Jankowski Jakub, Lee Hye Kyung, Liu Chengyu, Wilflingseder Julia, Hennighausen Lothar
Section of Genetics and Physiology, Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, 20892, USA.
Transgenic Core, National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, MD 20892, USA.
Res Sq. 2024 Apr 22:rs.3.rs-4188774. doi: 10.21203/rs.3.rs-4188774/v1.
Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.
转录增强子是调控常见基因和组织特异性基因的基因组序列，其破坏可导致人类疾病的发生和发展。α-klotho是一种在肾脏中特异性表达的性别二态性基因，与肾功能障碍密切相关，从小鼠基因组中删除该基因会导致早衰和死亡。然而，α-klotho的性别二态性调控机制尚不清楚。在此，我们利用H3K27ac表观遗传标记和转录因子结合来表征两个候选α-klotho增强子，并通过CRISPR-Cas9基因组工程单独和联合研究它们的功能。我们发现只有远端（E1）而非近端（E2）候选区域构成功能性增强子，双缺失不会导致α-klotho表达进一步降低。E1的活性依赖于增强子内的HNF1b转录因子结合位点。此外，qPCR和RNA测序表明E1控制着α-klotho的性别二态性。尽管α-klotho mRNA大幅减少，但与种系α-klotho基因敲除不同，突变小鼠表现出正常的表型，包括体重、寿命和血清生化指标。最后，只有缺乏E1的雄性小鼠表现出更明显的急性而非慢性肾损伤反应，这表明对单独的α-klotho缺失有显著的潜在适应范围，尤其是在雌性E1基因敲除小鼠中，尽管α-klotho减少了80%以上，但仍保留了肾脏保护作用。
