Epigenetic Changes during Hepatic Stellate Cell Activation

Background and Aims

Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC.

Methods and Results

The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.

Conclusions

In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

Plasticity of DNA methylation in mouse T cell activation and differentiation

Background

Circulating CD4⁺ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4⁺ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results

Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion

We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.     

Active DNA demethylation and DNA repair

DNA methylation on cytosine is an epigenetic modification and is essential for gene regulation and genome stability in vertebrates. Traditionally DNA methylation was considered as the most stable of all heritable epigenetic marks. However, it has become clear that DNA methylation is reversible by enzymatic “active” DNA demethylation, with examples in plant cells, animal development and immune cells. It emerges that “pruning” of methylated cytosines by active DNA demethylation is an important determinant for the DNA methylation signature of a cell. Work in plants and animals shows that demethylation occurs by base excision and nucleotide excision repair. Far from merely protecting genomic integrity from environmental insult, DNA repair is therefore at the heart of an epigenetic activation process.     

Identification of key genes and novel immune infiltration-associated biomarkers of sepsis

Sepsis is the major cause of mortality in the intensive care unit. The aim of this study was to identify the key prognostic biomarkers of abnormal expression and immune infiltration in sepsis. In this study, a total of 36 differentially expressed genes were identified to be mainly involved in a number of immune-related Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. The hub genes (MMP9 and C3AR1) were significantly related to the prognosis of sepsis patients. The immune infiltration analysis indicated a significant difference in the relative cell content of naive B cells, follicular Th cells, activated NK cells, eosinophils, neutrophils and monocytes between sepsis and normal controls. Weighted gene co-expression network analysis and a de-convolution algorithm that quantifies the cellular composition of immune cells were used to analyse the sepsis expression data from the Gene Expression Omnibus database and to identify modules related to differential immune cells. CEBPB is the key immune-related gene that may be involved in sepsis. Gene set enrichment analysis revealed that CEBPB is involved in the processes of T cell selection, B cell–mediated immunity, NK cell activation and pathways of T cells, B cells and NK cells. Therefore, CEBPB may play a key role in the biological and immunological processes of sepsis.     

Does DNA methylation pattern mark generative development in winter rape?

In this paper we report on changes in DNA methylation pattern in rape apices and leaves during transition from vegetative to reproductive stage due to grafting and/or vernalization. Grafted plants of winter rape (Brassica napus L., var. "Górczański") (stock from vernalized, scion from non-vernalized plants) were used together with vernalized non-grafted plants. In addition, methylation status was determined also in spring rape (var. "M?ochowski") grown under normal and low temperature. The methylation-sensitive amplification polymorphism (MSAP) method with EcoRI/MspI and EcoRII/HpaII restriction enzymes was employed. The majority (ca. 68%) of analyzed loci (566 in winter and 551 in spring rape) were monomorphic, i.e. did not undergo methylation. Both cultivars showed a similar degree of methylation. 188 loci in winter and 176 in spring cultivars expressed changes in the methylation pattern. All differentially amplified fragments resulted from either full methylation of an internal cytosine or from hemi-methylation of an external cytosine. A pair-wise comparison showed that a similar number of loci underwent development-related methylation changes in apices of the winter and spring rape. The majority (80%) of changes were demethylation events in generative (vernalized) apices of the winter cultivar. However, an increased number of demethylated loci was detected in vernalized apices in comparison with generative, non-vernalized ones. In apices of vegetative and generative grafted plants the same number of demethylation events was observed. Overall, 10 MSAP loci were detected that expressed methylation changes in vernalized apices only; among them 7 loci underwent demethylation after vernalization and remained methylated in both vegetative and generative non-vernalized stage. Only 1 locus was demethylated in generative non-vernalized apices. Thus, most of demethylation events can be ascribed to vernalization and not to the generative stage. In leaves of winter rape methylation and demethylation events occurred with similar frequency, while in the spring cultivar more demethylation events were detected. The results show that during vernalization and transition to the generative stage different sets of genes are activated.     

Interleukin-12 secreted by mature dendritic cells mediates activation of NK cell function

Dendritic cells (DCs) are known to modulate immune response by activating effector cells of both the innate and the adaptive immune system. In the present study, we demonstrate that co-culture of DCs with paraformaldehyde-fixed tumor cells augments the secretion of interleukin (IL)-12 by DCs and these activated DCs upon co-culture with naive NK cells enhance the cytolytic activity of NK cells against NK-sensitive target YAC-1. Similarly, DCs isolated from tumor-bearing animals also activated NK cells in vitro. For efficient activation of NK cells, the ratio of activated DCs to NK cells is crucial. Addition of anti-IL-12 antibody to the culture system completely abolished activation of NK cells by DCs, suggesting that IL-12 secreted by DCs is an essential factor in NK cell activation. Adoptive transfer of DCs isolated from tumor-bearing animals into normal rats also induced activation of NK cells in normal animals.     

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.     

First evidence of DNA methylation in insect Tribolium castaneum

DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.     

5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes

Background

Cytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements. Whereas DNA methylation patterns are generally inherited during replication, both embryonic and somatic differentiation processes require the removal of cytosine methylation at specific gene loci to activate lineage-restricted elements. However, the exact mechanisms facilitating the erasure of DNA methylation remain unclear in many cases.

Results

We previously established human post-proliferative monocytes as a model to study active DNA demethylation. We now show, for several previously identified genomic sites, that the loss of DNA methylation during the differentiation of primary, post-proliferative human monocytes into dendritic cells is preceded by the local appearance of 5-hydroxymethylcytosine. Monocytes were found to express the methylcytosine dioxygenase Ten-Eleven Translocation (TET) 2, which is frequently mutated in myeloid malignancies. The siRNA-mediated knockdown of this enzyme in primary monocytes prevented active DNA demethylation, suggesting that TET2 is essential for the proper execution of this process in human monocytes.

Conclusions

The work described here provides definite evidence that TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine initiates targeted, active DNA demethylation in a mature postmitotic myeloid cell type.     

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.     

CpG dinucleotide-specific hypermethylation of the TNS3 gene promoter in human renal cell carcinoma

Tensin3 is a cytoskeletal regulatory protein that inhibits cell motility. Downregulation of the gene encoding Tensin3 (TNS3) in human renal cell carcinoma (RCC) may contribute to cancer cell metastatic behavior. We speculated that epigenetic mechanisms, e.g., gene promoter hypermethylation, might account for TNS3 downregulation. In this study, we identified and validated a TNS3 gene promoter containing a CpG island, and quantified the methylation level within this region in RCC. Using a luciferase reporter assay we demonstrated a functional minimal promoter activity for a 500-bp sequence within the TNS3 CpG island. Pyrosequencing enabled quantitative determination of DNA methylation of each CpG dinucleotide (a total of 43) in the TNS3 gene promoter. Across the entire analyzed CpG stretch, RCC DNA showed a higher methylation level than both non-tumor kidney DNA and normal control DNA. Out of all the CpGs analyzed, two CpG dinucleotides, specifically position 2 and 8, showed the most pronounced increases in methylation levels in tumor samples. Furthermore, CpG-specific higher methylation levels were correlated with lower TNS3 gene expression levels in RCC samples. In addition, pharmacological demethylation treatment of cultured kidney cells caused a 3-fold upregulation of Tensin3 expression. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter occurring in human RCC, suggesting an epigenetic mechanism for aberrant Tensin downregulation in human kidney cancer.