A cell’s DNA is its most valuable treasure. So it only makes sense that cells keep their genetic material protected under lock and key, wrapped tightly around spools called histones. When it’s time to make proteins from their DNA recipes, the right bit of DNA is unraveled so that the cell’s transcriptional machinery can gain access to the correct genes. But the process of spooling and unspooling the DNA is under tight security, controlled by a complex array of enzymes and signals attached to the histone’s tail.
Studying this security system is an important part of epigenetics, the growing field researching the many factors that control gene expression. Where the basic components of DNA were figured out by Watson, Crick, Franklin and their successors in the 20th century, the proteins and enzymes involved in epigenetics are still largely unknown. With dozens of enzymes that each recognize their own unique combination of signals moving in and out and changing conformation, it’s a fluid, complicated system that’s hard to pin down using traditional techniques.
But the laboratory of Anthony Kossiakoff, professor of Biochemistry and Molecular Biology, thinks it might have the method to help crack part of that epigenetic code. With a new grant totaling $7 million over 5 years from the National Institutes of Health (part of their Protein Structure Initiative), Kossiakoff and colleagues will apply their Chaperone-Enabled Biology and Structure (CEBS) technology to the complex riddle of how dynamic regulation of histone modifications regulate gene expression.
Scientists have long used the specificity of the immune system’s antibodies to create reagents that recognize and tightly attach to a target protein. Once captured, that reagent can help researchers look at the location abundance of their target in the cell. But the “natural” method of creating antibodies, by essentially tricking the immune system of rabbits or mice into producing the highly specific antibody you need, is expensive and may not always produce antibodies that work as advertised.
“Over half of those you buy don’t work, others work maybe for one application but not another that you want, and so it’s buyer beware,” Kossiakoff said.
Kossiakoff’s CEBS technology produces customized antibody-like reagents called synthetic affinity binders, or sABs, created via a process called phage display mutagenesis – akin to “evolution in a test tube.” Billions of synthetic antibody-like proteins compete for their ability to bind with high affinity and specificity to a chosen target. Instead of having the very few candidates produced by traditional monoclonal antibody production , the CEBS selections can generate as many as fifty different “synthetic antibodies” that can be used as highly specific reagents. Over the last five years, the laboratories of Kossiakoff, UChicago’s Shohei Koide, and Sachdev Sidhu of the University of Toronto have refined that process and established a pipeline where super-specific reagents can be created for virtually any stable protein a researcher would like to study.
As part of the Protein Structure Initiative, the laboratory will become what Kossiakoff called a “mothership” for research on histone modification enzymes, producing sABs useful in studying their structure and function. Some of those reagents will be used for experiments in Kossiakoff’s lab, others will go out to biologists and structural biology collaborators around the world. One goal is to generate sAB reagents to act as “crystallization chaperones” to enable the “freezing” of complex proteins for crystallization structural studies – a task that has proved difficult to impossible previously.
“Proteins are dynamic, and they can be floppy, especially if they have multiple domains, as is the case for these histone modifying enzymes” Kossiakoff said. “So many times, even with heroic efforts, you can’t crystallize them to study.”
Previous studies have succeeded in characterizing small stretches of the enzymes, but not all the domains together. Capturing the entire organization of the enzyme rather than one section at a time is an important leap forward, due to their complexity – each enzyme must read several different positions on a histone simultaneously to determine whether or not to bind and activate. It’s a flurry of activity, like a janitor with a full key ring trying to unlock three different doors at once.
“These interactions are pretty transient, on and off, on and off,” Kossiakoff said. “By having all the domains clicking into the right place, binding becomes additive. It literally is a trial and error process, and there is just so much stuff going on.”
Slowing down this whirlwind long enough to determine these complex structures could lead to new insights and understanding into how histone modifications regulate gene expression. The enzymes themselves are also major targets for drug discovery – for example, certain forms of cancer have been traced back to errors in histone modification. Some potential drugs could even be derived from the sABs, with more precise targeting than the “dirtier,” less specific compounds currently used in most pharmaceuticals. With the keys to the DNA’s security system unlocked, laboratories developing new drugs could save far more money than the amount invested by the NIH in this grant.
“One of the major shortcomings in drug development is defining what the actual target is,” Kossiakoff said. “There are many cases where drugs have been produced that worked perfectly based on what was attempted to be done, but they don’t work in terms of what they were supposed to do. We can test things out before a lot of time and money are spent developing these drugs against incorrect targets.”