So-Called Junk DNA – Genetic “Dark Matter” – Is Actually Critical to Survival in Mammals

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So-Called Junk DNA – Genetic “Dark Matter” – Is Actually Critical to Survival in Mammals

Knocking out transposon promoter leads to pup death in mice; similar promoters found in many mammals.

Nearly half of our Evolutionary Tree of Transposons

Viral elements called transposons have invaded mammalian genomes for millions of years and currently make up nearly half the DNA in the genomes of all living mammals. The image depicts species-specific transposon integrations as unique events in the evolutionary history of each species. Credit: UC Berkeley image by Kerry Lin

In mice, this transposon regulates the proliferation of cells in the early fertilized embryo and the timing of implantation in the mother’s uterus. The researchers looked in seven other mammalian species, including humans, and also found virus-derived regulatory elements linked to cell proliferation and timing of embryo implantation, suggesting that ancient viral DNA has been domesticated independently to play a crucial role in early embryonic development in all mammals.

According to senior author Lin He, UC Berkeley professor of molecular and cell biology, the findings highlight an oft-ignored driver of evolution: viruses that integrate into our genome and get repurposed as regulators of host genes, opening up evolutionary options not available before.

“The mouse and humans share 99% of their protein coding genes in their genomes — we are very similar with each other,” He said. “So, what constitutes the differences between mice and humans? One of the major differences is gene regulation — mice and humans have the same genes, but they can be regulated differently. Transposons have the capacity to generate a lot of gene regulatory diversity and could help us to understand species-specific differences in the world.”

Colleague and co-senior author Ting Wang, the Sanford and Karen Loewentheil Distinguished Professor of Medicine in the Department of Genetics at the Washington University School of Medicine in St. Louis, Missouri, agrees.

“The real significance of this story is it tells us how evolution works in the most unexpected manner possible,” Wang said. “Transposons were long considered useless genetic material, but they make up such a big portion of the mammalian genome. A lot of interesting studies illustrate that transposons are a driving force of human genome evolution. Yet, this is the first example that I know of where deletion of a piece of junk DNA leads to a lethal phenotype, demonstrating that the function of specific transposons can be essential.”

The finding could have implications for human infertility. According to first author Andrew Modzelewski, a UC Berkeley postdoctoral fellow, nearly half of all miscarriages in humans are undiagnosed or don’t have a clear genetic component. Could transposons like this be involved?

“If 50% of our genome is non-coding or repetitive — this dark matter — it is very tempting to ask the question whether or not human reproduction and the causes of human infertility can be explained by junk DNA sequences,” he said.

Embryo implantation

He, the Thomas and Stacey Siebel Distinguished Chair Professor at UC Berkeley, studies the 98% or more of our genome that does not code for proteins. For most of He’s career, she has focused on microRNAs and longer pieces of non-coding RNAs, both of which are potent gene regulators. Five years ago, however, her team accidentally discovered a microRNA regulator for a transposon family called MERVL (mouse endogenous retroviral elements) that was involved in cell fate determination of early mouse embryos. The unexpected abundance of transposon transcription in mouse embryos led He’s team to investigate the developmental functions of transposons, which have taken up residence in the genomes of nearly every organism on Earth.

In a paper appearing this week in the journal Cell, He and her team identify the key regulatory DNA involved: a piece of a transposon — a viral promoter — that has been repurposed as a promoter for a mouse gene that produces a protein involved in cell proliferation in the developing embryo and in the timing of implantation of the embryo. A promoter is a short DNA sequence that is needed upstream of a gene in order for the gene to be transcribed and expressed.

Wild mice use this transposon promoter, called MT2B2, to initiate transcription of the gene Cdk2ap1 specifically in early embryos to produce a short protein “isoform” that increases cell proliferation in the fertilized embryo and speeds its implantation in the uterus. Using CRISPR-EZ, a simple and inexpensive technique that Modzelewski and He developed several years ago, they disabled the MT2B2 promoter and found that mice instead expressed the Cdk2ap1 gene from its default promoter as a longer form of the protein, a long isoform, that had the opposite effect: decreased cell proliferation and delayed implantation.

The result of this knockout was the death at birth of about half the pups.

Modzelewski said that the short form of the protein appears to make the many embryos of the mouse implant with a regular spacing within the uterus, preventing crowding. When the promoter is knocked out so that the long form is present only, the embryos implant seemingly randomly, some of them over the cervix, which blocks exit of the fully developed fetus and sometimes kills the mother during the birthing process.

They found that within a 24-hour period prior to embryo implantation, the MT2B2 promoter ramps up expression of the Cdk2ap1 gene so much that the short form of the protein makes up 95% of the two isoforms present in embryos. The long isoform is normally produced later in gestation when the default promoter upstream of the Cdk2ap1 gene becomes active.

Working with Wanqing Shao, co-first author of the study and a postdoctoral fellow in Wang’s group at Washington University, the team searched through published data on preimplantation embryos for eight mammalian species — human, rhesus monkey, marmoset, mouse, goat, cow, pig and opossum — to see whether transposons are turned on briefly before implantation in other species. These online data came from a technique called single cell DOI: 10.1016/j.cell.2021.09.021

Other co-authors of the study are Jingqi Chen, Angus Lee, Xin Qi, Mackenzie Noon, Kristy Tjokro and Anne Biton of UC Berkeley; Terry Speed of the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia; Aparna Anand of Washington University and Gabriele Sales of the University of Padua. The work was supported primarily by the Howard Hughes Medical Institute faculty scholar award and the National Institutes of Health.

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