Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

Elevated parkinsonism pathological markers in dopaminergic neurons with developmental exposure to atrazine.

Atrazine (ATZ) is one of the most used herbicides in the US and a known endocrine disruptor. ATZ is frequently detected in drinking water, especially in Midwestern regions of the United States, exceeding the EPA regulation of maximum contamination level (MCL) of 3 ppb. Epidemiology studies have suggested an association between ATZ exposure and neurodegeneration. Less, however, is known about the neurotoxic mechanism of ATZ, particularly for exposures at a developmental stage. Here, we exposed floor plate progenitors (FPPs) derived from human induced pluripotent stem cells (hiPSCs) to low concentrations of ATZ at 0.3 and 3 ppb for two days followed by differentiation into dopaminergic (DA) neurons in ATZ-free medium. We then examined the morphology, activity, pathological protein aggregation, and transcriptomic changes of differentiated DA neurons. We observed significant decrease in the complexity of neurite network, increase of neuronal activity, and elevated tau- and α-synuclein (aSyn) pathologies after ATZ exposure. The ATZ-induced neuronal changes observed here align with pathological characteristics in Parkinson's disease (PD). Transcriptomic analysis further corroborates our findings; and collectively provides a strong evidence base that low-concentration ATZ exposure during development can elicit increased risk of neurodegeneration.

Pre-differentiation exposure of PFOA induced persistent changes in DNA methylation and mitochondrial morphology in human dopaminergic-like neurons.

Perfluorooctanoic acid (PFOA) is abundant in environment due to its historical uses in consumer products and industrial applications. Exposure to low doses of PFOA has been associated with various disease risks, including neurological disorders. The underlying mechanism, however, remains poorly understood. In this study, we examined the effects of low dose PFOA exposure at 0.4 and 4 µg/L on the morphology, epigenome, mitochondrion, and neuronal markers of dopaminergic (DA)-like SH-SY5Y cells. We observed persistent decreases in H3K4me3, H3K27me3 and 5 mC markers in nucleus along with alterations in nuclear size and chromatin compaction percentage in DA-like neurons differentiated from SH-SY5Y cells exposed to 0.4 and 4 µg/L PFOA. Among the selected epigenetic features, DNA methylation pattern can be used to distinguish between PFOA-exposed and naïve populations, suggesting the involvement of epigenetic regulation. Moreover, DA-like neurons with pre-differentiation PFOA exposure exhibit altered network connectivity, mitochondrial volume, and TH expression, implying impairment in DA neuron functionality. Collectively, our results revealed the prolonged effects of developmental PFOA exposure on the fitness of DA-like neurons and identified epigenome and mitochondrion as potential targets for bearing long-lasting changes contributing to increased risks of neurological diseases later in life.

Atrazine exposure in zebrafish induces aberrant genome-wide methylation.

Atrazine (ATZ) is the second most common agricultural herbicide used in the United States and is an endocrine disrupting chemical (EDC). Developmental exposure to ATZ can lead to significant behavioral and morphological alterations in exposed animals and their progeny suggesting the involvement of an epigenetic mechanism. Specific epigenetic mechanisms responsible for these alterations, however, are yet to be elucidated. In this study, we exposed zebrafish embryos to 0, 0.3, 3, or 30 ppb (µg/L) of ATZ from 1 to 72 h post fertilization (hpf). Chemical exposure was ceased and zebrafish maintained until 9 months post fertilization (mpf), when whole-genome bisulfite sequencing (WGBS) was performed to assess the effects of embryonic ATZ exposure on DNA methylation in female fish brains. The number of differentially methylated genes (DMGs) increased with increasing treatment concentration. DMGs were enriched in neurological pathways with extensive methylation changes consistently observed in neuroendocrine pathways. Specifically, DMGs with methylation changes in promoter regions showed hypomethylation in estrogen receptor signaling and hypermethylation in androgen signaling. DMGs with methylation changes in genebody were primarily enriched for mitochondrion-related pathways associated with healthy aging. Integrated analysis with transcriptomic data at 9 mpf exhibited a similar trend identifying CABLES1 and NDUFA4 as shared targets at all concentrations. We then compared the predicted upstream regulators of transcriptomic changes with DMGs and identified CALML3 as a common upstream regulator at both 0.3 and 30 ppb that exhibit significant methylation changes. Collectively, our study identified long-lasting DNA methylation changes in genome after embryonic ATZ exposure and elucidated potential gene targets whose aberrant methylation features may drive alterations in gene transcription in long-term.

