Localization of the pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression [ChIP-Seq]

The eukaryotic genome is organized to enable the precise regulation of gene expression required for development. This organization is established during early development when the embryo transitions from a fertilized germ cell to the totipotent zygote. To understand the factors and processes that drive genomic organization, we focused on the pioneer factor GAGA factor (GAF) that is required for early embryonic development in Drosophila. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. We show that this non-uniform distribution is driven by binding to the highly abundant GA-satellite repeats. At GA-repeats, GAF is necessary to form heterochromatin and silence transcription. Thus, GAF is required to establish both active and silent regions. We propose that the foci formation enables GAF to have opposing transcriptional roles within a single nucleus. Our data support a model in which modulation of the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains that are essential for the robust regulation of gene expression. Overall design: ChIP-seq was performed using the anti-GFP antibody (Abcam #abcam290) on 2-3 hr AEL embryos from mothers expressing endogenously tagged sfGFP-GAF (sfGFP-GAF/+) or endogenously tagged sfGFP-GAF with the polyQ region of the short isoform deleted (sfGFP-GAFS-delta-PQ/sfGFP-GAFS-delta-PQ). DBD-sfGFP ChIP-seq was performed using the anti-GFP antibody on 2-2.5 hr AEL embryos laid by mothers expressing a truncated version of GAF from a transgene that contained only the DNA-binding domain (310-391aa) tagged with sfGFP. Anti-H3K9me3 ChIP-seq was performed using the anti-H3K9me3 antibody (Active motif #39161) on 2-2.5 hr AEL sfGFP-GAF homozygous controls and GAFdeGradFP embryos that have GAF knocked down. All experiments were done in duplicate, and sequencing was performed on immunoprecipitated samples as well as input controls.