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Science 324 (5923): 95-98

Copyright © 2009 by the American Association for the Advancement of Science

Nuclear Hormone Receptor Regulation of MicroRNAs Controls Developmental Progression

Axel Bethke1,2, Nicole Fielenbach1, Zhu Wang3, David J. Mangelsdorf3, and Adam Antebi1,4*

1 Huffington Center on Aging, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
2 University of Osnabrück, Fachbereich Biologie/Chemie, Barbarastrasse 11, 49069 Osnabrück, Germany.
3 Department of Pharmacology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
4 Max-Planck-Institute for Biology of Ageing, Gleulerstrasse 50a, D-50931 Cologne, Germany.

Figure 1 Fig. 1.. DAF-12 and dafachronic acid ({Delta}4-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 {Delta}4-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–{Delta}4-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. [View Larger Version of this Image (45K GIF file)]

Figure 2 Fig. 2.. DAF-12 and {Delta}4-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 ({Delta}4-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 {Delta}4-DA. In WT (B) or daf-12 null (D) animals, {Delta}4-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 {Delta}4-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 {Delta}4-DA–dependent. QPCR was carried out using the TaqMan system [see (13) for data analysis]. [View Larger Version of this Image (73K GIF file)]

Figure 3 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 {Delta}4-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, {Delta}4-DA (SEM). [View Larger Version of this Image (68K GIF file)]

Figure 4 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 {Delta}4-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 {Delta}4-DA–independent outputs onto microRNAs. [View Larger Version of this Image (73K GIF file)]

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