In August, Xiaojun Ren was nearly celebratory. Two days before, he’d heard that his application for an R01 National Institutes of Health (NIH) Research Project grant was competitive—“13th percentile,” he told me—which meant there was a good likelihood he’d get it, especially as a new researcher.
He also asked that we not talk about it in the article because, well, it hadn’t happened yet. He’s superstitious. And despite his ear-to-ear grin, Ren mentioned the word “stressful” more than a few times. Six years is a long time to work toward nearly $1.3 million of funding. But in that time, Ren, a CU Denver chemistry professor since 2012, has bent the conventions of the gene regulation field as others have scrambled to catch up.
This month, Ren’s speculation proved correct: the R01 funding came through.
Breakthrough of the Year
Last November, Ren and a team of undergraduate and graduate students published a paper in bioRxiv (pronounced “bio-archive”), an online distribution service for pre-publication critique. The study built on a decade-old discovery in the gene regulation world: in the nucleus of a cell, many proteins condense into droplets and separate, especially when responding to stress. This “liquid-liquid phase separation,” a physical process similar to the separation of oil and water, stimulates biochemical reactions and may underpin the selective expression of genes. Ren set out to understand the physiochemical principles behind droplet formation.
In the study, his team was able to separate and observe individual genes in a liquid droplet within the nucleus of a cell. From a very early point in the separation, the team found these liquid droplets assemble and dissemble in response to different environmental stimuli, like blowing colorful bubbles. They watched how genetic materials are incorporated into or released from the droplets in real time, and in doing so, pinpointed a driving force within the protein that encompassed specific genes within liquid droplets.
The discoveries explain why the same gene in different cell types of the human body functions distinctly—turning on in one cell type but shutting off in another. Ren and his team now had insight into how genes are controlled under healthy and diseased conditions. This was a giant leap in the field of epigenetics and the outside world took notice.
Within four days, journals began reaching out about the discovery. Within 20 days, the groundbreaking study was expedited and published in the Journal of Biological Chemistry. By the end of the year, the liquid-liquid phase separation in gene regulation was noted among the top 10 of Science’s 2018 Breakthroughs of the Year.
Observing a molecule in real time
“We are one of the first of a few labs to develop a single molecule technique to understand the order of the biochemical reactions within the cell,” said Ren. Only a few labs are within spitting distance of this kind of research—MIT, Harvard, National Institutes of Health, and University of California Berkeley among them.
In 2016, Ren and a team of undergraduate and graduate students developed a sophisticated technique called live-cell, single-molecule imaging, which allows the observation of single molecules in real time in a living cell. This pioneering technique was published in eLIFE. Two years after they developed the single-molecule technique, the team used it to view the genetic processes that lead to the formation of tumors, which led to a paper published in Nature Communications. By watching individual molecules, the team identified a single protein, Chromobox 7, capable of turning off genes that lead to tumors called diffuse intrinsic pontine gliomas (DIPGs)—a rare and devastating form of pediatric cancer.
Before then, most scientists and researchers guessed what was happening within a cell that causes diseases like DIPGs. They could narrow down and observe the molecular culprits in test tubes, but no one understood the physics principle that organized them. No one was searching for the driving force that led to the damaging structure change. The researchers acted as anthropologists when they needed someone wielding a scalpel and a physics background.
Enter Ren. Through his years in academia, Ren was interested in cell differentiation and cell assembly, but wanted to understand it from that physics perspective: what are the fundamentals of the assembly’s driving force? What is the energy source?
Changing the nature of chromatin
Ren focused on chromatin, the material that makes up the structure of chromosomes.
Chromatin acts like an unwieldy vine composed of DNA, RNA, and proteins called histones, which look like beads on a string. The DNA in one cell is approximately six feet long and chromatin wraps it so tightly that it crams those six feet into a space one-hundredth of a millimeter in diameter; it’s the twine that keeps the twisted ladder of the DNA helix from unraveling.
But chromatin is malleable and it exists in two forms: euchromatin (active) and heterochromatin (silent). Polycomb Group complexes, proteins that control a huge swath of genes that regulate several cellular functions and all developmental pathways, controls its structure. They can remodel chromatin and switch genes on and off, like the ones that lead to tumors. That’s why understanding the molecular details is so important.
The only problem? There was no easily accessible technique to study it.
Three years before his team discovered that the Chromobox 7 protein shuts off the growth of DIPGs, Ren and his team created what they called single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi). The group used an ultra-sensitive single-molecule technique that can observe Polycomb Group proteins binding to chromatin in action.
When Ren’s lab became the first to visualize a biochemical reaction of a single epigenetic molecule in a living cell in real time, it was a paradigm shift in epigenetics, the study of external modifications to DNA. With the technique, they could dissect molecules to understand their behavior. They could reprogram the epigenome and turn off the drivers of disease.
In their first study using Sm-ChIPi, the team found two protein complexes called PRC1 and PRC2 assembled differently on chromatin. Ren showed the scientific community that understanding the cell assembly process—and answering the why behind it—was possible. His group published their findings in the November 2015 issue of the Journal of Biological Chemistry. It won one of the journal’s Best of the Year designations and became one of its most read studies of 2015.
From physics to epigenetics
Ren credits his initial interest in chemistry to his middle school chemistry teacher in the small village of Tianxing in China. His interest in biology came in high school, but he chose to study polymer chemistry at Jilin University in China, where he went on to earn his masters in organic chemistry and his doctorate in macromolecular chemistry and physics under the supervision of Professor Jiacong Shen, member of the Chinese Academy of Sciences, and Professor Guimin Luo.
When he began his postdoctoral fellow at the University of Cambridge, he teamed up with Sir Shankar Balasubramanian and Sir David Klenerman, two knighted chemists who had created a new generation sequencing methodology called Solexa which led to an explosion of accurate, low-cost sequencing of human genomes. Ren’s advisors focused on telomerase, the end part of chromatin. Ren followed suit, developing an interest in chromatin and the single molecule.
During his time at Cambridge, Ren waffled between physics and biochemistry—which had bigger problems to solve?
“My advisors are pioneers in physics technology, but they focused on genetics,” said Ren. “I was interested in the cell itself. I thought I could develop a way for us to observe these molecules in living cells, in vivo. But to do it, I needed to leave.”
He left Cambridge for the University of Michigan in Ann Arbor to join Tom Kerppola for postdoctoral studies in epigenetics. There, Ren said, he focused on the molecular processes in living cells. He found his calling: Polycomb Group protein complexes and epigenetics.
“One day, I’d hoped to combine all of the epigenetics, cell biology, biophysics, chemistry in my own lab—that’s why I came to CU Denver,” said Ren. “I needed a place that would allow me to create a lab different from any other.”
The Xiaojun Ren Group
The Xiaojun Ren Group began in September 2012. He works alongside undergrads and graduates as they probe the mysteries of stem cells, chromatin biochemistry, and single-molecule biophysics.
Since its formation, the group has pushed the limits of single-molecule fluorescence microscopy methods to study epigenetic regulators in native states and in living cells using single-molecule imaging.
And now, with the R01 funding, the team can dive deeper into the liquid-liquid phase separation, hoping to understand the efficient and specific control of gene activity, which is essential for all life. They also hope to define how liquid droplets of Polycomb proteins organize the genome—a key to understanding how healthy cells function. Together, the studies will provide insight into epigenetic processes that govern normal development, physiology, and their dysregulation in cancer.
It’s something he could have only done with his own lab and he has CU Denver to thank.
“Here, I had the freedom to do what I wanted to do,” said Ren. “[Associate vice chancellor for research] Bob Damrauer encouraged me, and gave me the emotional support and funding support. He allowed me to take a risk, which is important when you try to build something that hasn’t been built before. And now we’re unique in this world of research.”
In addition to his research, Ren’s teaching graduate Biochemistry I and II, and Biochemistry of Gene Regulation and Cancer. He sees working with the students as his way of paying it forward.
“When I train and work with students, I feel excited and hope that I can inspire them, too,” said Ren. “I had one teacher in middle school tell me to stay in chemistry and it was central to my career. That’s why my students will always publish as the first author in our papers in top-tier journals.”
Like his instructors before him, Ren finds that his students challenge the way he approaches his science even now.
“It rewards me to think, ‘it’s simple, let’s find the answer,’ in research,” said Ren. “But students push me to think, to question our textbooks. As scientists, we have to quantify the world. In biochemistry, everything builds from the fact that every behavior is based on a biochemical reaction. It is our job to question everything.”