Assessing the impact of the R252W Charcot-Marie-Tooth disease mutation in MORC2 on HUSH-mediated repression
ABSTRACT: HeLa cells lacking MORC2 generated through CRISPR/Cas9-mediated gene disruption were reconstituted with either wild-type or R252W mutant MORC2, and re-repression of HUSH target genes assessed by RNA-seq Overall design: Total RNA-seq of MORC2 knockout cells, either 1) mock transduced, 2) transduced with lentiviral vector encoding wild-type MORC2 or 3) transduced with lentviral vector encoding R252W MORC2.
Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR-Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in ...[more]
Project description:By comparing wild-type SK-N-SH neuroblastoma cells to cells over-expressing either wild-type or R252W mutant MORC2, the goal of the experiment was to assess the effect of MORC2 over-expression on the transcriptome. Overall design: Total RNA-seq of SK-N-SH cells in triplicate, either 1) mock transduced, 2) transduced with lentiviral vector encoding wild-type MORC2 or 3) transduced with lentviral vector encoding R252W MORC2.
Project description:By comparing HeLa cells expressing V5 epitope-tagged MORC2 constructs, the goal of the experiment was to determine the genome-wide localisation of MORC2 in the presence or absence of the HUSH complex and upon inactivation of the CW domain. The occupancy of V5-MORC2 was also compared to that of endogenous TASOR. Overall design: ChIP-seq analysis of endogenous TASOR and IgG control in wild-type HeLa cells. ChIP-seq analysis of V5 epitope-tagged wild-type MORC2 and the CW domain mutant W505A expressed in MORC2 knockout HeLa cells generated through CRISPR/Cas9-mediated gene disruption. In parallel, ChIP-seq analysis of V5 epitope-tagged MORC2 in HUSH tirple knockout cells was also carried out.
Project description:We have generated mouse models of real CMT1B mutations in the gene encoding for myelin protein zero (P0). One of these mutants, P0S63del is retained in the ER where it elicits an unfolded protein response (UPR). Genetic ablation of the UPR factor CHOP restores the motor capacity in S63del mice. We used microarray to decipher the molecular mechanism undelying the P0S63del neuropathy and the rescue in S63del/Chop null nerves. Sciatic nerves were dissected from WT, S63del, Chop null and S63del/Chop null mice at three different time points: (i) postnatal day 5 (P5) when myelination has just started and only the primary effects of the presence of the mutant protein should be detected; (ii) P28, around the peak of myelination, when all the downstream targets of CHOP should be activated; and (iii) 4 months, to check for secondary effects of the disease and because this was the time-point when the motor and morphological rescue due to the ablation of CHOP were clearly detectable.
Project description:By comparing HeLa cells lacking MORC2 generated through CRISPR/Cas9-mediated gene disruption to wild-type HeLa cells, the goal of the experiment was to determine the effect of loss of MORC2 on the transcriptome. Overall design: Total RNA-seq of three independent knockout HeLa clones lacking MORC2.
Project description:By comparing HeLa cells lacking MORC2 or SETDB1 generated through CRISPR/Cas9-mediated gene disruption to wild-type HeLa cells, the goal of the experiment was to determine the effect of loss of MORC2 on the distribution of the repressive H3K9me3 histone modification. Overall design: H3K9me3 ChIP-seq analysis of wild-type HeLa cells compared to MORC2 knockout and SETDB1 knockout HeLa cells.
Project description:We used the Differential Viral Integration (DIVA) technique to compare chromatin accessibility in wild-type versus MORC2 knockout HeLa cells. Overall design: DIVA analysis of wild-type HeLa cells compared to MORC2 knockout HeLa cells.
Project description:Here, human induced pluripotent stem cells (control-hiPSCs, CMT1A-hiPSCs, and PMP22-hiPSCs) were induced to differentiate to Schwann cells (control-SCs, CMT1A-SCs, and PMP22-SCs) through neural crest stage (control-NCSCs, CMT1A-NCSCs, and PMP22-NCSCs). We sequenced mRNA samples from Schwann cell differentiation of human pluripotent stem cells at 3 different stage to generate the gene expression profiles of these cells. Overall design: 7 samples, including undifferentiated hiPSCs (control-hiPSCs and CMT1A-hiPSCs), freshly isolated p75+/HNK1+ NCSCs (control-NCSCs and CMT1A-NCSCs), and SCs (control-SCs, CMT1A-SCs, and PMP22-SCs) were analyzed.