Long Non-coding RNA Kancr Promotes Cortical Neurogenesis By Cis-activating The Long Isoform Of Kdm2b

The cerebral cortex in the central nervous system is the material basis of higher nervous function of the organism.As the functional units of brain,projection neurons is under tightly regulation during its differentiation,but the exact mechanism is unclear.As a consequence of cerebral cortical dysplasia,neurological diseases,such as dementia,autism and schizophrenia were brought out frequently.Long noncoding RNAs(lncRNAs)have gained widespread attention in recent years as a potentially new and crucial layer of biological regulation,such as embryonic stem cell maintenance and differentiation,organ development and diseases genesis.LncRNAs regulate complex biological processes,such as transcription,imprinting,chromatin modification,nuclear transport and cell signal transduction through interacting with many macromolecules,including proteins,DNA and RNA.However,the role of lncRNA on the development of cerebral cortex remains ambiguous.Through the high-throughput RNA-seq,we found thousands of divergent lncRNAs were expressed in a development-specific manner in the mouse cerebral cortex.Further identification of long noncoding RNA Kancr and its adjacent coding gene Kdm2 b revealed that they are expressed in the peak period of cortical neurogenesis in mice.Kdm2 b is a lysine demethylation enzyme that influences many aspects of cellular function involved in development,physiology,and disease.Such as,loss of Kdm2 b is relevant to syndromic intellectual disability.In situ hybridization and reporter mouse line showed the long isoform of Kdm2 b is highly expressed in the newborn neurons and some of the intermediate precursor cells,but expressed at very low levels in mature neurons of cortical plate,suggesting its role in the cortical neurogenesis and projection neural fate specification.Kdm2b-LF lineage tracing experiments further revealed that cells of the Kdm2b-LF lineage are fated to generate upper-layer neurons.In view of the sequence and genome location,Kancr is an evolutionarily conserved long noncoding RNA in mammals.In situ hybridization showed that Kancr and Kdm2b-LF have similar expression patterns,suggesting that Kancr may regulate Kdm2b-LF.To test this hypothesis,RNA fractionation of mouse primary neural progenitor cells were performed and date showed that about One-fifth of Kancr locates in nucleus.In addition,knockdown of Kancr in mouse neuroblastoma cell line Neuro-2a and mouse primary neural progenitor cells both resulted in the down-regulation of Kdm2b-LF on mRNA level.Nuclear run-on assay in Neuro-2a cells further showed that the loss of Kancr function suppresses the transcription of Kdm2b-LF nacent RNA,but has no effect on Kdm2b-SF nacent RNA,suggesting that Kancr positively regulate the transcription of Kdm2b-LF in cis.ChIP and chromosome conformation capture assays indicated that Kancr maintains activated state at the Kdm2b-LF promoter as well as promotes the interaction between the Kdm2b-LF promoter and cis-activating element T5,modulating the relative orientation of T5 at the Kdm2b-LF locus,which active and maintain the transcription of Kdm2b-LF.Moreover,in vivo electroporation in the embryonic mouse central nervous system showed that knockdown of Kancr enhances neural progenitor self-renewal and inhibits cortical projection neuron migration,consistent with the phenotypes caused by KDM2B-LF loss,even rescued by overexpression of KDM2B-LF.When KDM2B-LF was overexpressed in vivo,the self-renewal capacity of cortical neural progenitor cells was severely impaired,while the migration of projection neurons was promoted.On the contrary,Knockdown of Kdm2 b promotes the self-amplification of neural progenitor cells and inhibits neuronal migration.Further rescue experiments showed that KDM2B-LF and Kdm2b-SF are functional redundancy in the cortical neurogenesis.In order to elucidate the molecular mechanism of promoting the differentiation of projection neurons,we constructed a series truncated or substitution mutants of KDM2B-LF.In vivo electroporation in the mouse telencephalon showed that the loss of Kdm2 b function can be rescued by overexpression of KDM2B-LF mutants,including KDM2B-LF-mJmjC,KDM2B-LF-?CXXC,KDM2B-LF-mPHD or KDM2B-LF-?Fbox,but not KDM2B-LF-?LRR,indicating that the function of KDM2B-LF in cortical neurogenesis is dependent on LRR domain.As expected,overexpression of KDM2B-LF-?LRR mutant exerts a dominant negative effect.LRR domain of Kdm2 b has been reported to be essential for reconstitution of the catalytically active polycomb repressive complex 1(PRC1).However,our date shown that overexpression of RING1B(I53A),one of functional defect mutants of PRC1 core components in vivo does not recapitulate phenotypes overexpression of KDM2B-LF-?LRR mutant,implying that the function of KDM2B-LF in cortical neurogenesis dependents on LRR domain,but not involves in PRC1.In this study,we identified the specific expression patterns and functions of an lncRNA Kancr and its target gene Kdm2b-LF in mouse embryonic cortex.Further investigation showing that long non-coding RNA Kancr promotes cortical neurogenesis by cis-activates long isoform of Kdm2 b.Our work will contribute to understanding of the development of the cerebral cortex and the targeted treatment of some nervous system diseases.