Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila

<p>Abstract</p> <p>Background</p> <p>Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in <it>Schizosaccharomyces pombe </it>have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on <it>Drosophila </it>heterochromatin structure.</p> <p>Results</p> <p>The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (<it>dcr-2, ago1, ago2, piwi, Lip [D], aub </it>and <it>hls</it>). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of <it>white-mottled4h </it>position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using <it>Drosophila </it>embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.</p> <p>Conclusion</p> <p>Our experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in <it>Drosophila</it>. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in <it>Drosophila</it>.</p

HP1 Recruits Activity-Dependent Neuroprotective Protein to H3K9me3 Marked Pericentromeric Heterochromatin for Silencing of Major Satellite Repeats

H3 lysine 9 trimethylation (H3K9me3) is a histone posttranslational modification (PTM) that has emerged as hallmark of pericentromeric heterochromatin. This constitutive chromatin domain is composed of repetitive DNA elements, whose transcription is differentially regulated. Mammalian cells contain three HP1 proteins, HP1α, HP1β and HP1γ These have been shown to bind to H3K9me3 and are thought to mediate the effects of this histone PTM. However, the mechanisms of HP1 chromatin regulation and the exact functional role at pericentromeric heterochromatin are still unclear. Here, we identify activity-dependent neuroprotective protein (ADNP) as an H3K9me3 associated factor. We show that ADNP does not bind H3K9me3 directly, but that interaction is mediated by all three HP1 isoforms in vitro. However, in cells ADNP localization to areas of pericentromeric heterochromatin is only dependent on HP1α and HP1β. Besides a PGVLL sequence patch we uncovered an ARKS motif within the ADNP homeodomain involved in HP1 dependent H3K9me3 association and localization to pericentromeric heterochromatin. While knockdown of ADNP had no effect on HP1 distribution and heterochromatic histone and DNA modifications, we found ADNP silencing major satellite repeats. Our results identify a novel factor in the translation of H3K9me3 at pericentromeric heterochromatin that regulates transcription

Heterochromatin and the molecular mechanisms of 'parent-of-origin' effects in animals.

Twenty five years ago it was proposed that conserved components of constitutive heterochromatin assemble heterochromatinlike complexes in euchromatin and this could provide a general mechanism for regulating heritable (cell-to-cell) changes in gene expressibility. As a special case, differences in the assembly of heterochromatin-like complexes on homologous chromosomes might also regulate the parent-of-origin-dependent gene expression observed in placental mammals. Here, the progress made in the intervening period with emphasis on the role of heterochromatin and heterochromatin-like complexes in parent-of-origin effects in animals is reviewed

Attributions of adolescents with type 1 diabetes related to performing diabetes care around friends and peers: The moderating role of friend support

Objective: To examine the relationships among negative attributions of friend and peer reactions to diabetes management in social situations, anticipated adherence difficulties, friend support, diabetes stress, and metabolic control. Methods: A sample of 102 adolescents with Type 1 diabetes completed instruments measuring attribution of friend and peer reactions, anticipated adherence, friend support, and diabetes stress. Metabolic control was measured by the percentage of hemoglobin A1c. Results: Structural equation modeling demonstrated an excellent fit of two models depicting the mediating role of anticipated adherence difficulties and diabetes stress on the relationship between negative attributions of friend (first model) and peer (second model) reactions and metabolic control. Friend support was found to moderate the path between diabetes stress and metabolic control in an unexpected manner. That is, as friend support increased, so did the relationship between stress and metabolic control. Conclusions: Adolescents who make negative attributions about reactions of friends and/or non friend peers are likely to find adherence difficult in social situations and have increased stress, with the latter associated with metabolic control. Results are discussed in terms of a social information processing model of adjustment. © The Author 2006. Published by Oxford University Press on behalf of the Society of Pediatric Psychology. All rights reserved

Attributions of teacher reactions to diabetes self-care behaviors

Objective: This study had two objectives: examine relationships among negative attributions of teacher reactions, anticipated adherence difficulties, teacher support, diabetes stress, and metabolic control; and develop questionnaires to test these relationships. Methods: One hundred and two youths with type 1 insulin-dependent diabetes completed instruments measuring attribution of teacher reactions, anticipated adherence, teacher support, and diabetes stress. Metabolic control was measured by percentage of hemoglobin A 1c. Results: Structural equation modeling demonstrated that negative attributions had direct effects on anticipated adherence difficulties and diabetes stress. Diabetes stress had a direct effect on metabolic control. Negative attributions had a significant indirect effect on metabolic control through associations with diabetes stress. Teacher support moderated the path between negative attributions and anticipated adherence difficulties. Conclusions: Youths making negative attributions about teacher\u27s reactions are likely to find adherence difficult in school situations and have increased stress. Results are discussed in terms of a social information processing model of adjustment and practical applications. © The Author 2008. Published by Oxford University Press on behalf of the Society of Pediatric Psychology. All rights reserved