Establishment of a human induced stem cell line (FUi002-A) from Dravet syndrome patient carrying heterozygous R1525X mutation in SCN1A gene

De novo mutations in SCN1A are the most common cause of Dravet syndrome (DS), an infantile-onset epileptic encephalopathy. In this study, human induced pluripotent stem cell (hiPSC) line FUi002-A was generated from skin fibroblasts obtained from a clinically diagnosed 26-year-old male DS patient with the R1525X variant of the SCN1A gene. Skin fibroblasts were reprogrammed using OriP/EBNA-1 based episomal plasmids expressing reprogramming factors expressing OCT4, SOX2, KLF-4, L-MYC, LIN28, and p53 shRNA. The transgene-free FUi002-A showed pluripotency, three germ layer differentiation capacity in vitro, and a normal karyotype. The resulting hiPSCs were heterozygous for the mutation in the SCN1A gene

OTOF Encodes Multiple Long and Short Isoforms: Genetic Evidence That the Long Ones Underlie Recessive Deafness DFNB9

We have recently reported that OTOF underlies an autosomal recessive form of prelingual sensorineural deafness, DFNB9. The isolated 5-kb cDNA predicted a 1,230 amino acid (aa) C-terminus membrane–anchored cytosolic protein with three C2 domains. This protein belongs to a family of mammalian proteins sharing homology with the Caenorhabditis elegans fer-1. The two other known members of this family, dysferlin and myoferlin, both have six predicted C2 domains. By northern blot analysis, a 7-kb otoferlin mRNA could be detected in the human brain. We isolated the corresponding cDNA, which is expected to encode a 1,977-aa-long form of otoferlin with six C2 domains. A 7-kb cDNA derived from the murine orthologous gene, Otof, was also identified in the inner ear and the brain. The determination of the exon-intron structure of the human and murine genes showed that they are composed of 48 coding exons and extend ∼90 kb and ∼80 kb, respectively. Alternatively spliced transcripts could be detected that predict several long isoforms (six C2 domains) in humans and mice and short isoforms (three C2 domains) only in humans. Primers were designed to explore the first 19 OTOF exons, henceforth permitting exploration of the complete coding sequence of the gene in DFNB9 patients. In a southwestern Indian family affected by DFNB9, a mutation in the acceptor splice site of intron 8 was detected, which demonstrates that the long otoferlin isoforms are required for inner ear function

A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness

International audienceUsing a candidate gene approach, we identified a novel human gene, OTOF, underlying an autosomal recessive, nonsyndromic prelingual deafness, DFNB9. The same nonsense mutation was detected in four unrelated affected families of Lebanese origin. OTOF is the second member of a mammalian gene family related to Caenorhabditis elegans fer-1. It encodes a predicted cytosolic protein (of 1,230 aa) with three C2 domains and a single carboxy-terminal transmembrane domain. The sequence homologies and predicted structure of otoferlin, the protein encoded by OTOF, suggest its involvement in vesicle membrane fusion. In the inner ear, the expression of the orthologous mouse gene, mainly in the sensory hair cells, indicates that such a role could apply to synaptic vesicles

Transcription of the Geminin gene is regulated by a negative-feedback loop

Geminin performs a central function in regulating cellular proliferation and differentiation in development and also in stem cells. Of interest, down-regulation of Geminin induces gene transcription regulated by E2F, indicating that Geminin is involved in regulation of E2F-mediated transcriptional activity. Because transcription of the Geminin gene is reportedly regulated via an E2F-responsive region (E2F-R) located in the first intron, we first used a reporter vector to examine the effect of Geminin on E2F-mediated transcriptional regulation. We found that Geminin transfection suppressed E2F1- and E2F2-mediated transcriptional activation and also mildly suppressed such activity in synergy with E2F5, 6, and 7, suggesting that Geminin constitutes a negative-feedback loop for the Geminin promoter. Of interest, Geminin also suppressed nuclease accessibility, acetylation of histone H3, and trimethylation of histone H3 at lysine 4, which were induced by E2F1 overexpression, and enhanced tri­methylation of histone H3 at lysine 27 and monoubiquitination of histone H2A at lysine 119 in E2F-R. However, Geminin5EQ, which does not interact with Brahma or Brg1, did not suppress accessibility to nuclease digestion or transcription but had an overall dominant-negative effect. These findings suggest that E2F-mediated activation of Geminin transcription is negatively regulated by Geminin through the inhibition of chromatin remodeling

