The Amazing Brain: Tracking Molecular Events with Calling Cards
Posted on by Dr. Francis Collins
In days mostly gone by, it was fashionable in some circles for people to hand out calling cards to mark their arrival at special social events. This genteel human tradition is now being adapted to the lab to allow certain benign viruses to issue their own high-tech calling cards and mark their arrival at precise locations in the genome. These special locations show where there’s activity involving transcription factors, specialized proteins that switch genes on and off and help determine cell fate.
The idea is that myriad, well-placed calling cards can track brain development over time in mice and detect changes in transcription factor activity associated with certain neuropsychiatric disorders. This colorful image, which won first place in this year’s Show Us Your BRAINs! Photo and Video contest, provides a striking display of these calling cards in action in living brain tissue.
The image comes from Allen Yen, a PhD candidate in the lab of Joseph Dougherty, collaborating with the nearby lab of Rob Mitra. Both labs are located in the Washington University School of Medicine, St. Louis.
Yen and colleagues zoomed in on this section of mouse brain tissue under a microscope to capture dozens of detailed images that they then stitched together to create this high-resolution overview. The image shows neural cells (red) and cell nuclei (blue). But focus in on the neural cells (green) concentrated in the brain’s outer cortex (top) and hippocampus (two lobes in the upper center). They’ve been labelled with calling cards that were dropped off by adeno-associated virus .
Once dropped off, a calling card doesn’t bear a pretentious name or title. Rather, the calling card, is a small mobile snippet of DNA called a transposon. It gets dropped off with the other essential component of the technology: a specialized enzyme called a transposase, which the researchers fuse to one of many specific transcription factors of interest.
Each time one of these transcription factors of interest binds DNA to help turn a gene on or off, the attached transposase “grabs” a transposon calling card and inserts it into the genome. As a result, it leaves behind a permanent record of the interaction.
What’s also nice is the calling cards are programmed to give away their general locations. That’s because they encode a fluorescent marker (in this image, it’s a green fluorescent protein). In fact, Yen and colleagues could look under a microscope and tell from all the green that their calling card technology was in place and working as intended.
The final step, though, was to find out precisely where in the genome those calling cards had been left. For this, the researchers used next-generation sequencing to produce a cumulative history and map of each and every calling card dropped off in the genome.
These comprehensive maps allow them to identify important DNA-protein binding events well after the fact. This innovative technology also enables scientists to attribute past molecular interactions with observable developmental outcomes in a way that isn’t otherwise possible.
While the Mitra and Dougherty labs continue to improve upon this technology, it’s already readily adaptable to answer many important questions about the brain and brain disorders. In fact, Yen is now applying the technology to study neurodevelopment in mouse models of neuropsychiatric disorders, specifically autism spectrum disorder (ASD) . This calling card technology also is available for any lab to deploy for studying a transcription factor of interest.
This research is supported by the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative. One of the major goals of BRAIN Initiative is to accelerate the development and application of innovative technologies to gain new understanding of the brain. This award-winning image is certainly a prime example of striving to meet this goal. I’ll look forward to what these calling cards will tell us in the future about ASD and other important neurodevelopmental conditions affecting the brain.
 A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues. Cammack AJ, Moudgil A, Chen J, Vasek MJ, Shabsovich M, McCullough K, Yen A, Lagunas T, Maloney SE, He J, Chen X, Hooda M, Wilkinson MN, Miller TM, Mitra RD, Dougherty JD. Proc Natl Acad Sci U S A. 2020 May 5;117(18):10003-10014.
 A MYT1L Syndrome mouse model recapitulates patient phenotypes and reveals altered brain development due to disrupted neuronal maturation. Jiayang Chen, Mary E. Lambo, Xia Ge, Joshua T. Dearborn, Yating Liu, Katherine B. McCullough, Raylynn G. Swift, Dora R. Tabachnick, Lucy Tian, Kevin Noguchi, Joel R. Garbow, John N. Constantino. bioRxiv. May 27, 2021.
Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative (NIH)
Autism Spectrum Disorder (National Institute of Mental Health/NIH)
Dougherty Lab (Washington University School of Medicine, St. Louis)
Mitra Lab (Washington University School of Medicine)
Show Us Your BRAINs! Photo and Video Contest (BRAIN Initiative/NIH)
NIH Support: National Institute of Neurological Disorders and Stroke; National Institute of Mental Health; National Center for Advancing Translational Sciences; National Human Genome Research Institute; National Institute of General Medical Sciences
Tagging Essential Malaria Genes to Advance Drug Development
Posted on by Dr. Francis Collins
As a volunteer physician in a small hospital in Nigeria 30 years ago, I was bitten by lots of mosquitoes and soon came down with headache, chills, fever, and muscle aches. It was malaria. Fortunately, the drug available to me then was effective, but I was pretty sick for a few days. Since that time, malarial drug resistance has become steadily more widespread. In fact, the treatment that cured me would be of little use today. Combination drug therapies including artemisinin have been introduced to take the place of the older drugs , but experts are concerned the mosquito-borne parasites that cause malaria are showing signs of drug resistance again.
So, researchers have been searching the genome of Plasmodium falciparum, the most-lethal species of the malaria parasite, for potentially better targets for drug or vaccine development. You wouldn’t think such work would be too tough because the genome of P. falciparum was sequenced more than 15 years ago . Yet it’s proven to be a major challenge because the genetic blueprint of this protozoan parasite has an unusual bias towards two nucleotides (adenine and thymine), which makes it difficult to use standard research tools to study the functions of its genes.
Now, using a creative new spin on an old technique, an NIH-funded research team has solved this difficult problem and, for the first time, completely characterized the genes in the P. falciparum genome . Their work identified 2,680 genes essential to P. falciparum’s growth and survival in red blood cells, where it does the most damage in humans. This gene list will serve as an important guide in the years ahead as researchers seek to identify the equivalent of a malarial Achilles heel, and use that to develop new and better ways to fight this deadly tropical disease.