The release and nuclear translocation of the intracellular domain of Notch receptor (NICD) is the prerequisite for Notch signaling-mediated transcriptional activation. NICD is subjected to various posttranslational modifications including ubiquitination. Here, we surprisingly found that NUMB proteins stabilize the intracellular domain of NOTCH1 receptor (N1ICD) by regulating the ubiquitin–proteasome machinery, which is independent of NUMB’s role in modulating endocytosis. BAP1, a deubiquitinating enzyme (DUB), was further identified as a positive N1ICD regulator, and NUMB facilitates the association between N1ICD and BAP1 to stabilize N1ICD. Intriguingly, BAP1 stabilizes N1ICD independent of its DUB activity but relying on the BRCA1-inhibiting function. BAP1 strengthens Notch signaling and maintains stem-like properties of cortical neural progenitor cells. Thus, NUMB enhances Notch signaling by regulating the ubiquitinating activity of the BAP1–BRCA1 complex.
The mechanisms underlying spatial and temporal control of cortical neurogenesis of the brain are largely elusive. Long non-coding RNAs (lncRNAs) have emerged as essential cell fate regulators. Here we found LncKdm2b (also known as Kancr), a lncRNA divergently transcribed from a bidirectional promoter of Kdm2b, is transiently expressed during early differentiation of cortical projection neurons. Interestingly, Kdm2b's transcription is positively regulated in cis by LncKdm2b, which has intrinsic-activating function and facilitates a permissive chromatin environment at the Kdm2b's promoter by associating with hnRNPAB. Lineage tracing experiments and phenotypic analyses indicated LncKdm2b and Kdm2b are crucial in proper differentiation and migration of cortical projection neurons. These observations unveiled a lncRNA-dependent machinery in regulating cortical neuronal differentiation.
In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc.
13The mechanisms underlying spatial and temporal control of cortical neurogenesis of the 14 brain are largely elusive. Long non-coding RNAs (lncRNAs) have emerged as essential 15 cell fate regulators. Here we found LncKdm2b (also known as Kancr), a lncRNA 16 divergently transcribed from a bidirectional promoter of Kdm2b, is transiently expressed 17 during early differentiation of cortical projection neurons. Interestingly, Kdm2b's 18 transcription is positively regulated in cis by LncKdm2b, which has intrinsic-activating 19 function and facilitates a permissive chromatin environment at the Kdm2b's promoter by 20 associating with hnRNPAB. Lineage tracing experiments and phenotypic analyses 21 indicated LncKdm2b and Kdm2b are crucial in proper differentiation and migration of 22 cortical projection neurons. Moreover, KDM2B exerts its role relying on its leucine-rich 23 repeats (LRR) but independent of its PRC1-related function. These observations unveiled 24 a lncRNA-dependent machinery in regulating cortical neuronal differentiation.Recent studies indicate a few long non-coding RNAs could be essential cell fate regulators 50 in development (Grote et al., 2013; Klattenhoff et al., 2013). Long non-coding RNAs 51 3 (lncRNAs), defined as RNAs longer than 200 nucleotides but lacking protein-coding 52 potentials, are abundant in brain and display cell-type-, and developmental stage-specific 53 expression patterns compared to protein-coding transcripts (Aprea et al., 2013; Belgard 54 et al., 2011; Mercer et al., 2010; Molyneaux et al., 2015). LncRNAs may regulate gene 55 transcription by recruiting transcription factors, RNA-binding proteins and chromatin-56 remodeling machineries to the site of transcription and creating a locus-specific 57 environment (Lin et al., 2014; Ng et al., 2013; Wang et al., 2015). LncRNAs are often 58 derived from bidirectional promoters, such that initiating Pol II can generate divergently-59 oriented transcripts simultaneously, the sense (protein-coding mRNA) direction or the 60 upstream-antisense (divergent non-coding) direction, with these mRNA/divergent lncRNA 61 pairs having coordinated expression (Lepoivre et al., 2013; Scruggs and Adelman, 2015; 62 Sigova et al., 2013). Moreover, the transcription of divergent lncRNAs could affect the 63 expression of their neighboring protein-coding transcripts in cis (Luo et al., 2016; Ørom et 64 al., 2010). Anti-sense promoters could serve as platforms for transcription factor (TF) 65 binding and facilitate establishment of proper chromatin architecture to regulate sense-66 strand mRNA expression (Scruggs and Adelman, 2015; Scruggs et al., 2015). Although 67 divergent lncRNAs are prevalent in both embryonic and adult nervous system, only a few 68 functional divergent lncRNAs have been characterized, including roles of Emx2OS and in 69 regulating the expressions of their neighboring protein-coding transcripts Emx2, an 70 essential cortical RGPC gene (Noonan et al., 2003; Spigoni et al., 2010). Furthermore, 71 these are largely in vitro stu...
The hippocampus plays major roles in learning and memory. Similar to other parts of the brain, the development of hippocampus requires precise coordination of patterning, cell proliferation, differentiation, and migration, with both cell-intrinsic and extrinsic mechanisms involved. Here we genetically removed the chromatin-association capability of KDM2B - a key component of the variant Polycomb repressive complex 1 (PRC1) - in the progenitors of developing dorsal telencephalon (Kdm2bΔCxxC) to surprisingly discover that the size of Kdm2bΔCxxC hippocampus, particularly the dentate gyrus, became drastically smaller with disorganized cellular components and structure. Kdm2bΔCxxC mice displayed prominent defects in spatial memory, motor learning and fear conditioning. The differentiation trajectory of the developing Kdm2bΔCxxC hippocampus was greatly delayed, with a significant amount of TBR2-expressing intermediate progenitors stuck along the migratory/differentiation path. Transcriptome and chromatin immunoprecipitation studies of neonatal hippocampi and their progenitors indicated that genes implicated in stemness maintenance, especially components of canonical Wnt signaling, could not be properly silenced by PRC1 and PRC2. Activating the Wnt signaling disturbed hippocampal neurogenesis, recapitulating the effect of KDM2B loss. Together, we unveiled a previously unappreciated gene repressive program mediated by KDM2B that controls progressive fate specifications and cell migration, hence morphogenesis of hippocampus during development.
The hippocampus plays major roles in learning and memory, and its formation requires precise coordination of patterning, cell proliferation, differentiation, and migration. Here we removed the chromatin-association capability of KDM2B in the progenitors of developing dorsal telencephalon (Kdm2b∆CxxC) to discover that Kdm2b∆CxxC hippocampus, particularly the dentate gyrus, became drastically smaller with disorganized cellular components and structure. Kdm2b∆CxxC mice displayed prominent defects in spatial memory, motor learning and fear conditioning, resembling patients with KDM2B mutations. The migration and differentiation of neural progenitor cells was greatly impeded in the developing Kdm2b∆CxxC hippocampus. Mechanism studies revealed that Wnt signaling genes in developing Kdm2b∆CxxC hippocampi were de-repressed due to reduced enrichment of repressive histone marks by polycomb repressive complexes. Activating the Wnt signaling disturbed hippocampal neurogenesis, recapitulating the effect of KDM2B loss. Together, we unveiled a previously unappreciated gene repressive program mediated by KDM2B that controls progressive fate specifications and cell migration, hence morphogenesis of hippocampus.
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