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Painting human chromosomes in 3D

Collaborative genome study reveals human 3D chromosome organization using a combination of super-resolution imaging, structural sequencing, and integrative modeling approaches

By Benjamin Boettner

Human cells normally contain 23 pairs of chromosomes, the long stretches of DNA that encode the genetic information they need in order to develop tissues and organs and to carry out their functions — one chromosome in each pair inherited from the father and one from the mother. If attached to each other and stretched out, the chromosomes of a single human cell would be about six feet long, and a string made of the chromosomes from all of the five trillion cells in the human body would span about 100 times the distance between the earth and the sun.

This animation shows the step-wise addition of chromosome segments of the maternally and paternally inherited chromosomes 19, 5 and 3, resolved in a multi-chromosome walk by super-resolution imaging. Credit: Wyss Institute at Harvard University.

To fit all of their chromosomes into a tiny compartment known as the “nucleus,” while at the same time maintaining access to chromosomes’ DNA information, cells need to fold and package them up in a highly organized fashion. While the mechanisms and machineries that enable this highly complex natural origami still remain mysterious, researchers are making headway towards understanding how organized chromosome tangles look and in which ways they resemble each other between cells.

Genome biologists even speculate that each chromosome folds along a nonrandom path by creating specific contacts between distant chromosomal sequences. These contacts may also bring genes with their protein-encoding information close to regulatory elements in the DNA that they would not be able to touch in a linear, stretched-out DNA sequence. “By better understanding the secrets of higher order chromosome organization, we expect to get important new insights into how cells turn genes on and off. This could reveal entirely new types of signatures for differentiating cells in developing organisms and organs, and cells affected by diseases, as well as for distinguishing paternal and maternal chromosomes and their gene activities,” said Ting Wu, Ph.D., Associate Faculty member at the Wyss Institute for Biologically Inspired Engineering and Professor of Genetics at Harvard Medical School (HMS).

By better understanding the secrets of higher order chromosome organization, we expect to get important new insights into how cells turn genes on and off. This could reveal entirely new types of signatures for differentiating cells in developing organisms and organs, and cells affected by diseases.

Ting Wu

Techniques known as “chromosome conformation capture techniques” (3C) have been developed to freeze interactions between DNA sequences located in distant regions of a chromosome, and identify the exact sequences that touch each other. With advanced high-throughput versions of 3C known as Hi-C that use next-generation sequencing methods, genomicists can even produce maps containing nearly all the contacts present in the far-flung network of a chromosome’s touch points. However, these approaches do not allow them to visualize the physical 3D shapes that folded regions of a chromosome are assuming around and in between those touch points.

The researchers show in this image how equivalent domains on both copies of chromosome 19 can be tightly packed into small 3D territories of the nucleus and almost touch each other. Credit: Wyss Institute at Harvard University.

Wu and her team in close collaboration with other researchers has pioneered new approaches that are deeply rooted in super-resolution microscopy technology and known as OligoSTORM and OligoDNA-PAINT. Those approaches are able to distinguish fluorescent signals on the nanometer scale, using specifically designed fluorescent oligonucleotide-based “Oligopaint FISH probes” that bind to target sequences in the genome.

To integrate Oligopaint FISH probes with the single-molecule super-resolution technologies STORM and DNA-PAINT, Wu’s team had previously collaborated with Xiaowei Zhuang’s team at Harvard University, and Peng Yin’s team at the Wyss Institute and HMS. Zhuang, Ph.D., who invented STORM (short for Stochastic Optical Reconstruction Microscopy), is Professor at Harvard University and a Howard Hughes Medical Institute Investigator; and Yin’s team has been developing a series of technologies that are based on DNA-PAINT (short for Point Accumulation for Imaging in Nanoscale Topography) at the Wyss Institute and HMS, where Yin, Ph.D., is a Core Faculty member and Professor in the Department of Systems Biology, respectively.

In their new study published in PLoS Genetics, Wu’s team, headed by Postdoctoral Researcher Guy Nir, applied the combined forces of biochemical analysis and super-resolution imaging to visualize over 8 million bases of a region of human chromosome 19 in human PGP1 fibroblast cells, one of the best sequenced cell lines available. They designed a “genome walking” strategy in which, based on Hi-C data from a similar cell line, they generated large collections of Oligopaint FISH probes that blanketed intervals of genomic DNA located around contact points where distant sites of chromosome 19 have been shown to touch. “By imaging the genome throughout the depth of cells’ nuclei, one interval after another enabled us to visualize how the genome folds into units taking on distinct 3D shapes across the entire region,” said co-first author Nir, Ph.D.

