Biology subjects

Kanke, M.

Publications and source records attributed to Kanke, M..

At least 19 recordsLinked to original sources

bioRxiv2025

Basic-Leucine-Zipper Transcription Factors Regulate Selective Molecular Phenotypes in Regulatory T Cells During IL-2-Induced Activation

Regulatory T (Treg) cells have long been recognized as modulators of immunological tolerance and homeostasis. Previously, we used scRNA-seq to reveal significant Treg heterogeneity in response to IL-2-induced activation. Herein, we leveraged enrichment analyses, as well as bulk and single-nucleus multi-omics in splenic and lung Tregs, to uncover and confirm the importance of transcription factors (TFs) and chromatin remodeling in Treg activation. Multiple bZIP TF motifs showed increased chromatin accessibility post IL-2 treatment, with correlated transcriptional changes resembling Th1 and Th2 molecular phenotypes, further confirmed by spatial ATAC-seq. By combining gene perturbation and CUT&RUN assays before and after Treg stimulation, we show that bZIP TFs, such as BATF and BACH1, are critical to IL-2-induced Treg activation, coordinating epigenetic and transcriptional changes that selectively drive T-helper phenotypes and metabolic pathways.

genomics↗

bioRxiv2024

Single-cell RNA-sequencing reveals immune system compartmentalization under symbiosis in the stony coral Oculina arbuscula

Many cnidarians host single-celled algae within gastrodermal cells, yielding a mutually beneficial exchange of nutrients between host and symbiont, and dysbiosis can lead to host mortality. Previous research has uncovered symbiosis tradeoffs, including suppression of immune pathways in cnidarians hosting intracellular algae and correlations between symbiotic state and pathogen susceptibility. Here, we used a multiomic approach to characterize symbiotic states of the facultatively symbiotic coral Oculina arbuscula by generating genotype-controlled fragments of symbiotic and aposymbiotic tissue. 16S metabarcoding showed no difference in bacterial communities between symbiotic states. Whole-organism proteomics revealed differential abundance of proteins related to immunity, confirming immune suppression during symbiosis. Finally, single-cell RNAseq identified diverse cell clusters within seven cell types across symbiotic states. Specifically, the gastrodermal cell clusters containing algal-hosting cells from symbiotic tissue had higher expression of nitrogen cycling and sugar transport genes than aposymbiotic gastrodermal cells. Furthermore, differential enrichment of immune system gene pathways and lower expression of genes involved in immune regulation were observed in these gastrodermal cells from symbiotic tissue. However, no differences in immune gene expression in the immune cell cluster were observed between symbiotic states. This work reveals a compartmentalization of immune system regulation in specific gastrodermal cells in symbiosis, which may limit symbiosis tradeoffs by simultaneously dampening immunity in algal-hosting cells while maintaining general organismal immunity.

genomics↗

bioRxiv2023

A Highly Efficient Aorta-Gonad-Mesonephros-Like Definitive Hemogenic Endothelium From Human Pluripotent Stem Cells

Summary/AbstractHuman pluripotent stem cells are a tremendous tool to model early human development and disease including their use in the in vitro generation of blood cell fates. Hematopoietic progenitors and stem cells are the primary source of blood and the immune system from early development to adulthood and arise through successive waves of hemogenic mesoderm either in the yolk sac or embryo proper. Researchers have long sought a tractable human model for observing and distinguishing these waves of hematopoiesis in the dish for human developmental and disease modeling. Here we report a high-efficiency method for differentiating human pluripotent stem cells into an aorta-gonad-mesonephros-like definitive hemogenic mesoderm capable of giving rise to definitive hematopoietic progenitor and stem cells. The hematopoietic progenitor and stem cells exhibit robust multilineage in vitro colony forming potential. Gene expression analysis and single cell sequencing strongly support the developmental timing and notion that the pluripotent stem cell derived hematopoietic stem and progenitors are strikingly like bone fide hematopoietic stem cells. The hematopoietic progenitors can be subsequently differentiated into polarized macrophage and T-cells in vitro. Minimal silencing was observed upon differentiation of the pluripotent stem cells to hematopoietic lineages when conducting gene editing. Finally, upon engraftment into immunodeficient animals the hematopoietic progenitors and stem cells differentiate into multiple lineages including B-cells, T-cells, NK-cells, and monocytes.

