KMT9 Controls Stemness and Growth of Colorectal Cancer.

Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9ß that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9ß were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.

Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix.

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-L-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.

Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif.

DNA adenine methylation by Caulobacter crescentus Cell Cycle Regulated Methyltransferase (CcrM) is an important epigenetic regulator of gene expression. The recent CcrM-DNA cocrystal structure shows the CcrM dimer disrupts four of the five base pairs of the (5'-GANTC-3') recognition site. We developed a fluorescence-based assay by which Pyrrolo-dC tracks the strand separation event. Placement of Pyrrolo-dC within the DNA recognition site results in a fluorescence increase when CcrM binds. Non-cognate sequences display little to no fluorescence changes, showing that strand separation is a specificity determinant. Conserved residues in the C-terminal segment interact with the phospho-sugar backbone of the non-target strand. Replacement of these residues with alanine results in decreased methylation activity and changes in strand separation. The DNA recognition mechanism appears to occur with the Type II M.HinfI DNA methyltransferase and an ortholog of CcrM, BabI, but not with DNA methyltransferases that lack the conserved C-terminal segment. The C-terminal segment is found broadly in N4/N6-adenine DNA methyltransferases, some of which are human pathogens, across three Proteobacteria classes, three other phyla and in Thermoplasma acidophilum, an Archaea. This Pyrrolo-dC strand separation assay should be useful for the study of other enzymes which likely rely on a strand separation mechanism.

Structural insight into HEMK2-TRMT112-mediated glutamine methylation.

Post-translational modifications play important roles in mediating protein functions in a wide variety of cellular events in vivo. HEMK2-TRMT112 heterodimer has been reported to be responsible for both histone lysine methylation and eukaryotic release factor 1 (eRF1) glutamine methylation. However, how HEMK2-TRMT112 complex recognizes and catalyzes eRF1 glutamine methylation is largely unknown. Here, we present two structures of HEMK2-TRMT112, with one bound to SAM and the other bound with SAH and methylglutamine (Qme). Structural analyses of the post-catalytic complex, complemented by mass spectrometry experiments, indicate that the HEMK2 utilizes a specific pocket to accommodate the substrate glutamine and catalyzes the subsequent methylation. Therefore, our work not only throws light on the protein glutamine methylation mechanism, but also reveals the dual activity of HEMK2 by catalyzing the methylation of both Lys and Gln residues.

Single-molecule regulatory architectures captured by chromatin fiber sequencing.

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.

DNA Tetrahedra-Cross-linked Hydrogel Functionalized Paper for Onsite Analysis of DNA Methyltransferase Activity Using a Personal Glucose Meter.

Despite the recent developments on the construction of point-of-care testing (POCT) devices, it is still a big challenge to build portable POCT tools for simple, sensitive, selective, and quantitative determination of disease-related biomarkers. With this in mind, we developed a simple and user-friendly POCT tool for onsite analysis of DNA adenine methyltransferase (Dam) activity by using DNA tetrahedra-based hydrogel to trap glucose-producing enzymes for target recognition and signal transduction. The enzyme-encapsulated DNA hydrogel and the substrate of enzyme were separately modified on papers and then combined onto a commercial glucose test strip for the sensitive evaluation of Dam activity via using a personal glucose meter (PGM) for quantitative signal readout. Taking advantage of the great amount of enzyme entrapped in DNA hydrogel and the high signal amplification ability of enzyme, this POCT tool is capable of highly sensitive and selective determination of Dam activity with a direct detection limit down to 0.001 U/mL, which is superior to that of most previously reported biosensors. Furthermore, this device can also be applied to screen inhibitor and analyze Dam activity in spiked serum samples, indicating the great potential for clinical practice and diagnostic applications. Additionally, all the reactions for Dam assay are performed on paper, which is simple and deliverable to end-users for medical diagnostics at home or in-field.

Tetramerization at Low pH Licenses DNA Methylation Activity of M.HpyAXI in the Presence of Acid Stress.

