The intrinsic and extrinsic effects of TET proteins during gastrulation.

Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.

Role of Mammalian DNA Methyltransferases in Development.

DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.

2-Oxoglutarate-Dependent Oxygenases.

2-Oxoglutarate (2OG)-dependent oxygenases (2OGXs) catalyze a remarkably diverse range of oxidative reactions. In animals, these comprise hydroxylations and N-demethylations proceeding via hydroxylation; in plants and microbes, they catalyze a wider range including ring formations, rearrangements, desaturations, and halogenations. The catalytic flexibility of 2OGXs is reflected in their biological functions. After pioneering work identified the roles of 2OGXs in collagen biosynthesis, research revealed they also function in plant and animal development, transcriptional regulation, nucleic acid modification/repair, fatty acid metabolism, and secondary metabolite biosynthesis, including of medicinally important antibiotics. In plants, 2OGXs are important agrochemical targets and catalyze herbicide degradation. Human 2OGXs, particularly those regulating transcription, are current therapeutic targets for anemia and cancer. Here, we give an overview of the biochemistry of 2OGXs, providing examples linking to biological function, and outline how knowledge of their enzymology is being exploited in medicine, agrochemistry, and biocatalysis.

Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.

ALKBH1-Mediated tRNA Demethylation Regulates Translation.

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

CpG island reconfiguration for the establishment and synchronization of polycomb functions upon exit from naive pluripotency.

Polycomb group (PcG) proteins are essential for post-implantation development by depositing repressive histone modifications at promoters, mainly CpG islands (CGIs), of developmental regulator genes. However, promoter PcG marks are erased after fertilization and de novo established in peri-implantation embryos, coinciding with the transition from naive to primed pluripotency. Nevertheless, the molecular basis for this establishment remains unknown. In this study, we show that the expression of the long KDM2B isoform (KDM2BLF), which contains the demethylase domain, is specifically induced at peri-implantation and that its H3K36me2 demethylase activity is required for PcG enrichment at CGIs. Moreover, KDM2BLF interacts with BRG1/BRM-associated factor (BAF) and stabilizes BAF occupancy at CGIs for subsequent gain of accessibility, which precedes PcG enrichment. Consistently, KDM2BLF inactivation results in significantly delayed post-implantation development. In summary, our data unveil dynamic chromatin configuration of CGIs during exit from naive pluripotency and provide a conceptual framework for the spatiotemporal establishment of PcG functions.

Mechanism and function of oxidative reversal of DNA and RNA methylation.

The importance of eukaryotic DNA methylation [5-methylcytosine (5mC)] in transcriptional regulation and development was first suggested almost 40 years ago. However, the molecular mechanism underlying the dynamic nature of this epigenetic mark was not understood until recently, following the discovery that the TET proteins, a family of AlkB-like Fe(II)/α-ketoglutarate-dependent dioxygenases, can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since then, several mechanisms that are responsible for processing oxidized 5mC derivatives to achieve DNA demethylation have emerged. Our biochemical understanding of the DNA demethylation process has prompted new investigations into the biological functions of DNA demethylation. Characterization of two additional AlkB family proteins, FTO and ALKBH5, showed that they possess demethylase activity toward N(6)-methyladenosine (m(6)A) in RNA, indicating that members of this subfamily of dioxygenases have a general function in demethylating nucleic acids. In this review, we discuss recent advances in this emerging field, focusing on the mechanism and function of TET-mediated DNA demethylation.

UHRF2 commissions the completion of DNA demethylation through allosteric activation by 5hmC and K33-linked ubiquitination of XRCC1.

The transition of oxidized 5-methylcytosine (5mC) intermediates into the base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. We report here that UHRF2, the only paralog of UHRF1 in mammals that fails to rescue Uhrf1-/- phenotype, is physically and functionally associated with BER complex. We show that UHRF2 is allosterically activated by 5-hydroxymethylcytosine (5hmC) and acts as a ubiquitin E3 ligase to catalyze K33-linked polyubiquitination of XRCC1. This nonproteolytic action stimulates XRCC1's interaction with the ubiquitin binding domain-bearing RAD23B, leading to the incorporation of TDG into BER complex. Integrative epigenomic analysis in mouse embryonic stem cells reveals that Uhrf2-fostered TDG-RAD23B-BER complex is functionally linked to the completion of DNA demethylation at active promoters and that Uhrf2 ablation impedes DNA demethylation on latent enhancers that undergo poised-to-active transition during neuronal commitment. Together, these observations highlight an essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation.

