Apart from the ever rising and falling measurements of one’s waistline and bank balance, the three-dimensional (3D) chromatin conformation of the mammalian genome is one of the most dynamic and least understood factors in a scientist’s life. Luckily, the development of new high-definition techniques and enhanced computational analyses are helping us to understand how the 3D structure of histone-DNA complexes dynamically changes in response to a given situation, and what these changes may mean at the biological level.
Chromosome conformation capture (3C)-based techniques let you look past linear sequences and assess the spatial organization of chromatin by quantifying interactions that occur in 3-D space within the nucleus. Studies so far have identified several common architectural features of chromatin conformation within the nucleus – loops (created by interacting neighboring genomic regions), topologically associating domains (TADs, which are self-interacting insulated regions), and compartments (groups of expressed or silent TADs).
Due to technical limitations, previous studies achieved only brief snapshots of decondensed chromatin conformation in interphase cells, and this analysis superimposed all chromatin conformations into a “bulk” reading. Now, two fascinating Nature studies have employed modified versions of high-resolution 3C (or Hi-C) to visualize how chromatin conformation shifts and evolves during important events in mammalian cells.
Computing Chromatin Conformation during the Cell Cycle
In the first exciting study, researchers from the laboratories of Csilla Várnai (The Babraham Institute, Cambridge, UK), Peter Fraser (Florida State University, USA), and Amos Tanay (Weizmann Institute of Science, Rehovot, Israel) sought to visualize chromosome conformation dynamics during the various phases of the cell cycle.
To achieve this feat, Nagano et al. employed a scaled-up single-cell Hi-C protocol to collect data on nearly 2,000 flow cytometry-sorted single mouse embryonic stem cells from all stages of the cell cycle. The team hoped that this analysis would provide a continuum of chromatin conformation data that would allow them to patch together a properly-ordered cell cycle “movie” via a novel computational cell-cycle phasing method.
Interestingly, this new study indicated a high level of conformational heterogeneity and a lack of conformational stability at the single-cell level, although the team noted that each distinct architectural feature acted similarly as the cell cycle progressed. In general, loops remained stable during interphase (cell division preparation) and disappeared during subsequent mitosis (cell division), active TADs expanded as cells propelled themselves from mitosis back into interphase, with compartment formation reaching its peak by the end of the DNA replication step of interphase.
All this means that, contrary to previous thinking, the position of any given gene is in a positional flux as the cell passes through the cell cycle; the next step is to understand how the cell cycle drives this conformational flux and what this means at the biological level.
“We can now study other potentially dynamic processes such as development and differentiation time courses and observe how the genome organization of thousands of individual cells changes at each step, giving us unique insights that are hidden when one averages over populations of cells,” concludes Peter Fraser.
For all the dynamic details, see Nature, July 2017.
Chilled-out Conformation: Fertilization leads to Relaxed Chromatin Structure
In our next more “serene” study, the lab of Wei Xie (Tsinghua University, Beijing, China) employed Hi-C analysis to assess chromatin conformation dynamics during fertilization and subsequent development of the mouse embryo. The relative lack of cells for developmental analysis necessitated the development of a “low-input” Hi-C methodology called small-scale in situ Hi-C (sisHi-C) with the ability to generate high-quality conformational data from only ~500 cells.
Utilizing this new technique, Du et al. discovered that chromatin within oocytes lacks TADs and compartments and, following fertilization, loses higher-order structure and forms a “relaxed” chromatin state. Following chromatin conformation dynamics through preimplantation development (from 2-cell embryos to inner cell mass of the blastocyst), the authors discovered that TADs and compartments form only slowly, with the parental chromosome separation and separate compartmentalization found in the zygote (fertilized oocyte) extending up to the 8-cell embryo.
The mammalian embryonic genome, it seems, is in no rush to finds its final form! The authors suggest that the maturation of chromatin architecture to its final somatic-like form may occur progressively after each cell cycle, but, as for the previous study, the controlling factors and mechanisms remain unappreciated.
Feeling suitably chilled-out but in need of more of the particulars? Head over to Nature, July 2017 for all the fine print.
Looking to the Future of Chromatin Conformation Analyses
As previously mentioned, the who, the what, and the why of chromatin conformation changes during the mammalian cell cycle and embryo development remain to be discovered; however, the development of analytical tools such as those discussed here will inevitably lead to answers…….and yet more questions!