A cell cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia

Abstract

Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. How early signaling transcription factors (TF) activate signal transduction to initiate encystation remains an open question in Giardia. Here, we generate a CasRX-mediated knockdown system, together with an established CRISPRi system to screen early signaling TFs in Giardia lamblia. We identified an early response TF, GARP4 that regulates cyst wall protein (CWP) levels during encystation. Depletion of GARP4 increases encystation efficiency resulting in increased 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, indication that entry into the encystation pathway is cell cycle regulated. Key to this increase of CWP1 in G2/M cells is activation of MYB2, a TF commonly observed during 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 overexpression of GARP4 represses MYB2. Our findings provide the first molecular mechanism underlying the restriction point regulating differentiation during early signaling of encystation in Giardia lamblia.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

“…Therefore, the natural tetraploidy of G. intestinalis in combination with incomplete removal of gene alleles by CRISPR/Cas9 provides an experimental way to suppress gene expression by manipulation with gene dose. These data showed that CRISPR/Cas9 may actually represent an alternative strategy to CRISPRi [ 23 ] and CasRx [ 24 ] for gene silencing in G. intestinalis .

Figure 7 CRISPR/Cas9-mediated knockdown of tom40 .

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“…[18][19][20][21][22]. Recently, the catalytically inactive form of the CRISPR/Cas9 system, CRISPRi, has been introduced to G. intestinalis for the successful knockdown of several genes that encode cytoskeletal proteins [23] and recently, RNA-targeting Cas ribonuclease, CasRx, was experimentally used in G. intestinalis [24]. The 'classical' CRISPR/Cas9 approach was used to reduce the gene expression in G. intestinalis [25,26] but this strategy did not achieve full genomic knockout.…”