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SummaryCellular metabolism is linked to epigenetics, but the biophysical effects of metabolism on chromatin structure and implications for gene regulation remain largely unknown. Here, using a broken tricarboxylic acid (TCA) cycle and disrupted electron transport chain (ETC) exemplified by succinate dehydrogenase subunit C (SDHC) deficiency, we investigated the effects of metabolism on chromatin architecture over multiple distance scales [nucleosomes (∼102 bp), topologically-associated domains (TADs; ∼105 – 106 bp), and chromatin compartments (106 – 108 bp)]. Metabolically-driven hyperacylation of histones led to weakened nucleosome positioning in multiple types of chromatin, and we further demonstrate that lysine acylation directly destabilizes histone octamer-DNA interactions. Hyperacylation of cohesin subunits correlated with decreased mobility on interphase chromatin and increased TAD boundary strength, suggesting that cohesin is metabolically regulated. Erosion of chromatin compartment distinctions reveals metabolic regulation of chromatin liquid-liquid phase separation. The TCA cycle and ETC thus modulate chromatin structure over multiple distance scales.

Multiple sclerosis (MS) is a debilitating disease for which regenerative therapies are sought. We have previously described human antibodies and DNA aptamer-streptavidin conjugates that promote remyelination after systemic injection into mice infected by Theiler’s murine encephalomyelitis virus. Here, we report an in vitro assay of myelin binding with results that correlate with remyelination outcome in vivo, as shown for data from a set of DNA aptamer complexes of different size and formulation. This in vitro assay will be valuable for future screening of MS regenerative therapies targeting remyelination. Keywords: multiple sclerosis, aptamer, myelin, oligodendrocyte, regeneration

SUMMARY Succinylation is a post-translational protein acylation modification that converts the cationic lysine side chain to an anion with large potential impacts on protein structure and function. Here we characterize the epigenome-wide distribution of succinyllysine marks in chromatin using chromatin immuno-precipitation sequencing (ChIP-seq). We estimate that more than one-third of all nucleosomes contain lysine succinylation marks and demonstrate a potential role of chromatin succinylation in modulating gene expression. We further demonstrate that defective tricarboxylic acid (TCA) cycle metabolism perturbs the succinyllysine distribution in chromatin, correlating with transcriptional responses. This is consistent with previous observations linking nucleosome succinylation with enhanced in vitro transcription. We additionally demonstrate that defective TCA cycle metabolism results in a DNA repair defect and sensitivity to genotoxic agents, consistent with previously reported chromatin hypersuccinylation effects observed in the context of SIRT7 depletion. Chromatin succinylation may thus represent a mechanism by which metabolism modulates both genome-wide transcription and DNA repair activities., Graphical Abstract

Multiple sclerosis (MS) is a chronic and progressive inflammatory demyelinating disease of the human central nervous system (CNS) and is the most common disabling neurological condition in young adults, resulting in severe neurological defects. No curative or long-term progression-inhibiting therapy has yet been developed. However, recent investigation has revealed potential strategies that do not merely modulate potentially pathogenic autoimmune responses, but stimulate remyelination within CNS lesions.We discuss the history and development of natural human IgM-isotype immunoglobulins (HIgMs) and recently-identified aptamer-conjugates that have been shown to enhance endogenous myelin repair in animal models of demyelination by acting on myelin-producing oligodendrocytes (OLs) or oligodendrocyte progenitor cells (OPCs) within CNS lesions. We also discuss future development aims and applications for these important novel technologies.Aptamer conjugate Myaptavin-3064 and recombinant human IgM-isotype antibody rHIgM22 regenerate CNS myelin, thereby reducing axonal degeneration and offering the potential of recovery from MS relapses, reversal of disability and prevention of disease progression. Advancement of these technologies into the clinic for MS treatment is therefore a top priority. It remains unclear to what extent the therapeutic modalities of remyelinating antibodies and aptamers may synergize with other currently-approved therapies to yield enhanced therapeutic effects.

Succinate dehydrogenase (SDH)-loss pheochromocytoma and paraganglioma (PPGL) are tumors driven by metabolic derangement. SDH loss leads to accumulation of intracellular succinate, which competitively inhibits dioxygenase enzymes, causing activation of pseudohypoxic signaling and hypermethylation of histones and DNA. The mechanisms by which these alterations lead to tumorigenesis are unclear, however. In an effort to fundamentally understand how SDH loss reprograms cell biology, we developed an immortalized mouse embryonic fibroblast cell line with conditional disruption of Sdhc and characterize the kinetics of Sdhc gene rearrangement, SDHC protein loss, succinate accumulation, and the resultant hypoproliferative phenotype. We further perform global transcriptomic, epigenomic, and proteomic characterization of changes resulting from SDHC loss, identifying specific perturbations at each biological level. We compare the observed patterns of epigenomic derangement to another previously-described immortalized mouse chromaffin cell model of SDHB loss, and compare both models to human SDH-loss tumors. Finally, we perform analysis of SDHC synthetic lethality with lactate dehydrogenase A (LDHA) and pyruvate carboxylase (PCX), which are important for regeneration of NAD+ and aspartate biosynthesis, respectively. Our data show that SDH-loss cells are selectively vulnerable to LDH genetic knock-down or chemical inhibition, suggesting that LDH inhibition may be an effective therapeutic strategy for SDH-loss PPGL.

Combinatorial chemistry drives the biological generation of protein structural diversity in antibodies and T-cell receptors. When applied to nucleic acids, vast engineered random libraries of DNA and RNA strands allow selection of affinity reagents (“aptamers”) against molecular targets. Selection involves cycles rewarding target binding affinity with amplification. Despite the success of this approach, delivery of selected aptamers across cell membranes and to specific subcellular compartments is an unmet need in chemical biology. Here, we address this challenge, demonstrating in vitro selection of DNA aptamers capable of homing to nuclei of cultured cells without transfection agents or viral trans-duction. Selection of such folded karyophilic DNA aptamers (~100 nucleotides) is achieved by a biosensor strategy that rewards exposure to nuclear DNA ligase. Identified DNA molecules are preferentially delivered to cell nuclei within minutes. Related strategies can be envisioned to select aptamers that home to other subcellular compartments.

Objective To use whole exome sequencing (WES) of a family trio to identify a genetic cause for polyvalvular syndrome. Methods and Results A male child was born with mild pulmonary valve stenosis and mild aortic root dilatation, and an atrial septal defect, ventricular septal defect, and patent ductus arteriosus that were closed surgically. Subsequently, the phenotype of polyvalvular syndrome with involvement of both semilunar and both atrioventricular valves emerged. His family history was negative for congenital heart disease. Because of hypotonia, myopia, soft pale skin, joint hypermobility, and mild facial dysmorphism, either Noonan syndrome- or William syndrome-spectrum disorders were suspected clinically. However, chromosomal analysis was normal and commercially available Noonan syndrome and William syndrome genetic tests were negative. Whole exome sequencing of the patient and both parents was performed. Variants were analyzed by sporadic and autosomal recessive inheritance models. A sporadic mutation, annotated as c.1491 T > A, in TAB2, resulting in a nonsense mutation, p.Y497X, in the TAB2-encoded TGF-beta activated kinase 1 (TAK1) was identified as the most likely disease-susceptibility gene. This mutation results in elimination of the terminal 197 amino acids, including the C-terminal binding motif critical for interactions with TRAF6 and TAK1. Conclusions The combination of WES, genomic triangulation, and systems biology has uncovered perturbations in TGF-beta activated kinase 1 signaling as a novel pathogenic substrate for polyvalvular syndrome.

We previously reported the in vitro selection and characterization of a DNA aptamer capable of stimulating remyelination in a mouse model of multiple sclerosis. This aptamer was selected for its ability to bind to suspensions of crude murine myelin in vitro. Our initial studies in vitro and in vivo involved a 40-nucleotide derivative (LJM-3064) of the original 100-nucleotide aptamer. LJM-3064 retained robust myelin-binding properties. Structural characterization of LJM-3064 revealed that the guanosine-rich 5' half of the sequence forms different G-quadruplex-type structures that are variably stable in the presence of physiologically relevant ions. We hypothesized that this structured domain is sufficient for myelin binding. In this study, we confirm that a 20-nucleotide DNA, corresponding to the 5' half of LJM-3064, retains myelin-binding properties. We then optimize this minimal myelin-binding aptamer via systematic evolution of ligands by exponential enrichment after sparse rerandomization. We report a sequence variant (LJM-5708) of the 20-nucleotide myelin-binding aptamer with enhanced myelin-binding properties and the ability to bind cultured human oligodendroglioma cells in vitro, providing the first evidence of cross-species reactivity of this myelin-binding aptamer. As our formulation of DNA aptamers for in vivo remyelination therapy involves conjugation to streptavidin, we verified that the myelin-binding properties of LJM-5708 were retained in conjugates to avidin, streptavidin, and neutravidin. DNA aptamer LJM-5708 is a lead for further preclinical development of remyelinating aptamer technologies.

