Influences of HLH-2 stability on anchor cell fate specification duringCaenorhabditis elegansgonadogenesis

Abstract

The Caenorhabditis elegans E protein ortholog HLH-2 is required for the specification and function of the anchor cell (AC), a unique, terminally differentiated somatic gonad cell that organizes uterine and vulval development. Initially, four cells—two α cells and their sisters, the β cells—have the potential to be the AC. The β cells rapidly lose AC potential and invariably become ventral uterine precursor cells (VUs), while the two α cells interact via LIN-12/Notch in the “AC/VU decision” to resolve which will be the AC and which will become another VU. HLH-2 protein stability is dynamically regulated in cells with AC potential; initially present in all four cells, HLH-2 is degraded in presumptive VUs while remaining stable in the sole AC. Here, we demonstrate that stability of HLH-2 protein is regulated by the activity of lin-12/Notch in both α and β cells, providing evidence that activation of LIN-12 in an α cell promotes degradation of HLH-2 as part of a negative feedback loop during the AC/VU decision in α cells, and that ectopic stabilization of HLH-2 promotes AC fate in β cells. We also performed an RNAi screen of 232 ubiquitin-related genes and identified seven genes that contribute to HLH-2 degradation in VUs. When HLH-2 is stabilized by RNAi-depletion of ubiquitin ligases in a lin-12(+) background, there are no supernumerary ACs, suggesting that LIN-12 activation does not oppose hlh-2 activity solely by causing HLH-2 protein degradation. Furthermore, we provide evidence that hlh-2 transcription is negatively regulated in β cells independent of lin-12 activity. Together, our results indicate that hlh-2 activity is regulated at multiple levels to restrict the AC fate to a single cell.

Transcriptional regulation and repressive condensates modulate a proliferative-invasive cellular switch in vivo

“…In contrast, HLH-2 exhibits significant asymmetry in expression, as VU cells express merely 17% of HLH-2 levels observed in the AC on average (Figure 1C,D ). Previous studies have shown that post-translational, dimerization-driven degradation of HLH-2 is responsible for its downregulation in the VU cells (Benavidez et al, 2022;Karp and Greenwald, 2003;. Endogenously tagged NHR-67::GFP exhibits a similar pattern of expression with over three-fold enrichment in the AC, consistent with prior observations of transgenic reporters (Figure 1C,D ) (Verghese et al, 2011).…”

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch inC. elegans

“…In contrast, HLH-2 exhibits significant asymmetry in expression, as VU cells express merely 17% of HLH-2 levels observed in the AC on average (Figure 1C,D). Previous studies have shown that post-translational, dimerization-driven degradation of HLH-2 is responsible for its downregulation in the VU cells (Benavidez et al, 2022; Karp and Greenwald, 2003; Sallee and Greenwald, 2015). Endogenously tagged NHR-67::GFP exhibits a similar pattern of expression with over three-fold enrichment in the AC, consistent with prior observations of transgenic reporters (Figure 1C,D) (Verghese et al, 2011).…”

“…We observed that ectopic expression of HLH-2 resulted in elevated NHR-67 expression in VU cells (43% increase; n > 30) (Figure 2A,B). To control against potential dimerization-driven degradation of HLH-2 in the VU cells, which the heat shock inducible transgene would still be susceptible to, we disrupted UBA-1, an E1 ubiquitin-activating enzyme that has recently been shown to be necessary for HLH-2 degradation in VU cells (Benavidez et al, 2022). Following perturbation of UBA-1 through RNAi treatment, HLH-2 expression in the VU cells increased more than four-fold and NHR-67 expression increased by nearly 60% compared to the empty vector control (Figure 2–figure supplement 1B-D).…”

An expandable FLP-ON::TIR1 system for precise spatiotemporal protein degradation inC. elegans

“…Indeed, the ckb-3 promoter can drive transcriptional reporters in the gonad precursor cells Z1/Z4 and their decedents, but the level of promoter activity diminishes rapidly as the lineage progresses to more mature cell fates (Fig. S2A-B) (Benavidez et al 2022; Shaffer and Greenwald 2022).…”

“…We therefore sought to adapt the FLP-ON::TIR1 system in this context to determine if we could effectively deplete LIN-12 in the somatic gonad. To accomplish this, we generated FLP-ON ckb-3 ::TIR1 using the ckb-3 promoter (Benavidez et al 2022; Shaffer and Greenwald 2022). This system was then combined with LIN-12::mNG::AID and the LAG-2::P2A::H2B::mT2 reporter transgene (Fig.…”