Science

How a ‘Hmm That’s Strange’ Moment Led to a Therapeutic Discovery

Jyotsna Dhawan, a scientist talks about her research on quiet proteins which could have implications for therapeutic treatment of age and disease related muscle degeneration.

Jyotsna Dhawan in her lab. Credit: Jyotsna Dhawan

Jyotsna Dhawan in her lab. Credit: Jyotsna Dhawan

“I have a friend who says that in science, what usually precedes a discovery is not ‘eureka’, but a ‘hmm that’s strange.’,” said Jyotsna Dhawan, a scientist at Centre for Cellular and Molecular Biology, a CSIR research establishment situated in Hyderabad. Some years back Dhawan and her team had a ‘hmm that’s strange’ moment while studying the role of a protein in mice. In their quest to demystify the ‘hmm’, they ended up making an important discovery that has brought science one step closer to therapy for age and disease related muscle degeneration.

“In a way, skeletal muscle is a wonderful tissue – so contractile and stretchy – but it’s also a very human tissue. It forms about 40% of our body mass and allows us to move, breathe, and even express emotions through the 72 muscles in our face,” started Dhawan, softly introducing me to her complex research. “But because it is used so often, it often needs replacement.” Luckily for us, we have a batch of reserve cells that our body kept aside exactly for this purpose. These are called muscle stem cells.

Muscle tissue sections. Credit: Jyotsna Dhawan

Muscle tissue sections. Credit: Jyotsna Dhawan

A crash-course in stem cells

The very first cell formed from the fusion of an egg cell and a sperm cell is called the zygote. The zygote grows and multiplies rapidly to form a hollow ball of cells called a blastocyst. From this early stage of the embryo, cells start becoming specialised or differentiated into different kinds of tissues. “What you end up with at birth is a fully functioning organism that has all these different tissues and around 200 cell types,” explained Dhawan. “Usually there’s no stopping once you set the ball of development rolling. It’s a linear process, one-way path… no returns.”

But what is fascinating is that not all cells differentiate. Take muscle development for example. “At birth, the whole muscle tissue includes thousands of muscle fibres but in between, there are also satellite cells or muscle stem cells.” Stem cells are kept aside in an undifferentiated state at some point late in embryonic development. “And it’s not a musical chairs scenario where these are the cells that failed to develop – there’s a specific programme that sets them aside and tells them they are not going to differentiate,” stressed Dhawan. These cells stay quiet until there is a need for replacement muscle cells due to wear-and-tear, injury, disease or ageing. During such times, these stem cells develop into muscle cells and cater to the demand.

Here’s a nice intro about cell differentiation and stem cells:

Quiescence is golden

Cell and molecular biologists like Dhawan are interested in the program that makes these selected cells go quiet. A ‘program’ here refers to the set of rules that guide this process. These rules are usually in the form of specific genes and proteins. Dhawan’s lab is trying to understand all the genes, proteins and pathways involved in activating cell ‘quiescence’, the technical term for the silencing of these cells.

There are philosophical implications of this too, according to Dhawan. “Normally, when you think of a program that ‘activates’ something in a cell, you think of the cell being woken up to divide or induced to differentiate – very ‘active processes’,” she mused. “The idea of something ‘actively’ becoming quiet is not something that many people have looked at, and that’s what interested us.” For Dhawan and her peers in stem cell biology, this means going away from the idea of activity and inactivity to simply asking what are the processes that dominate in the quiet state and the ones that dominate the fully proliferative state.

Buzzing with inactivity

A Hindustani Musician. Credit: Wikimedia Commons

A Hindustani Musician. Credit: Wikimedia Commons

Dhawan uses the metaphor of the khali (empty) in Hindustani music to put the role of the quiescent cell into perspective. “The taal or the beat allows you to tell where you are in the rhythmic cycle. But you always have a khali (an empty or a void sound denoted by the wave of the hand), which is like the indexing to know where in the beat cycle you are. When you think of a cell, the taal is the cell cycle or cell division with very distinct phases. The quiescent/dormant cell is the khali – here, the cell division is stalled at a specific point in the cycle called G0. When it’s time to restart, the program doesn’t restart from the beginning but from that specific point. If the cell went quiet in random phases in the cell cycle, then you can envisage that the program to re-activate the cell would be very chaotic.” The importance of the G0 (G zero) phase is clear because it turns out that all stem cells (not just muscle stem cells, but blood stem cells, liver stem cells, umbilical cord stem cells, etc) are in this phase.

