Project description:Mutations in the minor spliceosome components such as RNU4atac, a small nuclear RNA (snRNA), are linked to primary microcephaly. We have reported that in the conditional knockout (cKO) mice for Rnu11, a minor spliceosome snRNA, minor intron splicing defect in minor intron-containing genes (MIGs) regulating cell cycle resulted in cell cycle defect with a concomitant increase in yH2aX+ cells and P53-mediated apoptosis. Trp53 ablation in the Rnu11 cKO mice did not prevent microcephaly. Although RNAseq analysis of the double knockout (dKO) pallium reflected transcriptomic shift towards the control from the cKO. We found elevated minor intron retention and alternative splicing across minor introns in the dKO. Disruption of these minor intron-containing genes (MIGs) resulted in cell cycle defect that was detected earlier and was more severe in the dKO, but with delayed detection of yH2aX+ DNA damage. Thus, P53 might also play a role in causing DNA damage in the developing pallium. In all, our findings further refine our understanding of the role of the minor spliceosome in cortical development and identify MIGs underpinning microcephaly in minor spliceosome-related disease.
Project description:SMPD4 (neutral sphingomyelinase-3/nSMase3) has recently been shown to be a new cause of microcephaly in a cohort of twenty-three pediatric patients. The function of nSMases in brain development and how SMPD4 variants cause human microcephaly and cerebellar hypoplasia was previously unknown.We developed an iPSC model to complement our mouse study. We found iPSC models from human SMPD4 patient and CRISPR/Cas9-induced SMPD4 knockout lines demonstrate a proliferation defect, increased cell death, loss of neural progenitors, and shortened primary cilia. Treatment with exogenous ceramide significantly rescues the cilia defect. SMPD4 patient and knockout cells have altered WNT signaling. We provide evidence that SMPD4 controls brain development by providing ceramide for primary ciliogenesis, suggesting a novel therapeutic strategy for SMPD4 mediated disease.
Project description:Hematopoiesis is maintained by a highly regulated and hierarchical system, whereas aberrant control of hematopoiesis is the underlying cause of severe hematological diseases. Here, we demonstrate the indispensable role of ARID4B in fetal hematopoiesis that recruits Ezh2 to transcriptionally downregulate the expression of KIT during erythroid cell differentiation. Functional analyses reveal that the aberration of Arid4b inhibits fetal hematopoiesis at the multipotent progenitors (MPPs) stage, which reactivates the KIT-Src-family kinase (Src) pathway and leads to pre-mature erythroblast proliferation. The differentiation defect caused by ARID4B aberration could be counteracted by the Src inhibitor PP2 or by KIT knockdown. In summary, we identify ARID4B as a master regulator in the KIT-Src pathway, thus providing a fundamental insight in hematopoiesis and stem cell regulation.
Project description:U4/U6 di-snRNPs were disrupted and singular U4 and U6 snRNPs accumulated in egy mutant embryos, establishing the recycling function of p110 in vivo. Based on microarray analyses, a subset of spliceosome components and splicing-related factors was coordinately upregulated in the egy mutant. This revealed an extensive network of coregulated components of the spliceosome cycle, compensating â albeit inefficiently â for the recycling defect. In contrast, another set of genes, many of them eye- and pancreas-specific, was downregulated in the egy mutant embryos. Experiment Overall Design: Zebrafish earl grey (egy) mutant embryos carry an autosomal recessive defect in the p110-orthologous gene which leads to microcephaly, microphthalmia, underdevelopment of the pharyngeal arches, and thymus hypoplasia by day 8 of development. To characterize the defect on the transcriptional level, egy whole embryos (n>100) were collected and morphologically separated into pools of mutant (mut) and wildtype (wt) sibling embryos. Pools of embryos were collected at 2 time points (3 and 4 days post fertilization) with 2 and 3 biological replicates, resp.. After RNA extraction, labelled cRNA was hybridized onto Affymetrix microarrays.
Project description:Hematopoiesis is maintained by a highly regulated and hierarchical system, whereas aberrant control of hematopoiesis is the underlying cause of severe hematological diseases. Here, we demonstrate the indispensable role of ARID4B in fetal hematopoiesis that recruits Ezh2 to transcriptionally downregulate the expression of KIT during erythroid cell differentiation. Functional analyses reveal that the aberration of Arid4b inhibits fetal hematopoiesis at the multipotent progenitors (MPPs) stage, which reactivates the KIT-Src-family kinase (Src) pathway and leads to pre-mature erythroblast proliferation. The differentiation defect caused by ARID4B aberration could be counteracted by the Src inhibitor PP2 or by KIT knockdown. In summary, we identify ARID4B as a master regulator in the KIT-Src pathway, thus providing a fundamental insight in hematopoiesis and stem cell regulation.
Project description:During early embryonic hematopoiesis, hematopoietic stem/progenitor cells (HSPCs) are considered to develop from hemogenic endothelial cells (HECs) though endothelial to hematopoietic transition (EHT). However, little is known about how EHT is regulated in human. Here, we report that GFI1 plays an essential role in ensuring EHT during hematopoietic differentiation of human embryonic stem cells (hESCs). GFI1 deletion in hESCs showed a complete EHT defect due to a closed chromatin state of hematopoietic genes in HECs. Further analysis revealed that GFI1 directly binds and maintains the open chromatin state of genes important for EHT, such as PI3K signaling. Together, our findings reveal an essential role of GFI1 mediated epigenetic mechanism underlying human EHT during hematopoiesis.
Project description:During early embryonic hematopoiesis, hematopoietic stem/progenitor cells (HSPCs) are considered to develop from hemogenic endothelial cells (HECs) though endothelial to hematopoietic transition (EHT). However, little is known about how EHT is regulated in human. Here, we report that GFI1 plays an essential role in ensuring EHT during hematopoietic differentiation of human embryonic stem cells (hESCs). GFI1 deletion in hESCs showed a complete EHT defect due to a closed chromatin state of hematopoietic genes in HECs. Further analysis revealed that GFI1 directly binds and maintains the open chromatin state of genes important for EHT, such as PI3K signaling. Together, our findings reveal an essential role of GFI1 mediated epigenetic mechanism underlying human EHT during hematopoiesis.
Project description:We performed a targeted NGS using the commercial gene panel design ClearSeq Inherited Disease (Agilent Technologies) to identify the pathogenic sequence variants in a girl presenting an apparent microcephaly with mild dysmorphic facial features, delayed psychomotoric development and central hypotonia.