MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties

The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor suppressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein complexes with different biochemical and functional properties.     

N6-methyladenosine demethylase FTO promotes M1 and M2 macrophage activation

Macrophage polarization is the driving force of various inflammatory diseases, especially those involved in M1/M2 imbalance. N6-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes that affects multiple biological processes, including those involved developmental arrest and immune response. However, the role of m⁶A in macrophage polarization remains unclear. This study found that FTO silencing significantly suppressed both M1 and M2 polarization. FTO depletion decreased the phosphorylation levels of IKKα/β, IκBα and p65 in the NF-κB signaling pathway. The expression of STAT1 was downregulated in M1-polarized macrophages while the expression of STAT6 and PPAR-γ decreased in M2 polarization after FTO knockdown. The actinomycin D experiments showed that FTO knockdown accelerated mRNA decay of STAT1 and PPAR-γ. Furthermore, the stability and expression of STAT1 and PPAR-γ mRNAs increased when the m⁶A reader YTHDF2 was silenced. In conclusion, our results suggest that FTO knockdown inhibits the NF-κB signaling pathway and reduces the mRNA stability of STAT1 and PPAR-γ via YTHDF2 involvement, thereby impeding macrophage activation. These findings indicated a previously unrecognized link between FTO and macrophage polarization and might open new avenues for research into the molecular mechanisms of macrophage polarization-related diseases.     

H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing

DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.     

Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.     

Lanosterol 14 alpha-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediate

Conditions have been identified which permit metabolic formation of the third oxidized intermediate in the lanosterol 14 alpha-methyl demethylase reaction cascade. Metabolism of either the immediate precursor substrate 3 beta-hydroxylanost-8-en-32-al or lanost-8-ene-3 beta,32-diol under mixed function oxidase conditions affords formation of the intermediate. It must be emphasized that the intermediate can only be detected if saponification procedures are omitted during sterol isolation. Comparative chemical and biochemical studies of the isolated metabolite with 3 beta,15 alpha-dihydroxylanost-8-en-32-al reveal that the metabolite is not the 15 alpha-hydroxylanosterol aldehyde, a putative demethylase intermediate. The metabolite is efficiently converted to the demethylated delta 8,14-diene sterol in the absence of molecular oxygen or NADPH, thus supporting its identity as the final oxidized intermediate in the lanosterol 14 alpha-methyl demethylase cascade. 1H NMR analysis shows a proton resonance at 7.86 ppm consistent with a formyloxy proton. Mass spectral and infrared analysis of the metabolite clearly establish oxygen insertion into the immediate precursor substrate, 3 beta-hydroxylanost-8-en-32-al. Collectively, the biochemical and chemical characteristics of the metabolite support a structural assignment for the metabolite as 14 alpha-formyloxy-lanost-8-en-3 beta-ol.     

Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation

The mammalian DNA glycosylase-methyl-CpG binding domain protein 4 (MBD4)-is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we present three structures of the MBD4 C-terminal glycosylase domain (wild-type and its catalytic mutant D534N), in complex with DNA containing a G:T or G:5hmU mismatch. MBD4 flips the target nucleotide from the double-stranded DNA. The catalytic mutant D534N captures the intact target nucleotide in the active site binding pocket. MBD4 specifically recognizes the Watson-Crick polar edge of thymine or 5hmU via the O(2), N(3) and O(4) atoms, thus restricting its activity to thymine/uracil-based modifications while excluding cytosine and its derivatives. The wild-type enzyme cleaves the N-glycosidic bond, leaving the ribose ring in the flipped state, while the cleaved base is released. Unexpectedly, the C(1)' of the sugar has yet to be hydrolyzed and appears to form a stable intermediate with one of the side chain carboxyl oxygen atoms of D534, via either electrostatic or covalent interaction, suggesting a different catalytic mechanism from those of other DNA glycosylases.     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.