Combined Effects of Clonal Hematopoiesis and Carotid Stenosis on Cardiovascular Mortality.

BACKGROUND: The expansion of hematopoietic stem cells caused by acquired somatic mutations (clonal hematopoiesis [CH]) is a novel cardiovascular risk factor. The prognostic value of CH in patients with carotid atherosclerosis remains to be evaluated. OBJECTIVES: This study assessed the prognostic significance of CH in patients with atherosclerosis as detected by ultrasound of the carotid artery. METHODS: We applied deep sequencing of selected genomic regions within the genes DNMT3A, TET2, ASXL1, and JAK2 to screen for CH in 968 prospectively collected patients with asymptomatic carotid atherosclerosis evaluated by duplex sonography. RESULTS: We detected clonal markers at variant allele frequency ≥2% in 133 (13.7%) of 968 patients (median age 69.2 years), with increasing prevalence at advanced age. Multivariate analyses including age and established cardiovascular risk factors revealed overall presence of CH to be significantly associated with increased risk of cardiovascular death (HR: 1.50; 95% CI: 1.12-2.00; P = 0.007), reflected also at the single gene level. The effect of CH was more pronounced in older patients and independent of the patients' inflammatory status as measured by high-sensitivity C-reactive protein. Simultaneous assessment of CH and degree of carotid stenosis revealed combined effects on cardiovascular mortality, depicted by a superior risk for patients with >50% stenosis and concomitant CH (adjusted HR: 1.60; 95% CI: 1.08-2.38; P = 0.020). CONCLUSIONS: CH status in combination with the extent of carotid atherosclerosis jointly predict long-term mortality. Determination of CH can provide additional prognostic information in patients with asymptomatic carotid atherosclerosis.

Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment.

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.

Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells.

DNA methyltransferase 3A (DNMT3A) and its catalytically inactive cofactor DNA methyltransferase 3-Like (DNMT3L) proteins form functional heterotetramers to deposit DNA methylation in mammalian germ cells. While both proteins have an ATRX-DNMT3-DNMT3L (ADD) domain that recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0), the combined and differential roles of the domains in the two proteins have not been fully defined in vivo. Here we investigate DNA methylation landscapes in female and male germ cells derived from mice with loss-of-function amino acid substitutions in the ADD domains of DNMT3A and/or DNMT3L. Mutations in either the DNMT3A-ADD or the DNMT3L-ADD domain moderately decrease global CG methylation levels, but to different degrees, in both germ cells. Furthermore, when the ADD domains of both DNMT3A and DNMT3L lose their functions, the CG methylation levels are much more reduced, especially in oocytes, comparable to the impact of the Dnmt3a/3L knockout. In contrast, aberrant accumulation of non-CG methylation occurs at thousands of genomic regions in the double mutant oocytes and spermatozoa. These results highlight the critical role of the ADD-H3K4me0 binding in proper CG and non-CG methylation in germ cells and the various impacts of the ADD domains of the two proteins.

Structure-guided functional suppression of AML-associated DNMT3A hotspot mutations.

DNA methyltransferases DNMT3A- and DNMT3B-mediated DNA methylation critically regulate epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote polymerization, leading to aberrant DNA methylation that may contribute to the pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the mutation-induced functional misregulation of DNMT3A remains unclear. Here, we report the crystal structures of the DNMT3A methyltransferase domain, revealing a molecular basis for its oligomerization behavior distinct to DNMT3B, and the enhanced intermolecular contacts caused by the R882H or R882C mutation. Our biochemical, cellular, and genomic DNA methylation analyses demonstrate that introducing the DNMT3B-converting mutations inhibits the R882H-/R882C-triggered DNMT3A polymerization and enhances substrate access, thereby eliminating the dominant-negative effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutations-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.

Epigenetic Activation of Cytochrome P450 1A2 Sensitizes Hepatocellular Carcinoma Cells to Sorafenib.

