FISHing for Insight

Geneticists just got a new pair of glasses.

By improving an imaging technology called FISH, they’ve made it possible to view genetic material in more detail than ever before.

One modification achieves “super resolution” imaging. The other can distinguish maternal from paternal chromosomes.

What the researchers are starting to see promises to help them better understand how DNA gets packaged into chromosomes, how that structure relates to health and disease, what the biological significance may be when genetic material is inherited from one parent versus the other—and more.

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“Geneticists have looked at chromosomes for over a century, but they’ve longed for higher resolution and an easier and more affordable strategy for looking at any part of any genome, not just the most accessible regions,” said Ting Wu, professor of genetics at Harvard Medical School and senior author of the study, published in Nature Communications. “With this new technology, we’ve enabled significant steps forward in all those regards.”

“Scientists have a lot of models where we draw in cartoon form what we think is happening,” said the study’s first author, Brian Beliveau, who conducted the work as a graduate student in the Wu lab. “When a region of the genome is silenced, we draw it as compacted. When a gene is expressed, we draw it as more open or active. But we don’t actually know yet what any of these look like.”

“Now we have a tool to start looking at this systematically, which is very exciting,” said Beliveau, currently a research fellow in the lab of HMS associate professor of systems biology Peng Yin at the Wyss Institute for Biologically Inspired Engineering.

Teach a person to FISH…

FISH, short for fluorescence in situ hybridization, is a decades-old visualization technique that locates and illuminates specific genes in the nuclei of cells.

Scientists use FISH to find out things like how many chromosomes a person has or where a particular gene is located on a chromosome.

But conventional FISH has its limits, and scientists want to look closer.

For instance, it hasn’t been able to show the details of how chromosomes are folded, whether there are different ways of folding or how two genome segments interact—all of which would help elucidate how our bodies work.

Researchers have had some success getting high-resolution FISH images of small segments of the genome. But the cost to look at such questions genome-wide has been “beyond anybody’s purse,” said Wu. “The flexibility to target a specific region on the genome has been very difficult also.”

In 2012, Beliveau devised an enhancement to FISH called Oligopaint that made this possible at low cost.

Traditional FISH works by attaching a fluorescent tag to a short, single strand of DNA called a probe, which has a sequence complementary to the segment of DNA a researcher wants to study. When released into a cell, the probe binds to the desired sequence in the nucleus and lights it up so it can be seen under a microscope.

A limiting factor to scaling up FISH has been that “it’s a ton of work to try to find those sequences in nature to make the necessary DNA probes and isolate them in a way that would be compatible with the tool,” said Beliveau.

He solved the problem by developing computer software that lets scientists design the probes they need and then build them with synthetic components—hundreds or thousands at a time.

Super resolution

In the new paper, Beliveau taught Oligopaint two new tricks.

First, he paired it up with two other technologies—STORM from the lab of Xiaowei Zhuang at Harvard University, and DNA-PAINT from Peng Yin’s lab—to zoom in on chromosomes at “super resolution.”

The DNA double helix is about 2 nanometers wide. When it gets wound around a bundle called a nucleosome, one of the simplest building blocks of chromosomes, it grows to about 10 nanometers. Since conventional microscopy can resolve images to only about 200 to 300 nanometers, it’s been impossible to see such infinitesimal structures.

The new Oligopaint combinations can bring chromosomes into focus down to about 20 nanometers. That’s possible because each DNA probe is able to bind to a second probe; the resulting fluorescent output blinks, enabling researchers to distinguish the fluorescent tags one at a time and achieve a finer resolution.

“This is an unprecedented level of detail,” said Wu.

“Right now when you do FISH on structures below a certain size, you get a spot,” said Beliveau. “With these technologies, we’re seeing interesting shapes and structures, loops and protrusions, start to drop out of these things.”

Wu, Beliveau and their colleagues have already begun to find “intriguing organizational themes” in the chromatin of mammalian and fruit fly cells. But they consider the current paper mostly a proof of concept.

“The goal is to communicate these techniques to the research community as quickly as possible so everyone can start making discoveries,” said Beliveau.

They hope researchers generate “a huge bolus of images” that will reveal more about what our genetic structure looks like, Wu added.

“Of course, naturally, if we get down to 20 nanometers then people will want 10 nanometers, and then 5,” she said wryly.

Distinguishing mom from dad

We normally inherit our chromosomes pairs: one from our mother and one from our father. The second trick Beliveau taught Oligopaint was to tell them apart.

“This was not thought possible but, to our surprise, our technology managed to break this barrier too,” said Wu.

Beliveau modified his Oligopaint probes so they could detect the presence or absence of the many single nucleotide variants, or SNPs, that distinguish maternal from paternal chromosomes.

As long as researchers know which SNPs to look for and have hundreds or thousands of them to target—just a few isn’t enough—Oligopaint can now light up a chromosome to identify “mom” or “dad.”

Wu, for one, is excited to use this new ability to study things like how one of two X chromosomes gets inactivated in female mammals. It’s also become clear to scientists that maternal and paternal genes don’t behave the same way, and that this might have effects on human health and disease. Wu is particularly interested in Down syndrome, in which children inherit three instead of two copies of chromosome 21.

“I’d love to look at a cell and ask, are two copies from the mother? From the father? How do they behave relative to each other?” she said.

This study was supported by the National Institutes of Health (R01GM61936, R01GM090278, 1R01EB018659, 5DP1GM106412, F32CA157188, 1DP2OD007292, 5R21HD072481, 1DP2OD004641), National Science Foundation (CCF1054898, CCF1162459), Office of Naval Research (N000141110914, N000141010827, N000141310593), Harvard Medical School, Wyss Institute for Biologically Inspired Engineering, Centre National de la Recherche Scientifique, Howard Hughes Medical Institute, Damon Runyon Cancer Research Foundation, Fulbright Visiting Scholar Program and Alexander von Humboldt-Foundation.