Hematologic DNMT3A reduction and high-fat diet synergize to promote weight gain and tissue inflammation.

During aging, blood cell production becomes dominated by a limited number of variant hematopoietic stem cell (HSC) clones. Differentiated progeny of variant HSCs are thought to mediate the detrimental effects of such clonal hematopoiesis on organismal health, but the mechanisms are poorly understood. While somatic mutations in DNA methyltransferase 3A (DNMT3A) frequently drive clonal dominance, the aging milieu also likely contributes. Here, we examined in mice the interaction between high-fat diet (HFD) and reduced DNMT3A in hematopoietic cells; strikingly, this combination led to weight gain. HFD amplified pro-inflammatory pathways and upregulated inflammation-associated genes in mutant cells along a pro-myeloid trajectory. Aberrant DNA methylation during myeloid differentiation and in response to HFD led to pro-inflammatory activation and maintenance of stemness genes. These findings suggest that reduced DNMT3A in hematopoietic cells contributes to weight gain, inflammation, and metabolic dysfunction, highlighting a role for DNMT3A loss in the development of metabolic disorders.

SRCAP mutations drive clonal hematopoiesis through epigenetic and DNA repair dysregulation.

Somatic mutations accumulate in all cells with age and can confer a selective advantage, leading to clonal expansion over time. In hematopoietic cells, mutations in a subset of genes regulating DNA repair or epigenetics frequently lead to clonal hematopoiesis (CH). Here, we describe the context and mechanisms that lead to enrichment of hematopoietic stem cells (HSCs) with mutations in SRCAP, which encodes a chromatin remodeler that also influences DNA repair. We show that SRCAP mutations confer a selective advantage in human cells and in mice upon treatment with the anthracycline-class chemotherapeutic doxorubicin and bone marrow transplantation. Furthermore, Srcap mutations lead to a lymphoid-biased expansion, driven by loss of SRCAP-regulated H2A.Z deposition and increased DNA repair. Altogether, we demonstrate that SRCAP operates at the intersection of multiple pathways in stem and progenitor cells, offering a new perspective on the functional impact of genetic variants that promote stem cell competition in the hematopoietic system.

Exercise reprograms the inflammatory landscape of multiple stem cell compartments during mammalian aging.

Exercise has the ability to rejuvenate stem cells and improve tissue regeneration in aging animals. However, the cellular and molecular changes elicited by exercise have not been systematically studied across a broad range of cell types in stem cell compartments. We subjected young and old mice to aerobic exercise and generated a single-cell transcriptomic atlas of muscle, neural, and hematopoietic stem cells with their niche cells and progeny, complemented by whole transcriptome analysis of single myofibers. We found that exercise ameliorated the upregulation of a number of inflammatory pathways associated with old age and restored aspects of intercellular communication mediated by immune cells within these stem cell compartments. Exercise has a profound impact on the composition and transcriptomic landscape of circulating and tissue-resident immune cells. Our study provides a comprehensive view of the coordinated responses of multiple aged stem cells and niche cells to exercise at the transcriptomic level.

DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells.

Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.

Cell-type-specific aging clocks to quantify aging and rejuvenation in neurogenic regions of the brain.

The diversity of cell types is a challenge for quantifying aging and its reversal. Here we develop 'aging clocks' based on single-cell transcriptomics to characterize cell-type-specific aging and rejuvenation. We generated single-cell transcriptomes from the subventricular zone neurogenic region of 28 mice, tiling ages from young to old. We trained single-cell-based regression models to predict chronological age and biological age (neural stem cell proliferation capacity). These aging clocks are generalizable to independent cohorts of mice, other regions of the brains, and other species. To determine if these aging clocks could quantify transcriptomic rejuvenation, we generated single-cell transcriptomic datasets of neurogenic regions for two interventions-heterochronic parabiosis and exercise. Aging clocks revealed that heterochronic parabiosis and exercise reverse transcriptomic aging in neurogenic regions, but in different ways. This study represents the first development of high-resolution aging clocks from single-cell transcriptomic data and demonstrates their application to quantify transcriptomic rejuvenation.

Constitutive loss of DNMT3A causes morbid obesity through misregulation of adipogenesis.

DNA Methyltransferase 3 A (DNMT3A) is an important facilitator of differentiation of both embryonic and hematopoietic stem cells. Heterozygous germline mutations in DNMT3A lead to Tatton-Brown-Rahman Syndrome (TBRS), characterized by obesity and excessive height. While DNMT3A is known to impact feeding behavior via the hypothalamus, here we investigated a role in adipocyte progenitors utilizing heterozygous knockout mice that recapitulate cardinal TBRS phenotypes. These mice become morbidly obese due to adipocyte enlargement and tissue expansion. Adipose tissue in these mice exhibited defects in preadipocyte maturation and precocious activation of inflammatory gene networks, including interleukin-6 signaling. Adipocyte progenitor cell lines lacking DNMT3A exhibited aberrant differentiation. Furthermore, mice in which Dnmt3a was specifically ablated in adipocyte progenitors showed enlarged fat depots and increased progenitor numbers, partly recapitulating the TBRS obesity phenotypes. Loss of DNMT3A led to constitutive DNA hypomethylation, such that the DNA methylation landscape of young adipocyte progenitors resemble that of older wild-type mice. Together, our results demonstrate that DNMT3A coordinates both the central and local control of energy storage required to maintain normal weight and prevent inflammatory obesity.

