Biology subjects

Johnson, A. M.

Publications and source records attributed to Johnson, A. M..

At least 19 recordsLinked to original sources

bioRxiv2026

An LRH-1 agonist reverses hepatic metabolic dysfunction in obesity

Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that controls metabolic gene programs important for liver health. Here, we address two challenges with dissecting ligand-driven LRH-1 metabolism in vivo. First, we generate ME610, a potent LRH-1 agonist with favorable pharmacokinetic properties. Second, we engineer Nr5a2humLBD mice, edited to recapitulate the ligand response of the human receptor. ME610 dose-dependently induces LRH-1 target genes and reduces triglycerides in livers of Nr5a2humLBD mice. In a mouse model of diet-induced obesity (DIO), ME610 dramatically improves insulin sensitivity and reduces liver fat. Multi-omics integration of transcriptomics, lipidomics, and metabolomics data demonstrates a coordinated reversal of DIO-induced hepatic metabolic stress via changes in lipid, amino acid, and energy metabolism, with peroxisomal lipid metabolism emerging as a central process correlated to efficacy. These studies uncover new pathways through which LRH-1 regulates metabolism and demonstrate the therapeutic potential of synthetic LRH-1 agonists in obesity-associated metabolic diseases.

physiology↗

bioRxiv2025

Characterization of an immunodeficiency-associated EZH2 variant

Regulation of gene expression is central to the development of immune cells and their ability to respond to infection. As part of a clinical evaluation, we identified two sisters with recurrent infections, hypogammaglobulinemia, and memory B cell deficiency, diagnosed as common variable immunodeficiency. Whole exome sequencing identified a heterozygous variant (Leu50Ser, L50S) in a conserved region of EZH2, the catalytic subunit of the epigenetic gene repressor Polycomb Repressive Complex 2 (PRC2). EZH2-catalyzed histone H3 lysine 27 methylation (H3K27me) in bulk was not overall significantly disrupted by this variant, in patient samples or cell lines expressing EZH2-L50S. EZH2-L50S protein is expressed similar to wild-type and can form PRC2. However, we find that specific genomic regions that normally have high wild-type levels of H3K27me3 are deficient in the L50S context, particularly around gene promoters. EZH2-L50S is still recruited to these sites, but is not as active. Using recombinant purified PRC2, we determine that L50S affects methylation of nucleosomes and disrupts allosteric stimulation that normally amplifies H3K27me3, consistent with the location of L50 in the allosteric regulatory region of PRC2. Thus, variation of EZH2 L50, occurring at low frequency in the population may interfere with normal B cell gene expression patterns, contributing to immunodeficiency. This study has implications for genetic variation in PRC2 in the general population.

molecular biology↗

bioRxiv2025

How do selective serotonin reuptake inhibitors effect metabolism? A lesson for teaching non-majors about enzymes and metabolic pathways

Understanding foundational concepts in molecular biology and biochemistry can be challenging for many college students. For non-science majors, an introductory biology course may be their sole exposure to college-level science and only opportunity to learn about enzymes and metabolism. Framing these topics within issues that are personally relevant can make the material more meaningful and impactful for non-majors. There is a need to develop curricula that instructors of large-enrollment introductory biology courses can use to teach non-biology majors about enzymes and metabolism. Here, we designed and implemented a lesson that integrates enzyme function and metabolism within the context of selective serotonin reuptake inhibitors (SSRIs). SSRIs are a widely prescribed class of antidepressants whose efficacy can be influenced by enzymes. Genetic polymorphisms in the genes encoding these enzymes can result in individual differences in metabolic responses and lead to medication-induced weight gain. Framing the content in this way allows non-major students to connect enzyme function to genetics, energy balance, and metabolic processes through a health-related, societally relevant issue. In our lesson, students engaged in collaborative learning activities that emphasized interpreting biological data, graphing, and scientific reasoning. Pre- and post-assessment data from a conceptual inventory showed learning gains that exceeded baseline data and were greater than those observed with the traditional curriculum. Students also reported a more coherent understanding of enzyme function and a greater appreciation of the contents relevance to their own lives. This lesson provides an example of embedding biochemical concepts in real-world contexts for students who are non-science majors.

scientific communication and education↗

bioRxiv2025

Klf4-dependent pivotal role of smooth muscle-derived Gli1+ lineage progenitor cells in lung cancer progression

