OperomeDB: database of condition specific transcription in prokaryotic genomes and genomic insights of convergent transcription in bacterial genomes

Indiana University-Purdue University Indianapolis (IUPUI)My thesis comprises of two individual projects: 1) we have developed a database for operon prediction using high-throughput sequencing datasets for bacterial genomes. 2) Genomics and mechanistic insights of convergent transcription in bacterial genomes. In the first project we developed a database for the prediction of operons for bacterial genomes using RNA-seq datasets, we predicted operons for bacterial genomes. RNA-seq datasets with different condition for each bacterial genome were taken into account and predicted operons using Rockhopper. We took RNA-seq datasets from NCBI with distinct experimental conditions for each bacterial genome into account and analyzed using tool for operon prediction. Currently our database contains 9 bacterial organisms for which we predicted operons. User interface is simple and easy to use, in terms of visualization, downloading and querying of data. In our database user can browse through reference genome, genes present in that genome and operons predicted from different RNA-seq datasets. Further in the second project, we studied the genomic and mechanistic insights of convergent transcription in bacterial genomes. We know that convergent gene pairs with overlapping head-to-head configuration are widely spread across both eukaryotic and prokaryotic genomes. They are believed to contribute to the regulation of genes at both transcriptional and post-transcriptional levels, although factors contributing to their abundance across genomes and mechanistic basis for their prevalence are poorly understood. In this study, we explore the role of various factors contributing to convergent overlapping transcription in bacterial genomes. Our analysis shows that the proportion of convergent overlapping gene pairs (COGPs) in a genome is affected due to endospore formation, bacterial habitat, oxygen requirement, GC content and the temperature range. In particular, we show that bacterial genomes thriving in specialized habitats, such as thermophiles, exhibit a high proportion of COGPs. Our results also conclude that the density distribution of COGPs across the genomes is high for shorter overlaps with increased conservation of distances for decreasing overlaps. Our study further reveals that COGPs frequently contain stop codon overlaps with the middle base position exhibiting mismatches between complementary strands. Further, for the functional analysis using cluster of orthologous groups (COGs) annotations suggested that cell motility, cell metabolism, storage and cell signaling are enriched among COGPs, suggesting their role in processes beyond regulation. Our analysis provides genomic insights into this unappreciated regulatory phenomenon, allowing a refined understanding of their contribution to bacterial phenotypes

Genomic and mechanistic insights of convergent transcription in bacterial genomes

Digitized for IUPUI ScholarWorks inclusion in 2021.Convergent gene pairs with overlapping head-to-head configuration are widely spread across both eukaryotic and prokaryotic genomes. They are believed to contribute to the regulation of genes at both transcriptional and post-transcriptional levels, although the factors contributing to their abundance across genomes and mechanistic basis for their prevalence are poorly understood. In this study, we explore the role of various factors contributing to convergent overlapping transcription in bacterial genomes. Our analysis shows that the proportion of convergent overlapping gene pairs (COGPs) in a genome is affected by endospore formation, bacterial habitat and the temperature range. In particular, we show that bacterial genomes thriving in specialized habitats such as thermophiles exhibit a high proportion of COGPs. Our results also show that the density distribution of COGPs across the genomes is high for shorter overlaps with increased conservation of distances for decreasing overlaps. Our study also reveals that COGPs frequently contain stop codon overlaps with the middle base exhibiting mismatches between complementary strands. Functional analysis using COGs (Cluster of Orthologous groups) annotations suggested that cell motility, cell metabolism, storage, and cell signaling are enriched among COGPs suggesting their role in processes beyond regulation. Our analysis provides genomic insights into this unappreciated regulatory phenomenon, allowing a refined understanding of their contribution to bacterial phenotypes

OperomeDB: A Database of Condition-Specific Transcription Units in Prokaryotic Genomes

Background. In prokaryotic organisms, a substantial fraction of adjacent genes are organized into operons—codirectionally organized genes in prokaryotic genomes with the presence of a common promoter and terminator. Although several available operon databases provide information with varying levels of reliability, very few resources provide experimentally supported results. Therefore, we believe that the biological community could benefit from having a new operon prediction database with operons predicted using next-generation RNA-seq datasets. Description. We present operomeDB, a database which provides an ensemble of all the predicted operons for bacterial genomes using available RNA-sequencing datasets across a wide range of experimental conditions. Although several studies have recently confirmed that prokaryotic operon structure is dynamic with significant alterations across environmental and experimental conditions, there are no comprehensive databases for studying such variations across prokaryotic transcriptomes. Currently our database contains nine bacterial organisms and 168 transcriptomes for which we predicted operons. User interface is simple and easy to use, in terms of visualization, downloading, and querying of data. In addition, because of its ability to load custom datasets, users can also compare their datasets with publicly available transcriptomic data of an organism. Conclusion. OperomeDB as a database should not only aid experimental groups working on transcriptome analysis of specific organisms but also enable studies related to computational and comparative operomics

Cxcr3-expressing leukocytes are necessary for neurofibroma formation in mice.

