Global Gene Expression Patterns Research Articles

Non-invasive evaluation of the equine gastrointestinal mucosal transcriptome.

Evaluating the health and function of the gastrointestinal tract can be challenging in all species, but is especially difficult in horses due to their size and length of the gastrointestinal (GI) tract. Isolation of mRNA of cells exfoliated from the GI mucosa into feces (i.e., the exfoliome) offers a novel means of non-invasively examining the gene expression profile of the GI mucosa. This approach has been utilized in people with colorectal cancer. Moreover, we have utilized this approach in a murine model of GI inflammation and demonstrated that the exfoliome reflects the tissue transcriptome. The ability of the equine exfoliome to provide non-invasive information regarding the health and function of the GI tract is not known. The objective of this study was to characterize the gene expression profile found in exfoliated intestinal epithelial cells from normal horses and compare the exfoliome data with the tissue mucosal transcriptome. Mucosal samples were collected from standardized locations along the GI tract (i.e. ileum, cecum, right dorsal colon, and rectum) from four healthy horses immediately following euthanasia. Voided feces were also collected. RNA isolation, library preparation, and RNA sequencing was performed on fecal and intestinal mucosal samples. Comparison of gene expression profiles from the tissue and exfoliome revealed correlation of gene expression. Moreover, the exfoliome contained reads representing the diverse array of cell types found in the GI mucosa suggesting the equine exfoliome serves as a non-invasive means of examining the global gene expression pattern of the equine GI tract.

Abstract A32: Characterizing patterns of cytokine coexpression with immune checkpoint markers in CD4 and CD8 tumor-infiltrating lymphocytes

Abstract In recent years, immunotherapy has become one of the most exciting and promising avenues to cancer treatment. Treatment with immune checkpoint inhibitors has managed to produce long-term remission of solid tumors in many patients. However, patients who respond well to such treatment are often a minority; this is particularly the case with some cancers such as renal cell carcinoma, non-small cell lung cancer, and glioblastoma, where many patients either derive no benefit or only a short-term benefit. In this analysis, we examined gene expression data from RNA sequencing experiments that compared tumor-infiltrating lymphocytes (TIL) with paired circulating lymphocytes from patients with renal cell carcinoma (RCC), bladder cancer (BLCA), prostate cancer (PRAD), and glioblastoma (GBM). Our analysis helped to characterize global CD4 and CD8 TIL gene expression patterns among these four cohorts. Further, using the expression profiles for known immune checkpoint markers PD-1, TIM-3, and LAG-3 in CD8 cells, we dichotomized the patient samples into potential checkpoint inhibitor responder and nonresponder groups. This model was then used to identify other genes that are associated with CD8 TIL exhaustion, which may lead to the identification of cytokines useful in discovering specific therapeutic targets. Citation Format: Christopher M. Jackson, Wikum Dinalankara, John Choi, Thomas R. Nirschl, Christina M. Kochel, Ayush Pant, Denis Routkevitch, Laura Saleh, Christina Jackson, Alyza M. Skaist, Anuj Gupta, Linda A. Snyder, Edward M. Schaeffer, Ashley E. Ross, Ballentine Carter, Mohamad E. Allaf, Trinity J. Bivalacqua, Angelo M. DeMarzo, Jon D. Weingart, Chetan Bettegowda, Henry Brem, Drew M. Pardoll, Luigi Marchionni, Charles G. Drake, Michael Lim. Characterizing patterns of cytokine coexpression with immune checkpoint markers in CD4 and CD8 tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A32.

Probing Carbon Utilization of Cordyceps militaris by Sugar Transportome and Protein Structural Analysis.

