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At least 613 records · Page 34

Combinatorial transcription factor binding encodes cis -regulatory wiring of mouse forebrain GABAergic neurogenesis

Transcription factors (TFs) bind combinatorially to cis-regulatory elements, orchestrating transcriptional programs. Although studies of chromatin state and chromosomal interactions have demonstrated dynamic neurodevelopmental cis-regulatory landscapes, parallel understanding of TF interactions lags. To elucidate combinatorial TF binding driving mouse basal ganglia development, we integrated chromatin immunoprecipitation sequencing (ChIP-seq) for twelve TFs, H3K4me3-associated enhancer-promoter interactions, chromatin and gene expression data, and functional enhancer assays. We identified sets of putative regulatory elements with shared TF binding (TF-pRE modules) that orchestrate distinct processes of GABAergic neurogenesis and suppress other cell fates. The majority of pREs were bound by one or two TFs; however, a small proportion were extensively bound. These sequences had exceptional evolutionary conservation and motif density, complex chromosomal interactions, and activity as in vivo enhancers. Our results provide insights into the combinatorial TF-pRE interactions that activate and repress expression programs during telencephalon neurogenesis and demonstrate the value of TF binding toward modeling developmental transcriptional wiring.

59 BASIC BIOLOGICAL SCIENCES↗

Human RNome Project draft human RNome sequence of GM12878, B-cell line, obtained by mass-spectrometry sequencing, long-read sequencing and short-read sequencing.

Here we report the first draft of the human RNome sequence, a reference map of RNA chemical modifications in a human B-cell line. RNA carries a diverse repertoire of chemical modifications that regulate gene expression, cellular function, and responses to physiological and pathological cues. Yet, unlike the genome, no reference map of RNA modifications is available for any human cell. To generate this resource, the Human RNome Project Consortium analyzed a shared RNA preparation from the well-characterized GM12878 B-cell line using short-read sequencing, long-read direct RNA sequencing, and mass spectrometry, generating more than 7.1 billion sequencing reads spanning approximately 1.2 trillion nucleotides. The resulting maps of the human RNome reveal that RNA modifications are organized according to function, transcript architecture, and cellular identity. Modifications concentrate at functional centers of ribosomal and transfer RNAs, follow the canonical topology of N6-methyladenosine in coding transcripts, and form coordinated hotspots in immune regulatory genes. This first reference human RNome provides a foundation for understanding how RNA chemistry shapes cellular identity, human disease, and the development of RNA-based therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗

VISTA Enhancer browser: an updated database of tissue-specific developmental enhancers

Regulatory elements (enhancers) are major drivers of gene expression in mammals and harbor many genetic variants associated with human diseases. Here, we present an updated VISTA Enhancer Browser (https://enhancer.lbl.gov), a database of transgenic enhancer assays conducted in developing mouse embryos in vivo. Since the original publication in 2007, the database grew nearly 20-fold from 250 to over 4500 experiments and currently harbors over 23 500 images. The updated database provides structured information on experiments conducted at different stages of embryonic development, including enhancer activities of human pathogenic and synthetic variants and sequences derived from a variety of species. In addition to manually curated results of thousands of individual experiments, the new database also features hundreds of manually curated comparisons between alleles. The VISTA Enhancer Browser provides a crucial resource for study of human genetic variation, gene regulation and developmental biology.

59 BASIC BIOLOGICAL SCIENCES↗

CRISPR-COPIES: Web Tool

CRISPR/Cas system has emerged as a powerful genome-editing tool for metabolic engineering and human gene therapy. However, the conundrum of where to integrate heterologous genes on the chromosome using the CRISPR/Cas system remains an open question. Selecting a site for gene integration requires incorporation of complex criteria such as factors involved in CRISPR/Cas-mediated integration, genetic stability, and gene expression and therefore, usually requires strenuous characterization of sites on particular or different chromosomal locations. To address these issues, we developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. The tool applies ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. This submission contains the code we developed to create a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate CRISPR-COPIES will serve as a useful tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, rapid strain construction to produce valuable biochemicals, and human gene and cell therapy.

Bioinformatics↗

An optimized ChIP-Seq framework for profiling histone modifications in Chromochloris zofingiensis

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high-quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein cross-linking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

59 BASIC BIOLOGICAL SCIENCES↗

Deletion of Carboxypeptidase E in β-Cells Disrupts Proinsulin Processing but Does Not Lead to Spontaneous Development of Diabetes in Mice

Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic β-cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic β-cell–specific Cpe knockout mice (βCpeKO; Cpe fl/fl x Ins1 Cre/+ ) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in βCpeKO islets remained intact. High-fat diet–fed βCpeKO mice showed weight gain and glucose tolerance comparable with those of Wt littermates. Notably, β-cell area was increased in chow-fed βCpeKO mice and β-cell replication was elevated in βCpeKO islets. Transcriptomic analysis of βCpeKO β-cells revealed elevated glycolysis and Hif1α-target gene expression. On high glucose challenge, β-cells from βCpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin injections, βCpeKO mice had accelerated development of hyperglycemia with reduced β-cell insulin and Glut2 expression. In conclusion, these findings suggest that Cpe and proper proinsulin processing are critical in maintaining β-cell function during the development of hyperglycemia.