developmental biology↗

bioRxiv2023

DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long non-coding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo models of disease. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, an indicator of mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease. HighlightsFibrolamellar carcinoma (FLC) is a lethal liver cancer lacking effective therapeutic options. Ma et al. demonstrate that primate-specific RNA LINC00473 is enriched in tumor epithelial cells and functions to promote FLC growth and dysregulate cellular energetics, unveiling an important mechanism downstream of the fusion oncogene, DNAJB1-PRKACA, in FLC pathogenesis. In BriefO_LILINC00473 is consistently elevated in primary FLC tumor tissue from different patient cohorts and in multiple disease models. C_LIO_LIDP fusion, the signature oncoprotein of FLC, drives LINC00473 expression. C_LIO_LILINC00473 promotes FLC growth via anti-apoptotic function. C_LIO_LILINC00473 modulates FLC energetics by promoting glycolysis and altering mitochondrial fitness. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/543290v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@20e0f2org.highwire.dtl.DTLVardef@904fbborg.highwire.dtl.DTLVardef@62af84org.highwire.dtl.DTLVardef@f114be_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

A Genetic Locus Mediating Attentional Processing

Attention is required for most higher-order cognitive functions. Prior studies have revealed functional roles for the prefrontal cortex and its extended circuits to enabling attention, but the underlying molecular processes and their impacts on cellular and circuit function remain poorly understood. To develop insights, we here took an unbiased forward genetics approach to identify single genes of large effect on attention. We studied 200 genetically diverse mice on measures of pre-attentive processing and through genetic mapping identified a small locus on chromosome 13 (95%CI: 92.22-94.09 Mb) driving substantial variation (19%) in this trait. Further characterization of the locus revealed a causative gene, Homer1, encoding a synaptic protein, where down-regulation of its short isoforms in prefrontal cortex (PFC) during early postnatal development led to improvements in multiple measures of attention in the adult. Subsequent mechanistic studies revealed that prefrontal Homer1 down-regulation is associated with GABAergic receptor up-regulation in those same cells. This enhanced inhibitory influence, together with dynamic neuromodulatory coupling, led to strikingly low PFC activity at baseline periods of the task but targeted elevations at cue onset, predicting short-latency correct choices. Notably high-Homer1, low-attentional performers, exhibited uniformly elevated PFC activity throughout the task. We thus identify a single gene of large effect on attention - Homer1 - and find that it improves prefrontal inhibitory tone and signal-to-noise (SNR) to enhance attentional performance. A therapeutic strategy focused on reducing prefrontal activity and increasing SNR, rather than uniformly elevating PFC activity, may complement the use of stimulants to improve attention.

neuroscience↗

bioRxiv2022

miR-29 is an important driver of aging-related phenotypes

Aging is a consequence of complex molecular changes, but the roles of individual microRNAs (miRNAs) in aging remain unclear. One of the few miRNAs that are upregulated during both normal and premature aging is miR-29. We confirmed this finding in our study in both mouse and monkey models. Follow-up analysis of the transcriptomic changes during normal aging revealed that miR-29 is among the top miRNAs predicted to drive the aging-related gene expression changes. We also showed that partial loss of miR-29 extends the lifespan of Zmpste24-/- mice, an established model of progeria, which indicates that miR-29 is functionally important in this accelerated aging model. To examine whether miR-29 upregulation alone is sufficient to promote aging-related phenotypes in vivo, we generated mice in which miR-29 can be conditionally overexpressed (miR-29TG). We found that miR-29 overexpression in mice is sufficient to drive aging-related phenotypes including alopecia, kyphosis, osteoporosis, senescence, and leads to early lethality. Transcriptomic analysis of both young miR-29TG and old WT mice revealed shared downregulation of genes enriched in extracellular matrix and fatty acid metabolism, and shared upregulation of genes in pathways linked to inflammation. Together, these results highlight the functional importance of miR-29 in controlling a gene expression program that drives agingrelated phenotypes.

physiology↗

bioRxiv2022

Integrative genome-scale analyses reveal post-transcriptional signatures of early human smallintestinal development in a directed differentiation organoid model

