ABSTRACT
Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus we found that ping-pong mechanism contributed to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. We found that observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.
Subject(s)
Adenosine Triphosphatases/metabolism , Argonaute Proteins/metabolism , Drosophila Proteins/metabolism , Peptide Initiation Factors/metabolism , RNA Helicases/metabolism , RNA, Small Interfering/genetics , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Animals , Argonaute Proteins/genetics , Base Sequence , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Male , Peptide Initiation Factors/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , RNA Helicases/genetics , RNA, Small Interfering/metabolismABSTRACT
Theoretical models suggest that gene silencing at the nuclear periphery may involve "closing" of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2 cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.
Subject(s)
Chromatin/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation , Histone Deacetylase 1/genetics , Histone Deacetylases/genetics , Histones/genetics , Nuclear Lamina/genetics , Acetylation , Animals , Blotting, Western , Cell Line , Chromatin/enzymology , Chromatin Immunoprecipitation , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , Gene Silencing , Histone Deacetylase 1/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Lamin Type B/antagonists & inhibitors , Lamin Type B/genetics , Lamin Type B/metabolism , Multigene Family , Nuclear Lamina/enzymology , RNA, Double-Stranded/genetics , Transcription, GeneticABSTRACT
In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes.
Subject(s)
DNA Replication/genetics , Drosophila melanogaster/genetics , Gene Rearrangement/genetics , Heterochromatin/genetics , X Chromosome/genetics , Animals , Blotting, Southern , Chromosome Mapping , Drosophila Proteins/genetics , In Situ Hybridization , Multigene Family/genetics , Protein Kinases/genetics , Salivary Glands/chemistryABSTRACT
The mechanism of silencing of testis expressed X-linked Stellate repeats by homologous Y-linked Suppressor of Stellate [Su(Ste)] repeats localized in the crystal locus was studied. The double stranded RNA as a product of symmetrical transcription of Su(Ste) repeat and small interference Su(Ste) siRNA were revealed suggesting the mechanism of RNA interference (RNAi) for Stellate silencing. The relief of Stellate silencing as a result of impaired complementarity between the sequences of putative target Stellate transcripts and Su(Ste) repeats was shown. The role of RNAi mechanism in the silencing of heterochromatic retrotransposon GATE inserted in Stellate cluster was revealed. The studies of cis-effects of Stellate tandem repeats causing variegated expression of juxtaposed reporter genes were extended and the lacZ variegation in imaginal disc was shown. The exceptional case of a non-variegated expression of mini-white gene juxtaposed to Stellate repeats in a construct inserted into the 39C region was shown to be accompanied by trans-activation in homozygous state. Trans-activation effect was retained after transposition of this construct into heterochromatic environment in spite of strong variegation of a mini-white gene.
