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Nuclear Hormone Receptor Regulation of MicroRNAs Controls Developmental Progression

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Science  03 Apr 2009:
Vol. 324, Issue 5923, pp. 95-98
DOI: 10.1126/science.1164899

Abstract

In response to small-molecule signals such as retinoids or steroids, nuclear receptors activate gene expression to regulate development in different tissues. MicroRNAs turn off target gene expression within cells by binding complementary regions in messenger RNA transcripts, and they have been broadly implicated in development and disease. Here we show that the Caenorhabditis elegans nuclear receptor DAF-12 and its steroidal ligand directly activate promoters of let-7 microRNA family members to down-regulate the microRNA target hbl-1, which drives progression of epidermal stem cells from second to third larval stage patterns of cell division. Conversely, the receptor without the ligand represses microRNA expression during developmental arrest. These findings identify microRNAs as components of a hormone-coupled molecular switch that shuts off earlier developmental programs to allow for later ones.

Lipophilic hormones coordinate organism-wide developmental progression in metazoans by binding to nuclear hormone receptors (NHRs), converting the presence or absence of ligand into changes in gene expression patterns (1). This regulation is conserved in the nematode Caenorhabditis elegans, where the nuclear hormone receptor DAF-12, a homolog of vertebrate liver X and vitamin D receptors, regulates developmental progression or arrest in response to the environment (2, 3). In favorable environments, activation of transforming growth factor-β (TGF-β) and insulin/insulin-like growth factor (IGF) signaling cascades results in production of the DAF-12 steroidal ligands, the dafachronic acids (e.g., Δ4-DA), which promote rapid progression through four larval stages (L1 to L4) to reproductive adults (4). In unfavorable environments, endocrine systems are suppressed, and DAF-12 without the ligand causes arrest at a stress-resistant, long-lived alternative third larval stage, called the dauer diapause (L3d) (5).

A more cell-intrinsic level of developmental control is exerted by microRNAs. MicroRNAs are ∼20- to 22-nucleotide-long RNA molecules that bind to the 3′ untranslated region (3′UTR) of target messenger RNAs (mRNAs) and decrease their expression (68). Null mutants for several microRNA genes show tissue-selective failure of progression from one stage-specific program to the next, generally described as heterochronic phenotypes. These phenotypes are most visible in the hypodermis, where hypodermal seam cells undergo invariant asymmetric stem cell division patterns, in which one daughter cell fuses to the hypodermal syncytium, whereas the other retains stem cell character and its capacity to divide (9). Only during L2 do seam cells undergo one proliferative division before stem cell division, and repetition or loss of this program leads to changes in overall seam cell number in later stages. Finally, seam cell division ceases altogether by adulthood. Seam cells in animals with mutation of the microRNA lin-4 repeat L1 programs of asymmetric cell division during L2 stage; a triple deletion of the let-7 microRNA homologs mir-48, -84, -241 (referred to as let-7s) repeat L2 programs of cell proliferation during L3 stage; and let-7 null mutants repeat larval stage divisions and molting behavior in adults (1012).

daf-12(rh61rh411) null mutants exhibit a heterochronic phenotype similar to triple deletion of let-7 family members mir-48,mir-241(nDf51); mir-84(n4037), resulting in extra seam cells at the L3 stage (table S1) (2, 11). This observation suggested that daf-12 might directly activate the let-7s microRNAs. To test this hypothesis, we fused the microRNA promoters mir-241p and mir-84p to the luciferase gene and co-transfected the reporters with DAF-12 into human cells (13). DAF-12 and Δ4-DA strongly activated mir-241p and mir-84p, whereas other promoters gave little or no signal (Fig. 1A). Deletion analysis of the mir-241p revealed that, whereas several deletion mutations retained transcriptional activity in the presence of DAF-12 and Δ4-DA, fragment 4 produced the highest fold induction, and its removal substantially reduced activity (Fig. 1B). This fragment contained two pairs of DAF-12 response elements (REs), as described by Shostak (14). In the full-length promoter context, mutation of one RE pair in mir-241p and two REs in mir-84p led to a decrease in activation of about seven- and three-fold, respectively. Gel mobility shift assays confirmed in vitro binding of DAF-12 to these REs, whereas mutated versions abolished the interaction (Fig. 1C).

