DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions Daniel Hochbaum1., Yue Zhang2., Carsten Stuckenholz3, Paul Labhart4, Vassili Alexiadis5, Rene´ Martin6,7, Hans-Joachim Kno¨ lker7, Alfred L. Fisher1* 1 Division of Geriatric Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America, 3 Division of Hematology/Oncology, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 4 Active Motif, Carlsbad, California, United States of America, 5 Biocept, San Diego, California, United States of America, 6 ChiroBlock GmbH, Wolfen, Germany, 7 Department Chemie, Technische Universita¨t Dresden, Dresden, Germany Abstract The nuclear receptor DAF-12 has roles in normal development, the decision to pursue dauer development in unfavorable conditions, and the modulation of adult aging. Despite the biologic importance of DAF-12, target genes for this receptor are largely unknown. To identify DAF-12 targets, we performed chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays. We identified 1,175 genomic regions to be bound in vivo by DAF-12, and these regions are enriched in known DAF-12 binding motifs and act as DAF-12 response elements in transfected cells and in transgenic worms. The DAF-12 target genes near these binding sites include an extensive network of interconnected heterochronic and microRNA genes. We also identify the genes encoding components of the miRISC, which is required for the control of target genes by microRNA, as a target of DAF-12 regulation. During reproductive development, many of these target genes are misregulated in daf-12(0) mutants, but this only infrequently results in developmental phenotypes. In contrast, we and others have found that daf-12 mutations enhance the phenotypes of many miRISC and heterochronic target genes. We also find that environmental fluctuations significantly strengthen the weak heterochronic phenotypes of daf-12 alleles. During diapause, DAF-12 represses the expression of many heterochronic and miRISC target genes, and prior work has demonstrated that dauer formation can suppress the heterochronic phenotypes of many of these target genes in post- dauer development. Together these data are consistent with daf-12 acting to ensure developmental robustness by committing the animal to adult or dauer developmental programs despite variable internal or external conditions. Citation: Hochbaum D, Zhang Y, Stuckenholz C, Labhart P, Alexiadis V, et al. (2011) DAF-12 Regulates a Connected Network of Genes to Ensure Robust Developmental Decisions. PLoS Genet 7(7): e1002179. doi:10.1371/journal.pgen.1002179 Editor: Stuart K. Kim, Stanford University Medical Center, United States of America Received November 26, 2010; Accepted May 23, 2011; Published July 21, 2011 Copyright: 2011 Hochbaum et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grant AG028977 to ALF (http://grants.nih.gov/grants/oer.htm), a pilot project from Pittsburgh Claude D Pepper Older Americans Independence Center P30 AG024827 (http://www.pepper.pitt.edu/), and a pilot grant from the University of Pittsburgh Institute Of Aging (http://www. aging.pitt.edu/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: afisher@pitt.edu . These authors contributed equally to this work. Introduction From bacteria to humans, organisms sense environmental cues and reprogram life history by altering gene expression patterns. In the nematode C. elegans, the nuclear receptor DAF-12 alters life history in response to the environment. During development, when conditions are unfavorable, due to starvation, crowding or high temperature, daf-12 promotes diapause and formation of dauer larvae [1–3]. In favorable conditions, the DAF-12 receptor is activated by a group of steroidal carboxylic acids, termed dafachronic acids (DA) [4–6], produced from cholesterol via a multi-step pathway involving the daf-36 Rieske-like oxygenase and the daf-9 cytochrome P450 enzyme [7–9]. The activation of DAF- 12 by DA leads to bypass of dauer arrest and expression of later larval developmental and reproductive programs [10]. On the other hand, in unfavorable conditions, DA is not produced, and DAF-12 binds to the co-repressor DIN-1 promoting dauer programs [11]. DAF-12 is also required for the normal lifespan of worms [12–14] and for the increased longevity of germline ablated animals [7,15,16]. DAF-12 also alters life history by regulation of the heterochronic circuit. The heterochronic pathway in C. elegans determines specific cell fate programs for each larval stage [17]. Several daf-12 alleles, especially those in phenotypic class 1, have heterochronic phenotypes [10]. These mutants repeat L2 programs instead of progressing to L3 programs, resulting in excessive hypodermal seam cell division in the L3 stage and defects in distal tip cell migration in the L4 stage [1,10]. The heterochronic phenotypes depend on the activity of the co-repressor din-1, as daf-12(rh61);din-1 mutants develop normally [11]. In part, DAF-12 regulates the heterochronic circuit by controlling expression of let-7 family miRNAs [18,19], which are necessary to promote L3 and adult programs through the down-regulation of the heterochronic genes hbl-1 and lin-41 respectively [20,21]. DAF-12 might also affect the heterochronic circuit via dauer formation, as many heterochronic phenotypes are suppressed in post dauer development [22–25]. PLoS Genetics | www.plosgenetics.org 1 July 2011 | Volume 7 | Issue 7 | e1002179