How events may be coordinated during embryogenesis
A new study reveals a mechanism by which gene timing may be controlled. This finding also highlights genetic mutations that can interfere with the timing of gene function.
Research conducted by Weill Cornell Medical College scientists reveals how mutations can affect the co-ordination of key events required for the coordinated development of an organism, and can also give rise to cancer by turning on genes at the wrong time.
The study, published October 29 in the open access journal 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 our development.
A crucial player in the development of the notochord is the Brachyury gene. The Brachyury gene encodes a DNA-binding protein that switches on all of the numerous notochord genes. This binding protein ensures the correct sequenced deployment of gene activity during the formation of the notochord — a pivotal structure in all chordates.
It was known that Brachyury directly binds distinct DNA sequences — known as cis-regulatory modules (CRMs) — that will then act as molecular switches and control when and where notochord genes are expressed or 'turned on.'
What this new study reveals is how CRM switches are flipped on by Brachyury in sequential intervals.
To clarify this, the laboratories of Dr. Anna Di Gregorio and Dr. Yutaka Nibu, both in the Department of Cell and Developmental Biology, analyzed the structure and activity of several notochord CRMs.
The model they propose suggests that some mutations don't appear to affect the ability of a CRM to be bound, and therefore activated, but do affect the timing of expression of the gene containing the CRM.
Di Gregorio and Nibu found that in the sea squirt, the timing of the Brachyury gene switch depends on the number of CRM binding sites on a particular notochord gene.
For genes that need to be turned on early in development, Brachyury binds to CRMs at multiple sites.
However, genes that need to be expressed later in notochord development are controlled through single binding Brachyury sites.
Finally, notochord genes needed for the last stages of notochord development are controlled by Brachyury indirectly, through a relay mechanism involving other transcription factors.
The paper goes into detail on the effects of a mutation in a CRM controlling notochord expression of the Ciona laminin gene. In sea squirts as well as in humans, laminins are major components in the basement membranes and extracellular matrix that hold cells together inside of tissues. Laminins are key regulators of cell migration, cell adhesion and cell proliferation.
The Ciona laminin notochord CRM, identified by the Di Gregorio lab, requires two cooperative binding sites in order for Brachyury to attach and institute full function of the Ciona laminin gene. When either one of these sites is mutated, Brachyury can still bind to the remaining unaltered site.
"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, adjunct assistant research professor and co-senior author of the study. "Such delay could 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, an associate professor. "To turn the light on, you need to flip the light switch – the CRM. A mutation that [completely] 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 humans, mutations in the Brachyury gene have been associated with spina bifida, vertebral malformations, and chordoma, a rare but insidious cancer.
"Our current study 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."
Anna Di Gregorio, Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York, United States of America
The appearance of the notochord represented a milestone in Deuterostome evolution. The notochord is necessary for the development of the chordate body plan and for the formation of the vertebral column and numerous organs. It is known that the transcription factor Brachyury is required for notochord formation in all chordates, and that it controls transcription of a large number of target genes. However, studies of the structure of the cis-regulatory modules (CRMs) through which this control is exerted are complicated in vertebrates by the genomic complexity and the pan-mesodermal expression territory of Brachyury. We used the ascidian Ciona, in which the single-copy Brachyury is notochord-specific and CRMs are easily identifiable, to carry out a systematic characterization of Brachyury-downstream notochord CRMs. We found that Ciona Brachyury (Ci-Bra) controls most of its targets directly, through non-palindromic binding sites that function either synergistically or individually to activate early- and middle-onset genes, respectively, while late-onset target CRMs are controlled indirectly, via transcriptional intermediaries. These results illustrate how a transcriptional regulator can efficiently shape a shallow gene regulatory network into a multi-tiered transcriptional output, and provide insights into the mechanisms that establish temporal read-outs of gene expression in a fast-developing chordate embryo.
Transcription factors control where and when gene expression is switched on by binding to specific stretches of DNA known as cis-regulatory modules (CRMs). In this study, we investigated the architecture and composition of CRMs that direct gene expression in the notochord—a transient rod-like structure found in all embryos that belong to the phylum chordata, which includes humans. Here we used the sea squirt Ciona, a simple chordate, and analyzed how the transcription factor Brachyury ensures the appropriate deployment of its target genes at specific times during the sequential steps of notochord formation. We compared CRMs found in different notochord genes downstream of Brachyury, expecting to find genes associated with greater numbers of Brachyury binding sites to be expressed at higher levels. To our surprise, we found instead that a higher number of functional Brachyury binding sites is typical of CRMs associated with genes that are expressed early in notochord development, while single-site CRMs are characteristic of genes that are turned on during the intermediate stages of this process. Finally, CRMs associated with genes expressed late in notochord development do not contain functional Brachyury binding sites but are controlled by Brachyury indirectly, through the action of intermediary transcription factors. These differences explain how a transcription factor that is present at all stages in a certain cell type can generate a sequential transcriptional output of gene expression.
Funding: This work was supported by grant NIH/NIGMS GM100466 along with supplemental funding from the American Recovery and Reinvestment Act award R01HD050704-05S1, by grant 1-FY11-468 from the March of Dimes Birth Defects Foundation, and a grant from the Charles A. Frueauff Foundation to ADG and by grants from the American Cancer Society (RSG-08-042-01-DDC) and the Charles A. Frueauff Foundation to YN. HA was supported in part by a postdoctoral fellowship from the Japan Society for the Promotion of Science (JSPS). DSJ-E was supported in part by NIH training grant T32 GM008539. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Citation: Katikala L, Aihara H, Passamaneck YJ, Gazdoiu S, Jose´-Edwards DS, et al. (2013) Functional Brachyury Binding Sites Establish a Temporal Read-out of Gene Expression in the Ciona Notochord. PLoS Biol 11(10): e1001697. doi:10.1371/journal.pbio.1001697
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Original press release:http://med.stanford.edu/ism/2013/october/liver.html