“Inspiration comes from discoveries”
In this interview, Jeannie T. Lee talks about the most important moments of her career, discusses her groundbreaking work in X chromosome inactivation and long non-coding RNAs, the challenges in academia, and her experiences in founding start-up companies. She also reveals why she feels she cannot retire yet and how she addresses skepticism about the functional roles of lncRNAs.
What were some highlight moments in your career?
I would highlight three moments in the last 25 years that stand out:
When starting my lab, we knew that X-inactive specific transcript (Xist) silences the X chromosome, but how did Xist get regulated? As a college student, I worked in Nancy Klekner’s lab on an antisense RNA regulator of Tn10 transposition, and the first thing that popped into my head was that Xist could have an antisense RNA regulating it. So, I designed some antisense probes to look into this hypothesis. I remember that day sitting at the microscope looking at the RNA fluorescence in situ hybridization experiment, and indeed, the antisense lit up beautifully. This result was very exciting as I realized this could be the key discovery for launching my career.
A couple of years later, I had a strong sense that the X chromosomes must communicate with each other right before inactivation. Otherwise, how would the cell know which X chromosome should be kept active and which to inactivate? For this communication, the X chromosomes could co-localize in 3D space. The second «aha moment» was when we saw them co-localizing under the microscope.
The third one I would like to mention was more recent, about eight years ago, when we were working on a project not linked to X-inactivation but around repetitive B2 and ALU retrotransposons and figuring out how these RNAs get cut by EZH2, a histone methyl transferase without known nuclease activity. Here, the «aha moment» was when we learned that EZH2 is not a nuclease, but a chaperone that directs the RNA to cleave itself.
“There could be no other way than for RNA to direct the silencing.”
Why does an RNA silence the X chromosome?
There could be no other way than for RNA to direct the silencing. How does a cell determine which X-chromosome is going to be active and which inactive, in a mutually exclusive fashion? And then, once the inactive one has been chosen, ensure that inactivation takes place only on that chromosome so that only the thousand protein-coding genes located on that chromosome are silenced? The answer is that it had to be RNA because it is the only macromolecule that can tether to a chromosome and mark the site of its synthesis.
What are the major open questions in the field of X chromosome silencing?
I cannot retire yet because none of the major questions are solved. How does X chromosome counting take place? There are several candidate factors, but I believe there will be many more.
X chromosome choice remains a big mystery. We believe that pairing is essential for X chromosome choice. However, how do the X chromosomes come together, what factors are exchanged in their communication, and what are the consequences at the molecular level on the chromosomes?
Many questions are open on the RNA side: How does Xist spread and stay localized on the X chromosome? What are the primary and secondary localization sites?
What are your thoughts on the association of RNA with chromatin and RNA-DNA triplexes?
It is not just RNA-DNA triplexes; R loops will also play an important role. There are more non-coding RNAs than there are coding RNAs. In terms of the sheer bulk of non-coding transcription, we know they will not all be junk. Even repetitive elements like B2, ALU and LINE retrotransposons, which were historically looked at as junk, turn out to have functions in epigenetic regulation.
RNAs are essential as guides and serve as scaffolds for chromatin. I think that all these epigenetic complexes that we know about cannot scaffold around and be directed to chromatin without RNAs. RNAs are the only players capable of cis-targeting as well as allele-specific targeting. RNAs can attach to chromosomes in several ways, e.g., they can be tethered to the chromosomes simply through their active transcription or by forming R loops.
RNA-DNA triplexes might also play essential roles. What I like about triplexes is that they could work not only in cis but also in trans. The evidence of such triplexes is not very well established, but I think that in the years to come, we will see more and more evidence for their roles not only in epigenetics but also in DNA damage response.
“In terms of the sheer bulk of non-coding transcription, we know they will not all be junk.”
https://doi.org/10.1073/pnas.1503690112
How did you deal with people saying that long non-coding RNAs are just noisy transcription?
They still do say that. I tend to ignore the naysayers and keep doing our work. If you believe that what you have discovered is correct and holds water, you keep plugging away. You keep researching until the truth is revealed. The truth can go either way. Sometimes, you or your ideas are disproven. Other times, they are validated. So, you have to have conviction in your ideas to continue but, simultaneously, be open to the possibility that they could be incorrect.
What is your fascination with long non-coding RNAs in general?
