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In mammalian cells, gene copy number is tightly controlled to maintain gene expression and genome stability. However, a common molecular feature across cancer types is oncogene amplification, which promotes cancer progression by drastically increasing the copy number and expression of tumor-promoting genes. For example, in tyrosine kinase inhibitor (TKI)-resistant lung adenocarcinoma (LUAD), oncogene amplification occurs in over 40% of patients' tumors. Despite the prevalence of oncogene amplification in TKI-resistant tumors, the mechanisms facilitating oncogene amplification are not fully understood. Here, we find that LUADs exhibit a unique chromatin signature demarcated by strong CTCF and cohesin deposition in drug-naïve tumors, which correlates with the boundaries of oncogene amplicons in TKI-resistant LUAD cells. We identified a global chromatin priming effect during the acquisition of TKI resistance, marked by a dynamic increase of H3K27Ac, cohesin loading, and inter-TAD interactions, which occurs before the onset of oncogene amplification. Furthermore, we have found that the METTL7A protein, which was previously reported to localize to the endoplasmic reticulum and inner nuclear membrane, has a novel chromatin regulatory function by binding to amplified loci and regulating cohesin recruitment and inter-TAD interactions. Surprisingly, we discovered that METTL7A remodels the chromatin landscape prior to large-scale copy number gains. Furthermore, while METTL7A depletion has little effect on the chromatin structure and proliferation of drug-naïve cells, METTL7A depletion prevents the formation and maintenance of TKI resistant-clones, highlighting the specific role of METTL7A as cells are becoming resistant. In summary, we discovered an unexpected mechanism required for the acquisition of TKI resistance regulated by a largely uncharacterized factor, METTL7A. This discovery sheds light into the maintenance of oncogene copy number and paves the way to the development of new therapeutics for preventing TKI resistance in LUAD., Competing Interests: Competing interests The authors declare no competing interests.

N 6 -methyladenosine (m 6 A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m 6 A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m 6 A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m 6 A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers., Competing Interests: Declaration of interests C.K. is the Scientific Founder, Scientific Advisor to the Board of Directors, Scientific Advisory Board member, shareholder, and consultant for Foghorn Therapeutics, Inc. (Cambridge, MA), serves on the Scientific Advisory Boards of Nereid Therapeutics, Nested Therapeutics, Accent Therapeutics, and Fibrogen, Inc. and is a consultant for Cell Signaling Technologies and Google Ventures. C.K. is also a member of the Molecular Cell and Cell Chemical Biology Editorial Boards. D.L. Rimm reports grants and personal fees from Amgen, Astra Zeneca, Cepheid, Konica – Minolta, Lilly, NextCure and personal fees from Cell Signaling Technology, Danaher, Fluidigm, GSK, Merck, Monopteros, NanoString, Odonate, Paige.AI, Regeneron, Roche, Sanofi, Ventana, Verily. K. Politi reports grants from the NCI/NIH; grants and personal fees from AstraZeneca; grants from Kolltan, Roche/Genentech, Boehringer Ingelheim, D2G Oncology and Symphogen; and personal fees from Janssen, Dynamo Therapeutics, Halda, Maverick Therapeutics, and Tocagen; and a patent for EGFR(T790M) mutation testing issued, licensed, and with royalties paid from Molecular Diagnostics/Memorial Sloan Kettering Cancer Center. P.A.J. is an equity owner in Gatekeeper Pharmaceuticals; consults for AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Chugai Pharmaceuticals, Eli Lilly Pharmaceuticals, Araxes Pharmaceuticals, SFJ Pharmaceuticals, Voronoi, Daiichi Sankyo, Biocartis, Novartis, Sanofi, Takeda Oncology, Mirati Therapeutics, Transcenta, Silicon Therapeutics, Syndax, Nuvalent, Bayer, Esai, Allorion Therapeutics, Accutar Biotech, and Abbvie; receives research support from AstraZeneca, Daiichi Sankyo, PUMA, Eli Lilly, Boehringer Ingelheim, Revolution Medicines, and Takeda Oncology and is a co-inventor and receives postmarketing royalties on a DFCI owned patent on EGFR mutations licensed to LabCorp. Q.Y. reports grants and personal fees from AstraZeneca, and is a Scientific Advisory Board member of AccuraGen Inc. D.X.N received research funding from AstraZeneca. The other authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In this issue of Cell Stem Cell, Xu et al. and Yu et al. use low-input epigenetic profiling techniques to map H3K9me3 deposition in early human development. They reveal stage-specific H3K9me3 deposition on retrotransposons, which may play crucial cis-regulatory roles in early development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Long non-coding RNAs (lncRNAs) are important regulators of development. In this issue of Developmental Cell, Wilson et al. utilize pluripotent stem cell models to demonstrate that a primate lncRNA, BANCR, is primarily expressed in fetal cardiomyocytes and promotes cell migration., (Copyright © 2020. Published by Elsevier Inc.)

The recent discovery of N 6 -methyladenine (N 6 -mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism 1 . However, the biological role of N 6 -mA and the molecular pathways that exert its function remain unclear. Here we show that N 6 -mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N 6 -mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD) 2-4 . Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures 3,5,6 . We show that the presence of N 6 -mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N 6 -mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N 6 -mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD-SATB1 interactions mediated by N 6 -mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N 6 -mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.

Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscore broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

DNA modifications are a major form of epigenetic regulation that eukaryotic cells utilize in concert with histone modifications. While much work has been done elucidating the role of 5-methylcytosine over the past several decades, only recently has it been recognized that N(6)-methyladenine (N 6 -mA) is present in quantifiable and biologically active levels in the DNA of eukaryotic cells. Unlike prokaryotes which utilize N 6 -mA to recognize "self" from "foreign" DNA, eukaryotes have been found to use N 6 -mA in varying ways, from regulating transposable elements to gene regulation in response to hypoxia and stress. In this review, we examine the current state of the N 6 -mA in research field, and the current understanding of the biochemical mechanisms which deposit and remove N 6 -mA from the eukaryotic genome.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N 6 -methyladenine (N 6 -mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N 6 -mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N 6 -mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N 6 -mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N 6 -mA., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The advance of iPS technology holds great promise for regenerative medicine. Despite their global similarity to ES cells, fully reprogrammed iPS cells generated by current procedures still display clone-to-clone variations in molecular properties and developmental potentials, which calls for the development of reliable quality control assays. The differences in developmental potentials in iPS cells may be caused by epigenetic variations, such as histone variant H2A.X deposition. In this review, we discuss the current understanding of molecular variations of iPS cells and their implication on quality assessments., (Copyright © 2017. Published by Elsevier Ltd.)

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N(6)-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.