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.
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