Hoxa9 Transduction Induces Hematopoietic Stem and Progenitor Cell Activity through Direct Down-Regulation of Geminin Protein

<div><p>Hoxb4, a 3′-located Hox gene, enhances hematopoietic stem cell (HSC) activity, while a subset of 5′-located Hox genes is involved in hematopoiesis and leukemogenesis, and some of them are common translocation partners for Nucleoporin 98 (Nup98) in patients with leukemia. Although these Hox gene derivatives are believed to act as transcription regulators, the molecular involvement of the Hox gene derivatives in hematopoiesis and leukemogenesis remains largely elusive. Since we previously showed that Hoxb4 forms a complex with a Roc1-Ddb1-Cul4a ubiquitin ligase core component and functions as an E3 ubiquitin ligase activator for Geminin, we here examined the E3 ubiquitin ligase activities of the 5′-located Hox genes, Hoxa9 and Hoxc13, and Nup98-Hoxa9. Hoxa9 formed a similar complex with the Roc1-Ddb1-Cul4a component to induce ubiquitination of Geminin, but the others did not. Retroviral transduction-mediated overexpression or siRNA-mediated knock-down of Hoxa9 respectively down-regulated or up-regulated Geminin in hematopoietic cells. And Hoxa9 transduction-induced repopulating and clonogenic activities were suppressed by Geminin supertransduction. These findings suggest that Hoxa9 and Hoxb4 differ from Hoxc13 and Nup98-Hoxa9 in their molecular role in hematopoiesis, and that Hoxa9 induces the activity of HSCs and hematopoietic progenitors at least in part through direct down-regulation of Geminin.</p> </div

Effects of Hoxb4, Hoxa9, Hoxc13 and Nup98-Hoxa9 transduction on BM.

<p>Retrovirally transduced cells were subjected to flow cytometry analysis as well as to real-time PCR analysis. (<b>A</b>) Cell populations in each phase of the cell cycle were analyzed by cell sorting analysis. (<b>B</b>) Relative expression of mRNA for Geminin. (<b>C</b>) Geminin protein expression in each phase of the cell cycle. Geomean of the fluorescence intensity was shown. MEP, an empty control vector.</p

Immunoprecipitation analysis of Hox derivatives and effect of Cul4a knock-down on Hoxa9-mediated down-regulation of Geminin protein in HEK-293 cells.

<p>(<b>A</b>) Either of Flag-Hoxa9, Flag-Hoxc13 or Flag-Nup98-Hoxa9 was transfected in HEK-293 cells, and the complex formation with endogenous Cul4a, Ddb1 and Roc1 was examined by means of immunoprecipitation analysis using an anti-Flag antibody. (<b>B</b>) Cul4a siRNA was transfected, and the effect on Hoxa9-mediated down-regulation of Geminin protein was examined. Down-regulation of Cul4a by siRNA was confirmed by immunoblot analysis, and the level was restored by transfection of myc-tagged Cul4a. Endogenous Cul4a was also detected in myc-tagged Cul4a-transfected cells even if cells were pre-treated with siRNA for Cul4a probably because exogenously overexpressed mRNA for Cul4a prevented siRNA from affecting endogenous Cul4a.</p

Effect on clonogenic activity.

<p>(<b>A</b>) Effect of transduction of Hox derivatives on clonogenic activity. Numbers and types of colonies are shown in the upper panel, and close-up photographs of representative colonies in the lower panel. (<b>B</b>) Effect of Geminin supertransduction on Hoxa9 transduction-mediated induction of clonogenic activity. Numbers and types of colonies are shown in the upper panel. Close-up photographs of representative colonies and images obtained with an inverted microscopy are shown in the lower panel. A cell cluster with more than 20 cells was counted as a colony under microscopy. (<b>C</b>) Effect of Geminin supertransduction on Hoxa9 transduction-mediated induction of replating activity. MEP and MPI, empty control vectors.</p