Fig. 1.

DAF-12 and dafachronic acid (Δ4-DA) activate microRNA promoters in vitro. (A) Activation of microRNA promoters in HEK293T cells. Promoters of let-7 homologs, mir-84 and mir-241, were strongly activated in the presence of DAF-12 and 400 nM Δ4-DA, whereas other microRNAs were relatively unaffected. Luciferase assays were measured in triplicate and are shown with SD. EtOH, ethanol vehicle control; ptk, empty luciferase vector. (B) Mutation analysis of mir-241p and mir-84p reveals DAF-12–Δ4-DA–activating elements. Deletion analysis of the mir-241p showed that the highest relative induction occurs with fragment 4, which contains four DAF-12 REs, 241a, b, c, and d. Deletion or point mutation of 241ab elements (in red) abolished activation (blue bars). Similarly, point mutation of DAF-12 REs in mir-84p, 84a and b, reduced expression (red bars). (C) Gel mobility shift assay of DAF-12 and mir-241p. 32P-radiolabeled oligomers containing the WT 241b element were shifted (s) by nuclear extracts expressing DAF-12::FLAG and supershifted (ss) in the presence of FLAG-specific antibody. Unlabeled WT 241b-oligomer prevented the shift, but addition of an oligomer with a point mutation did not.

To examine microRNA expression in vivo, we generated transgenic worms containing the microRNA promoters fused to green fluorescent protein (GFP). Wild-type (WT) worms containing mir-241p::GFP gave a broad expression pattern as described (15). However, daf-12 nulls showed decreased expression, most noticeably in the excretory cells (exc), as well as in muscles, pharynx, and intestine, although neuronal expression seemed less affected, which reveals the tissue selectivity of daf-12 regulation (Fig. 2, A to D). Null mutants for cytochrome P-450 (CYP450) daf-9(dh6) fail to produce dafachronic acids, and DAF-12 without the ligand interacts with its co-repressor DIN-1–SHARP to repress transcriptional targets; together they cause constitutive developmental arrest and dauer larvae formation (1618). In these hormone-deficient larvae, mir-241p::GFP expression was tightly repressed in most tissues (Fig. 2E). Supplementation with Δ4-DA rescued this arrest and brought mir-241p::GFP expression back to WT levels (Fig. 2F). Tight repression was also relieved in daf-9(dh6);din-1(dh149) double-null mutants lacking the corepressor, with mir-241p::GFP expression levels similar to daf-12 nulls (Fig. 2, G and H). Unlike daf-12 nulls, however, Δ4-DA supplementation of daf-9;din-1 worms restored mir-241p::GFP expression back to WT levels. Point mutation of all four daf-12-REs in mir-241p resulted in the same weak expression level in WT, daf-12 null with ligand, as well as daf-9 null, and daf-9;din-1 doubles with or without ligand (Fig. 2, I to K, and fig. S1), which revealed that these REs mediate both activation and repression. Thus, DAF-12 with the ligand works through its REs to mildly activate mir-241p in some tissues, whereas DAF-12 without the ligand, together with DIN-1, tightly represses expression in nearly all tissues.

Fig. 2.

DAF-12 and Δ4-DA regulate microRNA promoters in vivo. (A to K) mir-241p::GFP. Images show representative L3 animals, with indicated cell types (white arrowheads and exc, excretory cell; outlined arrowheads, neu, neuron; mus, muscle; int, intestine; ph, pharynx). Bar graphs alongside the images quantify the percentage of worms with excretory cell GFP expression as either strong (green), weak (yellow), or off (red) (two independent experiments, left and right, n = 10 animals each). For mir-241p::GFP expression level in worms grown without ligand (EtOH) or with ligand (Δ4-DA). (A and B) WT (N2-type). (C) Expression was decreased in daf-12(rh61rh411)–NHR null, (E) strongly repressed in daf-9(dh6)–CYP450 null, or (G) not activated in daf-9(dh6)–CYP450;din-1(dh149)–SHARP double null, but (F and H) was rescued nearly to WT level by growth on 250 nM Δ4-DA. In WT (B) or daf-12 null (D) animals, Δ4-DA had no effect. (I to K) Point mutation of all four DAF-12 REs abolished differences of tested genetic backgrounds or ligand [see also (fig. S1)]. (L to O), mir-84p::GFP. Epidermal seam cells (arrowheads) expressed mir-84p::GFP in (L) WT N2, but (M) not in daf-12 nulls. (N) Seam cell expression was absent in hormone-deficient daf-9;din-1 animals, but (O) restored to nearly WT levels by Δ4-DA supplementation. (Left) percentage of L3 animals showing weak or no seam cell expression (two independent experiments, n = 20 animals each). (P) Relative quantification of microRNAs by QPCR. MicroRNA expression was decreased in daf-12, daf-9;din-1 and repressed in daf-9 mutants. In daf-9 genotypes, expression was Δ4-DA–dependent. QPCR was carried out using the TaqMan system [see (13) for data analysis].

