Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.[Supplemental material is available for this article.]There can be little argument that genetic mapping has made a substantial contribution to our understanding of biology. For many years these studies used phenotypically defined markers, such as those used by Morgan in Drosophila and Haldane in mice (Morgan 1911;Haldane et al. 1915). The modern era of genetic analysis was heralded by the recognition that variation in genomic DNA sequence itself could be used as a facile assay for mapping (Botstein et al. 1980). This was initially accomplished using analysis of restriction fragmentlength polymorphisms, which were later replaced by microsatellites and subsequently by single nucleotide polymorphisms (SNPs). Despite the remarkable technological advances, these approaches hold in common with those of Morgan and Haldane the utilization of prespecified markers. Next-generation sequencing (NGS) technology enables simultaneous discovery of very dense sets of informative markers and actual gene mapping in the same experiment. Here, we present a strategy and computational tools to map genes in model organisms using sequencing of pooled samples. The approach can be applied to any model organism with a characterized genome and also to both spontaneous and induced mutants. We demonstrate the utility of the strategy and efficiency of the computational approach by mapping spontaneous and ethylnitrosourea (ENU)-induced developmental mutants in zebrafish and mouse.Large-scale forward mutagenesis screens in zebrafish have been used with success to investigate fundamental developmental processes. While the recent completion of the zebrafish genome has greatly aided in the identification of genes, mapping analyses continue to rely on the use of traditional microsatellite markers. However, the utilization of SNPs for mapping of zebrafish mutants was proposed almost a decade ago (Stickney et al. 2002), large numbers of SNPs have been identified (Guryev et al. 2006;Bradley et al. 2007), and the application of NGS for SNP discovery and mutat...

Edited by Joel M. GottesfeldErythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur

The essential functions of polycomb repressive complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs), but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs, which co-precipitate with PRC1 from chromatin and found candidates that impact polycomb group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen, CAT7, regulates expression and polycomb group binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain that shares high sequence similarity to a non-syntenic zebrafish analog, cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA, enhanced by interference of a PRC1 component, and suppressed by interference of a known PRC1 target gene, demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs and that CAT7/cat7l represents convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci.Cellular specification during development is an intricate process involving many layers of genomic regulation, including gene repression and chromatin modification by the PcG 9 complexes (1). Recent evidence suggests that a diverse class of lncRNAs may tune repression in a cell-type specific manner by initiating or stabilizing binding of PcG complexes to individual gene loci (2). Most of this work focuses on the polycomb repressive complex 2 (PRC2) family of PcG complexes, which methylates lysine 27 of histone H3 (H3K27me3) but whose activity is not sufficient to silence chromatin. The PRC2 family of complexes interacts with thousands of coding and non-coding transcripts in vivo, with varying degrees of specificity in vitro (3-6), making it difficult to discern which specific lncRNAs functionally impact PcG biology via interaction with PRC2.In contrast, the potential for lncRNAs to influence PRC1 function or targeting has been less studied despite the clear mechanistic importance of PRC1 to stable gene silencing. PRC1 family complexes are functionally and compositionally diverse (7,8) (Fig. 1A) and either ubiquitylate histone H2A or compact nucleosomes in vitro. This activity is necessary for stable repression in vivo, presumably limiting access of the transcriptional machinery to DNA (9 -11). The complexes are guided to chromatin by various mechanisms, including binding to H3K27me3 deposited by PRC2 (12) and PRC2-independent mechanisms (13). Recent studies have reported a handful of RNAs that functionally affect gene silencing and might directly impact PRC1 targeting to specific loci (14 -16). Defining additional lncRNAs that affect recruitment or silencing by PRC1 may provide novel insights into the regulation of PcG repression during development. * This work was supported, in whole or in part, by National Institutes of Health Grant RO1-GM043901 (to R. E. K.). The authors declare that they have no conflicts of interest with...