Poster Presentation 45th Lorne Genome Conference 2024

mTOR signalling regulates epigenetic factors during pluripotency state transitions  (#256)

Katherine Zyner 1 2 , Di Xiao 1 , Eren Ekmen 1 3 , V. Pragathi Masamsetti 4 , Hani Kim 1 , Raja Jothi 5 , Mark Graham 6 , Patrick Tam 2 4 , Pengyi Yang 1 7
  1. Computational Systems Biology Unit, Children's Medical Research Institute, Westmead, NSW, Australia
  2. School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, Australia
  3. School of Biomedical Engineering, Faculty of Engineering, The University of Sydney, Camperdown, NSW, Australia
  4. Embryology Unit, Children’s Medical Research Institute, Westmead, NSW, Australia
  5. Epigenetics & Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina, United States of America
  6. Biomedical Proteomics Facility, Children’s Medical Research Institute, Westmead, NSW, Australia
  7. Charles Perkins Centre, School of Mathematics and Statistics, The University of Sydney, Camperdown, NSW, Australia

During early development, embryonic stem cells (ESCs) transition through a pluripotency continuum marked by naïve, formative, and primed states. The pluripotency state transitions of ESCs is essential for cell-fate decisions and lineage allocation and are typically initiated by extracellular cues that trigger intracellular signalling cascades to culminate in the activation of downstream transcriptional and epigenetic regulations. While significant effort has gone into identifying the key kinases that propagate these signals intracellularly, how these signalling cascades impact the epigenome and translate to transcriptome changes and subsequent cellular differentiation is not fully defined. Identifying these trans-regulatory networks is essential for understanding the molecular mechanisms underpinning cell fate choices and lineage differentiation, which have translational implications for stem cell therapy. Mammalian target of rapamycin (mTOR) is a kinase that regulates many processes vital for overall cellular function, including metabolism, proliferation, survival, and stem cell self-renewal.  We have previously shown that mTOR signalling is enhanced during the transition of the pluripotency state in mouse naïve embryonic stem cells as they are reprogrammed to epiblast-like cells (EpiLCs)1, a process that epiblast cells in the embryo undertakes to acquire the formative state2 to launch the lineage differentiation program. Through large-scale multi-omic profiling of the phosphoproteome and proteome in the presence of multiple small molecule mTOR inhibitors coupled with our multi-step kinase scoring computational method, PhosR3, we have identified the direct targets of mTOR, which were further validated with in vitro kinase assays. Ongoing functional analyses using conditional mutant cell lines aim to dissect how these phosphorylation events contribute to the extensive epigenetic remodelling as cells transit into the developmental phase of lineage specification while still maintaining stemness/ pluripotency.

  1. P. Yang, S. J. Humphrey, S. Cinghu, R. Pathania, A. J. Oldfield, D. Kumar, D. Perera, J. Y. H. Yang, D. E. James, M. Mann & R. Jothi (2019) Multi-omic profiling reveals dynamics of the phased progression of pluripotency. Cell Systems, 8:427–445
  2. A. Smith (2017) Formative pluripotency: the executive phase in a developmental continuum. Development 144(3):365-373.
  3. H. J. Kim, T. J. Kim, T., J. N. Hoffman, D. Xiao, D. E. James, S. J. Humphrey, & P. Yang (2021) PhosR enables processing and functional analysis of phosphoproteomic data. Cell Rep 34, 108771 (2021).