Abstract
BackgroundThe 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.ResultsA 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.ConclusionsMed12 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0114-0) contains supplementary material, which is available to authorized users.
Highlights
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
Med12 regulates neural stem cell (NSC) gene expression programs linked to adhesion and cell cycle progression To investigate the global contribution of Med12 to NSC biology, we profiled the transcriptomes of mouse NS-5 NSCs following RNA interference (RNAi)-mediated Med12 depletion
The mouse NS-5 (mNS-5) cell line is a clonal embryonic stem (ES) cell-derived adherent neural stem cell line that is self-renewing, genetically stable, and multipotent with the capacity to differentiate into neurons and glia both in vitro and in vivo [43, 44]. mNS-5 NSCs were employed for transcriptome profiling in an effort to facilitate identification of target genes likely to mediate the function of Med12 in neural development
Summary
The RNA polymerase II transcriptional Mediator subunit Med is broadly implicated in vertebrate brain development, and genetic variation in human MED12 is associated with X-linked intellectual disability and neuropsychiatric disorders. Development of the mammalian brain is an intricate and protracted process that initiates with neurulation in the gastrulating embryo and extends postnatally to structural and experiential maturation in the adult. This process involves a highly orchestrated and spatiotemporally restricted series of stages, involving initial neurogenesis followed by neuronal migration, differentiation, synaptogenesis, and establishment of neural connectivity [1,2,3]. An initial pool of primary neural stem cells (NSCs), corresponding to neural tube-derived neuroepithelial cells, undergoes a gradual switch from symmetrical autoreplicative divisions to asymmetrical neurogenic divisions to produce a progressively restricted set of neural progenitors that, in turn, specify the final complement of neuronal subtypes and macroglia that populate individual brain structures [2, 7, 8]. Genetic or environmental perturbations that disrupt physiologic transcription controls can alter NSC fate leading to neurodevelopmental defects
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