GAGA factor, a positive regulator of global gene expression, modulates transcriptional pausing and organization of upstream nucleosomes

Abstract

Background:Genome-wide studies in higher eukaryotes have revealed the presence of paused RNA polymerase II (RNA-Pol) at about 30-50 bp downstream of the transcription start site of genes involved in developmental control, cell proliferation and intercellular signaling. Promoter-proximal pausing is believed to represent a critical step in transcriptional regulation. GAGA sequence motifs have frequently been found in the upstream region of paused genes in Drosophila, implicating a prevalent binding factor, GAF, in transcriptional pausing.Results: Using newly isolated mutants that retain only ~3 % normal GAF level, we analyzed its impacts on transcriptional regulation in whole animals. We first examined the abundance of three major isoforms of RNA-Pol on Hsp70 during heat shock. By cytogenetic analyses on polytene chromosomes and chromatin immunoprecipitation (ChIP), we show that paused RNA-Pol of Hsp70 is substantially reduced in mutants. Conversely, a global increase in paused RNA-Pol is observed when GAF is over-expressed. Coupled analyses of transcriptome and GAF genomic distribution show that 269 genes enriched for upstream GAF binding are down-regulated in mutants. Interestingly, ~15 % of them encode transcriptional factors, which might control ~2000 additional genes down-regulated in mutants. Further examination of RNA-Pol distribution in GAF targets reveals that a positive correlation exists between promoter-proximal RNA-Pol density and GAF occupancy in WT, but not in mutants. Comparison of nucleosome profiles indicates that nucleosome occupancy is preferentially attenuated by GAF in the upstream region that strongly favors nucleosome assembly. Using a dominant eye phenotype caused by GAF over-expression, we detect significant genetic interactions between GAF and the nucleosome remodeler NURF, the pausing factor NELF, and BAB1 whose binding sites are enriched specifically in genes displaying GAF-dependent pausing. Conclusion: Our results provide direct evidence to support a critical role of GAF in global gene expression, transcriptional pausing and upstream nucleosome organization of a group of genes. By cooperating with factors acting at different levels, GAF orchestrates a series of events from local nucleosome displacement to paused transcription. The use of whole animals containing broad tissue types attests the physiological relevance of this regulatory network.

Collaborative repressive action of the antagonistic ETS transcription factors Pointed and Yan fine-tunes gene expression to confer robustness inDrosophila

“…Furthermore, meta-analysis of genome-wide ChIP datasets revealed frequent overlap between Yan and Pnt occupancy patterns with those of components of the pausing machinery, including Trithorax-like (Trl, also known as GAGA factor) and Bric à brac 1 (Bab1) ( Fig. 5B; Contrino et al, 2012;Tsai et al, 2016). Focusing on eve, the locus bears hallmarks of pausing with high pol II occupancy near the TSS, together with low incidence of H3K4me3 and overlap with the pausing factor Trl ( Fig.…”

“…Focusing on eve, the locus bears hallmarks of pausing with high pol II occupancy near the TSS, together with low incidence of H3K4me3 and overlap with the pausing factor Trl ( Fig. 5C; Contrino et al, 2012;Lee et al, 2008;Fuda et al, 2015;Gaertner et al, 2012;Tsai et al, 2016).…”

Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP

“…The H3-H4p is highly conserved among 12 Drosophila species and contains two GA repeat cis elements (Salzler et al 2013). GA-rich cis elements have been implicated in a variety of nuclear processes in Drosophila, including RNA polymerase II pausing (Tsai et al 2016), zygotic genome activation (Chen et al 2013), three-dimensional genome organization (Quinn et al 2014), and DNA loop formation (Eagen et al 2017). Two known Drosophila zinc finger transcription factors directly interact with GA repeats.…”

Spatially uniform establishment of chromatin accessibility in the earlyDrosophilaembryo

“…While Zelda is clearly an important component of this ubiquitous chromatin opening system, there are almost certainly other players, including the ubiquitously expressed trithorax-like/GAGA Factor (or GAF) which plays an important role in establishing accessibility at promoters [22,51–53] and is likely associated with changes in the nucleo-cytoplasmic ratio [50,54].…”