DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is essential for genome regulation and development1, 2. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-Å crystal structure of the DNMT3A-DNMT3L-DNA complex where two DNMT3A monomers simultaneously attack two CpG dinucleotides, with the target sites separated by fourteen base pairs within the same DNA duplex. The DNMT3A–DNA interaction involves a target recognition domain (TRD), a catalytic loop and DNMT3A homodimeric interface. A TRD residue Arg836 makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Hematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of hematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its etiologic link to human disease.

SUMMARY Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3)-binding activity is harbored in the Tudor motifs of PRC2-associated polycomblike (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of ‘poised’ developmental genes, and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.

SUMMARY Direct reprogramming of induced cardiomyocytes (iCMs) suffers from low efficiency and requires extensive epigenetic repatterning, although the underlying mechanisms are largely unknown. To address these issues, we screened for epigenetic regulators of iCM reprogramming and found that reducing levels of the polycomb complex gene Bmi1 significantly enhanced induction of beating iCMs from neonatal and adult mouse fibroblasts. The inhibitory role of Bmi1 in iCM reprogramming is mediated through direct interactions with regulatory regions of cardiogenic genes, rather than regulation of cell proliferation. Reduced Bmi1 expression corresponded with increased levels of the active histone mark H3K4me3 and reduced levels of repressive H2AK199ub at cardiogenic loci, and de-repression of cardiogenic gene expression during iCM conversion. Furthermore, Bmi1 deletion could substitute for Gata4 during iCM reprogramming. Thus, Bmi1 acts as a critical epigenetic barrier to iCM production. Bypassing this barrier simplifies iCM generation and increases yield, potentially streamlining iCM production for therapeutic purposes.

During exocytosis, the evolutionarily conserved exocyst complex tethers Golgi-derived vesicles to the target plasma membrane, a critical function for secretory pathways. Here we show that exo70B1 loss-of-function mutants express activated defense responses upon infection and express enhanced resistance to fungal, oomycete and bacterial pathogens. In a screen for mutants that suppress exo70B1 resistance, we identified nine alleles of TIR-NBS2 (TN2), suggesting that loss-of-function of EXO70B1 leads to activation of this nucleotide binding domain and leucine-rich repeat-containing (NLR)-like disease resistance protein. This NLR-like protein is atypical because it lacks the LRR domain common in typical NLR receptors. In addition, we show that TN2 interacts with EXO70B1 in yeast and in planta. Our study thus provides a link between the exocyst complex and the function of a ‘TIR-NBS only’ immune receptor like protein. Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery. TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.

SUMMARY DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that DNMT3A mutational hotspot at Arg882 (DNMT3AR882H) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3AR882H directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1 and Hoxa gene cluster. DNMT3AR882H induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3AR882H-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3AR882H-induced gene expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

The Tudor domain comprises a family of motifs that mediate protein-protein interactions required for various DNA-templated biological processes. Emerging evidence demonstrates a versatility of the Tudor family domains by identifying their specific interactions to a wide variety of histone methylation marks. Here, we discuss novel functions of a number of Tudor-containing proteins (including JMJD2A, 53BP1, SGF29, Spindlin1, UHRF1, PHF1, PHF19 and SHH1) in ‘reading’ unique methylation events on histones in order to facilitate DNA damage repair or regulate transcription. This review covers our recent understanding of the molecular bases for histone-Tudor interactions and their biological outcomes. As deregulation of Tudor-containing proteins is associated with certain human disorders, pharmacological targeting of Tudor interactions could provide new avenues for therapeutic intervention.

Embryonic stem cells (ESCs) possess the capacity to proliferate indefinitely in an undifferentiated state and to differentiate into various cell types in an organism. However, the critical question of how self-renewal and differentiation are precisely regulated in ESCs is not entirely understood at present. Here, we report the essential role of Tbx3, a pluripotency-related transcription factor of the T-box gene family, for both the maintenance of self-renewal of mouse ESCs and for their differentiation into extraembryonic endoderm (ExEn). We show that Tbx3 is highly expressed in ExEn cells in addition to undifferentiated ESCs. Knockdown of Tbx3 expression using tetracycline-regulated Tbx3 siRNA resulted in the attenuation of ESC self-renewal ability and aberrant differentiation processes, including reduced ExEn differentiation but enhanced ectoderm and trophectoderm differentiation. Conversely, inducible forced expression of Tbx3 triggered ExEn lineage commitment. Mechanistically, Tbx3 directly activated the expression of Gata6, an essential regulator of ExEn. Interestingly, Tbx3 modulated H3K27me3 modification and the association of the PRC2 complex with the promoter region of Gata6. Taken together, the results of this study revealed a previously unappreciated role of a pluripotency factor in ExEn differentiation. Additionally, our data reveal that Tbx3 may function through direct binding and epigenetic modification of histones on the Gata6 promoter to maintain the ExEn differentiation potential of ESCs.The mouse blastocyst contains a trophectodermal epithelium surrounding an inner cell mass that later gives rise to primitive endoderm and epiblast cells. The primitive endoderm will develop into extraembryonic endoderm (ExEn), 2 which ultimately contributes to the yolk sac, whereas the epiblast contains pluripotent cells that generate all of the cell types of the three embryonic germ layers (ectoderm, endoderm, and mesoderm) and some extraembryonic tissues (1, 2). Mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocyst-stage embryos retain the capacity for unlimited self-renewal and their full developmental potential when cultured in vitro in the presence of leukemia inhibitory factor (LIF). Withdrawal of LIF leads to the disruption of ESC selfrenewal and extensive differentiation into various lineages. Understanding the mechanisms governing self-renewal and early lineage commitment is an essential step toward the future utilization of ESC derivatives in clinical applications. The unique properties of ESCs are conferred by a set of pluripotency-associated transcription factors, including Oct4, Sox2, and Nanog (3-6). Recent studies of the transcriptome and protein interactome of ESCs have further revealed the key role of Oct4/Sox2/Nanog-centered transcriptional and protein interaction networks in controlling ESC identity (7-11). However, the precise molecular mechanisms through which ESCs determine the cell fate between self-renewal and lineage commitment remain largely unknown. In additi...

Self-renewal and differentiation of embryonic stem cells (ESCs) are controlled by intracellular transcriptional factors and extracellular factor-activated signaling pathways. Transcription factor Oct4 is a key player maintaining ESCs in an undifferentiated state, whereas the Erk/MAPK pathway is known to be important for ESC differentiation. However, the manner in which intracellular pluripotency factors modulate extracellular factor-activated signaling pathways in ESCs is not well understood. Here, we report identification of a target gene of Oct4, serine/threonine kinase 40 (Stk40), which is able to activate the Erk/MAPK pathway and induce extraembryonicendoderm (ExEn) differentiation in mouse ESCs. Interestingly, cells overexpressing Stk40 exclusively contribute to the ExEn layer of chimeric embryos when injected into host blastocysts. In contrast, deletion of Stk40 in ESCs markedly reduces ExEn differentiation in vitro. Mechanistically, Stk40 interacts with Rcn2, which also activates Erk1/2 to induce ExEn specification in mouse ESCs. Moreover, Rcn2 proteins are specifically located in the cytoplasm of the ExEn layer of early mouse embryos. Importantly, knockdown of Rcn2 blocks Stk40-activated Erk1/2 and ESC differentiation. Therefore, our study establishes a link between the pluripotency factor Oct4 and the Erk/MAPK signaling pathway, and it uncovers cooperating signals in the Erk/ MAPK activation that control ExEn differentiation.embryonic stem cells | Rcn2 | Ras | Gata6