Six1 haploinsufficiency is associated with activation of NF-κB and TNF-related transcriptional signatures in aging mice.

The Six1 (SIX homeobox 1) gene is pivotal in renal and pulmonary development and differentiation. Its dysregulation is implicated in oncogenesis and tumor progression via enhancing cell proliferation and delaying senescence. However, whether or how it functions in the natural aging have not been investigated. To answer this question, we generated Six1 gene knockout mice using CRISPR-Cas9 technology. All Six1 biallelic knockout mice died at birth since the underdeveloped lungs. In Six1+/- mice, the developmental deficiencies in kidneys with vacuolar degeneration and epithelial disruption in renal tubules, as well as hematopoietic interstitial infiltration and lungs with interstitial condensation and alveolar hypoplasia were observed. These developmental deficiencies persist with age and age-dependent phenotypes become more pronounced in Six1+/- mice compared to the wild-type, with upregulation of senescence markers (p16, p53) and senescence-associated secreted factors (e.g., TNF-α, TIMP-2), increased α-SMA expression and collagen deposition, as well as susceptibility to pulmonary fibrosis. Transcriptomic sequencing coupled with bioinformatics analysis indicated that genes with altered expression in Six1+/- mouse lungs showed enrichment in pathways associated with senescence, including the NF-κB and TNF signaling pathways. These transcriptional patterns were also associated with gene sets involved in mitochondrial metabolic processes. Collectively, these findings suggest that Six1 haploinsufficiency is associated with transcriptional signatures linked to aging-related pathways under physiological conditions in mice, providing potential clues for future studies exploring mechanisms of aging.