MicroRNAs (miRNAs) are important post-transcriptional gene regulators in organ development. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Our analyses showed that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/499825v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ebf48eorg.highwire.dtl.DTLVardef@4a0d9org.highwire.dtl.DTLVardef@1dc3be9org.highwire.dtl.DTLVardef@1e14207_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2022

Human pancreatic islet microRNAs implicated in diabetes and related traits by large-scale genetic analysis

Genetic studies have identified [≥]240 loci associated with risk of type 2 diabetes (T2D), yet most of these loci lie in non-coding regions, masking the underlying molecular mechanisms. Recent studies investigating mRNA expression in human pancreatic islets have yielded important insights into the molecular drivers of normal islet function and T2D pathophysiology. However, similar studies investigating microRNA (miRNA) expression remain limited. Here, we present data from 63 individuals, representing the largest sequencing-based analysis of miRNA expression in human islets to date. We characterize the genetic regulation of miRNA expression by decomposing the expression of highly heritable miRNAs into cis- and trans-acting genetic components and mapping cis-acting loci associated with miRNA expression (miRNA-eQTLs). We find (i) 81 heritable miRNAs, primarily regulated by trans-acting genetic effects, and (ii) 5 miRNA-eQTLs. We also use several different strategies to identify T2D-associated miRNAs. First, we colocalize miRNA-eQTLs with genetic loci associated with T2D and multiple glycemic traits, identifying one miRNA, miR-1908, that shares genetic signals for blood glucose and glycated hemoglobin (HbA1c). Next, we intersect miRNA seed regions and predicted target sites with credible set SNPs associated with T2D and glycemic traits and find 32 miRNAs that may have altered binding and function due to disrupted seed regions. Finally, we perform differential expression analysis and identify 13 miRNAs associated with T2D status--including miR-187-3p, miR-21-5p, miR-668, and miR-199b-5p--and 4 miRNAs associated with a polygenic score for HbA1c levels--miR-216a, miR-25, miR-30a-3p, and miR-30a-5p.

genetics↗

bioRxiv2021

Genome edited colorectal cancer organoid models reveal distinct microRNA activity patterns across different mutation profiles

Somatic mutations drive colorectal cancer (CRC) by disrupting gene regulatory mechanisms. Distinct combinations of mutations can result in unique changes to regulatory mechanisms leading to variability in the efficacy of therapeutics. MicroRNAs are important regulators of gene expression, and their activity can be altered by oncogenic mutations. However, it is unknown how distinct combinations of CRC-risk mutations differentially affect microRNAs. Here, using genetically-modified mouse intestinal organoid (enteroid) models, we identify 12 different modules of microRNA expression patterns across distinct combinations of mutations common in CRC. We also show that miR-24-3p is aberrantly upregulated in genetically-modified mouse enteroids irrespective of mutational context. Furthermore, we identify an enrichment of miR-24-3p predicted targets in downregulated gene lists from various mutational contexts compared to WT. In follow-up experiments, we demonstrate that miR-24-3p promotes CRC cell survival in multiple cell contexts. Our novel characterization of genotype-specific patterns of miRNA expression offer insight into the mechanisms that drive inter-tumor heterogeneity and highlight candidate microRNA therapeutic targets for the advancement of precision medicine for CRC.

cancer biology↗

bioRxiv2021

Chemical, molecular, and single cell analysis reveal chondroitin sulfate proteoglycan aberrancy in fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is an aggressive liver cancer with no effective therapeutic options. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer growth and/or metastasis. To bridge this knowledge gap, we assessed pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and non-malignant liver tissue (n=27) to identify changes in glycosaminoglycan (GAG) biosynthesis pathways and found that genes associated with production of chondroitin sulfate, but not other GAGs, are significantly increased by 8-fold. We then implemented a novel LC-MS/MS based method to quantify the abundance of different types of GAGs in patient tumors (n=16) and found that chondroitin sulfate is significantly more abundant in FLC tumors by 6-fold. Upon further analysis of GAG-associated proteins we found that versican (VCAN) expression is significantly up-regulated at the mRNA and protein levels, the latter of which was validated by immunohistochemistry. Finally, we performed single-cell assay for transposon-accessible chromatin-sequencing on FLC tumors (n=3), which revealed for the first time the different cell types in FLC tumors and also showed that VCAN is likely produced not only from FLC tumor epithelial cells but also activated stellate cells. Our results reveal a pathologic aberrancy in chondroitin (but not heparan) sulfate proteoglycans in FLC and highlight a potential role for activated stellate cells. SignificanceThis study leverages a multi-disciplinary approach, including state-of-the-art chemical analyses and cutting-edge single-cell genomic technologies, to identify for the first time a marked chondroitin sulfate aberrancy in fibrolamellar carcinoma (FLC) that could open novel therapeutic avenues in the future.