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency.

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

Crystal Structure of the LSD1/CoREST Histone Demethylase Bound to Its Nucleosome Substrate.

LSD1 (lysine specific demethylase; also known as KDM1A), the first histone demethylase discovered, regulates cell-fate determination and is overexpressed in multiple cancers. LSD1 demethylates histone H3 Lys4, an epigenetic mark for active genes, but requires the CoREST repressor to act on nucleosome substrates. To understand how an accessory subunit (CoREST) enables a chromatin enzyme (LSD1) to function on a nucleosome and not just histones, we have determined the crystal structure of the LSD1/CoREST complex bound to a 191-bp nucleosome. We find that the LSD1 catalytic domain binds extranucleosomal DNA and is unexpectedly positioned 100 Å away from the nucleosome core. CoREST makes critical contacts with both histone and DNA components of the nucleosome, explaining its essential function in demethylating nucleosome substrates. Our studies also show that the LSD1(K661A) frequently used as a catalytically inactive mutant in vivo (based on in vitro peptide studies) actually retains substantial H3K4 demethylase activity on nucleosome substrates.

Demethylation of the Protein Phosphatase PP2A Promotes Demethylation of Histones to Enable Their Function as a Methyl Group Sink.

Dysregulation of chromatin methylation is associated with defects in cellular differentiation as well as a variety of cancers. How cells regulate the opposing activities of histone methyltransferase and demethylase enzymes to set the methylation status of the epigenome for proper control of gene expression and metabolism remains poorly understood. Here, we show that loss of methylation of the major phosphatase PP2A in response to methionine starvation activates the demethylation of histones through hyperphosphorylation of specific demethylase enzymes. In parallel, this regulatory mechanism enables cells to preserve SAM by increasing SAH to limit SAM consumption by methyltransferase enzymes. Mutants lacking the PP2A methyltransferase or the effector H3K36 demethylase Rph1 exhibit elevated SAM levels and are dependent on cysteine due to reduced capacity to sink the methyl groups of SAM. Therefore, PP2A directs the methylation status of histones by regulating the phosphorylation status of histone demethylase enzymes in response to SAM levels.

Nucleosome Turnover Regulates Histone Methylation Patterns over the Genome.

Recent studies have indicated that nucleosome turnover is rapid, occurring several times per cell cycle. To access the effect of nucleosome turnover on the epigenetic landscape, we investigated H3K79 methylation, which is produced by a single methyltransferase (Dot1l) with no known demethylase. Using chemical-induced proximity (CIP), we find that the valency of H3K79 methylation (mono-, di-, and tri-) is determined by nucleosome turnover rates. Furthermore, propagation of this mark is predicted by nucleosome turnover simulations over the genome and accounts for the asymmetric distribution of H3K79me toward the transcriptional unit. More broadly, a meta-analysis of other conserved histone modifications demonstrates that nucleosome turnover models predict both valency and chromosomal propagation of methylation marks. Based on data from worms, flies, and mice, we propose that the turnover of modified nucleosomes is a general means of propagation of epigenetic marks and a determinant of methylation valence.

The Set1 complex is dimeric and acts with Jhd2 demethylation to convey symmetrical H3K4 trimethylation.

Epigenetic modifications can maintain or alter the inherent symmetry of the nucleosome. However, the mechanisms that deposit and/or propagate symmetry or asymmetry are not understood. Here we report that yeast Set1C/COMPASS (complex of proteins associated with Set1) is dimeric and, consequently, symmetrically trimethylates histone 3 Lys4 (H3K4me3) on promoter nucleosomes. Mutation of the dimer interface to make Set1C monomeric abolished H3K4me3 on most promoters. The most active promoters, particularly those involved in the oxidative phase of the yeast metabolic cycle, displayed H3K4me2, which is normally excluded from active promoters, and a subset of these also displayed H3K4me3. In wild-type yeast, deletion of the sole H3K4 demethylase, Jhd2, has no effect. However, in monomeric Set1C yeast, Jhd2 deletion increased H3K4me3 levels on the H3K4me2 promoters. Notably, the association of Set1C with the elongating polymerase was not perturbed by monomerization. These results imply that symmetrical H3K4 methylation is an embedded consequence of Set1C dimerism and that Jhd2 demethylates asymmetric H3K4me3. Consequently, rather than methylation and demethylation acting in opposition as logic would suggest, a dimeric methyltransferase and monomeric demethylase cooperate to eliminate asymmetry and focus symmetrical H3K4me3 onto selected nucleosomes. This presents a new paradigm for the establishment of epigenetic detail.