What really happens in this G0 phase? “Even if you are a quiet cell, not doing a whole lot, how are you surviving? You have to be doing something actively, even though at a very low level. Are there processes which are boosted in the quiet cell which are not so active in the so-called active cell?” To answer these questions, Dhawan began looking for genes – specifically those that were more active in quiescent cells than other cells. The hypothesis was that these were likely the genes controlling quiescence.

Skeletal muscle fibres. Credit: Jyotsna Dhawan

Skeletal muscle fibres. Credit: Jyotsna Dhawan

A quietly working protein

They identified a whole bunch of such genes in mice (which is the preferred model organism for these kinds of studies) and from this bunch cherry-picked a single gene to study in more detail. “This gene, called PRDM2, produces a protein that belongs to a family known for playing different kinds of switching roles in cells – for example, switching between cell division and differentiation, switching between muscle cell to fat cell, etc. The PRDM2 protein seemed like the perfect candidate for a molecule that is going to say you are going to stay quiescent – not divide nor differentiate.”

Though their experiments caught PRDM2 in all the right places at all the right times, Jyotsna and her team wanted to be totally sure. They looked at whether PRDM2 binds to genes important in keeping a cell quiet. “The answer was an overwhelming yes. We found that the protein sat not only in cell cycle genes (important for cell division), but also on differentiation genes (important for differentiation of cells into skeletal muscle cells).”

prdm2 protein. Credit: Jyotsna Dhawan

prdm2 protein. Credit: Jyotsna Dhawan

With all these positive signs that PRDM2 is playing some crucial role in cell quiescence, the next question in front of Jyotsna was – what is this role? To probe further, Dhawan used every molecular biologists go-to toolkit – knock-out technology. She grew cells in the lab that were purposely engineered to have no PRDM2 protein and observed what happened to those cells. Such knock-out cells are used to predict what the role of a gene or protein is playing in an organism. As suspected, in the absence of PRDM2, nearly 1500 cell differentiation genes turned on in these cells. This strongly indicated that PRDM2 was inhibiting differentiation in quiescent cells. This fits, because quiescent cells do not differentiate.

‘Hmm, that’s strange’

What about cell division? This is where the puzzle began. Cell division and cell differentiation are typically antagonistic to each other – when a cell begins to differentiate, cell division shuts down. And the knock-down cells confirmed this. PRDM2-less cells that had all its differentiation genes turned on, had their cell division repressed. This suggested that PRDM2 is required for cell division. But quiescent cells can’t divide! So what is happening here? How can a protein be quieting a cell and promoting cell division at the same time? “Hmm, that’s strange,” thought Dhawan…

Further adding to this paradox was another existing feature of the PRDM2 protein. Previous studies have shown PRDM2 to be a tumour suppressor protein. Tumours involve rapidly dividing cells, hence tumour suppressors like PRDM2 should inhibit cell division. “If PRDM2 is a tumour-suppressor, then if you remove it, you should see an activation of the cell cycle, right?” she thought. But this was the opposite of what was happening in PRDM2-less cells, where cell division was repressed.

Figuring it out

But after much puzzling, Dhawan and her two graduate students realised they were looking at it all wrong. What was actually happening was this: it is true that quiescent cells do not divide. It is also true that PRDM2 sits on cell cycle genes in quiescent cells. But they had wrongly assumed that this meant that it is PRDM2 that represses the cell cycle. It turned out that repression happens by other means. PRDM2’s actual role is to keep the cell cycle alive, but on idle. Remember, a quiescent stem cell has to keep a memory of the fact that it is going to differentiate into a muscle cell at some point in the future and then begin to divide. Until then, the cell cycle has to be kept sort of idling. “The engine can’t be completely off, just on neutral. There must be something telling the cell you have to be on neutral, don’t shut off the engine (cell cycle) completely. That something is PRDM2,” explained Dhawan.

