Dataset Information

Analysis of temporal lens gene expression microarrays to expand the eye gene discovery tool iSyTE

ABSTRACT: Purpose: While the bioinformatics gene-discovery tool iSyTE (integrated Systems Tool for Eye gene discovery) has proved effective in identifying new cataract-linked genes, it primarily depended on lens-enriched expression of just three wild-type mouse embryonic stages. To increase iSyTE’s efficacy, a broad range of embryonic (E) and postnatal (P) lens microarray datasets from wild-type and specific gene-perturbation mutant mice were analyzed and a new web-interface was developed for their effective access and downstream-analysis. Methods: Five new mouse lens microarrays were generated and analyzed with all publicly available Affymetrix and Illumina lens datasets representing the stages E9.5, E10.25, E10.5, E11.5, E12.5, E15.5, E16.5, E17.5, E19.5, P0, P2, P4, P8, P12, P20, P28, P30, P42, P52, P56, P60 and mouse mutants for the genes: Brg1, CBP:p300, E2f1:E2f2:E2f3, Foxe3, Hsf4, Klf4, Mafg:Mafk, Notch2, Pax6, Sparc, Tdrd7. Results: The integrated lens microarray analysis accurately reflects established gene expression patterns and defines a new lens-signature gene-set for embryonic and postnatal stages that includes several novel candidates namely, Grifin, Ogn, Fabp5, Mboat1, Tmem40, Dhx32, Aldoc, Pgam2, Hmgn3, Mocs2, Gprc5b, Gstm1, Npl, and Zbtb8b. Combined analyses of 32 developmental/genetic data-conditions identifies several new transcription and signaling factors that potentially function in lens development and/or homeostasis. Conclusions: The updated iSyTE web-interface: (1) comprehensively informs on normal lens developmental and cataract-associated transcriptome dynamics and identifies new high-priority candidates for these processes, and (2) offers user-friendly visualization.

ORGANISM(S): Mus musculus

PROVIDER: GSE100136 | GEO | 2017/09/21

SECONDARY ACCESSION(S): PRJNA390783

REPOSITORIES: GEO

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Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants

Project description:Mutations of the RNA-granule component TDRD7 (OMIM: 611258) cause pediatric cataract in humans. Here, we applied an integrated approach to elucidate the molecular pathology of cataract in Tdrd7 targeted-knockout (Tdrd7-/-) mice. Tdrd7-/- animals precipitously develop lens fiber cell abnormalities early in life, suggesting a global-level breakdown/mis-regulation of key cellular processes. High-throughput RNA-sequencing followed by iSyTE-integrated bioinformatics-based analysis identified the molecular chaperone and cytoskeletal-modulator, HSPB1 (HSP27), among the high-priority down-regulated candidates in Tdrd7-/- lens. Moreover, a protein 2-D fluorescence difference gel electrophoresis-coupled mass spectrometry screen also identified HSPB1 to be reduced in Tdrd7-/- lens, offering independent support for focusing efforts on this factor to explain Tdrd7-/- cataract. Reduction of HSPB1 preceded lens morphological abnormalities, suggesting that cytoskeletal defects underlie the Tdrd7-/- cataract phenotype. In agreement, scanning electron microscopy revealed abnormal fiber cell membrane protrusions in Tdrd7-/- lenses. Significantly, abnormal F-actin staining was detected specifically in Tdrd7-/- fiber cells that exhibit nuclear degradation, thereby revealing that there are distinct mechanisms based on pre- or post-nuclear degradation differentiation stage for F-actin cytoskeletal maintenance in fiber cells. Further, RNA-immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA-imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in a specific pre-nuclear degradation area of differentiating fiber cells, indicating that TDRD7-ribonucleoprotein complexes are necessary for controlling optimal levels of key factors in lens development. Together, these data uncover a novel role for TDRD7 in regulating elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

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Analysis of temporal lens gene expression microarrays to expand the eye gene discovery tool iSyTE

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