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Provides information on the side population (SP) cells and multipotential stem cells on the basis of efficient Hoechst dye efflux. Possibility that tissue-restricted stem cells; Ability of SP cells to generate non-hematopoietic progeny; Introduction of stem cell population into injured tissue.

Abstract Cancer precursor/progenitor cells may initiate and sustain the growth of tumors, but evidence for their existence in human disease is indirect, relying on their in vitro properties and animal models. More directly, specific elimination of these rare cells from cancer patients should produce a delayed but progressive disappearance of differentiated malignant progeny. Here, we describe selective eradication of a putative precursor population in a patient with B-cell chronic lymphocytic leukemia, followed 6 months later by a progressive loss of mature tumor cells without further treatment. This outcome supports the presence of a rare population of precursor/progenitor cells in human malignancies, and suggests benefit from their removal.

Haematopoietic stem cells maintain blood production throughout life 1 . Although extensively characterized using the laboratory mouse, little is known about clonal selection and population dynamics of the haematopoietic stem cell pool during murine ageing. We isolated stem cells and progenitors from young and old mice, identifying 221,890 somatic mutations genome-wide in 1,845 single-cell-derived colonies. Mouse stem cells and progenitors accrue approximately 45 somatic mutations per year, a rate only approximately threefold greater than human progenitors despite the vastly different organismal sizes and lifespans. Phylogenetic patterns show that stem and multipotent progenitor cell pools are established during embryogenesis, after which they independently self-renew in parallel over life, evenly contributing to differentiated progenitors and peripheral blood. The stem cell pool grows steadily over the mouse lifespan to about 70,000 cells, self-renewing about every 6 weeks. Aged mice did not display the profound loss of clonal diversity characteristic of human haematopoietic ageing. However, targeted sequencing showed small, expanded clones in the context of murine ageing, which were larger and more numerous following haematological perturbations, exhibiting a selection landscape similar to humans. Our data illustrate both conserved features of population dynamics of blood and distinct patterns of age-associated somatic evolution in the short-lived mouse., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

In this issue, Waarts and colleagues developed an advanced ex vivo CRISPR screening platform to identify vulnerabilities in clonal hematopoiesis (CH). This unique system allowed the authors to identify a link between IDH2 and TET2 CH mutations, histone demethylases, and altered cytokine signaling, which enabled targeting by ruxolitinib leading to the elimination of CH clones, offering a possible path for preventing the development of malignancy. See related article by Waarts et al., p. 1860., (©2024 American Association for Cancer Research.)

Nearly every mammalian cell division is accompanied by a mutational event that becomes fixed in a daughter cell. When carried forward to additional cell progeny, a clone of variant cells can emerge. As a result, mammals are complex mosaics of clones that are genetically distinct from one another. Recent high-throughput sequencing studies have revealed that mosaicism is common, clone sizes often increase with age and specific variants can affect tissue function and disease development. Variants that are acquired during early embryogenesis are shared by multiple cell types and can affect numerous tissues. Within tissues, variant clones compete, which can result in their expansion or elimination. Embryonic mosaicism has clinical implications for genetic disease severity and transmission but is likely an under-recognized phenomenon. To better understand its implications for mosaic individuals, it is essential to leverage research tools that can elucidate the mechanisms by which expanded embryonic variants influence development and disease., (© 2024. Springer Nature Limited.)

Haematopoietic stem cells maintain blood production throughout life. While extensively characterised using the laboratory mouse, little is known about how the population is sustained and evolves with age. We isolated stem cells and progenitors from young and old mice, identifying 221,890 somatic mutations genome-wide in 1845 single cell-derived colonies, and used phylogenetic analysis to infer the ontogeny and population dynamics of the stem cell pool. Mouse stem cells and progenitors accrue ~45 somatic mutations per year, a rate only about 2-fold greater than human progenitors despite the vastly different organismal sizes and lifespans. Phylogenetic patterns reveal that stem and multipotent progenitor cell pools are both established during embryogenesis, after which they independently self-renew in parallel over life. The stem cell pool grows steadily over the mouse lifespan to approximately 70,000 cells, self-renewing about every six weeks. Aged mice did not display the profound loss of stem cell clonal diversity characteristic of human haematopoietic ageing. However, targeted sequencing revealed small, expanded clones in the context of murine ageing, which were larger and more numerous following haematological perturbations and exhibited a selection landscape similar to humans. Our data illustrate both conserved features of population dynamics of blood and distinct patterns of age-associated somatic evolution in the short-lived mouse.

