Genome binding properties of Zic transcription factors underlie their changing functions during neuronal maturation

Background The Zic family of transcription factors (TFs) promote both proliferation and maturation of cerebellar granule neurons (CGNs), raising the question of how a single, constitutively expressed TF family can support distinct developmental processes. Here we use an integrative experimental and bioinformatic approach to discover the regulatory relationship between Zic TF binding and changing programs of gene transcription during CGN differentiation. Results We first established a bioinformatic pipeline to integrate Zic ChIP-seq data from the developing mouse cerebellum with other genomic datasets from the same tissue. In newborn CGNs, Zic TF binding predominates at active enhancers that are co-bound by developmentally-regulated TFs including Atoh1, whereas in mature CGNs, Zic TF binding consolidates toward promoters where it co-localizes with activity-regulated TFs. We then performed CUT&RUN-seq in differentiating CGNs to define both the time course of developmental shifts in Zic TF binding and their relationship to gene expression. Mapping Zic TF binding sites to genes using chromatin looping, we identified the set of Zic target genes that have altered expression in RNA-seq from Zic1 or Zic2 knockdown CGNs. Conclusion Our data show that Zic TFs are required for both induction and repression of distinct, developmentally regulated target genes through a mechanism that is largely independent of changes in Zic TF binding. We suggest that the differential collaboration of Zic TFs with other TF families underlies the shift in their biological functions across CGN development. ### Competing Interest Statement The authors have declared no competing interest. * BART : Binding Analysis for Regulation of Transcription BP : Biological Process (reference to gene ontology enrichment) CGN : Cerebellar Granule Neuron CRISPR : Clustered Regularly Interspaced Short Palindromic Repeats DIV : Days in Vitro FIMO : Find Individual Motif Occurrences FDR : False discovery rate GO : Gene Ontology HOMER : Hypergeometric Optimization of Motif EnRichment KD : Knockdown RRHO : Rank-Rank Hypergeometric Overlap TF : Transcription Factor ZDD : Zic dependent and developmental regulated