GSK-3484862 targets DNMT1 for degradation in cells.

Maintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires the Dnmt1 accessory factor Uhrf1 and its E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner.

Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo.

Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs.

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2'O-methyladenosine (Am), generating N6, 2'O-dimethyladenosine (m6Am) when Am is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and Am), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2'O-methylated Am over unmodified A nucleosides is due mainly to increased binding affinity for Am. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5'-Am oligonucleotide with approximately the same affinity as that of a cap analog (KM = 0.4 versus 0.3 µM). In addition, PCIF1 methylates the uncapped 5'-Am with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.

RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro.

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoforms among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nt oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m7G capped ends. Second, among these 5'-capped nanopore reads, we screened for features consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds from human GM12878 cells, most of which (257,721) aligned to protein-coding genes. Of these, 4876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data for m7G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds.

Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist.

OBJECTIVE: Glucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study, we investigated the cellular and metabolic effects of modulating the balance between G protein and ß-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. METHODS: Dipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify the effects on glucose homeostasis and weight loss. RESULTS: Ligand-specific reductions in ß-arrestin-2 recruitment were associated with slower GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide despite a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and ß-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. CONCLUSIONS: Diminishing ß-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.

Genetic and biased agonist-mediated reductions in ß-arrestin recruitment prolong cAMP signaling at glucagon family receptors.

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of ß-arrestins. Indeed, recently described GLP-1R agonists with reduced ß-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both ß-arrestin isoforms the duration of G protein-dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, "biased" GLP-1, GCG, and GIP analogs with selective reductions in ß-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced ß-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

A ligand-based system for receptor-specific delivery of proteins.

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand's cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other.

Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3'-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3'-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5'-end (capped versus noncapped) in these biases. Our findings confirmed that the presence of a 5'-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5'-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells.

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

A Targeted RNAi Screen Identifies Endocytic Trafficking Factors That Control GLP-1 Receptor Signaling in Pancreatic ß-Cells.

The glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) is a key target for type 2 diabetes (T2D) treatment. Because endocytic trafficking of agonist-bound receptors is one of the most important routes for regulation of receptor signaling, a better understanding of this process may facilitate the development of new T2D therapeutic strategies. Here, we screened 29 proteins with known functions in G protein-coupled receptor trafficking for their role in GLP-1R potentiation of insulin secretion in pancreatic ß-cells. We identify five (clathrin, dynamin1, AP2, sorting nexins [SNX] SNX27, and SNX1) that increase and four (huntingtin-interacting protein 1 [HIP1], HIP14, GASP-1, and Nedd4) that decrease insulin secretion from murine insulinoma MIN6B1 cells in response to the GLP-1 analog exendin-4. The roles of HIP1 and the endosomal SNX1 and SNX27 were further characterized in mouse and human ß-cell lines and human islets. While HIP1 was required for the coupling of cell surface GLP-1R activation with clathrin-dependent endocytosis, the SNXs were found to control the balance between GLP-1R plasma membrane recycling and lysosomal degradation and, in doing so, determine the overall ß-cell incretin responses. We thus identify key modulators of GLP-1R trafficking and signaling that might provide novel targets to enhance insulin secretion in T2D.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing.

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.

Metabolic Incorporation of Azide Functionality into Cellular RNA.

Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2'- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.

Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression.

Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion.

Biochemical characterization of a Naegleria TET-like oxygenase and its application in single molecule sequencing of 5-methylcytosine.

Modified DNA bases in mammalian genomes, such as 5-methylcytosine ((5m)C) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of (5m)C to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like (5m)C oxygenase (NgTET1) from Naegleria gruberi, a single-celled protist evolutionarily distant from vertebrates. Here we show that NgTET1 is a 5-methylpyrimidine oxygenase, with activity on both (5m)C (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine ((5f)U) and 5-carboxyuridine ((5ca)U) in vitro. Mutagenesis studies reveal a delicate balance between choice of (5m)C or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to (5m)CpG and TpG dinucleotide sites in DNA. Intriguingly, NgTET1 displays higher T-oxidation activity in vitro than mammalian TET1, supporting a closer evolutionary relationship between NgTET1 and the base J-binding proteins from trypanosomes. Finally, we demonstrate that NgTET1 can be readily used as a tool in (5m)C sequencing technologies such as single molecule, real-time sequencing to map (5m)C in bacterial genomes at base resolution.

Multivalent site-specific phage modification enhances the binding affinity of receptor ligands.

High-throughput screening of combinatorial chemical libraries is a powerful approach for identifying targeted molecules. The display of combinatorial peptide libraries on the surface of bacteriophages offers a rapid, economical way to screen billions of peptides for specific binding properties and has impacted fields ranging from cancer to vaccine development. As a modification to this approach, we have previously created a system that enables site-specific insertion of selenocysteine (Sec) residues into peptides displayed pentavalently on M13 phage as pIII coat protein fusions. In this study, we show the utility of selectively derivatizing these Sec residues through the primary amine of small molecules that target a G protein-coupled receptor, the adenosine A1 receptor, leaving the other coat proteins, including the major coat protein pVIII, unmodified. We further demonstrate that modified Sec-phage with multivalent bound agonist binds to cells and elicits downstream signaling with orders of magnitude greater potency than that of unconjugated agonist. Our results provide proof of concept of a system that can create hybrid small molecule-containing peptide libraries and open up new possibilities for phage-drug therapies.

Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells.

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.

Probing homodimer formation of epidermal growth factor receptor by selective crosslinking.

Ligand binding promotes conformational rearrangement of the epidermal growth factor receptor (EGFR) leading to receptor autophosphorylation and downstream signaling. However, transient interactions between unstimulated EGFR molecules on the cell surface are not fully understood. In this report, we describe the investigation of homodimer formation of EGFR by means of an SNAP-tag based selective crosslinking approach (S-CROSS). EGFR homodimers were selectively captured in living cells and utilized for analysis of protein receptor interactions on the plasma membrane and ligand-induced activation. We showed that EGFR forms homodimers in unstimulated cells with efficiencies similar to those seen in cells treated with the epidermal growth factor ligand (EGF) supporting the existence of constitutive transient receptor-receptor interactions. EGFR crosslinked homodimers displayed a substantially increase in kinase activation upon ligand stimulation. Interestingly, in unstimulated cells the levels of spontaneous phosphorylation were found to correlate with the yields of the crosslinked homodimers species. In addition, we demonstrated that this crosslinking approach can be applied to interrogate the effect of small molecule inhibitors on receptor dimerization and kinase activity. Our crosslinking assay provides a new tool to dissect ligand-independent dimerization and activation mechanisms of receptor tyrosine kinases, many of which are important anticancer drug targets.

Methyllysine reader plant homeodomain (PHD) finger protein 20-like 1 (PHF20L1) antagonizes DNA (cytosine-5) methyltransferase 1 (DNMT1) proteasomal degradation.

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.