Background: NPM1 -mutated acute myeloid leukemia (AML) is the most frequent AML subtype. As wild-type NPM1 is known to orchestrate ribosome biogenesis, it has been hypothesized that altered translation may contribute to leukemogenesis and leukemia maintenance in NPM1 -mutated AML. However, this hypothesis has never been investigated. We reasoned that if mutant NPM1 (NPM1c) directly impacts translation in leukemic cells, loss of NPM1c would result in acute changes in the ribosome footprint., Methods: Here, we performed ribosome footprint profiling (Ribo-seq) and bulk messenger RNA (mRNA) sequencing in two NPM1 -mutated cell lines engineered to express endogenous NPM1c fused to the FKBP (F36V) degron tag (degron cells)., Results and Discussion: Incubation of degron cells with the small compound dTAG-13 enables highly specific degradation of NPM1c within 4 hours. As expected, RNA-sequencing data showed early loss of homeobox gene expression following NPM1c degradation, confirming the reliability of our model. In contrast, Ribo-seq data showed negligible changes in the ribosome footprint in both cell lines, implying that the presence of NPM1c does not influence ribosome abundance and positioning on mRNA. While it is predictable that NPM1c exerts its leukemogenic activity at multiple levels, ribosome footprint does not seem influenced by the presence of mutant NPM1., Competing Interests: The corresponding author holds a patent on NPM1 mutants (number 102004901256449). The remaining authors declare no conflict of interest., (© 2024 The Author(s). eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies, including acute myeloid leukemia. An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly comutated genes in myeloid malignancies, such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ER T2 murine model systems to study the effects of constitutively active Kras G12D -driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using posttransplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage. Specifically, 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras and only 13% of Kras mice. Here, 3aKO combined with Kras led to increased disease burden, multiorgan infiltration, and faster disease progression. DOT1L inhibition exerted profound antileukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell-cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we developed a robust model system for mechanistic and preclinical investigations of acute myeloid leukemia with DNMT3A and Ras-pathway lesions., Competing Interests: Conflicts of Interest Disclosure The authors declare no relevant competing financial interests., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg 2+ /Mn 2+ -dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D -mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D -mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D -mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D -mutant cancers., Competing Interests: LZ, JH, EB, CC, TP, AM, AG, KW, SW, HU, AT, EC, RM, RR, SJ, LV, Bd, AN, SH, CC, JA, KT, GV, MG No competing interests declared

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing., (© 2024. The Author(s).)

Loss of stem cell regenerative potential underlies aging of all tissues. Somatic mosaicism, the emergence of cellular patchworks within tissues, increases with age and has been observed in every organ yet examined. In the hematopoietic system, as in most tissues, stem cell aging through a variety of mechanisms occurs in lockstep with the emergence of somatic mosaicism. Here, we draw on insights from aging hematopoiesis to illustrate fundamental principles of stem cell aging and somatic mosaicism. We describe the generalizable changes intrinsic to aged stem cells and their milieu that provide the backdrop for somatic mosaicism to emerge. We discuss genetic and nongenetic mechanisms that can result in tissue somatic mosaicism and existing methodologies to detect such clonal outgrowths. Finally, we propose potential avenues to modify mosaicism during aging, with the ultimate aim of increasing tissue resiliency., (© 2024. Springer Nature America, Inc.)

