Gene regulatory mechanisms governed by the ASXL1/HCF1/OGT complex during neurogenesis

Project Summary
Mutation of HCFC1 causes a multiple congenital anomaly syndrome characterized by inborn errors of cobalamin
metabolism, intractable epilepsy, intellectual disability, and motor dysfunction. Despite an implication for HCFC1
in these neurological impairments, a mechanism describing the function of HCFC1 during brain development
has not been completely elucidated. HCFC1 encodes for a transcriptional co-factor protein known to regulate
cellular proliferation of various progenitor cells including neural precursor cells (NPC). NPCs undergo rapid
expansion during early brain development and differentiate into all the major cell types in the brain (i.e. neurons,
glia). To begin to elucidate a putative mechanism for HCFC1 in NPC expansion, we created the Co60 allele
which introduces a loss of function mutation in the zebrafish hcfc1a ortholog. Through immunohistochemical
labeling and cell counts, we demonstrated that heterozygous carriers of the Co60 allele (Co60/+) had increased
proliferation of NPCs. We next used transcriptomics of Co60/+ whole brain homogenates to reveal a 14-fold
increase in the expression of asxl1, a transcription factor critical for cell proliferation and activation of AKT
signaling. We found that inhibition of PI3K, an upstream activator of AKT, in Co60/+ mutants restored asxl1
dependent NPC over proliferation. Moreover, preliminary western blotting and densitometry analysis of our
mutants confirm hyperphosphorylation of AKT (Thr308). We next used chromatin immunoprecipitation to confirm
a direct binding of human HCFC1 to the zebrafish asxl1 promoter. Together, these findings indirectly link for the
first time hcfc1a function and asxl1 expression with AKT activation and NPC proliferation. What remains to be
understood is the level of AKT signaling in isolated NPCs derived from hcfc1a mutants and which AKT
downstream signaling molecules, like mTOR, regulate proliferation. Completion of this study will help to identify
novel molecular pathways that regulate brain development downstream of HCFC1 and pinpoint mechanisms as
to how its dysregulation contributes to neurodevelopmental disorders. During the F99 phase of this award, I seek
to gain working knowledge of techniques in molecular biology that include protein isolation, western
immunoblotting, fluorescence activated cell sorting (FACS), flow cytometry, advanced imaging, and behavioral
neuroscience. Additionally, I aim to refine my skills and obtain professional development in grantsmanship,
manuscript review and preparation, build and maintain my science network, and finalize my dissertation
research.

Grant Number: 5K00NS125690-04
NIH Institute/Center: NIH
Principal Investigator: Victoria Castro