Introduction: Metals in Biology: α-Ketoglutarate/Iron-Dependent Dioxygenases

Four minireviews deal with aspects of the α-ketoglutarate/iron-dependent dioxygenases in this eighth Thematic Series on Metals in Biology. The minireviews cover a general introduction and synopsis of the current understanding of mechanisms of catalysis, the roles of these dioxygenases in post-translational protein modification and de-modification, the roles of the ten-eleven translocation (Tet) dioxygenases in the modification of methylated bases (5mC, T) in DNA relevant to epigenetic mechanisms, and the roles of the AlkB-related dioxygenases in the repair of damaged DNA and RNA. The use of α-ketoglutarate (alternatively termed 2-oxoglutarate) as a co-substrate in so many oxidation reactions throughout much of nature is notable and has surprisingly emerged from biochemical and genomic analysis. About 60 of these enzymes are now recognized in humans, and a number have been identified as having critical functions.     

Novel extracellular enzymes (ligninases) of Phanerochaete chrysosporium

Abstract 3 New spectrophotometric enzyme assays were developed for the study of microbial lignin-degrading enzymes. The conversion of 2-methoxy-3-phenylbenzoic acid to 2-hydroxy-3-phenylbenzoic acid led to the discovery of an extracellular, aromatic methyl ether demethylase produced by the white-rot fungus Phanerochaete chrysosporium . The conversion of methyl 2-hydroxy-3-phenylbenzoate to 2-hydroxy-3-phenylbenzoic acid allowed the identification of an extracellular, aromatic methyl ester esterase produced by this fungus. The Phanerochaete sp. also excreted an enzyme complex that oxidized 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, probably to aliphatic products. All 3 novel enzyme activities were produced together with, and probably comprise a part of, the Phanerochaete ligninolytic enzyme complex. Unlike previously known ligninases, these enzymes did not oxidize 3,4-dimethoxybenzyl alcohol. All 3 were H₂O₂-dependent and were activated by Mn²⁺ ions.     

AlCl3 exposure regulates neuronal development by modulating DNA modification

BACKGROUNDAs the third most abundant element, aluminum is widespread in the environment. Previous studies have shown that aluminum has a neurotoxic effect and its exposure can impair neuronal development and cognitive function.AIMTo study the effects of aluminum on epigenetic modification in neural stem cells and neurons. METHODSNeural stem cells were isolated from the forebrain of adult mice. Neurons were isolated from the hippocampi tissues of embryonic day 16-18 mice. AlCl₃ at 100 and 200 μmol/L was applied to stem cells and neurons. RESULTSAluminum altered the differentiation of adult neural stem cells and caused apoptosis of newborn neurons while having no significant effects on the proliferation of neural stem cells. Aluminum application also significantly inhibited the dendritic development of hippocampal neurons. Mechanistically, aluminum exposure significantly affected the levels of DNA 5-hydroxy-methylcytosine, 5-methylcytosine, and N⁶-methyladenine in stem cells and neurons. CONCLUSIONOur findings indicate that aluminum may regulate neuronal development by modulating DNA modifications.     

DNA methylation and demethylation link the properties of mesenchymal stem cells: Regeneration and immunomodulation

Mesenchymal stem cells (MSCs) are a heterogeneous population that can be isolated from various tissues, including bone marrow, adipose tissue, umbilical cord blood, and craniofacial tissue. MSCs have attracted increasingly more attention over the years due to their regenerative capacity and function in immunomodulation. The foundation of tissue regeneration is the potential of cells to differentiate into multiple cell lineages and give rise to multiple tissue types. In addition,the immunoregulatory function of MSCs has provided insights into therapeutic treatments for immune-mediated diseases. DNA methylation and demethylation are important epigenetic mechanisms that have been shown to modulate embryonic stem cell maintenance, proliferation, differentiation and apoptosis by activating or suppressing a number of genes. In most studies, DNA hypermethylation is associated with gene suppression, while hypomethylation or demethylation is associated with gene activation. The dynamic balance of DNA methylation and demethylation is required for normal mammalian development and inhibits the onset of abnormal phenotypes. However, the exact role of DNA methylation and demethylation in MSC-based tissue regeneration and immunomodulation requires further investigation. In this review, we discuss how DNA methylation and demethylation function in multi-lineage cell differentiation and immunomodulation of MSCs based on previously published work. Furthermore, we discuss the implications of the role of DNA methylation and demethylation in MSCs for the treatment of metabolic or immune-related diseases.     

The function and regulation of TET2 in innate immunity and inflammation

TET2,a member of ten-eleven translocation(TET)family as a-ketoglutarate-and Fe2+-dependent dioxygenase catalyzing the iterative oxidation of 5-methylcytosine(5mC),has been widely recognized to be an important regulator for normal hematopoiesis especially myelopoiesis.Mutation and dysregulation of TET2 contribute to the development of multiple hematological malignancies.Recent studies reveal that TET2 also plays an important role in innate immune homeostasis by promoting DNA demethylation or independent of its enzymatic activity.Here,we focus on the functions of TET2 in the initiation and resolution of inflammation through epigenetic regulation and signaling network.In addition,we highlight regulation of TET2 at various molecular levels as well as the correlated inflammatory diseases,which will provide the insight to intervene in the pathological process caused by TET2 dysregulation.     

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.     

PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.