Chimeric antigen receptor (CAR) T-cells are an emerging therapy for refractory lymphomas. Clonal hematopoiesis (CH), the preferential outgrowth of mutated bone marrow progenitors, is enriched in lymphoma patients receiving CAR-T cells. CAR-T therapy requires conditioning chemotherapy and often induces systemic inflammatory reactions, both of which have been shown to promote expansion of CH clones. Thus, we hypothesized that pre-existing CH clones could expand during CAR-T cell treatment. We measured CH at 154 timepoints longitudinally sampled from 26 patients receiving CD30.CAR-T therapy for CD30+ lymphomas on an investigational protocol (NCT02917083). Pre-treatment CH was present in 54% of individuals and did not correlate with survival outcomes or inflammatory toxicities. Longitudinal tracking of single clones in individual patients revealed distinct clone growth dynamics. Initially small clones, defined as VAF <1%, expanded following CAR-T administration, compared with relatively muted expansions of larger clones (3.37-fold vs. 1.20-fold, P = 0.0014). Matched clones were present at low magnitude in the infused CD30.CAR-T product for all CH cases but did not affect the product's immunophenotype or transduction efficiency. As cellular immunotherapies expand to become frontline treatments for hematological malignancies, our data indicates CAR-T recipients could be enriched for CH, and further longitudinal studies centered on CH complications in this population are warranted., Competing Interests: Declaration of Competing Interest CAR has received research support from Athenex. HEH is a co-founder with equity in Allovir and Marker Therapeutics, has share options in Fresh Wind Biotechnologies and Coregen, has served on advisory boards for Tessa Therapeutics and Marker Therapeutics and received research support from Tessa Therapeutics and Athenex. PDL has received clinical trial funding from Allovir, Marker Therapeutics and Bristol Myers Squibb and has served on an advisory board for Janssen Therapeutics., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

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., Competing Interests: Declaration of interests A.S.S. receives consulting fees from Novartis and Roche. P.N. receives research grants from Allelica, Apple, Amgen, Boston Scientific, Genentech/Roche, and Novartis; receives personal fees from Allelica, Apple, AstraZeneca, Blackstone Life Sciences, Foresite Labs, Genentech/Roche, GV, HeartFlow, Magnet Biomedicine, and Novartis; has scientific advisory board membership in Esperion Therapeutics, Preciseli, and TenSixteen Bio; is a scientific co-founder of TenSixteen Bio; has equity in Preciseli and TenSixteen Bio; and has spousal employment at Vertex Pharmaceuticals—all unrelated to the present work. EpiCypher is a commercial developer and supplier of reagents and platforms used in this study. S.P. is employed by (and owns shares in) New England Biolabs. B.J.V. and M.-C.K. are employed by (and own shares in) EpiCypher. M.-C.K. is a board member of EpiCypher., (Copyright © 2023 Elsevier Inc. All rights reserved.)

