Mis-splicing of Mdm2 leads to Increased P53-Activity and Craniofacial Defects in a MFDM Eftud2 Mutant Mouse Model
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ABSTRACT:
PROVIDER: PRJNA661049 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12571332_1.fastq.gz | Fastqsanger.gz | |||
| SRR12571332_2.fastq.gz | Fastqsanger.gz | |||
| SRR12571333_1.fastq.gz | Fastqsanger.gz | |||
| SRR12571333_2.fastq.gz | Fastqsanger.gz | |||
| SRR12571334_1.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: EFTUD2, a GTPase and core component of the splicesome, is known to be mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). In this study, we examine the role of this gene in neural crest cells, the precursors of bones and cartilages affected in MFDM patients. Methods: We generated a mutant mouse line with conditional mutation in Eftud2 and used Wnt1-Cre2 to delete it in neural crest cells. Results: We show that homozygous deletion of Eftud2 leads to neural crest cell death and malformations in the brain and craniofacial region of embryos. RNAseq analysis of embryonic heads revealed a significant increase in exon skipping and retained introns in mutants and enriched levels of Mdm2 transcripts lacking exon 3. Mutants also had increased nuclear P53, higher expression of P53-target genes, and increased cell death. Craniofacial development was significantly improved when mutant embryos were treated with Pifithrin-alpha, an inihibitor of P53. Conclusion: We propose that craniofacial defects caused by mutations of EFTUD2 are a result of mis-splicing of Mdm2 and P53-associated cell death. Hence, drugs that reduce P53 activity may help prevent craniofacial defects associated with spliceosomopathies.
2021-09-03 | GSE157356 | GEO
Project description:EFTUD2 is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We generated a mutant mouse line with conditional mutation in Eftud2 and used Wnt1-Cre2 to delete it in neural crest cells. Homozygous deletion of Eftud2 causes brain and craniofacial malformations, affecting the same precursors as in MFDM patients. RNAseq analysis of embryonic heads revealed a significant increase in exon skipping and increased levels of an alternatively spliced Mdm2 transcript lacking exon 3. Exon skipping in Mdm2 was also increased in O9-1 mouse neural crest cells after siRNA knock-down of Eftud2 and in MFDM patient cells. Moreover, we found increased nuclear P53, higher expression of P53-target genes and increased cell death. Finally, overactivation of the P53 pathway in Eftud2 knockdown cells was attenuated by overexpression of non-spliced Mdm2, and craniofacial development was improved when Eftud2-mutant embryos were treated with Pifithrin-α, an inhibitor of P53. Thus, our work indicates that the P53-pathway can be targeted to prevent craniofacial abnormalities and shows a previously unknown role for alternative splicing of Mdm2 in the etiology of MFDM.
| S-EPMC8161524 | biostudies-literature
Project description:Mutations in the Notch1 receptor and delta-like 3 (Dll3) ligand cause global disruptions in axial segmental patterning. Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions. We examined Dll3-Notch1 compound mouse mutants to screen for potential gene interactions. While mice heterozygous at either locus appeared normal, 30% of Dll3-Notch1 double heterozygous animals exhibited localized, stochastic segmental anomalies similar to human congenital vertebral defects. Unexpectedly, double heterozygous mice also displayed statistically significant decreases in mandibular height and elongated maxillary hard palate. Examination of somite-stage embryos and perinatal anatomy and histology did not reveal any organ defects, so we used microarray-based analysis of Dll3 and Notch1 mutant embryos to identify gene targets that may be involved in notch-regulated segmental or craniofacial development. Therefore, Dll3-Notch1 double heterozygous mice model human congenital scoliosis and craniofacial disorders. Keywords: genotype comparison
2007-07-01 | GSE8027 | GEO
Project description:The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common physical properties such as length, splice site strengths and sequence motifs, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.