Methylation of genomic DNA can influence the transcription profile of an organism and may generate phenotypic diversity for rapid adaptation in a dynamic environment. M.HpyAXI is a Type III DNA methyltransferase present in Helicobacter pylori and is upregulated at low pH. This enzyme may alter the expression of critical genes to ensure the survival of this pathogen at low pH inside the human stomach. M.HpyAXI methylates the adenine in the target sequence (5'-GCAG-3') and shows maximal activity at pH 5.5. Type III DNA methyltransferases are found to form an inverted dimer in the functional form. We observe that M.HpyAXI forms a nonfunctional dimer at pH 8.0 that is incapable of DNA binding and methylation activity. However, at pH 5.5, two such dimers associate to form a tetramer that now includes two functional dimers that can bind and methylate the target DNA sequence. Overall, we observe that the pH-dependent tetramerization of M.HpyAXI ensures that the enzyme is licensed to act only in the presence of acid stress.

The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site.

The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson-Crick polar hydrogen bonds to ensure high enzymatic specificity.

The Common Partner of Several Methyltransferases TRMT112 Regulates the Expression of N6AMT1 Isoforms in Mammalian Cells.

Methylation is a widespread modification occurring in DNA, RNA and proteins. The N6AMT1 (HEMK2) protein has DNA N6-methyladenine as well as the protein glutamine and histone lysine methyltransferase activities. The human genome encodes two different isoforms of N6AMT1, the major isoform and the alternatively spliced isoform, where the substrate binding motif is missing. Several RNA methyltransferases involved in ribosome biogenesis, tRNA methylation and translation interact with the common partner, the TRMT112 protein. In this study, we show that TRMT112 regulates the expression of N6AMT1 isoforms in mammalian cells. Both isoforms are equally expressed on mRNA level, but only isoform 1 is detected on the protein level in human cells. We show that the alternatively spliced isoform is not able to interact with TRMT112 and when translated, is rapidly degraded from the cells. This suggests that TRMT112 is involved in cellular quality control ensuring that N6AMT1 isoform with missing substrate binding domain is eliminated from the cells. The down-regulation of TRMT112 does not affect the N6AMT1 protein levels in cells, suggesting that the two proteins of TRMT112 network, WBSCR22 and N6AMT1, are differently regulated by their common cofactor.

Integrated rate laws for processive and distributive enzymatic turnover.

Recently derived steady-state differential rate laws for the catalytic turnover of molecules containing two substrate sites are reformulated as integrated rate laws. The analysis applies to a broad class of Markovian dynamic models, motivated by the varied and often complex mechanisms associated with DNA modifying enzymes. Analysis of experimental data for the methylation kinetics of DNA by Dam (DNA adenine methyltransferase) is drastically improved through the use of integrated rate laws. Data that are too noisy for fitting to differential predictions are reliably interpreted through the integrated rate laws.

Structural insights into the critical DNA damage sensors DNA-PKcs, ATM and ATR.

ATM, ATR and DNA-PKCs are key effectors of DNA Damage response and have been extensively linked to tumourigenesis and survival of cancer cells after radio/chemotherapy. Despite numerous efforts, the structures of these proteins remained elusive until very recently. The resolution revolution in Cryo-EM allowed for molecular details of these proteins to be seen for the first time. Here we provide a comprehensive review of the structures of ATM, ATR and DNA-PKcs and their complexes and expand with observations springing from our own cryo-EM studies. These observations include a novel conformation of ATR and novel dimeric arrangements of DNA-PKcs.

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-ß-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.

Perylene-Based Photoactive Material as a Double-Stranded DNA Intercalating Probe for Ultrasensitive Photoelectrochemical Biosensing.

Photoelectrochemical (PEC) sensing techniques have attracted considerable concerns because of the intrinsic merit of complete separation between the excitation light and responsive current but still remain a great challenge for further potential application. It is assigned to the scarcity of photoactive materials with narrow band gap, good biosafety, and high photon-to-electron conversion efficiency and unfavorable processing methods for photoactive materials on indium tin oxide. Herein, we employed a perylene-based polymer (PTC-NH2) with exceptional photoelectrical properties to develop a red-light-driven PEC sensor for ultrasensitive biosensing based on its superior electrostatic intercalation efficiency in double-stranded DNA to that in single-stranded DNA, with DNA adenine methyltransferase (Dam MTase) as the model target. The prepared PTC-NH2 was characterized by Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, and PEC techniques, and the results demonstrated that PTC-NH2 rather than metal oxides/metal sulfides/C3N4/metal complexes enjoyed the prominent capacity of converting light to current. Benefiting from the unique PEC properties of PTC-NH2 and target-initiated hybridization chain reaction (HCR) signal amplification, ultrasensitive detection of Dam MTase was accessibly realized with the detection limit of 0.015 U/mL, which is lower than that of PEC, electrochemical, or fluorescent biosensors previously reported. Furthermore, the proposed PEC sensor has been also applied in screening Dam MTase activity inhibitors. Therefore, the perylene-based PEC sensor exhibits great potential in early accurate diagnosis of DNA methylation-related diseases.

