Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding inDrosophila melanogaster, we performed Hi-C and poly(A)⁺RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) inDrosophilaare only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However,Drosophilainter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwi ^Nt , removing the nuclear localization signal of the Piwi protein. piwi ^Nt females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwi ^Nt mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwi ^Nt ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells.

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.

Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.