Systematic Profiling of DNMT3A Variants Reveals Protein Instability Mediated by the DCAF8 E3 Ubiquitin Ligase Adaptor.

Clonal hematopoiesis is a prevalent age-related condition associated with a greatly increased risk of hematologic disease; mutations in DNA methyltransferase 3A (DNMT3A) are the most common driver of this state. DNMT3A variants occur across the gene with some particularly associated with malignancy, but the functional relevance and mechanisms of pathogenesis of the majority of mutations are unknown. Here, we systematically investigated the methyltransferase activity and protein stability of 253 disease-associated DNMT3A mutations, and found that 74% were loss-of-function mutations. Half of these variants exhibited reduced protein stability and, as a class, correlated with greater clonal expansion and acute myeloid leukemia development. We investigated the mechanisms underlying the instability using a CRISPR screen and uncovered regulated destruction of DNMT3A mediated by the DCAF8 E3 ubiquitin ligase adaptor. We establish a new paradigm to classify novel variants that has prognostic and potential therapeutic significance for patients with hematologic disease. SIGNIFICANCE: DNMT3A has emerged as the most important epigenetic regulator and tumor suppressor in the hematopoietic system. Our study represents a systematic and high-throughput method to characterize the molecular impact of DNMT3A missense mutations and the discovery of a regulated destruction mechanism of DNMT3A offering new prognostic and future therapeutic avenues.See related commentary by Ma and Will, p. 23.This article is highlighted in the In This Issue feature, p. 1.

Perturbed hematopoiesis in individuals with germline DNMT3A overgrowth Tatton-Brown-Rahman syndrome.

Tatton-Brown-Rahman syndrome (TBRS) is an overgrowth disorder caused by germline heterozygous mutations in the DNA methyltransferase DNMT3A. DNMT3A is a critical regulator of hematopoietic stem cell (HSC) differentiation and somatic DNMT3A mutations are frequent in hematologic malignancies and clonal hematopoiesis. Yet, the impact of constitutive DNMT3A mutation on hematopoiesis in TBRS is undefined. In order to establish how constitutive mutation of DNMT3A impacts blood development in TBRS we gathered clinical data and analyzed blood parameters in 18 individuals with TBRS. We also determined the distribution of major peripheral blood cell lineages by flow cytometric analyses. Our analyses revealed non-anemic macrocytosis, a relative decrease in lymphocytes and increase in neutrophils in TBRS individuals compared to unaffected controls. We were able to recapitulate these hematologic phenotypes in multiple murine models of TBRS and identified rare hematological and non-hematological malignancies associated with constitutive Dnmt3a mutation. We further show that loss of DNMT3A in TBRS is associated with an altered DNA methylation landscape in hematopoietic cells affecting regions critical to stem cell function and tumorigenesis. Overall, our data identify key hematopoietic effects driven by DNMT3A mutation with clinical implications for individuals with TBRS and DNMT3A-associated clonal hematopoiesis or malignancies.

Modeling IKZF1 lesions in B-ALL reveals distinct chemosensitivity patterns and potential therapeutic vulnerabilities.

IKAROS family zinc finger 1 (IKZF1) alterations represent a diverse group of genetic lesions that are associated with an increased risk of relapse in B-cell acute lymphoblastic leukemia. Due to the heterogeneity of concomitant lesions, it remains unclear how IKZF1 abnormalities directly affect cell function and therapy resistance, and whether their consideration as a prognostic indicator is valuable in improving outcome. CRISPR/Cas9 strategies were used to engineer multiple panels of isogeneic lymphoid leukemia cell lines with a spectrum of IKZF1 lesions to measure changes in chemosensitivity, gene expression, cell cycle, and in vivo engraftment that can be linked to loss of IKAROS protein. IKZF1 knockout and heterozygous null cells displayed relative resistance to a number of common therapies for B-cell acute lymphoblastic leukemia, including dexamethasone, asparaginase, and daunorubicin. Transcription profiling revealed a stem/myeloid cell-like phenotype and JAK/STAT upregulation after IKAROS loss. A CRISPR homology-directed repair strategy was also used to knock-in the dominant-negative IK6 isoform into the endogenous locus, and a similar drug resistance profile, with the exception of retained dexamethasone sensitivity, was observed. Interestingly, IKZF1 knockout and IK6 knock-in cells both have significantly increased sensitivity to cytarabine, likely owing to marked downregulation of SAMHD1 after IKZF1 knockout. Both types of IKZF1 lesions decreased the survival time of xenograft mice, with higher numbers of circulating blasts and increased organ infiltration. Given these findings, exact specification of IKZF1 status in patients may be a beneficial addition to risk stratification and could inform therapy.

