Poster Presentation Multi-Omics Conference 2024

Development of a single-cell multimodal assay reveals somatic NFI transcription factors as dosage-dependant pluripotency mediators (#128)

Ying Yang 1 , Ralph Patrick 1 , Xiaoli Chen 1 , Jingyu Zhang 1 , Stacey Andersen 2 , shivangi wani 2 , Yifei Huang 1 , Mohammadhosein Esmaeili 1 , Dominique Power 3 , Cassy Spiller 4 , Quan Nguyen 1 4 5 , Josephine Bowles 4 , Melanie White 1 4 , Shyuan Ngo 3 , Marina Naval Sanchez 1 , Christian Nefzger 1 6
  1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia
  2. Genome Innovation Hub, The University of Queensland, St Lucia, QLD, Australia
  3. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia
  4. School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, QLD, Australia
  5. Queensland Institute of Medical Research QIMR Berghofer, The University of Queensland, St Lucia, QLD, Australia
  6. School of Chemistry and Molecular Biosciences, Faculty of Science, The University of Queensland, St Lucia, QLD, Australia

Cellular identity is governed by transcription factors (TFs). The ectopic expression of four TFs (Oct4, Sox2, Klf4, and c-Myc) enables the conversion of fibroblasts into induced pluripotent stem cells (iPSCs). Traditionally, somatic TFs from the cell of origin, such as the NFI transcription factor family, are viewed as barriers to reprogramming. However, the interplay between somatic TFs and pluripotency TFs during reprogramming remains poorly studied. Single-cell RNA+ATAC-seq is a uniquely powerful assay to measure the impact of TF levels on regulatory architecture; however, no tools currently exist to directly study TF dosage effects on temporal cell state transitions. To address these gaps, we developed Dosage and Hashtag sequencing (DoseH-seq), an expansion of the 10x Genomics single-nucleus (sn)RNA+ATAC-seq assay that enables sensitive detection of lentiviral perturbations (e.g., TFs) linked to a heterogeneously expressed promoter. In combination with sample hash tagging, multiple temporal, and dosage states can be profiled. We used DoseH-seq to study the effect of Nfix overexpression during reprogramming. Unexpectedly, we showed that the reported reprogramming barrier Nfix can either inhibit or potently enhance (>20-fold) reprogramming, depending on the overexpression level. Our data indicates that low overexpression of Nfix beyond basal levels, works synergistically with the reprogramming factors Oct4 and Sox2 to de-repress closed chromatin, while high levels reinforce the somatic circuitry to block the process. This challenges the prevailing notion that somatic TFs are obligatory reprogramming barriers and shows that our multimodal DoseH-seq assay enables the discovery of dosage-dependent TF effects. This could enable more effective strategies to reshape cellular identity and enhance our understanding of somatic TFs in other relevant processes, such as malignant transformation. This may set the foundation for a novel framework to reconcile the paradoxical reports of somatic TFs, including NFI factors, in cancer development and progression in a dosage-dependent manner.