DNA methylation regulates gene expression in a variety of processes, including mouse embryonic development. Four catalytically active enzymes function in mice as DNA methyltransferases (Dnmts) and as transcriptional regulators. Inactivation of Dnmt3b results in mouse embryonic lethality, but which activities are involved is unclear. Here we show that catalytically inactive Dnmt3b restores a majority of methylation and expression changes deregulated in the absence of Dnmt3b, and as a result, mice survive embryonic development. Thus, Dnmt3b functions as an accessory cofactor supporting catalytic activities performed by other Dnmts. We further demonstrate that Dnmt3b is linked to a control of major developmental pathways, including Wnt and hedgehog signaling. Dnmt3b directly represses Wnt9b whose aberrant up-regulation contributes to embryonic lethality of Dnmt3b knockout embryos. Our results highlight that Dnmt3b is a multifaceted protein that serves as an enzyme, an accessory factor for other methyltransferases, and as a transcriptional repressor in mouse embryogenesis.

DNA methylation regulates gene transcription in many physiological processes in mammals including development and haematopoiesis. It is catalysed by several DNA methyltransferases, including Dnmt3b that mediates both methylation-dependant and independent gene repression. Dnmt3b is critical for mouse embryogenesis and functions as a tumour suppressor in haematologic malignancies in mice. However, the extent to which Dnmt3b's catalytic activity (CA) is involved in development and cancer is unclear. We used a mouse model expressing catalytically inactive Dnmt3b (Dnmt3bCI) to study a role of Dnmt3b's CA in development and cancer. We utilized global approaches including Whole-genome Bisulfite sequencing and RNA-seq to analyse DNA methylation and gene expression to identify putative targets of Dnmt3b's CA. To analyse postnatal development and haematopoiesis, we used tissue staining, histological and FACS analysis. To determine potential involvement of selected genes in lymphomagenesis, we used overexpression and knock down approaches followed by in vitro growth assays. We show that mice expressing Dnmt3bCI only, survive postnatal development and develop ICF (the immunodeficiency-centromeric instability-facial anomalies) -like syndrome. The lack of Dnmt3b's CA promoted fibroblasts transformation in vitro, accelerated MLL-AF9 driven Acute Myeloid Leukaemia and MYC-induced T-cell lymphomagenesis in vivo. The elimination of Dnmt3b's CA resulted in decreased methylation of c-Met promoter and its upregulation, activated oncogenic Met signalling, Stat3 phosphorylation and up-regulation of Lin28b promoting lymphomagenesis. Our data demonstrates that Dnmt3b's CA is largely dispensable for mouse development but critical to prevent tumourigenesis by controlling events involved in cellular transformation. This study was supported by Department of Anatomy and Cell Biology and Cancer Centre at the University of Florida start-up funds, NIH/NCI grant 1R01CA188561-01A1 (R.O.).

DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, Hairy cell leukemia and Diffuse Large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/- mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/- mice developed T-cell lymphomas, chronic lymphocytic leukemia and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/- mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1 and Trip13, and p53 down-regulation were major events contributing to Dnmt3b+/- lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo whereas accessory and/or methylation-independent repressive functions are important to prevent T-cell transformation.

Cytosine methylation contributes to the regulation of gene expression and normal hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases that include DNMT1, DNMT3A, and DNMT3B. Peripheral T–cell lymphomas (PTCLs) represent aggressive mature T–cell malignancies exhibiting a broad spectrum of clinical features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we profiled DNA methylation and gene expression of PTCLs. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples, suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose genetic and pharmacologic inactivation inhibited the proliferation of T–cell lines by inducing G2-M arrest and apoptosis. Our data thus show that human PTCLs have a significant number of recurrent methylation alterations that may affect the expression of genes critical for proliferation whose targeting might be beneficial in anti-lymphoma treatments.

Mutations in the DNA methyltransferase 3A ( DNMT3A ) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A (R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A , ectopically or endogenously, in vitro and in vivo . Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A (R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.