Enrichment of HP1a on Drosophila Chromosome 4 Genes Creates an Alternate Chromatin Structure Critical for Regulation in this Heterochromatic Domain Nicole C. Riddle1., Youngsook L. Jung2., Tingting Gu1., Artyom A. Alekseyenko 3 , Dalal Asker4,5, Hongxing Gui4, Peter V. Kharchenko 2 , Aki Minoda6,7, Annette Plachetka3, Yuri B. Schwartz4,8, Michael Y. Tolstorukov2, Mitzi I. Kuroda3, Vincenzo Pirrotta4, Gary H. Karpen6,7, Peter J. Park2*, Sarah C. R. Elgin1* 1 Department of Biology, Washington University in St. Louis, St. Louis, Missouri, United States of America, 2 Center for Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, United States of America, 3 Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America, 5 Food Science and Technology Department, Faculty of Agriculture, Alexandria University, Alexandria, Egypt, 6 Department of Molecular and Cell Biology, University of California Berkeley, California, United States of America, 7 Department of Genome Dynamics, Lawrence Berkeley National Lab, Berkeley, California, United States of America, 8 Department of Molecular Biology, Umea University, Umea, Sweden Abstract Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA. Citation: Riddle NC, Jung YL, Gu T, Alekseyenko AA, Asker D, et al. (2012) Enrichment of HP1a on Drosophila Chromosome 4 Genes Creates an Alternate Chromatin Structure Critical for Regulation in this Heterochromatic Domain. PLoS Genet 8(9): e1002954. doi:10.1371/journal.pgen.1002954 Editor: Jason D. Lieb, The University of North Carolina at Chapel Hill, United States of America Received January 12, 2012; Accepted July 31, 2012; Published September 20, 2012 Copyright: 2012 Riddle et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01 GM068388 (ARRA supplement) to SCRE and by the National Human Genome Research Institute of the National Institutes of Health under award number U01 HG004258 to GHK (lead PI). The GTAC is partially supported by NCI Cancer Center Support Grant #P30 CA91842 to the Siteman Cancer Center and by ICTS/CTSA Grant# UL1RR024992 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: peter_park@hms.harvard.edu (PJP); riddlenc@biology2.wustl.edu (SCRE) . These authors contributed equally to this work. Introduction In eukaryotes, initial gene regulation is achieved through the interaction of transcription factors and the transcriptional machinery with DNA packaged into chromatin. The basic unit of chromatin is the nucleosome, 147 bp of DNA wrapped around a histone octamer [1,2]. Post-translational modifications of histones, and the presence of core histone variants and additional chromosomal proteins, characterize various chroma- tin states that determine the accessibility of DNA for transcrip- tion. A subset of modifications, such as histone 3 lysine 9 (H3K9) methylation, is associated with gene silencing, while other modifications, such as histone 3 lysine 4 trimethylation (H3K4me3), correlate with gene activity. (For a recent review see [3].) Chromatin states and transcriptional activity are highly regulated to ensure gene activity at the proper developmental time and in the appropriate cell type while maintaining silencing at other, often tightly linked, sequences, including transposable elements (TEs). This need for regulation is particularly evident in genomes similar to the human, where TEs and genes are PLOS Genetics | www.plosgenetics.org 1 September 2012 | Volume 8 | Issue 9 | e1002954