Lung cancer remains the leading cause of cancer-related deaths worldwide. Despite the remarkable efficacy of immune checkpoint inhibitors, only a subset of lung adenocarcinoma (LUAD) patients respond and many eventually progress, underscoring the need to develop novel combination therapies. The interaction between cancer cells and the tumor microenvironment (TME), a complex niche including cells of the innate and adaptive immune system, cancer-associated fibroblasts (CAFs), vascular cells, and extracellular matrix (ECM) plays a vital role in tumorigenesis and cancer progression. Using fate mapping, we previously identified a novel subpopulation of resident vascular stem cells derived from vascular smooth muscle cells, designated AdvSca1-SM (vascular Adventitial location, Stem cell antigen-1 expression, SMooth muscle origin) cells. AdvSca1-SM cells are the predominant cell type responding to vessel wall dysfunction and their selective differentiation to myofibroblasts promotes vascular fibrosis. SMC-to-AdvSca1-SM cell reprogramming is dependent on SMC induction of the transcription factor KLF4, and KLF4 is essential for maintenance of the stem cell phenotype. The function of AdvSca1-SM cells in LUAD tumorigenesis has not been explored. Using an orthotopic immunocompetent mouse model of LUAD, we demonstrate that AdvSca1-SM cells are a major component of lung tumors, significantly contributing to cancer associated fibroblasts (CAFs). Compared to AdvSca1-SM cells, CAFs have altered ECM gene expression with a reduction of a stemness signature. AdvSca1-SM-specific genetic ablation of Klf4 altered their phenotype resulting in inhibition of communication between cancer cells and CAFs, decreases in innate immunosuppressive populations, and increased T cell infiltration into the tumor. Targeting this population may represent a novel strategy to improve the response to immunotherapy.

cancer biology↗

bioRxiv2024

Novel interactions within the silent information regulator heterochromatin complex potentiate inter-subunit communication and gene repression

Organisms with smaller genomes often perform multiple functions using one multi-subunit protein complex. The S. cerevisiae Silent Information Regulator complex (SIRc) carries out all of the core functions of heterochromatin. SIR complexes first drive the initiation and spreading of histone deacetylation in an iterative manner. Subsequently, the same complexes are incorporated stably with nucleosomes, driving compaction and repression of the underlying chromatin domain. These two distinct functions of SIRc have each been characterized in much detail, but the mechanism by which the dynamic spreading state switches to stable compaction is not well-understood. This incomplete knowledge of potential intra-complex communication is partly due to a lack of structural information of the complex as a whole; only structures of fragments have been determined to date. Using cross-linking mass spectrometry in solution, we identified a novel inter-subunit interaction that physically connects the two states of SIRc. The Sir2 deacetylase makes direct interactions with the scaffolding subunit Sir4 through its coiled-coil domain, which also interacts with the Sir3 compaction/repression subunit. Within the hub of interactions are conserved residues in Sir2 that can sense deacetylation state, as well as amino acids that likely diverged and co-evolved to interact with Sir4, promoting species-specific functions. Mutation of this interaction hub disrupts heterochromatic repression, potentially by disrupting a conserved mechanism that communicates completion of deacetylation to switch to compaction. Our work highlights how a single multi-functional chromatin regulatory complex can stage a step-wise mechanism that requires a major transition in activities to achieve epigenetic gene repression.

molecular biology↗

bioRxiv2024

Assessing the use of RIVPACS-derived invertebrate taxonomic predictions for river management

The River Invertebrate Prediction and Classification System (RIVPACS) is used widely in freshwater management to set targets for macroinvertebrate ecological health based on the expected scores of metrics such as WHPT or LIFE in the absence of anthropogenic stressors. An underutilised capability of RIVPACS-type models is the capability to predict expected macroinvertebrate community composition, which could function as a novel management metric for river health. We present a novel Monte-Carlo simulation approach that generates simulated expected communities for Englands rivers based on RIVPACS predictions. This allows for assessments of macroinvertebrate health using similarity calculations between observed and expected communities. We assess 10-year trends in similarity between 2010 and 2019 at 4172 sites in England, and contrast these trends with WHPT ASPT O/E trends in the same period. Similarity scores include both Chi-Squared and Hellinger methods, to prioritise rare and common species, respectively. We find that whilst most sites (63.3%) showed improvement in WHPT ASPT O/E in this period, most sites showed declines in similarity for Chi-Squared and Hellinger O/E (51.1% and 58.8%, respectively). We identified three case study regions showing contrasting trends and illustrate how the new RIVPACS-derived similarity calculations can track meaningful shifts in composition associated with water quality and multiple stressors including invasive species. RIVPACS-derived similarity calculations potentially provide a sensitive and practical management metric to assess ecosystem health, although further work is required to understand the composition of communities in changing environments with clear changes in stressor regimes.

ecology↗

bioRxiv2023

Dual role of Vascular Endothelial Growth Factor-C (VEGF-C) in post-stroke recovery

Using a mouse model of ischemic stroke, this study characterizes stroke-induced lymphangiogenesis at the cribriform plate (CP). While blocking CP lymphangiogenesis with a VEGFR-3 inhibitor improves stroke outcome, administration of VEGF-C induced larger brain infarcts. AbstractCerebrospinal fluid (CSF), antigens, and antigen-presenting cells drain from the central nervous system (CNS) into lymphatic vessels near the cribriform plate and dural meningeal lymphatics. However, the pathological roles of these lymphatic vessels surrounding the CNS during stroke are not well understood. Using a mouse model of ischemic stroke, transient middle cerebral artery occlusion (tMCAO), we show that stroke induces lymphangiogenesis near the cribriform plate. Interestingly, lymphangiogenesis is restricted to lymphatic vessels at the cribriform plate and downstream cervical lymph nodes, without affecting the conserved network of lymphatic vessels in the dura. Cribriform plate lymphangiogenesis peaks at day 7 and regresses by day 14 following tMCAO and is regulated by VEGF-C/VEGFR-3. These newly developed lymphangiogenic vessels transport CSF and immune cells to the cervical lymph nodes. Inhibition of VEGF-C/VEGFR-3 signaling using a blocker of VEGFR-3 prevented lymphangiogenesis and led to improved stroke outcomes at earlier time points but had no effects at later time points following stroke. Administration of VEGF-C after tMCAO did not further increase post-stroke lymphangiogenesis, but instead induced larger brain infarcts. The differential roles for VEGFR-3 inhibition and VEGF-C in regulating stroke pathology call into question recent suggestions to use VEGF-C therapeutically for stroke.

immunology↗

bioRxiv2023

Perilaryngeal Functional Muscle Network in Patients with Vocal Hyperfunction - A Case Study

Patients with both phonotraumatic and non-phonotraumatic dysphonia commonly present with vocal hyperfunction, defined as excessive perilaryngeal muscle activity and characterized by muscular pain and strain in the neck, increased vocal effort, and vocal fatigue. The inability to reliably measure vocal hyperfunction is a barrier to adequate evaluation and treatment of hyperfunctional voice disorders. We have recently demonstrated that the perilaryngeal functional muscle network can be a novel sensitive neurophysiological window to vocal performance in vocally healthy subjects. In this paper, for the first time, we evaluate the performance and symmetry of functional perilaryngeal muscle networks in three patients with voice disorders. Surface electromyography signals were recorded from twelve sensors (six on each side of the neck) using the wireless Trigno sEMG system (Delsys Inc., Natick, MA). Patient 1 was diagnosed with primary muscle tension dysphonia, Patient 2 was diagnosed with unilateral vocal fold paresis, and Patient 3 was diagnosed with age-related glottal insufficiency. This paper reports altered functional connectivity and asymmetric muscle network scan behavior in all three patients when compared with a cohort of eight healthy subjects. Our approach quantifies synergistic network activity to interrogate coordination of perilaryngeal and surrounding muscles during voicing and potential discoordination of the muscle network for dysphonic conditions. Asymmetry in muscle networks is proposed here as a biomarker for monitoring vocal hyperfunction.

bioengineering↗

bioRxiv2022

Adaptive immunity is required for durable responses to alectinib in murine models of EML4-ALK lung cancer

PurposeLung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g. alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). We previously demonstrated a positive association of a TKI-induced interferon gamma (IFN{gamma}) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used murine models of EML4-ALK lung cancer to test a role for host immunity in the therapeutic response to alectinib. Experimental DesignThree murine EML4-ALK cell lines (EA1, EA2, EA3) were implanted orthotopically into the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by CT. Immune cell content was measured by flow cytometry, multispectral immunofluorescence and CyTOF. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA. ResultsAll cell lines were sensitive to alectinib in vitro. EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of treatment while EA2 tumors were eliminated by TKI treatment. Alectinib induced inflammatory transcriptional programs and multiple chemokines in all cell lines while untreated tumors exhibited distinct baseline chemokine expression patterns and content of CD8+ T cells and myeloid subsets. When propagated in immune-deficient mice, all three cell line-derived lung tumor models exhibited significant shrinkage followed by prompt progression despite continuous alectinib treatment. ConclusionsThe findings support an hypothesis that host and TKI-stimulated production of chemokines by tumor cells promotes functional engagement of adaptive immune cells within the tumor microenvironment that enhances the durability and depth of TKI response. Statement of Translational RelevancePatients with metastatic lung cancer harboring ALK fusions are treated with targeted tyrosine kinase inhibitors (TKI) in the first line setting. Despite bearing the same driver oncogene, patients experience a range of tumor burden reduction and variable amounts of residual disease. Residual disease burden associates with patient survival and contributes to the emergence of drug resistance yielding treatment failure. The factors mediating this differential response to TKI and residual disease are incompletely understood. Our group has developed a panel of murine ALK driven lung cancer cell lines that reproducibly show differences in the depth and duration of response when implanted into immunocompetent mice. Data using this model indicate that the presence of CD8+ T cells is a major contributor to the depth and duration of response. These models will be critical in developing rational combination therapies to augment the immune microenvironment engagement along with TKIs to improve outcomes for these patients.

cancer biology↗

bioRxiv2021

Characterization of Cervical-Cranial Muscle Network in Correlation with Vocal Features

Objective evaluation of physiological responses using non-invasive methods has attracted great interest regarding the assessment of vocal performance and disorders. This paper, for the first time, demonstrates that the topographical features of the cervical-cranial intermuscular coherence network generated using surface electromyography (sEMG) have a strong potential for detecting subtle changes in vocal performance. For this purpose, in this paper, 12 sEMG signals were collected from six cervical and cranial muscles bilaterally. Data were collected from four subjects without a history of a voice disorder performing a series of vocal tasks. The vocal tasks were varied phonation (an /a/ sustained for the maximal duration with combinations of two levels of loudness and two levels of pitch), a pitch glide from low to high, singing a familiar song, spontaneous speech, and reading with different loudness levels. The varied phonation tasks showed the median degree, and weighted clustering coefficient of the coherence-based intermuscular network ascends monotonically, with a high effect size (|rrb| = 0.52). The set of tasks, including pitch glide, singing, and speech, was significantly distinguishable using the network features as both degree and weighted clustering coefficient had a very high effect size (|rrb| > 0.83) across these tasks. Also, pitch glide has the highest degree and weighted clustering coefficient among all tasks (degree > 0.6, weighted clustering coefficient > 0.6). Spectrotemporal features performed far less effective than the proposed functional muscle network metrics to differentiate the vocal tasks. The highest effect size for spectrotemporal features was only |rrb| = 0.19. In this paper, for the first time, the power of a cervical-cranial muscle network has been demonstrated as a neurophysiological window to vocal performance. The results also shed light on the tasks with the highest network involvement, which may be potentially used in monitoring vocal disorders and tracking rehabilitation progress.

neuroscience↗

bioRxiv2021

RNA-BINDING PROTEINS DIRECT MYOGENIC CELL FATE DECISIONS

RNA-binding proteins (RBPs) are essential for skeletal muscle regeneration and RBP dysfunction causes muscle degeneration and neuromuscular disease. How ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single cell RNA-sequencing of regenerating skeletal muscle reveals that RBP expression, including numerous neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates to stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discover that the neuromuscular disease associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis controlling myogenic cell fate transitions during terminal differentiation. The timing of RBP expression specifies cell fate transitions by providing a layer of post-transcriptional regulation needed to coordinate stem cell fate decisions during complex tissue regeneration.

molecular biology↗

bioRxiv2021

Cancer cell-specific MHCII expression as a determinant of the immune infiltrate organization and function in the non-small cell lung cancer tumor microenvironment

IntroductionIn patients with non-small cell lung cancer (NSCLC), the prognostic significance of the tumor microenvironment (TME) immune composition has been demonstrated using single or dual-marker staining on sequential tissue sections. While these studies show that relative abundance and localization of immune cells are important parameters, deeper analyses of the NSCLC TME are necessary to refine the potential application of these findings to clinical care. Currently, the complex spatial relationships between cells of the NSCLC TME and potential drivers contributing to its immunologic composition remain unknown. MethodsWe employed multispectral quantitative imaging on the lung adenocarcinoma TME in 153 patients with resected tumors. On a single slide per patient, we evaluated the TME with markers for CD3, CD8, CD14, CD19, major histocompatibility complex II (MHCII), cytokeratin, and DAPI. Image analysis including tissue segmentation, phenotyping, and spatial localization were performed. ResultsSpecimens where [≥]5% of lung cancer cells expressed MHCII (MHCIIhi TME) demonstrated increased levels of CD4+ and CD8+ T cell and CD14+ cell infiltration. In the MHCIIhi TME, the immune infiltrate was closer to cancer cells and expressed an activated phenotype. Morphologic image analysis revealed cancer cells in the MHCIIhi TME more frequently interfaced with CD4+ and CD8+ T cells. Patients with an MHCIIhi TME experienced improved overall survival (p=0.046). ConclusionsLung cancer cell-specific expression of MHCII associates with levels of immune cell infiltration, spatial localization, and activation status within the TME. This suggest cancer cell-specific expression of MHCII may represent a biomarker for the immune systems recognition and activation against the tumor.

