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. 2014 May 13:14:17.
doi: 10.1186/1471-213X-14-17.

Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6

Wan-Ju Liu, John S Reece-Hoyes, Albertha J M Walhout, David M Eisenmann  1
Affiliations

Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6

Wan-Ju Liu et al. BMC Dev Biol. .

Abstract

Background: Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo.

Results: We used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs.

Conclusions: These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

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Figures

Figure 1
Figure 1
LIN-39 regulators identified in yeast bind to lin-39 promoter regions in vitro. A - F) Gel mobility shift assays with proteins expressed and purified from E. coli. Arrowhead indicates free probe; arrow indicates protein DNA complexes. For each panel, the top line identifies the labeled probe used, the bottom line shows the amount of protein added in each lane; middle lines (panels A, B, D and F) indicate the identity and amount of competitor added. A) NHR-43 binds to YF1 (342 bp). Lanes 3–5 contain cold wild type YF1 as competitor, while lanes 6–8 contain cold YF1 with the TGAC site mutated as competitor; B) ALR-1 binds to ECR2 (40 bp); this binding is competed by wild type ECR2 but not by a scrambled oligonucleotide with the same nucleotide composition (ECR2S); C) ZTF-17 binds to YF4-2 (186 bp) and YF4-4 (110 bp); D) ZTF-17 binding to YF4-2 is competed by YF4-4 but not YF4-3; E) LIN-26 binds to YF4-3-1 (51 bp); F) LIN-26 binding to YF4-3-1 is competed by YF4-3-1 (51 bp) but not YF4-3-2 (52 b); G) Fragment YF4 with ECRs 7–10 is shown above, with smaller subfragments diagrammed below. Shading indicates fragments that were bound by ZTF-17 and/or LIN-26 in yeast one-hybrid assays and in vitro.
Figure 2
Figure 2
Seven transcription factors affect lin-39::GFP expression in the VPCs at early L3 stage. GFP expression in the VPCs P5.p - P8.p from smg-1; deIS4 (lin-39::GFP) animals treated for RNAi of individual transcription factors identified in yeast (panels B-H), or given control RNAi (empty vector; panel A). Early L3 stage animals are shown; anterior is left, ventral is down. These animals also express ajm-1::GFP, which outlines cell junctions of hypodermal cells and the pharynx (bright staining in the anterior seen in most panels). RNAi for lin-26, bed-3, and tbx-9 causes severe embryonic lethal and larva arrest phenotypes, so RNAi for these genes was performed by feeding newly-hatched L1 larvae on RNAi bacterial lawns and examining GFP expression in these same animals at the L3 stage. For all other genes, RNAi treatment was carried out on P0 animals, and their F1 progeny were examined as L3 animals. All pictures were taken under the same exposure.
Figure 3
Figure 3
Seven transcription factors affect lin-39::GFP expression in the VPCs at early L3 stage.smg-1; deIs4(lin-39::GFP) animals were treated for RNAi of individual transcription factors (panels A–G, dark bar), or control RNAi (panels A–G, light bar) as in Figure 2. Early L3 stage animals were photographed at the same exposure, and pixel counts in each VPC were determined using ImageJ (>20 animals for each strain). Bars show mean GFP pixel count in each cell with standard deviation. ‘*’ indicates P-value < 0.05. ‘**’ indicates P-value < 0.001, compared to control animals.
Figure 4
Figure 4
Regulation of lin-39 levels by transcription factors in vivo (qRT-PCR). A) Decrease in lin-39 transcript levels in nhr-43, lin-26, tbx-9, bed-3 mutant and ztf-17(RNAi) animals. B) Decrease in lin-39 transcript levels when activity of both elt-6 and egl-18 is reduced. C) Increase in lin-39 transcript levels when either elt-6 or egl-18 was overexpressed from the heat shock promoter. All analyses were done on L3 stage animals. The mean values for each genotype were obtained from at least two independent experiments and normalized to the housekeeping gene, gpd-2, as internal standard. The data was analyzed with unpaired t-test compared to the appropriate control. ‘*’ indicates P-value < 0.05.
Figure 5
Figure 5
NHR-43, LIN-26, ELT-6 and EGL-18 are necessary for lin-39::GFP expression in the embryo. A–D) GFP expression in embryos derived from smg-1; deIs4(lin-39::GFP) animals treated for control RNAi (empty vector, panel A) or for RNAi against transcription factor genes B)nhr-43;C)lin-26 or D)elt-6. E – H) GFP expression in embryos carrying pJW3.9::GFP in a wild-type background (E) or in animals carrying mutations in elt-6(F) or egl-18(G and H). Embryos shown are at the ‘bean’ stage of embryogenesis (~360 minutes). All photos were taken under identical exposure settings. Note that before the individual P cells interdigitate at the ventral midline, the cells are referred to by their possible fates (i.e., P5/6 L and P5/6R), and there are two GFP-expressing cells on the left side and two on the right.
Figure 6
Figure 6
ELT-6 interacts with pJW3.9 through a GATA binding site. A) Yeast strains containing HIS3 and lacZ reporters transformed with empty vector (Control) or ELT-6::GAL4AD plasmids (ELT-6) were diluted and replica plated to control (left), 10 mM 3aminotriazole (3AT, middle), and XGal (right) plates. Reporters had inserts of either JW3.9 (top) or JW3.9 in which GATA binding site S1 was mutated from TGATAA to GGTACC (‘Mut’ bottom). Mutation of the GATA site abolishes the interaction with ELT-6 based on lack of growth on 3AT and lack of blue color on XGal.; B) Increasing concentrations of ELT-6 interact with a 40 bp fragment around GATA site S1 (lanes 2-6). Mutation of the GATA site S1 from TGATAA to GGTACC (M1) abolishes interaction with ELT-6 (lane 8); C) 50 ng of cold wild type site S1 (W) can compete effectively for binding of ELT-6 to S1 (compare lanes 2 and 3). Mutation of GATA site S1 (M1) reduces but does not abolish the ability to compete (lane 4). Mutation of both site S1 and a second GATA site on the oligonucleotide (M2) drastically reduces the ability of the oligonucleotide to compete for ELT-6 binding (lane 5).
Figure 7
Figure 7
Direct transcriptional regulators of lin-39 in the embryo and larva. A) Horizontal lines represent 20 kb of genomic DNA surrounding the lin-39 locus. The lin-39 transcript is shown below the top line, with boxes representing exons. The next horizontal line shows evolutionarily-conserved regions (ECRs; thin vertical lines), the PCR fragments used in the yeast one hybrid assays containing the ECRs (boxes labeled 1–12), and two fragments (pJW3.9 shown, JW5 unlabeled) identified previously using an enhancerless GFP assay [47]. Transcription factors that bind the lin-39 gene are shown above the line (previously reported) or below the line (reported in this work). B) Model for positive feedback loop between egl-18/elt-6 and lin-39. EGL-18 and ELT-6 act via the GATA site in enhancer pJW3.9 to facilitate initiation of lin-39 expression in the embryo, and then LIN-39 acts to positively regulate egl-18/elt-6 expression via the Hox/Pbx binding site in the intron of egl-18[55].
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