cancer biology↗

bioRxiv2021

Single-cell analysis of colonic epithelium reveals unexpected shifts in cellular composition and molecular phenotype in treatment-naiïve adult Crohn's disease

The intestinal epithelial barrier is comprised of a monolayer of specialized intestinal epithelial cells (IECs) that are critical in maintaining gut mucosal homeostasis. Dysfunction within various IEC fractions can increase intestinal permeability, resulting in a chronic and debilitating condition known as Crohns disease (CD). Defining the molecular changes in each IEC type in CD will contribute to an improved understanding of the pathogenic processes and the identification of potential therapeutic targets. Here we performed, for the first time at single-cell resolution, a direct comparison of the colonic epithelial cellular and molecular landscape between treatment-naive adult CD and non-IBD control patients. Our analysis revealed that in CD patients there is a significant skew in the colonic epithelial cellular distribution away from canonical LGR5+ stem cells, located at the crypt-bottom, and toward one specific subtype of mature colonocytes, located at the crypt-top. Further analysis revealed unique changes to gene expression programs in every major cell type, including a previously undescribed suppression in CD of most enteroendocrine driver genes as well as L-cell markers including GCG. We also dissect a previously poorly understood SPIB+ cell cluster, revealing at least four sub-clusters that exhibit unique features. One of these SPIB+ sub-clusters expresses crypt-top colonocyte markers and is significantly up-regulated in CD, whereas another sub-cluster strongly expresses and stains positive for lysozyme (albeit no other canonical Paneth cell marker), which surprisingly is greatly reduced in expression in CD. Finally, through integration with data from genome-wide association studies, we show that genes implicated in CD risk exhibit heretofore unknown cell-type specific patterns of aberrant expression in CD, providing unprecedented insight into the potential biological functions of these genes.

genomics↗

bioRxiv2020

Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt

The role of individual miRNAs in small intestinal (SI) epithelial homeostasis is under-explored. In this study, we discovered that miR-375 is among the most enriched miRNAs in intestinal crypts and stem cells (ISCs), especially facultative ISCs. We then showed by multiple manipulations, including CRISPR/Cas9 editing, that miR-375 is strongly suppressed by Wnt-signaling. Single-cell RNA-seq analysis of SI crypt-enriched cells from miR-375 knockout (375-KO) mice revealed elevated numbers of tuft cells and increased expression of pro-proliferative genes in ISCs. Accordingly, the genetic loss of miR-375 promoted resistance to helminth infection and enhanced the regenerative response to irradiation. The conserved effects of miR-375 were confirmed by gain-of-function studies in Drosophila midgut stem cells in vivo. Moreover, functional experiments in enteroids uncovered a regulatory relationship between miR-375 and Yap1 that controls cell survival. Finally, analysis of mouse model and clinical data revealed an inverse association between miR-375 levels and intestinal tumor development. HighlightsO_LImiR-375 is one of the most enriched miRNAs in ISCs, especially facultative ISCs. C_LIO_LImiR-375 modifies tuft cell abundance and pro-proliferative gene expression in ISCs. C_LIO_LILoss of miR-375 in mice enhances the host response to helminth infection and crypt regeneration. C_LIO_LIMouse and human intestinal cancer are associated with reduced miR-375 expression. C_LI eTOC BlurbSethupathy and colleagues show that miR-375 is a Wnt-responsive, ISC-enriched miRNA that serves as a break on intestinal crypt proliferation. They also show that miR-375 modulates tuft cell abundance and pro-proliferative gene expression in ISCs, that miR-375 loss enhances the host response to helminth infection as well as crypt regeneration post-irradiation, and its reduced expression is associated with intestinal cancer.