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice.

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor-/- knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor-/-/Dnmt3a-/- knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.

Tissue-Biased Expansion of DNMT3A-Mutant Clones in a Mosaic Individual Is Associated with Conserved Epigenetic Erosion.

DNA methyltransferase 3A (DNMT3A) is the most commonly mutated gene in clonal hematopoiesis (CH). Somatic DNMT3A mutations arise in hematopoietic stem cells (HSCs) many years before malignancies develop, but difficulties in comparing their impact before malignancy with wild-type cells have limited the understanding of their contributions to transformation. To circumvent this limitation, we derived normal and DNMT3A mutant lymphoblastoid cell lines from a germline mosaic individual in whom these cells co-existed for nearly 6 decades. Mutant cells dominated the blood system, but not other tissues. Deep sequencing revealed similar mutational burdens and signatures in normal and mutant clones, while epigenetic profiling uncovered the focal erosion of DNA methylation at oncogenic regulatory regions in mutant clones. These regions overlapped with those sensitive to DNMT3A loss after DNMT3A ablation in HSCs and in leukemia samples. These results suggest that DNMT3A maintains a conserved DNA methylation pattern, the erosion of which provides a distinct competitive advantage to hematopoietic cells.

Large DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity.

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.

Factors motivating Mexico City residents to earthquake mass evacuation drills.

Evacuation drills may constitute a key activity for preparing for an emergency due to an earthquake. The paper presents the results of an analysis of participants' motivations on the factors leading to conducting drills on 19 September every year in Mexico City; the sample size considered for the analysis was N = 2400. In particular, the following research question has been addressed: What factors predict the likelihood that respondents would report that they agree on conducting mass evacuation drills yearly? The approach has been the application of logistic regression technique to identify these factors. Of the 19 initial explanatory variables, in the final model, only seven variables and one interaction term, were significantly associated with the outcome variable; i.e.: age (Odds Ratio (OR) = 1.366; 95% Confidence Interval (CI) = 1.039-1.795); occupation (OR = 3.378; CI = 1.457-7.830); frequency of drills: one/year (OR = 2.128; CI = 1.610-2.812); knowledge vs. drills (OR = 1.394; CI = 1.172-1.658); 'perception vulnerability city' (OR = 1.271; CI = 1.091-1.480); warning time (OR = 1.266; CI = 1.1036-1.548); usefulness of the SASMEX (OR = 0.783; CI = 0.615-0.998); and 'perception vulnerability city' by occupation interaction (OR = 0.786; CI = 0.643-0.961). Further research may be needed to gain a better understanding of people's motivations on evacuation drills taking place anytime during the day or at night, and whether evacuation drills should be unannounced.

Synthetic Lethal and Resistance Interactions with BET Bromodomain Inhibitors in Triple-Negative Breast Cancer.

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.

Mexico city's residents emotional and behavioural reactions to the 19 September 2017 earthquake.

The paper presents the results of a cross-sectional study conducted in Mexico City following the earthquake that occurred on September 19, 2017. The sample size of the study was N = 2400. The aim has been the study of people's emotional and behavioural responses during and immediately after the tremors. Some of the results and conclusions were the following: a). During the tremors, respondents coping reactions were related to escape, reaching and protecting people, and seeking shelter; b). The actions taken by the respondents immediately after the tremor were reuniting with family members, evacuation, and returning to houses; c). The emotional responses of the participants of the study were fear and surprise; d). The capital city's residents exhibited a relatively high percentage of prosocial behaviour during the seismic emergency response; e). More generally, the residents of the city should be trained on what to do during and after the tremors; moreover, key decision-makers should consider people's emotional and behavioural responses to tremors when designing plans for mass emergencies following earthquakes, such as the present case study.

High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program.

Systemic metabolic alterations associated with increased consumption of saturated fat and obesity are linked with increased risk of prostate cancer progression and mortality, but the molecular underpinnings of this association are poorly understood. Here, we demonstrate in a murine prostate cancer model, that high-fat diet (HFD) enhances the MYC transcriptional program through metabolic alterations that favour histone H4K20 hypomethylation at the promoter regions of MYC regulated genes, leading to increased cellular proliferation and tumour burden. Saturated fat intake (SFI) is also associated with an enhanced MYC transcriptional signature in prostate cancer patients. The SFI-induced MYC signature independently predicts prostate cancer progression and death. Finally, switching from a high-fat to a low-fat diet, attenuates the MYC transcriptional program in mice. Our findings suggest that in primary prostate cancer, dietary SFI contributes to tumour progression by mimicking MYC over expression, setting the stage for therapeutic approaches involving changes to the diet.

Trisomy of a Down Syndrome Critical Region Globally Amplifies Transcription via HMGN1 Overexpression.

Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted.