Department of Genetics and Penn Center for Bioinformatics, University of Pennsylvania, Philadelphia, PA 19104, USA.
Genome Biol. 2009;10(7):R72. doi: 10.1186/gb-2009-10-7-r72. Epub 2009 Jul 1.
Hundreds of proteins modulate neurotransmitter release and synaptic plasticity during neuronal development and in response to synaptic activity. The expression of genes in the pre- and post-synaptic neurons is under stringent spatio-temporal control, but the mechanism underlying the neuronal expression of these genes remains largely unknown.
Using unbiased in vivo and in vitro screens, we characterized the cis elements regulating the Rab3A gene, which is expressed abundantly in presynaptic neurons. A set of identified regulatory elements of the Rab3A gene corresponded to the defined Rab3A multi-species conserved elements. In order to identify clusters of enriched transcription factor binding sites, for example, cis-regulatory modules, we analyzed intergenic multi-species conserved elements in the vicinity of nine presynaptic genes, including Rab3A, that are highly and specifically expressed in brain regions. Sixteen transcription factor binding motifs were over-represented in these multi-species conserved elements. Based on a combined occurrence for these enriched motifs, multi-species conserved elements in the vicinity of 107 previously identified presynaptic genes were scored and ranked. We then experimentally validated the scoring strategy by showing that 12 of 16 (75%) high-scoring multi-species conserved elements functioned as neuronal enhancers in a cell-based assay.
This work introduces an integrative strategy of comparative genomics, experimental, and computational approaches to reveal aspects of a regulatory network controlling neuronal-specific expression of genes in presynaptic neurons.
在神经元发育过程中以及对突触活动的反应中,数百种蛋白质调节神经递质的释放和突触可塑性。 突触前和突触后神经元中基因的表达受到严格的时空控制,但这些基因在神经元中的表达机制在很大程度上仍然未知。
我们使用无偏的体内和体外筛选方法,对 Rab3A 基因的顺式元件进行了特征描述,该基因在突触前神经元中大量表达。 Rab3A 基因的一组已鉴定的调节元件与定义的 Rab3A 多物种保守元件相对应。为了识别富含转录因子结合位点的簇,例如顺式调控模块,我们分析了九个突触前基因(包括 Rab3A)附近的种间保守元件,这些基因在脑区中高度且特异性表达。在这些多物种保守元件中,有 16 个转录因子结合基序过表达。基于这些富集基序的组合出现,对 107 个先前鉴定的突触前基因附近的多物种保守元件进行了评分和排序。然后,我们通过实验验证了评分策略,结果表明,在基于细胞的测定中,16 个高评分多物种保守元件中的 12 个(75%)起神经元增强子的作用。
这项工作介绍了一种综合的比较基因组学、实验和计算方法策略,以揭示调节网络控制突触前神经元中基因的神经元特异性表达的某些方面。