A transcription regulator atlas identifies TOX3 as an Atoh1 coactivator 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 Thymocyte Selection-Associated High Mobility Group Box Family Member 3 (TOX3) as a potential coactivator of Atoh1 in cerebellar granule neuron progenitors (GNPs). <i>Tox3</i>-deficient mice display severe ataxia and cerebellar hypoplasia, driven by depletion of GNPs, diminished <i>Atoh1</i> expression, and impaired primary cilia. Single-nucleus RNA-sequencing analyses reveals compromised maintenance of the progenitor pool. <i>TOX3</i> 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-responsive E-box into a highly active enhancer that drives transcriptional activation by up to 120-fold, including at an ultraconserved E-box downstream of <i>Atoh1</i> itself. Cross-species single-cell comparisons further show an association between <i>Tox3</i> expression and cerebellum expansion during vertebrate evolution. Together, this work supports Tox3 as a critical Atoh1 coactivator 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.