The highly specific, cell cycle-regulated methyltransferase from Caulobacter crescentus relies on a novel DNA recognition mechanism.

Two DNA methyltransferases, Dam and ß-class cell cycle-regulated DNA methyltransferase (CcrM), are key mediators of bacterial epigenetics. CcrM from the bacterium Caulobacter crescentus (CcrM C. crescentus, methylates adenine at 5'-GANTC-3') displays 105-107-fold sequence discrimination against noncognate sequences. However, the underlying recognition mechanism is unclear. Here, CcrM C. crescentus activity was either improved or mildly attenuated with substrates having one to three mismatched bp within or adjacent to the recognition site, but only if the strand undergoing methylation is left unchanged. By comparison, single-mismatched substrates resulted in up to 106-fold losses of activity with α (Dam) and γ-class (M.HhaI) DNA methyltransferases. We found that CcrM C. crescentus has a greatly expanded DNA-interaction surface, covering six nucleotides on the 5' side and eight nucleotides on the 3' side of its recognition site. Such a large interface may contribute to the enzyme's high sequence fidelity. CcrM C. crescentus displayed the same sequence discrimination with single-stranded substrates, and a surprisingly large (>107-fold) discrimination against ssRNA was largely due to the presence of two or more riboses within the cognate (DNA) site but not outside the site. Results from C-terminal truncations and point mutants supported our hypothesis that the recently identified C-terminal, 80-residue segment is essential for dsDNA recognition but is not required for single-stranded substrates. CcrM orthologs from Agrobacterium tumefaciens and Brucella abortus share some of these newly discovered features of the C. crescentus enzyme, suggesting that the recognition mechanism is conserved. In summary, CcrM C. crescentus uses a previously unknown DNA recognition mechanism.

Role of Mg2+ Ions in DNA Hydrolysis by EcoRV, Studied by the 3D-Reference Interaction Site Model and Molecular Dynamics.

The role of Mg2+ ions during precursor formation in DNA hydrolysis by the homodimeric restriction enzyme EcoRV was elucidated based on the 3D-reference interaction site model (RISM) theory and the molecular dynamics (MD) simulation. From an analysis of the spatial distribution of Mg2+ in an active site using 3D-RISM, we identified a new position for Mg2+ in the X-ray EcoRV-DNA complex structure ( 1rvb ). We refer to the position as site IV†. Site IV† is almost at the same position as that of a Ca2+ ion in the superimposed X-ray crystallographic active-site structure of the PvuII-DNA complex ( 1f0o ). 3D-RISM was also used to locate the position of water molecules, including the water nucleophile at the active site. MD simulations were carried out with the initial structure having two Mg2+ ions at site IV† and at site I*, experimentally identified by Horton et al., to find a stable complex structure in which the DNA fragment was rearranged to orient the scissile bond direction toward the water nucleophile. The equilibrium active-site structure of the EcoRV-DNA complex obtained from the MD simulation was similar to the superimposed X-ray crystallographic structure of the BamHI-DNA complex ( 2bam ). In the active-site structure, two metal ions have almost the same position (≤1.0 Å) as that of 2bam , and the scissile phosphate is twisted to orient the scissile bond toward the water nucleophile, as is the case in 2bam . We propose the equilibrium active-site structure obtained in this study as a precursor for the hydrolysis reaction of EcoRV.

LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity.

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13°C and 37°C) and was stable after storage at -20°C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems.

Specificity versus Processivity in the Sequential Modification of DNA: A Study of DNA Adenine Methyltransferase.

A detailed analysis is carried out on both published experimental results and new experiments for the methylation kinetics of two-site DNA substrates (with site separations between 100 and 800 bp) catalyzed by bacterial DNA adenine methyltransferase (Dam). A previously reported rate enhancement for the second methylation event (relative to that of the first methylation) is shown to result from elevated substrate specificity for singly methylated DNA over that of unmethylated DNA and not processive turnover of both sites by the same copy of Dam. An elementary model is suggested that cleanly fits the experimental data over a broad range of intersite separations. The model hypothesizes a looping mediated interference between competing unmethylated Dam sites on the same DNA strand.