Beyond comparative genomics, we identified 85 sugar transporter genes in Cordyceps militaris, clustering into nine subfamilies as sequence- and phylogenetic-based functional classification, presuming the versatile capability of the fungal growths on a range of sugars. Further analysis of the global gene expression patterns of C. militaris showed 123 genes were significantly expressed across the sucrose, glucose, and xylose cultures. The sugar transporters specific for pentose were then identified by gene-set enrichment analysis. Of them, the putative pentose transporter, CCM_06358 gene, was highest expressed in the xylose culture, and its functional role in xylose transport was discovered by the analysis of conserved structural motifs. In addition, a battery of molecular modeling methods, including homology modeling, transport pathway analysis, residue interaction network combined with molecular mechanics Poisson–Boltzmann surface area simulation (MM-PBSA), was implemented for probing the structure and function of the selected pentose transporter (CCM_06358) as a representative of sugar transportome in C. militaris. Considering the network bottlenecks and structural organizations, we further identified key amino acids (Phe38 and Trp441) and their interactions with other residues, contributing the xylose transport function, as verified by binding free energy calculation. The strategy used herein generated remarkably valuable biological information, which is applicable for the study of sugar transportome and the structure engineering of targeted transporter proteins that might link to the production of bioactive compounds derived from xylose metabolism, such as cordycepin.

Molecular analysis of lymphoid tissue from rhesus macaque rhadinovirus-infected monkeys identifies alterations in host genes associated with oncogenesis.

Rhesus macaque (RM) rhadinovirus (RRV) is a simian gamma-2 herpesvirus closely related to human Kaposi’s sarcoma-associated herpesvirus (KSHV). RRV is associated with the development of diseases in simian immunodeficiency virus (SIV) co-infected RM that resemble KSHV-associated pathologies observed in HIV-infected humans, including B cell lymphoproliferative disorders (LPD) and lymphoma. Importantly, how de novo KSHV infection affects the expression of host genes in humans, and how these alterations in gene expression affect viral replication, latency, and disease is unknown. The utility of the RRV/RM infection model provides a novel approach to address these questions in vivo, and utilizing the RRV bacterial artificial chromosome (BAC) system, the effects of specific viral genes on host gene expression patterns can also be explored. To gain insight into the effects of RRV infection on global host gene expression patterns in vivo, and to simultaneously assess the contributions of the immune inhibitory viral CD200 (vCD200) molecule to host gene regulation, RNA-seq was performed on pre- and post-infection lymph node (LN) biopsy samples from RM infected with either BAC-derived WT (n = 4) or vCD200 mutant RRV (n = 4). A variety of genes were identified as being altered in LN tissue samples due to RRV infection, including cancer-associated genes activation-induced cytidine deaminase (AICDA), glypican-1 (GPC1), CX3C chemokine receptor 1 (CX3CR1), and Ras dexamethasone-induced 1 (RasD1). Further analyses also indicate that GPC1 may be associated with lymphomagenesis. Finally, comparison of infection groups identified the differential expression of host gene thioredoxin interacting protein (TXNIP), suggesting a possible mechanism by which vCD200 negatively affects RRV viral loads in vivo.

Influence of Micropillar Induced Deformation on Chromatin Architecture in Regulating Stem Cell Differentiation

Surface topography induced nuclear deformation in stem cells has previously shown to influence differentiation outcomes. Mechanical forces resulting from topographical interventions get transduced into the nucleus through the cytoskeleton, causing changes in chromatin architecture and gene expression. However, their effect on genome architecture and gene expression in regulating stem cell differentiation is not well understood. Here, we study the influence of nuclear deformation induced by micropillar surfaces composed of methacrylated poly (1,8-octamethylene citrate) on chromatin architecture and gene expression during human mesenchymal stem cell differentiation (hMSC) towards osteogenic and adipogenic lineages. Differentiation assays reveal that these micropillars facilitate osteogenic differentiation, and attenuate adipogenic differentiation. It has been shown that changes in chromatin nanoarchitecture regulate global gene expression patterns and that an increase in chromatin packing scaling corresponds to an increase in the dynamic range and variations in gene expression. We hypothesized that micropillars enhance osteogenic differentiation by limiting the transcriptional accessibility of the initially plastic stem cells to explore their genomic landscape, primarily, by modulating their chromatin packing scaling. Using Partial Wave Spectroscopic (PWS) microscopy, we show that hMSCs cultured on micropillars have decreased chromatin packing scaling compared to those cultured on flat surfaces. Moreover, corresponding RNA-Seq studies show significant downregulation of genes associated with cell proliferation, and, of those involved in inhibition of osteogenic differentiation in MSCs seeded on micropillars compared to the flat surface. Therefore, by combining nanoscale-imaging experiments involving PWS and Electron Microscopy with enhanced chromatin contrast (ChromEM), and sequencing performed on stem cells induced with osteogenic and adipogenic medium, this study aims to understand the relationship between nuclear deformation induced by micropillars, chromatin architecture, and expression of differentiation-specific transcriptional programs.

