title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei Taiwan, ROC ; Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
authors.forename: Shih-Ying
authors.initials: SY
authors.lastname: Tsai
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei Taiwan, ROC.
authors.forename: Yuh-Long
authors.initials: YL
authors.lastname: Chang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei Taiwan, ROC.
authors.forename: Krishna B S
authors.initials: KB
authors.lastname: Swamy
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei Taiwan, ROC.
authors.forename: Ruei-Lin
authors.initials: RL
authors.lastname: Chiang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei Taiwan, ROC.
authors.forename: Der-Hwa
authors.identifier: 0000-0003-2679-0867
authors.initials: DH
authors.lastname: Huang
keywords: GAGA factor; Nucleosome; RNA polymerase II; Transcription pausing