First person – Ajay Labade and Adwait Salvi Free

Ajay Labade (left) and Adwait Salvi (right)

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

A.L.: All cells in our body are genetically identical but nevertheless have diverse functional and physical identities, largely driven by a unique gene expression profile. How does a cell know which genes to express and when? The answer lies in epigenetics, which functions over and above the information encoded in the sequence of our genes. The epigenetics of every cell is defined by multidimensional structural and biochemical factors that regulate chromatin function. Our study sheds light on one such epigenetic mechanism that controls the temporal expression of HOXA genes, which are essential for the development of the embryo. What is unique about HOX genes is that their expression is precisely controlled in a time-dependent manner during development and differentiation. We found that the nuclear pore complex serves as a platform for regulating HOXA expression during differentiation. A stable nucleoporin, Nup93, helps anchor the HOXA gene locus to the nuclear periphery, thereby facilitating the repression of HOXA genes. However, upon receiving a green signal for expression from retinoic acid (RA; the active form of vitamin A), the HOXA chromatin undergoes a temporal ‘opening’ assisted by the well-known chromatin organizer CTCF. This remarkable scaffolding and re-arrangement of chromatin loops of the HOXA gene cluster is essential for their precise temporal activation. Upon differentiation, the HOXA gene cluster is silenced and re-tethered to the nuclear periphery by Nup93.

A.S.: The accurate differentiation of cells critically depends on the timing of gene expression, which in turn is regulated by the spatial positioning of genes in the interphase nucleus. The interplay between nuclear landmarks and regulatory proteins creates a permissive state for the spatial positioning of genes and therefore gene regulation. Nup93 functions as a stable anchor at the nuclear periphery, facilitated by the remarkable ability of CTCF as a master chromatin organizer, to direct HOXA genes into or out of the zone of repression. Fishing serves as a useful analogy to understand Nup93 function: to catch a big fish, one must be firmly grounded before reeling in the fishing line!!

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

A.L.: One specific challenge was while performing biological replicates of chromatin immunoprecipitation in NT2D1 cells with a 21-day-long differentiation time course. NT2D1 cells differentiate into various neuronal lineages upon RA treatment. Every lineage has its unique pattern of HOXA expression, and therefore, there is extensive cell-to-cell heterogeneity. It is therefore a challenge to replicate the exact same pattern of differentiation. Furthermore, RA is a fast-acting signaling molecule, and even the slightest change in treatment regimens can significantly alter HOXA expression. To overcome this, we took extreme care in maintaining passage number and cell viability, and carefully controlled RA dosage and treatment. We typically treated relatively large batches of cells for recovering cells and chromatin sufficient for ChIP even after 21 days of differentiation. Needless to say, performing multiple ChIP and qRT-PCR experiments together for all samples from all the time points was overwhelming.

A.S.: HOXA gene upregulation on RA treatment is a rapid event. Such massive upregulation is subject to heterogeneity based on the state of cells. Hence why the trend of HOXA gene expression is largely maintained. The relative levels vary between independent biological replicates. Experiments were performed as carefully as possible, while acknowledging intrinsic cell to cell heterogeneity.

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

A.L.: We built our hypothesis based on our observations and digging through literature. Curiosity led me throughout the process, and every result was a kind of eureka moment for me. Science is fascinating. I often tell myself that experiments might fail, but science always works. What is essential to know is what worked or failed, how and why? Finding answers to these elusive questions is always a eureka moment.

A.S.: While it was interesting to find a consensus motif of CTCF in the ChIP-Seq data derived from Nup93, we hardly expected that Nup93 or CTCF depletion would show antagonistic effects on the two regions of the HOXA gene cluster. Considering the presence of a topologically associated domain (TAD) boundary in the middle of the HOXA cluster, this result further motivated us to dig deep into the binding dynamics of Nup93 and CTCF to unravel the underlying mechanisms that regulate HOXA gene expression.