59 BASIC BIOLOGICAL SCIENCES↗

Transgenic Poplar Designed for Biofuels

Members of the genus Populus (i.e., cottonwood, hybrid poplar) represent a promising source of lignocellulosic biomass for biofuels. However, one of the major factors negatively affecting poplar’s efficient conversion to biofuel is the inherent recalcitrance to enzymatic saccharification due to cell wall components such as lignin. To this effect, there have been efforts to modify gene expression to reduce biomass recalcitrance by changing cell wall properties. Furthermore, we review recent genetic modifications of poplar that led to change cell wall properties and the resulting effects on subsequent pretreatment efficacy and saccharification. Although genetic engineering’s impacts on cell wall properties are not fully predictable, recent studies have shown promising improvement in the biological conversion of transgenic poplar to biofuels.

09 BIOMASS FUELS↗

Perfect and imperfect views of ultraconserved sequences

Across the human genome, there are nearly 500 'ultraconserved' elements: regions of at least 200 contiguous nucleotides that are perfectly conserved in both the mouse and rat genomes. Remarkably, the majority of these sequences are non-coding, and many can function as enhancers that activate tissue-specific gene expression during embryonic development. From their first description more than 15 years ago, their extreme conservation has both fascinated and perplexed researchers in genomics and evolutionary biology. The intrigue around ultraconserved elements only grew with the observation that they are dispensable for viability. Here, in this paper, we review recent progress towards understanding the general importance and the specific functions of ultraconserved sequences in mammalian development and human disease and discuss possible explanations for their extreme conservation.

59 BASIC BIOLOGICAL SCIENCES↗

Molecular subtyping reveals immune alterations in IDH wild‐type lower‐grade diffuse glioma

Abstract Isocitrate dehydrogenase ( IDH ) wild‐type diffuse lower‐grade glioma (LGG) is usually associated with poor outcome, but there have been disputes over its clinical outcome and classification. We present here a robust gene expression‐based molecular classification of IDH wild‐type diffuse LGG into two subtypes with distinct biological and clinical features. A discovery cohort of 49 IDH wild‐type diffuse LGGs from the Chinese Glioma Genome Atlas (CGGA) was subjected to clustering and function analysis. Seventy‐three tumors from The Cancer Genome Atlas (TCGA) were used to validate our findings. Consensus clustering of transcriptional data uncovered concordant classification of two robust and prognostically significant subtypes of IDH wild‐type LGG. Subtype 1, associated with poorer outcomes, was characterized by significantly higher immune and cytolytic scores, M2 macrophages, and up‐regulation of immune exhaustion markers, while Subtype 2, which had elevated lymphocytes and plasma cells, showed relatively favorable survival. Somatic alteration analysis revealed that Subtype 1 showed more frequently deleted regions, such as the locus of CDKN2A / CDKN2B , DMRTA1 , C9orf53 , and MTAP . Furthermore, we developed and validated a five‐gene signature for better application of this acquired stratification. Our data demonstrate the biological and prognostic heterogeneity within IDH wild‐type diffuse LGGs and deepen our molecular understandi‐g of this tumor entity. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Wu, Fan↗

Serendipita Fungi Modulate the Switchgrass Root Transcriptome to Circumvent Host Defenses and Establish a Symbiotic Relationship

The fungal family Serendipitaceae encompasses root-associated lineages with endophytic, ericoid, orchid, and ectomycorrhizal lifestyles. Switchgrass is an important bioenergy crop for cellulosic ethanol production owing to high biomass production on marginal soils otherwise unfit for food crop cultivation. The aim of this study was to investigate the host plant responses to Serendipita spp. colonization by characterizing the switchgrass root transcriptome during different stages of symbiosis in vitro. For this, we included a native switchgrass strain, Serendipita bescii, and a related strain, S. vermifera, isolated from Australian orchids. Serendipita colonization progresses from thin hyphae that grow between root cells to, finally, the production of large, bulbous hyphae that fill root cells during the later stages of colonization. We report that switchgrass seems to perceive both fungi prior to physical contact, leading to the activation of chemical and structural defense responses and putative host disease resistance genes. Subsequently, the host defense system appears to be quenched and carbohydrate metabolism adjusted, potentially to accommodate the fungal symbiont. In addition, prior to contact, switchgrass exhibited significant increases in root hair density and root surface area. Furthermore, genes involved in phytohormone metabolism such as gibberellin, jasmonic acid, and salicylic acid were activated during different stages of colonization. Both fungal strains induced plant gene expression in a similar manner, indicating a conserved plant response to members of this fungal order. Understanding plant responsiveness to Serendipita spp. will inform our efforts to integrate them into forages and row crops for optimal plant–microbe functioning, thus facilitating low-input, sustainable agricultural practices.

