Molecular mechanisms for Krüppel-like factor 13 actions in hippocampal neurons [ChSP-seq]

Krüppel-like factors (KLFs) play key roles in nervous system development and function. Several KLFs are known to promote, and then maintain neural cell differentiation. Our previous work focused on the actions of KLF9 in mouse hippocampal neurons. Here we investigated genomic targets and functions of KLF9’s paralog KLF13, with the goal of understanding how these two closely related transcription factors influence hippocampal cell function, proliferation, survival and regeneration. We engineered the adult mouse hippocampus-derived cell line HT22 to control Klf13 expression with doxycycline. We also generated HT22 Klf13 knock out cells, and we analyzed primary hippocampal cells from wild type and Klf13-/- mice. RNA sequencing showed that KLF13, like KLF9, acts predominantly as a transcriptional repressor in hippocampal neurons and can regulate other Klf genes. Pathway analysis revealed that genes regulated by KLF13 are involved in cell cycle, cell survival, cytoarchitecture regulation, among others. Chromatin-streptavidin sequencing conducted on chromatin isolated from HT22 cells expressing biotinylated KLF13 identified 9506 genomic targets; 79% were located 1 kilobase upstream of transcription start sites. Transfection-reporter assays confirmed that KLF13 can directly regulate transcriptional activity of its target genes. Comparison of the target genes of KLF9 and KLF13 found that they share some functions that were likely present in their common ancestor, but have also acquired distinct functions during evolution. Flow cytometry showed that KLF13 promotes cell cycle progression, and it protects cells from glutamate-induced excitotoxic damage. Taken together, our findings establish novel roles and molecular mechanisms for KLF13 actions in mammalian hippocampal neurons.

Krüppel样因子（Krüppel-like factors, KLFs）在神经系统发育与功能中发挥关键调控作用。已有研究证实，部分KLFs可促进并维持神经细胞的分化过程。我们前期的研究聚焦于KLF9在小鼠海马神经元中的功能与作用机制。本研究针对KLF9的旁系同源基因KLF13的基因组靶标与生物学功能展开探究，旨在阐明这两种亲缘关系紧密的转录因子如何影响海马细胞的功能、增殖、存活与再生过程。

我们通过基因工程手段构建了可经强力霉素（doxycycline）调控Klf13表达的成年小鼠海马来源细胞系HT22；同时生成了Klf13基因敲除的HT22细胞，并对野生型与Klf13基因敲除（Klf13-/-）小鼠的原代海马细胞进行了系统性分析。

RNA测序结果显示，与KLF9类似，KLF13在海马神经元中主要作为转录抑制因子发挥功能，且可调控其他Klf家族基因的表达。通路分析发现，KLF13所调控的基因广泛参与细胞周期、细胞存活、细胞结构调控等多种生物学过程。对表达生物素标记KLF13的HT22细胞开展染色质-链霉亲和素测序，共鉴定得到9506个基因组靶标，其中79%位于转录起始位点上游1千碱基（kb）范围内。转染报告基因实验证实，KLF13可直接调控其靶基因的转录活性。

对比KLF9与KLF13的靶基因谱发现，二者共享部分源自共同祖先的保守功能，但在进化过程中也各自获得了独特的生物学功能。流式细胞术检测结果表明，KLF13可促进细胞周期进程，并能保护细胞免受谷氨酸诱导的兴奋性毒性损伤。

综上，本研究的发现揭示了KLF13在哺乳动物海马神经元中全新的生物学功能与分子调控机制。