Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii

In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC.     

Small RNAs in pollen

In plants, each pollen mother cell undergoes two rounds of cell divisions to form a mature pollen grain, which contains a vegetative cell(VC) and two sperm cells(SC). As a companion cell, the VC carries the SCs to an ovule by germinating a pollen tube. In-depth sequencing analyses of mature pollen showed that micro RNAs(mi RNAs) and short interfering RNAs(si RNAs) are present in both the VC and SCs. Additionally, epigenetically-regulated transposable elements(TEs) are reactivated in the VC and these TE m RNAs are further processed into 21-nt epigenetically reactivated si RNA(easiR NA) in SCs, which prevent 24-nt si RNA accumulation and sequester mi RNA loading. Small RNAs are thought to move from the VC to SCs, where they regulate gene expression and reinforce TE silencing. Here, we summarize current knowledge of the biogenesis and function of mi RNAs, si RNAs, and easi RNAs in pollen, emphasizing how these different small RNAs coordinately contribute to sperm cell formation and TE silencing.     

The Role of Symmetric Stem Cell Divisions in Tissue Homeostasis

Successful maintenance of cellular lineages critically depends on the fate decision dynamics of stem cells (SCs) upon division. There are three possible strategies with respect to SC fate decision symmetry: (a) asymmetric mode, when each and every SC division produces one SC and one non-SC progeny; (b) symmetric mode, when 50% of all divisions produce two SCs and another 50%—two non-SC progeny; (c) mixed mode, when both the asymmetric and two types of symmetric SC divisions co-exist and are partitioned so that long-term net balance of the lineage output stays constant. Theoretically, either of these strategies can achieve lineage homeostasis. However, it remains unclear which strategy(s) are more advantageous and under what specific circumstances, and what minimal control mechanisms are required to operate them. Here we used stochastic modeling to analyze and quantify the ability of different types of divisions to maintain long-term lineage homeostasis, in the context of different control networks. Using the example of a two-component lineage, consisting of SCs and one type of non-SC progeny, we show that its tight homeostatic control is not necessarily associated with purely asymmetric divisions. Through stochastic analysis and simulations we show that asymmetric divisions can either stabilize or destabilize the lineage system, depending on the underlying control network. We further apply our computational model to biological observations in the context of a two-component lineage of mouse epidermis, where autonomous lineage control has been proposed and notable regional differences, in terms of symmetric division ratio, have been noted—higher in thickened epidermis of the paw skin as compared to ear and tail skin. By using our model we propose a possible explanation for the regional differences in epidermal lineage control strategies. We demonstrate how symmetric divisions can work to stabilize paw epidermis lineage, which experiences high level of micro-injuries and a lack of hair follicles as a back-up source of SCs.     

In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.

The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacterial modification enzymes HhaI methyl transferase (MTase) and HpaII MTase, alone or in combination with a human placental DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of HhaI and HpaII sites reduced CAT expression by approximately 70%-80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by greater than 95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.     

DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.     

The Role of DNA Methylation Reprogramming During Sex Determination and Transition in Zebrafish

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition.We reveal that primordial germ cells(PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization(9 dpf). When DNA methyltransferase(DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.     

DNA Methylation Landscapes of Human Fetal Development

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.