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Aging Jul 2023A hallmark of eukaryotic aging is a loss of epigenetic information, a process that can be reversed. We have previously shown that the ectopic induction of the Yamanaka...
A hallmark of eukaryotic aging is a loss of epigenetic information, a process that can be reversed. We have previously shown that the ectopic induction of the Yamanaka factors OCT4, SOX2, and KLF4 (OSK) in mammals can restore youthful DNA methylation patterns, transcript profiles, and tissue function, without erasing cellular identity, a process that requires active DNA demethylation. To screen for molecules that reverse cellular aging and rejuvenate human cells without altering the genome, we developed high-throughput cell-based assays that distinguish young from old and senescent cells, including transcription-based aging clocks and a real-time nucleocytoplasmic compartmentalization (NCC) assay. We identify six chemical cocktails, which, in less than a week and without compromising cellular identity, restore a youthful genome-wide transcript profile and reverse transcriptomic age. Thus, rejuvenation by age reversal can be achieved, not only by genetic, but also chemical means.
Topics: Animals; Humans; Cellular Reprogramming; Cellular Senescence; Aging; DNA Methylation; Induced Pluripotent Stem Cells; Mammals
PubMed: 37437248
DOI: 10.18632/aging.204896 -
Nature Oct 2023Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell...
Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration. Here, to explore whether metabolic reprogramming can overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b. We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia-reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b-mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref. ). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering their transcription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts.
Topics: Animals; Mice; Carnitine O-Palmitoyltransferase; Cell Hypoxia; Cell Proliferation; Cellular Reprogramming; Energy Metabolism; Enzyme Activation; Epigenesis, Genetic; Fatty Acids; Heart; Histone Demethylases; Ketoglutaric Acids; Mutation; Myocardium; Myocytes, Cardiac; Oxidation-Reduction; Regeneration; Reperfusion Injury; Transcription, Genetic
PubMed: 37758950
DOI: 10.1038/s41586-023-06585-5 -
Epigenomes Sep 2023Research in epigenetics has dramatically risen during the last decade to include aspects of environmental biology. However, many questions remain regarding the effects...
Research in epigenetics has dramatically risen during the last decade to include aspects of environmental biology. However, many questions remain regarding the effects of environmental stressors on the epigenome, incorporating the particular role of epigenetic mechanisms in the adaptation and evolution of organisms in changing environments. Epigenetics is commonly defined as mitotically and/or meiotically heritable changes in gene function that occur without altering the underlying DNA sequence. It encompasses DNA (hydroxy)methylation, histone modifications, chromatin structure, and non-coding RNAs that may be inherited across generations under certain circumstances. Epigenetic mechanisms are perfect candidates to extend our understanding of the impact of environmental stressors on organisms and to explain the rapid phenomenon of adaptive evolution. Existing evidence shows that environmental cues can affect the epigenome and modify gene expression accordingly. These changes can then induce phenotypic modifications that are morphological, physiological, or behavioral at the organismal level. In this Special Issue focusing on environmental epigenetics, we provide an overview of influences to the epigenome that are driven by various environmental and evolutionary factors, with a particular focus on DNA methylation (DNAm). Five research groups have contributed insightful studies or reviews on (1) DNAm and demethylation events affected by the exposome; (2) DNAm as a potential biomarker to determine cardiometabolic risk early in life; (3) consequences of DNAm across multiple generations; (4) DNAm variation within natural animal populations; and (5) epigenetic mechanisms in genetically uniform organisms. Collectively, the articles from this Special Issue consistently support that environmental changes can induce long-lasting epigenetic effects within a given organism pertaining to individual risk for disease, or multi-generational impacts that ultimately impact evolution.
PubMed: 37754273
DOI: 10.3390/epigenomes7030021 -
Proceedings of the National Academy of... Dec 2023Existing single-cell bisulfite-based DNA methylation analysis is limited by low DNA recovery, and the measurement of 5hmC at single-base resolution remains challenging....