Plexiform neurofibroma is a major contributor to morbidity in patients with neurofibromatosis type I (NF1). Macrophages and mast cells infiltrate neurofibroma, and data from mouse models implicate these leukocytes in neurofibroma development. Antiinflammatory therapy targeting these cell populations has been suggested as a means to prevent neurofibroma development. Here, we compare gene expression in Nf1-mutant nerves, which invariably form neurofibroma, and show disruption of neuron-glial cell interactions and immune cell infiltration to mouse models, which rarely progresses to neurofibroma with or without disruption of neuron-glial cell interactions. We find that the chemokine Cxcl10 is uniquely upregulated in NF1 mice that invariably develop neurofibroma. Global deletion of the CXCL10 receptor Cxcr3 prevented neurofibroma development in these neurofibroma-prone mice, and an anti-Cxcr3 antibody somewhat reduced tumor numbers. Cxcr3 expression localized to T cells and DCs in both inflamed nerves and neurofibromas, and Cxcr3 expression was necessary to sustain elevated macrophage numbers in Nf1-mutant nerves. To our knowledge, these data support a heretofore-unappreciated role for T cells and DCs in neurofibroma initiation

Maturation of heart valve cell populations during postnatal remodeling.

peer reviewedHeart valve cells mediate extracellular matrix (ECM) remodeling during postnatal valve leaflet stratification, but phenotypic and transcriptional diversity of valve cells in development is largely unknown. Single cell analysis of mouse heart valve cells was used to evaluate cell heterogeneity during postnatal ECM remodeling and leaflet morphogenesis. The transcriptomic analysis of single cells from postnatal day (P)7 and P30 murine aortic (AoV) and mitral (MV) heart valves uncovered distinct subsets of melanocytes, immune and endothelial cells present at P7 and P30. By contrast, interstitial cell populations are different from P7 to P30. P7 valve leaflets exhibit two distinct collagen- and glycosaminoglycan-expressing interstitial cell clusters, and prevalent ECM gene expression. At P30, four interstitial cell clusters are apparent with leaflet specificity and differential expression of complement factors, ECM proteins and osteogenic genes. This initial transcriptomic analysis of postnatal heart valves at single cell resolution demonstrates that subpopulations of endothelial and immune cells are relatively constant throughout postnatal development, but interstitial cell subpopulations undergo changes in gene expression and cellular functions in primordial and mature valves

Publisher Correction: MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Mast cell deficiency improves cognition and enhances disease-associated microglia in 5XFAD mice

Summary: Emerging evidence suggests that peripheral immune cells contribute to Alzheimer’s disease (AD) neuropathogenesis. Among these, mast cells are known for their functions in allergic reactions and neuroinflammation; however, little is known about their role in AD. Here, we crossed 5XFAD mice with mast cell-deficient strains and observed the effects on AD-related neuropathology and cognitive impairment. We found that mast cell depletion improved contextual fear conditioning in 5XFAD mice without affecting cued fear conditioning, anxiety-like behavior, or amyloid burden. Furthermore, mast cell depletion led to an upregulation of transcriptomic signatures for putatively protective disease-associated microglia and resulted in reduced markers indicative of reactive astrocytes. We hypothesize a system of bidirectional communication between dural mast cells and the brain, where mast cells respond to signals from the brain environment by expressing immune-regulatory mediators, impacting cognition and glial cell function. These findings highlight mast cells as potential therapeutic targets for AD

Fetal maturation revealed by amniotic fluid cell-free transcriptome in rhesus macaques

Accurate estimate of fetal maturity could provide individualized guidance for delivery of complicated pregnancies. However, current methods are invasive, have low accuracy, and are limited to fetal lung maturation. To identify diagnostic gestational biomarkers, we performed transcriptomic profiling of lung and brain, as well as cell-free RNA from amniotic fluid of preterm and term rhesus macaque fetuses. These data identify potentially new and prior-associated gestational age differences in distinct lung and neuronal cell populations when compared with existing single-cell and bulk RNA-Seq data. Comparative analyses found hundreds of genes coincidently induced in lung and amniotic fluid, along with dozens in brain and amniotic fluid. These data enable creation of computational models that accurately predict lung compliance from amniotic fluid and lung transcriptome of preterm fetuses treated with antenatal corticosteroids. Importantly, antenatal steroids induced off-target gene expression changes in the brain, impinging upon synaptic transmission and neuronal and glial maturation, as this could have long-term consequences on brain development. Cell-free RNA in amniotic fluid may provide a substrate of global fetal maturation markers for personalized management of at-risk pregnancies

TRAF6 Mediates Basal Activation of NF-κB Necessary for Hematopoietic Stem Cell Homeostasis

Summary: Basal nuclear factor κB (NF-κB) activation is required for hematopoietic stem cell (HSC) homeostasis in the absence of inflammation; however, the upstream mediators of basal NF-κB signaling are less well understood. Here, we describe TRAF6 as an essential regulator of HSC homeostasis through basal activation of NF-κB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient hematopoietic stem/progenitor cells (HSPCs) revealed changes in adaptive immune signaling, innate immune signaling, and NF-κB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection is required for HSC function. In addition, we established that loss of IκB kinase beta (IKKβ)-mediated NF-κB activation is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKβ similarly resulted in impaired HSC self-renewal and fitness. Taken together, TRAF6 is required for HSC homeostasis by maintaining a minimal threshold level of IKKβ/NF-κB signaling. : Fang et al. identify TRAF6 as an essential regulator of hematopoietic stem cell (HSC) self-renewal and quiescence. TRAF6 preserves HSC homeostasis by maintaining a minimal threshold level of NF-κB signaling in the absence of inflammation. Keywords: TRAF6, hematopoietic stem cell, NF-kB, innate immune signaling, toll-like receptor, IKKbet