Background:The classical hierarchical model of hematopoiesis has been revised based on single‐cell RNA sequencing (scRNAseq) data, which suggest a continuous rather than a step‐wise differentiation process. However, while differentiation trajectories can be inferred from scRNAseq data, it is impossible to discern whether discreet lineage commitment decisions occur at specific points along these trajectories. The existence of such commitment points could still be compatible with a step‐wise differentiation model and their characterization at the molecular level would be essential to accurately model the hematopoietic hierarchy.Aims:This study investigates DNA methylation programming in normal hematopoiesis to identify novel molecular commitment marks during hematopoietic differentiation. We hypothesized that whole genome DNA methylation analysis facilitates the identification of such molecular commitment marks due to the progressive nature of how this mark is programmed during differentiation.Methods:Using tagmentation‐based whole‐genome bisulfite sequencing, we generated a genome‐wide DNA methylation map of murine hematopoiesis. This map encompasses 26 murine hematopoietic cell populations throughout the hematopoietic hierarchy. Per cell population we sequenced at least three independent biological replicates. Differentially methylated regions (DMRs) were identified across all populations using DSS without smoothing. Multi‐tier single cell scRNAseq data were generated from murine bone marrow using the 10X Genomics Chromium Single Cell 3’ kit.Results:Across all cell populations studied, we identified 147,232 DMRs. Hierarchical clustering of these DMRs revealed co‐regulated epigenetic regions that show progressive DNA methylation programming during hematopoietic differentiation. These dynamically regulated regions can be interpreted as specific DNA methylation programs: pan‐hematopoietic‐, lineage‐ and cell type‐specific DNA methylation programs. The DNA methylation programs were strongly enriched in hematopoietic transcription factor binding sites as well as in lineage and cell type‐specific enhancer programs. To gain further insight into how the DNA methylation programming relates to regulation of gene expression, we generated a comprehensive multi‐tier single cell gene expression map of murine bone‐marrow cells. Integration of DNA methylome programs with single cell gene expression profiles revealed a strong anti‐correlation of promoter DNA methylation dynamics and cell‐state specific gene expression patterns. The progressive and lineage‐specific nature of DNA methylation programming occurring during hematopoietic differentiation allowed us to infer a phylogenetic tree of the hematopoietic system, which is solely based on DNA methylation dynamics. We demonstrated that previously defined subpopulations of common myeloid progenitors (CMPs) show distinct DNA methylation patterns: CD55+ CMPs showed megakaryocyte/erythrocyte patterns, while CD55‐ CMPs showed DNA methylation patterns compatible with granulocyte and monocyte differentiation. Furthermore, we showed that the identified DNA methylation programs are conserved across different mouse strains which underlines the broad applicability of this resource.Summary/Conclusion:We provide a novel view on the regulation of hematopoiesis based on DNA methylation dynamics which complements recent findings from scRNAseq studies. Our work provides a rich resource to investigate DNA methylation in normal as well as in abnormal hematopoiesis across a broad range of conditions and mouse strains.

BACKGROUND/OBJECTIVES:Adipose tissue is one of the main organs regulating energy homeostasis via energy storage as well as endocrine function. The adipocyte cell number is largely determined by adipogenesis. While the molecular mechanism of adipogenesis has been extensively studied, its role in dynamic DNA methylation plasticity remains unclear. Recently, it has been shown that Tet methylcytosine dioxygenase (TET) is catalytically capable of oxidizing DNA 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) toward a complete removal of the methylated cytosine. We investigate whether expression of the Tet genes and production of hydroxymethylcytosine are required for preadipocyte differentiation.SUBJECTS/METHODS:Murine 3T3-L1 preadipocytes were used to evaluate the role of Tet1 and Tet2 genes during adipogenesis. Changes in adipogenic ability and in epigenetic status were analyzed, with and without interfering Tet1 and Tet2 expression using small interfering RNA (siRNA). The adipogenesis was evaluated by Oil-Red-O staining and induced expression of adipogenic genes using quantitative polymerase chain reaction (qPCR). Levels of 5-hmC and 5-mC were measured by MassARRAY, immunoprecipitation and GC mass spectrometry at specific loci as well as globally.RESULTS:Both Tet1 and Tet2 genes were upregulated in a time-dependent manner, accompanied by increased expression of hallmark adipogenic genes such as Pparγ and Fabp4 (P<0.05). The TET upregulation led to reduced DNA methylation and elevated hydroxymethylcytosine, both globally and specifically at the Pparγ locus (P<0.05 and P<0.01, respectively). Knockdown of Tet1 and Tet2 blocked adipogenesis (P<0.01) by repression of Pparγ expression (P<0.05). In particular, Tet2 knockdown repressed conversion of 5-mC to 5-hmC at the Pparγ locus (P<0.01). Moreover, vitamin C treatment enhanced adipogenesis (P<0.05), while fumarate treatment inhibited it (P<0.01) by modulating TET activities.CONCLUSIONS:TET proteins, particularly TET2, were required for adipogenesis by modulating DNA methylation at the Pparγ locus, subsequently by inducing Pparγ gene expression.