Engineering a heterologously expressed fructosyltransferase from Aspergillus oryzae N74 in Komagataella phaffii (Pichia pastoris) for kestose production.

Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or ß-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme's tertiary structure modeled. More recently, residues that may be involved in protein-substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.

Lead exposure induces dysregulation of constitutive heterochromatin hallmarks in live cells.

Lead (Pb) is a heavy metal contaminant commonly found in air, soil, and drinking water due to legacy uses. Excretion of ingested Pb can result in extensive kidney damages due to elevated oxidative stress. Epigenetic alterations induced by exposure to Pb have also been implied but remain poorly understood. In this work, we assessed changes in repressive epigenetic marks, namely DNA methylation (meCpG) and histone 3 lysine 9 tri-methylation (H3K9me3) after exposure to Pb. Live cell epigenetic probes coupled to bimolecular fluorescence complementation (BiFC) were used to monitor changes in the selected epigenetic marks. Exposure to Pb significantly lowered meCpG and H3K9me3 levels in HEK293T cells suggesting global changes in constitutive heterochromatin. A heterodimeric pair of probes that tags chromatin regions enriched in both meCpG and H3K9me3 further confirmed our findings. The observed epigenetic changes can be partially attributed to aberrant transcriptional changes induced by Pb, such as overexpression of TET1 after Pb exposure. Lastly, we monitored changes in selected heterochromatin marks after removal of Pb and found that changes in these markers do not immediately recover to their original level suggesting potential long-term damages to chromatin structure.

Identifying distinct heterochromatin regions using combinatorial epigenetic probes in live cells.

The 3D spatial organization of the genome controls gene expression and cell functionality. Heterochromatin (HC), which is the densely compacted and largely silenced part of the chromatin, is the driver for the formation and maintenance of nuclear organization in the mammalian nucleus. It is functionally divided into highly compact constitutive heterochromatin (cHC) and transcriptionally poised facultative heterochromatin (fHC). Long regarded as a static structure, the highly dynamic nature of the heterochromatin is being slowly understood and studied. These changes in HC occur on various temporal scales during the cell cycle and differentiation processes. Most methods that capture information about the heterochromatin are static techniques that cannot provide a readout of how the HC organization evolves with time. The delineation of specific areas such as fHC are also rendered difficult due to its diffusive nature and lack of specific features. Another degree of complexity in characterizing changes in heterochromatin occurs due to the heterogeneity in the HC organization of individual cells, necessitating single cell studies. Overall, there is a need for live cell compatible tools that can stably track the heterochromatin as it undergoes re-organization. In this work, we present an approach to track cHC and fHC based on the epigenetic hallmarks associated with them. Unlike conventional immunostaining approaches, we use small recombinant protein probes that allow us to dynamically monitor the HC by binding to modifications specific to the cHC and fHC, such as H3K9me3, DNA methylation and H3K27me3. We demonstrate the use of the probes to follow the changes in HC induced by drug perturbations at the single cell level. We also use the probe sets combinatorically to simultaneously track chromatin regions enriched in two selected epigenetic modifications using a FRET based approach that enabled us tracking distinctive chromatin features in situ.

Evaluation of HIV-1 derived lentiviral vectors as transductors of Mucopolysaccharidosis type IV a fibroblasts.

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease produced by the deficiency of the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycans (GAGs) accumulation. Since currently available treatments remain limited and unspecific, novel therapeutic approaches are essential for the disease treatment. In an attempt to reduce treatment limitations, gene therapy rises as a more effective and specific alternative. We present in this study the delivery assessment of GALNS and sulfatase-modifying factor 1 (SUMF1) genes via HIV-1 derived lentiviral vectors into fibroblasts from MPS IVA patients. After transduction, we determined GALNS enzymatic activity, lysosomal mass change, and autophagy pathway impairment. Additionally, we computationally assessed the effect of mutations over the enzyme-substrate interaction and phenotypic effects. The results showed that the co-transduction of MPS IVA fibroblasts with GALNS and SUMF1 cDNAs led to a significant increase in GALNS enzyme activity and a reduction of lysosomal mass. We show that patient-specific differences in cellular response are directly associated with the set of mutations on each patient. Lastly, we present new evidence supporting autophagy impairment in MPS IVA due to the presence and changes in autophagy proteins in treated MPS IVA fibroblasts. Our results offer new evidence that demonstrate the potential of lentiviral vectors as a strategy to correct GALNS deficiency.

Pre-differentiation exposure to low-dose of atrazine results in persistent phenotypic changes in human neuronal cell lines.

Exposures to organic pesticides, particularly during a developmental window, have been associated with various neurodegenerative diseases later in life. Atrazine (ATZ), one of the most used pesticides in the U.S., is suspected to be associated with increased neurodegeneration later in life but few studies assessed the neurotoxicity of developmental ATZ exposure using human neuronal cells. Here, we exposed human SH-SY5Y cells to 0.3, 3, and 30 ppb of ATZ prior to differentiating them into dopaminergic-like neurons in ATZ-free medium to mimic developmental exposure. The differentiated neurons exhibit altered neurite outgrowth and SNCA pathology depending on the ATZ treatment doses. Epigenome changes, such as decreases in 5mC (for 0.3 ppb only), H3K9me3, and H3K27me3 were observed immediately after exposure. These alterations persist in a compensatory manner in differentiated neurons. Specifically, we observed significant reductions in 5mC and H3K9me3, as well as, an increase in H3K27me3 in ATZ-exposed cells after differentiation, suggesting substantial chromatin rearrangements after developmental ATZ exposure. Transcriptional changes of relevant epigenetic enzymes were also quantified but found to only partially explain the observed epigenome alteration. Our results thus collectively suggest that exposure to low-dose of ATZ prior to differentiation can result in long-lasting changes in epigenome and increase risks of SNCA-related Parkinson's Disease.

Low dose lead exposure induces alterations on heterochromatin hallmarks persisting through SH-SY5Y cell differentiation.

Lead (Pb) is a commonly found heavy metal due to its historical applications. Recent studies have associated early-life Pb exposure with the onset of various neurodegenerative disease. The molecular mechanisms of Pb conferring long-term neurotoxicity, however, is yet to be elucidated. In this study, we explored the persistency of alteration in epigenetic marks that arise from exposure to low dose of Pb using a combination of image-based and gene expression analysis. Using SH-SY5Y as a model cell line, we observed significant alterations in global 5-methycytosine (5 mC) and histone 3 lysine 27 tri-methylation (H3K27me3) and histone 3 lysine 9 tri-methylation (H3K9me3) levels in a dose-dependent manner immediately after Pb exposure. The changes are partially associated with alterations in epigenetic enzyme expression levels. Long term culturing (14 days) after cease of exposure revealed persistent changes in 5 mC, partial recovery in H3K9me3 and overcompensation in H3K27me3 levels. The observed alterations in H3K9me3 and H3K27me3 are reversed after neuronal differentiation, while reduction in 5 mC levels are amplified with significant changes in patterns as identified via texture clustering analysis. Moreover, correlation analysis demonstrates a strong positive correlation between trends of 5 mC alteration after differentiation and neuronal morphology. Collectively, our results suggest that exposure to low dose of Pb prior to differentiation can result in persistent epigenome alterations that can potentially be responsible for the observed phenotypic changes. Our work reveals that Pb induced changes in epigenetic repressive marks can persist through neuron differentiation, which provides a plausible mechanism underlying long-term neurotoxicity associated with developmental Pb-exposure.