Single Amino Acid Substitutions in the Cucumber Mosaic Virus 1a Protein Induce Necrotic Cell Death in Virus-Inoculated Leaves without Affecting Virus Multiplication.

When Arabidopsis thaliana ecotype Col-0 was inoculated with a series of reassortant viruses created by exchanging viral genomic RNAs between two strains of cucumber mosaic virus (CMV), CMV(Y), and CMV(H), cell death developed in the leaves inoculated with reassortant CMV carrying CMV(H) RNA1 encoding 1a protein, but not in noninoculated upper leaves. In general, cell death in virus-infected plants is a critical event for virus survival because virus multiplication is completely dependent on host cell metabolism. However, interestingly, this observed cell death did not affect either virus multiplication in the inoculated leaves or systemic spread to noninoculated upper leaves. Furthermore, the global gene expression pattern of the reassortant CMV-inoculated leaves undergoing cell death was clearly different from that in hypersensitive response (HR) cell death, which is coupled with resistance to CMV. These results indicated that the observed cell death does not appear to be HR cell death but rather necrotic cell death unrelated to CMV resistance. Interestingly, induction of this necrotic cell death depended on single amino acid substitutions in the N-terminal region surrounding the methyltransferase domain of the 1a protein. Thus, development of necrotic cell death might not be induced by non-specific damage as a result of virus multiplication, but by a virus protein-associated mechanism. The finding of CMV 1a protein-mediated induction of necrotic cell death in A. thaliana, which is not associated with virus resistance and HR cell death, has the potential to provide a new pathosystem to study the role of cell death in virus–host plant interactions.

Comparative Transcriptomic and Functional Assessments of Linezolid-Responsive Small RNA Genes in Staphylococcus aureus.

Staphylococcus aureus contains a repertoire of at least 50 and possibly 500 small RNAs (sRNAs). The functions of most sRNAs are not understood, although some are known to respond to environmental changes, including the presence of antibiotics. Here, in an effort to better understand the roles of sRNAs in the context of antibiotic exposure, we took a clinical methicillin-resistant S. aureus (MRSA) isolate and separately deleted eight sRNAs that were significantly upregulated in response to the last-line antibiotic linezolid as revealed by transcriptome sequencing (RNA-seq) comparisons. We also deleted an additional 10 sRNAs that were either highly expressed or previously found to respond to antibiotic exposure. There were no significant changes for any of the 18 mutants in a variety of phenotypic screens, including MIC screens, growth competition assays in the presence of linezolid, biofilm formation, and resistance to whole-blood killing. These data suggest sRNA functional redundancy, because despite their high expression levels upon antibiotic exposure, individual sRNA genes do not affect readily observable bacterial phenotypes. The sRNA transcriptional changes we measured during antibiotic exposure might also reflect sRNA "indifference," that is, a general stress response not specifically related to sRNA function. These data underscore the need for sensitive assays and new approaches to try and decipher the functions of sRNA genes in S. aureus IMPORTANCE Bacterial small RNAs (sRNAs) are RNA molecules that can have important regulatory roles across gene expression networks. There is a growing understanding of the scope and potential breadth of impact of sRNAs on global gene expression patterns in Staphylococcus aureus, a major human pathogen. Here, transcriptome comparisons were used to examine the roles of sRNA genes with a potential role in the response of S. aureus to antibiotic exposure. Although no measurable impact on key bacterial phenotypes was observed after deleting each of 18 sRNAs identified by these comparisons, this research is significant because it underscores the subtle modes of action of these sometimes abundant molecules within the bacterium.

Transcriptomic response to elevated water temperatures in adult migrating Yukon River Chinook salmon (Oncorhynchus tshawytscha).

