An early signaling GARP transcription factor regulates the entry point for differentiation in Giardia

Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. How early signaling transcription factors (TFs) activate signal transduction to initiate encystation remains an open question in Giardia lamblia. Here, we screened putative early signaling TFs with both a newly developed CasRX-mediated knockdown system and an established CRISPRi system to identify early signaling TFs that regulate encystation. We identified an early response TF, GARP4 that regulates Cyst Wall Protein (CWP) levels during encystation. Depletion of GARP4 increases cyst production. Interestingly, cyst viability and CWP1 trafficking are not altered in GARP4 knockdowns, suggesting GARP4 regulates the restriction point controlling the portion of cells that terminally differentiate into cysts. Consistent with previous studies, we find that stimulation of encystation shifts the distribution of cells to the G2/M phase and these cells exhibits higher levels of CWP1. Key to this increase of CWP1 in G2/M cells is activation of MYB2, a TF previously linked to the early phase of encystation in Giardia. Remarkably, activated GARP4 only exhibits in G1/S cells, suggesting it has a role in preventing encystation until G2/M. Furthermore, we demonstrate that depletion of GARP4 activates MYB2 and upregulates encystation while overexpression of GARP4 represses MYB2 and downregulates encystation. Our findings provide the first molecular mechanism underlying the restriction point regulating differentiation during early signaling of encystation in Giardia lamblia, which is a critical factor for optimizing parasite transmission.