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Creating cell type-specific mutants by enhancer mutagenesis.

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      Abstract

      Cell signaling plays an essential role in development, and knowledge of the identities of the cells sending the signal is critical. This can be a challenge, since signaling pathways and ligands are commonly used at multiple times and in multiple cell types during development. One solution to this problem is to create cell type-specific mutants using CRISPR/Cas9 to mutate enhancers that control different patterns of expression. In this issue ofGenes & Development, Rogers and colleagues (pp. 634-638) provide the first use of this method inDrosophilato solve a long-standing issue in patterning of the embryonic central nervous system.

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      Most cited references 10

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      Systematic localization of common disease-associated variation in regulatory DNA.

      Genome-wide association studies have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by deoxyribonuclease I (DNase I) hypersensitive sites (DHSs). Eighty-eight percent of such DHSs are active during fetal development and are enriched in variants associated with gestational exposure-related phenotypes. We identified distant gene targets for hundreds of variant-containing DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrated tissue-selective enrichment of more weakly disease-associated variants within DHSs and the de novo identification of pathogenic cell types for Crohn's disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease and provide pathogenic insights into diverse disorders.
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        Phenotypic robustness conferred by apparently redundant transcriptional enhancers

        Genes include cis-regulatory regions that contain transcriptional enhancers. Recent reports have shown that developmental genes often possess multiple discrete enhancer modules that drive transcription in similar spatio-temporal patterns1-4: primary enhancers located near the basal promoter and secondary, or “shadow”, enhancers located at more remote positions. It has been hypothesized that the seemingly redundant activity of primary and secondary enhancers contributes to phenotypic robustness1,5. We tested this hypothesis by generating a deficiency that removes two newly-discovered enhancers of shavenbaby (svb), a gene encoding a transcription factor that directs development of larval trichomes6. At optimal temperatures for embryonic development, this deficiency causes minor defects in trichome patterning. In embryos that develop at both low and high extreme temperatures, however, absence of these secondary enhancers leads to extensive loss of trichomes. These temperature-dependent defects can be rescued by a transgene carrying a secondary enhancer driving transcription of the svb cDNA. Finally, removal of one copy of wingless, a gene required for normal trichome patterning7, causes a similar loss of trichomes only in flies lacking the secondary enhancers. These results support the hypothesis that secondary enhancers contribute to phenotypic robustness in the face of environmental and genetic variability.
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          Shadow enhancers foster robustness of Drosophila gastrulation.

          Critical developmental control genes sometimes contain "shadow" enhancers that can be located in remote positions, including the introns of neighboring genes [1]. They nonetheless produce patterns of gene expression that are the same as or similar to those produced by more proximal primary enhancers. It was suggested that shadow enhancers help foster robustness in gene expression in response to environmental or genetic perturbations [2, 3]. We critically tested this hypothesis by employing a combination of bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging methods [2, 4]. Evidence is presented that the snail gene is regulated by a distal shadow enhancer located within a neighboring locus. Removal of the proximal primary enhancer does not significantly perturb snail function, including the repression of neurogenic genes and formation of the ventral furrow during gastrulation at normal temperatures. However, at elevated temperatures, there is sporadic loss of snail expression and coincident disruptions in gastrulation. Similar defects are observed at normal temperatures upon reductions in the levels of Dorsal, a key activator of snail expression (reviewed in [5]). These results suggest that shadow enhancers represent a novel mechanism of canalization whereby complex developmental processes "bring about one definite end-result regardless of minor variations in conditions" [6]. Copyright © 2010 Elsevier Ltd. All rights reserved.
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            Author and article information

            Affiliations
            [1 ] Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
            Journal
            Genes Dev.
            Genes & development
            Cold Spring Harbor Laboratory
            1549-5477
            0890-9369
            April 01 2017
            : 31
            : 7
            28446593
            31/7/629
            10.1101/gad.299586.117
            5411702

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