Chinook salmon (Oncorhynchus tshawytscha) declines are widespread and may be attributed, at least in part, to warming river temperatures. Water temperatures in the Yukon River and tributaries often exceed 18°C, a threshold commonly associated with heat stress and elevated mortality in Pacific salmon. Untangling the complex web of direct and indirect physiological effects of heat stress on salmon is difficult in a natural setting with innumerable system challenges but is necessary to increase our understanding of both lethal and sublethal impacts of heat stress on populations. The goal of this study was to characterize the cellular stress response in multiple Chinook salmon tissues after acute elevated temperature challenges. We conducted a controlled 4-hour temperature exposure (control, 18°C and 21°C) experiment on the bank of the Yukon River followed by gene expression (GE) profiling using a 3'-Tag-RNA-Seq protocol. The full transcriptome was analysed for 22 Chinook salmon in muscle, gill and liver tissue. Both the 21°C and 18°C treatments induced greater activity in genes associated with protein folding (e.g. HSP70, HSP90 mRNA) processes in all tissues. Global GE patterns indicate that transcriptomic responses to heat stress were highly tissue-specific, underscoring the importance of analyzing multiple tissues for determination of physiological effect. Primary superclusters (i.e. groupings of loosely related terms) of altered biological processes were identified in each tissue type, including regulation of DNA damage response (gill), regulation by host of viral transcription (liver) and regulation of the force of heart contraction (muscle) in the 21°C treatment. This study provides insight into mechanisms potentially affecting adult Chinook salmon as they encounter warm water during their spawning migration in the Yukon River and suggests that both basic and more specialized cellular functions may be disrupted.

Comparison of visceral adipose tissue DNA methylation and gene expression profiles in female adolescents with obesity

BackgroundEpigenetic changes in visceral adipose tissue (VAT) with obesity and their effects on gene expression are poorly understood, especially during emergent obesity in youth. The current study tested the hypothesis that methylation and gene expression profiles of key growth factor and inflammatory pathways are altered in VAT from obese compared to non-obese youth.MethodsVAT samples from adolescent females grouped as Lean (L; n = 15; age = 15 ± 3 years, BMI = 21.9 ± 3.0 kg/m2) or Obese (Ob; n = 15, age = 16 ± 2 years, BMI = 45.8 ± 9.8 kg/m2) were collected. Global methylation (n = 20) and gene expression (N = 30) patterns were profiled via microarray and interrogated for differences between groups by ANCOVA (p < 0.05), followed by biological pathway analyses.ResultsOverlapping differences in methylation and gene expression in 317 genes were found in VAT from obese compared to lean groups. PI3K/AKT Signaling (p = 1.83 × 10−6; 11/121 molecules in dataset/pathway) was significantly overrepresented in Ob VAT according to pathway analysis. Upregulations in the PI3K/AKT signaling pathway mRNAs TFAM (p = 0.03; fold change = 1.8) and PPP2R5C (p = 0.03, FC = 2.6) were confirmed via qRT-PCR.ConclusionOur analyses show obesity-related differences in DNA methylation and gene expression in visceral adipose tissue of adolescent females. Specifically, we identified methylation site/gene expression pairs differentially regulated and mapped these differences to pathways including PI3K/AKT signaling, suggesting that PI3K/AKT signaling pathway dysfunction in obesity may be driven in part by changes in DNA methylation.

Global Gene Expression and Mutation Signatures Are Preserved in PDX Models of Pediatric AML and Aid Discovery of Targeted Therapy for Cases with CBFA2T3/GLIS2 Rearrangement

