Eukaryotic genomes harbor transposable elements and other repetitive sequences that must be silenced. Small RNA interference pathways play a major role in their repression. Here, we reveal another mechanism for silencing these sequences in Drosophila. Depleting the linker histone H1 in vivo leads to strong activation of these elements. H1-mediated silencing occurs in combination with the heterochromatin-specific histone H3 lysine 9 methyltransferase Su(var)3-9. H1 physically interacts with Su(var)3-9 and recruits it to chromatin in vitro, which promotes H3 methylation. We propose that H1 plays a key role in silencing by tethering Su(var)3-9 to heterochromatin. The tethering function of H1 adds to its established role as a regulator of chromatin compaction and accessibility.

Knowledge of the role of RNA in affecting gene expression has expanded in the past several years. Small RNAs serve as homology guides to target messenger RNAs for destruction at the post-transcriptional level in the experimental technique known as RNA interference and in the silencing of some transgenes. These small RNAs are also involved in sequence-specific targeting of chromatin modifications for transcriptional silencing of transgenes, transposable elements, heterochromatin and some cases of Polycomb-mediated gene silencing. RNA silencing processes in Drosophila are described.

BackgroundCG11033 (dKDM2) is the Drosophila homolog of the gene KDM2B. dKDM2 has been known to possess histone lysine demethylase activity towards H3K36me2 in cell lines and it regulates H2A ubiquitination. The human homolog of the gene has dual activity towards H3K36me2 as well as H3K4me3, and plays an important role in cellular senescence.FindingsWe have used transgenic flies bearing an RNAi construct for the dKDM2 gene. The knockdown of dKDM2 gene was performed by crossing UAS-RNAi-dKDM2 flies with actin-Gal4 flies. Western blots of acid extracted histones and immunofluoresence analysis of polytene chromosome showed the activity of the enzyme dKDM2 to be specific for H3K4me3 in adult flies. Immunofluoresence analysis of polytene chromosome also revealed the presence of multiple nucleoli in RNAi knockdown mutants of dKDM2 and decreased H3-acetylation marks associated with active transcription.ConclusionOur findings indicate that dKDM2 is a histone lysine demethylase with specificity for H3K4me3 and regulates nucleolar organization.

BackgroundHeterochromatin 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 Schizosaccharomyces pombe 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 Drosophila heterochromatin structure.ResultsThe results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (dcr-2, ago1, ago2, piwi, Lip [D], aub and hls). 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 white-mottled4h 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 Drosophila 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.ConclusionOur experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in Drosophila. 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 Drosophila.

SummaryThe sex chromosomes of many species differ in dosage but the total gene expression output is similar, a phenomenon referred to as dosage compensation. Previously, diverse mechanisms were postulated to account for compensation in distantly related taxa. However, two recent papers present evidence that dosage compensation in Drosophila, mammals and nematodes share the property that there is an approximately two-fold upregulation of the single active X chromosome in each case.(1,2)The results suggest that a common mechanism might operate in these different cases.

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Linker histone H1 is among the most abundant components of chromatin. H1 has profound effects on chromosome architecture. H1 also helps to tether DNA-and histone-modifying enzymes to chromatin. Metazoan linker histones have a conserved tripartite structure comprising N-terminal, globular, and long, unstructured C-terminal domains. Here we utilize truncated Drosophila H1 polypeptides in vitro and H1 mutant transgenes in vivo to interrogate the roles of these domains in multiple biochemical and biological activities of H1. We demonstrate that the globular domain and the proximal part of the C-terminal domain are essential for H1 deposition into chromosomes and for the stability of H1-chromatin binding. The two domains are also essential for fly viability and the establishment of a normal polytene chromosome structure. Additionally, through interaction with the heterochromatin-specific histone H3 Lys-9 methyltransferase Su(var)3-9, the H1 C-terminal domain makes important contributions to formation and H3K9 methylation of heterochromatin as well as silencing of transposons in heterochromatin. Surprisingly, the N-terminal domain does not appear to be required for any of these functions. However, it is involved in the formation of a single chromocenter in polytene chromosomes. In summary, we have discovered that linker histone H1, similar to core histones, exerts its multiple biological functions through independent, biochemically separable activities of its individual structural domains.DNA in the eukaryotic nucleus is packaged into a compact nucleoprotein complex called chromatin (1, 2). The histones constitute a family of basic proteins that play a vital role in organizing chromatin. There are five major classes of histones: the core histones H2A, H2B, H3, and H4 and the linker histone H1. The nucleosome core particle, the fundamental unit of chromatin, consists of an octamer of two copies of each of the core histones around which about 146 bp of DNA is wrapped. H1 binds to the nucleosome core particle and the linker DNA between adjacent core particles. The stoichiometry of H1 to nucleosome core particles in cells of complex eukaryotes ranges from about 0.5 to approximately equimolar (3). Given this abundance, the linker histone H1 is expected to play important roles in chromatin structure. Indeed, numerous in vitro studies, as well as experiments in vivo, have shown that H1 has a profound effect on chromatin structure (reviewed in Refs. 4 and 5). For example, incorporation of H1 into chromatin leads to an increase in the spacing between nucleosome core particles. H1 also facilitates the folding of chromatin into more compact structures. In addition to these architectural roles, there are now an increasing number of reports describing interactions between H1 linker histones and a variety of other nuclear proteins (6), including functional interactions between H1, histone-modifying enzymes, and DNA methyltransferases that lead to modulation of epigenetic marking of chromatin (7,8). In this context, it is notable th...

The RNAi machinery is not only involved with post-transcriptional degradation of messenger RNAs, but also used for targeting of chromatin changes associated with transcriptional silencing. Two recent papers determine the global patterns of gene expression and chromatin modifications produced by the RNAi machinery in fission yeast.(9, 10) The major sites include the outer centromere repeats, the mating-type locus and subtelomeric regions. By comparison, studies of Arabidopsis heterochromatin also implicate transposons as a major target for silencing. Analyses of siRNA libraries from Drosophila, nematodes and Arabidopsis indicate that major repeats at centromeres, telomeres and transposable elements are likely targets of RNAi. Also, intergenic regions are implicated as targets in Arabidopsis.