p53 and TDG are dominant in regulating the activity of the human de novo DNA methyltransferase DNMT3A on nucleosomes.

DNA methylation and histone tail modifications are interrelated mechanisms involved in a wide range of biological processes, and disruption of this crosstalk is linked to diseases such as acute myeloid leukemia. In addition, DNA methyltransferase 3A (DNMT3A) activity is modulated by several regulatory proteins, including p53 and thymine DNA glycosylase (TDG). However, the relative role of histone tails and regulatory proteins in the simultaneous coordination of DNMT3A activity remains obscure. We observed that DNMT3A binds H3 tails and p53 or TDG at distinct allosteric sites to form DNMT3A-H3 tail-p53 or -TDG multiprotein complexes. Functional characterization of DNMT3A-H3 tail-p53 or -TDG complexes on human-derived synthetic histone H3 tails, mononucleosomes, or polynucleosomes shows p53 and TDG play dominant roles in the modulation of DNMT3A activity. Intriguingly, this dominance occurs even when DNMT3A is actively methylating nucleosome substrates. The activity of histone modifiers is influenced by their ability to sense modifications on histone tails within the same nucleosome or histone tails on neighboring nucleosomes. In contrast, we show here that DNMT3A acts on DNA within a single nucleosome, on nucleosomal DNA within adjacent nucleosomes, and DNA not associated with the DNMT3A-nucleosome complex. Our findings have direct bearing on how the histone code drives changes in DNA methylation and highlight the complex interplay between histone tails, epigenetic enzymes, and modulators of enzymatic activity.

A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A.

Screening of a small chemical library (Medicines for Malaria Venture Pathogen Box) identified two structurally related pyrazolone (inhibitor 1) and pyridazine (inhibitor 2) DNMT3A inhibitors with low micromolar inhibition constants. The uncompetitive and mixed type inhibition patterns with DNA and AdoMet suggest these molecules act through an allosteric mechanism, and thus are unlikely to bind to the enzyme's active site. Unlike the clinically used mechanism based DNMT inhibitors such as decitabine or azacitidine that act via the enzyme active site, the inhibitors described here could lead to the development of more selective drugs. Both inhibitors show promising selectivity for DNMT3A in comparison to DNMT1 and bacterial DNA cytosine methyltransferases. With further study, this could form the basis of preferential targeting of de novo DNA methylation over maintenance DNA methylation.

The R882H substitution in the human de novo DNA methyltransferase DNMT3A disrupts allosteric regulation by the tumor supressor p53.

A myriad of protein partners modulate the activity of the human DNA methyltransferase 3A (DNMT3A), whose interactions with these other proteins are frequently altered during oncogenesis. We show here that the tumor suppressor p53 decreases DNMT3A activity by forming a heterotetramer complex with DNMT3A. Mutational and modeling experiments suggested that p53 interacts with the same region in DNMT3A as does the structurally characterized DNMT3L. We observed that the p53-mediated repression of DNMT3A activity is blocked by amino acid substitutions within this interface, but surprisingly, also by a distal DNMT3A residue, R882H. DNMT3A R882H occurs frequently in various cancers, including acute myeloid leukemia, and our results suggest that the effects of R882H and other DNMT3A mutations may go beyond changes in DNMT3A methylation activity. To further understand the dynamics of how protein-protein interactions modulate DNMT3A activity, we determined that p53 has a greater affinity for DNMT3A than for DNMT3L and that p53 readily displaces DNMT3L from the DNMT3A:DNMT3L heterotetramer. Interestingly, this occurred even when the preformed DNMT3A:DNMT3L complex was actively methylating DNA. The frequently identified p53 substitutions (R248W and R273H), whereas able to regulate DNMT3A function when forming the DNMT3A:p53 heterotetramer, no longer displaced DNMT3L from the DNMT3A:DNMT3L heterotetramer. The results of our work highlight the complex interplay between DNMT3A, p53, and DNMT3L and how these interactions are further modulated by clinically derived mutations in each of the interacting partners.

Mutations in the DNMT3A DNA methyltransferase in acute myeloid leukemia patients cause both loss and gain of function and differential regulation by protein partners.

