Gland-specific expression of C. elegans hlh-6 requires the combinatorial action of three distinct promoter elements

AbstractThe pharyngeal glands of Caenorhabditis elegans are one of five cell types in the pharynx. The transcription factor HLH-6 is required for gland development and function, and is specifically expressed in pharyngeal glands. As a first step to understanding specification of pharyngeal glands, we analyzed the promoter of hlh-6 to identify the elements required for gland-specific expression. Our experiments identified three distinct regulatory elements required for hlh-6 expression: a PHA-4-binding site and two new elements, HRL1 and HRL2 (for hlh-6 regulatory elements 1 and 2). The three elements employ a simple logic for producing cell-type-specific expression: the PHA-4 site restricts expression to the pharynx, HRL2 restricts expression in both a position and lineage-dependent manner, and HRL1 restricts expression to a subset of cell types. In isolation, these three elements have little or no enhancer activity but in combination they produce robust, gland-specific expression. These findings describe a combinatorial code for gland-specific expression and suggest that similar codes may be employed for specification of other pharyngeal cell types

The C. elegans Snail homolog CES-1 can activate gene expression in vivo and share targets with bHLH transcription factors

Snail-type transcription factors (TFs) are found in numerous metazoan organisms and function in a plethora of cellular and developmental processes including mesoderm and neuronal development, apoptosis and cancer. So far, Snail-type TFs are exclusively known as transcriptional repressors. They repress gene expression by recruiting transcriptional co-repressors and/or by preventing DNA binding of activators from the basic helix-loop-helix (bHLH) family of TFs to CAGGTG E-box sequences. Here we report that the Caenorhabditis elegans Snail-type TF CES-1 can activate transcription in vivo. Moreover, we provide results that suggest that CES-1 can share its binding site with bHLH TFs, in different tissues, rather than only occluding bHLH DNA binding. Together, our data indicate that there are at least two types of CES-1 target genes and, therefore, that the molecular function of Snail-type TFs is more plastic than previously appreciated

The HLH-6 Transcription Factor Regulates C. elegans Pharyngeal Gland Development and Function

The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the “organ identity factor” PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen

Environmentally induced foregut remodeling by PHA-4/FoxA and DAF-12/NHR

Growth and development of the Caenorhabditis elegans foregut (pharynx) depends on coordinated gene expression, mediated by pharynx defective (PHA)-4/FoxA in combination with additional, largely unidentified transcription factors. Here, we used whole genome analysis to establish clusters of genes expressed in different pharyngeal cell types. We created an expectation maximization algorithm to identify cis-regulatory elements that activate expression within the pharyngeal gene clusters. One of these elements mediates the response to environmental conditions within pharyngeal muscles and is recognized by the nuclear hormone receptor (NHR) DAF-12. Our data suggest that PHA-4 and DAF-12 endow the pharynx with transcriptional plasticity to respond to diverse developmental and physiological cues. Our combination of bioinformatics and in vivo analysis has provided a powerful means for genome-wide investigation of transcriptional control

Early-1 and Early-2 Elements Are Required for K07C11.4 Expression

<div><p>(A) A portion of the promoter sequence of K07C11.4 from C. elegans (bottom) aligned with its ortholog from C. briggsae (top). Boxed regions show conserved predicted PHA-4 binding sites and Early-1 and Early-2 elements. Site-directed mutations that disrupt Early-1 and Early-2 (“E2 + E1 Mut”) are shown below their respective wild-type (“E2 + E1 WT”) sequence from K07C11.4.</p> <p>(B–E) Confocal images of mid-stage embryos expressing GFP under the control of the wild-type K07C11.4 promoter (B) or promoters with a mutation in Early-1 (C), Early-2 (D), or both Early-1 and Early-2 (E). Percentages are the fraction of transgenic embryos expressing GFP; the remainder of embryos do not express GFP.</p> <p>(F) Expression of the wild-type K07C11.4 reporter in a subset of somatic gonad cells in an L4 animal (arrowheads).</p> <p>(G) Mutation of the Early-1 element eliminates gonadal expression but does not strongly affect expression in other tissues, such as intestinal cells (arrows).</p> <p>Dashed lines indicate the outline of the developing pharynx.</p></div

Strategy to Identify Temporal Regulatory Elements

<div><p>(A) Flowchart of the strategy used.</p> <p>(B) Northern blot of <i>pha-4. pha-4</i> transcripts were approximately 25- to 100-fold enriched in <i>par-1</i> compared to <i>skn-1</i> embryos, but only approximately 5- to 10-fold enriched in wild-type compared to <i>skn-1</i> embryos. Arrowheads indicate the three different <i>pha-4</i> isoforms.</p> <p>(C) The same blot was probed with a fragment of the <i>act-1</i> gene to demonstrate equal loading of RNA between lanes.</p></div

High-Affinity PHA-4 Sites Activate Pharyngeal Expression Earlier Than Low-Affinity Sites

<div><p>Percent values indicate the percentage of transgenics exhibiting pharyngeal GFP expression. Dashed lines indicate the outline of the developing pharynx.</p> <p>(A–C) A reporter construct with three copies of a high-affinity PHA-4 site (TGTTTGC) upstream of the Δ<i>pes-10</i> promoter reproducibly activates pharyngeal expression from the time of pharynx primordium formation (“early”) through embryogenesis.</p> <p>(D–F) A reporter construct with three copies of a low-affinity PHA-4 site (TATTTGT) upstream of the Δ<i>pes-10</i> promoter activates pharyngeal expression from the time of attachment of the pharynx to the mouth (“mid”) through embryogenesis.</p></div

Temporal Elements Combined with PHA-4 Sites Regulate the Onset of Pharyngeal Expression

<p>“Early,” “mid,” and “late” are as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020352#pbio-0020352-g003" target="_blank">Figure 3</a>. E1, Early-1; L2, Late-2; High, high-affinity PHA-4 site (TGTTTGC); Low, low-affinity PHA-4 site (TATTTGT). Percent values indicate the percentage of transgenics exhibiting pharyngeal GFP expression; the remainder of embryos do not express GFP. A reporter construct with one copy of the Early-1 element and one copy of a high-affinity PHA-4 site is expressed in “early” to “late” embryos (A–C). In contrast, a reporter with one copy of the Early-1 element and one copy of a low-affinity PHA-4 site is not consistently expressed until the “mid” to “late” stages (D–F). Reporters with one copy of Late-2 and one copy of either a high-affinity (G–I) or low-affinity (J–L) PHA-4 site are expressed in “mid” and “late” stage embryos. Dashed lines indicate the outline of the developing pharynx.</p

Late-1 Represses Early PHA-4-Dependent Expression

<p>Percent values indicate the percentage of transgenic animals exhibiting pharyngeal GFP expression. A reporter construct with three copies of a high-affinity PHA-4 site (TGTTTGC) upstream of the Δ<i>pes-10</i> promoter (A) expresses GFP in pharyngeal cells of most transgenic embryos (B) and roughly one-third of transgenic larvae (C). The addition of three copies of the Late-1 element from R07B1.9 (CCTTGGCGGCGC) to this transgene (D) drastically reduces expression in transgenic embryos (E) but has no observable effect on transgenic larvae (F). Dashed lines indicate the outline of the pharynx.</p