CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations

In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population

A Simple and Efficient CRISPR Technique for Protein Tagging

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells

Human neurons carrying FMR1 unmethylated full mutations require large CGG repeat expansions for silencing and display neurodegenerative features

In Fragile X Syndrome (FXS), unmethylated full mutation (UFM) individuals carry a CGG expansion greater than 200 repeats in the FMR1 gene but lack the disease causing DNA methylation mediated silencing. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, we identify a subset of iPSC clones with large CGG expansions that silence FMR1. Furthermore, we demonstrate that de-novo silencing occurs upon expansion of CGG repeats. The silencing status of FMR1 is stable during neuronal differentiation. This indicates that UFM stem cells do not undergo developmental silencing as described for FXS. We also show that the expression of large unmethylated CGG repeats has direct phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the ability to silence FMR1 but require a higher CGG repeat size than the one described for FXS patients

Cdc42 Regulates Fcγ Receptor-mediated Phagocytosis through the Activation and Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) and Neural-WASP

Cdc42 is a key regulator of the actin cytoskeleton and activator of Wiskott-Aldrich syndrome protein (WASP). Although several studies have separately demonstrated the requirement for both Cdc42 and WASP in Fcγ receptor (FcγR)-mediated phagocytosis, their precise roles in the signal cascade leading to engulfment are still unclear. Reduction of endogenous Cdc42 expression by using RNA-mediated interference (short hairpin RNA [shRNA]) severely impaired the phagocytic capacity of RAW/LR5 macrophages, due to defects in phagocytic cup formation, actin assembly, and pseudopod extension. Addition of wiskostatin, a WASP/neural-WASP (N-WASP) inhibitor showed extensive inhibition of phagocytosis, actin assembly, and cell extension identical to the phenotype seen upon reduction of Cdc42 expression. However, using WASP-deficient bone marrow-derived macrophages or shRNA of WASP or N-WASP indicated a requirement for both WASP and N-WASP in phagocytosis. Cdc42 was necessary for WASP/N-WASP activation, as determined using a conformation-sensitive antibody against WASP/N-WASP and partial restoration of phagocytosis in Cdc42 reduced cells by expression of a constitutively activated WASP. In addition, Cdc42 was required for proper WASP tyrosine phosphorylation, which was also necessary for phagocytosis. These results indicate that Cdc42 is essential for the activation of WASP and N-WASP, leading to actin assembly and phagocytic cup formation by macrophages during FcγR-mediated phagocytosis

The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration

ETOC: Lgl1 interacts directly with NMII-A, inhibiting its filament assembly. It excludes NMII-A from the cell leading edge. Depletion of Lgl1 affects the size and number of focal adhesions, cell polarity, and the rate of migrating cells. These results indicate that Lgl1 regulates the polarity of migrating cells by controlling NMII-A filament assembly