molecular biology↗

bioRxiv2020

Ozone-induced changes in murine lung extracellular vesicle number and small RNA content

Inhalation exposure to ozone (O3) causes adverse respiratory health effects that result from airway inflammation, a complex response mediated by changes to airway cellular transcriptional programs. These programs may be regulated in part by a subset of microRNAs transferred between cells (e.g. epithelial cells and macrophages) via extracellular vesicles (EV miRNA). To explore this, we exposed female C57BL/6J mice to filtered air (FA), 1, or 2 ppm O3 by inhalation and collected bronchoalveolar lavage fluid (BALF) 21 hours later for markers of airway inflammation, EVs, and EV miRNA. Both concentrations of O3 significantly increased markers of inflammation (neutrophils and total protein) and the number of EVs in the BALF. Using high-throughput small RNA sequencing, we identified several differentially expressed (DE) BALF EV miRNAs after 1 ppm (16 DE miRNAs) and 2 ppm (99 DE miRNAs) O3 versus FA exposure. O3 concentration response patterns in EV miRNA expression were apparent, particularly for the two most highly expressed (miR-2137 and miR-126-3p) and lowly expressed (miR-378-3p and miR-351-5p) miRNAs. Integrative analysis of EV miRNA expression and airway cellular mRNA expression identified EV miR-22-3p as a candidate regulator of transcriptomic responses to O3 in airway macrophages. In contrast, we did not identify candidate miRNA regulators of mRNA expression data from conducting airways (predominantly composed of epithelial cells). In summary, our data show that O3 exposure alters EV release and EV miRNA expression, suggesting that further investigation of EVs may provide insight into their effects on airway macrophage function and other mechanisms of O3-induced respiratory inflammation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

AGO HITS-CLIP in Adipose Tissue Reveals miR-29 as a Post-Transcriptional Regulator of Leptin

MicroRNAs (miRNAs) are short, non-coding RNAs that associate with Argonaute (AGO) to regulate mRNA stability and translation. While individual miRNAs have been shown to play important roles in white and brown adipose tissue in normal physiology and disease1,2,3, a comprehensive analysis of miRNA activity in these tissues has not been performed. We used high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to comprehensively characterize the network of high-confidence, in vivo mRNA:miRNA interactions across white and brown fat, revealing over 100,000 unique miRNA binding sites. Targets for each miRNA were ranked to generate a catalog of miRNA binding activity, and the miR-29 family emerged as a top regulator of adipose tissue gene expression. Among the top targets of miR-29 was leptin, an adipocyte-derived hormone that acts on the brain to regulate food intake and energy expenditure4. Two independent miR-29 binding sites in the leptin 3-UTR were validated using luciferase assays, and miR-29 gain and loss-of-function modulated leptin mRNA and protein secretion in primary adipocytes. In mice, miR-29 abundance inversely correlated with leptin levels in two independent models of obesity. This work represents the only experimentally generated miRNA targetome in adipose tissue and identifies the first known post-transcriptional regulator of leptin. Future work aimed at manipulating miR-29:leptin binding may provide a therapeutic opportunity to treat obesity and its sequelae.

cell biology↗

bioRxiv2020

Hotspots of aberrant enhancer activity in fibrolamellar carcinoma reveal molecular mechanisms of oncogenesis and intrinsic drug resistance

Fibrolamellar carcinoma (FLC) is a rare, therapeutically intractable liver cancer that disproportionately affects youth. Although FLC tumors exhibit a distinct gene expression profile, the causative transcriptional mechanisms remain unclear. Here we used chromatin run-on sequencing to discover approximately 7,000 enhancers and 141 enhancer hotspots activated in FLC relative to non-malignant liver. Detailed bioinformatic analyses revealed aberrant ERK/MEK signaling and candidate master transcriptional regulators. We also defined the genes most strongly associated with aberrant FLC enhancer activity, including CA12 and SLC16A14. Treatment of FLC cell models with inhibitors of CA12 or SLC16A14 independently reduced cell viability and/or significantly enhanced the effect of MEK inhibitor cobimetinib. These findings highlight new molecular targets for drug development as well as novel drug combination approaches.