Cytochrome P450 1A2 (CYP1A2) is a known tumor suppressor in hepatocellular carcinoma (HCC), but its expression is repressed in HCC and the underlying mechanism is unclear. In this study, we investigated the epigenetic mechanisms of CYP1A2 repression and potential therapeutic implications. In HCC tumor tissues, the methylation rates of CYP1A2 CpG island (CGI) and DNA methyltransferase (DNMT) 3A protein levels were significantly higher, and there was a clear negative correlation between DNMT3A and CYP1A2 protein expression. Knockdown of DNMT3A by siRNA significantly increased CYP1A2 expression in HCC cells. Additionally, treating HCC cells with decitabine (DAC) resulted in a dose-dependent upregulation of CYP1A2 expression by reducing the methylation level of CYP1A2 CGI. Furthermore, we observed a decreased enrichment of H3K27Ac in the promoter region of CYP1A2 in HCC tissues. Treatment with the trichostatin A (TSA) restored CYP1A2 expression in HCC cells by increasing H3K27Ac levels in the CYP1A2 promoter region. Importantly, combination treatment of sorafenib with DAC or TSA resulted in a leftward shift of the dose-response curve, lower IC50 values, and reduced colony numbers in HCC cells. Our findings suggest that hypermethylation of the CGI at the promoter, mediated by the high expression of DNMT3A, and hypoacetylation of H3K27 in the CYP1A2 promoter region, leads to CYP1A2 repression in HCC. Epigenetic drugs DAC and TSA increase HCC cell sensitivity to sorafenib by restoring CYP1A2 expression. Our study provides new insights into the epigenetic regulation of CYP1A2 in HCC and highlights the potential of epigenetic drugs as a therapeutic approach for HCC. SIGNIFICANCE STATEMENT: This study marks the first exploration of the epigenetic mechanisms underlying cytochrome P450 (CYP) 1A2 suppression in hepatocellular carcinoma (HCC). Our findings reveal that heightened DNA methyltransferase expression induces hypermethylation of the CpG island at the promoter, coupled with diminished H3K27Ac levels, resulting in the repression of CYP1A2 in HCC. The use of epigenetic drugs such as decitabine and trichostatin A emerges as a novel therapeutic avenue, demonstrating their potential to restore CYP1A2 expression and enhance sorafenib sensitivity in HCC cells.

Chronic corticosterone exposure in rats induces sex-specific alterations in hypothalamic reelin fragments, MeCP2, and DNMT3a protein levels.

Women are disproportionately affected by stress-related disorders like depression. In our prior research, we discovered that females exhibit lower basal hypothalamic reelin levels, and these levels are differentially influenced by chronic stress induced through repeated corticosterone (CORT) injections. Although epigenetic mechanisms involving DNA methylation and the formation of repressor complexes by DNA methyl-transferases (DNMTs) and Methyl-CpG binding protein 2 (MeCP2) have been recognized as regulators of reelin expression in vitro, there is limited understanding of the impact of stress on the epigenetic regulation of reelin in vivo and whether sex differences exist in these mechanisms. To address these questions, we conducted various biochemical analyses on hypothalamic brain samples obtained from male and female rats previously treated with either 21 days of CORT (40 mg/kg) or vehicle (0.9 % saline) subcutaneous injections. Upon chronic CORT treatment, a reduction in reelin fragment NR2 was noted in males, while the full-length molecule remained unaffected. This decrease paralleled with an elevation in MeCP2 and a reduction in DNMT3a protein levels only in males. Importantly, sex differences in baseline and CORT-induced reelin protein levels were not associated with changes in the methylation status of the Reln promoter. These findings suggest that CORT-induced reelin decreases in the hypothalamus may be a combination of alterations in downstream processes beyond gene transcription. This research brings novel insights into the sexually distinct consequences of chronic stress, an essential aspect to understand, particularly concerning its role in the development of depression.

Curcumin regulates pulmonary extracellular matrix remodeling and mitochondrial function to attenuate pulmonary fibrosis by regulating the miR-29a-3p/DNMT3A axis.

