H3K9 and H4K20 methyltransferases are directly involved in the heterochromatinization of the paternal chromosomes in male Planococcus citri embryos.

Using a new method for bulk preparation of early stage embryos, we have investigated the role played by putative Planococcus citri H3K9 and H4K20 histone methyl transferases (HMTases) in regulating heterochromatinization of the imprinted paternal chromosomal set in male embryos. We found that H3K9 and H420 HMTases are required for heterochromatinization of the paternal chromosomes. We present evidence that both HMTases maintain the paternal "imprint" during the cleavage divisions when both parental chromosome sets are euchromatic. A testable model that accommodates our findings is proposed.

Comparison of genome architecture at two stages of male germline cell differentiation in Drosophila.

Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). Here, we identified LADs and TADs at two stages of Drosophila spermatogenesis - in bamΔ86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). We found that initiation of SpC-specific transcription correlates with promoters' detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. We also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, we uncovered the specific X chromosome organization in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.

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 SU(VAR)3-9 distribution in chromosomes of Drosophila melanogaster.

Histone modifications represent one of the key factors contributing to proper genome regulation. One of histone modifications involved in gene silencing is methylation of H3K9 residue. Present in the chromosomes across different eukaryotes, this epigenetic mark is controlled by SU(VAR)3-9 and its orthologs. Despite SU(VAR)3-9 was discovered over two decades ago, little is known about the details of its chromosomal distribution pattern. To fill in this gap, we used DamID-seq approach and obtained high-resolution genome-wide profiles for SU(VAR)3-9 in two somatic (salivary glands and brain ganglia) and two germline (ovarian nurse cells and testes) tissues of Drosophila melanogaster. Analysis of tissue and developmental expression of SU(VAR)3-9-bound genes indicates that in the somatic tissues tested, as well as in the ovarian nurse cells, SU(VAR)3-9 tends to associate with transcriptionally silent genes. In contrast, in the testes, SU(VAR)3-9 shows preferential association with testis-specific genes, and its binding appears dynamic during spermatogenesis. In somatic cells, the mere presence/absence of SU(VAR)3-9 binding correlates with lower/higher expression. No such correlation is found in the male germline. Interestingly, transcription units in piRNA clusters (particularly flanks thereof) are frequently targeted by SU(VAR)3-9, and Su(var)3-9 mutation affects the expression of select piRNA species. Our analyses suggest a context-dependent role of SU(VAR)3-9. In euchromatin, SU(VAR)3-9 may serve to fine-tune the expression of individual genes, whereas in heterochromatin, chromosome 4, and piRNA clusters, it may act more broadly over large chromatin domains.

Data analysis algorithm for DamID-seq profiling of chromatin proteins in Drosophila melanogaster.

Analysis of gene expression regulation typically requires identification of genomic sites bound by regulatory proteins. For this purpose, chromatin immunoprecipitation (ChIP) and Dam identification (DamID) methods can be applied to cell lines, whole organisms, or enriched cell populations. In this work, we present modifications to the experimental DamID protocol, as well as a custom data processing algorithm, that allow to confidently identify genomic sites enriched with the proteins of interest. This algorithm is implemented in Perl and is also available as executable files, thereby making DamID analysis relatively straightforward. Finally, we demonstrate how this pipeline performs when fed with real experimental data.

ASIP Promoter Variants Predict the Sesame Coat Color in Shiba Inu Dogs.

Animals exhibit a wide variety of genetically determined coat colors and pigmentation patterns that serve important roles in adaptation and communication. Although the genetics of the main coat colors in dogs have been studied extensively, there are types of coat pigmentation that have not been explained yet. Recently, an association between the variants in the ASIP gene Ventral (VP) and Hair Cycle (HCP) promoters with different coat colors in dogs has been established. Here, we used the new findings as a basis to investigate the genetics of the red sesame coat color in Shiba Inu dogs. Our study revealed that red sesame dogs carry a specific heterozygous ASIP promoter diplotype, VP2-HCP1/VP2-HCP3, where VP2-HCP1 is responsible for the red coat with a dark overlay, and VP2-HCP3 for a tan point-like pattern. This finding explains the inheritance of this coat color pattern and can be used by breeders to produce dogs with this rare phenotype. A comparison of sesame dogs (VP2-HCP1/VP2-HCP3) to a dog homozygous for the VP2-HCP1 promoter haplotype suggests that the incomplete dominance between the ASIP alleles may be involved in the sesame coat formation. These results are in good agreement with the new model explaining how different levels of ASIP gene expression affect the regulation of pigment synthesis in melanocytes.