As reported, mir-84p::GFP was expressed in pharynx, somatic gonad, seam cells, vulva cells, and, occasionally, in the intestine (15, 19). Seam cell expression was absent in daf-12 null and daf-9;din-1 null worms, and expression was increased almost to WT levels in daf-9;din-1 animals by addition of Δ4-DA (Fig. 2, L to O), which explains daf-12 heterochronic phenotypes as a failure to activate the microRNAs in temporally patterned tissues. By contrast, expression in other tissues, such as the pharynx, was less affected, which shows again tissue-specific daf-12 regulation (fig. S2). The transcriptional regulation of the let-7s is also reflected in the abundance of total mature microRNAs as measured by TaqMan fluorescence-based quantitative real-time polymerase chain reaction (QPCR). daf-12 mutants and daf-9;din-1 animals showed decreased levels compared with WT, whereas daf-9 nulls showed tight repression of let-7 family of microRNAs (Fig. 2P and figs. S3 to S5). As expected, expression in daf-9, daf-9;din1, but not daf-12, mutants was rescued by Δ4-DA.

The dauer signaling pathways work upstream of daf-12 to govern organismal developmental progression and Δ4-DA production. We therefore wanted to see if let-7s expression was affected by dauer constitutive mutant backgrounds of daf-7–TGF-β, daf-2–insulin/IGF-I receptor, and daf-9–CYP450. mir-241p::GFP expression was nearly completely repressed in these dauer larvae, whereas mir-84p::GFP was down-regulated in some tissues, such as the pharynx (Fig. 3, A to D), but consistently up-regulated and more penetrant in others such as the seam (Fig. 3, E to H). Even in reproductively growing L3 larvae, seam cell expression was more penetrant in daf-2(e1368) and daf-7(e1372) mutants than in WT (Fig. 3G). Δ4-DA supplementation largely reversed this effect in daf-7, but not in the daf-2 background (Fig. 3, I to M), which suggests that insulin/IGF and TGF-β signaling distinctly regulate mir-84p expression in a Δ4-DA–independent and Δ4-DA–dependent manner.

Fig. 3.

MicroRNA regulation by dauer signaling pathways. mir-241p::GFP showed high expression in continuously growing WT (A), but low expression in daf-7(e1372) dauer larvae (B). mir-84p::GFP showed high expression in the pharynx of continuously growing WT (C) but low expression in daf-2(e1368) dauers (D). mir-84p::GFP seam cell expression (E) was elevated in daf-2 and daf-9 mutants during dauer stage (F and H) and was even higher in daf-7 mutants during reproductive growth at 20°C (G). Penetrant seam expression was reversed by 500 nM Δ4-DA in daf-7, but not daf-2, during reproductive growth (I to M). Animals were assayed during L3 and/or L3d stages, n > 20. Red bars, ethanol vehicle, green bars, Δ4-DA (SEM).