Studies like this have great potential. “Eventually, we will be able to perturb these programs and overcome some problems associated to loss of muscle stem cells during muscle dystrophies and other muscle diseases which are quite common,” informed Dhawan. That’s the long-term goal of muscle stem cell biologists, she added. Dhawan credits her two “phenomenal graduate students” Sirisha Cheedipudi and Deepika Puri for the discovery of this mechanism, which went on to be described in a paper they published in Nucleic Acids Research journal in 2015.

For Dhawan, this was the culmination of a 15-year journey that began when she submitted a proposal for funding this project. The proposal had outlined her hypothesis that such a class of proteins existed. “(Being proven right) was very pleasing from that perspective,” she said.

Science, science, everywhere

Dhawan’s foray into the sciences was not completely out-of-the-blue. She grew up in the intellectual haven of the Indian Institute of Sciences in Bengaluru; her father is the famous aerospace engineer Satish Dhawan after whom India’s rocket launch centre in Sriharikota is named; her mother Nalini Dhawan was a botanist, and her brother is an astronomer. But growing up in school Dhawan was more drawn to history and the arts. In fact, she initially had her sights set on graphic design, but she did not get into the design school she preferred. So she pursued a basic sciences degree instead.

“School was great but quite conventional so anything unconventional had to come from home. I was very lucky in that regard,” said Dhawan. Plants were a big part of her life. “My mother’s family was completely plant-mad. They dealt anyone who didn’t obsess over plants with great pity and were more likely to know the latin names of the plants around them than their own relatives,” she laughed. Dhawan believes that from such an upbringing she and her siblings began to understand that a life of ideas is a life worth living. “But at some point, it occurred to us that we need to earn a living!”

A crisis, a mistake, and finding her way

Though she went on to a Master’s in Delhi University, Dhawan was not very clear about what she wanted to do with the degree. She recalled having a crisis during one of her final exams and almost deciding to drop out. Her father, who had come by for a meeting, suggested that she write the exams anyway. “He said, ‘look, you do what you want but I think you might feel bad about quitting if you don’t do your exams. Do your exams. It doesn’t matter what the outcome is if you’ve decided to quit’.”

The decision to stick on paid off because in the subsequent year, during a summer internship with TIFR, Dhawan truly fell in love with biology. “It was 1983, and there was a huge explosion of information about molecular biology. My background until then was more oriented to ecology and botany, but in TIFR I studied yeast genetics. It was difficult for me but I was absolutely hooked – fascinated by the puzzle solving that you had to do. I decided I’m definitely doing a PhD.”

She started her PhD coursework at home ground in IISc. During a conference in her first year, Dhawan was offered a scholarship by an Indian geneticist working in Canada to join his lab. “I decided this was a huge opportunity to do science and see the world – what a great idea!” But just three days after she arrived, Dhawan realised with dread that she’d made a mistake. “It was a really dull and run-of-the-mill place compared to IISc.” She panicked a little bit but then decided she had to be accountable for her choices. “I decided that now that I’m here, I’m going to take as many courses as I can this year and then transfer.”

Luckily for her, her brother was doing his PhD in the educational hub of Boston, US. During the holidays she visited him and began scouting for universities. She got a position at Boston University and transferred there. “I had to start afresh but, as I often tell my students today, there’s no such thing as wasted time if you’ve actually reflected on what you’ve been doing. This notion of ‘oh my god, I’m 24, life has passed me by because I haven’t done ‘x’ is so mistaken.”

In Boston, Dhawan thrived. “Everybody in Boston is a student. There are nine universities, classes happening everywhere and they are very flexible. What I encountered there for the first time was real cell biology. The fact that you could culture cells, look at them under a microscope and watch them divide was just a huge revelation for me.” In her five-and-a-half years there, she began to get interested in muscle.

Dhawan went on to spend another five years in Stanford University for post-doctoral work. While on a vacation trip home during Stanford, she connected with her future husband Imran Siddiqi while on a vacation home and married him on her return. Imran is also a scientist at CCMB. “He’s a plant biologist. Naturally, my mother loved him!”

