title: Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia

city: Neuherberg

first: Johannes

last: Beckers

address: Ingolstaedter Landstr. 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum Muenchen

summary: During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358A->C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71T->C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development.

overall_design: Time course analysis of mouse embryonic tissues from Aey069 mice and controls

first: Sharmilee

last: Vetrivel

first: Natascia

last: Tiso

first: Andrea

last: Richter-Kügler

first: Martin

last: Irmler

first: Daniela

last: Hladik

first: Florian

last: Giesert

first: Helmut

last: Fuchs

first: Sibylle

last: Sabrautzki

first: Jochen

last: Graw

first: Marion

last: Horsch

first: Valerie

last: Gailus-Durner

last: Hrabê de Angelis


title: Microarray analysis of mouse skeletal muscle atrophied by denervation.

status: Public on Apr 01 2019

city: Kyoto

first: Yukino

last: Hatazawa

address: 1-5 Hangi-cho, Shimogamo, Sakyo-ku

country: Japan

institute: Kyoto Prefectural University

summary: Muscle atrophy is associated with aging (sarcopenia) and chronic unloading (such as bed confinement and immobilization with casts), as well as various pathological conditions such as type 1 diabetes and nerve injury (denervation). C57BL/6 mice (7 weeks old, male) were denervated. After 14 days, skeletal muscle was collected and RNA extracted. Expression of Dnmt3a was reduced while expression of Gdf5 was increased by denervation.

overall_design: WT mice (7 weeks old, male) were denervated for 14 days. For microarray analysis, RNA was isolated from the gastrocnemius skeletal muscle of control mice as well as denervated mice. Samples from control mice and denervated mice (N = 6 for each group) were pooled before use.

first: Yasutomi

last: Kamei


title: Mediator complex interaction partners organize the transcriptional network that defines neural stem cells

city: Rotterdam

first: Raymond

middle: A

last: Poot

address: Wytemaweg 80

department: Cell Biology

country: Netherlands

institute: Erasmu MC

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The Mediator complex regulates transcription by connecting enhancers to promoters. High Mediator binding density is one defining feature of so-called super enhancers, which regulate cell-identity genes and oncogenes. Protein interactions of Mediator may explain its role in these processes but have not been identified comprehensively. Here we purify Mediator from neural stem cells (NSCs) and identify 77 novel protein-protein interaction partners. We identified super enhancers in NSCs and show that Mediator-interacting chromatin modifiers colocalise with Mediator at enhancers and super enhancers. Three transcription factor families with high affinity for Mediator dominate enhancers and super enhancers in NSCs and can explain genome-wide Mediator localization. We identify E-box transcription factor Tcf4 as a key regulator in NSCs. Tcf4 interacts with Mediator, colocalises and correlates with Mediator at super enhancers and regulates other neural transcription factors via super enhancers. Our data suggest that high binding-affinity for Mediator is an important organizing feature in the transcriptional network that determines NSC identity.

overall_design: Examination of Med1, Jmjd1c, Carm1 and control IgG genomic occupancy in mouse neural stem cells (mNSCs) by ChIP-seq

first: M

last: Quevedo

first: R

last: Fornerod

first: Z

last: Ozgür

first: W

middle: J

last: van Ijcken


title: Microarray analysis of satellite cells in Dnmt3a knockout mice

summary:
DNA methylation occurs as 5-methylcytosines mainly at cytosine-guanine dinucleotides, so-called CpG sites, and such methylation is a well-studied epigenetic mechanism for transcriptional regulation. Generally, DNA methylation of the gene promoter is correlated with transcriptional repression. Genomic DNA methylation patterns are established by the actions of the de novo methyltransferases Dnmt3a and Dnmt3b, and are maintained by the methyltransferase Dnmt1. Expression of Dnmt3a mRNA is relatively high in skeletal muscle, suggesting a major role in transcriptional regulation. Also, denervation of skeletal muscle decreased expression of Dnmt3a mRNA, suggesting involvement of Dnmt3a in pathophysiology during atrophy.
Decreased regeneration capacity in atrophied skeletal muscle hampers muscle mass recovery after injury and the impaired muscle function, lowers the quality of life. We found that in skeletal muscle of atrophy models, Dnmt3a was decreased. Muscle regeneration was delayed in the skeletal muscle-specific Dnmt3a knockout (Dnmt3a-KO) mice, in which Dnmt3a was deleted in skeletal muscle as well as in satellite cells, important for muscle regeneration. In this study, we used Dnmt3a-KO mice, and attempt to clarify the mechanism of reduced muscle regeneration during atrophy. The microarray data shows that expression of a Gdf/Bmp family protein Gdf5, which suppressed differentiation of satellite cells, is activated in Dnmt3a-KO mice compared with wild-type mice.

overall_design: Satellite cells were isolated from the EDL of Dnmt3a-KO mice and age- and sex-matched WT littermates (8 weeks of age, male). After isolation, satellite cells were cultured in growth medium for 5 days, replated with cell number matching, and cultured in differentiation medium for 2, 4, 5, and 6 days. For microarray analysis, RNA was isolated from the satellite cells from wild-type control mice as well as Dnmt3a-KO mice.


title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis [ChIP-seq]

status: Public on Mar 31 2019

city: Bath

first: Mark

middle: Andrew

last: Lindsay

address: Claverton Down

department: Pharmacy and Pharmacology

country: United Kingdom

institute: University of Bath

summary: Phenotypic changes in lung fibroblasts are believed to contribute to the development of Idiopathic Pulmonary Fibrosis (IPF), a progressive and fatal lung disease.  Long intergenic non-coding RNAs (lincRNAs) have been identified as novel regulators of gene expression and protein activity. In non-stimulated cells, we observed reduced proliferation and inflammation but no difference in the fibrotic response of IPF fibroblasts. These functional changes in non-stimulated cells were associated with changes in the expression of the histone marks, H3K4me1, H3K4me3 and H3K27ac indicating a possible involvement of epigenetics. Following activation with TGF-b1 and IL-1b, we demonstrated an increased fibrotic but reduced inflammatory response in IPF fibroblasts. There was no significant difference in proliferation following PDGF exposure. The lincRNAs, LINC00960 and LINC01140 were upregulated in IPF fibroblasts. Knockdown studies showed that LINC00960 and LINC01140 were positive regulators of proliferation in both control and IPF fibroblasts but had no effect upon the fibrotic response. Knockdown of LINC01140 but not LINC00960 increased the inflammatory response, which was greater in IPF compared to control fibroblasts. Overall, these studies demonstrate for the first time that lincRNAs are important regulators of proliferation and inflammation in human lung fibroblasts and that these might mediate the reduced inflammatory response observed in IPF-derived fibroblasts.

overall_design: Chromatin immunoprecipitation (ChIP) using a H3K4me1 antibody on the 5 control and 5 IPF fibroblast samples. In addition, we performed ChIP on a single control and IPF using an IgG antibody to provide a background control.


title: The IRE1-XBP1 pathway promotes natural killer cell responses against viral infection and cancer by regulation of c-Myc

city: New York

first: Yichi

last: Xu

state: NY

address: 1275 York Avenue

country: USA

institute: MSKCC

type: Expression profiling by high throughput sequencing

summary: Natural killer (NK) cells are critical mediators of host immunity against infectious disease and cancer. The intrinsic regulators of NK cells are not fully understood. Here, we demonstrate that the ER stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) critically drive NK cell-mediated responses against viral infection and tumors. IRE1α and XBP1 were essential for the robust expansion of activated mouse and human NK cells. Transcriptome analysis revealed the transcription factor c-Myc as a novel and direct target of XBP1 for downstream regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, whereas overexpression of XBP1 resulted in c-Myc hyperactivation. NK cells lacking c-Myc demonstrated a functional deficit comparable to IRE1α or XBP1 deficiency. Overall, our study identifies a novel IRE1-XBP1-cMyc axis in NK cell immunity, providing new insight into the host immune response against infection and cancer.

overall_design: Transcriptome analysis of activated and quiescent NK cells (WT and IRE1 KO)

first: Han

last: Dong

first: Nicholas

middle: M

last: Adams

first: Jin

last: Cao

first: David

last: Allan

first: James

middle: R

last: Carlyle

first: Xi

last: Chen

first: Joseph

middle: C

last: Sun

first: Laurie

middle: H

last: Glimcher


title: Single-cell integrative analysis of CAR-T cell activation reveals a predominantly TH1/TH2 mixed response independent of differentiation

first: Kam

last: Leong

address: 3600 Broadway, RM450C

department: Biomedical Engineering

institute: Columbia University

summary: We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19 4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4+ ‘helper’ and CD8+ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of TH1 and TH2 signature cytokines (e.g., IFNγ, TNFα, IL5, and IL13), as confirmed by the expression of master transcription factors TBX21 (Tbet) and GATA-3. However, rather than conforming to stringent TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell and the regulatory T cell (Treg) activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 population. GM-CSF is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status although all major differentiation subsets such as naïve, central memory, effector memory and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that ‘polyfunctional’ CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights to the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

overall_design: Single-cell analysis of CAR-T activation states (unstimulated control, Raji-stimulated)

first: Iva

last: Xhangolli

first: Yang

last: Xiao

first: Rong

last: Fan


title: Microarray analysis of mouse skeletal muscle atrophied by aging

first: Yukino

last: Hatazawa

summary: Muscle atrophy is associated with aging (sarcopenia) and chronic unloading (such as bed confinement and immobilization with casts), as well as various pathological conditions such as type 1 diabetes and nerve injury (denervation). Skeletal muscle tissues of young mice (13 weeks old, male) and old mice (25 months old, male) were collected and RNA extracted. Expression of Dnmt3a was reduced while expression of Gdf5 was increased in old mice compared to young mice.

overall_design: For microarray analysis, Skeletal muscle tissues of young mice (13 weeks old, male) and old mice (25 months old, male) were collected and RNA extracted. Samples from young mice and old mice (young: N = 3, old: N = 3) were use.

first: Yasutomi

last: Kamei


title: Microarray analysis of mouse skeletal muscle atrophied by a plaster cast

summary: Muscle atrophy is associated with aging (sarcopenia) and chronic unloading (such as bed confinement and immobilization with casts), as well as various pathological conditions such as type 1 diabetes and nerve injury (denervation). The hindlimb skeletal muscles of C57BL/6 mice (9 weeks old, male) were immobilized (unloaded) by a plaster cast. After 11 days, skeletal muscle was collected and RNA extracted. Expression of Dnmt3a was reduced while expression of Gdf5 was increased by plaster cast immobilization compared to age-matched control mice.

