cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. Here we searched for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. We propose that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for approximately 10 kb or until encountering competition from a euchromatic determinant.     

HP2-like protein: a new piece of the facultative heterochromatin puzzle

In Drosophila melanogaster, the two chromosomal proteins HP1 and HP2 colocalize on heterochromatic and euchromatic sites in polytene chromosomes. Mutations in the HP2 gene act as dominant suppressors of position effect variegation, demonstrating a role for HP2 in the formation or maintenance of heterochromatin. In this paper, we investigated whether a putative homolog of the D. melanogaster HP2 is involved in the facultative heterochromatinization process in mealybugs. Using an antibody raised against the Drosophila HP2, we identified in the mealybug Planococcus citri a cross-reactive epitope, which we refer to as HP2-like. We investigated the HP2-like pattern during the male embryo development where the entire paternal haploid chromosome set becomes heterochromatic. The HP2 antibody heavily decorates the chromocenters, where it localizes with HP1, and marks the chromatin before it acquires the full cytological characteristics of the male-specific heterochromatin. In euchromatic chromosomes, HP2-like is mainly concentrated at telomeric sites. The interplay between HP2-like and HP1-like was studied by dsRNA interference experiments. Extinguishing HP1-like expression by RNAi does not prevent the association of HP2-like with facultative heterochromatin, implying that HP2-like binds to chromatin in a HP1-independent manner. Our results confirm and extend the structural and functional conservation of proteins involved in heterochromatin assembly. Silvia Volpi and Silvia Bongiorni contributed equally to the work.     

Inputs and outputs for chromatin-targeted RNAi

Plant gene silencing is targeted to transposons and repeated sequences by small RNAs from the RNA interference (RNAi) pathway. Like classical RNAi, RNA-directed chromatin silencing involves the cleavage of double-stranded RNA by Dicer endonucleases to create small interfering RNAs (siRNAs), which bind to the Argonaute protein. The production of double-stranded RNA (dsRNA) must be carefully controlled to prevent inappropriate silencing. A plant-specific RNA polymerase IV (Pol IV) initiates siRNA production at silent heterochromatin, but Pol IV-independent mechanisms for making dsRNA also exist. Downstream of siRNA biogenesis, multiple chromatin marks might be targeted by Argonaute-siRNA complexes, yet mechanisms of chromatin modification remain poorly understood. Genomic studies of siRNA target loci promise to reveal novel biological functions for chromatin-targeted RNAi.     

Maternal Depletion of Piwi,a Component of the RNAi System,Impacts Heterochromatin Formation in Drosophila

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.