Project 1: Elucidating the functions of chromatin remodelers during meiosis in yeast S. cerevisiae.

Abstract: Eukaryotic DNA is tightly wrapped around nucleosomes for its compaction to form chromosomes (Fig. 1A). Although this compaction makes it easier to transport DNA (chromosomes) within a dividing cell, it also makes DNA less accessible for the DNA-protein interactions essential for DNA synthesis, repair and transcription. ATP-dependent Chromatin Remodelers (CRs) selectively mobilize nucleosomes leading to nucleosome-depleted chromatin (Fig. 1A) to favor DNA-protein interactions. There are four subfamilies of ATP-dependent CRs in eukaryotes: INO80, CHD/Mi-2, SWI/SNF, ISW1. Several lines of evidence suggest the role of CRs in meiotic recombination. However, the relative contribution of each of the CR complexes and how their absence affects various stages of the meiotic recombination (such as double-strand break (DSB) formation, 5’ end resection, strand invasion and D-loop formation, second end capture and DNA synthesis) is not yet quantified. From the existing literature, it can also be envisioned that CRs may be involved in the other aspects of meiotic chromosome segregation such as cohesin loading, sister chromatid cohesion, centromere function, kinetochore-microtubule attachments, however, it still needs experimental evidence. CRs are also known for regulating the transcription of many genes by making gene promoters accessible for transcription factor binding. Hence, it would be interesting to know how CRs affect the precise timing of the meiotic transcription cascade and possibly delay the meiotic progression in their absence. Recent reports suggest that ~190 meiotic genes have extended transcripts. As CRs are known to regulate promoter accessibility and transcription, it would be interesting to know how CRs maintain the critical balance between the extended and regular transcripts of meiosis-specific genes to regulate the protein levels. We will use a combination of fluorescence microscopy, biochemistry, cell and molecular biology and yeast genetics to explore the functions of CRs in meiosis, a process by which germ cells are produced in humans. As 20% of all human tumors contain mutations in one of the CR complexes, targeting CR pathways is currently evolving as a major therapeutic strategy in the treatment of cancers. Understanding the role of CRs in meiosis has the potential for therapeutic development for treating infertility, stillbirth, genetic disorders and cancers.

Figure 1: A) Multisubunit CR complexes make the chromatin accessible either by evicting or by sliding or by exchanging nucleosomes, B) Spindle dynamics over time during meiosis (immunofluorescence with Tub1 antibodies and DAPI) represents pace of meiotic progression. Scale: 5 µm C) Chromatin spread to quantify the defects in homolog pairing (scale: 2 µm, Ref. 2) and D) synaptonemal complex formation. Scale: 5 µm

Key References:

1) Mehta et al. 2014, Molecular Microbiology 91(6): 1179-1199

2) Hong et al. 2019, Nucleic Acids Research 47(22): 11691-11708

3) Clapier et al. 2017, Nature Reviews Molecular and Cell Biology 18, 407-422

Project 2: Single-Molecule dynamics of the cancer therapy target aurora kinase B.

Abstract: Aurora kinase B (AKB) is one of the key regulators of cell division and erroneous cell division leads to developmental disorders and cancers. Overexpression or underperformance of AKB has been observed in many human cancers and hence it is currently one of the most promising targets for cancer therapy. Despite AKB’s multi-faceted functions in mitotic regulation, the molecular mechanism of establishing and maintaining the phosphorylation level of its substrates (cell cycle regulators) remains unknown. Human has three Aurora kinases (A, B, and C) with significant overlap in localization and functions, making it difficult to study their functions in isolation. Yeast Saccharomyces cerevisiae has only a single aurora kinase (Ipl1) with significant structural and functional homology to human AKB. Like humans, yeast AKB (Ipl1) localizes to two distinct locations: at the kinetochores during metaphase for checkpoint regulation and kinetochore assembly; and on the spindle/spindle midzones during anaphase for spindle assembly/disassembly, respectively. Ipl1 phosphorylates different substrates at these locations: kinetochore proteins Ndc10, Dam1, Ndc80, Dsn1 at the kinetochore and Ase1 at the spindle midzone. We hypothesized that Ipl1 interacts with the kinetochores and the spindles dynamically. Their dynamic localization creates their clouds around the kinetochores and the spindles to maintain a critical level of phosphorylation of AKB substrates. Preliminary experiments using FRAP validated this hypothesis by showing the fast recovery of Ipl1 (half recovery in ~30 s) at the spindle midzones (Fig. 2B). Using single-molecule biophysics and quantitative proteomics, I will quantify the dynamics of the Ipl1 to reveal its target-search mechanism, how long does it interact with the kinetochores and spindles for specific phosphorylation events and how it maintains a critical level of phosphorylation for various cell cycle regulators (substrates). Using Single-Molecule Tracking (SMT), we will measure the diffusion constants, mean squared displacements, bound fractions and residence times of Ipl1 at the kinetochores (during metaphase) and over the spindles (during anaphase). To correlate the Single-Molecule dynamics of Ipl1 with the level of phosphorylation, we will purify entire yeast kinetochores using an affinity purification approach (a well-established method from Sue Biggins lab) from metaphase and anaphase synchronized cells, followed by phospho-enrichment and LC-MS/MS analysis for the quantitative comparison of the phosphorylated Ipl1 substrates. To make the conclusions more relevant to the human cells, we will perform single-molecule tracking of AKB in human cells and compare its dynamics with yeast Ipl1 to learn how AKBs explore the different volume of the cell nuclei to find their target sites, how long do they take for each phosphorylation events and how they differ in their mode of action between yeast and human cells. Additionally, we will test how cancer therapeutic AKB inhibitor (Barasertib AZD1152-HQPA) and overexpression of AKB (a cause for many cancers) affect the single-molecule dynamics of AKB and eventually phosphorylation in human cells. Understanding the molecular dynamics of AKB in normal and cancerous cells would allow us to find therapies and design better drugs that would preferentially modulate AKB function in cancerous cells.