overall_design: The hindlimb skeletal muscles of WT mice (9 weeks old, male) were immobilized (unloaded) by a plaster cast for 11 days. For microarray analysis, RNA was isolated from the gastrocnemius skeletal muscle of control mice as well as plaster cast mice. Samples from control mice and plaster cast mice (N = 6 for each group) were pooled before use.


title: Microarray analysis of atrophied skeletal muscle models

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: The role of IFN-I and IL-27 in the expansion of virus-specific CD8+ T cell subtypes during persistent infection

city: San Diego

first: John

last: Teijaro

state: CA

address: 10550 North Torrey Pines Rd

department: Immunology and Microbial Science

institute: The Scripps Research Institute

summary: Chronic infection and cancer are associated with suppressed T cell responses in the presence of activating antigen. Recent work identified memory-like CD8+ T cells termed follicular cytotoxic T cells (TFC) which sustain T cell responses during persistent infection and are essential for the T-cell proliferative burst following PD1 blockade. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to TFC expansion in an IL-27-and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell-intrinsic IL-27 signaling safeguards the ability of TCF1-high cells to sustain proliferation and prevents premature cell cycle exit and programmed cell death. Transcriptome analysis revealed an IL-27-regulated gene module controlling survival of activated CD8+ T cells which is enhanced in IFNAR-attenuated conditions. We further demonstrated a cell-intrinsic requirement for the IL-27 target IRF1 in TFC expansion through promoting sustained division. These findings reveal that IL-27 opposes IFN-I to uncouple cell division from effector differentiation, and suggest IL-27 signaling could be exploited to augment self-renewing T cell populations in patients with chronic infections.

overall_design: Cells were isolated from mouse spleens, sorted using flow cytometry and RNA isolated for sequencing. 3 replicates per biological group were used, each sample represents cells from an individual mouse.

first: Jaroslav

last: Zak

first: Zhe

last: Huang

first: Isaraphorn

last: Pratumchai


title: Differentially expressed genes in hematopoietic stem cells after loss of Mettl3

first: Heather

last: Lee

summary: Hematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation according to physiological demands, but how different facets of these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) mRNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deleting the m6A methyltransferase, Mettl3, from the adult hematopoietic system led to an accumulation of HSCs in the bone marrow and marked reduction of HSC reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not have any discernable impact on myeloid cell number or function. m6A sequencing on purified HSCs revealed 2,073 genes with significant m6A modification. In particular, Myc, a key regulator of HSC differentiation, was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to up-regulate Myc expression upon stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results thus revealed a key role of m6A in governing HSC differentiation by regulating Myc expression. This data includes RNA-Seq analysis to showing only minor gene expression changes in adult bone marrow murine hematopoietic stem cells 10 days after Mettl3 deletion by pIpC administration compared to pIpC treated controls.

overall_design: 7 samples were sequenced, 3 control and 4 Mettl3-deficient mutants from different biological replicates of Mx1-cre; Mettl3fl/fl mice and cre-negative control littermates 10 days after Mettl3 deletion by pIpC administration.

first: Suying

last: Bao

first: Yingzhi

last: Qian

first: Shay

last: Geula

first: Juliana

last: Leslie

first: Chaolin

last: Zhang

first: Jacob

last: Hanna

first: Lei

last: Ding


title: Ikaros tumor suppression and epigenetic regulation

city: Hershey

first: Sinisa

last: Dovat

state: PA

address: 500 University Dr

institute: Penn State University


title: Stage-specific requirement for Mettl3-dependent m6A mRNA methylation during hematopoietic stem cell differentiation

type: Methylation profiling by high throughput sequencing


title: Ikaros tumor suppression and epigenetic regulation [ATAC-seq]

summary: Ikaros encodes a transcription factor that functions as a tumor suppressor in T-ALL. The mechanisms through which Ikaros regulates gene expression and cellular proliferation in T-ALL are unknown. Re-introduction of Ikaros into Ikaros-null T-ALL cells results in cessation of cellular proliferation and induction of T-cell differentiation. We performed dynamic global epigenomic and gene expression analyses to determine the role of Ikaros in tumor suppression during this process. Our results identified novel Ikaros functions in the epigenetic regulation of gene expression: Ikaros directly regulates de novo formation and depletion of enhancers, as well as de novo formation of active enhancers and activation of poised enhancers; Ikaros directly induces the formation of super-enhancers; and Ikaros demonstrates pioneering activity by directly regulating chromatin accessibility. Dynamic analyses demonstrate the long-lasting effect of Ikaros DNA binding on enhancer activation, de novo formation of enhancers and super-enhancers, and chromatin accessibility. Ikaros induces profound, global re-distribution of HDAC1 via recruitment of HDAC1 to promoter and enhancer regions of different target genes. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and Ikaros-induced enhancers, and that are responsible for the cessation of proliferation and induction of T-cell differentiation in T-ALL cells.

overall_design: DN3 cells were transduced with retrovirus that contains HA-tagged wild-type murine Ikaros for 1 day, 2 days and 3 days. ATAC-seqs were performed for cells from these four time points.

first: Yali

first: Bo


title: Ikaros tumor suppression and epigenetic regulation [ChIP-seq]

overall_design: DN3 cells were transduced with retrovirus that contains HA-tagged wild-type murine Ikaros for 1 day, 2 days and 3 days. ChIP-seqs (Ikaros, H3K4me3, H3K4me1 and H3K27Ac) were performed for cells from these four time points.


title: Adventitial stromal cells define group 2 innate lymphoid cell tissue niches

city: San Francisco

first: Ari

middle: B

last: Molofsky

state: California

address: 513 Parnassus Ave, HSW1201

department: Laboratory Medicine

institute: UCSF

summary:
We identify a subset of IL-33 expressing adventitial stromal cells as local regulators of ILC2s

Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. In this study, we used quantitative imaging together with scRNAseq to define tissue niches of group 2 innate lymphoid cells (ILC2s), critical instigators of type 2 immunity. We identified a dominant adventitial cuff niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASC), a mesenchymal fibroblast-like subset that expressed Interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) and supported ILC2s and tissue-resident Th2 cells.

overall_design: Analysis of CD45- stromal cells in lung and adipose tissue

first: Madelene

middle: W

last: Dahlgren


title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors [ABE-max]

first: Caleb

last: Lareau

state: New York

address: 417 E 68th St, Zuckerman - ZRC 1132

institute: Memorial Sloan Kettering

summary: CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.

overall_design: HEK293T cells were transfected with pCMV-ABEmax-P2A-EGFP or control pCMV-bpNLS-32AAlinker-nCas9(D10A)-bpNLS constructs with HEK site 2 gRNA as described below. Cells were sorted for top 5% GFP based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs.

first: Julian

last: Grunewald


title: Comparative expression analysis of wild-type and OsCAld5H1-knockout rice plant using Agilent Rice Gene Expression 44k Array

city: Tsuruoka

first: Yuri

last: Takeda-Kimura

state: Yamagata

address: 1-23 Wakabamachi

department: Faculty of Agriculture

institute: Yamagata University

summary: Knockout of a gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1) leads to modulate increase guaiacyl (G) lignin aromatic composition in rice. We compared the global gene expression profiles of wild-type and OsCAld5H1-knockout mutants by microarray to examine the possible effects of OsCAld5H1 disruption on gene expression. We subsequently applied the up- and down-regulated gene sets to gene ontology term enrichment analysis. The results demonstrated that no GO biological process categories enriched significantly, indicating that minimal perturbation of gene expression were caused by OsCAld5H1-knockout.

overall_design: Gene expression in wild-type and OsCAld5H1-knockout rice internode was measured at the heading stage. Three biological replicates were prepared for each genotype.

last: Takeda

first: Shiro

last: Suzuki

first: Yuki

last: Tobimatsu

first: Keishi

last: Osakabe

first: Yuriko

first: Safendrri

middle: K

last: Ragamustari

first: Masahiro

last: Sakamoto

first: Toshiaki

last: Umezawa


title: The transcriptome profile of the stomach fibroblasts with high-metastatic or low-metastatic gastric cancer cell-derived extracellular vesicles.

city: Chuo-ku

first: Yutaka

last: Naito

state: Tokyo

address: 5-1-1, Tsukiji

institute: National cancer center research institute

summary: Cancer-associated fibroblasts (CAFs) have an important role in the tumor progression. CAFs are heterogeneous, and its subpopulation with distinct functions have been regarded as the major obstacle to CAF targeting therapy. However, it is still unclear how cancer cells mediate CAF subpopulations and create preferable tumor microenvironment for their metastasis. In this study, by using the metastatic cancer cell line models of diffuse-type gastric cancer (DGC), we demonstrate that high-metastatic cancer cell-derived extracellular vesicles (EVs) contribute to the formation of activated fibroblast subpopulations. High-metastatic DGC cells create at least two types of CAF subpopulations: myofibroblastic feature and chemokine producing feature. The CAF subpopulation with chemokine producing feature was selectively induced by high-metastatic DGC cell-derived EVs. We also found that high-metastatic DGC cell-derived EVs contain various miRNAs to induce chemokine expression in the fibroblasts. Our findings suggest that intercellular communications via EVs contribute to establishing the appropriate tumor microenvironment toward cancer metastasis. To study the effect of EVs derived from GC cell lines on gene expression in the stomach fibroblasts, transcriptome analysis for the stomach fibroblasts with EVs derived from GC cell lines was performed.

overall_design: EVs were isolated from the conditioned media of two DGC cell lines: high-metastatic (44As3) and low-metastatic (HSC-44PE). The stomach fibroblast was derived from the “normal” non-neoplastic primary tumor site of DGC patient tissue sample. The “primary” stomach fibroblast was immortalized by following infection with retroviruses expressing mutant Cdk4, cyclin D1 and human telomerase reverse transcriptase. DGC cell-derived EVs were treated to the immortalize stomach fibroblasts. PBS (-) treatment and no treated samples were also prepared as a control. After five days incubation, total RNAs were extracted from them. We used Agilent microarray chip (SurePrint G3 Human GE v3).