DNA methylation deregulation at partially methylated domains (PMDs) represents an epigenetic signature of aging and cancer, yet the underlying molecular basis and resulting biological consequences remain unresolved. We report herein a mechanistic link between disrupted DNA methylation at PMDs and the spatial relocalization of H3K9me3-marked heterochromatin in aged hematopoietic stem and progenitor cells (HSPCs) or those with impaired DNA methylation. We uncover that TET2 modulates the spatial redistribution of H3K9me3-marked heterochromatin to mediate the upregulation of endogenous retroviruses (ERVs) and interferon-stimulated genes (ISGs), hence contributing to functional decline of aged HSPCs. TET2-deficient HSPCs retain perinuclear distribution of heterochromatin and exhibit age-related clonal expansion. Reverse transcriptase inhibitors suppress ERVs and ISGs expression, thereby restoring age-related defects in aged HSPCs. Collectively, our findings deepen the understanding of the functional interplay between DNA methylation and histone modifications, which is vital for maintaining heterochromatin function and safeguarding genome stability in stem cells., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: Clonal hematopoiesis (CH), the expansion of clones in the hematopoietic system, has been linked to different internal and external features such as aging, genetic ancestry, smoking, and oncologic treatment. However, the interplay between mutations in known cancer predisposition genes and CH has not been thoroughly examined in patients with solid tumors., Methods: We used prospective tumor-blood paired sequencing data from 46,906 patients who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) testing to interrogate the associations between CH and rare pathogenic or likely pathogenic (P/LP) germline variants., Results: We observed an enrichment of CH-positive patients among those carrying P/LP germline mutations and identified a significant association between P/LP germline variants in ATM and CH. Germline and CH comutation patterns in ATM , TP53 , and CHEK2 suggested biallelic inactivation as a potential mediator of clonal expansion. Moreover, we observed that CH- PPM1D mutations, similar to somatic tumor-associated PPM1D mutations, were depleted in patients with P/LP germline mutations in the DNA damage response (DDR) genes ATM , CHEK2 , and TP53 . Patients with solid tumors and harboring P/LP germline mutations, CH mutations, and mosaicism chromosomal alterations might be at an increased risk of developing secondary leukemia while germline variants in TP53 were identified as an independent risk factor (hazard ratio, 36; P < .001) for secondary leukemias., Conclusion: Our results suggest a close relationship between inherited variants and CH mutations within the DDR genes in patients with solid tumors. Associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities in patients with cancer harboring these germline mutations.

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., Competing Interests: Declaration of interests J.C.W. is a co-founder of Greenstone Biosciences but has no competing interests as the work presented was performed independently., (Published by Elsevier Inc.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© 2022. The Author(s).)

Most adult organs contain regenerative stem cells, often organized in specific niches. Stem cell function is critical for tissue homeostasis and repair upon injury, and it is dependent on interactions with the niche. During ageing, stem cells decline in their regenerative potential and ability to give rise to differentiated cells in the tissue, which is associated with a deterioration of tissue integrity and health. Ageing-associated changes in regenerative tissue regions include defects in maintenance of stem cell quiescence, differentiation ability and bias, clonal expansion and infiltration of immune cells in the niche. In this Review, we discuss cellular and molecular mechanisms underlying ageing in the regenerative regions of different tissues as well as potential rejuvenation strategies. We focus primarily on brain, muscle and blood tissues, but also provide examples from other tissues, such as skin and intestine. We describe the complex interactions between different cell types, non-cell-autonomous mechanisms between ageing niches and stem cells, and the influence of systemic factors. We also compare different interventions for the rejuvenation of old regenerative regions. Future outlooks in the field of stem cell ageing are discussed, including strategies to counter ageing and age-dependent disease., (© 2022. Springer Nature Limited.)

In the face of constant genomic insults, the DNA damage response (DDR) is initiated to preserve genome integrity; its disruption is a classic hallmark of cancer. Protein phosphatase Mg2+/Mn2+-dependent 1D (PPM1D) is a central negative regulator of the DDR that is mutated or amplified in many solid cancers. PPM1D overexpression is associated with increased proliferative and metastatic behavior in multiple solid tumor types and patients with PPM1D-mutated malignancies have poorer prognoses. Recent findings have sparked an interest in the role of PPM1D in hematologic malignancies. Acquired somatic mutations may provide hematopoietic stem cells with a competitive advantage, leading to a substantial proportion of mutant progeny in the peripheral blood, an age-associated phenomenon termed "clonal hematopoiesis" (CH). Recent large-scale genomic studies have identified PPM1D to be among the most frequently mutated genes found in individuals with CH. While PPM1D mutations are particularly enriched in patients with therapy-related myeloid neoplasms, their role in driving leukemic transformation remains uncertain. Here, we examine the mechanisms through which PPM1D overexpression or mutation may drive malignancy by suppression of DNA repair, cell-cycle arrest, and apoptosis. We also discuss the divergent roles of PPM1D in the oncogenesis of solid versus hematologic cancers with a view to clinical implications and new therapeutic avenues., (©2022 The Authors; Published by the American Association for Cancer Research.)