cancer biology↗

bioRxiv2019

Contrasting population genetic structure in Acropora coral hosts and their algal symbionts across multiple spatial scales

Many broadly-dispersing corals acquire their algal symbionts (Symbiodiniaceae) horizontally from their environment upon recruitment. Horizontal transmission could promote coral fitness across diverse environments provided that corals can associate with divergent algae across their range and that these symbionts exhibit reduced dispersal potential. Here we quantified community divergence of Cladocopium algal symbionts in two coral host species (Acropora hyacinthus, Acropora digitifera) across two spatial scales (reefs on the same island, and between islands) across the Micronesian archipelago using microsatellites. We find that both hosts associated with two genetically distinct Cladocopium lineages (C40, C21), confirming that Acropora coral hosts associate with a range of Cladocopium symbionts across this region. Both C40 and C21 exhibited extensive clonality. Clones not only existed across host conspecifics living on the same reef, but also spanned host species, reef sites within islands, and even different islands. Both Cladocopium lineages exhibited moderate host specialization and divergence across islands. In addition, within every island, algal symbiont communities were significantly clustered by both host species and reef site, highlighting that coral-associated Cladocopium communities are structured across small spatial scales and within hosts on the same reef. This is in stark contrast to their coral hosts, which never exhibited significant genetic divergence between reefs on the same island. These results support the view that horizontal transmission could improve local fitness for broadly dispersing Acropora coral species.

ecology↗

bioRxiv2018

EndoC-βH1 multi-genomic profiling defines gene regulatory programs governing human pancreatic β cell identity and function

EndoC-{beta}H1 is emerging as a critical human beta cell model to study the genetic and environmental etiologies of beta cell function, especially in the context of diabetes. Comprehensive knowledge of its molecular landscape is lacking yet required to fully take advantage of this model. Here, we report extensive chromosomal (spectral karyotyping), genetic (genotyping), epigenetic (ChIP-seq, ATAC-seq), chromatin interaction (Hi-C, Pol2 ChIA-PET), and transcriptomic (RNA-seq, miRNA-seq) maps of this cell model. Integrated analyses of these maps define known (e.g., PDX1, ISL1) and putative (e.g., PCSK1, mir-375) beta cell-specific chromatin interactions and transcriptional cis-regulatory networks, and identify allelic effects on cis-regulatory element use and expression.\n\nImportantly, comparative analyses with maps generated in primary human islets/beta cells indicate substantial preservation of chromatin looping, but also highlight chromosomal heterogeneity and fetal genomic signatures in EndoC-{beta}H1. Together, these maps, and an interactive web application we have created for their exploration, provide important tools for the broad community in the design and success of experiments to probe and manipulate the genetic programs governing beta cell identity and (dys)function in diabetes.

genomics↗

bioRxiv2018

The SMAC Mimetic AZD5582 is a Potent HIV Latency Reversing Agent

The leading strategy towards eradication of human immunodeficiency virus (HIV) infection is the depletion of viral reservoirs through reversal of viral latency, followed by clearance of persistently infected cells. To date, a latency reversing agent (LRA) that reactivates a majority of the quiescent provirus population, without significant off-target effects, has not been identified. We show here that molecules mimicking the active N-terminal tetrapeptide of the second mitochondrial-derived activator of caspases (SMACm) potently reverse HIV latency in vitro and ex vivo without the pleotropic cellular effects seen with other LRA. We verified that SMACm facilitate latency reversal through activation of the non-canonical NFkB pathway as exemplified by rapid degradation of cellular inhibitor of apoptosis protein 1 (cIAP1), followed by a slower conversion of the inactive p100 form of NF{kappa}B2 into the active p52 transcription factor. A potent representative of this class, AZD5582, increases cell-associated HIV RNA expression in resting CD4+ T cells from ART-suppressed, HIV-infected donors while altering the expression of a restricted number of human genes. These findings represent the first demonstration that SMACm have single agent latency reversal activity in patient-derived cells and support evaluation of this class of molecules in preclinical animal models.

microbiology↗