Volume 1, Issue 7
Fly Guy
Nestled inside the human genome, there may be another secret code waiting to be deciphered. The human genome is now thought to contain 22,000 or so genes that code for proteins, the building blocks of life. But how are such a small number of genes programmed to embark on widely different paths of development? In other words, says UC Berkeley molecular and cell biologist Michael Levine, "what puts your head on top of your shoulders and not in your rear end?" To answer that question, Levine has spent several decades reigning over his genetics laboratory as lord of the flies.
Michael Levine is a professor of Genetics and Development and is also affiliated with the Division of Cell and Developmental Biology.
"Flies may be small, but they're up there with vertebrates in terms of complexity," Levine says. "I'm a fly guy for life."
Indeed, Drosophila, the common fruit fly, is ideally-suited for Levine's experiments that provide insight into the mind-boggling intricacies of fetal development. The researchers focus on the second hour of a fly's embryonic life. That's when the cells begin to differentiate into various parts of the fly tissue and the signs of regulatory DNA are first revealed. Regulatory DNA, Levine explains, controls how and where a gene is expressed in a cell. Of the three types of regulatory DNA--enhancer, silencer, and insulator--"enhancers are king, activating gene expression in specific cell types for specific tissues," he says. Scientists conservatively estimate that while the human genome has less than 30,000 genes, it may contain 100,000 enhancers at the minimum. So far, just 50 or so have been identified.
This confocal microscopy image shows a potential enhancer-promoter loop. The two copies of the gene are closely linked. Red identifies the promoter, blue the proximal (or nearby enhancer), and green the distal (or remote) enhancer. The copy of the gene on the right shows the expected linear order: promoter, nearby enhancer, distant enhancer. However, the gene copy on the left shows the remote enhancer (green) near the promoter (red), with the nearby enhancer (blue) displaced away from the promoter. The simplest interpretation of this structure is that the distant enhancer has formed a looped structure to the promoter. (courtesy the researchers)
"It's hard to come up with an accurate estimate because they're so elusive," Levine says. "You can take an unknown genome and find a protein-coding gene just by reading the code. You may not know a thing about the gene, but at least you can identify it. So far though, we haven't found the code for regulatory DNA, if one even exists."
In recent years, Levine has leveraged computational methods to sift through genetic data in search of binding sites within certain genes that may be indicative of a "landing pad" for enhancers. As his research group continues to build a dataset of possible enhancers, they've also begun to examine enhancers function. Specifically, how does an enhancer activate a gene when it maps thousands of base pairs of DNA away?
For several years, scientists have believed that the DNA between the two genes loops outward, enabling the enhancer to physically land near the appropriate promoter, the part of the gene that kicks off the protein synthesis process by triggering the transcription of mRNA. Still, the evidence of this flopping mechanism behind long-range enhancer-promoter communication was indirect. Scientists could see the promoter region of a gene, but not the enhancer. Until recently.
In this confocal microscopy image, the two copies of the gene are closely linked. The copy on top displays an extended organization of the promoter (red), nearby enhancer (blue), and distant enhancer (green). The copy below shows a compact organization. Note that the distant green enhancer (green) on the compact chromosome is in very close proximity with the promoter (red) on the extended chromosome. This might represent a direct visualization of transvection. (courtesy the researchers)
Levine and postdoctoral researcher Matt Ronshaugen devised a way to visualize the remote enhancer. The researchers' trick was to leverage another known but mysterious phenomena in the genome. The enhancers themselves are coated with RNA. Nobody understands why, Levine says. The RNA could be an unimportant byproduct of a nearby promoter or it could help recruit proteins that are essential in the looping process. In any case, the non-coding RNA enabled the researchers to apply a time-tested technique of visualizing genetic material called RNA-FISH (RNA-Fluorescent In Situ Hybridization). Using RNA-FISH, the researchers tagged the RNA associated with the enhancers with a green fluorescent molecule. Then, using a confocal microscope, they literally watched the green-tagged enhancer loop over to the red-tagged promoter gene.
A serendipitous surprised followed. In 1954, Nobel Laureate Ed Lewis, whom Levine calls the "Einstein of flies," proposed that a gene on one chromosome can directly affect the expression of its homologue gene on another chromosome, a process called transvection. However, the frequency of this "crisscross" was unclear. Levine and Ronshaugen observed that, at least in the case of fruit flies, transvection is quite common.
"One possible explanation for transvection is maybe that it's used as a homeostasis mechanism," Levine says. "If an enhancer fails on one chromosome, the other chromosome can compensate. That way you make sure to get the right levels of expression."
Understanding the myriad molecular processes of fetal development, Levine says, could someday help scientists realize the promise of the post-genome era.
"Could we determine that a certain gene will get turned on in the prostate of a 50-year-old man just by reading off the sequence?" he asks. "It's possible, but we're nowhere near that. The fly embryos are a simple model that helps us determine if there's any underlying code that we can crack."