Description

During C. elegans development, two cells, Z1 and Z4, give rise to the entire somatic gonad, including populations of dorsal uterine (DU) and ventral uterine (VU) cells, as well as the postmitotic anchor cell (AC), which invades the underlying basement membrane during uterine-vulval morphogenesis (Kimble & Hirsh, 1979; Sherwood & Sternberg, 2003). The helix-loop-helix transcription factor hlh-2 (E/Daughterless) is expressed in these three cell types, among others, and is particularly enriched in the AC following post-transcriptional down-regulation of HLH-2 in the VU through dimerization-driven degradation (Karp & Greenwald, 2003; Sallee & Greenwald, 2015). At the L2 stage, HLH-2 regulates the Notch signaling event that specifies AC and VU fates, with its initial onset predicting the lineage ultimately giving rise to the presumptive VU (Karp & Greenwald, 2004; Attner et al., 2019). Following AC/VU specification, HLH-2 functions to regulate cell cycle arrest and invasion of the AC (Medwig-Kinney et al., 2020; Schindler & Sherwood, 2011). We and others have shown that HLH-2 does so by regulating expression of the nuclear hormone receptor and transcription factor NHR-67 (Tailless/TLX/NR2E1), and likely cell-cycle independent, pro-invasive targets as well (Bodofsky et al., 2018; Medwig-Kinney et al., 2020).

In addition to hlh-2 and nhr-67, the histone deacetylase (HDAC) hda-1 was also identified as a regulator of AC invasion through a reverse genetic screen (Matus et al., 2010). It was later shown that hda-1 function is necessary for AC invasive fate differentiation and plays a role in the pro-invasive pathway encompassing NHR-67 (Matus et al., 2015). In order to further elucidate the mechanism by which HDA-1 regulates AC invasion, we sought to test we sought to test whether HDA-1 regulated expression of hlh-2. Previous studies of the hlh-2 promoter identified a proximal regulatory element (hlh-2(prox)) that confers expression in the AC and VU cells specifically (Figure 1A) (Sallee & Greenwald, 2015). Approximately 3.3 kb downstream of this hlh-2(prox) element is an HDA-1 binding motif that lies within a putative HDA-1 binding site based on ChIP-sequencing data generated by the modENCODE Project (Figure 1A) (Shao et al., 2020; Celniker et al., 2009). Using CRISPR/Cas9 genome engineering, we edited the endogenous hlh-2 locus to introduce a deletion mutation (hlh-2(bmd231)) from 1303 to 702 base pairs (bp) upstream of the GFP start codon, hereafter referred to as hlh-2(Δ-1303-702) (Figure 1A).

Consistent with findings using immunostaining, we find that endogenous GFP::hlh-2 expression is normally enriched in the AC compared to the VU and DU (Karp & Greenwald, 2003) (Figure 1B). When compared to wild type, the hlh-2(Δ-1303-702) mutant showed a modest but statistically significant reduction in HLH-2 expression in the AC and VU cells (Figure 1C-D). The endogenous GFP::hlh-2 allele paired with high resolution microscopy allowed us to detect subtle regulation of HLH-2 expression by hda-1 in the AC and VU cells that was not previously reported using a transgenic reporter (Ranawade et al., 2013). DU expression, however, was virtually eliminated in the mutant when accounting for camera-derived background noise (Figure 1C-D). We also observed that cytoplasmic expression of HLH-2 in the AC was modestly reduced in the mutant as well (Figure D-E). However, the alterations in HLH-2 expression in the AC and VU do not seem to be functionally significant, as expression of a NHR-67, a downstream target of HLH-2 in the cell cycle dependent pro-invasive gene regulatory network (Medwig-Kinney et al., 2020), was not significantly reduced (Figure 1F-G). Furthermore, no defects in AC specification or invasion were observed in any of the strains containing the hlh-2(Δ-1303-702) allele (n > 50) based on both the presence of a single AC and an underlying gap in the basement membrane, in all animals examined (visualized by cdh-3p::mCherry::moeABD and laminin::GFP, respectively). RNAi-induced knockdown of hda-1 resulted in reduced levels of HLH-2 in the DU, providing further evidence of this regulatory interaction (Figure 1H-I).

In summary, here we generate and characterize a new mutant allele of hlh-2. Deletion of the genomic region containing a putative HDA-1 binding site from the hlh-2 promoter appears to primarily affect expression in the DU cells and is recapitulated through RNAi-induced knockdown of hda-1. Although HDA-1 is traditionally thought of as a repressor of transcription through chromatin deacetylation (Kadosh & Struhl, 1997; Kadosh & Struhl, 1998; Rundlett et al., 1998; Gui et al., 2003), this data adds to the growing evidence that HDACs can also function as activators of transcription (Vidal & Gaber, 1991; De Nadal et al., 2004; Wang et al., 2014). Whether there is a functional consequence of hlh-2 depletion in the DU cells is currently unknown. It is our hope that this reagent will be useful to the C. elegans community to further study the roles of hda-1 and hlh-2.