A Custom EOG-Based HMI Using Neural Network Modeling to Real-Time for the Trajectory Tracking of a Manipulator Robot.

Although different physiological signals, such as electrooculography (EOG) have been widely used in the control of assistance systems for people with disabilities, customizing the signal classification system remains a challenge. In most interfaces, the user must adapt to the classification parameters, although ideally the systems must adapt to the user parameters. Therefore, in this work the use of a multilayer neural network (MNN) to model the EOG signal as a mathematical function is presented, which is optimized using genetic algorithms, in order to obtain the maximum and minimum amplitude threshold of the EOG signal of each person to calibrate the designed interface. The problem of the variation of the voltage threshold of the physiological signals is addressed by means of an intelligent calibration performed every 3 min; if an assistance system is not calibrated, it loses functionality. Artificial intelligence techniques, such as machine learning and fuzzy logic are used for classification of the EOG signal, but they need calibration parameters that are obtained through databases generated through prior user training, depending on the effectiveness of the algorithm, the learning curve, and the response time of the system. In this work, by optimizing the parameters of the EOG signal, the classification is customized and the domain time of the system is reduced without the need for a database and the training time of the user is minimized, significantly reducing the time of the learning curve. The results are implemented in an HMI for the generation of points in a Cartesian space (X, Y, Z) in order to control a manipulator robot that follows a desired trajectory by means of the movement of the user's eyeball.

Lysosomal storage diseases: current therapies and future alternatives.

Lysosomal storage disorders (LSDs) are a group of monogenic diseases characterized by progressive accumulation of undegraded substrates into the lysosome, due to mutations in genes that encode for proteins involved in normal lysosomal function. In recent years, several approaches have been explored to find effective and successful therapies, including enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, hematopoietic stem cell transplantation, and gene therapy. In the case of gene therapy, genome editing technologies have opened new horizons to accelerate the development of novel treatment alternatives for LSD patients. In this review, we discuss the current therapies for this group of disorders and present a detailed description of major genome editing technologies, as well as the most recent advances in the treatment of LSDs. We will further highlight the challenges and current bioethical debates of genome editing.

Role of Connexins 30, 36, and 43 in Brain Tumors, Neurodegenerative Diseases, and Neuroprotection.

Gap junction (GJ) channels and their connexins (Cxs) are complex proteins that have essential functions in cell communication processes in the central nervous system (CNS). Neurons, astrocytes, oligodendrocytes, and microglial cells express an extraordinary repertory of Cxs that are important for cell to cell communication and diffusion of metabolites, ions, neurotransmitters, and gliotransmitters. GJs and Cxs not only contribute to the normal function of the CNS but also the pathological progress of several diseases, such as cancer and neurodegenerative diseases. Besides, they have important roles in mediating neuroprotection by internal or external molecules. However, regulation of Cx expression by epigenetic mechanisms has not been fully elucidated. In this review, we provide an overview of the known mechanisms that regulate the expression of the most abundant Cxs in the central nervous system, Cx30, Cx36, and Cx43, and their role in brain cancer, CNS disorders, and neuroprotection. Initially, we focus on describing the Cx gene structure and how this is regulated by epigenetic mechanisms. Then, the posttranslational modifications that mediate the activity and stability of Cxs are reviewed. Finally, the role of GJs and Cxs in glioblastoma, Alzheimer's, Parkinson's, and Huntington's diseases, and neuroprotection are analyzed with the aim of shedding light in the possibility of using Cx regulators as potential therapeutic molecules.

Profiling epigenetic changes in human cell line induced by atrazine exposure.

