of California, Berkeley, USA) sought to explore the macro potential of this micro-protocol to study the links between fine-scale chromatin organization and gene activity, transcriptional regulation, and gene silencing; this time in mouse ESCs.
So, what did Hseih and colleagues discover when they moved past large scale epigenomic compartments and explored fine-scale chromatin architecture:
- Combinatorial binding of transcription factors, cofactors, and chromatin modifiers segregate TAD regions into fine-scale structures with distinct regulatory features
- Overall, nested structures represent the prevalent folding feature within TADs
- Fine-scale structures include stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions
- Micro-C reveals that E-P and P-P interactions occur as stripes extending from the borders of fine-scale chromatin domains below the TAD level that link multiple genes or genes and enhancers together
- The authors hypothesize that such structures can form due to a “sticky” RNA Polymerase II enzyme
- Of significant note, only a subset of fine-scale structures appears to be CTCF- and cohesin-specific
- Transcription drives short-range interactions that connect enhancers and promoters
- Furthermore, transcriptional inhibition disrupts small-scale gene-related architectural features without altering higher-order chromatin structures
While this fascinating study has used Micro-C to establish some functional links between genome organization and gene regulation, the authors point to the next steps that may further our understanding. The hope that nucleosome-resolution chromatin maps created by Micro-C may synergize with live-cell single-molecule imaging or single-cell technologies to further refine our understanding of how chromatin architecture regulates gene regulation.
High Praise for Single-cell Hi-C: Chromosome Organization Dynamics in Early Embryogenesis
While Micro-C may represent the future of chromosome conformation capture analysis, Hi-C-based strategies still deserve high praise, as they continue to provide exciting new findings from the micro perspective of single-cells. As an example, researchers led by a veritable dream team of Peter Fraser (Babraham Institute, Cambridge, UK), Katia Ancelin (Institut Curie, Paris, France), and Edith Heard (Institut Curie, Paris, France/EMBL, Heidelberg, Germany) recently sought to understand the genome organization events that occur during early mouse embryogenesis employing an optimized single-cell Hi-C protocol.
Here are all the exciting new findings from Collombet and colleagues following their integration of Hi-C chromosome conformation maps with allelic expression states and chromatin marks:
- Higher-order chromatin structural alterations after fertilization coincide with an allele-specific enrichment in methylation at lysine 27 histone H3 and Polycomb proteins
- Such early-stage parent-specific domains associate with gene repression and allow for parent-specific gene expression, including transiently imprinted loci
- TADs comprised of domains of active chromatin arise in a non-parental-specific manner during a second wave of genome re-organization
- Investigations into structural changes to the paternal X chromosome before and during inactivation in preimplantation female embryos provided insights into the relationship between TADs and gene expression
- Gene silencing on the paternal X chromosome leads to TAD loss and progressive chromosome compaction without megadomain formation
- However, TADs remain in regions that escape X chromosome inactivation, suggesting that an active chromatin state and/or transcription influences local structure
Overall, single-cell Hi-C can also provide much-needed insight into the dynamics of genome organization and gene expression during early development, suggesting that higher-order chromatin structure matures from parental-specific and early repressive compartments towards the progressive establishment of TADs during development.
Chromosome Conformation Capture: Where to Next?
The findings revealed by the newly developed Micro-C and single-cell Hi-C protocols continue to provide macro insights into the organization of the mammalian genome, but where to next? Next steps could include the application of these exciting techniques to study any potential links between genome organization and diseases such as cancer and normal processes such as aging. Furthermore, can we apply these techniques to study the reprogramming on somatic cells to induced pluripotent stem cells and delve further into the links between genome architecture and pluripotency?
For all macro insights from Micro-C, see the protocol description in Molecular Cell, March 2020, and an exploration of the protocol’s full potential in Molecular Cell, March 2020. However, don’t forget all about Hi-C yet; see what single-cell methods can do for you at Nature, March 2020.