A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants.

Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses DamID (Escherichia coli DNA adenine methylase as a reporter coupled with methylation-sensitive restriction enzyme digestion and high-throughput sequencing) to enable in vivo quantitative analyses of upstream regulatory sequences. Using the genome-reduced bacterium Mycoplasma pneumoniae, we show that ELM-seq has a large dynamic range and causes minimal toxicity. We use ELM-seq to determine key sequences (known and putatively novel) of promoter and untranslated regions that influence transcription and translation efficiency. Applying ELM-seq to other organisms will help us to further understand gene expression and guide synthetic biology.Quantitative analysis of how DNA sequence determines transcription and translation regulation is of interest to systems and synthetic biologists. Here the authors present ELM-seq, which uses Dam activity as reporter for high-throughput analysis of promoter and 5'-UTR regions.

N6-Adenosine DNA Methyltransferase from H. pylori 98-10 Strain in Complex with DNA and AdoMet: Structural Insights from in Silico Studies.

Helicobacter pylori is a primitive Gram-negative bacterium that resides in the acidic environment of the human gastrointestinal tract, and some strains of this bacterium cause gastric ulcers and cancer. DNA methyltransferases (MTases) are promising drug targets for the treatment of cancer and other diseases that are also caused by epigenetic alternations of the genome. The N6-adenine-specific DNA MTase from H. pylori (M. Hpy N6mA) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) to the flipped adenine of the substrate DNA. In this work, we report the sequence analyses, three-dimensional structure modeling, and molecular dynamics simulations of M. Hpy N6mA, when complexed with AdoMet as well as DNA. We analyzed the protein-DNA interactions prominently established by the flipped cytosine and the interactions between protein cofactors in the active site. The comparable orientation of AdoMet in both systems confirms that AdoMet is in a catalytically competent orientation in the bimolecular system that is retained upon DNA binding in the termolecular system of M. Hpy N6mA. In both systems, AdoMet is stabilized in the binding pocket by hydrogen bonding (Thr84, Glu99, Asp122, and Phe123) as well as van der Waals (Ile100, Phe160, Arg104, and Cys76) interactions. We propose that the contacts made by flipped adenine DA6 with Asn138 (N6 and N1 atom of DA6) and Pro139 (N6) and π-stacking interactions with Phe141 and Phe219 play an important role in the methylation mechanism at the N6 position in our N6mA model. Specific recognition of DNA is mediated by residues 143-155, 183-189, 212-220, 280-293, and 308-325. These findings are further supported by alanine scanning mutagenesis studies. The conserved residues in distantly related sequences of the small domain are important in DNA binding. Results reported here elucidate the sequence, structure, and binding features necessary for the recognition between cofactor AdoMet and substrate DNA by the vital epigenetic enzyme, M. Hpy N6mA.

Mechanisms of Protein Translocation on DNA Are Differentially Responsive to Water Activity.

Water plays important but poorly understood roles in the functions of most biomolecules. We are interested in understanding how proteins use diverse search mechanisms to locate specific sites on DNA; here we present a study of the role of closely associated waters in diverse translocation mechanisms. The bacterial DNA adenine methyltransferase, Dam, moves across large segments of DNA using an intersegmental hopping mechanism, relying in part on movement through bulk water. In contrast, other proteins, such as the bacterial restriction endonuclease EcoRI, rely on a sliding mechanism, requiring the protein to stay closely associated with DNA. Here we probed how these two mechanistically distinct proteins respond to well-characterized osmolytes, dimethyl sulfoxide (DMSO), and glycerol. The ability of Dam to move over large segments of DNA is not impacted by either osmolyte, consistent with its minimal reliance on a sliding mechanism. In contrast, EcoRI endonuclease translocation is significantly enhanced by DMSO and inhibited by glycerol, providing further corroboration that these proteins rely on distinct translocation mechanisms. The well-established similar effects of these osmolytes on bulk water, and their differential effects on macromolecule-associated waters, support our results and provide further evidence of the importance of water in interactions between macromolecules and their ligands.