Orc4 spatiotemporally stabilizes centromeric chromatin

Dr. Lakshmi Sreekumar’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Lakshmi was born in Cochin and was brought up in different parts of India. Due to her father’s transferable job (in the Army), Lakshmi got to travel around India, changing schools almost every two years. She did her Bachelors’ in Microbiology from Fergusson College, Pune. She joined JNCASR, Bangalore, for the Integrated PhD program, where she did her Masters’ thesis and later her Ph.D. in the Molecular Mycology Laboratory under Prof Kaustuv Sanyal. She earned her Ph.D. on her thesis titled, “Crosstalk of DNA replication and chromosome segregation machinery ensures genome stability in Candida albicans” in 2019. Her research problem focused on examining factors for centromere specification and function in Candida albicans. She currently works as a postdoctoral researcher with Prof. Julia P. Cooper at the University of Colorado, Anschutz Medical Campus, Aurora, Colorado, since March 2020. Her research focuses on elucidating the mechanism for a non-reciprocal translocation that facilitates fission yeast cells to maintain linear chromosomes in the absence of the telomere synthesizing enzyme telomerase. When she is not working at the bench, she enjoys traveling (restricted due to the pandemic), reading fiction novels and dabbling on her ukulele and guitar. Here, Lakshmi talks about her work titled “Orc4 spatiotemporally stabilizes centromeric chromatin”, published as the first author in Genome Research, 2021.

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

Our genetic material is organized within the nucleus of every cell as chromosomes. Chromosomes duplicate and are equally partitioned (or segregated) from one generation to the next. On every chromosome there is a specialized region called the centromere, which helps in accurate chromosome segregation. Chromosomes are arranged within the three-dimensional nuclear space distinctly. In many organisms, all the centromeres are clustered close to the nuclear envelope just beneath the spindle pole bodies/SPB (organelles that produce spindle fibers to pull chromosomes apart), and the chromosome arms are located away from this cluster (see schematic). If you look at centromeres within a particular organism, they exhibit several conserved features, e.g., a common DNA sequence or a specific pattern of 3D folding. It is known that a centromere protein, CENPA, recognizes these sequences/ regions and helps the chromosomes to be pulled apart by spindle fibers during cell division. How a cell decides a particular location of the centromere preferentially over any other site is still a puzzling question. Moreover, the mode of centromere recognition by CENPA in an organism where none of the conserved centromeric features are present is still unanswered. Our work sheds light on how a cell chooses the centromere location on a chromosome, how CENPA recognizes centromeres and how centromeres are stabilized throughout the cell cycle.

We carried out our study in a single-celled pathogenic yeast, Candida albicans. Apart from its immense clinical relevance, this yeast has peculiar genomic features. It can seemingly alter its chromosome number when exposed to stress, a strategy that it efficiently uses to evade antifungal drugs. In contrast to other organisms, all eight centromeres of C. albicans have different DNA sequences. Through our work, we have discovered a protein, Orc4, to be a crucial factor in determining the location of a centromere. While this protein is conventionally known to be important for DNA replication and is present at hundreds of sites on chromosomes, we found that the highest Orc4 concentrations occur at the centromeres. The way Orc4 is distributed on chromosomes in space and time (hence the name “spatiotemporal” in the title) creates a pattern that helps the cells memorize where to form centromeres in the subsequent cell cycle. We also found that if you perturb Orc4 levels using genetic tools, you compromise centromere integrity and get rid of CENPA protein molecules, thereby adversely affecting centromere structure and function. In short, such an Orc4 concentration gradient helps cells to duplicate and segregate their chromosomes efficiently. I call this the “ORCanization” of the centromere J. This “centromeric signature” imparted by the Orc4 concentration gradient is the main discovery presented in our paper. We found additional proteins (Mcm2 and Scm3) that help protect centromeres and ensure that CENPA is deposited at centromeres in a timely manner. Additionally, we determined the precise time of CENPA deposition at centromeres with the help of extensive microscopic analyses and photobleaching experiments.