How environmental chemicals can affect and exert their toxic effect at a molecular level has gained significant interest in recent years, not only for understanding their immediate health implications over exposed individuals, but also for their subsequent progeny. Atrazine (ATZ) is a commonly used herbicide in the U.S. and a long-suspected endocrine disrupting chemical. The molecular mechanism conferring long-term adverse health outcomes, however, remain elusive. Here, we explored changes in epigenetic marks that arise after exposure to ATZ at selected doses using image-based analysis coupled with data clustering. Significant decreases in methylated CpG (meCpG) and histone 3 lysine 9 tri-methylated (H3K9me3) were observed in the selected human cell line with a clear spatial preference. Treating cells with ATZ leads to the loss of a subpopulation of cells with high meCpG levels as identified in our clustering and histogram analysis. A similar trend was observed in H3K9me3 potentially attributing to the cross-talking between meCpG and H3K9me3. Changes in meCpG are likely to be associated with alterations in epigenetic enzyme expression levels regulating meCpG and persist after the removal of ATZ source which collectively provide a plausible mechanism for long-term ATZ-induced toxicity.

Monitoring Histone Methylation (H3K9me3) Changes in Live Cells.

H3K9me3 (methylation of lysine 9 of histone H3) is an epigenetic modification that acts as a repressor mark. Several diseases, including cancers and neurological disorders, have been associated with aberrant changes in H3K9me3 levels. Different tools have been developed to enable detection and quantification of H3K9me3 levels in cells. Most techniques, however, lack live cell compatibility. To address this concern, we have engineered recombinant protein sensors for probing H3K9me3 in situ. A heterodimeric sensor containing a chromodomain and chromo shadow domain from HP1a was found to be optimal in recognizing H3K9me3 and exhibited similar spatial resolution to commercial antibodies. Our sensor offers similar quantitative accuracy in characterizing changes in H3K9me3 compared to antibodies but claims single cell resolution. The sensor was applied to evaluate changes in H3K9me3 responding to environmental chemical atrazine (ATZ). ATZ was found to result in significant reductions in H3K9me3 levels after 24 h of exposure. Its impact on the distribution of H3K9me3 among cell populations was also assessed and found to be distinctive. We foresee the application of our sensors in multiple toxicity and drug-screening applications.

In Silico Analysis of the Structure of Fungal Fructooligosaccharides-Synthesizing Enzymes.

Fructooligosaccharides (FOS) are prebiotics commonly manufactured using fungal fructosyltransferases (FTases) or ß-fructofuranosidases. Several reports have attempted to optimize FOS production by changing operational conditions. Nevertheless, there is a lack of information related to the molecular enzyme-substrate interaction. In this study, we present an in silico evaluation of the interactions between substrates (i.e., glucose, sucrose, GF2, GF3, and GF4) and native FOS-synthesizing enzymes from fungi, with reported FOS production yield. In addition, a molecular dynamic simulation was conducted to assess the stability of these interactions. Six fungal enzymes with reported data of FOS production were selected: sucrose-sucrose 1-fructosyltransferase from A. foetidus (GenBank No. CAA04131); intracellular invertase from A. niger (GenBank No. ABB59679); extracellular invertase from A. niger (GenBank No. ABB59678); ß-fructofuranidase from A. japonicus ATCC 20611 (GenBank No. BAB67771); fructosyltransferase from A. oryzae N74 (GenBank No. ACZ48670); and fructosyltransferase from A. japonicus (PDB ID 3LF7). These enzymes shared an identity between 15 and 96 %, but have a highly conserved folding, and the characteristic FTases domains. Docking results showed that these enzymes also share a similar protein-ligand interaction profile. It was observed that the production yield of total FOS correlated with the sum of affinity energies for GF2, GF3, and GF4. Finally, we present the first molecular dynamic simulation for FOS and fungal FOS-synthesizing enzymes, showing that the protein-ligand interaction does not induce significant changes on the enzyme stability. Overall, these results represent valuable information to continue understanding the FOS synthesis process by fungal FOS-synthesizing enzymes, and they can have a significant impact toward the improvement in their catalytic properties and the synthesis of specific FOS.

Anaerobic sulfatase maturase AslB from Escherichia coli activates human recombinant iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS).

