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A team of scientists at Harvard University has developed new imaging technology to visualize the organization of chromatin, a substance within a chromosome made up of DNA and proteins, at multiple scales in single cells with high genomic throughput.
his et al. report a multiplex MERFISH (Error Resistant In Situ Fluorescence In Situ Hybridization) method for genomic-scale chromatin tracking and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes along with nuclear structures of reference. Image credit: Su et al., doi: 10.1016 / j.cell.2020.07.032.
“It is very important to determine the 3D organization to understand the molecular mechanisms underlying the organization and also to understand how this organization regulates the function of the genome,” said senior author, Professor Xiaowei Zhuang, a researcher at Howard. Hughes Medical Institute, the Department of Chemistry and Chemical Biology and the Department of Physics of Harvard University.
With their new imaging method, Professor Zhuang and colleagues began building a chromosome map from both wide-angle images of all 46 chromosomes and close-ups of a section of a chromosome.
To visualize something that is still too small to visualize, they captured linked points – genomic loci – along each DNA chain.
By connecting many dots, they could form a complete picture of the chromatin structure.
“But there was a hitch. Previously, the number of points we could view and identify was limited by the number of colors they could represent together – three. Three points cannot create a complete picture, “Professor Zhuang noted.
So, the researchers came up with a sequential approach: imagine three different loci, extract the signal, and then imagine three more in quick succession. With this technique, each point gets two identifying marks: color and round image.
‘We now actually have 60 loci simultaneously identified and located and, importantly, identified,’ said Professor Zhuang.
However, to cover the entire genome, the authors needed more – thousands – so they turned to a language already used to organize and store huge amounts of information: binary.
By imprinting binary barcodes on different chromatin loci, they could visualize many more loci and decode their identities at a later time. For example, a molecule that is imaged in the first round but not in the second round gets a barcode that starts with 10.
With 20-bit barcodes, the team could differentiate 2,000 molecules in just 20 imaging rounds.
‘In this combinatorial way, we can increase the number of molecules that are displayed and identified much faster,’ said Professor Zhuang.
With this technique, the team imaged about 2,000 chromatin loci per cell, a more than ten-fold increase from previous work and enough to form a high-resolution image of what the structure of chromosomes looks like in its natural habitat.
They also envisioned transcription activity – when RNA replicates genetic material from DNA – and nuclear structures such as nuclear dots and nucleoli.
With their high-resolution images, Professor Zhuang and co-authors determined that areas with many genes tend to cluster in similar areas on any chromosome. But areas with few genes only join if they share the same chromosome.
One theory is that gene-rich areas, which are active sites for gene transcription, come together like a factory to allow for more efficient production.
Although more research is needed before confirming this theory, one thing is now certain: the local chromatin environment affects transcription activity. Structure influences function.
The team also found that no two chromosomes are alike, even in otherwise identical cells.
Finding out what every chromosome looks like in every cell of the human body will take a lot more work than a laboratory can do alone.
“It will not be possible to build on our work alone. We have to rely on the work of many, many labs to have a complete understanding, ”said Professor Zhuang.
The team’s findings were published in the journal Cell.
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Jun-Han Su et al. Genomic scale imaging of the 3D organization and transcriptional activity of chromatin. Cell, published online on 20 August 2020; doi: 10.1016 / j.cell.2020.07.032
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