Sexually dimorphic renal expression ofKlothois directed by a kidney-specific distal enhancer responsive to HNF1b

Abstract

ABSTRACTTranscription 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 ofKlothois not understood. Here, we characterize two candidateKlothoenhancers 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 causingKlothoexpression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism ofKlothoas evidenced by qPCR and RNA- seq. Despite the sharp reduction ofKlothomRNA, unlike germlineKlothoknockouts, 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 isolatedKlotholoss, especially in female E1 knockouts, retaining renoprotection despite over 80%Klothoreduction.