Figure 2: Ipl1/AK-B spatiotemporally changes its localization during mitosis in yeast (top panel) and human/HeLa cells (bottom panel) to phosphorylate various substrates. Cellular localization of Ipl1/AK-B are depicted in blue fonts and its substrates are depicted in red fonts. CEN: centromeres, KT: kinetochores, MCAK: mitotic centromere-associated kinesin, MRLC: myosin II regulatory light chain, GFAP: glial fibrillary acidic protein. Images of HeLa cells are adapted from (Ref. 5). Scale: 5 µm.

Key References:

1) Saurin AT 2018, Frontiers in Cell and Developmental Biology 19(6):62

2) Schmidt JC et al. 2016, Cell 166(5):1188-1197.e1189

3) Mehta GD et al. 2018, Molecular Cell 72(5):875-887.e9

4) Buvelot et al. 2003, The Journal of Cell Biology 160(3):329-339

5) Fu J et al. 2007, Molecular Cancer Research 5(1):1-10

Project 3: Unraveling the mechanism of mitosis to meiotic transition at the levels of kinetochore composition, 3D genome organization and transcriptome.

Abstract: Meiosis is a specialized cell division process that leads to the production of gametes with half the number of chromosomes from their mother cells. In humans, errors in meiosis are the leading cause of aneuploidy, stillbirth, developmental defects, mental retardation and infertility. Hence, it is essential to know how germ cells decide to leave the mitotic cell cycle and begin progression through the meiotic cell cycle to produce gametes. From the existing literature, it is poorly known how a germ cell decides to leave the mitotic cell cycle and begin progression through the meiotic cell cycle for the production of gametes. Using cutting-edge genomics, proteomics and transcriptomics, we will address the following questions: 1) How kinetochore composition changes from mitosis to meiosis? Why it changes and what additional functions it provides? 2) How does the transcription program change from mitosis to meiosis? 3) How the 3D genome organization changes from mitosis to meiosis? To address the first question, we will purify the entire kinetochores from yeast mitotic and meiotic cultures, followed by LC-MS/MS analysis to quantify kinetochore composition at different stages. A quantitative imaging approach will be used to validate those findings. Structural biology and computational modeling approaches will be used to understand how those changes in composition translate into microtubule binding and force generation. To address the second question, transcriptome profiles of the mitotic and meiotic cultures arrested at different stages will be quantitatively compared. The resulting targets will be verified using RT-qPCR and smFISH. Recent studies have identified an important role of transcription regulation for faithful meiosis, hence it is essential to identify more mRNA targets that regulate meiosis. To address the fourth question, Hi-C (High Throughput-Chromosome Conformation Capture) will be used to identify differences in the Topologically Associating Domains (TADs) between mitosis and meiosis. TADs represent physically isolated units of genome organization, manifesting “co-regulation” of genes within TADs and the blocking of the “spread” of activity between neighboring TADs. This project will elucidate the molecular mechanism for germ cell decision to switch from mitotic to meiotic cell division. This understanding is critical for developing therapies for infertility, aneuploidy and cancers and it promises to impact key issues in human reproduction.

Figure 3: A process of gamatogenesis. Primordial germ cells produce spermatogonia and oogonia which divide by mitosis for several generations. These cells make decision at one point to start meiotic cell cycle to produce gametes. This project aims to understand how cells decide to switch mitotic cell cycle to meiotic cell cycle by modulating kinetochore composition, 3D-genome organization and transcriptome. This figure was modified from Prof. A. Cuschieri, University of Malta website.

Key References:

1) Mehta GD et al. 2014, Molecular Microbiology 91(6): 1179-1199

2) Borek et al. 2021, Current Biology 31:1-14

3) Belton and Dekker 2015, Cold Spring Harbor Protocol 7:649-61

4) Gupta et al. 2018, Methods in Cell Biology 144:349-370

Indian Institute of Technology Hyderabad, India

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