Epigenetic regulation and mitochondrial dysfunction are essential to the progression of idiopathic pulmonary fibrosis (IPF). Curcumin (CCM) in inhibits the progression of pulmonary fibrosis by regulating the expression of specific miRNAs and pulmonary fibroblast mitochondrial function; however, the underlying mechanism is unclear. C57BL/6 mice were intratracheally injected with bleomycin (5 mg/kg) and treated with CCM (25 mg/kg body weight/3 times per week, intraperitoneal injection) for 28 days. Verhoeff-Van Gieson, Picro sirius red, and Masson's trichrome staining were used to examine the expression and distribution of collagen and elastic fibers in the lung tissue. Pulmonary fibrosis was determined using micro-computed tomography and transmission electron microscopy. Human pulmonary fibroblasts were transfected with miR-29a-3p, and RT-qPCR, immunostaining, and western blotting were performed to determine the expression of DNMT3A and extracellular matrix collagen-1 (COL1A1) and fibronectin-1 (FN1) levels. The expression of mitochondrial electron transport chain complex (MRC) and mitochondrial function were detected using western blotting and Seahorse XFp Technology. CCM in increased the expression of miR-29a-3p in the lung tissue and inhibited the DNMT3A to reduce the COL1A1 and FN1 levels leading to pulmonary extracellular matrix remodeling. In addition, CCM inhibited pulmonary fibroblasts MRC and mitochondrial function via the miR-29a-3p/DNMT3A pathway. CCM attenuates pulmonary fibrosis via the miR-29a-3p/DNMT3A axis to regulate extracellular matrix remodeling and mitochondrial function and may provide a new therapeutic intervention for preventing pulmonary fibrosis.

Natural history of clonal haematopoiesis seen in real-world haematology settings.

Recursive partitioning of healthy consortia led to the development of the Clonal Hematopoiesis Risk Score (CHRS) for clonal haematopoiesis (CH); however, in the practical setting, most cases of CH are diagnosed after patients present with cytopenias or related symptoms. To address this real-world population, we characterize the clinical trajectories of 94 patients with CH and distinguish CH harbouring canonical DNMT3A/TET2/ASXL1 mutations alone ('sole DTA') versus all other groups ('non-sole DTA'). TET2, rather than DNMT3A, was the most prevalent mutation in the real-world setting. Sole DTA patients did not progress to myeloid neoplasm (MN) in the absence of acquisition of other mutations. Contrastingly, 14 (20.1%) of 67 non-sole DTA patients progressed to MN. CHRS assessment showed a higher frequency of high-risk CH in non-sole DTA (vs. sole DTA) patients and in progressors (vs. non-progressors). RUNX1 mutation conferred the strongest risk for progression to MN (odds ratio [OR] 10.27, 95% CI 2.00-52.69, p = 0.0053). The mean variant allele frequency across all genes was higher in progressors than in non-progressors (36.9% ± 4.62% vs. 24.1% ± 1.67%, p = 0.0064). This analysis in the post-CHRS era underscores the natural history of CH, providing insight into patterns of progression to MN.

EBV+ nodal T/NK-cell lymphoma associated with clonal hematopoiesis and structural variations of the viral genome.

ABSTRACT: Epstein-Barr virus (EBV)-positive (EBV+) nodal T- and natural killer (NK)-cell lymphoma is a peripheral T-cell lymphoma (EBV+ nPTCL) that presents as a primary nodal disease with T-cell phenotype and EBV-harboring tumor cells. To date, the genetic aspect of EBV+ nPTCL has not been fully investigated. In this study, whole-exome and/or whole-genome sequencing was performed on 22 cases of EBV+ nPTCL. TET2 (68%) and DNMT3A (32%) were observed to be the most frequently mutated genes whose presence was associated with poor overall survival (P = .004). The RHOA p.Gly17Val mutation was identified in 2 patients who had TET2 and/or DNMT3A mutations. In 4 patients with TET2/DNMT3A alterations, blood cell-rich tissues (the bone marrow [BM] or spleen) were available as paired normal samples. Of 4 cases, 3 had at least 1 identical TET2/DNMT3A mutation in the BM or spleen. Additionally, the whole part of the EBV genome was sequenced and structural variations (SVs) were found frequent among the EBV genomes (63%). The most frequently identified type of SV was deletion. In 1 patient, 4 pieces of human chromosome 9, including programmed death-ligand 1 gene (PD-L1) were identified to be tandemly incorporated into the EBV genome. The 3' untranslated region of PD-L1 was truncated, causing a high-level of PD-L1 protein expression. Overall, the frequent TET2 and DNMT3A mutations in EBV+ nPTCL seem to be closely associated with clonal hematopoiesis and, together with the EBV genome deletions, may contribute to the pathogenesis of this intractable lymphoma.

Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma.

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.

Downregulation of UBB potentiates SP1/VEGFA-dependent angiogenesis in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC.

Skeletal abnormalities in mice with Dnmt3a missense mutations.