On marriage and hiring couples

Did marriage change a lot? “I had my fears, maybe that’s why I married very late, at 35-36. I was quite clear that for me, I had to be convinced that this was going to be the right person. I wasn’t interested in a relationship for its own sake. But I have to say no (not much changed) because, in many ways, we’re just pretty useless when it comes to anything family-oriented,” she laughed. “In the sense, we don’t have kids – though this was not a choice, it just so happened. Of course, home is an important domestic space for us, our lives are not focused at home.”

Dhawan feels lucky to have got a place in the same institute has her husband. “There are all kinds of structures against couples being hired. It’s an unwritten rule and I have argued against it because I think it’s not correct. Many places across the world actually hire couples specifically. It’s nobody’s argument that this should be done against merit. Of course, merit should be the primary factor but if you are trying to build places where people are happy and going to be contributing, you have to look to their personal lives as well. I was lucky to not face the issue.”

Dhawan's current lab members. Credit: Jyotsna Dhawan

Dhawan’s current lab members. Credit: Jyotsna Dhawan

Being true with female students

Among Dhawan’s postdoc students, the gender ratio is pretty even. Though her experience with women students has been really good, she has seen them go through challenging times, most rooting from family pressures. Dhawan has seen a pattern. “Though most of their families are pleased about them going for higher education, during the third of fourth years, there comes this feeling that you are 28, when are you gonna settle down, have children… the women start feeling enormous pressure.” Dhawan has no doubt that this kind of pressure does affect them. “It affects their ability to be able to see themselves as free agents, to see that self-determination is actually an important goal. Sometimes you have to be able to say to your family – not now.”

How does she deal with these situations? With the truth. “I would be failing in my duty as a mentor if I didn’t say it’s important to see yourself as a free agent. I’m not saying you should abandon your responsibilities. if you have somebody to look after, a parent or a sibling or a cousin, you need to do that. But sometimes you need to be responsible to yourself as well. You cannot put that on the backburner. Sometimes you have to say ‘for six months, you have to leave me alone’.”

Dhawan says that the strength of some of the women in her lab has inspired even her. “One of my students has adopted a child as a single mother. There are also others who are faculty members and still single, some married.”

“One thing I learnt from when I was a postdoc was that the married postdocs with me were very very efficient. They had to work under much more constrained conditions than others who could spend the whole day in the lab not necessarily doing productive work. When you have a kid to pick up at 5.30 p.m. you’re not going to be hanging out drinking chai.” Jyotsna suspects her strong feelings on this matter may be coming from the fact that her mother did not get the opportunity to advance her career after marriage. “She had every enabling factor but still found it difficult. There were just too many responsibilities.”

Stem cells – a note of caution against stem cell therapy and banking

“One of my biggest challenges in these exciting times for stem cells is sounding a note of caution. We have way too much hype and when you try to clamp down on the hype people think you are against it. Hahaha, how can I be against it when I spend my life studying it! We need caution because we have way too many clinics operating under the radar providing what they call therapy. These are essentially unproven treatments with unproven sources of cells. They are fleecing the public. There is no gene therapy that is even approved in India. There’s no regulatory frame. Some of my colleagues are pushing for a regulatory framework because these are clinicians who see the value of gene therapy especially for conditions like haemophilia. We do need to have the biotech companies alongside us. There has been much progress, but the public needs to know that there are many people and clinics making hay while the sun shines. They are providing services like stem cell banking which is highly dubious. With private stem cell banks (which offer to store your baby’s umbilical cord stem cells as a ‘biological insurance’), what you’re paying for is liquid nitrogen. Nobody is going to use those cells. The companies cannot even guarantee that the cells will be functional at the end. They have no assays, they don’t even know how to test them. I think this is something we have to be proactive about. I’m not a clinician so i don’t like to advise anyone but I have made a set of guidelines for people seeking cord blood banking or stem cell banking to go and ask their doctors. But I think we need to do more.

This piece was originally published by The Life of Science. The Wire is happy to support this project by Aashima Dogra and Nandita Jayaraj, who are travelling across India to meet unsung women scientists.