59 BASIC BIOLOGICAL SCIENCES↗

MONTI: A Multi-Omics Non-negative Tensor Decomposition Framework for Gene-Level Integrative Analysis

Multi-omics data is frequently measured to enrich the comprehension of biological mechanisms underlying certain phenotypes. However, due to the complex relations and high dimension of multi-omics data, it is difficult to associate omics features to certain biological traits of interest. For example, the clinically valuable breast cancer subtypes are well-defined at the molecular level, but are poorly classified using gene expression data. Here, we propose a multi-omics analysis method called MONTI (Multi-Omics Non-negative Tensor decomposition for Integrative analysis), which goal is to select multi-omics features that are able to represent trait specific characteristics. Here, we demonstrate the strength of multi-omics integrated analysis in terms of cancer subtyping. The multi-omics data are first integrated in a biologically meaningful manner to form a three dimensional tensor, which is then decomposed using a non-negative tensor decomposition method. From the result, MONTI selects highly informative subtype specific multi-omics features. MONTI was applied to three case studies of 597 breast cancer, 314 colon cancer, and 305 stomach cancer cohorts. For all the case studies, we found that the subtype classification accuracy significantly improved when utilizing all available multi-omics data. MONTI was able to detect subtype specific gene sets that showed to be strongly regulated by certain omics, from which correlation between omics types could be inferred. Furthermore, various clinical attributes of nine cancer types were analyzed using MONTI, which showed that some clinical attributes could be well explained using multi-omics data. We demonstrated that integrating multi-omics data in a gene centric manner improves detecting cancer subtype specific features and other clinical features, which may be used to further understand the molecular characteristics of interest. The software and data used in this study are available at: https://github.com/inukj/MONTI.

59 BASIC BIOLOGICAL SCIENCES↗

CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

Abstract The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

59 BASIC BIOLOGICAL SCIENCES↗

Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo- versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4 cis -elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.

59 BASIC BIOLOGICAL SCIENCES↗

Deconstructing the Soil Microbiome into Reduced-Complexity Functional Modules

The soil microbiome is an invaluable component of the biosphere and critical for ecosystem functions, including biogeochemical cycling, soil-atmosphere gas exchange, degradation of toxic compounds, and promotion of plant growth and stress resistance/resilience. An improved understanding of the soil microbiome will help with predicting how these processes respond to external perturbations, and with harnessing beneficial aspects of the soil microbiome for agronomic applications such as crop amendments. However, the extensive taxonomic and functional diversity inherent within the soil microbiome hinders efficient analysis of this system. Microbial biodiversity in soils is orders of magnitude greater than other commonly studied systems such as the human gut microbiome (Blum, Zechmeister-Boltenstern, and Keiblinger 2019; Berendsen, Pieterse, and Bakker 2012). Thousands of microbial taxa may be found in a single gram of soil (Roesch et al. 2007), and high rates of gene flow and mutation further promote microbial diversification (Sergaki et al. 2018). Concomitantly, functional diversity in soil is similarly extensive. Soil is a heterogeneous mixture of microenvironments with defined physical and chemical attributes (Bach et al. 2018), within which numerous microbial guilds of distinct life-strategies and metabolic capacities can be found (Perez-Garcia, Lear, and Singhal 2016; H.-S. Song et al. 2014). Furthermore, the soil microbiome harbors a significant fraction of rare and/or quiescent members (Blagodatskaya and Kuzyakov 2013) that exist below the threshold of detection of current technologies. Such rare taxa may make significant contributions to process rates (Shade and Gilbert 2015; Dawson et al. 2017), but their scarcity complicates their identification and analysis. Finally, the vast amounts of information generated through holistic analyses of soil communities represent an intense computational burden (Scholz, Lo, and Chain 2012; Prosser 2015) that precludes assessment of the complete functional and taxonomic diversity contained in this ecosystem.