Eukaryotic DNA methylation prevents genomic instability by regulating the expression of oncogenes and tumor-suppressor genes. The negative effects of dysregulated DNA methylation are highlighted by a strong correlation between mutations in the de novo DNA methyltransferase gene DNA methyltransferase 3α (DNMT3A) and poor prognoses among acute myeloid leukemia (AML) patients. We show here that clinically observed DNMT3A mutations dramatically alter enzymatic activity, including mutations that lead to 6-fold hypermethylation and 3-fold hypomethylation of the human cyclin-dependent kinase inhibitor 2B (CDKN2B or p15) gene promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN-AML) constitutes 40-50% of all AML cases and is the most epigenetically diverse AML subtype with pronounced changes in non-CpG DNA methylation. We identified a subset of DNMT3A mutations that enhance the enzyme's ability to perform non-CpG methylation by 2-8-fold. Many of these mutations mapped to DNMT3A regions known to interact with proteins that themselves contribute to AML, such as thymine DNA glycosylase (TDG). Using functional mapping of TDG-DNMT3A interactions, we provide evidence that TDG and DNMT3-like (DNMT3L) bind distinct regions of DNMT3A. Furthermore, DNMT3A mutations caused diverse changes in the ability of TDG and DNMT3L to affect DNMT3A function. Cell-based studies of one of these DNMT3A mutations (S714C) replicated the enzymatic studies and revealed that it causes dramatic losses of genome-wide methylation. In summary, mutations in DNMT3A lead to diverse levels of activity, interactions with epigenetic machinery components and cellular changes.

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.

Improved in vivo targeting of BCL-2 phenotypic conversion through hollow gold nanoshell delivery.

Although new cancer therapeutics are discovered at a rapid pace, lack of effective means of delivery and cancer chemoresistance thwart many of the promising therapeutics. We demonstrate a method that confronts both of these issues with the light-activated delivery of a Bcl-2 functional converting peptide, NuBCP-9, using hollow gold nanoshells. This approach has shown not only to increase the efficacy of the peptide 30-fold in vitro but also has shown to reduce paclitaxel resistant H460 lung xenograft tumor growth by 56.4%.

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.

Light-Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near-Infrared Light.

A light-activated genome editing platform based on the release of enzymes from a plasmonic nanoparticle carrier when exposed to biocompatible near-infrared light pulses is described. The platform relies on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of copper which is attached to double-stranded nucleic acids self-assembled on the gold nanoparticle surface. A protein fusion of the Cre recombinase containing a TAT internalization peptide sequence to achieve endosomal localization is also employed. High-resolution gene knock-in of a red fluorescent reporter is observed using a commercial two-photon microscope. High-throughput irradiation is described to generate useful quantities of edited cells.

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the Km, kcat, kmethylation, and Kd for single-stranded and hemimethylated substrates, revealing discrimination of 107-fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

Affinity-Based Assembly of Peptides on Plasmonic Nanoparticles Delivered Intracellularly with Light Activated Control.

We report a universal strategy for functionalizing near-infrared light-responsive nanocarriers with both a peptide "cargo" and an orthogonal cell-penetrating peptide. Modularity of both the cargo and the internalization peptide attachment is an important feature of these materials relying on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of nickel as well as the affinity of biotin labeled peptides to streptavidin. Attachment to the gold surface uses thiol-labeled scaffolds terminated with the affinity partner. These materials allow for unprecedented spatiotemporal control over the release of the toxic α-helical amphipathic peptide (KLAKLAK)2 which disrupts mitochondrial membranes and initiates apoptotic cell death. Laser treatment at benign near-infrared wavelengths releases peptide from the gold surface as well as breaches the endosome barrier for cytosolic activity (with 105-fold improved response to peptide activity over the free peptide) and can be monitored in real time.

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.

De novo DNA methyltransferase DNMT3A: Regulation of oligomeric state and mechanism of action in response to pH changes.

BACKGROUND: The oligomeric state of the human DNMT3A is functionally important and cancer cells are known to undergo changes in pH (intracellular). METHODS: Light scattering, gel filtration, and fluorescence anisotropy. Also, methylation and processivity assays. CONCLUSIONS: Physiologically relevant changes in pH result in changes in DNMT3A oligomer composition which have dramatic consequences on DNMT3A function. GENERAL SIGNIFICANCE: The pH changes which occur within cancer cells alter the oligomeric state and function of DNMT3A which could contribute to changes in genomic DNA methylation observed in vivo.

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine.

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 ß-elimination contributes to the rate-limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59-1.83 fold change in the rate of catalysis, thus suggesting that ß-elimination is partly rate-limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing ß-elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.

DNA adenine methyltransferase facilitated diffusion is enhanced by protein-DNA "roadblock" complexes that induce DNA looping.

