John T. Lis, Molecular Biology and Genetics, has always been eager to see around the next bend. As a kid, he climbed trees to gain a new vantage; he spent all day exploring the woods. “I had this burning desire to see what was around me,” Lis says. “And it’s the same approach now. Finding new ways of looking and seeing is to me one of the more exciting parts of scientific discovery—to get that first look at something that you’d only ever dreamt of seeing.”
Lis has devoted much of his career to developing the technologies, both optical and biochemical, needed to observe cellular mechanisms, with a particular interest in how genes are first regulated when transcribed into mRNA. In developing these tools, Lis has helped reveal—both for his own lab and for the field at large—new ways to observe the transcription of genes and entire genomes, all happening beyond what the eye can see.
Cellular Mechanisms—Watching and Teasing Out the Details
The process of transcription in higher eukaryotic cells goes something like this: an enzyme, RNA Polymerase II (Pol II) is directed by DNA sequences and a collection of core proteins to the beginning sequence of a gene called the promoter. There, it initiates transcription and transcribes a short distance to a pause site. Certain proteins called transcription factors then signal Pol II to begin productive elongation—where it moves along the strand of DNA like a zipper, copying the DNA code into an mRNA that is translated into protein.
But questions about this process abound: how is it turned on and off? When turned on, how is the regulation tuned to the required level? What do the transcription factors—and there are many—do exactly?
“There’s a lot of interesting molecular biology taking place here that we really want to understand,” Lis says. A full understanding of gene regulatory mechanisms for normal development and homeostasis, he adds, is critical to the engineering of precision diagnoses and therapies when regulation goes awry.
One approach to observing these processes directly in live cells is to use microscopy, but the tools were limiting a decade ago. So, in collaboration with Professor Emeritus Watt W. Webb, Applied and Engineering Physics, Lis and his team developed new microscopy techniques to examine polytene chromosomes—special chromosomes in fruit flies that are over-sized and therefore more visible. For the first time, he actually watched transcription happen in live cells, in real time, a dream come true.
“We were able to watch chromosomes undergo this tremendous change,” Lis says. “We could tag the transcription factors and actually watch them come into the DNA, see what order they come in and when. This puts critical limits in generating hypotheses about what these factors were doing.” Lis continues to collaborate with Warren R. Zipfel, Biomedical Engineering, on microscopy technologies, with the aim of seeing and understanding transcription in human diploid cells.
But microscopy is not the only way Lis sees. His group has also pioneered biochemical technologies for teasing out the details of cellular mechanisms. Lis developed the foundations for Ultraviolet (UV) crosslinking methods—using UV light to stably link proteins to DNA. The DNA can then be broken apart and purified to see exactly when and where a protein, like a transcription factor, is binding to it. These methods served as a precursor to now widely used chromatin immunoprecipitation (ChIP) techniques. More recently, Lis has evolved these methods to map RNA polymerases, those key catalysts of transcription, over the entire genome.
A specialized technique, called PRO-seq, allows Lis to gather a wide range of data with base pair resolution. “PRO-seq tells us where every polymerase is in the genome, its orientation, which way it’s pointing, and the level of transcription,” he says. This information has led to breakthroughs in understanding transcription and its regulation.
Eureka! Pausing Pol II—Essential to Transcription
Lis’ methods are not designed to see only for the sake of seeing. “We develop these technologies to answer questions,” Lis says.
“Even if it’s an old problem, something you think you understand, if you look at it with new tools, more often than not you learn exciting things.”
One night in 2008, Lis and a group of his students were shouting for joy while looking at the first read-out of GRO-seq data. “It had been previously thought that when you activate a gene, the cell brings in the Pol II—but the polymerase was already there, producing a small amount of mRNA that we could detect. It was primed and ready to go,” Lis says. “We had seen this in targeted experiments of specific genes years ago, but we considered that these genes might be odd balls. But with the genome-wide technique, we saw it in spades, these peaks from one gene to the next.
“This changed the paradigm for our understanding of how genes are regulated, so it was very exciting—one of those yahoo, eureka kinds of moments,” Lis adds.
Further studies by Lis and others have shown that this “pausing” of Pol II is widespread and essential to transcription—and therefore essential to development and differentiation. “We know now that there are transcription factors that open up and allow the polymerase to enter, and that, as soon as it enters, it goes to the pause site. So the polymerase is ready to go, holding open the whole promoter for new regulatory signals,” Lis says.
Lis’ lab is currently focused on uncovering the function and dynamics of the various transcription factors, those that bring the Pol II in to the promoter and others that stimulate its release from the pause and entry into productive transcription.
Observe, Perturb, Observe—Studying RNA Aptamers
A key strategy in studying these factors is to go beyond observing the normal processes—to perturb those processes and re-observe.
There are many ways of doing this, but Lis has a favorite that his lab is working to develop: RNA aptamers. RNA aptamers are strands of RNA that can be selected to bind to particular proteins. This is important for studying transcription because the interactions of proteins, those transcription factors, are highly complex, sometimes with different surfaces of the proteins performing different functions. The specificity of the RNA aptamers allows Lis and his group to mask very precise protein interactions. “We can produce the mask in cells quickly, and then begin teasing apart what each protein surface does without having to disrupt the whole complex,” he says.
In collaboration with Harold G. Craighead, Applied and Engineering Physics, among others, Lis and his team have built RNA aptamer libraries that allow them to find strands of RNA that bind to specific proteins. One of Lis’ postdoctoral fellows, Abdullah Ozer, created a library with 1016 RNAs to choose from. “Imagine you have 1016 keys in your possession,” Lis says. “You can probably open any door. That’s the idea behind this. You can find which of those RNAs have affinity for a particular target and select for those keys.”
Lis hopes to demonstrate how the aptamers can interfere with macromolecular interactions and to use them to tease apart the biological mechanisms. “If we can do that for transcription, great,” he says. “But it would also be a model for showing that it can be done generally.”
The aptamers could have widespread implications for molecular research as well as for gene therapy. “A lot of these factors are essential to how genes work normally and in diseased states,” Lis says.
As Lis moves forward, most importantly, he keeps an open mind about what he knows and how he knows it. He says, “Even if it’s an old problem, something you think you understand, if you look at it with new tools, more often than not you learn exciting things.”