We previously identified two structurally related pyrazolone (compound 1) and pyridazine (compound 2) allosteric inhibitors of DNMT3A through screening of a small chemical library. Here, we show that these compounds bind and disrupt protein-protein interactions (PPIs) at the DNMT3A tetramer interface. This disruption is observed with distinct partner proteins and occurs even when the complexes are acting on DNA, which better reflects the cellular context. Compound 2 induces differentiation of distinct myeloid leukemia cell lines including cells with mutated DNMT3A R882. To date, small molecules targeting DNMT3A are limited to competitive inhibitors of AdoMet or DNA and display extreme toxicity. Our work is the first to identify small molecules with a mechanism of inhibition involving the disruption of PPIs with DNMT3A. Ongoing optimization of compounds 1 and 2 provides a promising basis to induce myeloid differentiation and treatment of diseases that display aberrant PPIs with DNMT3A, such as acute myeloid leukemia.

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., Competing Interests: AT, JR, LZ, YH, CR, AD, AG, RR, CC, TG, SG, LO, DP, AC, RR, SH, MK, MG No competing interests declared, (© 2022, Tovy et al.)

DNA methyltransferase 3a (DNMT3A) plays a crucial role during mammalian development. Two isoforms of DNMT3A are differentially expressed from stem cells to somatic tissues, but their individual functions remain largely uncharacterized. Here we report that the long isoform DNMT3A1, but not the short DNMT3A2, is essential for mouse postnatal development. DNMT3A1 binds to and regulates bivalent neurodevelopmental genes in the brain. Strikingly, Dnmt3a1 knockout perinatal lethality could be partially rescued by DNMT3A1 restoration in the nervous system. We further show that the intrinsically disordered N terminus of DNMT3A1 is required for normal development and DNA methylation at DNMT3A1-enriched regions. Mechanistically, a ubiquitin-interacting motif embedded in a putative α-helix within the N terminus binds to mono-ubiquitinated histone H2AK119, probably mediating recruitment of DNMT3A1 to Polycomb-regulated regions. These data demonstrate an isoform-specific role for DNMT3A1 in mouse postnatal development and reveal the N terminus as a necessary regulatory domain for DNMT3A1 chromatin occupancy and functions in the nervous system., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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.

Aging is characterized by increased mortality, functional decline, and exponential increases in the incidence of diseases such as cancer, stroke, cardiovascular disease, neurological disease, respiratory disease, etc. Though the role of aging in these diseases is widely accepted and considered to be a common denominator, the underlying mechanisms are largely unknown. A significant age-related feature observed in many population cohorts is somatic mosaicism, the detectable accumulation of somatic mutations in multiple cell types and tissues, particularly those with high rates of cell turnover (e.g., skin, liver, and hematopoietic cells). Somatic mosaicism can lead to the development of cellular clones that expand with age in otherwise normal tissues. In the hematopoietic system, this phenomenon has generally been referred to as "clonal hematopoiesis of indeterminate potential" (CHIP) when it applies to a subset of clones in which mutations in driver genes of hematologic malignancies are found. Other mechanisms of clonal hematopoiesis, including large chromosomal alterations, can also give rise to clonal expansion in the absence of conventional CHIP driver gene mutations. Both types of clonal hematopoiesis (CH) have been observed in studies of animal models and humans in association with altered immune responses, increased mortality, and disease risk. Studies in murine models have found that some of these clonal events are involved in abnormal inflammatory and metabolic changes, altered DNA damage repair and epigenetic changes. Studies in long-lived individuals also show the accumulation of somatic mutations, yet at this advanced age, carriership of somatic mutations is no longer associated with an increased risk of mortality. While it remains to be elucidated what factors modify this genotype-phenotype association, i.e., compensatory germline genetics, cellular context of the mutations, protective effects to diseases at exceptional age, it points out that the exceptionally long-lived are key to understand the phenotypic consequences of CHIP mutations. Assessment of the clinical significance of somatic mutations occurring in blood cell types for age-related outcomes in human populations of varied life and health span, environmental exposures, and germline genetic risk factors will be valuable in the development of personalized strategies tailored to specific somatic mutations for healthy aging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Walsh, Raghavachari, Kerr, Bick, Cummings, Druley, Dunbar, Genovese, Goodell, Jaiswal, Maciejewski, Natarajan, Shindyapina, Shuldiner, Van Den Akker and Vijg.)