first: Yusuke

last: Yamamoto

first: Masakazu

last: Yashiro

first: Tohru

last: Kiyono

first: Kazuyoshi

last: Yanagihara

first: Takahiro

last: Ochiya


title: Mettl3-dependent m6A targets in adult murine hematopoietic stem cells

first: Heather

last: Lee

summary: Hematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation according to physiological demands, but how different facets of these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) mRNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deleting the m6A methyltransferase, Mettl3, from the adult hematopoietic system led to an accumulation of HSCs in the bone marrow and marked reduction of HSC reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not have any discernable impact on myeloid cell number or function. m6A sequencing on purified HSCs revealed 2,073 genes with significant m6A modification. In particular, Myc, a key regulator of HSC differentiation, was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to up-regulate Myc expression upon stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results thus revealed a key role of m6A in governing HSC differentiation by regulating Myc expression. This data includes m6A sequencing data on HSCs from wild-type or Mettl3-deficient mice, revealing the Mettl3-depedent m6A targets in HSCs.

overall_design: HSCs were sorted from wild-type mice and Mx1-cre; Mettl3fl/fl mice 10 days after Mettl3 deletion by pIpC administration. mRNA was isolated and subject to m6A RNA immunoprecipitation, fractioning each mRNA pool into input, anti-m6A-antibody-tagged, and antibody-unbound samples. Libraries were prepared and sequenced from each fraction, with 3 biological replicates per fraction per wild-type or Mettl3-deficient mice. 18 samples were sequenced in total.

first: Suying

last: Bao

first: Yingzhi

last: Qian

first: Shay

last: Geula

first: Juliana

last: Leslie

first: Chaolin

last: Zhang

first: Jacob

last: Hanna

first: Lei

last: Ding


title: The transcriptome profile of the stomach fibroblasts with high-metastatic or low-metastatic gastric cancer cell.

summary: Cancer-associated fibroblasts (CAFs) have an important role in the tumor progression. CAFs are heterogeneous, and its subpopulation with distinct functions have been regarded as the major obstacle to CAF targeting therapy. However, it is still unclear how cancer cells mediate CAF subpopulations and create preferable tumor microenvironment for their metastasis. Because the CAF subpopulations are likely depending on the cancer cell subtypes and aggressiveness, it is conceivable the possibility that high-metastatic cancer cells have the capacity to induce the appropriate CAF subpopulation compared with low-metastatic cancer cells. To make it clear, by using the metastatic cancer cell line models of diffuse-type gastric cancer (DGC), we cultured the immortalized stomach fibroblasts with high-metastatic and low-metastatic DGC cell lines. And then, we compared the transcriptome profile of them by microarray analysis. It has shown that high-metastatic DGC cells can induce at least two types of CAF-like phenotypes: myofibroblastic feature and chemokine producing feature. We also found that these two features of fibroblasts were basically distributed in the different fibroblasts. These data indicated that high-metastatic DGC cells form the distinct CAF subpopulations.

overall_design: We used two DGC cell lines: high-metastatic (44As3) and low-metastatic (HSC-44PE). The stomach fibroblasts (NF-58 and NF-60) were derived from the “normal” non-neoplastic primary tumor site of DGC patient tissue samples. These “primary” stomach fibroblasts were immortalized by following infection with retroviruses expressing mutant Cdk4, cyclin D1 and human telomerase reverse transcriptase. These immortalized fibroblasts were cultured with 44As3 or HSC-44PE. Mono-cultured fibroblasts were also prepared as a control. After one week incubation, total RNAs were extracted from fibroblasts. We used Agilent microarray chip (SurePrint G3 Human GE v3).


title: The Pleiotropic Effects of c-di-GMP content in Pseudomonas syringae

city: Hong Kong

first: Xin

last: Deng

address: To Yuen Building

department: Biomedical Sciences

country: Hong Kong

institute: City University of Hong Kong

summary: Although the ubiquitous bacterial secondary messenger cyclic diguanylate (c-di-GMP) plays important roles in various cellular functions including the formation of biofilm in a wide range of bacteria, its function in model plant pathogen Pseudomonas syringae is largely elusive. In order to test this in P. syringae, we overexpressed a diguanylate cyclase (YedQ) and a phosphodiesterase (YhjH) that are originally from Escherichia coli, resulting in high and low c-di-GMP levels in P. syringae, respectively. Through performing genome-wide RNA sequencing of these two strains, we found that c-di-GMP regulates (i) fliN, fliE and flhA genes, which are associated with flagellar assembly, (ii) alg8 and alg44, which are related to exopolysaccaride biosynthesis pathway, (iii) pvdE, pvdP and pvsA genes, related to siderophore biosynthesis pathway, and (iv) sodA, which is a superoxide dismutase. In particular, we identified five genes sensitive to elevated c-di-GMP level and constructed five luciferase-based reporters that effectively respond to intracellular level of c-di-GMP in P. syringae, which can be used to measure c-di-GMP levels in vivo in the future. Based on the RNA-seq results, phenotypic assays confirmed that c-di-GMP regulated many important biological pathways in P. syringae, such as negative regulation of type III secretion system (T3SS) and motility as well as positive regulation of EPS production, siderophore production and oxidative stress resistance. Taken together, the present study demonstrated that c-di-GMP is closely related to virulence and stress response in P. syringae, suggesting that tuning its level can be a new strategy to protect plants from the attack of this pathogen in the future.

overall_design: Detecting the effects of c-di-GMP by changing c-di-GMP content in Pseudomonas syringae


title: The profile of miRNAs in extracellular vesicles derived from high-metastatic or low-metastatic gastric cancer cell lines.

summary: Cancer-associated fibroblasts (CAFs) have an important role in the tumor progression. CAFs are heterogeneous, and its subpopulation with distinct functions have been regarded as the major obstacle to CAF targeting therapy. However, it is still unclear how cancer cells mediate CAF subpopulations and create preferable tumor microenvironment for their metastasis. In this study, by using the metastatic cancer cell line models of diffuse-type gastric cancer (DGC), we demonstrate that high-metastatic cancer cell-derived extracellular vesicles (EVs) contribute to the formation of activated fibroblast subpopulations. High-metastatic DGC cells create at least two types of CAF subpopulations: myofibroblastic feature and chemokine producing feature. The CAF subpopulation with chemokine producing feature was selectively induced by high-metastatic DGC cell-derived EVs. We also found that high-metastatic DGC cell-derived EVs contain various miRNAs to induce chemokine expression in the fibroblasts. Our findings suggest that intercellular communications via EVs contribute to establishing the appropriate tumor microenvironment toward cancer metastasis.To ask how high-metastatic DGC cell-derived EVs induced chemokines in the stomach fibroblasts, miRNA microarray analysis of DGC cell-derived EVs was performed.

overall_design: EVs were isolated from the conditioned media of two DGC cell lines: high-metastatic (44As3) and low-metastatic (HSC-44PE). Total RNAs were extracted from these EVs. Microarray analysis was performed using 20ng of EV miRNA.


title: MicroRNA-223 ameliorates nonalcoholic steatohepatitis and cancer by targeting multiple inflammatory and oncogenic genes in hepatocytes

status: Public on Mar 30 2019

city: Rockville

first: Yong

last: He

state: MD

address: 5625 Fishers lane Room 2S-12

institute: NIAAA/NIH

summary: Here, we found that microRNA-223 (miR-223) was highly elevated in hepatocytes after high fat diet (HFD) feeding in mice and in human nonalcoholic steatohepatitis (NASH) samples. Genetic deletion of the miR-223 induced a full spectrum of nonalcoholic fatty liver disease (NAFLD) in mice after long-term (up to one year) HFD feeding including NASH-related steatosis, inflammation, fibrosis and HCC. To better explore the mechanisms underlying the abnormalities observed in HFD-fed miR-223KO mice, we examined hepatic gene expression in 3-month-HFD-fed WT and miR-223KO mice by microarray analysis. Finally, we revealed that miR-223 plays a key role in controlling steatosis-to-NASH progression by inhibiting hepatic Cxcl10 and Taz expression.

overall_design: Two-condition experiment, WT and miR-223KO.

first: Yan

last: Cai

first: Bin

last: Gao


title: Cell type-dependent differential activation of ERK by oncogenic KRAS in human colon cancer and mouse intestinal epithelium

first: Florian

last: Uhlitz

department: Computational Oncology

institute: Memorial Sloan Kettering Cancer Center

summary: Mutations activating the KRAS GTPase or the BRAF kinase are frequent in colorectal cancer and are thought to constitutively activate the terminal mitogen-activated protein kinase, ERK. Using mass cytometry, we found graded phosphorylation of ERK anti-correlated with cell differentiation in patient-derived colorectal cancer organoids, and unexpectedly this gradient was observed independently of KRAS mutational status. We therefore investigated differentiation-dependent signal transduction elicited by oncogenic KRAS or BRAF in transgenic mouse organoid models. Reporter, single cell transcriptome and mass cytometry analyses showed that transgenic expression of KRASG12V activated ERK in a cell type-specific pattern. Furthermore, expression of oncogenic KRAS induced the formation of RAS-ERK-responsive cells. In contrast, transgenic expression of BRAFV600E triggered high ERK activity and downstream gene expression in all intestinal cell types, followed by epithelial disorganisation. We analysed signal transduction to ERK using single cell-resolved network perturbation data in transgenic organoids. Network reconstruction followed by quantitative modelling revealed that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of MEK to ERK signal transduction. Our experiments highlight key differences between ERK activity elicited by the BRAF or KRAS oncogenes in colorectal cancer and find unexpected functional heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.

overall_design: Transgene-inducible mouse intestinal organoids for KRAS, BRAF and CTNNB1 were subjected to single cell RNA-sequencing.

first: R

last: Brandt

first: T

last: Sell

first: M

last: Lüthen

first: F

first: B

last: Klinger

first: P

last: Riemer

first: C

last: Giesecke

first: S

last: Schulze

first: I

last: El-Shimy

first: D

last: Kunkel

last: Fauler

last: Mielke

first: N

last: Mages

middle: G

last: Herrmann

last: Sers

last: Blüthgen

last: Morkel


title: scRNA-seq of Ciona juvenile heart

first: wei

last: wang

address: 100 washington square east

department: Biology

institute: New York University

summary: scRNA-seq on 386 FACS-purified cardiopharyngeal lineage cells dissociated from stage 38 juveniles.

