12x HWT or 12x K9R ∆HisC ∆HisC Increased chromatin accessibility and gene expression in K9R mutants. A-B) Ratio of K9R to HWT FAIRE-seq (A) or RNA-seq (B) signal prepared from third larval wandering (3LW) imaginal wing discs. Darker color indicates higher density of peaks. Published in Penke et al. (2016). A WT K9R H3 H3K9me3 H3K9me2 WT K9R Reduced H3K9me3 in K9R mutants. A) Immunofluorescence of polytene chromosomes prepared from 3rd instar larval (3LW) salivary glands. Blue, DAPI; Green H3K9me2; red, H3K9me3. Published in Penke et al. (2016). B) Western blot from total cellular histones from whole 3LW larvae. Published in Penke et al. (2016). RNA-seq signal K9R/HWT (log2) B RNA-seq FAIRE-seq A B H3K9me3 Cut-n-Run C B A A chr3R chr3L chr2R chr2R chr2L Future Directions Interrogating the role of the histone mark H3K9me3 in D. melanogaster Genome Organization and Gene Regulation Abstract H3K9R mutation results in loss of H3K9me3 and HP1 binding at pericentromeres Methodology Purpose and Hypotheses Acknowledgements UNIVERSITY of NORTH CAROLINA at CHAPEL HILL Dowen Jill & McKay Dan Labs Telomere architecture is maintained in H3K9R HP1 Background: H3K9me3 and HP1 The histone replacement platform. We delete the endogenous histone locus from chr2L (∆HisC) and insert a designer histone cassette harboring 12 copies of the replication-independent histone gene cassette to a chr3L. Mutant or wild type histones are expressed from the designer histone cassette (12x WT or 12x K9R) to replace all endogenous replication-dependent histones in the fly (McKay 2015). We investigate the role of H3K9me3-mediated HP1 binding in establishing architectural features by performing HiC in H3K9me3-deficient D. melanogaster. Given the roles for HP1 and H3K9me3 in heterochromatin structure, we hypothesize that H3K9me3-deficient flies will exhibit dramatic changes to genome architecture, particularly in transcriptionally silent (aka B) compartments and known regions of constitutive heterochromatin. Eukaryotic genomes are packaged into basic units known as nucleosomes, which are comprised of DNA wrapped around a bundle of 8 core histone protiens. Histone tails are post-translationally modified (PTM) to act as a substrate for effector proteins to bind. One such PTM, tri-methylation, occurs at lysine 9 on histone H3 (H3K9me3). H3K9me3 is canonically referred to as a heterochromatic histone mark, and has been implicated in repressing gene expression. H3K9me3 recruits an effector protein, HP1a, which has strong self-associating properties. It has been hypothesized that engagement of H3K9me3 by HP1 is responsible for establishing heterochromatin. Organization of chromatin in three-dimensional (3D) space impacts DNA-templated events by controlling the accessibility of transcriptional machinery to the underlying DNA sequence. Despite its importance in regulating genome function, the mechanisms controlling 3D architecture remain poorly understood. Here, we examine the contribution of histone post-translational modifications (PTMs), a feature of chromatin that is highly correlated with 3D genome organization, but whose role has not been directly tested. In particular, we interrogate the role for the heterochromatin-correlate, histone H3 lysine 9 trimethylation (H3K9me3), in governing 3D organization of the D. melanogaster genome. We directly test the contribution of H3K9me3 by using a genetic platform in which the endogenous histone gene locus has been deleted and replaced with transgenic versions encoding non modifiable histone H3K9. H3K9me3 provides a binding site for heterochromatin protein 1 (HP1), which has been implicated in liquid-liquid phase separation, a biophysical property that separates molecules in 3D space. Given this property of HP1 and it’s localization to H3K9me3, we hypothesize H3K9me3 demarcates regions of the genome that become physically separated from other regions of the genome. We perform a comprehensive analysis of genomics data, including Hi