TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation.

DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.

Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes.

Templated chemistry offers the prospect of addressing specificity challenges occurring in bioconjugation reactions. Here, we show two peptide-templated amide-bond forming reactions that enable the concurrent labelling of two different membrane proteins with two different peptide nucleic acid (PNA) barcodes. The reaction system is based on the mutually selective coiled coil interaction between two thioester-linked PNA-peptide conjugates and two cysteine peptides serving as genetically encoded peptide tags. Orthogonal coiled coil templated covalent labelling is highly specific, quantitative and proceeds within a minute. To demonstrate the usefulness, we evaluated receptor internalisation of two membranous receptors EGFR (epidermal growth factor) and ErbB2 (epidermal growth factor receptor 2) by first staining PNA-tagged proteins with fluorophore-DNA conjugates and then erasing signals from non-internalized receptors via toehold-mediated strand displacement.

Loss-of-function mutations in the histone methyltransferase EZH2 promote chemotherapy resistance in AML.

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.

Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins.

DNA nanotechnology is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labelling by way of toehold-mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells.

Staying true to yourself: mechanisms of DNA methylation maintenance in mammals.

DNA methylation is essential to development and cellular physiology in mammals. Faulty DNA methylation is frequently observed in human diseases like cancer and neurological disorders. Molecularly, this epigenetic mark is linked to other chromatin modifications and it regulates key genomic processes, including transcription and splicing. Each round of DNA replication generates two hemi-methylated copies of the genome. These must be converted back to symmetrically methylated DNA before the next S-phase, or the mark will fade away; therefore the maintenance of DNA methylation is essential. Mechanistically, the maintenance of this epigenetic modification takes place during and after DNA replication, and occurs within the very dynamic context of chromatin re-assembly. Here, we review recent discoveries and unresolved questions regarding the mechanisms, dynamics and fidelity of DNA methylation maintenance in mammals. We also discuss how it could be regulated in normal development and misregulated in disease.

METTL6 is a tRNA m3C methyltransferase that regulates pluripotency and tumor cell growth.

Recently, covalent modifications of RNA, such as methylation, have emerged as key regulators of all aspects of RNA biology and have been implicated in numerous diseases, for instance, cancer. Here, we undertook a combination of in vitro and in vivo screens to test 78 potential methyltransferases for their roles in hepatocellular carcinoma (HCC) cell proliferation. We identified methyltransferase-like protein 6 (METTL6) as a crucial regulator of tumor cell growth. We show that METTL6 is a bona fide transfer RNA (tRNA) methyltransferase, catalyzing the formation of 3-methylcytidine at C32 of specific serine tRNA isoacceptors. Deletion of Mettl6 in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, Mettl6 knockout results in reduced energy expenditure. We reveal a previously unknown pathway in the maintenance of translation efficiency with a role in maintaining stem cell self-renewal, as well as impacting tumor cell growth profoundly.

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma.

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.

Loss of KDM6A confers drug resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.

DNA methylation analysis on purified neurons and glia dissects age and Alzheimer's disease-specific changes in the human cortex.

BACKGROUND: Epigenome-wide association studies (EWAS) based on human brain samples allow a deep and direct understanding of epigenetic dysregulation in Alzheimer's disease (AD). However, strong variation of cell-type proportions across brain tissue samples represents a significant source of data noise. Here, we report the first EWAS based on sorted neuronal and non-neuronal (mostly glia) nuclei from postmortem human brain tissues. RESULTS: We show that cell sorting strongly enhances the robust detection of disease-related DNA methylation changes even in a relatively small cohort. We identify numerous genes with cell-type-specific methylation signatures and document differential methylation dynamics associated with aging specifically in neurons such as CLU, SYNJ2 and NCOR2 or in glia RAI1,CXXC5 and INPP5A. Further, we found neuron or glia-specific associations with AD Braak stage progression at genes such as MCF2L, ANK1, MAP2, LRRC8B, STK32C and S100B. A comparison of our study with previous tissue-based EWAS validates multiple AD-associated DNA methylation signals and additionally specifies their origin to neuron, e.g., HOXA3 or glia (ANK1). In a meta-analysis, we reveal two novel previously unrecognized methylation changes at the key AD risk genes APP and ADAM17. CONCLUSIONS: Our data highlight the complex interplay between disease, age and cell-type-specific methylation changes in AD risk genes thus offering new perspectives for the validation and interpretation of large EWAS results.

CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes.

While the epidermal growth factor receptor (EGFR) is an established regulator of skin development and homeostasis, the functions of the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue have only recently been examined. Previously reported, skin-specific deletion of each of these receptors in mice resulted in similar defects in keratinocyte proliferation and migration, resulting in impaired wound healing and tumorigenesis. Because both ERBB2 and ERBB3 are targets for treating an array of cancer types, it is important to examine the consequences of receptor inhibition in human keratinocytes. Here, we employed the CRISPR/Cas9 technology to generate HaCaT cells (an established human keratinocyte cell line) lacking ERBB2 or ERBB3. HaCaT clones lacking ERBB2 or ERBB3 showed comparable reductions in cell proliferation as assessed by BrdU staining. Apoptosis, in contrast, was reduced in ERBB3-deficient HaCaT cells only. Assessment of cell migration using a wound healing (scratch) assay showed that the closure of the wound gaps was completed by 48 h in mock and in ERBB3 knockout clones. In contrast, this process was considerably delayed in ERBB2 knockout clones, and a complete closure of the gap in the latter cells did not occur before 72 h. In conclusion, both ERBB2 and ERBB3 are essential for normal proliferation of skin keratinocytes, but in contrast to ERBB3, ERBB2 is essential for migration of human keratinocytes. These observations might bear significance to patient adverse effects of therapeutic agents targeting ERBB2 and ERBB3.

Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in FLT3-ITD AML with concurrent epigenetic mutations.

Effectively targeting leukemia-initiating cells (LIC) in FLT3-ITD-mutated acute myeloid leukemia (AML) is crucial for cure. Tyrosine kinase inhibitors (TKI) have limited impact as single agents, failing to eradicate LIC in the bone marrow. Using primary AML samples and a patient-derived xenograft model, we investigated whether combining the FLT3-selective TKI crenolanib with the hypomethylating agent azacitidine (AZA) eliminates FLT3-ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show FLT3-ITD occurs within the most primitive Lin-/CD33(+)/CD45dim/CD34+CD38- LIC compartment. Crenolanib alone could not target FLT3-ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC in vitro and severely impaired engraftment in NSG mice. Strikingly, FLT3-mutated samples harboring TET2 mutations were completely resistant to crenolanib whereas neither NPM1 nor DNMT3A mutations influenced response. Conversely, primary AML LIC harboring either TET2, DNMT3A or NPM1 mutations did not show increased sensitivity to AZA. In summary, resistance of FLT3-ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of FLT3-ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.

The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage.

Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo.

Fluorescence microscopy-based high-throughput screening for factors involved in gene silencing.

Gene silencing in eukaryotes is a highly controlled process. It involves the concerted action of histone and DNA-modifying enzymes as well as transcription factors and chromatin-associated proteins. To understand how epigenetic gene silencing is regulated, it is important to identify the factors involved in this process. Here we describe an assay that allows high-throughput screening for factors involved in gene silencing. This assay exploits the susceptibility of the viral cytomegalovirus (CMV) promoter to epigenetic silencing in embryonic stem cells (ESCs) and uses reporter constructs with an optical readout to determine the gene silencing potential of candidate factors. This approach allows to study mechanisms and kinetics of gene silencing in living cells and to evaluate the role of DNA methyltransferases, histone-modifying enzymes, and other chromatin-associated factors during gene silencing.

Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state.

DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1⁻/⁻) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1⁻/⁻;Dnmt3a⁻/⁻;Dnmt3b⁻/⁻ or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1⁻/⁻ and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1⁻/⁻ and TKO EBs. Despite late wild type and Dnmt1⁻/⁻ EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1⁻/⁻ and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation.

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1.

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1⁻/⁻ embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1⁻/⁻ ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA.

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.