ChIP-seq data from fkh-Gal4::UAS-rib-GFP and sage-Gal4::UAS-rib-GFP mid- through late-stage Drosophila embryos

Transcription factors, which regulate the spatiotemporal patterns of gene expression during organogenesis, often regulate multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape changes during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the abnormal cell/organ shape in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib that control cell and organ shape, we performed ChIP-seq analysis in embryos driving rib expression specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression through transcriptional activation, repression, or both, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites of bound Rib most likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell shape change in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that a critical component of the SG morphogenetic gene network involving Rib is its autoregulation. chromatin from three independent collections of stage 11 – 16 fkh-Gal4::UAS-rib-GFP embryos and three of sage-Gal4::UAS-rib-GFP embryos were cross-linked at room temperature in 1.8% formaldehyde in 2ml of homogenization buffer (60mM KCl, 15mM NaCl, 15mM HEPES {pH7.6}, 4mM MgCl2, 0.5mM DTT, 0.5% Triton X-100 and cOmplete™ protease inhibitor cocktail {1 tablet per 50ml buffer}). The cross-linked material was resuspended in 0.1%SDS and 0.5% N-lauroylsarcosine in 0.5ml lysis buffer (140mM NaCl, 15mM HEPES {pH 7.6}, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 1% Triton X-100, 0.5mM DTT and cOmplete™ protease inhibitor cocktail {1 tablet per 50 ml buffer}). Chromatin was sonicated thrice at 4°C using the Sonic Dismembrator Model 100 (Fisher Scientific) under the following conditions: power setting 3, 20s ON, 20s OFF. Immediately after sonication, the chromatin extract was stored at -80°C before it was used for immunoprecipitation. Immunoprecipitations were performed using a polyclonal goat anti-GFP antibody (a gift from Kevin White). Immunoprecipitated DNA was prepared for Illumina sequencing using the TruSeq ChIP Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2000 (50 bp reads) according to the manufacturer’s standard protocols. Library preparation and Illumina sequencing were performed at the University of Minnesota Genomics Center.