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 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

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., (© 2021 by The American Society of Hematology.)

Mutations in the isocitrate dehydrogenase 1 ( IDH1 ) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2 R140Q transgenic mice to examine the role of the Idh2 R140Q mutation in leukemia. No leukemia developed in Idh2 R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion ( NHD13 ) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2 R140Q /NHD13 double transgenic mice. Idh2 R140Q /NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2 R140Q /NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2 R140Q /NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3 , and EZH2 were also found in Idh2 R140Q /NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2 R140Q /NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2 R140Q /NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy. SIGNIFICANCE: T-cell leukemia induced in Idh2 R140Q /NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment., (©2021 American Association for Cancer Research.)

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.

The discovery of clonal hematopoiesis (CH) in older individuals has changed the way hematologists and stem cell biologists view aging. Somatic mutations accumulate in stem cells over time. While most mutations have no impact, some result in subtle functional differences that ultimately manifest in distinct stem cell behaviors. With a large pool of stem cells and many decades to compete, some of these differences confer advantages under specific contexts. Approximately 20 genes are recurrently found as mutated in CH, indicating they confer some advantage. The impact of these mutations has begun to be analyzed at a molecular level by modeling in cell lines and in mice. Mutations in epigenetic regulators such as DNMT3A and TET2 confer an advantage by enhancing self-renewal of stem and progenitor cells and inhibiting their differentiation. Mutations in other genes involved in the DNA damage response may simply enhance cell survival. Here, we review proposed mechanisms that lead to CH, specifically in the context of stem cell biology, based on our current understanding of the function of some of the CH-associated genes., (© 2020 by The American Society of Hematology.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chronic inflammation has been implicated in myelodysplastic syndrome (MDS); however, its role in disease progression is unclear. In a new study by Muto et al., MDS stem cells sparked with TRAF6-activated innate immune signaling were found to outcompete normal counterparts only when fueled by environmental inflammatory stimuli. Non-canonical NF-κB signaling is implicated in inflammatory synergy and MDS progression., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Several acute myeloid leukemia genetic sub-types converge on high expression of HOX genes, critical for their self-renewal. A new orally bioavailable Menin-MLL inhibitor (VTP-50469) appears to promote their differentiation through direct effects on the HOX cofactor MEIS1, paving the way for clinical trials., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mutations in the epigenetic regulators DNMT3A and IDH1/2 co-occur in patients with acute myeloid leukemia and lymphoma. In this study, these 2 epigenetic mutations cooperated to induce leukemia. Leukemia-initiating cells from Dnmt3a-/- mice that express an IDH2 neomorphic mutant have a megakaryocyte-erythroid progenitor-like immunophenotype, activate a stem-cell-like gene signature, and repress differentiated progenitor genes. We observed an epigenomic dysregulation with the gain of repressive H3K9 trimethylation and loss of H3K9 acetylation in diseased mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). HDAC inhibitors rapidly reversed the H3K9 methylation/acetylation imbalance in diseased mouse HSPCs while reducing the leukemia burden. In addition, using targeted metabolomic profiling for the first time in mouse leukemia models, we also showed that prostaglandin E2 is overproduced in double-mutant HSPCs, rendering them sensitive to prostaglandin synthesis inhibition. These data revealed that Dnmt3a and Idh2 mutations are synergistic events in leukemogenesis and that HSPCs carrying both mutations are sensitive to induced differentiation by the inhibition of both prostaglandin synthesis and HDAC, which may reveal new therapeutic opportunities for patients carrying IDH1/2 mutations., (© 2020 by The American Society of Hematology.)