Existing single-cell bisulfite-based DNA methylation analysis is limited by low DNA recovery, and the measurement of 5hmC at single-base resolution remains challenging. Here, we present a bisulfite-free single-cell whole-genome 5mC and 5hmC profiling technique, named Cabernet, which can characterize 5mC and 5hmC at single-base resolution with high genomic coverage. Cabernet utilizes Tn5 transposome for DNA fragmentation, which enables the discrimination between different alleles for measuring hemi-methylation status. Using Cabernet, we revealed the 5mC, hemi-5mC and 5hmC dynamics during early mouse embryo development, uncovering genomic regions exclusively governed by active or passive demethylation. We show that hemi-methylation status can be used to distinguish between pre- and post-replication cells, enabling more efficient cell grouping when integrated with 5mC profiles. The property of Tn5 naturally enables Cabernet to achieve high-throughput single-cell methylome profiling, where we probed mouse cortical neurons and embryonic day 7.5 (E7.5) embryos, and constructed the library for thousands of single cells at high efficiency, demonstrating its potential for analyzing complex tissues at substantially low cost. Together, we present a way of high-throughput methylome and hydroxymethylome detection at single-cell resolution, enabling efficient analysis of the epigenetic status of biological systems with complicated nature such as neurons and cancer cells.
Topics: Animals; Mice; 5-Methylcytosine; DNA Methylation; Sulfites; Sequence Analysis, DNA; Cytosine
PubMed: 38011566
DOI: 10.1073/pnas.2310367120 -
Gastroenterology Aug 2023Immune checkpoint blockade therapy benefits only a small subset of patients with colorectal cancer (CRC), and identification of CRC-intrinsic events modulating immune...
BACKGROUND & AIMS
Immune checkpoint blockade therapy benefits only a small subset of patients with colorectal cancer (CRC), and identification of CRC-intrinsic events modulating immune checkpoint blockade efficacy is an unmet need. We found that AlkB homolog 5 (ALKBH5), an RNA N-methyladenosine eraser, drives immunosuppression and is a molecular target to boost immune checkpoint blockade therapy in CRC.
METHODS
Clinical significance of ALKBH5 was evaluated in human samples (n = 205). Function of ALKBH5 was investigated in allografts, CD34 humanized mice, and Alkbh5 knockin mice. Immunity change was determined by means of flow cytometry, immunofluorescence, and functional investigation. Methylated RNA immunoprecipitation sequencing and RNA sequencing were used to identify ALKBH5 targets. Vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA was constructed for targeting ALKBH5 in vivo.
RESULTS
High ALKBH5 expression predicts poor prognosis in CRC. ALKBH5 induced myeloid-derived suppressor cell accumulation but reduced natural killer cells and cytotoxic CD8 T cells to induce colorectal tumorigenesis in allografts, CD34 humanized mice, and intestine-specific Alkbh5 knockin mice. Mechanistically, AXIN2, a Wnt suppressor, was identified as a target of ALKBH5. ALKBH5 binds and demethylates AXIN2 messenger RNA, which caused its dissociation from N-methyladenosine reader IGF2BP1 and degradation, resulting in hyperactivated Wnt/β-catenin. Subsequently, Wnt/β-catenin targets, including Dickkopf-related protein 1 (DKK1) were induced by ALKBH5. ALKBH5-induced DKK1 recruited myeloid-derived suppressor cells to drive immunosuppression in CRC, and this effect was abolished by anti-DKK1 in vitro and in vivo. Finally, vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA, or anti-DKK1 potentiated anti-PD1 treatment in suppressing CRC growth by enhancing antitumor immunity.
CONCLUSIONS
This study identified an ALKBH5-N-methyladenosine-AXIN2-Wnt-DKK1 axis in CRC, which drives immune suppression to facilitate tumorigenesis. Targeting of ALKBH5 is a promising strategy for sensitizing CRC to immunotherapy.
Topics: Humans; Mice; Animals; beta Catenin; CD8-Positive T-Lymphocytes; Immune Checkpoint Inhibitors; Carcinogenesis; Cell Transformation, Neoplastic; RNA, Small Interfering; Immunotherapy; Immunosuppression Therapy; Colorectal Neoplasms; Axin Protein; AlkB Homolog 5, RNA Demethylase
PubMed: 37169182
DOI: 10.1053/j.gastro.2023.04.032 -
Nature Aug 2023Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human...
Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
Topics: Humans; Cellular Reprogramming; Chromatin; DNA Demethylation; DNA Methylation; DNA Transposable Elements; Epigenesis, Genetic; Induced Pluripotent Stem Cells; Human Embryonic Stem Cells; Lamin Type B
PubMed: 37587336
DOI: 10.1038/s41586-023-06424-7 -
Signal Transduction and Targeted Therapy Aug 2023Ten-eleven translocation (TET) family proteins (TETs), specifically, TET1, TET2 and TET3, can modify DNA by oxidizing 5-methylcytosine (5mC) iteratively to yield... (Review)
Review
Ten-eleven translocation (TET) family proteins (TETs), specifically, TET1, TET2 and TET3, can modify DNA by oxidizing 5-methylcytosine (5mC) iteratively to yield 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), and then two of these intermediates (5fC and 5caC) can be excised and return to unmethylated cytosines by thymine-DNA glycosylase (TDG)-mediated base excision repair. Because DNA methylation and demethylation play an important role in numerous biological processes, including zygote formation, embryogenesis, spatial learning and immune homeostasis, the regulation of TETs functions is complicated, and dysregulation of their functions is implicated in many diseases such as myeloid malignancies. In addition, recent studies have demonstrated that TET2 is able to catalyze the hydroxymethylation of RNA to perform post-transcriptional regulation. Notably, catalytic-independent functions of TETs in certain biological contexts have been identified, further highlighting their multifunctional roles. Interestingly, by reactivating the expression of selected target genes, accumulated evidences support the potential therapeutic use of TETs-based DNA methylation editing tools in disorders associated with epigenetic silencing. In this review, we summarize recent key findings in TETs functions, activity regulators at various levels, technological advances in the detection of 5hmC, the main TETs oxidative product, and TETs emerging applications in epigenetic editing. Furthermore, we discuss existing challenges and future directions in this field.
Topics: Dioxygenases; Epigenesis, Genetic; DNA Methylation; Gene Expression Regulation; Oxidation-Reduction
PubMed: 37563110
DOI: 10.1038/s41392-023-01537-x -
Experimental & Molecular Medicine Aug 2023Improving health and delaying aging is the focus of medical research. Previous studies have shown that mesenchymal stem cell (MSC) senescence is closely related to...
Improving health and delaying aging is the focus of medical research. Previous studies have shown that mesenchymal stem cell (MSC) senescence is closely related to organic aging and the development of aging-related diseases such as osteoarthritis (OA). m6A is a common RNA modification that plays an important role in regulating cell biological functions, and ALKBH5 is one of the key m6A demethylases. However, the role of m6A and ALKBH5 in MSC senescence is still unclear. Here, we found that the m6A level was enhanced and ALKBH5 expression was decreased in aging MSCs induced by multiple replications, HO stimulation or UV irradiation. Downregulation of ALKBH5 expression facilitated MSC senescence by enhancing the stability of CYP1B1 mRNA and inducing mitochondrial dysfunction. In addition, IGF2BP1 was identified as the m6A reader restraining the degradation of m6A-modified CYP1B1 mRNA. Furthermore, Alkbh5 knockout in MSCs aggravated spontaneous OA in mice, and overexpression of Alkbh5 improved the efficacy of MSCs in OA. Overall, this study revealed a novel mechanism of m6A in MSC senescence and identified promising targets to protect against aging and OA.
Topics: Animals; Mice; Demethylation; Hydrogen Peroxide; Mesenchymal Stem Cells; Osteoarthritis; RNA Stability; RNA, Messenger; AlkB Homolog 5, RNA Demethylase; Cytochrome P-450 CYP1B1
PubMed: 37524872
DOI: 10.1038/s12276-023-01059-0 -
Nature Immunology Aug 2023Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune...
Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N-methyladenosine (mA) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. mA RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly mA methylated in ILC2s. Targeted mA demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of mA for ILC2 responses.
Topics: Cytokines; Homeostasis; Immunity, Innate; Lymphocytes; RNA; Animals; Mice
PubMed: 37400674
DOI: 10.1038/s41590-023-01548-4 -
Nature Nov 2023Microsatellite repeat expansions within genes contribute to a number of neurological diseases. The accumulation of toxic proteins and RNA molecules with repetitive...
Microsatellite repeat expansions within genes contribute to a number of neurological diseases. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N-methyladenosine (mA) by TRMT61A, and that mA can be demethylated by ALKBH3. We also observed that the mA/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with mA in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, mA in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of mA in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.
Topics: Animals; Humans; Adenosine; Caenorhabditis elegans; DNA-Binding Proteins; Drosophila melanogaster; Neurodegenerative Diseases; RNA; Trinucleotide Repeat Expansion; Cytoplasm; Disease Models, Animal
PubMed: 37938769
DOI: 10.1038/s41586-023-06701-5