Expansion and diversification of the signaling capabilities of the CD2/SLAM family in Xenopodinae amphibians.

We studied the evolution of the CD2 family in tetrapods by extracting and analyzing CD2-like genes from the genome of the amphibian species Silurana (Xenopus) tropicalis. An exhaustive analysis of the genomic and cDNA databases resulted in the identification of at least 70 CD2-like genes. The predicted receptors mostly maintain the typical VC2 ectodomains, but are highly diverse in their C-termini, which suggests a broad range of signaling capacities. Apart from the presumed monomeric receptors with ITSM and/or ITIM motifs, the Silurana family includes secreted proteins. Furthermore, a fraction of the receptors contain a conserved TM subtype with the NxxR motif that is known to promote an association with the FcRγ subunit and that was previously found in the members of the FcR- and KIR-related receptors. The expression analysis of a sample of the genes showed broad tissue distribution and gene-specific expression patterns. Phylogenetic analysis predicted that the CD58, CD150/SLAM, and SLAMF8 genes were maintained as single-copy genes in both mammals and amphibians, while others expanded/contracted in a lineage-specific manner.

Biology and Physics of Heterochromatin-Like Domains/Complexes.

The hallmarks of constitutive heterochromatin, HP1 and H3K9me2/3, assemble heterochromatin-like domains/complexes outside canonical constitutively heterochromatic territories where they regulate chromatin template-dependent processes. Domains are more than 100 kb in size; complexes less than 100 kb. They are present in the genomes of organisms ranging from fission yeast to human, with an expansion in size and number in mammals. Some of the likely functions of domains/complexes include silencing of the donor mating type region in fission yeast, preservation of DNA methylation at imprinted germline differentially methylated regions (gDMRs) and regulation of the phylotypic progression during vertebrate development. Far cis- and trans-contacts between micro-phase separated domains/complexes in mammalian nuclei contribute to the emergence of epigenetic compartmental domains (ECDs) detected in Hi-C maps. A thermodynamic description of micro-phase separation of heterochromatin-like domains/complexes may require a gestalt shift away from the monomer as the "unit of incompatibility" that determines the sign and magnitude of the Flory-Huggins parameter, χ. Instead, a more dynamic structure, the oligo-nucleosomal "clutch", consisting of between 2 and 10 nucleosomes is both the long sought-after secondary structure of chromatin and its unit of incompatibility. Based on this assumption we present a simple theoretical framework that enables an estimation of χ for domains/complexes flanked by euchromatin and thereby an indication of their tendency to phase separate. The degree of phase separation is specified by χN, where N is the number of "clutches" in a domain/complex. Our approach could provide an additional tool for understanding the biophysics of the 3D genome.

Comparative gene expression profiling of human primary endotheliocytes cultivated on polyurethane-based electrospun 3D matrices and natural decellularized vein.

The formation of a continuous layer of normally functioning endothelium on the lumen surface of small diameter vascular grafts is considered a prerequisite of their long-term functioning without stenosis. Thus, materials supporting not only endothelialization but also the normal functioning state of endotheliocytes are demanded. In this study, we have evaluated the functional state of human umbilical vein endothelial cells (HUVEC) cultivated on the surface of autologous decellularized human umbilical vein and electrospun polyurethane-based matrices by next generation sequencing gene expression profiling. Three types of matrices produced by electrospinning from hexafluoroisopropanol solutions of pure TECOFLEX™ EG-80A polyurethane, polyurethane with gelatin and polyurethane with gelatin and bivalirudin were studied. Cells cultivated on different supports were subjected to RNA-Seq profiling on an Illumina HiSeq platform. The data demonstrated that the structure of 3D matrices and the chemical composition of the fibers have a significant effect on the gene expression profiles of HUVEC. The results suggest that protein-enriched polyurethane-based 3D matrices represent a convenient surface for obtaining a normally functioning endothelial layer.

Deconstructing age reprogramming.