first: Wei

last: Wang

first: William

last: Mauck


title: Phenotypic variation between stromal cells differentially impacts engineered cardiac tissue function

first: Todd

last: McDevitt

address: 1650 Owens St

institute: Gladstone Institutes

summary: Understanding the relationship between parenchymal and supporting cell populations is paramount to recapitulate the multicellular complexity of native tissues. Incorporation of stromal cells is widely recognized to be necessary for the stable formation of stem cell-derived cardiac tissues, yet the types of stromal cells used has varied widely. This study systematically characterized several stromal populations and found that stromal phenotype and morphology was highly variable depending on cell source and exerted differential impacts on cardiac tissue function and iPSC-CM phenotype. Therefore, the choice of supporting stromal population can differentially impact the phenotypic or functional performance of engineered cardiac tissues.

overall_design: RNA-sequencing experiment to compare transcriptomes of multiple stromal cell populations commonly used in cardiac tissue engineering studies


title: Identifying circRNA-associated-ceRNA networks in the hippocampus of Aß1-42-induced Alzheimer's disease-like rats using microarray analysis (miRNA)

city: Changsha

first: Weijun

last: Peng

address: No.139 Middle Renmin Road

department: The Second Xiangya Hospital

country: China

institute: Central South University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: In the present study, we used microarray analysis technology to characterize the expression patterns of circular RNAs (circRNAs), microRNAs (miRNAs), and mRNAs in hippocampal tissue from Aβ1‐42‐induced AD model rats, to integrate interaction data and thus provide novel insights into the mechanisms  underlying AD.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Hippocampal circRNA,miRNA and mRNA profiles of the AD group and the sham group. Each group included 3 hippocampal tissue were submitted to microarray analysis.


title: Identifying circRNA-associated-ceRNA networks in the hippocampus of Aß1-42-induced Alzheimer's disease-like rats using microarray analysis (mRNA)

overall_design: Hippocampal circRNA,miRNA and mRNA profiles of the AD group and the control group. Each group included 3 hippocampal tissue were submitted to microarray analysis.


title: Identifying circRNA-associated-ceRNA networks in the hippocampus of Aß1-42-induced Alzheimer's disease-like rats using microarray analysis (circRNA)


title: Increased β-cell proliferation prior to immune-cell invasion prevents progression of type 1 diabetes

status: Public on Mar 29 2019

city: Boston

first: Hui

last: Pan

address: 1 Joslin Place

department: Bioinformatics and Biostatistics Core

institute: Joslin Diabetes Center

summary: Type 1 diabetes (T1D) is characterized by pancreatic islet infiltration by autoreactive immune cells and a near-total loss of β-cells. Restoration of insulin-producing β-cells coupled with immunomodulation to suppress the autoimmune attack has emerged as a potential approach to counter T1D. Here we report that enhancing β-cell mass in female NOD mice early in life (prior to weaning) results in immunomodulation of T-cells, reduced islet infiltration and lower β-cell apoptosis, that together protect them from developing T1D. We observed that a model exhibiting β-cell hyperplasia on the NOD background (NOD-LIRKO) displayed altered β-cell antigens, and islet transplantation studies showed prolonged graft survival of NOD-LIRKO islets even upon exposure to diabetogenic splenocytes in vivo. Adoptive transfer of splenocytes from the NOD-LIRKOs prevented diabetes development in pre-diabetic NOD mice, while conversely, similar protective outcomes were obtained when NOD-LIRKO splenocytes were adoptively transferred after mixing them with diabetogenic NOD splenocytes in a dose-dependent manner. A significant increase in the splenic CD4+CD25+FoxP3+ regulatory T-cell (Treg) population in the NOD-LIRKO mice was observed to drive the protected phenotype since Treg depletion rendered NOD-LIRKO mice diabetic. The increase in Tregs coupled with a downregulation of key mediators of cellular function, upregulation of apoptosis and activation of TGF-β/SMAD3 signaling pathway in pathogenic T-cells favored reduced ability to kill β-cells. These data provide novel evidence that initiating β-cell proliferation, alone, prior to islet infiltration by immune cells alters the identity of β-cells, decreases pathologic self-reactivity of effector cells and increases Tregs to prevent progression of T1D.

overall_design: Microarray was performed by the Molecular Genetic Core Facility at Children’s Hospital Boston. CD8+ T-cells and Treg from both NOD-Lox and NOD-LIRKO at eight and twelve weeks of age were analyzed. Briefly, the microarray data was analyzed by correcting for background and normalizing, and estimating empirical array quality weights. We performed differential expression of the microarray data using the R package limma. Analysis was performed in R/Bioconductor.

first: Ercument

last: Dirice

first: Rohit

middle: N

last: Kulkarni


title: Proteomics identifies a marker signature of MAPKi resistance in melanoma

status: Public on Mar 28 2019

city: Geneva

first: Phil

middle: Fang

last: Cheng

address: Rue Gabrielle-Perret-Gentil 4

department: Oncology

country: Switzerland

institute: Hopitaux universitaires de Geneve

summary: Combined proteomic and RNAseq analysis on 6 melanoma cell cultures reveals a signature for resistance to MAPKi resistance.

overall_design: 2 sensitive and 4 resistant melanoma cell cultures were RNAseqed

first: Verena

last: Paulitschke


title: Expression data from mouse brain mononuclear cells

status: Public on Mar 22 2019

city: Maebashi

first: Hiroshi

last: Ohnishi

state: Gunma

address: 3-39-22 Showa-machi

department: Department of Laboratory Sciences

institute: Gunma University Graduate School of Health Sciences

summary:
A membrane protein SIRPα (CD172a) interacts with another membrane protein CD47 and thereby constitutes a cell–cell contact signal.
To investigate the functional role of CD47-SIRPα signal in the brain, we analyzed the effect of genetic ablation of CD47-SIRPα signal on the gene expression profile in specific brain cells by the use of microarrays.

overall_design: Total RNAs were prepared from the brain mononuclear cells of wild-type or CD47 knockout mice. Brain mononuclear cells were isolated by the use of Percoll gradient method. Total RNAs from seven (wild-type) and six (CD47 knockout) different animals were pooled and subjected to the analysis on Affymetrix Gene Chip.


title: Stochastic Changes in Gene Expression Promote Chaotic Dysregulation of Homeostasis in Clonal Breast Tumors

status: Public on Mar 26 2019

city: Rochester

first: Larry

last: Pease

state: MN

address: 200 First St SW

department: Immunology

institute: Mayo Clinic

summary: Much remains unknown about the mechanisms contributing to molecular and cellular diversity in cancer.  This diversity enables the evolutionary processes that govern tumor progression and contributes to treatment failures.  In a previous study, we described atypical losses of heterozygosity at the DNA level in Her2/neu-driven breast tumors from first generation (F1) intercrossed mice.  We now describe similar losses of heterozygosity at the level of gene expression.  By quantitating each parental allele for genes expressed across a population of clonal tumors, we found variegated patterns of expression outliers in hundreds of genes, enriched in pathways typically co-opted by tumors.  The frequency of outliers was correlated with transcriptional repression of a large set of homozygous genes.  These findings reveal the potential for sporadic epigenetic errors that cause dysregulated cellular homeostasis and generate phenotypic variations available for clonal selection.

overall_design: Quantitation and characterization of germline-encoded BALB/c versus FVB/J allelic expression in breast tumors from [BALB/c x FVB/J] F1 mice.

first: Sara

last: Felts


title: Metabolic remodeling during Bacillus subtilis biofilm development

status: Public on Mar 27 2019

city: Madison

first: Tippapha

last: Pisithkul

state: Wisconsin

address: 1550 Linden Drive

department: Bacteriology

institute: UW-Madison

summary: Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report a surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm-associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following these results, we demonstrate that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This study represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful roadmap for future studies on biofilm physiology.

overall_design: Transcriptome analysis of B. subtilis NCIB3610 grown in a biofilm-inducing medium from 8 to 32 hours at 37℃. Sampling was done at 8, 12, 16, 20, 24, 28, and 32 hours of growth (every 4 hours). The experiment was repeated in 4 biological replicates.

first: Jeremy

last: Schroeder

first: Daniel

last: Amador-Noguez


title: Expression data from mouse optic nerve and optic tract

summary:
A membrane protein SIRPα (CD172a) interacts with another membrane protein CD47 and thereby constitutes a cell–cell contact signal.
To investigate the functional role of CD47-SIRPα signal in the brain, we analyzed the effect of genetic ablation of CD47-SIRPα signal on the gene expression profile in specific brain regions by the use of microarrays.

overall_design: Total RNAs were prepared from the white matter (optic nerve and optic tract) of wild-type or CD47 knockout mice. Total RNAs from five (wild-type) and four (CD47 knockout) different genotype-matched animals were pooled and subjected to the analysis on Affymetrix Gene Chip.


title: SNHG15 is a bifunctional MYC-regulated noncoding locus encoding a lncRNA that promotes cell proliferation, invasion and drug resistance in colorectal cancer by interacting with AIF

city: Pamplona

first: Morvarid

last: Saeinasab

state: Navarra

address: Avda. Pío XII, 55

department: Gene Therapy

country: Spain

institute: Center for Applied Medical Research (CIMA)

summary:
Thousands of long noncoding RNAs (lncRNAs) are aberrantly expressed in various types of cancers, however our understanding of their role in the disease is still very limited.   We applied RNAseq analysis from patient-derived data with validation in independent cohort of patients. We followed these studies with gene regulation analysis as well as experimental dissection of the role of the identified lncRNA by multiple in vitro and in vivo methods.
We analyzed RNA-seq data from tumors of 456 CRC patients compared to normal samples, and identified SNHG15 as a potentially oncogenic lncRNA that encodes a snoRNA in one of its introns.  The processed SNHG15 is overexpressed in CRC tumors and its expression is highly correlated with poor survival of patients. Interestingly, SNHG15 is more highly expressed in tumors with high levels of MYC expression, while MYC protein binds to two E-box motifs on SNHG15 sequence, indicating that SNHG15 transcription is directly regulated by the oncogene MYC. The depletion of SNHG15 by siRNA or CRISPR-Cas9 inhibits cell proliferation and invasion, decreases colony formation as well as the tumorigenic capacity of CRC cells, whereas its overexpression leads to opposite effects. Gene expression analysis performed upon SNHG15 inhibition showed changes in multiple relevant genes implicated in cancer progression, including MYC, NRAS, BAG3 or ERBB3. Several of these genes are functionally related to AIF, a protein that we found to specifically interact with SNHG15, suggesting that the SNHG15 acts, at least in part, by regulating the activity of AIF. Interestingly, ROS levels, which are directly regulated by AIF, show a significant reduction in SNHG15-depleted cells. Moreover, knockdown of SNHG15 increases the sensitiveness of the cells to 5-FU, while its overexpression renders them more resistant to the chemotherapeutic drug.
Altogether, these results describe an important role of SNHG15 in promoting colon cancer and mediating drug resistance, suggesting its potential as prognostic marker and target for RNA-based therapies.