Therapies for pediatric acute myeloid leukemia (AML) remain unsatisfactory and generally do not incorporate molecularly-targeted agents aside from FLT3 inhibitors outside of the relapse setting. Patient-derived xenograft (PDX) models of AML are increasingly accessible for the preclinical evaluation of targeted therapies, though the degree to which these systems recapitulate the disease state as found in patients has not been well defined for AML. Gene expression profiling of patient blasts has been successfully used to discriminate distinct subtypes of AML, to uncover sub-type specific vulnerabilities, and to predict response to therapy and outcomes. We sought to systematically examine PDX models of pediatric AML for their ability to replicate global gene expression patterns and preserve mutational signatures found in patients. In addition, we conducted in-depth bioinformatic analyses of samples with cryptic CBA2T3-GLIS2 fusion generated by the inv(16)(p13.3q24.3) for identification of potential novel targeted therapies. We performed detailed analyses of RNA sequencing data from a diverse series of 24 pediatric AML PDX models established from samples obtained from patients with relapse and refractory disease. Initially we compared our PDX data against 49 selected relapse and refractory patient sample data files found in the NCI TARGET dataset of pediatric AML. When applying unsupervised hierarchical clustering to the PDX samples, we found that clustering was associated with MLL status. Clustering of the combined sets of samples by MLL status showed integration of samples according to mutation profile, regardless of data source (PDX or patient). The expression levels of all detectable transcripts were highly conserved between PDX and patient MLL-r samples. Separate analysis of each dataset yielded MLL specific gene lists that included a subset of overlapping genes which may point to a unique relapse and refractory pediatric MLL-r signature. This list contains several interesting new targets for further study. A subset of 12 PDX models were compared directly to the matched patient sample from which they were established. This analysis revealed strong similarity, with each PDX most closely related to its matched patient sample, suggesting retention of sample-specific gene expression in immune deficient mice. We set up our PDX models in NSG mice with transgenic expression of human myelo-supportive cytokines SCF, GM-CSF, and IL-3 in order to promote the most efficient and robust engraftment of precious patient material. In order to detect any skewing effects due to the host mouse strain, we compared NSGS PDX RNA sequencing data to 10 matched NSG PDX models. This comparison revealed consistent differences in only 9 transcripts, which were almost entirely related to increased JAK/STAT signaling and macrophage activation pathways in NSGS mice relative to NSG mice. Interestingly, during this analysis we observed a distinct PCA-driven clustering of a pair of PDX samples with previously clinically unidentified driver mutations. Reanalysis of the RNA sequencing data revealed evidence of a cryptic GLIS2 rearrangement (found in ~1% of pediatric AML cases) as the driver mutation, which was subsequently confirmed by RT-PCR in both samples. The unique CBFA2T3/GLIS2 RNA signature was mined to guide the composition of a focused 75-molecule in vitro drug screen against ex vivo PDX samples with an emphasis on the SHH, WNT, and BCL2 pathways. This screen identified the Wnt-C59 PORCN inhibitor as having specific activity against CBFA2T3/GLIS2+ AMLs. Further testing of C-59 in combinatorial studies revealed enhanced effects with the addition of the BCL2 inhibitor, venetoclax. In vivo experiments are currently underway to determine the pre-clinical efficacy of this novel combination. In summary, we found highly significant fidelity of gene expression in PDX models of relapse and refractory pediatric AML. Analysis of this dataset has led to several insights, including potential targeted therapies, highlighting how this system could be a valuable tool for discovery of novel targeted therapies, especially for very rare, distinct subtypes of disease. Disclosures Perentesis: Kurome Therapeutics: Consultancy.

Transcriptome-Enabled Network Inference Revealed the GmCOL1 Feed-Forward Loop and Its Roles in Photoperiodic Flowering of Soybean.

Photoperiodic flowering, a plant response to seasonal photoperiod changes in the control of reproductive transition, is an important agronomic trait that has been a central target of crop domestication and modern breeding programs. However, our understanding about the molecular mechanisms of photoperiodic flowering regulation in crop species is lagging behind. To better understand the regulatory gene networks controlling photoperiodic flowering of soybeans, we elucidated global gene expression patterns under different photoperiod regimes using the near isogenic lines (NILs) of maturity loci (E loci). Transcriptome signatures identified the unique roles of the E loci in photoperiodic flowering and a set of genes controlled by these loci. To elucidate the regulatory gene networks underlying photoperiodic flowering regulation, we developed the network inference algorithmic package CausNet that integrates sparse linear regression and Granger causality heuristics, with Gaussian approximation of bootstrapping to provide reliability scores for predicted regulatory interactions. Using the transcriptome data, CausNet inferred regulatory interactions among soybean flowering genes. Published reports in the literature provided empirical verification for several of CausNet's inferred regulatory interactions. We further confirmed the inferred regulatory roles of the flowering suppressors GmCOL1a and GmCOL1b using GmCOL1 RNAi transgenic soybean plants. Combinations of the alleles of GmCOL1 and the major maturity locus E1 demonstrated positive interaction between these genes, leading to enhanced suppression of flowering transition. Our work provides novel insights and testable hypotheses in the complex molecular mechanisms of photoperiodic flowering control in soybean and lays a framework for de novo prediction of biological networks controlling important agronomic traits in crops.

