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Not As Simple as It Looks

JUPITER, FL, September 16, 2009 – Scientists have known for some time that proteins called CREB regulated transcription co-activators (CRTCs) help regulate many biological processes by integrating and converting environmental stimuli into responses that modify gene expression.

But now it turns out that these co-activators alter gene expression in ways that no one had suspected, according to a study from Assistant Professor Michael Conkright and his colleagues in the Department of Cancer Biology at Scripps Florida. These proteins may even play a role in the spread of genetic diversity across the animal kingdom.

The article was published in the volume 28, number 18 (September 16, 2009) issue of The Embo Journal.

What CRTCs do is provide alternative splicing to what might be considered the standard DNA transcription process—which produces an RNA copy of DNA (messenger RNA or mRNA), which is then used to produce the specific protein that fulfills the purpose of that gene.

But what if you could modify transcription of that mRNA by just a few DNA base pairs instead of the entire underlying DNA strand? And what if that changed gene expression in only a slightly different way? It would mean that what we thought we knew about how and why gene expression is handled might not be as simple as once thought.

"We have lots of hormonal signals and those signals target specific cells with specific receptors," Conkright said. "Those signals turn genes on and off, or so we believed. As it turns out, it's not that simple. The signal can turn on the gene—and then modify it by alternative splicing and even by what tissue the cells are located in. The outcomes aren't just binary, on and off. Instead, gene expression can be changed subtly. Frankly, we've been marveling at the levels of fine tuning that biology has created for itself."

How It Works

In response to environmental stimuli, cells call on mechanisms that integrate these extracellular signals with specific changes in gene expression. Some transcriptional co-activators like the CRTCs play a critical regulatory role as integrators of various signaling pathways. These co-activators bind to an activator or transcription factor with a DNA binding domain such as the CREB protein itself (cAMP response element binding); co-activators cannot bind to DNA by themselves.

The messenger molecule cAMP itself is involved in intracellular signal transduction, and influences several biochemical processes, including the regulation of sugar and fat metabolism. It also activates certain protein kinases involved in the regulation of adrenaline; kinases transfer a phosphorous group to other molecules and are involved in signal transmission and control of complex cellular processes.

Although the mechanisms by which CRTCs sense cellular signals have been characterized, little was known regarding how CRTCs actually contribute to the regulation of gene expression.

The study shows that these dynamic regulators independently direct either pre-mRNA splice-site selection or transcriptional activation, depending on the cell type.

"After studying these transcriptions factors and co-activators, we now know that genes are no longer making one messenger RNA and one protein," said Research Associate Antonio L. Amelio, a member of Conkright's laboratory and first author of the study. "As it turns out, the CREB protein regulates roughly 10 percent of the genome by binding to co-activators such as the CRTCs, so a huge number of genes are regulated by this one factor."

The CRTC CREB co-activators don't just turn these genes on, Amelio pointed out, they actually help dictate what transcript is being made and, ultimately, what protein is expressed.

"As a result, you can get several different shades of the protein expression," he said. "Up to this point, we've only documented the fact that environmental signals can dictate specific transcript patterns, but the question is, do these minute variants in gene expression mean anything? Our data leads us to believe that they have some biological significance."

Amelio, who joined Conkright's laboratory in 2006 from the University of Florida, thinks that regulation of these selective splice variants results in gene expression patterns that contribute to diversity within the animal kingdom but may also play key roles in human diseases such as cancer as well.

"With advances in genome sequencing, we have found there is very little difference in the absolute number of genes between various animal groups yet there are so many different phenotypes [observable characteristics]," he said. "How can you get so much complexity with not that many more genes? But the fact that genes can be alternatively spliced offers the possibility to make several different proteins from the same gene, which could result in significant changes in phenotypes including development of disease."

The process is even more complex because gene expression is also tissue specific. While they have relatively few answers as to how these co-activators discriminate in terms of splicing, they do know that cell type can influence the ultimate effect — CRTC co-activators in the brain may splice differently than the same co-activator in the liver.

Interestingly, uncovering the phenomenon of alternative splicing was really a lucky accident and relates to an earlier study published by Conkright and Amelio in the volume 104, number 51 (December 18, 2007) issue of The Proceeding of the National Academy of Sciences.

"We just wanted to know how these co-activators turned on genes and fell into the splicing findings by serendipity," Conkright said. "We weren‘t out to prove this. It's just what happened." In addition to Conkright and Amelio, "Bipartite Functions of the CREB Co-Activators Selectively Direct Alternative Splicing or Transcriptional Activation" was authored by Massimo Caputi of Florida Atlantic University. For more information, see http://www.nature.com/emboj/journal/v28/n18/abs/emboj2009216a.html .

The study was supported by the National Institutes of Health.


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