overall_design: 3 replicates of siRNA SNHG15-transfected LoVo cells, and 3 replicates of siRNA scramble-transfected LoVo cells

first: Ahmad

last: Bahrami

first: Jovanna

last: González

first: Francesco

middle: P

last: Marchese

first: Dannys

last: Martinez

first: Seyed

last: Mowla

first: Maryam

last: Matin

first: Maite

last: Huarte


title: Cdx2 ChIP-seq performed in E8.5 whole mouse embryos

city: Ottawa

first: Tanya

middle: Elizabeth

last: Foley

state: ON

address: 451 Smyth Road

department: Cellular and Molecular Medicine

country: Canada

institute: University of Ottawa

summary: We identified genomic regions bound by Cdx2 in E8.5 whole embryos relative to an IgG control.

overall_design: Cdx2 and IgG ChIP-seq in E8.5 whole embryos.

first: Bradley

last: Hess

first: Joanne

last: Savory

first: Randy

last: Ringuette

first: David

last: Lohnes


title: Gene expression data of nasopharyngeal carcinoma (NPC) cell and Podoplanin (PDPN) siRNA treatment

status: Public on Mar 19 2019

city: New Taipei City

first: Yu-Lun

last: Kuo

address: 7F-3, No.99, Sec. 1, Xintai 5th Rd., Xizhi Dist.

country: Taiwan

institute: BIOTOOLS

summary:
Podoplanin, a transmembrane sialomucin-like glycoprotein, is now widely used as a marker for lymphatic endothelial cells and fibroblastic reticular cells in lymphoid organs, but its study in nasopharyngeal carcinoma (NPC) is still limited. The aims of this study were t characterize the role of PDPN in NPC. Our results showed that PDPN was expressed in most TW01 NPC cells. PDPN knockdown by siRNA decreased NPC cell proliferation, migration, and invasion.
Knocking down PDPN results in suppression of NPC cell proliferation, migration, and invasion. PDPN may serve as a potential chemotherapeutic target for NPC treatment in the future.

overall_design: Total RNA from NPC cells treated with or without200 nM PDPN siRNA for 48 hours at 37°C was extracted using an illustra RNAspin Mini RNA isolation kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. Amplification and biotinylation of the RNA and hybridization of the fragmented biotin-labeled cRNA with Affymetrix HG-U133 Plus 2.0 GeneChip microarrays.

first: Yen-Bin

last: Hsu

first: Ming-Chin

last: Lan

first: Kuan-Ting

last: Lin

first: Chi-Ying

middle: F

last: Huang

first: Ming-Ying


title: First Degree Relatives (FDRs) of Patients with Celiac Disease Harbour an Intestinal Transcriptomic Signature that Might Protect Them from Enterocyte Damage

status: Public on Mar 21 2019

city: New Delhi

first: Mitali

last: Mukerji

state: Delhi

address: Near Mathura Road, Sukhdev Vihar

department: CSIR TRISUTRA UNIT

country: India

institute: CSIR-Institute of Genomics and Integrative Biology

summary: Patients with Celiac Disease, first degree relatives of celiac patients and control groups displayed significant differential gene expression.

overall_design: All subjects were underwent upper GI endoscopic examination. Multiple mucosal biopsies were obtained using spike biopsy forceps. Four bits of tissues in 10% formal saline were sent for histological assessment for villous architecture assessment. Other 4-6 bites of mucosal biopsies were taken in RNA later for gene expression analysis. In this study, FDRs were selected randomly and none of the FDRs were related to Celiac patients.

first: Rajesh

last: Pandey

first: Govind

last: Makharia

first: Rintu

last: Kutum


title: Expression data from FIRE deficient mice

status: Public on Mar 18 2019

city: Edinburgh

first: Clare

last: Pridans

address: 47 Little France Crescent

department: University of Edinburgh

institute: Centre for Inflammation Research

summary: We used microarrays to examine changes in gene expression in the absence of the FIRE enhancer of Csf1r in mice.

overall_design: RNA was isolated from hippocampus and a section of ileum containing a Peyer's patch, from saline-perfused WT and KO animals.

last: Hume

first: Rocio

last: Rojo


title: Gene expression data from sorted primary human acute myeloid leukemia (AML) samples

status: Public on Mar 15 2019

city: Basel

first: Claudia

last: Lengerke

address: Hebelstr. 20

department: Department of Biomedicine

institute: University Hospital Basel and University of Basel

summary: In vivo studies indicate that human acute myeloid leukaemia (AML) originates from subpopulations of neoplastic cells with stem cell properties, thereby laying the foundation for the cancer stem cell concept. For clinically apparent disease to occur, cancer-initiating cells must also escape immune control. Here we show that stemness and immune evasion are closely intertwined in AML. Patients with AML harbour leukaemic subpopulations that lack expression of ligands for the danger detector NKG2D, a critical mediator of immune surveillance, which stimulates anti-tumour immunity by cytotoxic lymphocytes such as natural killer (NK) cells. We compared NKG2DLneg and NKG2DLpos subfractions from n=5 patients (each measured in triplicates) and found that NKG2DLneg cells displayed amongst others stemness characteristics.Our findings identify immune privilege as a general feature of LSCs, and provide a conceptual framework for the integration of two central hypotheses of cancer development – the concepts of cancer stem cells and immune escape

overall_design: Analysis of gene expression in FACS sorted AML fractions separated into NKG2DLpos and NKG2DLneg fractions from n=5 AML patients (3 CD34 expressing and 2 CD34 non-expressing AML patients) each performed in triplicates

first: Martina

last: Konantz


title: Gene expression in the ileum of Cftr null mice

city: Rotterdam

first: Marcel

last: Bijvelds

address: PO Box 2040

department: Gastroenterology and Hepatology

institute: Erasmus MC University Medical Center

summary: Transcriptome sequencing was performed on ileum of Cftr null mice and wildtype littermates

overall_design: Tissue from the distal small intestine (<6 cm proximal to the ileocecal valve) was collected from Cftr null mice (Cftrtm1Cam; congenic FVB) and littermate controls (Cftr N/N). In some cases, animals had been treated with the antibiotics ciprofloxacin (0.3 g/L) and metronidazole (0.5 g/L). For transcriptome analysis, mRNA was isolated from tissue RNA extracts, fragmented, and, subsequently used for cDNA synthesis. The cDNA libraries were sequenced using an HiSeq 2000 sequencer (TruSeq SBS KIT-HS V3; Illumina) and the Ion Proton System (4476610). Data analysis was performed using CLC genomic workbench 7.5 (CLC Bio), and the sequence reads were mapped to the Genome Reference Consortium (GRC) genome data set GRCm38.76, using default parameters.

first: M

first: K

last: Dulla


title: Physiological and transcriptomic characterization of white lupin response to Fe deficiency

city: Udine

first: Fabio

last: Marroni

address: Via delle Scienze 208

country: Italy

institute: Università di Udine

summary: Iron (Fe) and phosphorus (P) are essential nutrients for plants growth. Despite their abundance in soils, they are barely available for plants. In order to overcome these nutritional stresses, plants have evolved strategies including physiological, biochemical and morphological adaptations. Biosynthesis and release of low molecular weight compounds from the roots play a crucial role in P and Fe mobilization. White lupin (Lupinus albus L.) is considered a model plant for studying root exudates and for P-deficient adaptation. White lupin is able to markedly modify its root architecture by forming special structures called cluster roots, and modifies the rhizospheric soil characteristics by biosynthesising and releasing great amounts of exudates. These phenomena are quite well described in response to P deficiency, but there is few information on the adaptation of a cluster-root producing plant species to Fe deficiency. This prompted this work, aimed to characterize the physiological and transcriptomic responses of white lupin plants to Fe deficiency. Occurrence of Strategy I components and interactions with P nutrition has been also investigated in this work. Results showed a physiological and transcriptional link between the responses to Fe and P deficiency in white lupin roots. Phosphorus-deficient plants activated the Strategy I Fe acquisition mechanisms that lead to an enhanced Fe mobilization and translocation and that might help the P acquisition process. On the other hand, also the Fe deficiency enhanced the phosphate acquisition and some P-deficient-responsive genes were overexpressed.

overall_design: RNA of Roots of Control Plants, Root Apices of Fe Deprived Roots, Root Clusters of Fe Deprived Roots and Root Clusters of P Deprived Roots were sequenced in triplicate on an Illimina HiSeq2000 Sequencer

first: Silvia

last: Venuti

first: Laura

last: Zanin

first: Alessandro

last: Franco

first: Michele

last: Morgante

first: Roberto

last: Pinton

first: Nicola

last: Tomasi


title: Gene expression profiling of mouse BMPR1B+ chondrocytes

status: Public on Mar 12 2019

city: Los Angeles

first: Denis

last: Evseenko

address: 1450 Biggy St

department: Orthopaedic Surgery

institute: University of Southern  California

summary: We profiled gene expression of mouse chondrocytes from embryonic and adult samples.

overall_design: 3 biological replicates from embryonic and adult mouse knee cartilage

first: Gabriel

last: Ferguson

first: Ben

last: Van Handel


title: Transcriptomic profiling in a Drosophila model of diet induced transgenerational epigenetic inheritance.

first: Manoj

middle: Ramesh

last: Teltumbade

address: Mathura Road, Near Sukhdev Vihar, bus depo

institute: Institute of Genomics and Integrative Biology

summary: The study was conducted to identify differentially expressed genes in 1) whole flies that were fed a high sugar diet (HSD), in comparison to flies treated with control diet (CD), and 2) HSD male line derived F1 and F2 progenies, compared to that derived through CD male line. The ancestral HSD exposure of F1 or F2 flies was thus limited only to F0 males. However, besides comparing F1 and F2 flies raised on CD, the F1 and F2 flies treated with HSD were also compared. F0 HSD females were profiled but not used for the mating purpose.

