RNAseq analysis of Tox3 dependent gene expression in the mouse cerebellum

Organ development relies on precise transcriptional control, yet how lineage-defining factors like Atoh1/Math1 drive robust gene expression despite weak intrinsic transactivation activity remains unclear. Here, we present a comprehensive atlas profiling 1,904 transcription regulators across organs, uncovering TOX3 as a potential co-activator of Atoh1 in cerebellar granule neuron progenitors. Tox3-deficient mice display severe ataxia and cerebellar hypoplasia, driven by depletion of granule neuron progenitors, diminished Atoh1 expression, and impaired primary cilia. Single-nucleus RNA-seq analyses reveals compromised maintenance of the progenitor pool. TOX3 is also highly expressed in subsets of medulloblastoma, and its deletion reduces cerebellar neoplasia and prolongs survival in a mouse model. Mechanistically, Tox3 and Atoh1 co-occupy and synergistically activate E-boxes in shared target genes by up to 120-fold, including an ultra-conserved E-box downstream of Atoh1. These findings establish Tox3 as a critical Atoh1 co-activator in cerebellar development, tumorigenesis and evolution, and provides a resource for exploring novel transcriptional regulators in progenitor maintenance and organogenesis.