RESUMEN
Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from â¼127 Mb in Drosophila mercatorum to â¼400 Mb in Drosophila cyrtoloma. However, the assembled portion of the Muller F element (orthologous to the fourth chromosome in Drosophila melanogaster) shows a nearly 14-fold variation in size, ranging from â¼1.3 Mb to >18 Mb. Here, we present chromosome-level long-read genome assemblies for 4 Drosophila species with expanded F elements ranging in size from 2.3 to 20.5 Mb. Each Muller element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.
Asunto(s)
Drosophila melanogaster , Drosophila , Animales , Drosophila/genética , Drosophila melanogaster/genética , Cromosomas/genética , GenomaRESUMEN
Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from â¼127 Mb in Drosophila mercatorum to â¼400 Mb in Drosophila cyrtoloma . However, the assembled portion of the Muller F Element (orthologous to the fourth chromosome in Drosophila melanogaster ) shows a nearly 14-fold variation in size, ranging from â¼1.3 Mb to > 18 Mb. Here, we present chromosome-level long read genome assemblies for four Drosophila species with expanded F Elements ranging in size from 2.3 Mb to 20.5 Mb. Each Muller Element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.
RESUMEN
Scientists are sequencing new genomes at an increasing rate with the goal of associating genome contents with phenotypic traits. After a new genome is sequenced and assembled, structural gene annotation is often the first step in analysis. Despite advances in computational gene prediction algorithms, most eukaryotic genomes still benefit from manual gene annotation. This requires access to good genome browsers to enable annotators to visualize and evaluate multiple lines of evidence (e.g., sequence similarity, RNA sequencing [RNA-Seq] results, gene predictions, repeats) and necessitates many volunteers to participate in the work. To address the technical barriers to creating genome browsers, the Genomics Education Partnership (GEP; https://gep.wustl.edu/) has partnered with the Galaxy Project (https://galaxyproject.org) to develop G-OnRamp (http://g-onramp.org), a web-based platform for creating UCSC Genome Browser Assembly Hubs and JBrowse genome browsers. G-OnRamp also converts a JBrowse instance into an Apollo instance for collaborative genome annotations in research and educational settings. The genome browsers produced can be transferred to the CyVerse Data Store for long-term access. G-OnRamp enables researchers to easily visualize their experimental results, educators to create Course-based Undergraduate Research Experiences (CUREs) centered on genome annotation, and students to participate in genomics research. In the process, students learn about genes/genomes and about how to utilize large datasets. Development of G-OnRamp was guided by extensive user feedback. Sixty-five researchers/educators from >40 institutions participated through in-person workshops, which produced >20 genome browsers now available for research and education. Genome browsers generated for four parasitoid wasp species have been used in a CURE engaging students at 15 colleges and universities. Our assessment results in the classroom demonstrate that the genome browsers produced by G-OnRamp are effective tools for engaging undergraduates in research and in enabling their contributions to the scientific literature in genomics. Expansion of such genomics research/education partnerships will be beneficial to researchers, faculty, and students alike.
Asunto(s)
Biología Computacional/educación , Biología Computacional/métodos , Genoma , Genómica/educación , Genómica/métodos , Anotación de Secuencia Molecular , Programas Informáticos , Algoritmos , Animales , Secuencia de Bases , Gráficos por Computador , Bases de Datos Genéticas , Drosophila melanogaster , Humanos , Análisis de Secuencia de ARN , Estudiantes , Interfaz Usuario-ComputadorRESUMEN
BACKGROUND: Chromatin-based transcriptional silencing is often described as a stochastic process, largely because of the mosaic expression observed in position effect variegation (PEV), where a euchromatic reporter gene is silenced in some cells as a consequence of juxtaposition with heterochromatin. High levels of variation in PEV phenotypes are commonly observed in reporter stocks. To ascertain whether background mutations are the major contributors to this variation, we asked how much of the variation is determined by genetic variants segregating in the population, examining both the level and pattern of expression using the fruit fly, Drosophila melanogaster, as the model. RESULTS: Using selective breeding of a fourth chromosome PEV reporter line, 39C-12, we isolated two inbred lines exhibiting contrasting degrees of variegation (A1: low expression, D1: high expression). Within each inbred population, remarkable similarity is observed in the degree of variegation: 90% of the variation between the two inbred lines in the degree of silencing can be explained by genotype. Further analyses suggest that this result reflects the combined effect of multiple independent trans-acting loci. While the initial observations are based on a PEV phenotype scored in the fly eye (hsp70-white reporter), similar degrees of silencing were observed using a beta-gal reporter scored across the whole fly. Further, the pattern of variegation becomes almost identical within each inbred line; significant pigment enrichment in the same quadrant of the eye was found for both A1 and D1 lines despite different degrees of expression. CONCLUSIONS: The results indicate that background genetic variants play the major role in determining the variable degrees of PEV commonly observed in laboratory stocks. Interestingly, not only does the degree of variegation become consistent in inbred lines, the patterns of variegation also appear similar. Combining these observations with the spreading model for local heterochromatin formation, we propose an augmented stochastic model to describe PEV in which the genetic background drives the overall level of silencing, working with the cell lineage-specific regulatory environment to determine the on/off probability at the reporter locus in each cell. This model acknowledges cell type-specific events in the context of broader genetic impacts on heterochromatin formation.