Maturation of type I sulfatases requires the conversion of the cysteine (Cys) or serine (Ser) present in the active site to formylglycine (FGly). This activation represents a limiting step during the production of recombinant sulfatases in bacteria and eukaryotic hosts. AslB, YdeM and YidF have been proposed to participate in the activation of sulfatases in Escherichia coli. In this study, we combined in-silico and experimental approaches to study the interaction between Escherichia coli BL21(DE3) AslB and human sulfatases, more specifically iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In-silico results show that AslB has a higher affinity for the residual motif of GALNS (-9.4kcalmol-1), Cys- and Ser-type, than for the one of IDS (-8.0kcalmol-1). However, the distance between the AslB active residue and the target motif favors the interaction with IDS (4.4Å) more than with GALNS (5.5Å). Experimental observations supported in-silico results where the co-expression of AslB with GALNS Cys- and Ser-type presented an activity increment of 2.0- and 1.5-fold compared to the control cultures, lacking overexpressed AslB. Similarly, IDS activity was increased in 4.6-fold when co-expressed with AslB. The higher sulfatase activity of AslB-IDS suggests that the distance between the AslB active residue and the motif target is a key parameter for the in-silico search of potential sulfatase activators. In conclusion, our results suggest that AslB is involve in the maturation of heterologous human sulfatases in E. coli BL21(DE3), and that it can have important implications in the production of recombinant sulfatases for therapeutic purposes and research.

Atrazine exposure decreases the activity of DNMTs, global DNA methylation levels, and dnmt expression.

Atrazine, a herbicide used on agricultural crops is widely applied in the Midwestern United States as well as other areas of the globe. Atrazine frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. Previous studies have reported morphological, hormonal, and molecular alterations due to developmental and adulthood atrazine exposure; however, studies examining epigenetic alterations are limited. In this study, the effects of atrazine exposure on DNA methyltransferase (DNMT) activity and kinetics were evaluated. Global DNA methylation levels and dnmt expression in zebrafish larvae exposed to 0, 3, or 30 parts per billion (ppb) atrazine throughout embryogenesis was then assessed. Results indicate that atrazine significantly decreased the activity of maintenance DNMTs and that the inhibition mechanism can be described using non-competitive Michaelis-Menten kinetics. Furthermore, results show that an embryonic atrazine exposure decreases global methylation levels and the expression of dnmt4 and dnmt5. These findings indicate that atrazine exposure can decrease the expression and activity of DNMTs, leading to decreased DNA methylation levels.

Engineering Recombinant Protein Sensors for Quantifying Histone Acetylation.

H3K14ac (acetylation of lysine 14 of histone H3) is one of the most important epigentic modifications. Aberrant changes in H3K14ac have been associated with various diseases, including cancers and neurological disorders. Tools that enable detection and quantification of H3K14ac levels in cell extracts and in situ are thus of critical importance to reveal its role in various biological processes. Current detection techniques of specific histone modifications, however, are constrained by tedious sample pretreatments, lack of quantitative accuracy, and reliance on high quality antibodies. To address this issue, we engineered recombinant sensors that are suitable for probing histone acetylation levels using various biological samples. The protein sensor contains recongition domain(s) with sequences derived from the bromodomain of human polybromo-1 (PB1), a natural H3K14ac reader domain. Various sensor designs were tested using nuclear extracts and live cells. The sensor containing dimeric repeats of bromodomain was found most effective in quantifying H3K14ac level in both in vitro and in situ assays. The sensor has a linear detection range of 0.5-50 nM when mixed with nuclear extracts. The sensor colocalizes with H3K14ac antibodies in situ when transfected into human embryonic kidney 293T (HEK293T) cells and is thus capable of providing spatial details of histone modification within the nucleus. Corrected nuclear fluorescence intensity was used to quantify the modification level in situ and found to correlate well with our in vitro assays. Our sensor offers a novel tool to characterize the histone modification level using nuclear extracts and probe histone modification change in live cells.