Schematic

With the findings from this paper and previous work in our group, we have established a model for how centromeres are replicated, protected, and regulated throughout the cell cycle. We achieved our goals by combining genetic, biochemical, and cell biological approaches and collaborated with labs with expertise in polymer modeling (IIT Bombay) and computational biology (IMSc, Chennai; NCL, Pune).

We have also added to the existing reservoir of molecules that can crosstalk between two essential DNA metabolic processes: DNA replication and chromosome segregation.

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

The association of Orc4 at the centromere will pave the way to discover additional non-canonical factors and other such signature molecules in centromere regulation. These findings can be extrapolated to multicellular organisms, which have diverse and often more complex forms of regulation. We have progressed the field by understanding how these varied interactions regulate conserved biological processes like chromosome segregation. We have also added to the existing reservoir of molecules that can crosstalk between two essential DNA metabolic processes: DNA replication and chromosome segregation.

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

I have had precisely two eureka moments during my PhD. Pertaining to this paper, I distinctly remember the first time I observed Orc4 enrichment at one of the centromeres. I made the observation using a technique called chromatin immunoprecipitation (ChIP), followed by a PCR reaction. We later confirmed this result using high-throughput sequencing. But the day I got my PCR result in the lab (which happened to be a Sunday, and hence I had no one to share the excitement with!) is etched on my memory.

What do you hope to do next?

As an immediate follow-up to this work, I wish to know the exact molecular mechanisms of centromere regulation by Orc4 —does it physically interact with the centromere or is it indirectly mediated through some other protein (complexes)? We have promising leads in that regard, which are being taken forward by enthusiastic graduate researchers in the lab. I am eagerly waiting to hear where this project leads to.

I am currently doing my postdoc in telomere biology. Telomeres are yet another critical chromosomal landmarks that maintain chromosomal linearity and length. My current project involves studying the survival mechanism of cells that lack telomerase, the enzyme that adds telomeres at the ends of chromosomes. For that, I am using fission yeast as a model, which elegantly maintains linear chromosomes even in the absence of telomerase by employing certain other sequences from its chromosomes to make “new telomeres.” This survival trick has been observed in many types of cancers. My goal is to find the mechanism for this specific translocation reaction using various biochemical, genetic and cell biological tools and shedding light on this remarkable phenomenon.

Where do you seek scientific inspiration?

In my high school and later in my undergrad, I was introduced to bacterial and phage genetics, which captured my attention and curiosity. I do not have a science-hero per se, but some of the earlier experiments in biology, like the Lederberg experiment, Benzer’s rII phage mutant analyses, and Meselson-Stahl’s experiments, made a mark on me and inspired me to pursue research. I often wonder how those scientists came up with their landmark experiments. To be honest, no amount of high-throughput data can beat the brilliance and elegance of some of these classical papers. These experiments inspire me because I love working at the bench—it is a very immersive experience for me, and I would not trade it for anything!   

How do you intend to help Indian science improve?

When I look at my peers, I feel encouraged and hopeful about the future of scientific research in our country. That said, I do think we can do better! We need to design a more inclusive undergraduate curriculum, be it for basic science or other vocations. Students should be encouraged to study diverse subjects and not be confined to, e.g., only biology or only physics. Being a strong proponent of interdisciplinary sciences, I would love to be involved in building systems/ channels to help mentor both undergrads and postgrads embarking on their scientific journeys, drawing from my experiences and training acquired during my PhD and postdoc. Another point I feel strongly about: our graduate research programs need to build holistic admissions processes that look beyond just previous academic scores. The grades you get in high school have very little to do with your research capability, or for that matter, even your interest in science. I could go on a rant about it, but you get the point.

Reference

Sreekumar L, Kumari K, Guin K, et al. Orc4 spatiotemporally stabilizes centromeric chromatin. Genome Research. 2021 Apr;31(4):607-621. DOI: 10.1101/gr.265900.120

Prof. Kaustuv Sanyal group: https://molecularmycologylab.wixsite.com/kaustuv

Edited by: Vikramsingh Gujar

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