The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein-DNA complexes (roadblocks) diminish and-interestingly-enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA adenine methyltransferase (Dam, recognizes 5'-GATC-3') with tightly bound EcoRV ENase-DNA complexes, which bend DNA. A single EcoRV roadblock does not alter processive (multiple modifications) methylation by Dam. This result disfavors a reliance on heavily touted mechanisms involving sliding or short hops for Dam. Specific conformations of two EcoRV roadblocks cause an increase in processivity. The histone-like leucine-responsive regulatory protein (Lrp) binds DNA nonspecifically as an octamer, and also increases Dam's processivity. These results can be explained by our prior demonstration that Dam moves over large regions (>300 bp) within a single DNA molecule using an "intersegmental hopping" mechanism. This mechanism involves the protein hopping between looped DNA segments. Both roadblock systems can cause the DNA to loop and therefore facilitate intersegmental hopping. For Lrp, this only occurs when the Dam sites are separated (by >134bp) such that they can be looped around the protein. Intersegmental hopping may well be a general mechanism for proteins that navigate long distances along compacted DNA. Unlike Dam, EcoRI ENase (recognizes 5'-GAATTC-3') relies extensively on a sliding mechanism, and as expected, Lrp decreases its processivity. Our systematic use of protein roadblocks provides a powerful strategy to differentiate between site location mechanisms.

Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

Targeted intracellular delivery of proteins with spatial and temporal control.

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

Extracting enzyme processivity from kinetic assays.

A steady-state analysis for the catalytic turnover of molecules containing two substrate sites is presented. A broad class of Markovian dynamic models, motivated by the action of DNA modifying enzymes and the rich variety of translocation mechanisms associated with these systems (e.g., sliding, hopping, intersegmental transfer, etc.), is considered. The modeling suggests an elementary and general method of data analysis, which enables the extraction of the enzyme's processivity directly and unambiguously from experimental data. This analysis is not limited to the initial velocity regime. The predictions are validated both against detailed numerical models and by revisiting published experimental data for EcoRI endonuclease acting on DNA.

STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition.

The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design.

Modular plasmonic nanocarriers for efficient and targeted delivery of cancer-therapeutic siRNA.

We have combined a versatile and powerful route to deliver nucleic acids with peptide-based cell-specific targeting. siRNA targeting the polo-like kinase gene is in clinical trials for cancer treatment, and here we deliver this RNA selectively to cancer cells displaying the neuropilin-1 epitope using gold nanoshells. Release of the siRNA from the nanoparticles results from irradiation with a pulsed near-infrared laser, which also provides efficient endosomal escape within the cell. As a result, our approach requires 10-fold less material than standard nucleic acid transduction materials and is significantly more efficient than other particle-based methods. We also describe a particle-nucleic acid design that does not rely on modified RNA, thereby making the preparation of these materials more efficient and much less expensive. These improvements, when combined with control over when and where the siRNA is released, could provide the basis for diverse cell biological studies.

Distinct facilitated diffusion mechanisms by E. coli Type II restriction endonucleases.

The passive search by proteins for particular DNA sequences involving nonspecific DNA is essential for gene regulation, DNA repair, phage defense, and diverse epigenetic processes. Distinct mechanisms contribute to these searches, and it remains unresolved as to which mechanism or blend of mechanisms best suits a particular protein and, more importantly, its biological role. To address this, we compare the translocation properties of two well-studied bacterial restriction endonucleases (ENases), EcoRI and EcoRV. These dimeric, magnesium-dependent enzymes hydrolyze related sites (EcoRI ENase, 5'-GAATTC-3'; EcoRV ENase, 5'-GATATC-3'), leaving overhangs and blunt DNA segments, respectively. Here, we demonstrate that the extensive sliding by EcoRI ENase, involving sliding up to ∼600 bp prior to dissociating from the DNA, contrasts with a larger reliance on hopping mechanism(s) by EcoRV ENase. The mechanism displayed by EcoRI ENase results in a highly thorough search of DNA, whereas the EcoRV ENase mechanism results in an extended, yet less rigorous, interrogation of DNA sequence space. We describe how these mechanistic distinctions are complemented by other aspects of these endonucleases, such as the 10-fold higher in vivo concentrations of EcoRI ENase compared to that of EcoRV ENase. Further, we hypothesize that the highly diverse enzyme arsenal that bacteria employ against foreign DNA involves seemingly similar enzymes that rely on distinct but complementary search mechanisms. Our comparative approach reveals how different proteins utilize distinct site-locating strategies.