Multiple sclerosis (MS) is a debilitating disease for which regenerative therapies are sought. We have previously described human antibodies and DNA aptamer-streptavidin conjugates that promote remyelination after systemic injection into mice infected by Theiler’s murine encephalomyelitis virus. Here, we report an in vitro assay of myelin binding with results that correlate with remyelination outcome in vivo, as shown for data from a set of DNA aptamer complexes of different size and formulation. This in vitro assay will be valuable for future screening of MS regenerative therapies targeting remyelination.

To use whole exome sequencing (WES) of a family trio to identify a genetic cause for polyvalvular syndrome.A male child was born with mild pulmonary valve stenosis and mild aortic root dilatation, and an atrial septal defect, ventricular septal defect, and patent ductus arteriosus that were closed surgically. Subsequently, the phenotype of polyvalvular syndrome with involvement of both semilunar and both atrioventricular valves emerged. His family history was negative for congenital heart disease. Because of hypotonia, myopia, soft pale skin, joint hypermobility, and mild facial dysmorphism, either Noonan syndrome- or William syndrome-spectrum disorders were suspected clinically. However, chromosomal analysis was normal and commercially available Noonan syndrome and William syndrome genetic tests were negative. Whole exome sequencing of the patient and both parents was performed. Variants were analyzed by sporadic and autosomal recessive inheritance models. A sporadic mutation, annotated as c.1491 T A, in TAB2, resulting in a nonsense mutation, p.Y497X, in the TAB2-encoded TGF-beta activated kinase 1 (TAK1) was identified as the most likely disease-susceptibility gene. This mutation results in elimination of the terminal 197 amino acids, including the C-terminal binding motif critical for interactions with TRAF6 and TAK1.The combination of WES, genomic triangulation, and systems biology has uncovered perturbations in TGF-beta activated kinase 1 signaling as a novel pathogenic substrate for polyvalvular syndrome.

Background: Succinate dehydrogenase (SDH) loss and mastermind-like 3 (MAML3) translocation are two clinically important genetic alterations that occur in subtypes of human paraganglioma and pheochromocytoma (PPGL) neuroendocrine tumors that correlate with increased rates of metastasis. Although hypotheses exist for how global activation of pseudohypoxia and epigenomic hypermethylation are driven via dioxygenase poisoning in the context of SDH loss, it remains unclear how these defects drive tumorigenesis. Additionally, MAML3 translocation has recently been identified as a genetic alteration in PGL and is poorly understood. We hypothesize that a key to understanding the tumorigenic basis for these genetic alterations is the deconvolution of the gene expression profiles in these tumors and identification of the transcription factors responsible for the observed transcriptional changes. Methods: In the present work, we leverage publicly-available human tumor gene expression profiling experiments (N=179) to reconstruct a PPGL tumor-specific transcriptional network. Inferred network structure was subsequently validated by examining the frequency of known transcription factor DNA-binding motifs in the inferred regulons. We subsequently use the inferred transcriptional networks and known gene expression signatures for specific PPGL subtypes to perform a master regulator analysis nominating specific transcription factors that may control observed oncogenic patterns of gene expression. The findings of the master regulator analysis were confirmed by duplication of the analysis on a second PPGL-specific inferred transcriptional network (N=188 specimens). Results: A small number of master regulator transcription factors are likely drivers of the observed subtype-specific gene expression patterns in SDH loss- and MAML3 translocation-positive PPGL. Unexpectedly, hypoxia-inducible transcription factors were not identified as master regulators in SDH-loss tumors. Additionally, analysis of the MAML3 translocation subtype master regulators revealed that a single transcription factor, IRX4, may control 37% of the deferentially expressed genes in this highly malignant tumor subtype. Conclusions: These observations from unbiased analyses suggest a basis for future development of targeted therapies for specific PPGL molecular subtypes. Citation Format: John A. Smestad, Louis J. Maher. Master regulator analysis of paragangliomas carrying SDH, VHL, or MAML3 genetic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1291.

Background Putative G-quadruplex-forming sequences (PQS) have long been implicated in regulation of transcription, though the actual mechanisms are not well understood. One proposed mechanism involves the activity of PQS-specific helicases belonging to the RecQ helicase family. However, patterns of PQS that correlate with transcriptional sensitivity to RecQ helicases are not well studied, and no adequate transcriptional model exists to account for PQS effects. Methods To better understand PQS transcriptional effects, we analyze PQS motifs in genes differentially-transcribed in Bloom Syndrome (BS) and Werner Syndrome (WS), two disorders resulting in loss of PQS-interacting RecQ helicases. We also correlate PQS genome-wide with transcription in multiple human cells lines while controlling for epigenetic status. Finally, we perform neural network clustering of PQS motifs to assess whether certain motifs are over-represented in genes sensitive to RecQ helicase loss. Results By analyzing PQS motifs in promoters of genes differentially-transcribed in BS and WS, we demonstrate that abundance of promoter PQS is generally higher in down-regulated genes and lower in up-regulated genes, and show that these effects are position-dependent. To interpret these correlations we determined genome-wide PQS correlations with transcription while controlling for epigenetic status. Our results identify multiple discrete transcription start site-proximal positions where PQS are correlated with either increased or decreased transcription. Finally, we report neural network clustering analysis of PQS motifs demonstrating that genes differentially-expressed in BS and WS are significantly biased in PQS motif composition. Conclusions Our findings unveil unappreciated detail in the relationship between PQS, RecQ helicases, and transcription. We show that promoter PQS are generally correlated with reduced gene expression, and that this effect is relieved by RecQ helicases. We also show that PQS at certain positions on the downstream sense strand are correlated with increased transcription. We therefore propose a new transcriptional model in which promoter PQS have at least two distinct types of transcriptional regulatory effects. Electronic supplementary material The online version of this article (doi:10.1186/s12881-015-0236-4) contains supplementary material, which is available to authorized users.

Putative G-quadruplex-forming sequences (PQS) have long been implicated in regulation of DNA replication and transcription, though their actual roles are unknown. To gain insight into potential PQS transcriptional function, we map and analyze PQS motifs in promoters of genes differentially-expressed in Bloom Syndrome (BS) and Werner Syndrome (WS), two human genetic disorders resulting in loss of PQS-interacting RecQ helicases. Non-B-DNA structures at PQS might be stabilized in these syndromes. For BS and WS we demonstrate that PQS promoter abundance is generally higher in down-regulated genes and lower in up-regulated genes, and show that these effects are position-dependent. To interpret these correlations we determined genome-wide PQS correlations with transcription using epigenetic information to predict gene expression. We report that 33% and 35% of analyzed PQS positions in promoter antisense and sense strands, respectively, displayed statistically-significant correlation with gene expression. Of these statistically-significant positions, 100% and 84% on antisense and sense strands, respectively, were correlated with reduced expression. This suggests that promoter PQS repress transcription. Finally, we report neural network clustering analysis of PQS motifs to demonstrate that genes differentially-expressed in BS and WS are significantly biased in their PQS motifs, suggesting an unappreciated biological relationship between PQS, RecQ helicases, and transcription.

Background & Aims Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor ( Lepr db/db ), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene ( Sdhc ). Methods Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase–Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. Results In obese, hyperglycemic, hyperinsulinemic female Lepr db/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female Kit K641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1—a master transcription factor for ICCs—and consequent up-regulation of v-kit Hardy–Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc . Conclusions Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.

DNA aptamer oligonucleotides and their protein conjugates show promise as therapeutics in animal models of diseases such as multiple sclerosis. These molecules are large and highly charged, raising questions about their biodistribution and pharmacokinetics in mammals. Here we exploit the power of quantitative polymerase chain reaction to accurately quantitate the tissue distribution of 40-nucleotide DNA aptamers and their streptavidin conjugates after intraperitoneal injection in mice. We show remarkably rapid distribution to peripheral tissues including the central nervous system. Modeling of tissue distribution data reveals the importance of DNA aptamer sequence, 3′ modification, and protein conjugation in enhancing tissue exposure. These data help to interpret the previously observed effectiveness of aptamer conjugates, as opposed to free aptamers, in stimulating central nervous system remyelination in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler’s encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination in vivo. Here we show that a 40-nucleotide single-stranded DNA aptamer selected for affinity to murine myelin shows this property. This aptamer binds multiple myelin components in vitro. Peritoneal injection of this aptamer results in distribution to CNS tissues and promotes remyelination of CNS lesions in mice infected by Theiler’s virus. Interestingly, the selected DNA aptamer contains guanosine-rich sequences predicted to induce folding involving guanosine quartet structures. Relative to monoclonal antibodies, DNA aptamers are small, stable, and non-immunogenic, suggesting new possibilities for MS treatment.