Stress-induced nuclear condensation of NELF mediates transcriptional downregulation and cell survival upon heat shock

Prashant Rawat’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Prashant is originally from Delhi, India, where he did his undergraduate and postgraduate education He did his bachelor’s in Biomedical Science honors from Bhaskaracharya College of Applied Sciences, University of Delhi, and then did a master’s in Biotechnology from Jawaharlal Nehru University. He holds junior research fellowships (JRF) from CSIR (AIR-15) and DBT. Besides, he also qualified GATE Life Sciences with AIR-14 in 2017. He started his Ph.D. in the Laboratory of Dr. Ritwick Sawarkar at Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany, in September 2017 where he works in the field of molecular and cellular biology. His research work includes identification of novel chromatin based regulatory pathways of heat shock response and their molecular and cellular consequences. His hobbies include reading primarily scientific biographies, cooking Indian food, traveling to different countries, and exploring their culture. Here, Prashant talks about his co-first author paper on “Stress-Induced Nuclear Condensation of NELF Drives Transcriptional Downregulation” published in Molecular Cell.

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How would you explain your paper’s key results to the non-scientific community?

All living organisms face numerous environmental stimuli constantly, many of which are harmful to them. Therefore, the idea behind this project was straightforward – how do our cells act when they experience sudden deleterious changes in their environment. We used heat shock as a model to answer this question, where we just put human cells at higher temperatures. It is like experiencing a fever of 109 degree Fahrenheit. Others and we have observed previously that cells have a pre-wired program to combat all these changes, which gets activated upon these emergency stress situations and helps them ensure their survival.

The cells completely reprogram and channelize their resources under these conditions. Imagine this like the current corona pandemic. All the regular day-to-day activities came to a halt. All the resources are invested in essential programs like making masks and sanitizers and preparing more healthcare services and staff to minimize the impact of such pandemics and ensure minimum life loss.

Cells do the same thing; upon stress such as elevated temperatures, oxidative stress, or toxic metal exposures, cells completely shut off genes required for cell growth and metabolism. Instead, cells turn on the genes necessary to produce rescue proteins called Heat Shock Proteins (HSPs) or molecular chaperones. These proteins are critical for cell survival under stress conditions. The molecular mechanisms for induction of these chaperones are well known, but how cells turn off hundreds and thousands of genes very rapidly upon stress was unknown at the chromatin level.

This is where this study becomes so essential. We elucidated a novel molecular mechanism by which transcriptional regulator protein Negative Elongation Factor (NELF) attenuates transcription upon stress. We discovered that NELF undergoes phase separation to form nuclear condensates or droplet-like structures very rapidly upon stress. This condensation facilitates its recruitment to chromatin at promoters of downregulated genes and shut off transcription of hundreds of genes within minutes. We also report a detailed characterization of how NELF forms these condensates. We showed three distinct and complementary mechanisms that emerge at NELF condensation. The NELF phosphorylation counteracts its condensation, whereas stress-induced NELF SUMOylation facilitates its condensation. NELF has low complexity or structurally disordered regions in addition to these external forces, which drive this condensation process. None of the three pathways are sufficient to cause NELF condensation, but all of them are necessary. More importantly, disrupting any of these components leads to loss of NELF condensation and compromised cell survival upon stress.

Figure 1: NELF undergoes biomolecular condensation upon stress, such as heat shock. The NELF condensation is driven by its disordered domains or tentacles, reduced NELF phosphorylation, and stress-induced SUMOylation. 

This study has implications and impressions upon three significant domains of molecular biology research – transcription, proteostasis, and phase separation

What are the possible consequences of these findings for your research area?

This study has implications and impressions upon three significant domains of molecular biology research – transcription, proteostasis, and phase separation. Transcriptional regulation is one of the most crucial and fundamental pathways in every living organism. Our findings identified and characterized a novel regulatory layer of gene transcription. The role of SUMOylation in transcriptional repression upon stress is another prominent finding arising from our study. Our paper also characterizes a less studied branch of heat shock response, i.e., global transcription shutdown of housekeeping genes. We showed that this global transcriptional shutoff is necessary to ensure cellular survival. These results also have real-life implications, for instance, when we get a fever or exposure to heavy metals like arsenate in our drinking water.​

What was the exciting moment (eureka moment) during your research?

During my initial months in the lab, I started expressing all of our favorite proteins involved in transcriptional regulation and imaging them at high resolution with the confocal microscope. And suddenly, I realized that NELF has the property of forming these beautiful tiny droplets only after heat shock (stress). They were perfectly spherical. This observation really stunned us. This was our EUREKA moment in the project. Once we confirmed that these droplets are not just heat shock artifacts and can be modulated, the project was up and running.

What do you hope to do next?

After my great four years as a Ph.D student in the lab of Dr. Ritwick Sawarkar, I’m looking forward to submitting my thesis soon and graduate by autumn. The rigorous scientific training in the lab made me ready for my future step, which is to do a post-doc now and expand my theoretical and hands-on knowledge.

Where do you seek scientific inspiration?

My teachers and professors have been my biggest scientific inspiration and motivation throughout my career. I am fortunate to have great teachers in mentors who helped shape my academic and scientific career. I want to mention my scientific mentors from my undergraduate days, Dr. Uma Chaudhry and Dr. Anshu Bhardwaj. They were encouraging and inspiring, indeed. Both of them helped me engage in real-life scientific processes from the very beginning of my college days. This early practical experience has a profound impact on my scientific career. Also, excellent teachers and mentors from SBT, JNU were instrumental in giving me rigorous training to build a strong foundation for my Ph.D. overseas.

How do you intend to help Indian science improve?

Science is a global phenomenon and any advancement made helps the entire humankind. The science behind Covid-19 vaccines is one such example. The key to improving science in any country is to engage and encourage young minds. I hope that at some point, I can contribute to the enhancement of research activities at undergraduate and postgraduate levels across various universities in India. Also, recruiting well-trained scientists worldwide to India will help expand the knowledge base and technical expertise for significant benefits. If given a chance, I will be willing to take the opportunity to start my lab in India to contribute in more direct ways to help improve Indian science.

Reference

Prashant Rawat, Marc Boehning, Barbara Hummel, Fernando Aprile-Garcia, Anwit S. Pandit, Nathalie Eisenhardt, Ashkan Khavaran, Einari Niskanen, Seychelle M. Vos, Jorma J. Palvimo, Andrea Pichler, Patrick Cramer and Ritwick Sawarkar. “Stress-Induced Nuclear Condensation of NELF Drives Transcriptional Downregulation.” Molecular Cell 81.5 (2021): 1013-1026.e11.  DOI: https://doi.org/10.1016/j.molcel.2021.01.016

DOI: https://doi.org/10.1016/j.molcel.2021.02.010

Edited by: Manveen K Sethi

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