GADD45 promotes locus-specific DNA demethylation and 2C cycling in embryonic stem cells.

Mouse embryonic stem cell (ESC) cultures contain a rare cell population of "2C-like" cells resembling two-cell embryos, the key stage of zygotic genome activation (ZGA). Little is known about positive regulators of the 2C-like state and two-cell stage embryos. Here we show that GADD45 (growth arrest and DNA damage 45) proteins, regulators of TET (TET methylcytosine dioxygenase)-mediated DNA demethylation, promote both states. Methylome analysis of Gadd45a,b,g triple-knockout (TKO) ESCs reveal locus-specific DNA hypermethylation of ∼7000 sites, which are enriched for enhancers and loci undergoing TET-TDG (thymine DNA glycosylase)-mediated demethylation. Gene expression is misregulated in TKOs, notably upon differentiation, and displays signatures of DNMT (DNA methyltransferase) and TET targets. TKOs manifest impaired transition into the 2C-like state and exhibit DNA hypermethylation and down-regulation of 2C-like state-specific genes. Gadd45a,b double-mutant mouse embryos display embryonic sublethality, deregulated ZGA gene expression, and developmental arrest. Our study reveals an unexpected role of GADD45 proteins in embryonic two-cell stage regulation.

Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A.

Repair of DNA double-strand breaks (DSBs) must be orchestrated properly within diverse chromatin domains in order to maintain genetic stability. Euchromatin and heterochromatin domains display major differences in histone modifications, biophysical properties, and spatiotemporal dynamics of DSB repair. However, it is unclear whether differential histone-modifying activities are required for DSB repair in these distinct domains. We showed previously that the Drosophila melanogaster KDM4A (dKDM4A) histone demethylase is required for heterochromatic DSB mobility. Here we used locus-specific DSB induction in Drosophila animal tissues and cultured cells to more deeply interrogate the impact of dKDM4A on chromatin changes, temporal progression, and pathway utilization during DSB repair. We found that dKDM4A promotes the demethylation of heterochromatin-associated histone marks at DSBs in heterochromatin but not euchromatin. Most importantly, we demonstrate that dKDM4A is required to complete DSB repair in a timely manner and regulate the relative utilization of homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways but exclusively for heterochromatic DSBs. We conclude that the temporal kinetics and pathway utilization during heterochromatic DSB repair depend on dKDM4A-dependent demethylation of heterochromatic histone marks. Thus, distinct pre-existing chromatin states require specialized epigenetic alterations to ensure proper DSB repair.

PHF2-mediated H3K9me balance orchestrates heterochromatin stability and neural progenitor proliferation.

Heterochromatin stability is crucial for progenitor proliferation during early neurogenesis. It relays on the maintenance of local hubs of H3K9me. However, understanding the formation of efficient localized levels of H3K9me remains limited. To address this question, we used neural stem cells to analyze the function of the H3K9me2 demethylase PHF2, which is crucial for progenitor proliferation. Through mass-spectroscopy and genome-wide assays, we show that PHF2 interacts with heterochromatin components and is enriched at pericentromeric heterochromatin (PcH) boundaries where it maintains transcriptional activity. This binding is essential for silencing the satellite repeats, preventing DNA damage and genome instability. PHF2's depletion increases the transcription of heterochromatic repeats, accompanied by a decrease in H3K9me3 levels and alterations in PcH organization. We further show that PHF2's PHD and catalytic domains are crucial for maintaining PcH stability, thereby safeguarding genome integrity. These results highlight the multifaceted nature of PHF2's functions in maintaining heterochromatin stability and regulating gene expression during neural development. Our study unravels the intricate relationship between heterochromatin stability and progenitor proliferation during mammalian neurogenesis.

Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm.

