SetDB1 and Su(var)3-9 are essential for late stages of larval development of Drosophila melanogaster.

Methylation of H3K9 histone residue is a marker of gene silencing in eukaryotes. Three enzymes responsible for adding this modification - G9a, SetDB1/Egg, and Su(var)3-9 - are known in Drosophila. To understand how simultaneous mutations of SetDB1 and Su(var)3-9 may affect the fly development, appropriate combinations were obtained. Double mutants egg; Su(var)3-9 displayed pronounced embryonic lethality, slower larval growth and died before or during metamorphosis. Analysis of transcription in larval salivary glands and wing imaginal disks indicated that the effect of double mutation is tissue-specific. In salivary gland chromosomes, affected genes display low H3K9me2 enrichment and are rarely bound by SetDB1 or Su(var)3-9. We suppose that each of these enzymes directly or indirectly controls its own set of gene targets in different organs, and double mutation results in an imbalanced developmental program. This also indicates that SetDB1 and Su(var)3-9 may affect transcription via H3K9-independent mechanisms. Unexpectedly, in double and triple mutants, amount of di- and tri-methylated H3K9 is drastically reduced, but not completely absent. We hypothesize that this residual methylation implies the existence of additional H3K9-specific methyltransferase in Drosophila.

Age Prediction Using DNA Methylation Heterogeneity Metrics.

Dynamic changes in genomic DNA methylation patterns govern the epigenetic developmental programs and accompany the organism's aging. Epigenetic clock (eAge) algorithms utilize DNA methylation to estimate the age and risk factors for diseases as well as analyze the impact of various interventions. High-throughput bisulfite sequencing methods, such as reduced-representation bisulfite sequencing (RRBS) or whole genome bisulfite sequencing (WGBS), provide an opportunity to identify the genomic regions of disordered or heterogeneous DNA methylation, which might be associated with cell-type heterogeneity, DNA methylation erosion, and allele-specific methylation. We systematically evaluated the applicability of five scores assessing the variability of methylation patterns by evaluating within-sample heterogeneity (WSH) to construct human blood epigenetic clock models using RRBS data. The best performance was demonstrated by the model based on a metric designed to assess DNA methylation erosion with an MAE of 3.686 years. We also trained a prediction model that uses the average methylation level over genomic regions. Although this region-based model was relatively more efficient than the WSH-based model, the latter required the analysis of just a few short genomic regions and, therefore, could be a useful tool to design a reduced epigenetic clock that is analyzed by targeted next-generation sequencing.

SetDB1 and Su(var)3-9 play non-overlapping roles in somatic cell chromosomes of Drosophila melanogaster.

We explored functional roles of two H3K9-specific histone methyltransferases of Drosophila melanogaster, SetDB1 (also known as Eggless) and Su(var)3-9. Using the DamID approach, we generated the binding profile for SetDB1 in Drosophila salivary gland chromosomes, and matched it to the profile of Su(var)3-9. Unlike Su(var)3-9, SetDB1 turned out to be an euchromatic protein that is absent from repeated DNA compartments, and is largely restricted to transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of ubiquitously expressed genes. Significant SetDB1 association is also observed at binding sites for the insulator protein CP190. SetDB1 and H3K9 di- and tri-methylated (me2 and me3)-enriched sites tend to display poor overlap. At the same time, SetDB1 has a clear connection with the distribution of H3K27me3 mark. SetDB1 binds outside the domains possessing this modification, and about half of the borders of H3K27me3 domains are decorated by SetDB1 together with actively transcribed genes. On the basis of poor correlation between the distribution of SetDB1 and H3K9 methylation marks, we speculate that, in somatic cells, SetDB1 may contribute to the methylation of a broader set of chromosomal proteins than just H3K9. In addition, SetDB1 can be expected to play a role in the establishment of chromatin functional domains.

Genome-wide analysis of gene regulation mechanisms during Drosophila spermatogenesis.

BACKGROUND: During Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types. RESULTS: Our study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes. CONCLUSIONS: Discovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated.