# A transcription regulator atlas identifies TOX3 as an Atoh1 coactivator in cerebellar development and tumorigenesis

## TL;DR

This study identifies TOX3 as a coactivator of Atoh1 in cerebellar development and cancer, using a large-scale transcription regulator atlas in mice.

## Contribution

TOX3 is newly identified as a coactivator of Atoh1, revealing a mechanism for robust gene activation in cerebellar development and tumorigenesis.

## Key findings

- Tox3 loss disrupts cerebellar development and reduces medulloblastoma tumor formation in mice.
- TOX3 and Atoh1 synergistically activate genes by converting weak E-boxes into strong enhancers.
- TOX3 expression correlates with cerebellum expansion in vertebrate evolution.

## Abstract

The formation and function of organs depend on tightly controlled gene networks, yet we lack a comprehensive understanding of the transcription factors that drive organ development and disease. Here, we profile nearly 2,000 transcriptional regulators across multiple mouse organs and identify Tox3 as a key coactivator of Atoh1 in the cerebellum. Loss of Tox3 disrupts cerebellar development, impairs progenitor maintenance, and reduces tumor formation in a mouse model of medulloblastoma. Mechanistically, Tox3 and Atoh1 synergistically activate target genes, revealing how weak transcription factors achieve robust gene expression. These findings uncover a fundamental mechanism of neuronal development and tumorigenesis and provide a resource and strategy for discovering novel transcriptional regulators in organogenesis and tissue function.

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). Tox3-deficient mice display severe ataxia and cerebellar hypoplasia, driven by depletion of GNPs, diminished Atoh1 expression, and impaired primary cilia. Single-nucleus RNA-sequencing 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-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 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 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.