Project Summary
Mutations affecting the activity or expression of cell type-specific DNA-binding transcription factors (TFs) are a
common cause for a range of human diseases and congenital disorders. Despite the established relevance of
TF function for human health, the inaccessibility of the developing embryo and the complexity of tissue patterning
in vivo has resulted in significant gaps in our basic molecular understanding on how mutations in TFs result in
organ-specific pathologies. An example for this is haploinsufficiency of OTX2 – an essential homeobox TF
expressed in pluripotent tissues of the early embryo and throughout nervous system development – which results
in several rare, poorly understood congenital disorders that affect neuronal, sensory, and endocrine components
of the central and peripheral nervous system (NS). An assessment of clinical case reports and the published
literature on OTX2 function in animal models let us to hypothesize that NS-specific pathologies downstream of
OTX2 haploinsufficiency are caused by dysregulation of gene loci controlled by neurodevelopmental cis-
regulatory elements and chromatin-associated protein complexes that are uniquely sensitive to OTX2 dosage.
Our long-term goal is to understand how molecular and cellular pathologies caused by OTX2 deficiency in the
nervous system can be effectively reversed. The objective of this proposal is to implement a tractable
experimental platform that combines custom modifications of the endogenous OTX2 locus with the directed
differentiation of human pluripotent stem cells (hPSCs) to identify gene regulatory mechanisms employed by this
TF during NS development. To allow discerning gene regulatory elements such as distal enhancers and
associated gene loci controlled by OTX2 during human NS development we have engineered human pluripotent
stem cells to harbor a multipurpose degron allele for rapid depletion and efficient immunoprecipitation of
endogenous OTX2, which we will leverage to identify OTX2-dependent enhancers during early stages of ex vivo
human NS specification (Aim 1). In parallel, we will engineer a second multipurpose allele that will allow
identification of OTX2 interaction partners by proximity ligation and utilize it to discern OTX2 containing gene
regulatory protein complexes active in hPSCs and derivative NS (Aim 2). Together, our proposed experiments
will provide a workable molecular framework for OTX2-dependent gene regulatory networks in the developing
human nervous system, thereby helping to close important gaps in our understanding of OTX2 function in
physiological and pathological conditions.
