TCF4 Mediates PHF6 Transcriptional Regulation of Neural Stem Cells in the Developing Brain, a Mechanism Disrupted in Börjeson-Forssman-Lehmann Syndrome

Abstract

Intellectual disability (ID) comprises a heterogeneous group of neurodevelopmental disorders characterized by impairments in cognitive function and adaptive behavior. Börjeson Forssman-Lehmann syndrome (BFLS) is a rare X-linked form of ID caused by mutations in plant homeodomain finger protein 6 (PHF6). Individuals with BFLS exhibit moderate-to-severe ID, characteristic craniofacial abnormalities, and seizure susceptibility. Although PHF6 has been implicated in brain development, the mechanisms through which PHF6 deficiency contributes to BFLS pathogenesis remain poorly understood.

Using integrated chromatin immunoprecipitation sequencing (ChIP-seq), transcriptomic analyses, molecular and functional assays in embryonic and adult neural stem cells (NSCs), this thesis identifies transcription factor 4 (TCF4), a basic helix-loop-helix transcription factor essential for brain development, as a previously unrecognized downstream target of PHF6. PHF6 was found to directly occupy the Tcf4 regulatory region, promoting Tcf4 transcription. Consistent with this mechanism, TCF4 expression was significantly reduced in multiple BFLS mouse models. Functional studies demonstrated that Tcf4 knockdown phenocopies PHF6 deficiency by enhancing NSC self-renewal, increasing neurosphere formation, and elevating expression of the stem cell markers Nestin and Sox2. Importantly, restoration of TCF4 expression rescued the abnormal NSC phenotypes observed following PHF6 loss, demonstrating that TCF4 is a major functional mediator of PHF6 during neurogenesis. Furthermore, this regulatory relationship persisted in adult NSCs, indicating that PHF6-dependent regulation of TCF4 contributes to stem cell homeostasis beyond embryonic development.

Together, these findings establish the PHF6-TCF4 signaling axis as a critical transcriptional pathway regulating NSC fate during corticogenesis and provide a mechanistic insight for how PHF6 mutations disrupt neurodevelopment in BFLS. Beyond identifying TCF4 as a key downstream effector of PHF6, this work expands our understanding of the transcriptional networks governing cortical development and provides a foundation for future studies investigating whether targeting the PHF6-TCF4 pathway may represent a therapeutic strategy for BFLS and other TCF4-associated neurodevelopmental disorders.