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Unbiased transcriptome profiling of mNS-5 (mouse embryonic stem cell-derived neural stem cells) following RNAi-mediated MED12 depletion.

ABSTRACT: The RNA polymerase II transcriptional Mediator subunit MED12 is broadly implicated in vertebrate brain development, and genetic variation in human MED12 is associated with X-linked intellectual disability and neuropsychiatric disorders. Although prior studies have begun to elaborate the functional contribution of MED12 within key neurodevelopmental pathways, a more complete description of MED12 function in the developing nervous system, including the specific biological networks and cellular processes under its regulatory influence, remains to be established. Herein, we sought to clarify the global contribution of MED12 to neural stem cell (NSC) biology through unbiased transcriptome profiling of mouse embryonic stem (ES) cell-derived NSCs following RNAi-mediated MED12 depletion. A total of 240 genes (177 up, 73 down) were differentially expressed in MED12-knockdown versus control mouse NS-5 (mNS-5) NSCs. Gene set enrichment analysis revealed MED12 to be prominently linked with “cell-to-cell interaction” and “cell cycle” networks, and subsequent functional studies confirmed these associations. Targeted depletion of MED12 led to enhanced NSC adhesion and upregulation of cell adhesion genes, including Syndecan 2 (SDC2). Concomitant depletion of both SDC2 and MED12 reversed enhanced cell adhesion triggered by MED12 knockdown alone, confirming that MED12 negatively regulates NSC cell adhesion by suppressing the expression of cell adhesion molecules. MED12-mediated suppression of NSC adhesion is a dynamically regulated process in vitro, enforced in self-renewing NSCs and alleviated during the course of neuronal differentiation. Accordingly, MED12 depletion enhanced adhesion and prolonged survival of mNS-5 NSCs induced to differentiate on gelatin, effects that were bypassed completely by growth on laminin. On the other hand, MED12 depletion in mNS-5 NSCs led to reduced expression of G1/S phase cell cycle regulators and a concordant G1/S phase cell cycle block without evidence of apoptosis, resulting in a severe proliferation defect. These findings suggest that MED12 contributes to the maintenance of NSC identity through a functionally bipartite role in suppression and activation of gene expression programs dedicated to cell adhesion and G1/S phase cell cycle progression, respectively. MED12 may thus contribute to the regulatory apparatus that controls the balance between NSC self-renewal and differentiation, with important implications for MED12-linked neurodevelopmental disorders.

ORGANISM(S): Mus musculus

PROVIDER: GSE80624 | GEO | 2016/05/19

SECONDARY ACCESSION(S): PRJNA319584

REPOSITORIES: GEO

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Project description:Stem cells are a potential key strategy for treating neurodegenerative diseases in which the generation of new neurons is critical. A better understanding of the characteristics and molecular properties of neural stem cells (NSC) and differentiated neurons can help in assessing neuronal maturity and possibly in devising better therapeutic strategies. We have therefore performed an in-depth gene expression profiling study of the C17.2 NSC line and primary neurons (PN) derived from embryonic mouse brains. Microarray analysis revealed a neuron-specific gene expression signature that distinguishes PN from NSCs, with elevated levels of transcripts involved in neuronal functions such as neurite development, axon guidance, in PN. The same comparison revealed decreased levels of multiple cytokine transcripts such as IFN, TNF, TGF, and IL. Among the differentially expressed genes, we found a statistically significant enrichment of genes in the ephrin, neurotrophin, CDK5 and actin pathways which control multiple neuronal-specific functions. Furthermore, genes involved in cell cycle were among the most significantly changed in PN. In order to better understand the role of cell cycle arrest in mediating NSCs differentiation, we blocked the cell cycle of NSCs with Mitomycin C (MMC) and examined cellular morphology and gene expression signatures. Although these MMC-treated NSCs displayed a neuronal morphology and expressed some neuronal differentiation marker genes, their gene expression patterns was very different from primary neurons. We conclude that: 1) Fully differentiated primary neurons display a specific neuronal gene expression signature; 2) cell-cycle block in NSC does not induce the formation of fully differentiated neurons; 3) Cytokines such as IFN, TNF, TGF and IL are part of normal NSC function and/or physiology; 4) Signaling pathways of ephrin, neurotrophin, CDK5 and actin, related to major neuronal features, are dynamically enriched in genes showing changes in expression level. Gene expression profiles in neuronal stem cell, mitomycin-treated neuronal stem cells and primary neuronal cultures were compared to examine cellular morphology and gene expression signatures during neuronal differentiation.

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Unbiased transcriptome profiling of mNS-5 (mouse embryonic stem cell-derived neural stem cells) following RNAi-mediated MED12 depletion.

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