Naylor, Dan T.↗

Molecular basis of crosstalk in nuclear receptors: heterodimerization between PXR and CAR and the implication in gene regulation

The 48 human nuclear receptors (NRs) form a superfamily of transcription factors that regulate major physiological and pathological processes. Emerging evidence suggests that NR crosstalk can fundamentally change our understanding of NR biology, but detailed molecular mechanisms of crosstalk are lacking. Here, we report the molecular basis of crosstalk between the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), where they form a novel heterodimer, resulting in their mutual inhibition. PXR and CAR regulate drug metabolism and energy metabolism. Although they have been broadly perceived as functionally redundant, a growing number of reports suggests a mutual inhibitory relation, but their precise mode of coordinated action remains unknown. Using methods including RNA sequencing, small-angle X-ray scattering and crosslinking mass spectrometry we demonstrate that the mutual inhibition altered gene expression globally and is attributed to the novel PXR–CAR heterodimerization via the same interface used by each receptor to heterodimerize with its functional partner, retinoid X receptor (RXR). These findings establish an unexpected functional relation between PXR, CAR and RXR, change the perceived functional relation between PXR and CAR, open new perspectives on elucidating their role and designing approaches to regulate them, and highlight the importance to comprehensively investigate nuclear receptor crosstalk.

59 BASIC BIOLOGICAL SCIENCES↗

Temporal change in chromatin accessibility predicts regulators of nodulation in Medicago truncatula

Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules. We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

59 BASIC BIOLOGICAL SCIENCES↗

Building a FAIR data ecosystem for incorporating single-cell transcriptomics data into agricultural genome to phenome research

Introduction The agriculture genomics community has numerous data submission standards available, but the standards for describing and storing single-cell (SC, e.g., scRNA- seq) data are comparatively underdeveloped. Methods To bridge this gap, we leveraged recent advancements in human genomics infrastructure, such as the integration of the Human Cell Atlas Data Portal with Terra, a secure, scalable, open-source platform for biomedical researchers to access data, run analysis tools, and collaborate. In parallel, the Single Cell Expression Atlas at EMBL-EBI offers a comprehensive data ingestion portal for high-throughput sequencing datasets, including plants, protists, and animals (including humans). Developing data tools connecting these resources would offer significant advantages to the agricultural genomics community. The FAANG data portal at EMBL-EBI emphasizes delivering rich metadata and highly accurate and reliable annotation of farmed animals but is not computationally linked to either of these resources. Results Herein, we describe a pilot-scale project that determines whether the current FAANG metadata standards for livestock can be used to ingest scRNA-seq datasets into Terra in a manner consistent with HCA Data Portal standards. Importantly, rich scRNA-seq metadata can now be brokered through the FAANG data portal using a semi-automated process, thereby avoiding the need for substantial expert curation. We have further extended the functionality of this tool so that validated and ingested SC files within the HCA Data Portal are transferred to Terra for further analysis. In addition, we verified data ingestion into Terra, hosted on Azure, and demonstrated the use of a workflow to analyze the first ingested porcine scRNA-seq dataset. Additionally, we have also developed prototype tools to visualize the output of scRNA-seq analyses on genome browsers to compare gene expression patterns across tissues and cell populations. This JBrowse tool now features distinct tracks, showcasing PBMC scRNA-seq alongside two bulk RNA-seq experiments. Discussion We intend to further build upon these existing tools to construct a scientist-friendly data resource and analytical ecosystem based on Findable, Accessible, Interoperable, and Reusable (FAIR) SC principles to facilitate SC-level genomic analysis through data ingestion, storage, retrieval, re-use, visualization, and comparative annotation across agricultural species.

Genetics & Heredity↗

Single-cell RNA-seq revealed diverse cell types in the mouse placenta at mid-gestation

The mammalian placenta consists of a set of cells to ensure normal placental functions throughout gestation. Dysfunctional placentae are considered as the origin of a series of pregnancy complications. Therefore, it is urgent for detailed information about the molecular recipes of the cell types within the normal placenta. In the past years, gene expression analysis via single-cell RNA-seq (scRNA-seq) offers opportunities to identify new cell types in a variety of organs and tissues. In this study, scRNA-seq was used to explore the cell heterogeneity within the E10.5 mouse placenta and unravel their discrepancies in cell composition and communications. We identified sixteen cell clusters, including some cell clusters that originated from the maternal tissue. Moreover, we traced the developmental trajectories of trophoblasts and Hofbauer-like cells. Further analysis revealed cell connections between the endothelial cells and pericytes, syncytiotrophoblasts, as well as decidual cells. Besides, we highlighted several signaling pathways, such as the EGF, FGF, canonical, and non-canonical WNT signaling pathways, which mediated the potential crosstalk between different cell types within placenta. Our research provides an in-depth understanding of placental development, cellular composition, and communications at the maternal-fetal interface.

60 APPLIED LIFE SCIENCES↗