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Efectos de la Posición Cromosómica , Drosophila melanogaster/genética , Animales , Linaje de la Célula , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Expresión Génica , Genes Reporteros , Antecedentes Genéticos , Variación Genética , Proteínas HSP70 de Choque Térmico/genética , Heterocromatina/metabolismo , Modelos Genéticos , Fenotipo , Regiones Promotoras GenéticasRESUMEN
SUMMARY: G-OnRamp provides a user-friendly, web-based platform for collaborative, end-to-end annotation of eukaryotic genomes using UCSC Assembly Hubs and JBrowse/Apollo genome browsers with evidence tracks derived from sequence alignments, ab initio gene predictors, RNA-Seq data and repeat finders. G-OnRamp can be used to visualize large genomics datasets and to perform collaborative genome annotation projects in both research and educational settings. AVAILABILITY AND IMPLEMENTATION: The virtual machine images and tutorials are available on the G-OnRamp web site (http://g-onramp.org/deployments). The source code is available under an Academic Free License version 3.0 through the goeckslab GitHub repository (https://github.com/goeckslab). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Eucariontes , Genoma , Genómica , Alineación de Secuencia , Programas InformáticosRESUMEN
The F element of the Drosophila karyotype (the fourth chromosome in Drosophila melanogaster) is often referred to as the "dot chromosome" because of its appearance in a metaphase chromosome spread. This chromosome is distinct from other Drosophila autosomes in possessing both a high level of repetitious sequences (in particular, remnants of transposable elements) and a gene density similar to that found in the other chromosome arms, â¼80 genes distributed throughout its 1.3-Mb "long arm." The dot chromosome is notorious for its lack of recombination and is often neglected as a consequence. This and other features suggest that the F element is packaged as heterochromatin throughout. F element genes have distinct characteristics (e.g, low codon bias, and larger size due both to larger introns and an increased number of exons), but exhibit expression levels comparable to genes found in euchromatin. Mapping experiments show the presence of appropriate chromatin modifications for the formation of DNaseI hypersensitive sites and transcript initiation at the 5' ends of active genes, but, in most cases, high levels of heterochromatin proteins are observed over the body of these genes. These various features raise many interesting questions about the relationships of chromatin structures with gene and chromosome function. The apparent evolution of the F element as an autosome from an ancestral sex chromosome also raises intriguing questions. The findings argue that the F element is a unique chromosome that occupies its own space in the nucleus. Further study of the F element should provide new insights into chromosome structure and function.
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Cromosomas de Insectos/genética , Drosophila/genética , Genes de Insecto/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Animales , Evolución Molecular , Recombinación GenéticaRESUMEN
Although bioinformatics is becoming increasingly central to research in the life sciences, bioinformatics skills and knowledge are not well integrated into undergraduate biology education. This curricular gap prevents biology students from harnessing the full potential of their education, limiting their career opportunities and slowing research innovation. To advance the integration of bioinformatics into life sciences education, a framework of core bioinformatics competencies is needed. To that end, we here report the results of a survey of biology faculty in the United States about teaching bioinformatics to undergraduate life scientists. Responses were received from 1,260 faculty representing institutions in all fifty states with a combined capacity to educate hundreds of thousands of students every year. Results indicate strong, widespread agreement that bioinformatics knowledge and skills are critical for undergraduate life scientists as well as considerable agreement about which skills are necessary. Perceptions of the importance of some skills varied with the respondent's degree of training, time since degree earned, and/or the Carnegie Classification of the respondent's institution. To assess which skills are currently being taught, we analyzed syllabi of courses with bioinformatics content submitted by survey respondents. Finally, we used the survey results, the analysis of the syllabi, and our collective research and teaching expertise to develop a set of bioinformatics core competencies for undergraduate biology students. These core competencies are intended to serve as a guide for institutions as they work to integrate bioinformatics into their life sciences curricula.
