In a significant breakthrough published in the world-leading journal Science researchers in Queensland and the United States have discovered that the human X chromosome attracts an unusual kind of DNA mutation, potentially doubling the associated risk of certain genetic disorders, including haemophilia and muscular dystrophy.
The study shows that the process of X-chromosome inactivation (XCI) – the natural mechanism ensuring that X chromosome genes are expressed equally in males and females – makes the X chromosome a target for L1 retrotransposons, a type of ‘jumping gene’.
Led by Professor Geoffrey J. Faulkner from Mater Research and The University of Queensland and Professor John V. Moran from The University of Michigan Medical School, the team’s findings also resolve a longstanding mystery as to why L1 retrotransposons are particularly abundant on the human X chromosome.
“It has been thought for nearly 30 years that our X chromosome is exceptionally rich in L1 retrotransposons because these genetic elements help XCI and, for this reason, are preserved by evolution,” Prof Faulkner said.
“However, our results suggest the opposite relationship is true - XCI attracts L1 retrotransposon insertions to the X chromosome.”
“This finding is important because L1 mutations can destroy genes, meaning that XCI increases the global rate of X chromosome-linked genetic disorders, such as haemophilia A, haemophilia B, and Duchenne muscular dystrophy.”
Haemophilia, which prevents normal blood clotting, is believed to affect around 1 million people globally. Various types of muscular dystrophy, which cause loss of muscle mass and physical disability, affect hundreds of thousands of people globally.
Using old and new technologies to re-examine L1 insertion preferences
In the 1990s, Prof Moran pioneered systems where L1 retrotransposons are engineered to carry antibiotic resistance or fluorescent marker genes, allowing new L1 mutations to be traced to the chromosome upon which they landed. They since used that system to elucidate the mechanism by which L1s move and how their insertion sculpts the structure, function, and evolution of the human genome.
“By combining engineered L1s tagged with a reporter gene and targeted long-read DNA sequencing, we previously characterised nearly 30,000 L1 mutations in a cultured human PA-1 embryonic cancer cell line. We noticed that far more L1s inserted on the X chromosome that expected by chance but at that time we could not explain how this occurred,” said Prof Moran.
“Once we could distinguish the active and inactive X chromosomes using long-read DNA sequencing, we could go back and count how many L1 insertions were present on each PA-1 X chromosome, which allowed us to conclude that the inactive X chromosome was an obvious hotspot for L1 mutations.”
A unique cell type to study X-chromosome inactivation
Females typically inherit two X chromosomes (XX), whereas most males inherit one X and one Y chromosome (XY). During human development, XCI switches off one X chromosome copy around the time XX embryos implant in the uterine lining. However, when stem cells obtained from XX embryos are grown in a laboratory, XCI quickly erodes, making it very difficult to accurately model XCI. In their Science paper, the team discovered that PA-1 cells support near-perfect XCI.
“We were pleasantly surprised to find that XCI was exceptionally stable and virtually identical amongst PA-1 cells, making them a unique model to study human XCI” Prof Faulkner said.
Using state-of-the-art long-read DNA sequencing developed by Oxford Nanopore Technologies, the researchers were able to distinguish the active and inactive X chromosomes in PA-1 cells and then count how many L1 mutations each chromosome acquired over time.
“Our results flipped the previous script and reinforce the idea that L1 is a selfish element – preferentially inserting on the inactive X chromosome may benefit L1 by allowing it to evade host defence processes.” Prof Moran said.
The team, which included 19 researchers from Australia, Spain and the United States, is now further investigating how L1 mutations are drawn to the inactive X chromosome, including the possibility that it is a ‘safe haven’ where L1 retrotransposons can continue to jump from in future generations.
