Dynamics of DNA methylation in differentiating hematopoietic cells

The methylation of CpG dinucleotides represents an epigenetic mark that is crucial for regulating the normal progression of numerous biological processes including development and cell differentiation. During the last decade, it became increasingly clear that methylation patterns are not static but may adapt to various cellular requirements. Regarding normal somatic cells, the dynamic of DNA methylation including its extent throughout the genome as well as its implication in cellular differentiation is largely unknown. In the context of the present thesis, it was demonstrated that several cell type or cell lineage specific genes harboured a specific methylation profile. Interestingly, those differences in DNA methylation were mostly confined to regions upstream or downstream of the core promoter and preferentially affected CpG poor DNA regions. The gene regulatory relevance of DNA sequences affected by dynamical alterations in the methylation pattern, may be studied by means of transient transfection assays. For this purpose, a novel CpG free luciferase reporter vector was designed that provides a simple and robust tool for analysing effects of DNA methylation within CpG poor as well as CpG rich DNA stretches on gene expression. As particularly the regulated and active removal of methyl CpG marks still remains controversial, the major aim of the present work was the characterization of this epigenetic phenomenon in a natural setting of post mitotic cells: the proliferation independent differentiation of human peripheral blood monocytes into dendritic cells or macrophages, respectively. Using a global, comparative CpG methylation profiling approach that was directed to detect differentially methylated regions in CpG rich as well as CpG poor DNA stretches, 45 examples for active demethylation were identified. The validation by a bisulfite conversion based technique and the characterization of a selected subset revealed that DNA demethylation was not restricted to promoter regions and that the time course varied for individual CpGs. Irrespective of their location, the removal of methylated cytosines strictly coincided with the appearance of activating histone marks indicating the presence of cis acting elements. Since demethylation events were highly reproducible between monocyte derived dendritic cells from distinct donors, the present data suggest that active demethylation is a precisely targeted process. The comparison of the global methylation data with the genome wide mRNA expression profiles demonstrated that active DNA demethylation is not always directly followed by transcriptional activation. Probably, gene activation is a multilevel process that is dependent on various genetic and epigenetic factors. Thereby, CpG demethylation seems to be a necessary prerequisite for priming the chromatin structure for transcription factor binding

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

Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia

The methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. Because aberrant hypermethylation may be used as a marker for disease, a sensitive method for the global detection of DNA methylation events is of particular importance. We describe a novel and robust technique, called methyl-CpG immunoprecipitation, which allows the unbiased genome-wide profiling of CpG methylation in limited DNA samples. The approach is based on a recombinant, antibody-like protein that efficiently binds native CpG-methylated DNA. In combination with CpG island microarrays, the technique was used to identify >100 genes with aberrantly methylated CpG islands in three myeloid leukemia cell lines. Interestingly, within all hypermethylation targets, genes involved in transcriptional regulation were significantly overrepresented. More than half of the identified genes were absent in microarray expression studies in either leukemia or normal monocytes, indicating that hypermethylation in cancer may be largely independent of the transcriptional status of the affected gene. Most individually tested genes were also hypermethylated in primary blast cells from acute myeloid leukemia patients, suggesting that our approach can identify novel potential disease markers. The technique may prove useful for genome-wide comparative methylation analysis not only in malignancies

Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity

DNA methylation participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. With few exceptions, research thus far has focused on gene promoters, and little is known about the extent, functional relevance, and regulation of cell type-specific DNA methylation at promoter-distal sites. Here, we present a comprehensive analysis of differential DNA methylation in human conventional CD4+ T cells (Tconv) and CD4+CD25+ regulatory T cells (Treg), cell types whose differentiation and function are known to be controlled by epigenetic mechanisms. Using a novel approach that is based on the separation of a genome into methylated and unmethylated fractions, we examined the extent of lineage-specific DNA methylation across whole gene loci. More than 100 differentially methylated regions (DMRs) were identified that are present mainly in cell type-specific genes (e.g., FOXP3, IL2RA, CTLA4, CD40LG, and IFNG) and show differential patterns of histone H3 lysine 4 methylation. Interestingly, the majority of DMRs were located at promoter-distal sites, and many of these areas harbor DNA methylation-dependent enhancer activity in reporter gene assays. Thus, our study provides a comprehensive, locus-wide analysis of lineage-specific methylation patterns in Treg and Tconv cells, links cell type-specific DNA methylation with histone methylation and regulatory function, and identifies a number of cell type-specific, CpG methylation-sensitive enhancers in immunologically relevant genes

Dap12 expression in activated microglia from retinoschisin-deficient retina and its PU.1-dependent promoter regulation

Several alterations in the expression of immune-related transcripts were identified recently in the degenerating retina of the retinoschisin knockout (Rs1h(-/Y)) mouse, including the strong expression of the adaptor protein Dap12. As Dap12 is found in leukocytes, we hypothesized that its disease-related expression may be confined to activated retinal microglia cells. To test this hypothesis, we established a procedure for isolation and culture of retinal microglia cells and performed genome-wide expression profiling from Rs1h(-/Y) and control microglia. While retaining their activated state in culture, ex vivo microglia expressed high levels of Dap12 and the transcription factor PU.1. The activation-dependent induction of Dap12 was also confirmed in the microglia cell line BV-2 following in vitro stimulation. To examine the transcriptional regulation of Dap12 further, macrophage cell lines were transfected with several Dap12 reporter constructs. Promoter deletion assays and site-directed mutagenesis experiments demonstrated an essential role of evolutionarily conserved PU.1 consensus sites in the proximal -104/+118 Dap12 promoter. In vitro and in vivo binding of PU.1 to this promoter region was demonstrated using EMSA and chromatin immunoprecipitation. Knockdown of PU.1 by RNA interference caused a significant reduction of endogenous Dap12 expression and re-expression, and activation of PU.1 in PU.1(-/-) progenitor cells induced Dap12 transcription. Taken together, our results indicate that activated microglia from degenerating retinae express high levels of Dap12 and PU.1, and PU.1 controls the myeloid-specific regulation of Dap12 directly and may also play a general role in microglia gene expression during retinal degeneration

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers

Contains fulltext : 118733.pdf (publisher's version ) (Open Access)Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 x 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer