Hepatocyte mARC1 promotes fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)-siRNA to understand the in vivo impacts of MTARC1. Results: We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and in vivo GalNAc-siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD. Impact and implications: We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 for NAFLD.

MLX plays a key role in lipid and glucose metabolism in humans: Evidence from in vitro and in vivo studies.

BACKGROUND AND AIM: Enhanced hepatic de novo lipogenesis (DNL) has been proposed as an underlying mechanism for the development of NAFLD and insulin resistance. Max-like protein factor X (MLX) acts as a heterodimer binding partner for glucose sensing transcription factors and inhibition of MLX or downstream targets has been shown to alleviate intrahepatic triglyceride (IHTG) accumulation in mice. However, its effect on insulin sensitivity remains unclear. As human data is lacking, the aim of the present work was to investigate the role of MLX in regulating lipid and glucose metabolism in primary human hepatocytes (PHH) and in healthy participants with and without MLX polymorphisms. METHODS: PHH were transfected with non-targeting or MLX siRNA to assess the effect of MLX knockdown on lipid and glucose metabolism, insulin signalling and the hepatocellular transcriptome. A targeted association analysis on imputed genotype data for MLX on healthy individuals was undertaken to assess associations between specific MLX SNPs (rs665268, rs632758 and rs1474040), plasma biochemistry, IHTG content, DNL and gluconeogenesis. RESULTS: MLX knockdown in PHH altered lipid metabolism (decreased DNL (p < 0.05), increased fatty acid oxidation and ketogenesis (p < 0.05), and reduced lipid accumulation (p < 0.001)). Additionally, MLX knockdown increased glycolysis, lactate secretion and glucose production (p < 0.001) and insulin-stimulated pAKT levels (p < 0.01) as assessed by transcriptomic, steady-state and dynamic measurements. Consistent with the in vitro data, individuals with the rs1474040-A and rs632758-C variants had lower fasting plasma insulin (p < 0.05 and p < 0.01, respectively) and TG (p < 0.05 and p < 0.01, respectively). Although there was no difference in IHTG or gluconeogenesis, individuals with rs632758 SNP had notably lower hepatic DNL (p < 0.01). CONCLUSION: We have demonstrated using human in vitro and in vivo models that MLX inhibition favored lipid catabolism over anabolism and increased glucose production, despite increased glycolysis and phosphorylation of Akt, suggesting a metabolic mechanism that involves futile cycling.

Central androgen action reverses hypothalamic astrogliosis and atherogenic risk factors induced by orchiectomy and high-fat diet feeding in male mice.

Hypogonadism in males confers elevated cardiovascular disease (CVD) risk by unknown mechanisms. Recent radiological evidence suggests that low testosterone (T) is associated with mediobasal hypothalamic (MBH) gliosis, a central nervous system (CNS) cellular response linked to metabolic dysfunction. To address mechanisms linking CNS androgen action to CVD risk, we generated a hypogonadal, hyperlipidemic mouse model with orchiectomy (ORX) combined with hepatic PCSK9 overexpression. After 4 wk of high-fat, high-sucrose diet (HFHS) consumption, despite equal body weights and glucose tolerance, androgen-deficient ORX mice had a more atherogenic lipid profile and increased liver and leukocyte inflammatory signaling compared with sham-operated control mice. Along with these early CVD risk indicators, ORX markedly amplified HFHS-induced astrogliosis in the MBH. Transcriptomic analysis further revealed that ORX and high-fat diet feeding induced upregulation of inflammatory pathways and downregulation of metabolic pathways in hypothalamic astrocytes. To interrogate the role of sex steroid signaling in the CNS in cardiometabolic risk and MBH inflammation, central infusion of T and dihydrotestosterone (DHT) was performed on ORX mice. Central DHT prevented MBH astrogliosis and reduced the liver inflammatory signaling and monocytosis induced by HFHS and ORX; T had a partial protective effect. Finally, a cross-sectional study in 41 adult men demonstrated a positive correlation between radiological evidence of MBH gliosis and plasma lipids. These findings demonstrate that T deficiency in combination with a Western-style diet promotes hypothalamic gliosis concomitant with increased atherogenic risk factors and provide supportive evidence for regulation of lipid metabolism and cardiometabolic risk determinants by the CNS action of sex steroids.NEW & NOTEWORTHY This study provides evidence that hypothalamic gliosis is a key early event through which androgen deficiency in combination with a Western-style diet might lead to cardiometabolic dysregulation in males. Furthermore, this work provides the first evidence in humans of a positive association between hypothalamic gliosis and LDL-cholesterol, advancing our knowledge of CNS influences on CVD risk progression.

RNA G-quadruplex organizes stress granule assembly through DNAPTP6 in neurons.

Consecutive guanine RNA sequences can adopt quadruple-stranded structures, termed RNA G-quadruplexes (rG4s). Although rG4-forming sequences are abundant in transcriptomes, the physiological roles of rG4s in the central nervous system remain poorly understood. In the present study, proteomics analysis of the mouse forebrain identified DNAPTP6 as an RNA binding protein with high affinity and selectivity for rG4s. We found that DNAPTP6 coordinates the assembly of stress granules (SGs), cellular phase-separated compartments, in an rG4-dependent manner. In neurons, the knockdown of DNAPTP6 diminishes the SG formation under oxidative stress, leading to synaptic dysfunction and neuronal cell death. rG4s recruit their mRNAs into SGs through DNAPTP6, promoting RNA self-assembly and DNAPTP6 phase separation. Together, we propose that the rG4-dependent phase separation of DNAPTP6 plays a critical role in neuronal function through SG assembly.

NMDA Receptor Antagonists Increase the Release of GLP-1 From Gut Endocrine Cells.

Type 2 diabetes mellitus (T2DM) remains one of the most pressing health issues facing modern society. Several antidiabetic drugs are currently in clinical use to treat hyperglycaemia, but there is a need for new treatments that effectively restore pancreatic islet function in patients. Recent studies reported that both murine and human pancreatic islets exhibit enhanced insulin release and ß-cell viability in response to N-methyl-D-aspartate (NMDA) receptor antagonists. Furthermore, oral administration of dextromethorphan, an over-the-counter NMDA receptor antagonist, to diabetic patients in a small clinical trial showed improved glucose tolerance and increased insulin release. However, the effects of NMDA receptor antagonists on the secretion of the incretin hormone GLP-1 was not tested, and nothing is known regarding how NMDA receptor antagonists may alter the secretion of gut hormones. This study demonstrates for the first time that, similar to ß-cells, the NMDA receptor antagonist MK-801 increases the release of GLP-1 from a murine L-cell enteroendocrine model cell line, GLUTag cells. Furthermore, we report the 3' mRNA expression profiling of GLUTag cells, with a specific focus on glutamate-activated receptors. We conclude that if NMDA receptor antagonists are to be pursued as an alternative, orally administered treatment for T2DM, it is essential that the effects of these drugs on the release of gut hormones, and specifically the incretin hormones, are fully investigated.

Decitabine increases neoantigen and cancer testis antigen expression to enhance T-cell-mediated toxicity against glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Despite maximal treatment, median survival remains dismal at 14-24 months. Immunotherapies, such as checkpoint inhibition, have revolutionized management of some cancers but have little benefit for GBM patients. This is, in part, due to the low mutational and neoantigen burden in this immunogenically "cold" tumor. METHODS: U87MG and patient-derived cell lines were treated with 5-aza-2'-deoxycytidine (DAC) and underwent whole-exome and transcriptome sequencing. Cell lines were then subjected to cellular assays with neoantigen and cancer testis antigen (CTA) specific T cells. RESULTS: We demonstrate that DAC increases neoantigen and CTA mRNA expression through DNA hypomethylation. This results in increased neoantigen presentation by MHC class I in tumor cells, leading to increased neoantigen- and CTA-specific T-cell activation and killing of DAC-treated cancer cells. In addition, we show that patients have endogenous cancer-specific T cells in both tumor and blood, which show increased tumor-specific activation in the presence of DAC-treated cells. CONCLUSIONS: Our work shows that DAC increases GBM immunogenicity and consequent susceptibility to T-cell responses in vitro. Our results support a potential use of DAC as a sensitizing agent for immunotherapy.

Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection.

Whole genome base-resolution methylome sequencing allows for the most comprehensive analysis of DNA methylation, however, the considerable sequencing cost often limits its applications. While reduced representation sequencing can be an affordable alternative, over 80% of CpGs in the genome are not covered. Building on our recently developed TET-assisted pyridine borane sequencing (TAPS) method, we here described endonuclease enrichment TAPS (eeTAPS), which utilizes dihydrouracil (DHU)-cleaving endonuclease digestion of TAPS-converted DNA to enrich methylated CpG sites (mCpGs). eeTAPS can accurately detect 87% of mCpGs in the mouse genome with a sequencing depth equivalent to 4× whole genome sequencing. In comparison, reduced representation TAPS (rrTAPS) detected less than 4% of mCpGs with 2.5× sequencing depth. Our results demonstrate eeTAPS to be a new strategy for cost-effective genome-wide methylation analysis at single-CpG resolution that can fill the gap between whole-genome and reduced representation sequencing.

Identification of Binding Targets of a Pyrrole-Imidazole Polyamide KR12 in the LS180 Colorectal Cancer Genome.

Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as seco-CBI. These molecules also provide an alternative for oncogenes deemed "undruggable" at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide's possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.

Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer Pyrrole-Imidazole Polyamide Probes.

Synthetic molecules that bind sequence-specifically to DNA have been developed for varied biological applications, including anticancer activity, regulation of gene expression, and visualization of specific genomic regions. Increasing the number of base pairs targeted by synthetic molecules strengthens their sequence specificity. Our group has been working on the development of pyrrole-imidazole polyamides that bind to the minor groove of DNA in a sequence-specific manner without causing denaturation. Recently, we reported a simple synthetic method of fluorescent tandem dimer polyamide probes composed of two hairpin moieties with a linking hinge, which bound to 12 bp in human telomeric repeats (5'-(TTAGGG)n-3') and could be used to specifically visualize telomeres in chemically fixed cells under mild conditions. We also performed structural optimization and extension of the target base pairs to allow more specific staining of telomeres. In the present study, we synthesized tandem tetramer polyamides composed of four hairpin moieties, targeting 24 bp in telomeric repeats, the longest reported binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding molecules. The novel tandem tetramers bound with a nanomolar dissociation constant to 24 bp sequences made up of four telomeric repeats. Fluorescently labeled tandem tetramer polyamide probes could visualize human telomeres in chemically fixed cells with lower background signals than polyamide probes reported previously, suggesting that they had higher specificity for telomeres. Furthermore, high-throughput sequencing of human genomic DNA pulled down by the biotin-labeled tandem tetramer polyamide probe confirmed its effective binding to telomeric repeats in the complex chromatinized genome.

Genome-Wide Assessment of the Binding Effects of Artificial Transcriptional Activators by High-Throughput Sequencing.

One of the major goals in DNA-based personalized medicine is the development of sequence-specific small molecules to target the genome. SAHA-PIPs belong to such class of small molecule. In the context of the complex eukaryotic genome, the differential biological effects of SAHA-PIPs are unclear. This question can be addressed by identifying the binding regions across the genome; however, it is a challenge to enrich small-molecule-bound DNA without chemical crosslinking. Here, we developed a method that employs high-throughput sequencing to map the binding area of small molecules throughout the chromatinized human genome. Analysis of the sequenced data confirmed the presence of specific binding sites for SAHA-PIPs from the enriched sequence reads. Mapping the binding sites and enriched regions on the human genome clarifies the reason for the distinct biological effects of SAHA-PIP. This approach will be useful for identifying the function of other small molecules on a large scale.

Comparative Analysis of DNA-Binding Selectivity of Hairpin and Cyclic Pyrrole-Imidazole Polyamides Based on Next-Generation Sequencing.

Many long pyrrole-imidazole polyamides (PIPs) have been synthesized in the search for higher specificity, with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind-n-Seq analysis revealed that both bound to the intended sequences. However, adenines in the data analyzed by the previously reported Bind-n-Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method based on Bind-n-Seq motif identification with a reference sequence (Bind-n-Seq-MR). High-throughput sequence analysis of the PIP-enriched DNA data by Bind-n-Seq-MR presented A and T comparably. Surface plasmon resonance assays were performed to validate the new method.

Sequence-specific DNA binding by long hairpin pyrrole-imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit.

