Neurogenomics Group, Hospital del Mar Research Institute, Parc de Recerca Biomèdica de Barcelona (PRBB), Dr. Aiguader, 88, Barcelona 08003, Catalonia, Spain.
Structural Bioinformatics Lab (GRIB-IMIM), Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Dr. Aiguader, 88, Barcelona 08003 Catalonia, Spain.
Nucleic Acids Res. 2024 Nov 27;52(21):12895-12917. doi: 10.1093/nar/gkae950.
Proneural factors of the basic helix-loop-helix family coordinate neurogenesis and neurodifferentiation. Among them, NEUROG2 and NEUROD2 subsequently act to specify neurons of the glutamatergic lineage. Disruption of these factors, their target genes and binding DNA motifs has been linked to various neuropsychiatric disorders. Proneural factors bind to specific DNA motifs called E-boxes (hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands). While corticogenesis heavily relies on E-box activity, the collaboration of proneural factors on different E-box types and their chromatin remodeling mechanisms remain largely unknown. Here, we conducted a comprehensive analysis using chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for NEUROG2 and NEUROD2, along with time-matched single-cell RNA-seq, ATAC-seq and DNA methylation data from the developing mouse cortex. Our findings show that these factors are highly enriched in transiently active genomic regions during intermediate stages of neuronal differentiation. Although they primarily bind CAG-containing E-boxes, their binding in dynamic regions is notably enriched in CAT-CAT E-boxes (i.e. CATATG, denoted as 5'3' half sites for dimers), which undergo significant DNA demethylation and exhibit the highest levels of evolutionary constraint. Aided by HT-SELEX data reanalysis, structural modeling and DNA footprinting, we propose that these proneural factors exert maximal chromatin remodeling influence during intermediate stages of neurogenesis by binding as homodimers to CAT-CAT motifs. This study provides an in-depth integrative analysis of the dynamic regulation of E-boxes during neuronal development, enhancing our understanding of the mechanisms underlying the binding specificity of critical proneural factors.
碱性螺旋-环-螺旋家族的神经前体细胞因子协调神经发生和神经分化。其中，NEUROG2 和 NEUROD2 随后作用于指定谷氨酸能谱系的神经元。这些因子、它们的靶基因和结合 DNA 基序的破坏与各种神经精神疾病有关。神经前体细胞因子与特定的 DNA 基序结合，称为 E 盒（形式为 CANNTG 的六核苷酸，由相反链上的两个 CAN 半位点组成）。虽然皮质发生强烈依赖于 E 盒活性，但不同 E 盒类型的神经前体细胞因子的协作及其染色质重塑机制在很大程度上仍然未知。在这里，我们使用 NEUROG2 和 NEUROD2 的染色质免疫沉淀测序（ChIP-seq）数据以及来自发育中的小鼠皮质的时间匹配的单细胞 RNA-seq、ATAC-seq 和 DNA 甲基化数据进行了全面分析。我们的研究结果表明，这些因子在神经元分化的中间阶段高度富集在短暂活跃的基因组区域中。尽管它们主要结合含有 CAG 的 E 盒，但它们在动态区域的结合明显富含 CAT-CAT E 盒（即 CATATG，用二聚体的 5'3' 半位点表示），这些 E 盒经历了显著的 DNA 去甲基化，并表现出最高水平的进化约束。借助 HT-SELEX 数据的重新分析、结构建模和 DNA 足迹分析，我们提出这些神经前体细胞因子通过与 CAT-CAT 基序形成同源二聚体，在神经发生的中间阶段发挥最大的染色质重塑作用。这项研究提供了对神经元发育过程中 E 盒动态调节的深入综合分析，增强了我们对关键神经前体细胞因子结合特异性背后机制的理解。
