This reduced expression supports the idea that this threshold Dorsal concentration required for repression is sensitive to Gro concentration [1, 23]. that, contrary to what has been observed for another Gro family member, Drosophila Gro is probably a dedicated repressor. This analysis also allows us to define a set of high confidence Gro repression targets. Using publically available data regarding the physical and genetic interactions between these targets, we are able to place them in the regulatory network controlling development. Through analysis of chromatin associated pre-mRNA levels at these targets, we find that genes regulated by Gro in the embryo are enriched for characteristics of promoter proximal paused RNA polymerase II. Conclusions Our findings are inconsistent with a one-dimensional distributing model for long-range repression and suggest that Gro-mediated repression must be regulated at a post-recruitment step. They also show that Gro is likely a dedicated repressor that sits at a prominent highly interconnected regulatory hub in the developmental network. Furthermore, our findings suggest a role for RNA polymerase II pausing in Gro-mediated repression. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3589-6) contains supplementary material, which is available to authorized users. embryonic development. We find that Gro associates with chromatin in discrete usually transient peaks often clustered upstream of or within regulated genes in a pattern that is not compatible with a simple distributing model for long-range repression. By combining genome-wide chromatin binding and gene expression analysis, we have also recognized a set of high-confidence Gro targets, allowing more confident positioning of Gro within the developmentally-regulated gene network. These high confidence targets are highly enriched for promoter-proximal paused RNA Mouse monoclonal to HER2. ErbB 2 is a receptor tyrosine kinase of the ErbB 2 family. It is closely related instructure to the epidermal growth factor receptor. ErbB 2 oncoprotein is detectable in a proportion of breast and other adenocarconomas, as well as transitional cell carcinomas. In the case of breast cancer, expression determined by immunohistochemistry has been shown to be associated with poor prognosis. polymerase II (Pol II), suggesting a role for Pol II pausing in Gro-mediated repression. Methods Travel strains Flies were maintained on standard medium at 25?C. UAS-transgenic flies were explained previously [23]. Embryos for overexpression studies were obtained from staged embryos collected from crosses of UAS-with a maternal driver, [23]. Control embryos for RNA sequencing (RNA-seq) were obtained from crossing flies with this driver. Germ PROTAC Sirt2 Degrader-1 collection clones of the mutant travel allele MB36 (a null allele) were utilized for Groucho loss-of-function studies [24]. These clones were generated using the standard dominant female sterile FLP/FRT protocol [25]. Groucho chromatin immunoprecipitation (ChIP) and sequencing Chromatin immunoprecipitation (ChIP) was carried out as explained previously [26]. Embryos were collected in three successive 2.5?h windows beginning 1.5?h post-deposition from OregonR population cages PROTAC Sirt2 Degrader-1 and crosslinked with formaldehyde prior to sonication (Diagenode Bioruptor). Immunoprecipitation was carried out using rabbit PROTAC Sirt2 Degrader-1 polyclonal antibodies raised against the Gro-GP domain name GST fusion protein affinity purified against the Halo-tagged GP domain name. Libraries for multiplex sequencing were prepared using the Nugen Ovation Ultralow System V2 kit (catalog # 0344C32). Groucho ChIP sequencing (ChIP-seq) data analysis Multiplexed libraries were sequenced on Illumina HiSeq 2000 sequencing platforms (High Throughput Sequencing Facility, Broad Stem Cell Research Center, UCLA). Reads were demultiplexed via custom scripts. Demultiplexed libraries were filtered for go through quality and PCR duplicates. The number of non-redundant mapped reads varied from PROTAC Sirt2 Degrader-1 ~7.1 million to ~9.3 million for the six experiments (two from each of three timepoints (Additional file 1: Table S1)). Alignment was performed against the genome (iGenomes BDGP 5.25 assembly) with Bowtie2 (v2.2.5) using the following parameters: ?[27]. Peak calling was performed using MACS2 (v2.1.0) with default parameters [28]. Peak visualizations were generated with Integrated Genome Browser (v8.4.2) [29]. Peaks with a minimum 1?bp overlap between replicates were used for further analysis, unless otherwise noted (ChIPpeakAnno) [30]. Motif enrichment analysis was performed with the DREME software suite (v4.10.1) on 500 base pair regions centered on ChIP-seq peaks identified by MACS2 [31]. Embryonic.