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 of the 5'-untranslated region at +298 and +351 represses BACE1 expression in mouse BV-2 microglial cells.

BACE1, which cleaves the amyloid precursor protein, is the rate-limiting enzyme for ß-amyloid peptide production, leading to the pathogenesis of Alzheimer's disease (AD). A high plasma level of homocysteine, acting as a potent methyltransferase inhibitor, is assumed to be a risk factor for AD onset. Using the demethylating drug 5-aza-2'-deoxycytidine (5-Aza), we tested whether and how BACE1 expression is regulated in mouse BV-2 microglial cells. 5-Aza increased both BACE1 mRNA and protein levels in a dose-dependent manner. Bisulfite-sequencing analysis revealed that two CpG sites at positions +298 and +351 in the 5'-untranslated region (5'-UTR) of the BACE1 gene were specifically demethylated in BV-2 cells treated with 5-Aza. In silico analysis showed that the +351 site is the STAT3/CTCF-binding site; the function of the +298 site has not been identified. To assess whether these two CpG sites play an important role in 5-Aza-induced transcriptional activation of BACE1, we constructed a BACE1 gene promoter including the 5'-UTR (-1136 to +500) fused to a CpG-free luciferase gene (pCpGL-BACE1) and its mutant pCpGL-BACE1-AA, which has substituted CG dinucleotides at the two CpG sites of pCpGL-BACE1 to AA. Promoter analysis showed a significant decrease (∼30%) in the activity of pCpGL-BACE1-AA compared with that of pCpGL-BACE1. Furthermore, in vitro methylation of these two reporter constructs showed a complete silencing of their promoter activities. Our data demonstrate that BACE1 gene expression is regulated by DNA methylation of at least two CpG sites at positions +298 and +351 in the 5'-UTR in BV-2 microglial cells.

Active DNA demethylation in human postmitotic cells correlates with activating histone modifications, but not transcription levels.

BACKGROUND: In mammals, the dynamics of DNA methylation, in particular the regulated, active removal of cytosine methylation, has remained a mystery, partly due to the lack of appropriate model systems to study DNA demethylation. Previous work has largely focused on proliferating cell types that are mitotically arrested using pharmacological inhibitors to distinguish between active and passive mechanisms of DNA demethylation. RESULTS: We explored this epigenetic phenomenon in a natural setting of post-mitotic cells: the differentiation of human peripheral blood monocytes into macrophages or dendritic cells, which proceeds without cell division. Using a global, comparative CpG methylation profiling approach, we identified many novel examples of active DNA demethylation and characterized accompanying transcriptional and epigenetic events at these sites during monocytic differentiation. We show that active DNA demethylation is not restricted to proximal promoters and that the time-course of demethylation varies for individual CpGs. Irrespective of their location, the removal of methylated cytosines always coincided with the appearance of activating histone marks. CONCLUSIONS: Demethylation events are highly reproducible in monocyte-derived dendritic cells from different individuals. Our data suggest that active DNA demethylation is a precisely targeted event that parallels or follows the modification of histones, but is not necessarily coupled to alterations in transcriptional activity.

General transcription factor binding at CpG islands in normal cells correlates with resistance to de novo DNA methylation in cancer cells.

Aberrant DNA methylation at CpG islands is thought to contribute to cancer initiation and progression, but mechanisms that establish and maintain DNA methylation status during tumorigenesis or normal development remain poorly understood. In this study, we used methyl-CpG immunoprecipitation to generate comparative DNA methylation profiles of healthy and malignant cells (acute leukemia and colorectal carcinoma) for human CpG islands across the genome. While searching for sequence patterns that characterize DNA methylation states, we discovered several nonredundant sequences in CpG islands that were resistant to aberrant de novo methylation in cancer and that resembled consensus binding sites for general transcription factors (TF). Comparing methylation profiles with global CpG island binding data for specific protein 1, nuclear respiratory factor 1, and yin-yang 1 revealed that their DNA binding activity in normal blood cells correlated strictly with an absence of de novo methylation in cancer. In addition, global evidence showed that binding of any of these TFs to their consensus motif depended on their co-occurrence with neighboring consensus motifs. In summary, our results had two major implications. First, they pointed to a major role for cooperative binding of TFs in maintaining the unmethylated status of CpG islands in health and disease. Second, our results suggest that the majority of de novo methylated CpG islands are characterized by the lack of sequence motif combinations and the absence of activating TF binding.

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.

CCAAT enhancer-binding protein beta regulates constitutive gene expression during late stages of monocyte to macrophage differentiation.

Human monocyte to macrophage differentiation is accompanied by pronounced phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are poorly understood. Here, we studied the regulation of the macrophage-specific chitotriosidase (CHIT1) gene promoter to gain insights into the mechanisms of transcriptional control during the differentiation of human blood monocytes into macrophages. We used transient transfections to define a cell type-specific minimal promoter that was mainly dependent on a proximal C/EBP motif that bound multiple C/EBP factors in gel shift assays. In depth analysis of occupied promoter elements using in vivo footprinting and chromatin immunoprecipitation analyses demonstrated the differentiation-associated recruitment of C/EBPbeta and PU.1 at the proximal promoter in parallel with CHIT1 mRNA induction. Notably, the induction of C/EBPbeta promoter binding strongly correlated with increased nuclear levels of Thr-235-phosphorylated C/EBPbeta protein during the differentiation process, whereas C/EBPbeta mRNA and total protein expression remained relatively stable. Our data suggest an important constitutive gene regulatory function for C/EBPbeta in differentiated macrophages but not in human blood monocytes.

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.

Functional analysis of promoter CpG methylation using a CpG-free luciferase reporter vector.

Methylation of CpG dinucleotides within proximal promoters is often associated with transcriptional silencing. Methylation-dependent repression is well established for hypermethylated CpG island promoters that are characterized by a high density of CpG residues. The effect of CpG DNA methylation on CpG-poor promoters is less well characterized, probably due to the lack of convenient assay systems to test promoter activities in vitro. In this report, we describe a novel luciferase reporter vector, pCpGL, which completely lacks CpG dinucleotides and can be used to study the effect of promoter DNA methylation in transfection assays. Whereas a traditional reporter vector that contains a large number of backbone CpG residues significantly represses a CpG-free promoter when methylated, our new reporter vector is only repressed due to the presence of functionally important, methylated CpG residues. The pCpGL vector provides a useful tool to study the effects of CpG methylation on CpG-rich and CpG-poor promoters.