A transcription regulator atlas identifies TOX3 as an Atoh1 co-activator in cerebellar development and tumorigenesis

Organ development and function are orchestrated by intricate transcriptional circuits. Here, we present a comprehensive atlas profiling 1,904 transcription regulators in the brain, cerebellum, heart, kidney, liver, ovary, and testis of fetal, neonatal and adult mice. Using this dataset, we uncover TOX High Mobility Group Box Family Member 3 (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, how lineage-defining factors such as Atoh1 drive robust gene expression despite weak intrinsic transactivation activity remains unclear. We show that Tox3 physically associates with Atoh1 and co-occupies shared regulatory elements, converting an otherwise weak single-copy Atoh1-reponsive E-box into a highly active enhancer that drives transcriptional activation by up to 120-fold, including at an ultra-conserved E-box downstream of Atoh1 itself. Cross-species single-cell comparisons further show an association between Tox3 expression and cerebellum expansion during vertebrate evolution. Together, this work supports Tox3 as a critical Atoh1 co-activator in cerebellar development, tumorigenesis and evolution, while providing an atlas and screening strategy as a valuable resource for exploring novel transcriptional regulators in organogenesis and tissue physiology.