First person – Hardik P. Gala and Debarya Saha Free

Hardik P. Gala

How would you explain the main findings of your paper in lay terms?

H.P.G.: Adult stem cells are often maintained in quiescence or a state of inactivity (at both metabolic and transcriptional levels). Upon external cues, such as injury, adult stem cells can transition rapidly into a state of activity, kickstart biosynthesis pathways, and contribute to replenishing or repairing the tissue. The central focus of this manuscript is to characterize what constitutes this cellular state of inactivity and gain understanding of the molecular mechanisms that allow quiescent stem cells to be maintained in a state of ‘inactivity’ and yet retain the ability to rapidly respond to kick start cellular activity when needed. Here, we identify a transcriptional poising mechanism controlling a group of biosynthesis genes that are crucial for maintaining stem cells in quiescent state of inactivity and is permissive of rapid induction upon favorable cues.

D.S.: Our muscle exhibits substantial regenerative capacity. This is due to the presence of muscle stem cells, which are in a reversibly arrested cell cycle state in which they do not divide, but form which they can become activated upon muscle injury to form new tissue. Such cells have been described as having a ‘sleeping beauty’ state. However, we found these cells to be maintained in a state of readiness to enter the cell division cycle. This state of readiness can be compared to a sprinter waiting at start line for the race to start. We report mechanisms that keep the transcription machinery engaged and ready on a network of genes in these cells. Upon activation, these genes are quickly expressed without delay, resulting in coordinated and timely entry into the cell division cycle. Unexpectedly, our study also revealed non-canonical functions of two well-known transcription regulators, Nelf and Aff4.

Were there any specific challenges associated with this project? If so, how did you overcome them?

H.P.G.: Our genome-wide molecular studies including transcriptomics and RNA Pol2 occupancy data corroborated primary cellular observations of reduced RNA and RNA Pol2 levels and its implications on quiescence. Although this integration of global analysis gave a long list of candidate genes to be tested, it was conceptually challenging to set up a panel of assays for a functional analysis screen. This was difficult for the following reason – in an ideal scenario, I really wanted to test the implications of RNA Pol2 pausing on quiescence for several candidate genes but there was no way to perturb Pol2 pausing at individual genes without affecting pausing/transcription of other genes. It took me a long time to figure out the next best thing – to simulate reduced RNA levels (the outcome of RNA Pol2 pausing in quiescence) in the proliferating condition and test whether we could achieve quiescence-like properties.

On a side note, it was during the tail end of my graduate program and the start of that for incoming graduate student Debarya Saha that we started to screen for upstream regulators of RNA Pol2 pausing. It was a steep learning curve for me to get into a ‘mentor’ role, but I think it was a fruitful collaboration in terms of project and conclusion.

D.S.: Depletion of Aff4 in quiescent cells resulted in increased expression of target genes. This was surprising, considering the known role of Aff4 in promoting transcription. Since this was an unexpected finding, we had to employ multiple different approaches to prove the restraining of Aff4 role in quiescence. This part of the project was challenging since we ourselves were not able to appreciate the results until later.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

H.P.G.: The reduced levels of Ser2-p, Ser5-p and Total RNA Pol2 in quiescence was a key observation forming the framework on which the following study and experiments were devised. I recall the excitement and train of thoughts and questions we had shortly after sharing the outcome of this experiment with Jyotsna.

D.S.: Knockdown of Aff4 in proliferating cells increased the frequency of S-phase cells in the population thus, showing that Aff4 has an anti-proliferative role in muscle cells. This is in sharp contrast with what is known about the role of Aff4 from the literature. This information holds value, since targeting of induction of Aff4 has been suggested as a therapeutic strategy for cancer treatment.

Why did you choose Journal of Cell Science for your paper?

H.P.G.: Outcomes of this work have implications for transcriptional regulation, cell cycle regulation and muscle development. Past experience from the lab has often shown that JCS is a good fit for such studies wherein a wide field of sciences in cell biology are explored in a single manuscript, contrary to other field- or niche-specific journals. This aspect is particularly highlighted by virtue of active biology researchers involved throughout the review process thereby making reviewing and publishing a seamless process.

D.S.: Our study is an overlap of two fields – quiescence/stem cells and transcription biology. We uncovered the transcriptional regulation of a particular cell state and cell state transitions by employing cell biology approaches. Journal of Cell Science is an appropriate journal for this kind of multi-dimensional study. Moreover, the readership of JCS perfectly overlaps with our target audience.

Debarya Saha

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

H.P.G.: The interactions with my PhD supervisor Jyotsna have greatly influenced my scientific thought process. She has been extremely supportive throughout my graduate work and has guided me at crucial junctures. Since I moved across institutes (and cities) during my PhD, I have seen the shrinking and swelling of lab sizes but this also gave me the opportunity to meet many individuals. I would like to give a shout out to Ramkumar Sambasivan and Dr Dasaradhi Palakodeti for having been instrumental in giving constructive criticism throughout my graduate program.

D.S.: I had two mentors who influenced me. My PhD supervisor, Jyotsna Dhawan, patiently mentored me through my PhD. The most important lesson I learnt from her was not to neglect negative results. Because, what we perceive as negative, might actually be a very significant new finding. Additionally, the freedom she provided me in undertaking new projects and experiments was invaluable. The other person is Gunjan Purohit, who was a post-doc in our lab (also a co-author on this paper). It was those odd-hour discussions with him that shaped my understanding of the muscle stem cell field and helped me troubleshoot numerous experiments related to this paper.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

H.P.G.: My Bachelor’s course has transformed my approach and empowered me to view curiosity with the lens of scientific enquiry. This, along with discussions with the teachers and my cohort, has initiated my career in science.

D.S.: From my school days, I have been interested in science. In fact, I was in high school when I decided to pursue research. Part of the credit goes to my location, close to a renowned University, which provided me with the opportunity to imbibe a bit of the scientific vibe early in life. Accordingly, I enrolled myself in a Bachelor's course in microbiology. It turned out to be a significant step in my career, since microbiology taught me the basics of biology, which can be applied to almost any other biological system.

Who are your role models in science? Why?

D.S.: This is a difficult one. I admire many scientists from my field or otherwise. But if I must choose then it has to be Marie Curie. She broke boundaries and societal stereotypes to follow her passion.

What's next for you?

H.P.G.: After my PhD, my research interests grew towards understanding developmental plasticity during organ formation. For this, I transitioned to root development in the plant model system as it exhibits potential to form diverse lateral organs originating from the same tissue type and employing developmental pathways. During my post-doc, I used single-cell approaches to study lateral roots in Arabidopsis and am now looking for positions to lead a research group and extend this line of enquiry on formation of lateral organs.

D.S.: I have had a long PhD tenure, but I still get the same amount of adrenaline rush when I am about to get a new result as I used to in my first year. I am quite sure I want to stay in academia. I have just submitted my thesis and currently I am looking for post-doctoral positions.

Tell us something interesting about yourself that wouldn't be on your CV

H.P.G.: I enjoy landscape photography and traveling, which fuel each other.

D.S.: I am someone who loves to stay updated with what is happening around the world. I prefer watching news over movies and TV series. I am also a map aficionado and a mass transit enthusiast.