Background & Aims: The molecular mechanisms, in particular epigenetic alterations, underlying the genesis of cholangiocarcinomas (CC) are poorly understood. We have previously shown data of global methylation changes in CC. Here, we focus on specific microRNAs (miRNAs) that showed drastic methylation alterations in our methylome screen.

Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here, we determine the contribution of epigenetically silenced genes to the development of PDAC. We analyzed enriched, highly methylated DNAs from PDACs, chronic pancreatitis (CP) and normal tissues using CpG island microarrays and identified WNK2 as a prominent candidate tumor suppressor gene being downregulated early in PDAC development. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanIN), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in tumor than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expressions were lower in PDAC and CP compared with normal tissues both in patients and mouse models. Overexpression of WNK2 led to reduced cell growth, and WNK2 expression in tissues correlated negatively with pERK1/2 expression, a downstream target of WNK2 responsible for cell proliferation. Downregulation of WNK2 by promoter hypermethylation occurs early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway.

negative feedback loop due to the induction of AHR pathway by dioxin-like compounds.Our results regarding a key gene in the AHR pathway will have relevance beyond tobacco smoke exposure given the key role played by this pathway in the metabolism of many environmental carcinogens.

High-grade glioma is the most common brain tumor in adults, and the prognosis for patients diagnosed with this type of cancer is still poor. The biological behavior of the tumors is correlated to t ...

A previously isolated cDNA sequence with homology to the long-range repeat (LRR) cluster in chromosome 1 of the house mouse, Mus musculus, was identified as derived from a 1.3 kb polyadenylated RNA. This transcript belongs to a family of polyadenylated RNAs which are synthesized from a multicopy gene included in the LRR copies. The representation of the 1.3 kb transcript in genomic DNA was studied in lambda and cosmid clones from the LRR cluster. Two different types of LRRs were detected with respect to the arrangement of coding regions. In the type-1 arrangement, the sequence is split into five exons, and in the type-2 arrangement, into six exons. The respective exons with their flanking regions were sequenced. The analysis of splice signals revealed that LRR copies with a type-1 arrangement are presumably the source of the 1.3 kb transcript. The 1.3 kb transcript has sequence homology to a human gene encoding Sp100, a nuclear antigen recognized by autoantibodies from patients suffering from some autoimmune diseases including primary biliary cirrhosis. Mouse exons II and III exhibit 71% homology at the nucleotide level and 56% homology at the amino acid level to the human Sp100 cDNA. We mapped the human Sp100 gene to chromosome 2. This location corroborates the assumption that the human Sp100 gene and the mouse LRR gene are homologous, as the human chromosome 2 contains the segment which is homologous to the mouse LRR region.

To facilitate the use of large-insert bacterial clones for functional analysis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein–Barr virus replicon, oriP, is included to ensure stable episomal propagation of the large insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable selection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeting in mammalian chromosomes are also present. In addition, we demonstrate that the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been included in the vector to enable linearization of the large-insert clones, e.g., for optical mapping studies. The pPAC4 vector has been used to generate libraries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experiments, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene into mutant cells or transgenic or knock-out animals.