Overgrowth and intellectual disability disorders in humans are typified by length/height and/or head circumference ≥ 2 standard deviations above the mean as well as intellectual disability and behavioral comorbidities, including autism and anxiety. Tatton-Brown-Rahman Syndrome is one type of overgrowth and intellectual disability disorder caused by heterozygous missense mutations in the DNA methyltransferase 3A (DNMT3A) gene. Numerous DNMT3A mutations have been identified in Tatton-Brown-Rahman Syndrome patients and may be associated with varying phenotype severities of clinical presentation. Two such mutations are the R882H and P904L mutations which result in severe and mild phenotypes, respectively. Mice with paralogous mutations (Dnmt3aP900L/+ and Dnmt3aR878H/+) exhibit overgrowth in their long bones (e.g., femur, humerus), but the mechanisms responsible for their skeletal overgrowth remain unknown. The goal of this study is to characterize skeletal phenotypes in mouse models of Tatton-Brown-Rahman Syndrome and identify potential cellular mechanisms involved in the skeletal overgrowth phenotype. We report that mature mice with the Dnmt3aP900L/+ or Dnmt3aR878H/+ mutation exhibit tibial overgrowth, cortical bone thinning, and weakened bone mechanical properties. Dnmt3aR878H/+ mutants also contain larger bone marrow adipocytes while Dnmt3aP900L/+ mutants show no adipocyte phenotype compared to control animals. To understand the potential cellular mechanisms regulating these phenotypes, growth plate chondrocytes, osteoblasts, and osteoclasts were assessed in juvenile mutant mice using quantitative static histomorphometry and dynamic histomorphometry. Tibial growth plates appeared thicker in mutant juvenile mice, but no changes were observed in osteoblast activity or osteoclast number in the femoral mid-diaphysis. These studies reveal new skeletal phenotypes associated with Tatton-Brown-Rahman Syndrome in mice and provide a rationale to extend clinical assessments of patients with this condition to include bone density and quality testing. These findings may be also informative for skeletal characterization of other mouse models presenting with overgrowth and intellectual disability phenotypes.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

DNMT3A Cooperates with YAP/TAZ to Drive Gallbladder Cancer Metastasis.

Gallbladder cancer (GBC) is an extremely lethal malignancy with aggressive behaviors, including liver or distant metastasis; however, the underlying mechanisms driving the metastasis of GBC remain poorly understood. In this study, it is found that DNA methyltransferase DNMT3A is highly expressed in GBC tumor tissues compared to matched adjacent normal tissues. Clinicopathological analysis shows that DNMT3A is positively correlated with liver metastasis and poor overall survival outcomes in patients with GBC. Functional analysis confirms that DNMT3A promotes the metastasis of GBC cells in a manner dependent on its DNA methyltransferase activity. Mechanistically, DNMT3A interacts with and is recruited by YAP/TAZ to recognize and access the CpG island within the CDH1 promoter and generates hypermethylation of the CDH1 promoter, which leads to transcriptional silencing of CDH1 and accelerated epithelial-to-mesenchymal transition. Using tissue microarrays, the association between the expression of DNMT3A, YAP/TAZ, and CDH1 is confirmed, which affects the metastatic ability of GBC. These results reveal a novel mechanism through which DNMT3A recruitment by YAP/TAZ guides DNA methylation to drive GBC metastasis and provide insights into the treatment of GBC metastasis by targeting the functional connection between DNMT3A and YAP/TAZ.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.

DNA methyltransferase 3a-induced hypermethylation of the fructose-1,6-bisphosphatase-2 promoter contributes to gastric carcinogenesis.

BACKGROUND: Aberrant DNA methylation has been implicated in the development of gastric cancer (GC). In our previous study, we demonstrated that fructose-1,6-bisphosphatase-2 (FBP2), an enzyme that suppresses cell glycolysis and growth, is downregulated in GC due to promoter methylation. However, the precise mechanism underlying this process remains unknown. Thus, this study aimed to elucidate the mechanisms involved in FBP2 promoter hypermethylation. METHODS AND RESULTS: The methylation levels in GC and normal adjacent tissues were quantified using methylation-specific polymerase chain reaction. FBP2 promoter was frequently hypermethylated in primary GC tissues compared to adjacent normal tissues. To explore the functional consequences of this hypermethylation, we employed small interfering RNA-mediated knockdown of DNA methyltransferase 3a (DNMT3a) in GC cells. FBP2 expression increased following DNMT3a knockdown, suggesting that reduced methylation of the FBP2 promoter contributed to this upregulation. To further investigate this interaction, chromatin immunoprecipitation assays were conducted. The results confirmed an interaction between DNMT3a and the FBP2 promoter region, providing evidence that DNMT3a-mediated hypermethylation of the FBP2 promoter promotes GC progression. CONCLUSIONS: This study provides evidence that DNMT3a is involved in the hypermethylation of the FBP2 promoter and regulation of GC cell metabolism. Hypermethylation of the FBP2 promoter may be a promising prognostic biomarker in GC.