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Biología Computacional/educación , Competencia Mental , Aprendizaje Basado en Problemas , Adolescente , Adulto , Femenino , Humanos , Masculino , Estados UnidosRESUMEN
The era of 'big data' is also the era of abundant data, creating new opportunities for student-scientist research partnerships. By coordinating undergraduate efforts, the Genomics Education Partnership produces high-quality annotated data sets and analyses that could not be generated otherwise, leading to scientific publications while providing many students with research experience.
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Biología Computacional/educación , Ciencia/educación , Estadística como Asunto , Colaboración de las Masas , Educación de Pregrado en Medicina , HumanosRESUMEN
Heterochromatin is a common DNA packaging form employed by eukaryotes to constitutively silence transposable elements. Determining which sequences to package as heterochromatin is vital for an organism. Here, we use Drosophila melanogaster to study heterochromatin formation, exploiting position-effect variegation, a process whereby a transgene is silenced stochastically if inserted in proximity to heterochromatin, leading to a variegating phenotype. Previous studies identified the transposable element 1360 as a target for heterochromatin formation. We use transgene reporters with either one or four copies of 1360 to determine if increasing local repeat density can alter the fraction of the genome supporting heterochromatin formation. We find that including 1360 in the reporter increases the frequency with which variegating phenotypes are observed. This increase is due to a greater recovery of insertions at the telomere-associated sequences (â¼50% of variegating inserts). In contrast to variegating insertions elsewhere, the phenotype of telomere-associated sequence insertions is largely independent of the presence of 1360 in the reporter. We find that variegating and fully expressed transgenes are located in different types of chromatin and that variegating reporters in the telomere-associated sequences differ from those in pericentric heterochromatin. Indeed, chromatin marks at the transgene insertion site can be used to predict the eye phenotype. Our analysis reveals that increasing the local repeat density (via the transgene reporter) does not enlarge the fraction of the genome supporting heterochromatin formation. Rather, additional copies of 1360 appear to target the reporter to the telomere-associated sequences with greater efficiency, thus leading to an increased recovery of variegating insertions.
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Elementos Transponibles de ADN , Drosophila melanogaster/genética , Expresión Génica , Genes Reporteros , Heterocromatina/genética , Elementos de Respuesta , Animales , Secuencia de Bases , Mapeo Cromosómico , Análisis por Conglomerados , Femenino , Dosificación de Gen , Perfilación de la Expresión Génica , Genómica/métodos , Masculino , Datos de Secuencia Molecular , Mutagénesis Insercional , Fenotipo , Alineación de Secuencia , Secuencias Repetidas en Tándem , Sitio de Iniciación de la Transcripción , TransgenesRESUMEN
Heterochromatin protein 1 (HP1a in Drosophila) is a conserved eukaryotic chromosomal protein that is prominently associated with pericentric heterochromatin and mediates the concomitant gene silencing. Mechanistic studies implicate HP1 family proteins as 'hub proteins,' able to interact with a variety of chromosomal proteins through the chromo-shadow domain (CSD), as well as to recognize key histone modification sites [primarily histone H3 di/trimethyl Lys9 (H3K9me2/3)] through the chromodomain (CD). Consequently, HP1 has many important roles in chromatin architecture and impacts both gene expression and gene silencing, utilizing a variety of mechanisms. Clearly, HP1 function is altered by context, and potentially by post-translational modifications (PTMs). Here, we report on recent ideas as to how this versatile protein accomplishes its diverse functions.
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Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Transcripción Genética , Animales , Homólogo de la Proteína Chromobox 5 , Silenciador del Gen , Humanos , Procesamiento Proteico-Postraduccional/genética , Activación TranscripcionalRESUMEN
UNLABELLED: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE: Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.
