Transcriptional regulation of HLH-6-independent and subtype-specific genes expressed in the Caenorhabditis elegans pharyngeal glands

The Caenorhabditis elegans pharyngeal glands represent one of five cell types in the pharynx. We have previously shown that the bHLH transcription factor, HLH-6, is required for gland development and for expression of many, but not all, gland genes (Smit et al., 2008). Here, we have identified additional gland-expressed genes and find that transcriptional regulatory inputs other than HLH-6 are necessary for their regulation. We demonstrate that at least two hlh-6 independent gland genes, nas-12 and Y8A9A.2, require a cis-acting motif (HRL3- Hlh-6 Regulatory eLement 3), previously described based on its requirement for hlh-6 expression (Ghai and Gaudet, 2008). We also show that expression of the gland-expressed genes, ZK596.1, scl-3, wrt-3, and Y76B12C.3, rely on cis-elements and trans-acting factor(s) other than HLH-6 and HRL3. In addition, we show that negative regulatory mechanisms are employed to refine the spatial expression of some genes, resulting in expression in only a subset of the five gland cells. We show that one of these genes, Y8A9A.2, is negatively regulated by the NHR transcription factor encoded by nhr-48, which represses Y8A9A.2 expression in the g1A cells. We also show that another gene expressed in the reciprocal subset of gland cells, phat-5, is negatively regulated in the g1P and g2 cells by an unknown factor acting through a conserved cis-element in the phat-5 promoter. Overall, this work reveals levels of regulation of gene expression in a single cell type beyond that previously known, and suggests mechanisms by which the different gland sub-types are distinguished.