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Methylated lncRncr3-MeCP2 limits neuroepithelial progenitor differentiation

ABSTRACT: Long non-coding RNAs are often not conserved and their specific functions remain enigmatic. Our studies identify independent and opposing roles for two neural-specific and differentially expressed non-coding RNAs derived from the same locus: the evolutionarily conserved lncRNA Rncr3 and the embedded microRNA miR124a-1. In this study, we found that Rncr3 is expressed in and necessary for embryonic neuroepithelial progenitor proliferation and survival whereas miR124a expression initiates at the onset of and promotes neuronal differentiation. Exons 2 and 3 are also highly conserved between mouse lncRncr3 and human RNCR3. We used CRISPR/Cas9 gene editing of NE-4C cells, a neuroepithelial progenitor cell line from cerebral vesicles of embryonic day 9 (E9) mouse embryos, to create full-length deletion of the Rncr3-miR124a locus (exons 1-4) and specifically delete conserved exons 2 and 3. Both knock-out (KO) and exon2/3 deletion reduced proliferation and increased apoptosis. To comprehensively evaluate the effects of full-length Rncr3-miR124a KO and exons 2/3 deletion, we performed RNA-seq. Genes whose expression was consistently up- or down-regulated in full-length KO and exons 2/3 deletion clones relative to wildtype were aimed. Gene ontology (GO) functional annotation showed enrichment for negative regulation of cell proliferation; cell fate commitment; cell cycle arrest; negative regulation of apoptotic process. We also found that expression of miR124a encoded in exon 4 is limited by Rncr3 conserved exons 2 and 3. The RNA-seq data showed that the GO Biological Process (BP) enrichment of genes significantly upregulated in exons 2/3 deletion cells but not significantly changed in full-length deletion cells (where miR124a is deleted, and hence more similar to wildtype proliferating NPCs with very low miR124a expression) are associated with neuronal differentiation, consistent with miR124a function in differentiation. Our studies illustrate that two non-coding RNAs, Rncr3 and miR124a, are differentially generated from the same primary transcript and have opposing functions in NPC maintenance versus promotion of neuronal differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE191133 | GEO | 2024/04/16

REPOSITORIES: GEO

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Project description:Background: Altered neuronal development is discussed as underlying pathogenic mechanism of Autism Spectrum Disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during in-vitro neuronal differentiation. We hypothesized a causal relation between this tryptophan related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on differentiation of a neuronal cell line and specifically focused on neuronal morphology, metabolites of the tryptophan pathway and the neurodevelopmental transcriptome. Methods: The gene dosage dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical inhibition, and (iii) complete CRISPR/Cas9 induced knockout (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain. Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with neurite maturation in SH-SY5Y cells. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT associated tryptophan pathway was not affected by KO. At the transcriptome level neuronal processes like “synapse organization” were affected. Differentially regulated genes were enriched for ASD-candidates and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala. Conclusions: In this study QPRT was causally related to in vitro neuronal differentiation and affected the regulation of genes and gene-networks that were previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.

Project description:Signaling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs) by opposing MAP kinase signaling. This contrasts with most cell types where STAT3 signaling induces differentiation. We find that STAT3 binding across the pluripotent genome is dependent upon Brg, the ATPase subunit of a specialized chromatin remodeling complex (esBAF) found in ESCs. Brg is required to establish chromatin accessibility at STAT3 binding targets, in essence preparing these sites to respond to LIF signaling. Moreover, Brg deletion leads to rapid Polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg-activated targets genome-wide, including the target genes of the LIF signaling pathway. Hence, one crucial role of Brg in ESCs involves its ability to potentiate LIF signaling by opposing PcG. Contrary to expectations, Brg also facilitates PcG function at classical PcG target including all four Hox loci, reinforcing their repression in ESCs. These findings reveal that esBAF does not simply antagonize PcG, but rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency. Brg conditional embryonic stem cell (ESC) line was prepared from Brg(lox/lox);Actin-CreER mice that allow conditional deletion of Brg upon addition of 4-hydroxytamoxifen (4OHT, Sigma). Brg conditional ESCs were treated with 4-OHT for 72 hours to delete Brg. Brg protein levels completely abolished only at 48 hours (Brg-KO). RNA from Brg conditional ESCs (WT), Brg-KO ESCs, Brg conditional ESCs starved of LIF (Lif-WD) for 48 hours were extracted using Trizol reagent (Invitrogen), followed by RNeasy kit with DNaseI digestion to eliminate genomic DNA. Purified RNA was processed and hybridized onto Affymetrix Mouse MOE 4.0 3’ expression arrays according to the manufacturer's instructions. ChIP-Seq was used to detect and measure STAT3 occupancy and levels of H3K27me3 in the genome before and after the removal of the SWI/SNF ATPase Brg in mouse ESCs. Total of 4 samples (WT and KO STAT3; WT and KO H3K27me3). Input DNA was sequenced as control.

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Methylated lncRncr3-MeCP2 limits neuroepithelial progenitor differentiation

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