FTO, the first RNA demethylase discovered, mediates the demethylation of internal N6-methyladenosine (m6A) and N6, 2-O-dimethyladenosine (m6Am) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m6A and cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am in snRNAs, and N1-methyladenosine (m1A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m6A than the ones with cap m6Am in the tested cells. We also show that FTO can directly repress translation by catalyzing m1A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m6A and m6Am in mRNA and snRNA as well as m1A in tRNA.

The HP1α protein is mandatory to repress the circadian clock and its output genes during the 12 h period of transcriptional repression.

The transcriptional repressions driven by the circadian core clock repressors RevErbα, E4BP4, and CRY1/PER1 involve feedback loops which are mandatory for generating the circadian rhythms. These repressors are known to bind to cognate DNA binding sites, but how their circadian bindings trigger the cascade of events leading to these repressions remain to be elucidated. Through molecular and genetic analyses, we now demonstrate that the chromatin protein HP1α plays a key role in these transcriptional repressions of both the circadian clock (CC) genes and their cognate output genes (CCGs). We show that these CC repressors recruit the HP1α protein downstream from a repressive cascade, and that this recruitment is mandatory for the maintenance of both the CC integrity and the expression of the circadian genes. We further show that the presence of HP1α is critical for both the repressor-induced chromatin compaction and the generation of "transcriptionally repressed biomolecular hydrophobic condensates" and demonstrates that HP1α is mandatory within the CC output genes for both the recruitment of DNA methylating enzymes on the intronic deoxyCpG islands and their subsequent methylation.

The circadian demethylation of a unique intronic deoxymethylCpG-rich island boosts the transcription of its cognate circadian clock output gene.

We demonstrate that there is a tight functional relationship between two highly evolutionary conserved cell processes, i.e., the circadian clock (CC) and the circadian DNA demethylation-methylation of cognate deoxyCpG-rich islands. We have discovered that every circadian clock-controlled output gene (CCG), but not the core clock nor its immediate-output genes, contains a single cognate intronic deoxyCpG-rich island, the demethylation-methylation of which is controlled by the CC. During the transcriptional activation period, these intronic islands are demethylated and, upon dimerization of two YY1 protein binding sites located upstream to the transcriptional enhancer and downstream from the deoxyCpG-rich island, store activating components initially assembled on a cognate active enhancer (a RORE, a D-box or an E-box), in keeping with the generation of a transcriptionally active condensate that boosts the initiation of transcription of their cognate pre-mRNAs. We report how these single intronic deoxyCpG-rich islands are instrumental in such a circadian activation/repression transcriptional process.

Lack of mismatch repair enhances resistance to methylating agents for cells deficient in oxidative demethylation.

The human alkylation B (AlkB) homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct reversal repair enzymes that remove 1-methyladenine (1meA) and 3-methylcytosine (3meC) lesions commonly generated by alkylating chemotherapeutic agents. Thus, the existence of deficiencies in ALKBH proteins can be exploited in synergy with chemotherapy. In this study, we investigated possible interactions between ALKBH2 and ALKBH3 with other proteins that could alter damage response and discovered an interaction with the mismatch repair (MMR) system. To test whether the lack of active MMR impacts ALKBH2 and/or ALKBH3 response to methylating agents, we generated cells deficient in ALKBH2, ALKBH3, or both in addition to Mlh homolog 1 (MLH1), another MMR protein. We found that MLH1koALKBH3ko cells showed enhanced resistance toward SN1- and SN2-type methylating agents, whereas MLH1koALKBH2ko cells were only resistant to SN1-type methylating agents. Concomitant loss of ALKBH2 and ALKBH3 (ALKBH2ko3ko) rendered cells sensitive to SN1- and SN2-agents, but the additional loss of MLH1 enhanced resistance to both types of damage. We also showed that ALKBH2ko3ko cells have an ATR-dependent arrest at the G2/M checkpoint, increased apoptotic signaling, and replication fork stress in response to methylation. However, these responses were not observed with the loss of functional MLH1 in MLH1koALKBH2ko3ko cells. Finally, in MLH1koALKBH2ko3ko cells, we observed elevated mutant frequency in untreated and temozolomide treated cells. These results suggest that obtaining a more accurate prognosis of chemotherapeutic outcome requires information on the functionality of ALKBH2, ALKBH3, and MLH1.