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Bacterias/virología , Bacteriófagos/genética , Genómica/educación , Microbiología/educación , Adulto , Femenino , Humanos , Masculino , Estudiantes , Adulto JovenRESUMEN
Heterochromatin assembly and its associated phenotype, position effect variegation (PEV), provide an informative system to study chromatin structure and genome packaging. In the fruit fly Drosophila melanogaster, the Y chromosome is entirely heterochromatic in all cell types except the male germline; as such, Y chromosome dosage is a potent modifier of PEV. However, neither Y heterochromatin composition, nor its assembly, has been carefully studied. Here, we report the mapping and characterization of eight reporter lines that show male-specific PEV. In all eight cases, the reporter insertion sites lie in the telomeric transposon array (HeT-A and TART-B2 homologous repeats) of the Y chromosome short arm (Ys). Investigations of the impact on the PEV phenotype of mutations in known heterochromatin proteins (i.e., modifiers of PEV) show that this Ys telomeric region is a unique heterochromatin domain: it displays sensitivity to mutations in HP1a, EGG and SU(VAR)3-9, but no sensitivity to Su(z)2 mutations. It appears that the endo-siRNA pathway plays a major targeting role for this domain. Interestingly, an ectopic copy of 1360 is sufficient to induce a piRNA targeting mechanism to further enhance silencing of a reporter cytologically localized to the Ys telomere. These results demonstrate the diversity of heterochromatin domains, and the corresponding variation in potential targeting mechanisms.
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Efectos de la Posición Cromosómica , Drosophila melanogaster/genética , Heterocromatina/química , Telómero/química , Cromosoma Y/química , Animales , Ensamble y Desensamble de Cromatina , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Mapeo Cromosómico , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Masculino , Mutación , Fenotipo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Both the RNAi system and heterochromatin components HP1 (Swi6) and H3K9me2/3 are required for initial establishment of heterochromatin structures in S. pombe. Here we utilize both loss of function alleles and the newly developed Drosophila melanogaster transgenic shRNA lines to deplete proteins of interest at specific development stages to dissect their roles in heterochromatin assembly in early zygotes and in maintenance of the silencing chromatin state during development. Using reporters subject to Position Effect Variegation (PEV), we find that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. HP1a profiles in piwi mutant animals confirm that Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult.
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Proteínas Argonautas/genética , Efectos de la Posición Cromosómica/genética , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Epigénesis Genética , Interferencia de ARN , Alelos , Animales , Animales Modificados Genéticamente , Proteínas Argonautas/metabolismo , Blastodermo/crecimiento & desarrollo , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero , Silenciador del Gen , Heterocromatina/genética , Histona Demetilasas/metabolismoRESUMEN
Position-effect variegation (PEV) results when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition. When heterochromatin packaging spreads across the heterochromatin/euchromatin border, it causes transcriptional silencing in a stochastic pattern. PEV is intensely studied in Drosophila using the white gene. Screens for dominant mutations that suppress or enhance white variegation have identified many conserved epigenetic factors, including the histone H3 lysine 9 methyltransferase SU(VAR)3-9. Heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9, creating a core memory system. Genetic, molecular, and biochemical analysis of PEV in Drosophila has contributed many key findings concerning establishment and maintenance of heterochromatin with concomitant gene silencing.
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Transportadoras de Casetes de Unión a ATP/genética , Efectos de la Posición Cromosómica/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas del Ojo/genética , Reordenamiento Génico/genética , Silenciador del Gen/fisiología , Heterocromatina/genética , Modelos Genéticos , Animales , Efectos de la Posición Cromosómica/fisiología , Drosophila/fisiología , Reordenamiento Génico/fisiología , Heterocromatina/fisiología , Mutación/genéticaRESUMEN
Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is an essential protein critical for heterochromatin assembly and regulation. Its chromo shadow domain (CSD) homodimerizes, a requirement for binding protein partners that contain a PXVXL motif. How does HP1a select among its many different PXVXL-containing partners? HP1a binds tightly to Heterochromatin Protein 2 (HP2), but weakly to PIWI. We investigated differences in homodimerization and the impact of the C-terminal extension (CTE) by contrasting HP1a to its paralogue, HP1b. HP1a and HP1b differ in the dimerization interface, with HP1a having an Arg at position 188 rather than Glu. We find that while this substitution reduces the dimerization constant, it does not impact the binding surface as demonstrated by unchanged partner binding affinities. However, the CTE (only 4 residues in HP1a as compared with 87 residues in HP1b) is critical; the charged residues in HP1a are necessary for tight peptide binding. Examining a panel of amino acid substitutions in the HP1a CSD, we find that Leu-165 in HP1a interacts with HP2 but not PIWI, supporting the conclusion that different sites in the binding surface provide discrimination for partner selection. Partner sequence is also critical for affinity, as the remaining difference in binding between HP2 and PIWI polypeptides is eliminated by swapping the PXVXL motifs between the two. Taken together, these studies indicate that the binding surface of the HP1a CSD plus its short CTE provide the needed discrimination among HP1a's partners, and that the CTE is important for differentiating the interactions of the Drosophila HP1 paralogs.
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Aminoácidos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Dimerización , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
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 T(m) 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.