Systems-level network modeling of Small Cell Lung Cancer subtypes identifies master regulators and destabilizers.

Adopting a systems approach, we devise a general workflow to define actionable subtypes in human cancers. Applied to small cell lung cancer (SCLC), the workflow identifies four subtypes based on global gene expression patterns and ontologies. Three correspond to known subtypes (SCLC-A, SCLC-N, and SCLC-Y), while the fourth is a previously undescribed ASCL1+ neuroendocrine variant (NEv2, or SCLC-A2). Tumor deconvolution with subtype gene signatures shows that all of the subtypes are detectable in varying proportions in human and mouse tumors. To understand how multiple stable subtypes can arise within a tumor, we infer a network of transcription factors and develop BooleaBayes, a minimally-constrained Boolean rule-fitting approach. In silico perturbations of the network identify master regulators and destabilizers of its attractors. Specific to NEv2, BooleaBayes predicts ELF3 and NR0B1 as master regulators of the subtype, and TCF3 as a master destabilizer. Since the four subtypes exhibit differential drug sensitivity, with NEv2 consistently least sensitive, these findings may lead to actionable therapeutic strategies that consider SCLC intratumoral heterogeneity. Our systems-level approach should generalize to other cancer types.

Microbe-host interplay in atopic dermatitis and psoriasis

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

Comprehensive Transcriptomic Analysis of Mouse Gonadal Development Involving Sexual Differentiation, Meiosis and Gametogenesis

BackgroundMammalian gonadal development is crucial for fertility. Sexual differentiation, meiosis and gametogenesis are critical events in the process of gonadal development. Abnormalities in any of these events may cause infertility. However, owing to the complexity of these developmental events, the underlying molecular mechanisms are not fully understood and require further research.ResultsIn this study, we employed RNA sequencing to examine transcriptome profiles of murine female and male gonads at crucial stages of these developmental events. By bioinformatics analysis, we identified a group of candidate genes that may participate in sexual differentiation, including Erbb3, Erbb4, and Prkg2. One hundred and two and 134 candidate genes that may be important for female and male gonadal development, respectively, were screened by analyzing the global gene expression patterns of developing female and male gonads. Weighted gene co-expression network analysis was performed on developing female gonads, and we identified a gene co-expression module related to meiosis. By alternative splicing analysis, we found that cassette-type exon and alternative start sites were the main forms of alternative splicing in developing gonads. A considerable portion of differentially expressed and alternatively spliced genes were involved in meiosis.ConclusionTaken together, our findings have enriched the gonadal transcriptome database and provided novel candidate genes and avenues to research the molecular mechanisms of sexual differentiation, meiosis, and gametogenesis.

Short- and long-term impact of hyperoxia on the blood and retinal cells\u2019 transcriptome in a mouse model of oxygen-induced retinopathy

BackgroundWe aimed to identify global blood and retinal gene expression patterns in murine oxygen-induced retinopathy (OIR), a common model of retinopathy of prematurity, which may allow better understanding of the pathogenesis of this severe ocular prematurity complication and identification of potential blood biomarkers.MethodsA total of 120 C57BL/6J mice were randomly divided into an OIR group, in which 7-day-old pups were maintained in 75% oxygen for 5 days, or a control group. RNA was extracted from the whole-blood mononuclear cells and retinal cells on days 12, 17, and 28. Gene expression in the RNA samples was evaluated with mouse gene expression microarrays.ResultsThere were 38, 1370 and 111 genes, the expression of which differed between the OIR and control retinas on days 12, 17, and 28, respectively. Gene expression in the blood mononuclear cells was significantly altered only on day 17. Deptor and Nol4 genes showed reduced expression both in the blood and retinal cells on day 17.ConclusionThere are sustained marked changes in the global pattern of gene expression in the OIR mice retinas. An altered expression of Deptor and Nol4 genes in the blood mononuclear cells requires further investigation as they may indicate retinal neovascularization.