Clonal hematopoiesis (CH) has emerged as an important factor linked to adverse health conditions in the elderly. CH is characterized by an overrepresentation of genetically distinct hematopoietic stem cell clones in the peripheral blood. Whereas the genetic mutations that underlie CH have been closely scrutinized, relatively little attention has been paid to the environmental factors that may influence the emergence of one dominant stem cell clone. As there is huge individual variation in latency between acquisition of a genetic mutation and emergence of CH, environmental factors likely play a major role. Indeed, environmental stressors such as inflammation, chemotherapy, and metabolic syndromes are known to affect steady-state hematopoiesis. To date, epidemiologic studies point toward smoking and prior chemotherapy exposure as likely contributors to some forms of CH, though the impact of other environmental factors is also being investigated. Mechanistic studies in murine models indicate that the role of different environmental factors in CH emergence may be highly specific to the mutation that marks each stem cell clone. For instance, recent studies have found that clones with mutations in the PPM1D gene are more resistant to genotoxic stress induced by chemotherapy. These clones thus have a competitive advantage in the setting of chemotherapy, but not in other types of stress. Here we review currently available literature on the interplay between environment and the genetic landscapes in CH and highlight critical areas for future study. Improved understanding of the effects of environmental stress on emergence of CH with mutation-specific clarity will guide future efforts to provide preventive medicine to individuals with CH., (Copyright © 2019 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Primary and acquired drug resistance imposes a major threat to achieving optimized clinical outcomes during cancer treatment. Aberrant changes in epigenetic modifications are closely involved in drug resistance of tumor cells. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on the CRISPR interference technology, we identified the second intron of IncRNA, PVT1, as a unique bona fide gained enhancer that restored MYC transcription independent of BRD4 recruitment in leukemia. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYC locus in BETi-resistant leukemia cells. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies.

The phosphoinositide-3-kinase like kinases (PIKK) such as ATM and ATR play a key role in initiating the cellular DNA damage response (DDR). One key ATM target is the cyclin-dependent kinase inhibitor p27 Kip1 that promotes G1 arrest. ATM activates p27 Kip1 -induced arrest in part through phosphorylation of p27 Kip1 at Serine 140. Here we show that this site is dephosphorylated by the type 2C serine/threonine phosphatase, WIP1 (Wildtype p53-Induced Phosphatase-1), encoded by the PPM1D gene. WIP1 has been shown to dephosphorylate numerous ATM target sites in DDR proteins, and its overexpression and/or mutation has often been associated with oncogenesis. We demonstrate that wildtype, but not phosphatase-dead WIP1, efficiently dephosphorylates p27 Kip1 Ser140 both in vitro and in cells and that this dephosphorylation is sensitive to the WIP1-specific inhibitor GSK 2830371. Increased expression of wildtype WIP1 reduces stability of p27 Kip1 while increased expression of similar amounts of phosphatase-dead WIP1 has no effect on p27 Kip1 protein stability. Overexpression of wildtype p27 Kip1 reduces cell proliferation and colony forming capability relative to the S140A (constitutively non-phosphorylated) form of p27. Thus, WIP1 plays a significant role in homeostatic modulation of p27 Kip1 activity following activation by ATM.

