Characterization of Enhancers Active in the Mouse Embryonic Cerebral Cortex Suggests Sox/Pou cis-Regulatory Logics and Heterogeneity of Cortical Progenitors

Abstract

We aimed to identify cis-regulatory elements that control gene expression in progenitors of the cerebral cortex. A list of 975 putative enhancers were retrieved from a ChIP-Seq experiment performed in NS5 mouse stem cells with antibodies to Sox2, Brn2/Pou3f2, or Brn1/Pou3f3. Through a selection pipeline including gene ontology and expression pattern, we reduced the number of candidate enhancer sequences to 20. Ex vivo electroporation of green fluorescent pProtein (GFP) reporter constructs in the telencephalon of mouse embryos showed that 35% of the 20 selected candidate sequences displayed enhancer activity in the developing cortex at E13.5. In silico transcription factor binding site (TFBS) searches and mutagenesis experiments showed that enhancer activity is related to the presence of Sox/Pou TFBS pairs in the sequence. Comparative genomic analyses showed that enhancer activity is not related to the evolutionary conservation of the sequence. Finally, the combination of in utero electroporation of GFP reporter constructs with immunostaining for Tbr2 (basal progenitor marker) and phospho-histoneH3 (mitotic activity marker) demonstrated that each enhancer is specifically active in precise subpopulations of progenitors in the cortical germinal zone, highlighting the heterogeneity of these progenitors in terms of cis-regulation.

Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation

“…In general, transcription factors interact with other transcription factor(s) or co‐factor(s) and exert synergistic transcriptional activation of the target gene. It has been previously reported that four class III POU transcription factors interact with each other in vitro . This study demonstrated that the mutual inducible ability of class III/IV POU transcription factors varied among three NSCLC cell lines and corresponded to the alterations of neuroendocrine marker and proneural cell‐specific transcription factor expression.…”

“…Among them, class III POU genes ( POU3F1 , POU3F2 , POU3F3 , and POU3F4 ) and class IV POU genes ( POU4F1 , POU4F2 , and POU4F3 ) are thought to be indispensable for the proper generation of central nervous systems and/or sensory organs . Furthermore, some class III/IV POU genes are expressed in specific parts of the body and show similar functions . These similarities in the expressional loci and function of classIII/IV POU genes suggest the possibility that not only POU3F2 but also other classIII/IV POU genes may function as key factors for proneural/neuroendocrine differentiation of lung cancers.…”

Recent advances in histogenesis research of lung neuroendocrine cancers: Evidence obtained from functional analyses of primitive neural/neuroendocrine cell‐specific transcription factors

“…POU transcription factors play essential roles in organ development and cellular differentiation. Among the six classes of POU transcription factors, class III/IV POU transcription factors are indispensable for the proper generation of the central nervous systems and sensory organs . Class III POU transcription factors consist of POU3F1, POU3F2, POU3F3, and POU3F4, and class IV POU transcription factors consist of POU4F1, POU4F2, and POU4F3.…”

“…Class III POU transcription factors consist of POU3F1, POU3F2, POU3F3, and POU3F4, and class IV POU transcription factors consist of POU4F1, POU4F2, and POU4F3. The class III POU transcription factors co‐localize and interact each other . We recently reported that all class III/IV POU transcription factors are specifically expressed in SmCC cells, in which they induce the expression of not only neuroendocrine markers (i.e., synaptophysin, neural cell adhesion molecule 1, and chromogranin A) but also proneural transcription factors (i.e., ASCL1 and ND1), and also mutually induce the other class III/IV transcription factors .…”

“…20 Nishikawa et al demonstrated that ASCL1 directly activates miR-375, which functions as a sufficient inducer of neuroendocrine differentiation. 14 Repressor MYBPH TGF-β-mediated epithelial-to-mesenchymal transition 36,37 POU-III/IV POU Activator POU-III/IV, ASCL1, NeuroD1, TTF1, neuroendocrine markers (NCAM1, SYP, CGA) Organigenesis of brain and nervous systems Unknown 35,[38][39][40][41][42][43] POU-III/IV: class III/IV POU; bHLH, basic helix-loop-helix; SYP, synaptophysin; ALDH1A1 aldehyde dehydrogenase 1A1; CIITA, class II transactivator; DKK1, Dikkopf WNT signaling pathway inhibitor 1; CDH1, cadherin 1 (E-cadherin); NCAM1, neural cell adhesion molecule 1; IGFBP2, insulin-like growth factor binding protein 2; CGA, chromogranin A; MYBPH, myosin binding protein H. ASCL1 was also reported to upregulate synaptophysin and increase tumor-initiating capacity via upregulation of the candidate stem cell markers CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1). 15,16 Neuronal differentiation 1 (NeuroD1) NeuroD1 (also known as BETA2, BHF-1, MODY6, NeuroD, or bHLHa3) is also a member of the basic helix-loop-helix family of transcription factors and plays crucial roles in the development of pancreatic islets and the cerebellum, hippocampus, inner ear sensory ganglia, and retinal photoreceptor cells.…”