Oligopaint FISH probes in combination with additional oligonucleotides produce a fluorescent blinking signal at their target sites under the microscope. Essentially, it is the blinking feature of the signal that allowed Wu and her collaborators in their 2015 study to distinguish Oligopaint FISH probes that bind merely 25 nm apart from each other. In comparison, one turn of the DNA double helix resembling a spiral staircase measures 3.4 nm and its diameter about 2 nm. This unprecedented resolution of genomic features would have been impossible to achieve with more conventional high-resolution microscopy, which is unable to differentiate between targets that are closer than 200 nm apart from each other, the barrier known as the “diffraction limit” set by the natural wavelength of light.

“Walking along chromosomes with super-resolution imaging and Hi-C contact maps enabled us to visualize 3D structures across a considerable range of sizes within in a larger chromosomal interval, to assign different chromosomal segments to distinct compartments of the nucleus, and even to observe differences in organization between a paternally and maternally inherited chromosome,” said Wu. This high definition of chromosomal features and differences critically depended on the sequential imaging of different structures via a microfluidic system and a capacity to image through entire nuclei, both achieved through Wu’s group’s collaboration with Jeff Stuckey and Carl Ebeling at Bruker Nano Inc., who also provided a super-resolution microscope versatile enough to accommodate the technical demands of the project. Jeff Stuckey, Ph.D., is a co-corresponding author, and Carl Ebeling, Ph.D., a co-first author on the study.

Her team also collaborated with Marc Marti-Renom and Irene Farabella from the Barcelona Institute of Science and Technology, who integrated the study’s super-resolution images with Hi-C data, which were generated in a collaborative effort with Erez Lieberman Aiden and Cynthia Pérez Estrada. This integration allowed the imaged regions to be modeled in silico with an additional increase in resolution, and to separately present the maternally and paternally inherited segments of chromosome 19. Marti-Renom, Ph.D., who co-corresponded the study, is a group leader at the Institute’s Centre for Genomic Regulation, and co-first author Farabella, Ph.D., is a Postdoctoral Researcher working with him. Lieberman Aiden, Ph.D., who also co-corresponded the study, is a Principle Investigator at Baylor College of Medicine, and co-first author Pérez Estrada, is a Postdoctoral Researcher on his team.

We are excited about the possibilities that DNA-PAINT technology has opened for this new level of genome research and will continue to develop and apply it to elucidate chromosome organization together with Ting’s team.

Peng Yin

The researchers think that in future research, especially OligoDNA-PAINT technology, has the potential to drive super-resolution capabilities down to an even smaller scale. It uses fluorescent dye-labeled oligonucleotides called “imager strands” to localize Oligopaint FISH probes, whose blinking frequencies can be precisely programmed by varying their length and composition. This makes the signal at a particular target site highly unique and distinguishable from a neighboring signal. “We are excited about the possibilities that DNA-PAINT technology has opened for this new level of genome research and will continue to develop and apply it to elucidate chromosome organization together with Ting’s team,” said Yin. “There is much more to be learned about human biology and disease from this approach.”

The study was also authored by past and present members from Yin’s team, including Brian Beliveau, Ph.D., Hiroshi Sasaki, Ph.D., and Jocelyn Kishi , Ph.D.; past and present members of from Wu’s team S. Dean Lee, Son Nguyen, Ph.D., Ruth McCole, Ph.D., Shyamtanu Chattoraj, Ph.D., Jelena Erceg, Ph.D., Jumana Alhaj Abed, Ph.D., Nuno Martins, Ph.D., Huy Nguyen, Ph.D., and Mohammed Hannan; Sheikh Russell, Neva Durand, Ph.D., and Suhas Rao from Lieberman Aiden’s team; Paula Soler-Vila from Marti-Renom’s team; José Onuchic, Ph.D., Professor, and Michele Di Pierro, Ph.D., at Rice University; and Steven Callahan, Ph.D., and John Schreiner, Ph.D., from Zero Epsilon, LLC.

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