2032 - DYNAMICS QUANTIFICATION OF TRANSCRIPTION FACTOR SWITCHES IN HSPC LINEAGE CHOICE

Abstract

Transcription factor (TF) networks regulate the lineage choice of hematopoietic stem and progenitor cells (HSPCs). Previous studies suggested a functional antagonism between the core hematopoietic TFs PU.1 and GATA1, and their autoregulation as the basis for a cell-intrinsic stochastic switch regulating PU.1 expression in lineage choice of HSPCs. Using long-term quantitative time-lapse imaging of HSCs from the PU.1eYFP/GATA1mCherry reporter mouse line, we previously showed that a random PU.1 and GATA1 switch does not initiate myeloid lineage choice. To further analyze the regulation of PU.1, we generated a novel mouse line with an inducible PU.1-ERT2 allele co-expressed with the PU.1eYFP/GATA1mCherry reporter alleles. Using single-cell reprogramming assays, microfluidics facilitated reversible PU.1 induction by TNF and single-cell quantification in response to transgenic PU.1-ERT2 activation, we quantified PU.1 expression in thousands of single HSPCs to measure the short- and long- term autoregulation dynamics. Surprisingly, we do not observe direct autoregulation of PU.1 at many different stages of hematopoietic differentiation. We find PU.1 autoregulation not to be involved in initiating differentiation, but to be an indirect consequence of it. Finally, to dissect the molecular triad of PU.1, GATA1 and GATA2 in regulating HSC lineage choice, we have generated a novel reporter mouse line for GATA2 protein and continuously quantify its expression dynamics, simultaneous with that of GATA1 in living HSPCs along the megakaryocyte-erythrocyte and granulocyte-monocyte differentiation trajectories. These data disprove several previous assumptions about the regulation of HSPC fate control, identify novel regulators, and demonstrate vacancy for other cell-extrinsic and -intrinsic players in regulating hematopoietic TFs specifically and lineage choice of HSCs in general.