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New Understanding of Timing of Gene Expression Provides Insight Into Development and Disease

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A new study by Weill Cornell Medical College scientists reveals a mechanism through which the expression of genes is controlled - a finding that calls attention to genetic mutations that can impair the timing of gene expression. Such mutations can affect the development of an organism and can also give rise to cancer by turning on the right genes at the wrong time.

Sea squirt Ciona embryo

The study, published in the Oct. 29 issue of PLoS Biology, analyzes how the timing of gene expression is regulated in the notochord, the evolutionary and developmental precursor of the backbone. The notochord is the main feature that sets humans, mice, sea squirts and related animals (collectively known as chordates) apart from flies, worms and mollusks, and is essential for their development.

A crucial player in the development of the notochord is the Brachyury gene, whose product is a DNA-binding protein. In humans, mutations in the BRACHYURY gene have been associated with spina bifida, vertebral malformations, and chordoma, a rare but insidious cancer. Brachyury controls the expression of numerous notochord genes and ensures their sequential deployment during the formation of this pivotal structure. It was known that Brachyury directly binds distinct DNA sequences - known as cis-regulatory modules (CRMs) - that act as molecular switches and control when and where genes are expressed. The temporal regulation of gene expression is usually achieved through separate switches -physically distinct early and late CRMs - that are controlled by different sets of regulators of gene expression.

How these switches could be flipped on by Brachyury at different times was still unknown.

To clarify this point, scientists in the laboratories of Dr. Anna Di Gregorio and Dr. Yutaka Nibu, both in the Department of Cell and Developmental Biology, systematically analyzed the structure and function of several notochord CRMs. The model they proposed - using funding from the NIH National Institute of General Medical Sciences, the March of Dimes Birth Defects Foundation and the American Cancer Society - suggests that some mutations can leave the spatial properties of a CRM intact while affecting the timing of gene expression. "The work calls attention to a mostly unexplored area of gene regulation, and suggests that elusive mutations in CRMs, particularly those that alter the timing of gene expression, might explain the molecular origins of birth defects and diseases that have not been previously linked to mutations in protein-coding regions," says Dr. Di Gregorio, an associate professor.

They found that in the sea squirt, Brachyury binds the CRMs associated with early-onset genes in multiple regions, called binding sites. However, genes that are expressed later during notochord development are controlled by Brachyury through individual binding sites, while the notochord genes with the latest onsets are controlled by Brachyury indirectly, through a relay mechanism that involves other transcription factors. Brachyury and these additional transcription factors are part of a large gene regulatory network, one that studies in the Di Gregorio lab have dissected for the past decade.

The paper shows an example of this kind of mutation for the CRM that controls notochord expression of a laminin gene. "Studies of CRMs' structure and function are expensive and time-consuming in most animal models," Dr. Di Gregorio says. "We employed an out-of-the-box approach, consisting in the use of a non-mainstream model organism, the sea squirt Ciona. In addition to having an experimentally accessible notochord, Ciona features compact CRMs, a fast developmental timeline and an easy method for introducing DNA synthesized in the laboratory into Ciona's living embryos."

In sea squirts as well as in humans, laminins are major components of the basement membranes and extracellular matrix that hold cells together within tissues, and are key regulators of cell migration, cell adhesion and cell proliferation. The Ciona laminin notochord CRM, which was identified in the Di Gregorio lab, requires two cooperative binding sites for Brachyury. When either one of these sites is mutated, Brachyury is unable to bind to that site, but can still bind to the remaining one.

"As a consequence, the CRM is still active in the notochord, but the full onset of its activity is delayed by a few hours, which is a crucial interval during the development of an organism," says Dr. Yutaka Nibu, an adjunct assistant research professor and co-senior author of the study. "Such delay can cause a birth defect by slowing down the synthesis of a building block necessary for the organism to form properly, or might postpone the activation of a tumor suppressor gene and trigger cancer formation."

"Imagine that the gene is the light in a specific room of your house, and that the room is a specific cell in your body," adds Dr. Di Gregorio. "To turn the light on, you need to flip the light switch - the CRM. A mutation that inactivates your switch would keep your room in the dark. However, a mutation that delays the onset of activity of your switch would still let you turn on the light, but only at a later time. If you had to perform any task in that room that required a certain level of light, you would have to wait for a few hours until the light came on. During those precious hours, you would not be able to complete your tasks in that room, or you could have a burglar taking advantage of the darkness to wreak havoc in there."

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