多重 CRISPRi 揭示小鼠新皮层成熟中 KLF 激活因子与抑制因子的转录切换
Multiplexed CRISPRi reveals a transcriptional switch between KLF activators and repressors in the maturing neocortex
研究整合新生与成熟小鼠新皮层两类锥体神经元的染色质可及性和基因表达数据，预测 Krüppel 样因子（KLF）家族参与出生后表达基因的发育调控。作者用多重 CRISPRi 敲低策略发现，出生后早期发育中表达从激活因子 Klf6、Klf7 转向抑制因子 Klf9、Klf13，形成一个转录切换，先激活、后抑制包括 Tubb2b 和 Dpysl3 在内的一组细胞骨架功能共享靶基因。研究显示该切换受 KLF 旁系同源基因之间的冗余缓冲，而多重 CRISPRi 能够克服并研究这种冗余。
来源：eLife · doi.org
A critical phase of mammalian brain development takes place after birth. Neurons of the mouse neocortex undergo dramatic changes in their morphology, physiology, and synaptic connections during the first postnatal month, while properties of immature neurons, such as the capacity for robust axon outgrowth, are lost. The genetic and epigenetic programs controlling prenatal development are well studied, but our understanding of the transcriptional mechanisms that regulate postnatal neuronal maturation is comparatively lacking. By integrating chromatin accessibility and gene expression data from two subtypes of neocortical pyramidal neurons in the neonatal and maturing brain, we predicted a role for the Krüppel-like factor (KLF) family of transcription factors in the developmental regulation of neonatally expressed genes. Using a multiplexed CRISPR interference (CRISPRi) knockdown strategy, we found that a shift in expression from KLF activators (Klf6, Klf7) to repressors (Klf9, Klf13) during early postnatal development functions as a transcriptional ‘switch’ to first activate, then repress a set of shared targets with cytoskeletal functions including Tubb2b and Dpysl3 . We demonstrate that this switch is buffered by redundancy between KLF paralogs, which our multiplexed CRISPRi strategy is equipped to overcome and study. Our results indicate that competition between activators and repressors within the KLF family regulates a conserved component of the postnatal maturation program that may underlie the loss of intrinsic axon growth in maturing neurons. This could facilitate the transition from axon growth to synaptic refinement required to stabilize mature circuits.