With the aim of improving aqueous solubility, we designed and synthesized five N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides capable of recognizing 9-bp sequences. Their DNA-binding affinities and sequence specificities were evaluated by SPR and Bind-n-Seq analyses. The design of polyamide 1 was based on a conventional model, with three consecutive Py or Im rings separated by a ß-alanine to match the curvature and twist of long DNA helices. Polyamides 2 and 3 contained an 8-amino-3,6-dioxaoctanoic acid (AO) unit, which has previously only been used as a linker within linear Py-Im polyamides or between Py-Im hairpin motifs for tandem hairpin. It is demonstrated herein that AO also functions as a linker element that can extend to 2-bp in hairpin motifs. Notably, although the AO-containing unit can fail to bind the expected sequence, polyamide 4, which has two AO units facing each other in a hairpin form, successfully showed the expected motif and a KD value of 16nM was recorded. Polyamide 5, containing a ß-alanine-ß-alanine unit instead of the AO of polyamide 2, was synthesized for comparison. The aqueous solubilities and nuclear localization of three of the polyamides were also examined. The results suggest the possibility of applying the AO unit in the core of Py-Im polyamide compounds.

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing.

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2: showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing.

Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing.

Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein.

Selective Targeting of the KRAS Codon 12 Mutation Sequence by Pyrrole-Imidazole Polyamide seco-CBI Conjugates.

Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon 12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon 12 mutation in a mouse model by using an alkylating hairpin N-methylpyrrole-N-methylimidazole polyamide seco-1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one conjugate (conjugate 4) designed to target the KRAS codon 12 mutation sequence. Herein, we have compared the alkylating activity of 4 against three other conjugates that were also designed to target the KRAS codon 12 mutation sequence. Conjugate 4 displayed greater affinity for the G12D mutation sequence than for the G12V sequence. A computer-minimized model suggested that conjugate 4 could bind more efficiently to the G12D match sequence than to a one-base-pair mismatch sequence. Conjugate 4 was modified for next-generation sequencing. Bind-n-Seq analysis supported the evidence showing that conjugate 4 could target the G12D mutation sequence with exceptionally high affinity and the G12V mutation sequence with much higher affinity than that for the wild-type sequence.

A Synthetic DNA-Binding Domain Guides Distinct Chromatin-Modifying Small Molecules to Activate an Identical Gene Network.

Synthetic dual-function ligands targeting specific DNA sequences and histone-modifying enzymes were applied to achieve regulatory control over multi-gene networks in living cells. Unlike the broad array of targeting small molecules for histone deacetylases (HDACs), few modulators are known for histone acetyltransferases (HATs), which play a central role in transcriptional control. As a novel chemical approach to induce selective HAT-regulated genes, we conjugated a DNA-binding domain (DBD) "I" to N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB), an artificial HAT activator. In vitro enzyme activity assays and microarray studies were used to demonstrate that distinct functional small molecules could be transformed to have identical bioactivity when conjugated with a targeting DBD. This proof-of-concept synthetic strategy validates the switchable functions of HDACs and HATs in gene regulation and provides a molecular basis for developing versatile bioactive ligands.

A Synthetic Transcriptional Activator of Genes Associated with the Retina in Human Dermal Fibroblasts.

Small molecules capable of modulating epigenetic signatures can activate the transcription of tissue-restricted genes in a totally unrelated cell type and have potential use in epigenetic therapy. To provide an example for an initial approach, we report here on one synthetic small-molecule compound-termed "SAHA-PIP X"-from our library of conjugates. This compound triggered histone acetylation accompanied by the transcription of retinal-tissue-related genes in human dermal fibroblasts (HDFs).

Targeted suppression of EVI1 oncogene expression by sequence-specific pyrrole-imidazole polyamide.

Human ectopic viral integration site 1 (EVI1) is an oncogenic transcription factor known to play a critical role in many aggressive forms of cancer. Its selective modulation is thought to alter the cancer-specific gene regulatory networks. Pyrrole-imidazole polyamides (PIPs) are a class of small DNA binders that can be designed to target any destined DNA sequence. Herein, we report a sequence-specific pyrrole-imidazole polyamide, PIP1, which can target specific base pairs of the REL/ELK1 binding site in the EVI1 minimal promoter. The designed PIP1 significantly inhibited EVI1 in MDA-MB-231 cells. Whole-transcriptome analysis confirmed that PIP1 affected a fraction of EVI1-mediated gene regulation. In vitro assays suggested that this polyamide can also effectively inhibit breast cancer cell migration. Taken together, these results suggest that EVI1-targeted PIP1 is an effective transcriptional regulator in cancer cells.