overall_design: Throughout the experiment, 3-4 days old unmated male or virgin female flies were fed either HSD or CD for two weeks. The HSD and CD contained 1M and 0.15M sucrose, in that order. At the end of treatment, the flies were collected and homogenized in Trizol for RNA extraction. Transcriptomic profiling was performed using Affymetrix Drosophila Genome 2.0 array.

first: Abhay

last: Sharma


title: Plasma fatty acid levels and gene expression related to lipid metabolism in peripheral blood mononuclear cells

status: Public on Mar 08 2019

city: OSLO

first: Mari

last: Myhrstad

address: Postboks 4, St. Olavs plass

department: Department of health

country: Norway

institute: Oslo and Akershus University College

summary: Solid evidence indicates that intake of marine n-3 fatty acids lower serum triglycerides, and that replacing saturated fatty acids (SFA) with polyunsaturated fatty acids (PUFA) reduces plasma total cholesterol and LDL-cholesterol. The molecular mechanisms underlying these health beneficial effects are however not completely elucidated. The aim of this study was to investigate the expression of genes related to lipid metabolism in peripheral blood mononuclear cells (PBMC) depending on the plasma levels of n-6 and n-3 fatty acids and the SFA to PUFA ratio.

overall_design: This study is a cross-sectional sub-study of a randomised controlled trial designed to investigate the health effects of fish oil intake (Ottestad el al, 2012, Myhrstad et al, 2014). The study population was grouped into tertiles three times according to the plasma fatty acid levels of n-6 and n-3 fatty acids and the plasma SFA to PUFA ratio at the end of study by arranging samples from the highest to the lowest value. Peripheral blood monnucelar cell gene expression between subjects in the highest (n=18) and the lowest (n=18) tertile within each arrangement in groups were further compared. A total of 285 genes encoding proteins related to cholesterol and triglyceride metabolism were selected for this explorative analysis. 161 genes were defined as expressed on the HumanHT-12 v4 microarray and included in the statistical analyses.

first: Sunniva

middle: V

last: Larsen

first: Kirsten

last: Holven

first: Inger

last: Ottestad

first: Kine

last: Dagsland

first: Stine

last: Ulven


title: Screening of Candida albicans GRACE library revealed a unique pattern of biofilm construction under repression of the essential gene ILS1

city: London

first: Vanessa

last: Dumeaux

state: Ontario

address: Elgin Dr

department: Anatomy and Cell Biology

institute: Western University

summary: ILS1 mutated Candida albicans  was unable to grow as yeast-phase cells but was capable of producing a tridimensional biofilm structure in spite of reduced metabolic activity. This biofilm still relied on the classical biofilm genes, while it differentially induced groups of genes involved in adhesion, protein synthesis, cell wall organization, and protein folding. Although the conditional mutant repressed genes annotated for morphology and homeostasis processes affecting morphology and metabolism, the dynamic cell growth enabled the construction of a complex biofilm community independent of ILS1.

overall_design: The wild-type (WT) planktonic cells were grown in YPD media at 30 degrees celcius. The wild-type (WT) and the ILS1 GRACE strain biofilms  were grown in RPMI medium buffered with MOPS.  We conduted RNAseq profiling  for 2 replicates in each condition (WT planktonic, WT biofilm and WT ILS1 strains)

first: Anna

last: Costa

first: Malcolm

last: Whiteway


title: Characterization of functional relationships of R-loops with gene transcription and epigenetic modifications in rice

status: Public on Mar 07 2019

city: Nanjing

first: Fang

last: Yuan

state: Jiangsu

address: No.1, Weigang Street, Xuanwu District, Nanjing

institute: NanJing Argricultural University

summary: We conducted genome-wide identification of R-loops followed by integrative analyses of R-loops with relation to gene expressionand epigenetic signatures in the rice genome. We found that the correlation between gene expression levels and profiled R-looppeak levels was dependent on the positions of R-loops within gene structures (hereafter named 'genic position'). Both antisenseonly (ASO)-R-loops and sense/antisense(S/AS)-R-loops sharply peaked around transcription start sites (TSSs), and these peaklevels corresponded positively with transcript levels of overlapping genes. In contrast, sense only (SO)-R-loops were generallyspread over the coding regions, and their peak levels corresponded inversely to transcript levels of overlapping genes. Inaddition, integrative analyses of R-loop data with existing RNA-seq, chromatin-immunoprecipitation (ChIP-seq), deoxyribonucleaseI-seq (DNase-seq) and whole-genome bisulfite sequencing (BS-seq) data revealed interrelationships and intricate connectionsamong R-loops, gene expression and epigenetic signatures. Experimental validation provided evidence that the demethylationof both DNA and histone marks can influence R-loop peak levels on a genome-wide scale. This is the first study in plants thatreveals novel functional aspects of R-loops, their interrelations with epigenetic methylation, and roles in transcriptionalregulation.

overall_design: Examination of  two tissues in rice including seedlings and calli

first: Yuan

last: Fang

first: Wenli

last: Zhang


title: Sorghum axillary bud transcriptome response to leaf removal

status: Public on Mar 05 2019

city: College Station

first: Scott

last: Finlayson

state: Texas

address: 370 Olsen Blvd

department: SOil and Crop Sciences

institute: Texas A&M University

summary: Basal branching in grasses, or tillering, is an important trait determining both form and function of crops.  While similarities exist between eudicot and grass branching programs, one notable difference is that the tiller buds of grasses are covered by the subtending leaf, whereas eudicot buds are typically unconstrained.  The current study shows that contact with the leaf sheath represses sorghum bud growth by providing a mechanical signal that cues the bud to refrain from rapid growth.  Leaf removal resulted in massive reprogramming of the bud transcriptome that included signatures of epigenetic modifications, and also implicated several hormones in the response.  Bud abscisic acid (ABA) transiently increased, then decreased following leaf removal relative to controls, and ABA was necessary to repress bud growth in the presence of the leaf.  Jasmonic acid (JA) levels and signaling increased in buds following leaf removal.  Remarkably, application of JA to buds in situ promoted growth.  The repression of bud growth by leaf contact shares characteristics of thigmomorphogenic responses in other systems, including the involvement of JA, though the JA effect is opposite.  The repression of bud growth by leaf contact may represent a mechanism to time tillering to an appropriate developmental stage of the plant.

overall_design: Analysis of bud transcriptomes at 1, 3 and 6 h with and without removal of the overlying leaf.

middle: A


title: TCP-seq of yeast initiation factor mutants

status: Public on Mar 06 2019

city: Clayton

first: Stuart

middle: Kenneth

last: Archer

state: VIC

address: 15 Innovation Walk, Monash University

department: Monash Bioinformatics Platform

country: Australia

institute: Monash University

summary: Each mutant was cultured in duplicate, and from each culture two fractions were sequenced: Total fragmented RNA, and small ribosomal subunit footprints.

first: Neelam

middle: D

last: Sen

first: Alan

last: Hinnebusch


title: Osteoarthritis RNA Sequencing from ground cartilage samples

status: Public on Mar 01 2019

city: Newcastle upon Tyne

first: Andrew

middle: James

last: Skelton

state: Tyne and Wear

address: Framlington Place

department: Bioinformatics

institute: Newcastle University

summary: Osteoarthritis (OA) is the most prevalent joint disease with the typifying feature being the progressive degradation of articular cartilage during disease progression. In this study we used whole transcriptome RNA-seq as a tool to compare gene expression changes between age-matched osteoarthritic human hip OA cartilage (n=10) compared to control (neck of femur fracture) cartilage (n=6) [GSE107308]. All cartilage was from patients undergoing acetabulofemoral joint replacement. Cartilage RNA was isolated from cartilage within 2 hr of joint replacement surgery, mRNA was polyA purified and transcript expression was analysed using 78-base paired-end sequencing generating on average 28 million reads/sample sequencing.  The data shows excellent correlation with our previous microarray data but identifies significantly more differentially expressed transcripts plus novel transcript variants, several of which have been validated by real-time qPCR. Our work sheds further light on chondrocyte transcriptome expression and highlights gene expression changes and novel transcripts potentially important in osteoarthritis progression

overall_design:
osteoarthritic human hip cartilage (n=10) RNA was isolated from cartilage within 2 hr of joint replacement surgery, mRNA was polyA purified and transcript expression was analysed using 78-base paired-end sequencing
Please note that processed data was generated from both NOF samples (GSE107308) and OA samples.

first: David

last: Young

middle: J

first: Matt

last: Barter


title: A Suv39H1-low chromatin state drives migratory cell populations in cervical cancer [ChIP-Seq]

city: Bangalore

first: Calvin

last: Rodrigues

address: GKVK, Off Bellary Road

institute: National Centre for Biological Sciences

summary: The evolution of migratory populations of cells within solid tumours represents a critical stage during cancer metastasis. We have previously reported the enhanced migratory properties of one such population, marked by the expression of CD66, within cervical cancers. However, it is not clear what mechanisms drive such migratory populations, and how these cells would retain a “memory” of initial migration-inducing cues during metastasis. Here, we describe the role of a Suv39H1-low heterochromatin state as a driver of cervical cancer migratory populations. Cervical cancer cells were sorted based on migratory ability in vitro. These migrated populations show low Suv39H1, and Suv39H1 knockdown enhances cell migration. Using histopathology to examine clinical carcinoma progression, initial progression showed expansion of a proliferative Suv39H1high population, followed by the emergence of a sarcomatoid, migratory Suv39H1low cell population in advanced stages. Additionally, meta-analysis of TCGA data revealed that low tumoural Suv39H1 also correlated with lower patient survival, and with expression signatures of migration, TGF-β signalling, and links with a CD66 cell signature. Lastly, using RNA-Seq and H3K9me3 ChIP-Seq on migrated populations in vitro, it was observed that these populations show Suv39H1-linked transcriptome alterations and broad scale H3K9me3 remodelling. This Suv39H1-linked heterochromatin regulation could explain plasticity in migratory and metastatic populations, and allows for a “memory” of migration inducing cues, through metastable changes in chromatin conformation. The understanding of such regulatory mechanisms in migratory populations may prove valuable in efforts to develop anti-metastatic strategies.

