In continuation of the 2014 “Advanced Workshop on Interdisciplinary Views on Chromosome Structure and Function,” this year’s “Conference on Genome Architecture in Space and Time” united an excellent group of students and scientist at the ICTP in Trieste, Italy.
The conference was made possible by organizers Angelo Rosa, Mario Nicodemi, Ana Pombo, François Kepes, Matteo Marsili and co-sponsor SISSA.
Attendees immersed themselves in interdisciplinary topics on structure/function relationships of genomes from both prokaryotes and eukaryotes. Cutting-edge results were presented by groups with state-of-the art expertise in super-resolution microscopy, in vivo fluorescence imaging, and chromosome conformation capture-based methods, including methods for large-scale physical modeling and simulation of chromosome structure such as new approaches for analyzing bioinformatics.
Some of the hottest topics within this rapidly growing research area were captured in the following selected talks:
Localization Microscopy of Nuclear Structure: Imaging the Epigenetic Landscape at the Nanoscale
Christoph Cremer | Institute of Molecular Biology (IMB), Mainz and Kirchhoff-Institute for Physics (KIP), Heidelberg, Germany
“Numerical models for nuclear genome structures allow quantitative predictions on chromatin domain distributions, but reality is more complex,” explains Christoph Cremer. Overcoming the conventional resolution of far-field light microscopy (the Abbe/Rayleigh limit), he presented the latest quantitative imaging results of the epigenetic nuclear landscape based on spectral precision distance microscopy/spectral position determination microscopy (SPDM). As a single-molecule localization method (SMLM), this process allowed for a resolution of tens of nanometers within the cell nucleus.
In murine cardiomyocytes, he then induced short-term oxygen and nutrient deprivation (OND) to analyze the environmental effects of ischemia on the chromatin nanostructure. He found that OND caused chromatin compaction within the nucleus, but also that thus compaction was reversed upon restitution of normoxia and nutrients. By comparing cells under OND to undisturbed cells, he showed that the nuclear genome architecture can dynamically respond to changes in environmental conditions. Further, his group visualized chromosomes in murine oocytes that were undergoing the pachytene stage of meiosis; to view them, the group used dual localization microscopy combined with statistical evaluation methods.
In addition to validating high-throughput sequencing methods, these light microscopic approaches can also resolve the nuclear genome structures of an individual cell at unique optical resolution, down the level of a single molecule.
The Major Chromatin Classes Blueprint the Nuclear Architecture
Irina Solovei | Ludwig-Maximilians-Universität (LMU) Munich, Biocenter Martinsried, Germany
To understand the major principles of nuclear organization, Irina Solovei’s group studied spatial arrangements of the eu- and heterochromatic chromosomal subregions. Specifically, they investigated the relationship between hierarchically organized chromatin domains and primary genomic sequences. Hereby, they used murine retinal neurons and fibroblasts that contained linear and circular human artificial chromosomes (HAC) and showed that chromosome subregions faithfully locate in the rod nuclear zones occupied by the same chromatin classes. This observation was found to be irrespective of the chromosomal context and xenospecific background.
Further, she showed that segregation of the three major chromatin classes (gene-rich and SINE-rich euchromatin, gene-poor and LINE-rich heterochromatin, and gene-depleted constitutive heterochromatin consisting of satellite DNA) is highly autonomous. Therefore, these genome sequences and their spatial distributions may be linked to each other.
Deep Imaging to Probe Genome Organization and Function
Tom Mistelli | Center for Cancer Research, National Cancer Institute, Bethesda, USA
Tom Mistelli’s group investigated eukaryotic cells using Deep Imaging methods to gain insight into their genome architecture and function. To understand the three-dimensional organization of genes inside the cell nucleus, he analysed molecular mechanisms using high-throughput imaging position mapping (HIPMap).
The technology, based on high-precision automated microscopes, was applied to detect chromosome breaks and translocations, such as non-random gene positions. He found that repositioning was not affected by gene activity alone; instead, a major contributor to positioning is replication. In addition to detecting gene positions, this method can be used to validate chromosome conformation capture-based sequencing data, probe DNA-protein interactions, and interrogate the relationship between gene expression and localization.
Herewith, in combination with computational image analysis, it is possible to detect molecular mechanisms and functions of genome organizations to gain novel insights into cell biology.
Single Cell and Single Molecule Chromosome Conformation Capture
Amos Tanay | Weizmann Institute of Science, Rehovot, Israel
To understand how transcription factors find their enhancers and how enhancers find their targets within a transactivation domain (TAD), Amos Tanay’s group used state-of-the-art single cell Hi-C as well as single molecule chromosome conformation capture-based techniques.
Investigations of dynamic contact structures between promoters and enhancers ranged from chromosomal territories to intra-loop interactions. Analyzing pairwise (transient) and multi-way hubs within TADs, they found stable hubs to be in repressive coherence, while transient chromosomal interactions were shown in transcriptionally active domains.
He clearly pointed out how important dynamic chromosome conformation studies are in order to understand complex transcriptional programs.
Genome Architecture Mapping: A Spatial Approach to Map Chromatin Contacts
Ana Pombo | Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany
Ana Pombo and her colleagues have dedicated their time to investigating the interactions of genes with their regulatory elements to better understand how genome folding relates to gene activity during development and in disease states. To overcome the limitations of 3C-based methods, she developed the novel genome-wide Genome Architecture Mapping (GAM) technique. This allows for the analysis of multiple chromatin interactions and their positions within the genome architecture at the single-cell level. Hereby, cryosections of the nucleus were taken to capture the three-dimensional chromatin topology without ligation. In combination with DNA sequencing, GAM was applied to quantify a large collection of thin nuclear sections to capture the frequency of locus co-segregation.
Proof of principle for this technique was done in murine embryonic stem cells, where TADs and A/B compartments were identified. Interestingly, using a statistical model (SLICE) in combination with GAM, they found that active genes and their enhancers span large regions of the genome. In addition to their high occupation by pluripotency transcription factors, these genomic regions are also highly transcribed. Further, multiple contacts are mostly located further away from the nuclear lamina.
Taken together, GAM gave new insights into the genome architecture and served as novel tool to detect interaction partners that are important for gene expression and specific for genome organizations.
**EpiGenie would like to thank Daniel Schuetzmann of WWU Muenster for covering this great conference.