Title: Regulation and function of GAGA factor mitotic retention in the early Drosophila embryo
Abstract: Development depends on the faithful maintenance of gene-expression programs as cells proliferate. During mitosis, chromatin condenses, transcription arrests, and most transcription factors (TFs) dissociate from the genome. It remains unclear how, despite these substantial disruptions, the cell rapidly and robustly re-establishes the transcriptional program to maintain cell identity. A subset of TFs remains associated with mitotic chromosomes, suggesting that they may function to preserve transcriptional memory. Due to technical challenges to specifically disrupt protein activity during mitosis, the functional relevance of mitotic retention of TFs remains unresolved. To address this, we are investigating GAGA factor (GAF), a TF essential for promoting gene expression in the early Drosophila embryo. GAF is retained on chromatin throughout the 13 rapid, synchronous mitotic divisions of early development, but mitotic retention is lost at gastrulation. To understand the impact of mitotic retention on development and gene expression, we engineered two gain-of-function GAF mutants. By extending the C-terminus or mutating phosphoserines in the DNA-binding domain, we increased GAF mitotic retention at both heterochromatin and euchromatin and prolonged its mitotic association into gastrulation. These data may indicate that chromatin affinity promotes mitotic retention and chromatin maturation limits it. Despite dramatic changes in mitotic retention, the GAF phosphoserine mutant occupies largely the same genomic loci as wildtype, and embryos develop normally. However, quantitative image analysis of labeled GAF molecules demonstrated increased mitotic retention results in greater nuclear concentration and altered binding kinetics as compared to wildtype. To determine the impact on gene expression, we compared the transcriptomes of both gain-of-function GAF mutants to wildtype controls. Contrary to the current model suggesting mitotic retention promotes transcription of cell-identity genes, we did not identify increased GAF-target gene expression. Ongoing live imaging experiments will determine the effects of increased GAF mitotic retention on transcriptional reactivation following mitosis. Our findings demonstrate that increased GAF mitotic retention does not influence target gene expression or inhibit development, challenging the prevailing assumption that mitotic retention reflects a function in mitotic memory.
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