overall_design: H3K9me3 ChIP Sequencing of migrated and non migrated fractions of SiHa cells isolated from a transwell set up, 2 paired replicate sets

first: Sudhir

last: Krishna


title: A Suv39H1-low chromatin state drives migratory cell populations in cervical cancer [RNA-Seq]

overall_design: RNA Sequencing of migrated and non migrated fractions of SiHa cells isolated from a transwell set up, 3 paired replicate sets


title: Gene expression changes in the supraorbital cranial mesenchyme after beta-catenin deletion in vivo

city: Cleveland

first: Radhika

middle: Paresh

last: Atit

state: Ohio

address: 2074 Adelbert Road, Millis 316

department: Biology

institute: Case Western Reserve University

summary: Goal of this study was to examine gene expression changes upon conditional deletion of beta-catenin at e13.5 cranial mesenchyme.

overall_design:
En1Cre;b-cateninfl/fl and En1Cre;b-cateninfl/+ supraorbital cranial mesenchyme was manually dissected from E13.5 embryos (4 mutants and 3 controls). RNA was isolated using Trizol and a Qiagen RNeasy Mini Kit. Libraries were prepared in the CWRU Genomics sequencing core using the Illumina TruSeq Stranded Total RNA kit-with Ribo Zero Gold. Paired-end sequencing was performed on an Illumina HiSeq 2500  v2 Rapid Run flowcell. The resulting 100bp reads were aligned to the mouse genome release mm9 genome using TopHat. Genomic assembly was completed using Cufflinks v1.3 (Roberts et al., 2011a, Roberts et al., 2011b, Trapnell et al., 2010, Trapnell et al., 2013). mm9_reFlat was used to annotate the data with a max intron length of 20,000 and genomic bias correction. Differential gene expression was determined with CuffDiff using the default settings plus genomic bias correction.
contributor: Case Western Reserve University

first: James

last: Ferguson


title: Supraorbital cranial mesenchyme with ectoderm upon the deletion of β-catenin

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: AmpliSeq transcriptome profiling of human adipose tissue progenitor cell types

status: Public on Feb 27 2019

city: Chilly Mazarin

first: Veeranagouda

last: Yaligara

address: Sanofi R&D

country: France

institute: Sanofi

summary: Three different progenitor cell subsets in subcutaneous and visceral adipose tissues derived from 5 obese patients were subjected to AmpliSeq transcriptome profiling. Transcriptomic profiles were analyzed to compare progenitor cell subsets and the impact of subcutaneous and visceral adipose tissue location.

overall_design: Transcriptomic profiling of 3 different progenitor cell types in subcutaneous and visceral adipose tissues derived from 5 obese patients (3X2X5=30 samples).

first: N

last: Boulet

first: A

last: Zakaroff-Girard

first: Y

last: Veeranagouda

first: M

last: Didier

last: Bouloumié

first: J

last: Galitzky


title: ADAMTSL2, a missing link in Wnt/ß-catenin regulated CNS vascular development

status: Public on Feb 22 2019

city: Stockholm

first: Daniel

last: Ramsköld

address: Nobels väg 3

country: Sweden

institute: Karolinska Institutet

summary: Context dependent molecular cues shape the formation of the cerebral vascular network and the function of the blood-brain barrier (BBB). The Wnt/ß-catenin pathway is orchestrating CNS vascular development, but downstream mediators have not been characterized. Here we generated an endothelial cell-specific R26-Axin1 overexpression (AOE) mouse model to inhibit Wnt/ß-catenin signaling. In AOE mice we discovered that blockade of Wnt/ß-catenin pathway leads to premature regression and remodeling without compromising BBB integrity. Importantly, by comparing transcriptomes of endothelial cells from wildtype and AOE mice, we identified ADAMTSL2 as a novel Wnt/ß-catenin-induced, secreted factor, important for stabilizing the BBB during development. Zebrafish loss-of-function and gain-of-function models, further demonstrated that ADAMTSL2 is crucial for normal vascular development and could rescue vascular phenotypes in AOE zebrafish brains. In conclusion, the studies presented here reveal a hitherto unrecognized role of ADAMTSL2 as an endothelial cell-specific mediator of Wnt/ß-catenin signaling during CNS vascular development and BBB-formation.

overall_design: Examination of expression changes in mouse brain endothelial cells when overexpressing Axin1

first: Belma

last: Salagic Hot

first: Mike

last: Hupe

first: Jan

last: Stenman

first: Julianna

last: Kele


title: mitomanip microarray experiment 4-ANR MITOMANIP

status: Public on Mar 04 2019

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

institute: INRA

summary: rs13-02_mitomanip - mitomanip microarray experiment 4 - Mitochondrial transcriptome regulation and coordination with the nucleus - Following a previously established strategy (Val et al., 2011, Nucleic Acids Res. 39, 9262–9274), we express, from an estradiol-inducible nuclear transgene, a trans-cleaving ribozyme directed against the nad9 mitochondrial mRNA and associated as a trailor sequence to a tRNA mimic. The latter serves as a shuttle and ensures mitochondrial uptake of the chimeric RNA through the natural tRNA import pathway. In mitochondria, the ribozyme triggers cleavage and degradation of the target mRNA. The impact of the nad9 mRNA knockdown on the overall plant transcriptome is analyzed over 4 successive days. Control plants express the shuttle RNA combined with a ribozyme that has no specific target in A. thaliana.

overall_design: 15 dye-swap - trans-ribozyme-mediated knockdown of a specific mitochondrial rna vs control, time course.

first: Ludivine

last: Taconnat

first: André

last: Dietrich

first: Etienne

last: Delannoy

first: Sandrine

last: Balzergue

first: Marie-Laure

last: Martin-Magniette


title: Heterozygous and homozygous knock-in of PIK3CA-H1047R into human iPSCs

status: Public on Feb 15 2019

city: Cambridge

first: Ralitsa

middle: Radostinova

last: Madsen

address: Addenbrookes Treatment Centre, Keith Day Road

department: Metabolic Research Laboratories, Clinical Biochemistry

institute: University of Cambridge

summary: We used CRISPR/Cas9 to knock in the cancer "hotspot" mutation PIK3CA-H1047R into one or both alleles of a wild-type induced pluripotent stem cell (iPSC) line (WTC11; Coriell # GM25256; P37-P38). Three clones from each genotype (wild-type, heterozygous, homozygous) were subjected to single-end mRNA sequencing (mean read depth per sample: 20 million) to determine whether PIK3CA-H1047R exerts allele dose-dependent transcriptional effects. Multidimensional scaling demonstrated distinct transcriptomic signatures of wild-type, heterozygous and homozygous cells. The transcriptome of heterozygous cells was nearly identical to wild-type controls, with only 131 differentially-expressed transcripts (FDR = 0.05). In contrast, homozygosity for PIK3CA-H1047R led to differential expression of 1,914 genes. This indicates widespread transcriptional remodeling with a sharp allele dose-dependency, suggestive of a threshold effect.

overall_design: 3 independent clones per genotype (WT/WT, WT/H1047R, H1047R/H1047R) collected 3 hours following medium replenishment

middle: R

first: Robert

last: Semple


title: Transcriptome stability profiling using 5'-bromouridine IP chase (BRIC-seq) identifies novel and functional microRNA targets in human melanoma cells.

status: Public on Feb 20 2019

city: St. Petersburg

first: Piyush

last: Joshi

state: FL

address: 600 5th St S

department: Oncolog/JHACH

institute: Johns Hopkins University

summary: RNA half-life is closely related to its cellular physiological function, so stability determinants may have regulatory functions. Micro(mi)RNAs have primarily been studied with respect to post-transcriptional mRNA regulation and target degradation. Here we study the impact of the tumour suppressive melanoma miRNA miR-211 on transcriptome stability and phenotype in the non-pigmented melanoma cell line, A375. Using 5ʹ-bromouridine IP chase (BRIC)-seq, transcriptome-wide RNA stability profiles revealed highly regulated genes and pathways important in this melanoma cell line. By combining BRIC-seq, RNA-seq and in silico predictions, we identified both existing and novel direct miR-211 targets. We validated DUSP3 as one such novel miR-211 target, which itself sustains colony formation and invasion in A375 cells via MAPK/PI3K signalling. miRNAs have the capacity to control RNA turnover as a gene expression mechanism, and RNA stability profiling is an excellent tool for interrogating functionally relevant gene regulatory pathways and miRNA targets when combined with other high-throughput and in silico approaches.

overall_design: Total RNA was isolated from A375 and A375/211 cell line for BRIC-seq and RNA-seq. For BRIC-seq, cells were labelled with BrU for 24 h and collected at 0, 1, 3 and 6 h post-labelling. For RNA-seq cells were collceted from A375 and A375/211 cell lines. Reads were aligned to hg38 genome using HISAT2 and quantified using StringTie. DESeq2 was used to perform differential gene expression analysis.

first: Ranjan

last: Perera


title: H3K27me3 enrichment in the supraorbital cranial mesenchyme with ectoderm upon the deletion of β-catenin

first: Radhika

last: Atit

overall_design:
e13.5 supraorbital cranial mesenchyme was manually dissected. The sample collected consisted of all the tissue above the brain which included the cranial mesenchyme plus the ectoderm. Chromatin immunoprecipitation and sequencing (ChIP-seq) was then performed by Active Motif. 14 µg chromatin was immunoprecipitated with 4 µg rabbit anti-H3K27me3 (Millipore #07-449). Sequencing was performed on an Illumina NextSeq 500 producing 75-nucleotide, single end reads. Drosophila DNA was “spiked in” and used to normalize the number of reads in the samples. Reads were aligned using Bowtie2 to the mouse mm9 genome and peaks were called using Macs 1.4 at default settings.
contributor: Case Western Reserve University

first: James

last: Ferguson


title: Roles of HDAC1 and HDAC2 in the developing mouse brain

status: Public on Feb 12 2019

city: Shanghai

first: Yunli

last: Xie

state: Shanghai

address: 131 Dong'an Road, Shanghai

department: Institutes of Brain Science

institute: Fudan University

summary: In this study, we investigated the role of HDAC1 and HDAC2 in the developing mouse brain. We found that the removal of HDAC1 and HDAC2 caused severe malformation. To examine the molecular mechanisms underlying the cortical malformation, we examined the transcriptome in both WT and HDAC1/2 deficient brains. We found that many genes that are associated with transcription were misregulated in HDAC1 and HDAC2 mutant brains. This result provides a resource for studying gene expression controlled by HDAC1 and HDAC2.

