show Abstracthide AbstractPioneer transcription factors initiate changes in gene expression by binding to cis-regulatory modules and promoting chromatin accessibility. It is suggested that this activity is an essential first step in priming these sites for the binding of other factors. Nonetheless, it is unclear whether pioneering activity is always required when these factors are present during development. The highly conserved, essential transcription factor Grainy head was recently identified as a pioneer factor in Drosophila larval imaginal discs, yet Grainy head is present throughout development. To determine whether Grainy head maintains its pioneering role throughout development, we performed ATAC-seq on embryos lacking either maternal or zygotic Grainy head at three stages of development (stage 5, stage 6, and stage 15). Surprisingly, we discovered that neither maternal nor zygotic Grainy head are required for chromatin accessibility in early embryogenesis. Nonetheless, we find that Grainy head binding is correlated with some cis-regulatory modules that gain chromatin accessibility at gastrulation. Intriguingly, later in embryogenesis Grainy head gains a role in promoting chromatin accessibility. Additionally, we determined that Grainy head is able to remain bound to mitotic chromatin, like other pioneer factors, yet this ability is separate from its pioneering activity. Our data reveal that Grainy head pioneering activity is temporally, suggesting that other unidentified pioneer factors compensate for the loss of Grainy head. Overall design: ATAC-seq chromatin accessibility profiling of Drosophila melanogaster embryos either wild-type or lacking maternal or zygotically expressed Grainy head at embryonic stages 5, 6, and 15 Please note that each narrowpeak data file was generated from all replicates and is linked to the corresponding rep1 sample records.