2020-08-05 | GSE149369 | GEO
Project description:Mutations in the Notch1 receptor and delta-like 3 (Dll3) ligand cause global disruptions in axial segmental patterning. Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions. We examined Dll3-Notch1 compound mouse mutants to screen for potential gene interactions. While mice heterozygous at either locus appeared normal, 30% of Dll3-Notch1 double heterozygous animals exhibited localized, stochastic segmental anomalies similar to human congenital vertebral defects. Unexpectedly, double heterozygous mice also displayed statistically significant decreases in mandibular height and elongated maxillary hard palate. Examination of somite-stage embryos and perinatal anatomy and histology did not reveal any organ defects, so we used microarray-based analysis of Dll3 and Notch1 mutant embryos to identify gene targets that may be involved in notch-regulated segmental or craniofacial development. Therefore, Dll3-Notch1 double heterozygous mice model human congenital scoliosis and craniofacial disorders. Experiment Overall Design: Given the reduced penetrance and stochastic nature of the segmental and craniofacial defects in Dll3-Notch1 double heterozygous animals, we next sought to identify candidate genes that may be down or up-regulated in Dll3 and Notch1 homozygous mutant embryos during development of these structures. We carried out microarray analysis using Affymetrix MOE430 microarrays, comparing 9.5 dpc homozygous embryos in duplicate to littermates with wild-type alleles at both loci, and comparing with heterozygous littermate embryos. Affymetrix analysis software (Microarray Suite 5.0) was used to determine whether gene probes were present, marginal or absent. Probes with present flags in replicates of Dll3, Notch1, or embryos with wild-type alleles at both loci were considered present for further analysis. Altogether, out of 22,690 probe sets on the MOE430A array, we identified 12,820 probes that were present in replicates of at least one genotypic group. To identify genes that were increased or decreased in expression in mutant embryos, we carried out robust multichip average (RMA) normalization of microarray data sets using the RMA module of Genespring GX 7.3 (Agilent). After RMA normalization, housekeeping genes such as Gapdh showed steady expression (probe AFFX-GapdhMur/M32599_M_at, normalized expression 1.00 ± 0.02). As a general indicator of variability between samples, we calculated Pearsonâs correlation coefficients. We found that the correlations between duplicates were moderately high: wild-type embryos (0.623), Dll3 embryos (0.584), Notch1 embryos (0.531).
2008-06-15 | E-GEOD-8027 | biostudies-arrayexpress
Project description:Craniofacial dysmorphisms are among the most common birth defects. Proteasome mutations frequently result in craniofacial dysmorphisms including lower jaw malformations; however, the underlying mechanisms are unknown. Here we use a zebrafish proteasome subunit beta 1 (psmb1) mutant to define the cellular mechanisms underlying proteasome mutation-induced craniofacial dysmorphisms. psmb1 mutants exhibit a flattened ceratohyal and smaller Meckel’s and palatoquadrate cartilages. Ceratohyal flattening is a result of failed chondrocyte convergent extension, accompanied by reduced numbers of chondrocytes in the lower jaw due to defects in chondrocyte differentiation. Morphogenesis of craniofacial muscles and tendons is similarly perturbed. psmb1 mutants lack the hyohyal muscles and craniofacial tendons are shortened and disorganized. We additionally identify a critical period for proteasome function in craniofacial development, specifically during chondrocyte and muscle differentiation. psmb1 overexpression in sox10+ cells of mutant embryos rescued both cartilage and tendon phenotypes but induced only a partial rescue of the muscle phenotype, indicating that psmb1 is required in both tissue-autonomous and non-autonomous fashions during craniofacial development. Overall, our work demonstrates that psmb1 is required for craniofacial cartilage, tendon, and muscle differentiation and morphogenesis.
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Project description:Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a non-canonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA-seq data from colorectal cancer cell lines (HCT116, RKO and SW48) in the presence or absence of DNA damage. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs, only a few circRNAs were induced from the p53-induced genes. Circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2. Knocking down circ-MDM2 with siRNAs that targeted the circ-MDM2 junction and had no effect on linear MDM2 mRNA, resulted in increased basal p53 levels and growth defects in vitro and in vivo. Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced Rb phosphorylation and defects in G1-S progression upon silencing circ-MDM2. Our results reveal the role of a novel circRNA by which the MDM2 locus suppresses p53 levels and cell cycle progression.
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Project description:A majority of craniofacial development occurs early in pregnancy and to fully understand how craniofacial defects arise, it is essential to observe gene expression during this critical time period. To address this, we performed bulk and single-cell RNA-seq on human craniofacial tissue from embryonic development -4 to 8 weeks post conception. To observe how genes are organized in this system, we have constructed co-expression networks from the bulk RNA-seq. Strong disease candidates are likely genes that are co-expressed with many other genes, serving as regulatory hubs within these networks. We have identified 29 modules of co-expressed genes and ~1800 hub genes and found enrichment of craniofacial relevant biology suggesting that these networks could reveal new disease genes and potential targets of previously identified disease genes. We leveraged large functional genomics databases including GTEx and GnomAD to reveal genes that are specifically expressed in craniofacial tissue. We integrate these data with our co-expression networks in order to prioritize the genes in this study. Our analysis revealed 268 novel or underappreciated disease candidate genes.
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