It has been proposed that age reprogramming enables old cells to be rejuvenated without passage through an embryonic stage (Singh and Zacouto in J. Biosci. 35 315-319, 2010). As such, age reprogramming stands apart from the induced pluripotent stem (iPS) and nuclear transfer-embryonic stem (NT-ES) cell therapies where histo-compatible cells are produced only after passage through an embryonic stage. It avoids many of the disadvantages associated with iPS and NT-ES cell therapies. Experimental evidence in support of age reprogramming is burgeoning. Here, we discuss possible new approaches to enhance age reprogramming, which will have considerable benefits for regenerative therapies.

General Study and Gene Expression Profiling of Endotheliocytes Cultivated on Electrospun Materials.

Endothelization of the luminal surface of vascular grafts is required for their long-term functioning. Here, we have cultivated human endothelial cells (HUVEC) on different 3D matrices to assess cell proliferation, gene expression and select the best substrate for endothelization. 3D matrices were produced by electrospinning from solutions of poly(D,L-lactide-co-glycolide) (PLGA), polycaprolactone (PCL), and blends of PCL with gelatin (Gl) in hexafluoroisopropanol. Structure and surface properties of 3D matrices were characterized by SEM, AFM, and sessile drop analysis. Cell adhesion, viability, and proliferation were studied by SEM, Alamar Blue staining, and 5-ethynyl-2'-deoxyuridine (EdU) assay. Gene expression profiling was done on an Illumina HiSeq 2500 platform. Obtained data indicated that 3D matrices produced from PCL with Gl and treated with glutaraldehyde provide the most suitable support for HUVEC adhesion and proliferation. Transcriptome sequencing has demonstrated a minimal difference of gene expression profile in HUVEC cultivated on the surface of these matrices as compared to tissue culture plastic, thus confirming these matrices as the best support for endothelization.

Isolation, culturing and gene expression profiling of inner mass cells from stable and vulnerable carotid atherosclerotic plaques.

The connective tissue components that form the atherosclerotic plaque body are produced by the plaque inner mass cells (PIMC), located inside the plaque. We report an approach to isolate and culture cells from the connective tissue of stable and vulnerable human atherosclerotic plaques based on elimination of non-connective tissue cells such as blood and non-plaque intima cells with a lysis buffer. The resulting plaque cells were characterized by growth capacity, morphology, transcriptome profiling and specific protein expression. Plaque cells slowly proliferated for up to three passages unaffected by the use of proliferation stimulants or changes of culture media composition. Stable plaques yielded more cells than vulnerable ones. Plaque cell cultures also contained several morphological cellular types. RNA-seq profiles of plaque cells were different from any of the cell types known to be involved in atherogenesis. The expression of the following proteins was observed in cultured plaque cells: smooth muscle cells marker α-SMA, macrophage marker CD14, extracellular matrix proteins aggrecan, fibronectin, neovascularisation markers VEGF-A, CD105, cellular adhesion receptor CD31 and progenitor/dedifferentiation receptor CD34. Differential expression of several notable transcripts in cells from stable and vulnerable plaques suggests the value of plaque cell culture studies for the search of plaque vulnerability markers.

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.

Functional dissection of Drosophila melanogaster SUUR protein influence on H3K27me3 profile.

BACKGROUND: In eukaryotes, heterochromatin replicates late in S phase of the cell cycle and contains specific covalent modifications of histones. SuUR mutation found in Drosophila makes heterochromatin replicate earlier than in wild type and reduces the level of repressive histone modifications. SUUR protein was shown to be associated with moving replication forks, apparently through the interaction with PCNA. The biological process underlying the effects of SUUR on replication and composition of heterochromatin remains unknown. RESULTS: Here we performed a functional dissection of SUUR protein effects on H3K27me3 level. Using hidden Markow model-based algorithm we revealed SuUR-sensitive chromosomal regions that demonstrated unusual characteristics: They do not contain Polycomb and require SUUR function to sustain H3K27me3 level. We tested the role of SUUR protein in the mechanisms that could affect H3K27me3 histone levels in these regions. We found that SUUR does not affect the initial H3K27me3 pattern formation in embryogenesis or Polycomb distribution in the chromosomes. We also ruled out the possible effect of SUUR on histone genes expression and its involvement in DSB repair. CONCLUSIONS: Obtained results support the idea that SUUR protein contributes to the heterochromatin maintenance during the chromosome replication. A model that explains major SUUR-associated phenotypes is proposed.