Long non-coding RNAs are the secret glue that holds the nucleus together and mediates epigenetic regulation. Nowadays, many believe that Xist is a paradigm for how non-coding RNAs work, but Xist is not unique. Many other cell transcripts will have a mechanism of action similar to Xist.
The name «long non-coding RNAs» was initially chosen because of their length and because they seemed to not code for proteins. Is there a need for better characterization, and if yes, what would that characterization look like?
I was never a big fan of that categorization, as grouping any factors by their size is generally not a good idea. I prefer to group by function. However, we still know so little about the function of most non-coding RNAs that I believe we are not at the stage where we can have a Linnaean-like classification system of long non-coding RNAs.
At a very fundamental level, you could group them into structural versus functional. This categorization might also not be a very good one, but this came to my mind right now from the top of my head.
Can you tell us about your efforts to develop small molecules targeting RNA?
We are very interested in compounds that target RNAs for therapeutic purposes. However, I think this area is still in its nascent stages, and we are far from being able to design such RNA-targeting small compounds rationally. Mainly because we still know so little about RNA structure. Methodologies are being developed, but we are far from translating what we see in vitro to in vivo. In vivo, there are too many players, like protein interactions and RNA helicases, which dynamically change the shape of RNAs. So, what you measure in vitro will not necessarily be there in vivo.
Our approach to identifying small molecules that target RNAs has been based on structure-agnostic methods. The methodology is a high throughput affinity screen for small molecules and requires that you know a little bit about the function of an RNA and a little bit about its important domains. So, you do not need to understand how an RNA folds overall, but you have to know which pieces of the RNA are important to limit the screen to the dedicated portion of the RNA.
It was fascinating that the molecule we found apparently does not intercalate but still changes the RNA structure and abolishes two fundamental interactions at the same time. We believe it binds to some folded structure within a repeat, disrupting the structure and thereby abolishing the ability of the RNA to recognize two of its cognate essential interactions.
“I came from a background where being a woman was not considered a weakness.”
What was your experience with the companies RaNA Therapeutics/TranslateBio that you co-founded?
RaNA’s initial mission was to treat human diseases by de-repressing genes. The approach was to use oligonucleotides to target protein-RNA interactions. That was the original concept, and RaNA then grew into a dual-modality company when 2017 it acquired Shire’s messenger RNA therapy technology, which put it in direct competition with Moderna. Also that year, RaNA relaunched as Translate Bio. Then came the pandemic and in 2021, Sanofi, without its own mRNA technology, bought the company. So that was that.
From that journey with RaNA Therapeutics/TranslateBio, what advice would you give to somebody considering founding a company?
First, you need a good idea, and then you need to weaponize it, meaning enable it. Once you have done that, you need a proper narrative around it, a sales pitch, if you will, to bring it to potential investors. And it could be a long road, as talking to investors takes up much time. Unless your heart is in it and you are motivated to bring the technology to the clinic, I would say you should not do it because it is very time-consuming.
Would you do it again?
As for the case of RaNA Therapeutics/TranslateBio, I would absolutely do it again. Also, we started Fulcrum Therapeutics, which had a slightly different beginning and was also a tremendously rewarding experience. Now, we have two drugs in the clinic, one for facioscapulohumeral muscular dystrophy and another one for sickle cell disorder and other hemoglobinopathies. That company was based on small molecules to upmodulate or downregulate expression, depending on the case.
So, two very different companies and two really great experiences there.
With an MD-PhD, have you regretted not taking the MD route with likely a more stable position earlier in your career and better salary options?
More stability and higher salary would have been the case. However, I never looked back because as soon as I started as an independent investigator, we made several exciting discoveries. Based on these, I knew I could grow my career as a scientist in the coming years. One thing led to another, but because of my physician-scientist training, this desire to do something for the medical community and patients has stayed with me.
The MD-PhD training took a long time, but I do not think I would do things differently now. I have come full circle by doing not only basic research in RNA biology and epigenetics but also translating what we know about how RNA and the X chromosome work to treat patients with X-linked diseases.
Is an academic career nowadays less attractive than it used to be?
There are different challenges at every stage of the career. If I were today a graduate student thinking about careers, I would say there are many more options than a group leader, like journalist, patent attorney or industry scientist. When I counsel graduate students, I do not necessarily try to direct them toward an academic career, as this holds more and more challenges.