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

A.L.: Selection of the right journal with high research standards is a challenging part of research. Therefore, selecting Journal of Cell Science for our manuscript was a collective decision of the entire team. JCS has been at the forefront of publishing high quality findings on the ‘cell biology of our genomes’ – a remarkable field that combines the power of high-resolution imaging and omics. In addition, the quality of research papers published in JCS is exceptional. Moreover, the charge-free and format-free publication, and previous experience of my PhD advisor with the author-friendly publication process of JCS were major factors for why we unanimously selected JCS as the most suitable journal for submitting our manuscript.

A.S.: Journal of Cell Science is a reputed journal and with my previous experience of reading manuscripts published in JCS, it seemed the best fit for our work. I was also captivated by the stringent quality controls demanded by JCS, which I believe is crucial for the progress of science.

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

A.L.: I feel that research is the process of constant learning and documentation. I like to learn from my own experiences and the experiences of more experienced people. Therefore, I gathered knowledge throughout my PhD from multiple sources, including people and research papers. Beyond science, my mentor Dr Kundan Sengupta and lab members were always there as friends to discuss many non-scientific topics to ease work stress. Many thanks to Adwait Salvi for co-driving this research. I had a great network of friends who helped maintain the required happiness in my PhD. Everyone is special in some way or another.

A.S.: I completed my Master's thesis under the guidance of Dr Kundan Sengupta. The freedom to choose my question and critical feedback from him brought rigor to my scientific approach. Working with Dr Ajay Labade was an amazing experience as I saw how having practical knowledge beyond just protocols and a thorough knowledge base makes great science. Special thanks to Dr Maithilee Khot and other CBL members for their help and support during my years in lab.

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?

A.L.: I think that my motivation came from my ability to ask rational questions. Why and how do things inside, outside, and around us work? Every attempt to find answers to these basic questions pulled me towards science. I was always in search of the best place that could fulfill my thirst for scientific explanations. IISER Pune provided the right platform to start my journey in science. And now Harvard is constantly nourishing my journey in science.

A.S.: Growing up, I was fascinated by stories of scientists and efforts which led to great discoveries. For me, science is the search for truth. It's this idea which motivates me to ask questions and perform experiments. During my formative years, I was awestruck reading about DNA (and other biomolecules), which notwithstanding its miniscule dimensions and organization within the nucleus, is the basis of life. This was undoubtedly the most fascinating moment for me that led me to pursue a career in molecular and cellular biology and life sciences.

Who are your role models in science? Why?

A.L.: I am uncomfortable with the concept of a role model in science. In my opinion, the research we do is not ours. We leave it behind as a source of knowledge so that future generations can make the best of it. We build new scientific concepts while refining older ones. In a way, we contribute to the growth of science for a better understanding of many known and unknown scientific phenomena. Every small or large contribution made by every scientist in the past enlightens us about scientific principles which play a profound role in research. I am respectful towards those who are loyal to science and maintain the highest level of scientific integrity.

A.S.: My role model is the late Dr A.P.J. Abdul Kalam (scientist and former president of India). Science has many challenges, including resources and one's personal life, irrespective of one's place or position. It has its ups and downs and yet, one must face challenges so that we do our best for humanity to progress. This was exemplified in the illustrious life of Dr A.P.J. Abdul Kalam – a true motivation for all of us.

What's next for you?

A.L.: Currently, I am thoroughly involved in postdoctoral research in the lab of Dr Jason Buenrostro here at Harvard, USA. In the future, I am interested in basic research. It could be academia or industry. But for now, I don't have any plans to leave academia, provided I get to do what interests me the most. The freedom to do basic research could be one reason for my inclination towards academia. I want to contribute to the growth of science in India.

A.S.: I would like to continue research in molecular biology. I would like to determine the fundamental principles that govern 3D genome organization and gene expression. I hope to have my own lab someday and focus on 3D genome organization and gene regulation, which can potentially be translated to find appropriate therapeutic interventions.

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

A.L.: I have the vision of an artist. I love sketching and photography. Maybe that's the reason I keep talking to every component involved in my experiment when I am alone. I have long conversations with cells, bacteria, DNA, histones, enzymes, pipettes, tips, chemicals, microscopes, and even, for that matter, I talk to the invisible authors of research papers that I am reading. Science is joyful even in failures.

A.S.: I am also interested in drama. In my previous institute, I was part of the drama club, where I contributed to writing and directing plays.