CrebA regulation of secretory capacity: Genome-wide transcription profiling coupled with in vivo DNA binding studies

DNA binding assays, expression analyses, and binding site mutagenesis revealed that the Drosophila CrebA transcription factor (TF) boosts secretory capacity in the embryonic salivary gland (SG) through direct regulation of secretory pathway component genes (SPCGs). The mammalian orthologues of CrebA, the Creb3L-family of leucine zipper TFs, not only activate SPCG expression in a variety of mammalian tissues but can also activate SPCG expression in Drosophila embryos, suggesting a highly conserved role for this family of proteins in boosting secretory capacity. However, in vivo assays reveal that CrebA binds far more genes than it regulates, and it remains unclear what distinguishes functional binding. It is also unclear if CrebA is the major factor driving SPCG gene expression in all Drosophila embryonic tissues and/or if CrebA also regulates other tissue-specific functions. Thus, we did single cell RNA sequencing (scRNA-seq) of wild-type (WT) and CrebA null embryos to explore the relationship between CrebA binding and gene regulation. We find that CrebA binds the proximal promoters of its targets, that SPCGs are the major class of genes regulated by CrebA across tissues, and that CrebA is sufficient to activate SPCG expression even in cells that do not normally express the protein. A comparison of scRNA-Seq to other methods for capturing regulated transcripts reveals that the different methodologies identify overlapping but distinct sets of CrebA targets. CrebA23w-/TM3, twi-GFP and CrebAR3576/TM3, twi-GFP were crossed to generate CrebA protein-null embryos for processing using the same single-cell RNA-sequencing pipeline previously established in the lab (PENG et al. 2024). Briefly, embryos were collected and aged to capture stages 10-12 of embryonic development (“early organogenesis”) or stages 13-16 (“late organogenesis”). Embryos were dechorionated in a 50% bleach solution and sorted using a COPAS Flow Pilot sorter to isolate GFP- negative (CrebA protein null) embryos. Live embryos were visually inspected under a fluorescent microscope to confirm both stage and absence of GFP-positive individuals. Samples were immediately prepared for single cell isolation and 10X Genomics single-cell RNA sequencing.