cancer biology↗

bioRxiv2020

Mainly on the Plane: Deep Subsurface Bacterial Proteins Bind and Alter Clathrate Structure

Gas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a low-pressure analog for structure II gas clathrate. When grown in the presence of CbpAs, THF clathrate was polycrystalline and plate-like instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a plate-like structure during gas clathrate transportation. Table of Contents Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2020

A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells

N6-methyladenosine (m6A) modification of RNA plays important roles in normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we investigate whether m6A regulates the function of the human HOTAIR lncRNA, which contributes to multiple pro-tumor phenotypes in triple-negative breast cancer (TNBC) cells. We identify at least 8 individual m6A sites within HOTAIR, with a single site (A783) consistently methylated. Mutation of A783 impairs cellular proliferation and invasion in HOTAIR-overexpressing TNBC cells. m6A at A783 regulates HOTAIRs ability to localize to chromatin and induce gene pathways that affect tumor progression. In contrast, A783U mutant HOTAIR demonstrates loss-of-function and antimorph behaviors by impairing gene expression changes induced by WT HOTAIR and, in some cases, inducing opposite changes in gene expression. HOTAIR interacts with nuclear m6A reader YTHDC1 and high HOTAIR is significantly associated with shorter overall patient survival, particularly in the context of high YTHDC1. At the molecular level, YTHDC1-HOTAIR interactions are required for chromatin localization and regulation of gene repression. Our work demonstrates how modification of one base in a lncRNA can elicit a distinct gene regulation mechanism and drive disease-associated phenotypic changes such as proliferation and invasion.

molecular biology↗

bioRxiv2020

Cancer Cell Intrinsic Expression of MHCII Regulates the Immune Microenvironment and Response to Anti-PD-1 Therapy in Lung Adenocarcinoma

MHC class II (MHCII) expression is usually restricted to antigen presenting cells, but can be expressed by cancer cells. We examined the effect of cancer cell-intrinsic MHC class II (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy. The functional significance of altering csMHCII expression was explored using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis Lung Carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, while LLC tumors are resistant. RNA-seq analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, while resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII, and converted tumors from anti-PD-1-sensitive to anti-PD-1-resistant. This was associated with decreased T cell infiltration, lower levels of Th1 cytokines, increased B cell number and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.

cancer biology↗

bioRxiv2020

RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target genes can relieve RNA inhibition of PRC2. RNA matchmaking is promoted by heterogenous nuclear ribonucleoprotein (hnRNP) B1, which uses multiple protein domains to bind regions of HOTAIR via multi-valent protein-RNA interactions. Chemical probing demonstrates that RNA matchmaking changes HOTAIR RNA structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

biochemistry↗

bioRxiv2020

Development of a new class of liver receptor homolog-1 (LRH-1) agonists by photoredox conjugate addition

LRH-1 is a nuclear receptor that regulates lipid metabolism and homeostasis, making it an attractive target for the treatment of diabetes and non-alcoholic fatty liver disease. Building on recent structural information about ligand binding from our labs, we have designed a series of new LRH-1 agonists that further engage LRH-1 through added polar interactions. While the current synthetic approach to this scaffold has, in large part, allowed for decoration of the agonist core, significant variation of the bridgehead substituent is mechanistically precluded. We have developed a new synthetic approach to overcome this limitation, identified that bridgehead substitution is necessary for LRH-1 activation, and described an alternative class of bridgehead substituents for effective LRH-1 agonist development. We determined the crystal structure of LRH-1 bound to a bridgehead-modified compound, revealing a promising opportunity to target novel regions of the ligand-binding pocket to alter LRH-1 target gene expression.

biochemistry↗

bioRxiv2020

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP) based approaches that rely on inducing specific mutations during reverse transcription via UV crosslinking of the anti-m6A antibody to methylated RNA have been employed to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

genomics↗