Rapid and accurate remethylation of DNA in Dnmt3a-deficient hematopoietic cells with restoration of DNMT3A activity.

Here, we characterize the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic deficiency of Dnmt3a or Dnmt3b (or both enzymes) or expressing the dominant-negative Dnmt3aR878H mutation [R882H in humans; the most common DNMT3A mutation found in acute myeloid leukemia (AML)]. Using these cells as substrates, we defined DNA remethylation after overexpressing wild-type (WT) DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive "chaperone" for DNMT3A and DNMT3B in early embryogenesis). Overexpression of DNMT3A for 2 weeks reverses the hypomethylation phenotype of Dnmt3a-deficient cells or cells expressing the R878H mutation. Overexpression of DNMT3L (which is minimally expressed in AML cells) also corrects the hypomethylation phenotype of Dnmt3aR878H/+ marrow, probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele. DNMT3L reactivation may represent a previously unidentified approach for restoring DNMT3A activity in hematopoietic cells with reduced DNMT3A function.

DNMT3B PWWP mutations cause hypermethylation of heterochromatin.

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here, we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1α and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3-marked heterochromatin in a PWWP-independent manner that is facilitated by the protein's N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome.

DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts.

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.

Intergenerational inheritance induced by a high-fat diet causes hyperphagia and reduced hypothalamic sensitivity to insulin and leptin in the second-generation of rats.

OBJECTIVE: The aim was to investigate the intergenerational inheritance induced by a high-fat diet on sensitivity to insulin and leptin in the hypothalamic control of satiety in second-generation offspring, which were fed a control diet. METHODS: Progenitor rats were fed a high-fat or a control diet for 59 d until weaning. The first-generation and second-generation offspring were fed the control diet until 90 d of age. Body mass and adiposity index of the progenitors fed the high-fat diet and the second-generation offspring from progenitors fed the high-fat diet were evaluated as were the gene expression of DNA methyltransferase 3a, angiotensin-converting enzyme type 2, angiotensin II type 2 receptor, insulin and leptin signaling pathway (insulin receptor, leptin receptor, insulin receptor substrate 2, protein kinase B, signal transducer and transcriptional activator 3, pro-opiomelanocortin, and neuropeptide Agouti-related protein), superoxide dismutase activity, and the concentration of carbonyl protein and satiety-regulating neuropeptides, pro-opiomelanocortin and neuropeptide Agouti-related protein, in the hypothalamus. RESULTS: The progenitor group fed a high-fat diet showed increased insulin resistance and reduced insulin-secreting beta-cell function and reduced food intake, without changes in caloric intake. The second-generation offspring from progenitors fed a high-fat diet, compared with second-generation offspring from progenitors fed a control diet group, had decreased insulin-secreting beta-cell function and increased food and caloric intake, insulin resistance, body mass, and adiposity index. Furthermore, second-generation offspring from progenitors fed a high-fat diet had increased DNA methyltransferase 3a, neuropeptide Agouti-related protein, angiotensin II type 1 receptor, and nicotinamide adenine dinucleotide phosphate oxidase p47phox gene expression, superoxide dismutase activity, and neuropeptide Agouti-related protein concentration in the hypothalamus. In addition, there were reduced in gene expression of the insulin receptor, leptin receptor, insulin receptor substrate 2, pro-opiomelanocortin, angiotensin II type 2 receptor, angiotensin-converting enzyme type 2, and angiotensin-(1-7) receptor and pro-opiomelanocortin concentration in the second-generation offspring from progenitors fed the high-fat diet. CONCLUSIONS: Overall, progenitors fed a high-fat diet induced changes in the hypothalamic control of satiety of the second-generation offspring from progenitors fed the high-fat diet through intergenerational inheritance. These changes led to hyperphagia, alterations in the hypothalamic pathways of insulin, and leptin and adiposity index increase, favoring the occurrence of different cardiometabolic disorders in the second-generation offspring from progenitors fed the high-fat diet fed only with the control diet.