441 Overlapping functions of PARP1 and p63 in cutaneous squamous cell carcinoma

Cutaneous SCC (cSCC) is the second most frequent type of human malignancy worldwide primarily caused by chronic long-term sun exposure. Somatic mutations in the tumor suppressor gene TP53 have been shown to occur frequently in aged sun-exposed skin and to favor clonal expansion. In contrast, p63, a p53 family member, is rarely mutated in cancer and rather is overexpressed and/or amplified in squamous cell carcinomas. Several studies have shown that p63 overexpression leads to higher proliferation rates and an increased ability to contrast differentiation, cellular senescence and apoptosis. Using a proteomic screening, we identified PARP1 as a p63 protein partner. PARP1 is an enzyme that add PAR moieties onto target proteins and regulates different cellular processes including DNA repair and transcriptional regulation. Furthermore, it is a known therapeutic target for several tumor types including breast and ovarian cancer. Similar to p63, PARP1 is abundantly expressed in a large cohort of cSCC samples as compared to normal skin and is upregulated in cSCC cell lines. To investigate whether PARP1 and p63 coordinately regulate global patterns of gene expression, either PARP1 or p63 were knocked down in SCC cells followed by RNA-seq. Correlation analyses of the transcriptomic profile under basal condition, showed that a large proportion of genes affected by the depletion of one factor was similarly affected by the depletion of the other, causing a highly significant coordinated perturbation in transcription. Furthermore, most of the downregulated genes in PARP1 and p63 depleted cells are involved specifically in cell cycle progression and mitosis. Taken together these data reveal a previously unrecognized functional interaction between p63 and PARP1 in regulating transcription of a large set of cell cycle genes during early stages of skin tumorigenesis, potentially opening a window for novel therapeutic intervention.

An 'eFP-Seq Browser' for visualizing and exploring RNA sequencing data.

SummaryImprovements in next‐generation sequencing technologies have resulted in dramatically reduced sequencing costs. This has led to an explosion of ‘‐seq’‐based methods, of which RNA sequencing (RNA‐seq) for generating transcriptomic data is the most popular. By analysing global patterns of gene expression in organs/tissues/cells of interest or in response to chemical or environmental perturbations, researchers can better understand an organism's biology. Tools designed to work with large RNA‐seq data sets enable analyses and visualizations to help generate hypotheses about a gene's function. We present here a user‐friendly RNA‐seq data exploration tool, called the ‘eFP‐Seq Browser’, that shows the read map coverage of a gene of interest in each of the samples along with ‘electronic fluorescent pictographic’ (eFP) images that serve as visual representations of expression levels. The tool also summarizes the details of each RNA‐seq experiment, providing links to archival databases and publications. It automatically computes the reads per kilobase per million reads mapped expression‐level summaries and point biserial correlation scores to sort the samples based on a gene's expression level or by how dissimilar the read map profile is from a gene splice variant, to quickly identify samples with the strongest expression level or where alternative splicing might be occurring. Links to the Integrated Genome Browser desktop visualization tool allow researchers to visualize and explore the details of RNA‐seq alignments summarized in eFP‐Seq Browser as coverage graphs. We present four cases of use of the eFP‐Seq Browser for ABI3,SR34,SR45a and U2AF65B, where we examine expression levels and identify alternative splicing. The URL for the browser is https://bar.utoronto.ca/eFP-Seq_Browser/.Open research badges This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. Tool is at http://sps:urlprefix::https; RNA‐seq data at http://sps:urlprefix::https and http://sps:urlprefix::https. Code is available at http://sps:urlprefix::https

1024 - EPIGENETIC CONTROL OF MYELOID CELL DEVELOPMENT BY THE TRANSCRIPTION FACTOR IRF8

Differentiation of hematopoietic stem and progenitor cells to various types of blood cells is a process of establishing cell type-specific gene expression patterns. We have been investigating the mechanism of myeloid cell development from a viewpoint of gene expression control by transcription factors, particularly IRF8. In Irf8–/– mice, mononuclear phagocyte progenitors are accumulated, and these progenitors do not efficiently differentiate into monocytes (Mos) or dendritic cells (DCs) but instead, give rise to a large number of neutrophils. Accordingly, the loss of IRF8 causes immunodeficiency and chronic myeloid leukemia-like neutrophilia in mice and humans. Recently, we found that a novel 3’ enhancer is responsible for high Irf8 expression in the DC lineage. Deletion of this enhancer in vivo resulted in the loss of classical DC1s (cDC1s) and somehow surprisingly, caused a significant increase in Mo counts. Thus, the expression level of IRF8 determines the fate of myeloid progenitors; absence, low, or high expression of IRF8 promotes differentiation towards neutrophils, Mos, or DCs, respectively. We also analyzed enhancer landscape dynamics and the genome-wide behavior of IRF8 during the development of mononuclear phagocytes in vivo. While IRF8 does not immediately change the global gene expression pattern in the mononuclear phagocyte progenitors, it does establish their enhancer landscapes by cooperating or antagonizing with other transcription factors, thereby preparing for future gene expression. Furthermore, we found that IRF8 is weakly expressed in a subpopulation of lymphoid-primed multipotent progenitors and leads to early lineage specification towards cDC1s by regulating chromatin states before inducing major transcriptional changes. We are now investigating the 3D chromatin structure dynamics and the role of IRF8 during myeloid development. Collectively, these results illustrate that the epigenetic changes induced by key transcription factors such as IRF8 determine the fate of hematopoietic progenitor cells before establishing gene expression patterns.

THE TRANSCRIPTION FACTOR IRF8 EPIGENETICALLY REGULATES EARLY DENDRITIC CELL SPECIFICATION

Dendritic cells (DCs) are vital for immune responses to pathogens. These cells are produced from bone marrow hematopoietic stem cells via common DC progenitors (CDPs). Recently, DC lineage fate decisions occurring at stages much earlier than CDPs have been recognized, yet the mechanism remains unknown. In this study, we demonstrate that IRF8 regulates chromatin at the LMPP stage to induce early commitment towards DCs. Single-cell RNA-seq revealed that the expression of IRF8, a transcription factor essential for DC and monocyte development, was initiated in a subpopulation within LMPPs. By in vivo transfer experiments, we showed that these IRF8 expressing LMPP (IRF8+ LMPPs) were derived from IRF8- LMPPs and predominantly produced DCs, especially classical DC1s (cDC1s). On the other hand, IRF8+ LMPPs did not generate significant numbers of monocytes, neutrophils or lymphocytes. RNA-seq and an assay for transposase-accessible chromatin (ATAC)-seq revealed that IRF8- and IRF8+ LMPPs displayed very similar global gene expression patterns; however, the chromatin of enhancers near DC lineage genes was more accessible in IRF8+ LMPPs than in IRF8- LMPPs, an epigenetic change dependent on IRF8. The majority of the genes epigenetically primed by IRF8 were still transcriptionally inactive at the LMPP stage, but were highly expressed in the downstream DC lineage populations such as CDPs and cDC1s. Therefore, early expression of the key transcription factor IRF8 changes chromatin states in otherwise multipotent progenitors, thereby biasing their fate decision towards DCs.

Quantitative modelling predicts the impact of DNA methylation on RNA polymerase II traffic

Patterns of gene expression are primarily determined by proteins that locally enhance or repress transcription. While many transcription factors target a restricted number of genes, others appear to modulate transcription levels globally. An example is MeCP2, an abundant methylated-DNA binding protein that is mutated in the neurological disorder Rett syndrome. Despite much research, the molecular mechanism by which MeCP2 regulates gene expression is not fully resolved. Here, we integrate quantitative, multidimensional experimental analysis and mathematical modeling to indicate that MeCP2 is a global transcriptional regulator whose binding to DNA creates "slow sites" in gene bodies. We hypothesize that waves of slowed-down RNA polymerase II formed behind these sites travel backward and indirectly affect initiation, reminiscent of defect-induced shockwaves in nonequilibrium physics transport models. This mechanism differs from conventional gene-regulation mechanisms, which often involve direct modulation of transcription initiation. Our findings point to a genome-wide function of DNA methylation that may account for the reversibility of Rett syndrome in mice. Moreover, our combined theoretical and experimental approach provides a general method for understanding how global gene-expression patterns are choreographed.