As faculty, we are expected to spend more and more time on non-science tasks like serving on committees, taking certifications and a lot more administrative paperwork. A lot of regulation was not there 30 years ago when I thought about becoming a group leader. When I started my group, I spent 90% of my time doing science, 10% on teaching and very little on administrative responsibilities.
With the increase in regulations, you have committees set up to study and enforce them and so on and so forth, and I spend about 25% of my time in such tasks.
At the same time, we are expected to bring in research funding, and the cost of doing science is more expensive now than ever.
Salaries at all levels, research consumables and services have gone up tremendously. However, the NIH funding levels have not considerably increased in the last 10 or 15 years and the maximum tops off between 250,000 to 500,000 US dollars. These developments create challenges for both senior and junior people who are just starting their independent scientific careers.
“I would almost say there is a compulsion to get to an answer.”
https://doi.org/10.1073/pnas.1503690112
Which particular challenges have you faced and which of those would you attribute to being a woman?
Again, every stage comes with its particular challenges. As a graduate student, I did not feel that I faced any specific challenges because I came from a background where being a woman was not considered a weakness. My father promoted the idea of me being a physician-scientist or going into surgery, which was undoubtedly then not a female-friendly discipline. My father was almost blind to all these male-female differences. He thought that being a woman should not stop me from doing anything I set my mind to.
Although it is interesting that I actually did have a mentor (not the PI whose group I worked in), who commented when it came to the choice of postdoc field that I should not follow my interest and work on fly and worm genetics but should go into something a bit less competitive like mouse genetics. This is a most interesting comment because first, I think he was alluding to the challenges of being a woman in science, and second, the thought that mouse genetics was less competitive than fly and worm genetics. Fast forward 10-20 years and the opposite has become the case.
Do you think science would be done differently with equal gender representation at the full professor level?
Yes and no. It is crucial to have better representation of women in leadership positions, certainly at the full professor level. Promoting more women to positions of power will only work if the women are informed and have proper leadership skills. Putting somebody in a leadership position simply because they have two X chromosomes, e.g., to lead an institute that did not have a woman president in the past, is not a good policy. Man or woman, that person needs to be qualified, fair, and informed.
How would you describe your leadership style?
Young scientists do best when given sufficient freedom to explore, but not so much that they get lost. My approach to running the lab is hinged on this model of guided freedom. I neither like nor do I have the time for micromanagement with a lab of 25 people. However, I also do not believe that benign neglect is a good leadership style, so I play a reasonably strong hand in guiding the interpretation of experiments and what to do next in the projects. This guidance is probably even stronger when formulating the narrative around the publication.
Where do you find inspiration?
Inspiration comes from discoveries. I get excited when I see data that flies in the face of what we know and is the exact opposite of what we were expecting. When that happens, I tell myself and the person showing me that piece of data that there is something exciting and that it is our job to find out why these results are unexpected.
Scientifically, besides unforeseen results, it is the unknown and unsolved problems. I would almost say there is a compulsion to get to an answer. I do not know how else to describe it because I have come to know myself as somebody who needs to finish. Over 25 years ago, I started asking questions about how X chromosome inactivation works. Today, we still do not know the answer, and I feel that my career and life would be incomplete without a satisfying answer.
Another key driver is that we may be just around the corner to helping girls with Rett syndrome, which is one of the most devasting congenital pediatric disorders. These girls develop seemingly normal after birth and then they developmentally regress. So, these girls, at some point, could talk or even stand up and walk. They lose those developmental abilities to develop milestones and end up in a wheelchair and never speak another word. What this does to affected families and how much they suffer is a powerful motivating force not only for me but also for the people in the lab working on X-linked disorders.
Biography Jeannie T. Lee
Jeannie T. Lee obtained her undergraduate degree from Harvard University, during which she researched retrotransposition in the lab of Nancy Kleckner. Subsequently, she received her MD-PhD degree from the University of Pennsylvania, doing her thesis research in the lab of Robert Nussbaum. She then moved back to Boston first as a resident in clinical pathology and then for postdoctoral research in the lab of Rudolf Jaenisch at the Whitehead Institute and the Massachusetts Institute of Technology. She then joined the Department of Molecular Biology at Massachusetts General Hospital and Harvard Medical School as a faculty member. She is a member of the National Academy of Sciences and the National Academy of Medicine and a Fellow of the American Association for the Advancement of Science. She is currently Chair of Molecular Biology at the Massachusetts General.
Interview was conducted by Dominik Theler on November 28, 2022.