Mechanisms of Zic2 function in zebrafish.

PROJECT SUMMARY Holoprosencephaly (HPE) is a congenital malformation of the cerebral cortex linked to genetic disruption in Hedgehog (Hh) signaling. Hh signaling acts early in the developing forebrain (future cerebral cortex) to control transcription of key target genes by zinc-finger transcription factors GLIs. ZIC2, a transcription factor with homology to GLIs, is also linked to HPE. Our work in zebrafish showed that ZIC2 homologs zic2a and zic2b pattern the forebrain by modulating Hh signaling, but the mechanism of this regulatory relationship is poorly understood. Recently, Zic2 and Glis were shown to function as co-factors of the nucleosome remodeling and histone deacetylase complex (NuRD) in mammalian embryonic stem cells. Our preliminary evidence shows that zic2a and hdac1 interact genetically in zebrafish. These data have led us to hypothesize that Zic2 regulates the transcriptional readout of Hh signaling in the forebrain primordium through regulation of chromatin dynamics. To test this hypothesis, we will ask if Zic2 controls Hh target gene transcription by modulating histone dynamics and if it physically interacts with NuRD in the zebrafish forebrain. We have designed an innovative strategy that combines in vivo genomics and proteomics with unique transgenic zebrafish lines to answer these questions at the stage of development when the forebrain is most vulnerable to disruptions that result in HPE. Aim 1: Identify candidate Zic2 targets in the developing forebrain using CUT&RUN and RNAseq. No direct transcriptional targets of zic2 in the developing forebrain have been identified to date. We will use CUT&RUN, recently optimized for use in zebrafish, to map DNA binding sites of Zic2a, and to ask if zic2 function regulates chromatin remodeling and transcription of Hh target genes. To do this efficiently, we will adapt a CUT&RUN modification which relies on transgenic GFP-tagged Zic2a and a GFP nanobody fused to a micrococcal nuclease. Aim 2: Identify protein partners of Zic2a using in vivo proximity labeling. A role for Zic2 in regulating chromatin dynamics, suggested by recent findings in mammals, has not been tested in the context of the embryonic forebrain. We will use proximity-directed biotinylation and mass spectrometry to identify in vivo binding partners of Zic2a in the zebrafish embryo. Completion of these aims will shed light on an important, poorly understood regulatory relationship between Hh signaling and Zic2 in the developing vertebrate forebrain, removing a critical barrier toward deciphering the genetic basis of HPE.