The zinc finger protein hbl-1 (hunchback) is responsible for L2 proliferative programs of seam cells (11). hbl-1 loss leads to a loss of proliferative programs and, hence, a decreased seam cell number (table S1). let-7s are proposed to inhibit hbl-1, through microRNA-mediated repression of its 3′UTR, because let-7s mutants have both increased seam number and hbl-1::GFP expression at L3 (11, 20). Given that DAF-12 activates let-7s, we examined interactions with hbl-1. Consistent with the notion that hbl-1 inhibition is promoted by NHR signaling, daf-12 mutants and daf-9;din-1 double-mutants have extra seam cells, the latter reversed by Δ4-DA (table S1). Moreover, the penetrant extra seam phenotype of the daf-12(rh61) ligand–binding domain mutant was dependent on functional hbl-1(+), placing daf-12 upstream of hbl-1 by genetic epistasis. Accordingly, we observed consistent up-regulated hypodermal expression of hbl-1p::GFP::hbl-1–3′UTR during L3 in daf-12(rh61), but not in WT (Fig. 4, A and B). This up-regulation was likely due to loss of UTR-mediated repression, as exchange of the hbl-1–3′UTR for a nonrepressed unc-54–3′UTR led to equal hypodermal expression levels in both WT and the daf-12(rh61) background (Fig. 4, C and D).

Fig. 4.

let-7s repression target, hbl-1, is regulated by DAF-12. A GFP-fusion to hbl-1 promoter and 3′UTR was repressed in the hypodermis at mid L3 (28 hours) in WT (A). In the daf-12(rh61) mutant, reporter signal was up-regulated in the hypodermis (arrows) and other tissues (B) (exposure 250 ms). A GFP-fusion to the hbl-1 promoter, containing the unc-54–3′UTR lacked substantial up-regulation in the hypodermis (C and D), although body muscles showed modest reporter up-regulation (D) (exposure time 50 ms). (E) Model for NHR-microRNA signaling cascades (E). In response to favorable environmental signals, activated insulin/IGF and TGF-β pathways induce Δ4-DA biosynthesis through DAF-9–CYP450. (Right) DAF-12 with the ligand activates L3 programs and expression of let-7s and thereby inhibits HBL-1 and genes of L2 programs, which result in developmental progression (E). (Left) During unfavorable conditions, DAF-12 without the ligand, together with DIN-1, repress L3 programs and let-7s, which allows derepression of L2 programs or developmental arrest. Dauer signaling also has Δ4-DA–independent outputs onto microRNAs.

In this work, we show that the NHR DAF-12 directly regulates the let-7 relatives, mir-84 and mir-241, and connects organism-wide commitments to cell intrinsic programs (Fig. 4E). These studies suggest a model whereby, in unfavorable environments, down-regulated insulin/IGF-1 and TGF-β pathways suppress dafachronic acid production and DAF-12 without the ligand together with co-repressor DIN-1 repress microRNA expression in most tissues and specify developmental arrest. Conversely, in favorable environments, stimulation of insulin/IGF-1 and TGF-β growth signaling pathways results in dafachronic acid production. DAF-12 with the ligand activates let-7 homologs, which, in turn, down-regulate their target, hbl-1, and allows L2 to L3 transitions in the hypodermis. The use of this NHR-microRNA–coupled molecular switch to turn off earlier programs to allow for later ones is a function likely to be conserved and may be a paradigm for understanding hormone-dependent developmental progression, stem cell differentiation, maturation, or tumor formation in metazoans. In particular, activation of new programs and inhibition of earlier ones are critical for the fidelity of distinct developmental states, which may be less apparent in more complex animals whose cell lineages are unknown. In fact, DAF-12 itself is down-regulated by let-7 at later stages, which suggests that both feedforward and feedback loops drive transitions (21). In Drosophila melanogaster, the steroid hormone ecdysone and its cognate receptor regulate developmental progression in part via mir-14, but it is not known whether regulation is direct or indirect (22). Our studies also reveal the intricacy of NHR signaling in an organismal context. They give visible evidence that receptors with the ligand can activate their targets, whereas receptors without the ligand can repress them, with vastly different outcomes for progression or arrest. This hormone-dependent modulation of target gene expression around basal transcription mirrors that seen with the vertebrate homolog LXR (23). Finally, DAF-12–NHR regulation of the microRNAs is highly tissue- and stage-specific, implicating other transcription factors, coregulators, and chromatin factors in the control of microRNA expression.

Supporting Online Material

www.sciencemag.org/cgi/content/full/324/5923/95/DC1

Materials and Methods

Figs. S1 to S5

Tables S1 and S2

References

References and Notes

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