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Identification and Functional Characterization of a New Splicing Variant of EZH2 in the Central Nervous System.

ABSTRACT: EZH2 plays vital roles in epigenetic regulation, neuronal development and cancer progression. Here a novel EZH2 variant, namely EZH2-X9 (X9 for short) resulting from alternative splicing, was isolated, identified and functionally characterized. X9 was highly expressed in the brains of SD rats, indicating a potentially distinguished role in the central nervous system (CNS). Owing to a transcript profiling, X9 was enriched in multiple brain regions at very early stage of life. Immunostaining validated the presence of the protein form of X9, which was localized similarly with the wild-type form, EZH2-WT. To investigate the functional consequence of X9, genetic intervention was performed in PC-12 cell line, a classic cellular model for neuronal development. It revealed that the depletion of either variant was sufficient to impair neuronal proliferation and differentiation significantly, an evidence that roles of X9 could not be complemented by EZH2-WT. Considering epigenetic regulation, X9 lost the capability to recruit the histone mark H3K27me3, but retained the cooperation with EED, as well as the repressive aspects in governing gene expression. Nonetheless, through profiling the genes affected, it's discovered that EZH2-WT and X9 markedly differed in their regulatory targets, as X9 intended to repress cell cycle- and autophagy-related genes, like GSK and MapILC3. Overall, a novel Ezh2 variant was characterized in the mammal CNS, providing insight with the structural and functional delineation of this key developmental switch, Ezh2.

SUBMITTER: Li D

PROVIDER: S-EPMC6329929 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:It has been widely accepted that mammalian neural stem cells (NSCs) only exist in central nervous system (CNS). Here, we challenge this concept and show that peripheral NSCs (pNSCs) could be isolated out of CNS from mouse embryonic limbs, postnatal tail and adult lung tissues. Derived-pNSCs express multiple NSC-specific markers, exhibit cell morphology, self-renewing capacity, genome-wide transcriptional profile, epigenetic features similar to those of brain control NSCs. Derived-pNSCs can differentiate into astrocytes, electrophysiologically functional neurons, and oligodendrocytes in vitro and in vivo, indicating multipotency. Lineage-tracing results reveal that pNSCs are progeny cells of neural epithelial cells, but not neural crest cells. Our finding of pNSCs out of CNS expands the field of developmental neuroscience and presents an alternative potential strategy for neural regenerative therapy. Generation of pNSCs: To generate pNSCs, embryonic limbs cells, adult lung cells and postnatal tail cells were cultured in simple defined NSC medium: DMEM/F-12 [1:1] with 2% B27 w/o Vitamin A, 1% N2 (both Gibco), 1x Glutmax (Gibco), 1x penicillin-streptomycin (Sigma), supplemented with 10 ng/ml EGF and 10 ng/ml human bFGF (both from Invitrogen). Medium was changed every 24h. After pNSCs clusters were observed, each single cluster colony was manually picked and cultured in 96 well individually. Microarray and data analysis: RNA samples to be analysed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion) that involved synthesis of T7-linked double-stranded cDNA and 12 h of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer’s instructions. After washing as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.

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Identification and Functional Characterization of a New Splicing Variant of EZH2 in the Central Nervous System.

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