A short upstream promoter region mediates transcriptional regulation of the mouse <it>doublecortin </it>gene in differentiating neurons

• Main Authors: Baudouin Gregory, Bodson Morgan, Muller Marc, Piens Marie, Plumier Jean-Christophe
• Format: Article
• Language: English
• Published: BMC 2010-05-01
• Series: BMC Neuroscience
• Online Access: http://www.biomedcentral.com/1471-2202/11/64

<p>Abstract</p> <p>Background</p> <p>Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced <it>Dcx </it>expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling <it>Dcx </it>gene expression by studying its transcriptional regulation during neuronal differentiation.</p> <p>Results</p> <p>To determine and analyze important regulatory sequences of the <it>Dcx </it>promoter, we studied a putative regulatory region upstream from the mouse <it>Dcx </it>coding region (<it>pdcx</it>2kb) and several deletions thereof. These different fragments were used <it>in vitro </it>and <it>in vivo </it>to drive reporter gene expression. We demonstrated, using transient expression experiments, that <it>pdcx</it>2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of <it>Dcx </it>promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the <it>Dcx </it>gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of <it>Dcx </it>promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y).</p> <p>Conclusions</p> <p>Studies of <it>Dcx </it>gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the <it>Dcx </it>coding sequence are sufficient to induce specific Dcx expression <it>in vitro</it>: in heterogeneous differentiated neurons from mESC, in primary mouse cerebellar neurons (PND3) and in organotypic slice cultures. Furthermore, a region in the 3'-end region of the <it>Dcx </it>promoter is highly conserved across several species and exerts positive control on <it>Dcx </it>transcriptional activation. Together, these results indicate that the proximal 3'-end region of the mouse <it>Dcx </it>regulatory sequence is essential for <it>Dcx </it>gene expression during differentiation of neuronal precursors.</p>