TET2 is among the most frequently mutated genes in hematological malignancies, as well as in healthy individuals with clonal hematopoiesis. Inflammatory stress is known to promote the expansion of Tet2-deficient hematopoietic stem cells, as well as the initiation of pre-leukemic conditions. Infection is one of the most frequent complications in hematological malignancies and antibiotics are commonly used to suppress infection-induced inflammation, but their application in TET2 mutation-associated cancers remained underexplored. In this study, we discovered that Tet2 depletion led to aberrant expansion of myeloid cells, which was correlated with elevated serum levels of pro-inflammatory cytokines at the pre-malignant stage. Antibiotics treatment suppressed the growth of Tet2-deficient myeloid and lymphoid tumor cells in vivo. Transcriptomic profiling further revealed significant changes in the expression of genes involved in the TNF-α signaling and other immunomodulatory pathways in antibiotics-treated tumor cells. Pharmacological inhibition of TNF-α signaling partially attenuated Tet2-deficient tumor cell growth in vivo. Therefore, our findings establish the feasibility of targeting pro-inflammatory pathways to curtail TET2 inactivation-associated hematological malignancies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acute myeloid leukemia (AML), the most common acute leukemia in adults, increases exponentially with age. While a number of recent advances have improved treatment, high cure rates have not yet been achieved. Nucleophosmin (NPM1) is found mutated in nearly one-third of newly diagnosed cases and leads to NPM1 protein that is mislocalized to the cytoplasm instead of the nucleolus. If the mechanistic basis through which this mislocalization leads to malignancy could be revealed, this AML subtype may be targetable with new drugs. Here, we review the structure and functions of the normal and mutant forms of nucleophosmin. We discuss several recent studies that have shed light on the pathophysiology of NPM1 mutations. We discuss the importance of HOX gene misregulation in NPM1-mutated leukemias, as well as evidence for the reliance of mutated NPM1 on its continued nuclear export. Together, these aspects, as well as new tools to manipulate and study NPM1, open the door to new therapeutic strategies that may ultimately improve treatment of this common subtype of AML.

Hematologic diseases may be among the first clinical targets for therapeutic gene editing. In this issue of Cell Stem Cell, Schiroli et al. (2019) report a thorough, unbiased investigation of the impact of gene editing on hematopoietic stem and progenitor function and offer a clear path forward for editing in multiple tissues and conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Eukaryotic DNA methylation prevents genomic instability by regulating the expression of oncogenes and tumor-suppressor genes. The negative effects of dysregulated DNA methylation are highlighted by a strong correlation between mutations in the de novo DNA methyltransferase gene DNA methyltransferase 3 α ( DNMT3A ) and poor prognoses among acute myeloid leukemia (AML) patients. We show here that clinically observed DNMT3A mutations dramatically alter enzymatic activity, including mutations that lead to 6-fold hypermethylation and 3-fold hypomethylation of the human cyclin-dependent kinase inhibitor 2B ( CDKN2B or p15 ) gene promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN-AML) constitutes 40-50% of all AML cases and is the most epigenetically diverse AML subtype with pronounced changes in non-CpG DNA methylation. We identified a subset of DNMT3A mutations that enhance the enzyme's ability to perform non-CpG methylation by 2-8-fold. Many of these mutations mapped to DNMT3A regions known to interact with proteins that themselves contribute to AML, such as thymine DNA glycosylase (TDG). Using functional mapping of TDG-DNMT3A interactions, we provide evidence that TDG and DNMT3-like (DNMT3L) bind distinct regions of DNMT3A. Furthermore, DNMT3A mutations caused diverse changes in the ability of TDG and DNMT3L to affect DNMT3A function. Cell-based studies of one of these DNMT3A mutations (S714C) replicated the enzymatic studies and revealed that it causes dramatic losses of genome-wide methylation. In summary, mutations in DNMT3A lead to diverse levels of activity, interactions with epigenetic machinery components and cellular changes., (© 2019 Sandoval et al.)

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. Here, we characterized global epigenomic changes during the direct reprogramming of mouse fibroblasts to neurons using whole-genome base-resolution DNA methylation (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming., Competing Interests: CL, QL, OW, RC, JN, MM, MK, SC, MG, HC, MW, JE No competing interests declared, (© 2019, Luo et al.)

DNA methylation plays important roles in determining cellular identity, disease, and environmental responses, but little is known about the mechanisms that drive methylation changes during cellular differentiation and tumorigenesis. Meanwhile, the causal relationship between DNA methylation and transcription remains incompletely understood. Recently developed targeted DNA methylation manipulation tools can address these gaps in knowledge, leading to new insights into how methylation governs gene expression. Here, we summarize technological developments in the DNA methylation editing field and discuss the remaining challenges facing current tools, as well as potential future directions.