overall_design: Mouse embryonic for brains were isolated from E12.5 embryos. Total RNAs were extracted using Trizol reagent and applied for RNA sequencing subsequently.

first: Tianxiang

last: Tang


title: Molecular analysis of gastric cancer identifies discrete subtypes associated with distinct clinical characteristics and survival outcomes: the ACRG (Asian Cancer Research Group) study [normal tissue]

city: West Point

first: Michael

last: Nebozhyn

state: PA

address: Mail Stop WP53B-120, 770 Sumneytown Pike, Building 53, P.O. Box 4

department: GpGx/Computational System biology

institute: Merck, Inc

summary: Gastric cancer, a leading cause of cancer-related deaths, is a heterogeneous disease, with little consensus on molecular subclasses and their clinical relevance. We describe four molecular subtypes linked with distinct patterns of molecular alterations, disease progression and prognosis viz. a) Microsatellite Instable: hypermutated intestinal subtype tumors occurring in antrum, best overall prognosis, lower frequency of recurrence (22%), with liver metastasis in 23% of recurred cases; b) Mesenchymal-like: diffuse tumors with worst prognosis, a tendency to occur at an earlier age and highest recurrence (63%) with peritoneal seeding in 64% of recurred cases, low frequency of molecular alterations; c) TP53-inactive with TP53 loss, presence of focal amplifications and chromosomal instability; and d) TP53-active marked by EBV infection and PIK3CA mutations. The key molecular mechanisms and associated survival patterns are validated in multiple independent cohorts, to provide a consistent and unified framework for further preclinical and clinical research.

overall_design: ACRG Gastric cohort: microarray profiles from 100 patient-matched normal tissues from gastric cancer patients.

first: R

last: Cristescu

first: J

last: Lee

first: M

first: K

last: Kim

middle: C

last: Ting

first: S

middle: S

last: Wong

last: Liu

first: Y

last: Yue

last: Wang

last: Yu

first: X

last: Ye

first: I

last: Do

first: L

last: Gong

last: Fu

last: Jin

last: Choi

first: T

last: Sohn

middle: H

middle: M

last: Bae

middle: T

last: Park

first: P

last: Tan

first: C

last: Ronghua

last: Hardwick

first: W

middle: K

last: Kang

last: Ayers

first: D

last: Hongyue

last: Reinhard

first: A

last: Aggarwal

last: Loboda


title: A Suv39H1-low chromatin state drives migratory cell populations in cervical cancer

first: Calvin

last: Rodrigues


title: A novel CRISPR-engineered prostate cancer cell line defines the AR-V transcriptome and identifies PARP inhibitor sensitivities.

status: Public on Feb 09 2019

first: Evangelia

last: Kounatidou

department: Northern Institute for Cancer Research

summary:
Development of a novel CRISPR-derived cell line which is a derivative of CWR22Rv1 cells, called CWR22Rv1-AR-EK, that has lost expression of FL-AR, but retains all endogenous AR-Vs.
AR-Vs act unhindered by loss of FL-AR to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrate that AR-Vs drive expression of a cohort of DNA damage response genes and depletion of AR-Vs sensitizes cells to ionizing radiation.

overall_design: Transcriptomic profile (mRNA) of AR splice variants in CWR22Rv1 AR-EK cells was generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.

first: Sirintra

last: Nakjang

first: Stuart

last: McCracken

first: Craig

middle: N

last: Robson

first: Scott

last: Dehm

first: Dominic

last: Jones

first: Luke

last: Gaughan


title: Egg transcriptome reveals molecular signature to predict developmental success and novel maternal-effect genes

status: Public on Feb 01 2019

city: Rennes

first: Aurelie

last: LE CAM

address: Campus de Beaulieu

department: PHASE

summary: Good quality or developmentally competent eggs result in high survival of progeny. Previous research has shed light on factors that determine egg quality, however, large gaps remain. Initial development of the embryo relies on maternally-inherited molecules, such as transcripts, deposited in the egg, thus, they would likely reflect egg quality. We performed microarray analysis on zebrafish fertilized eggs of different quality from unrelated, wildtype couples to obtain a global portrait of the egg transcriptome to determine its association with developmental competence and to identify new candidate maternal-effect genes. Fifteen of the most differentially expressed genes (DEGs) were validated by quantitative real-time PCR. Gene ontology analysis showed that enriched terms included ribosomes and translation. In addition, statistical modeling using partial least squares regression and genetics algorithm also demonstrated that gene signatures from the transcriptomic data can be used to predict reproductive success. Among the validated DEGs, otulina and slc29a1a were found to be increased in good quality eggs and to be predominantly localized in the ovaries. CRISPR/Cas9 knockout mutants of each gene revealed remarkable subfertility whereby the majority of their embryos were unfertilizable.Our novel findings suggested that even in varying quality of eggs due to heterogeneous causes from unrelated wildtype couples, gene signatures exist in the egg transcriptome, which can be used to predict developmental competence. Further,  transcriptomic profiling revealed two new potential maternal-effect genes that have essential roles in vertebrate reproduction.

overall_design: This study compared the expression in two groups of samples each containing N=16.

first: Caroline

last: Cheung

first: Thao-vi

last: Nguyen

first: Aurélie

last: Le Cam

first: Amélie

last: Patinote

first: Laurent

last: Journot

first: Christelle

last: Reynes

first: Julien

last: Bobe


title: Expression data from rat liver

city: Osaka

first: Ayako

last: Ohara

address: 1-98, Kasugade-naka 3-chome, Konohana-ku

institute: Sumitomo Chemical Co., Ltd.

summary:
BrlHan:WIST@Jcl(GALAS) male rats were fed diets containing 0 (control) and 3000 ppm epsilon-momfluorothrin for periods of 2 weeks.
We used microarrays to evaluate gene expression profiling in rat liver at the early phase of treatment with epsilon-momfluorothrin.

overall_design: After treatment periods, livers of 6 rats (3 rats in each group) were excised for RNA extraction and hybridization on Affymetrix microarrays.


title: Polysome profiling and mRNA-seq to quantify translational gene regulation in dietary restricted C. elegans and in a long-lived daf-2:rsks-1 double mutant.

city: Bar Harbor

first: Jarod

middle: Alton

last: Rollins

state: ME

address: 159 Old Bar Harbor Rd

institute: MDI Biological Laboratory

summary: Dietary restriction (DR) and loss of the genes rsks-1 and daf-2 increase longevity in C. elegans.  Polysome profiling allows actively translated mRNA bound by multiple ribosomes to be isolated and compared to the total mRNA present. In this project, we differentiate transcriptional and translational changes in gene expression in C. elegans by combining polysome profiling and mRNA-sequencing. By comparing gene abundance between the two RNA pools, genes with altered translational regulation under DR and in the mutant can be identified.

overall_design: Total and polysome bound RNA was isolated from well-fed daf-2;rsks-1 double mutants, well-fed N2, and dietary restricted N2 C. elegans in biological quadruplicate. Age syncronized worms were maintained at 20°C and subject to DR starting at day one of adulthood in the presence of FuDR to prevent contamination of progeny.  Whole worms were lysed at day four of adulhood and the lysate was subject to polysome profiling to isolate RNA bound to 2 or more ribosomes. A aliquot of unprocessed lysate was used for RNA extraction of total RNA. RNA was enriched for polyadenylated transcripts and used for unstranded paired-end library synthesis with a Tru-seq RNA kit. Read length of 100 bp were generated using Illumina HiSeq 2000. Reads were aligned to the C.elegans genome as guided by gene annotations from ensemble version 66. Processed data contains the unfiltered counts per million (CPM) of normalized reads aligning to each gene for each of the samples submitted.

first: J

first: D

last: Chen

first: A

last: Rogers

first: P

last: Kapahi


title: Examination of gene expression in beta-cells isolated from either wildtype or alms1 mutant zebrafish

city: Baltimore

first: Norann

last: Zaghloul

state: MD

address: 670 W. Baltimore Street; HSF3 Room 4176

department: Medicine

institute: University of Maryland School of Medicine

overall_design: RNA-seq of beta-cells isolated from either wildtype or alms1 mutant zebrafish

first: Jessica

last: Dunleavey


title: Improving fibroblast characterization using single-cell RNA sequencing: an optimized tissue disaggregation and data processing pipeline

status: Public on Feb 06 2019

city: Southampton

first: Sara

last: Waise

state: Hampshire

address: Somers Building MP284, Southampton General Hospital, Tremona Road

department: Cancer Sciences

institute: University of Southampton

summary: Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. We also assess the impact of in vitro culture conditions on stromal cell gene expression and proliferation, showing that altering these conditions can skew phenotypes.

overall_design: mRNA profiles of human lung samples (tumour, inflamed and normal lung) from 3 patients

first: Christopher

last: Hanley


title: A conserved seed-pairing domain affords small RNA-mediated stress resistance in enterobacteria

status: Public on Jan 22 2019

city: Jena

first: Kathrin

last: Fröhlich

state: Thuringia

address: Winzerlaer Straße 2

department: Fakultät für Biowissenschaften, Bacterial RNA Biology

country: Germany

institute: Friedrich-Schiller-Universität Jena

type: Non-coding RNA profiling by high throughput sequencing


title: Oocyte-specific deletion of Mitofusin1 causes female infertility and accelerated follicular depletion

status: Public on Jan 26 2019

city: Gainesville

first: Zongliang

last: Jiang

address: 2033 Mowry Rd

department: Animal Sciences

institute: University of Florida

summary: We performed RNAseq analysis to determine the effect of MFN1 deletion on oocyte global gene expression profile. RNAseq revealed a total of 982 genes were significantly differentially expressed (p<0.05) in Mfn1-/-  oocytes compared to WT (654 up-regulated and 337 down-regulated). Pathway analysis indicated significant over-representation of elements involved in regulation of ceramide biosynthesis, death receptor signaling and adherens junction signaling. Differential expression of these genes (Bad, G2e3, Cdh17 and Myh2) was also confirmed by qRT-PCR.Our findings provide new insight into the role of MFN1 in the oocytes, and may help understand the potential mechanism of infertility and reproductive aging associated with MFN1-deficiency.

overall_design: Secondary follicle-enclosed oocytes were collected from 8-week-old Mfn1-/- and WT mice (n=3 for each group) and 5 oocytes from each group were pooled for RNA sequencing analysis.

first: Man

last: Zhang

first: Muhammed

middle: B

last: Bener

first: Emre

last: Seli