title: Genome-wide collaboration of canonical and non-canonical STAT1 complexes with NF-κB to control signal integration between Interferons and TLR4 in vascular and immune cells [RNA-seq]

city: Poznań

first: Anna

last: Piaszyk-Borychowska

address: ul. Umultowska 89

department: Department of Human Molecular Genetics

country: Poland

institute: Adam Mickiewicz University

summary:
Atherosclerosis is a disease of large and medium-sized muscular arteries and is characterized by vascular inflammation and lipid-laden plaque formation within the intima of the vessel wall. Atherosclerosis is initiated by recruitment of blood leukocytes to the injured vascular endothelium and leads to altered contractility of Vascular Smooth Muscle Cells (VSMCs), acute and chronic luminal obstruction, abnormalities of blood flow and diminished oxygen supply to target organs. The pro-inflammatory pathways activated by Toll-like Receptors (TLRs), and Interferons (IFNs) have been identified as key components of atherogenesis. These pathways culminate in the activation of Signal Transducers and Activator of Transcription (STAT)1-containing complexes ISGF3 and GAF, and Nuclear factor-κB (NFκB) that coordinate expression of multiple chemokines, adhesion molecules, antiviral and antimicrobial proteins in vascular and immune cells. IFN-I and IFN-II participate in signaling cross-talk with TLR4 through combinatorial actions of ISGF3 and GAF with NF-kB leading to enhanced gene expression. Currently it is not clear how this signal integration is regulated at the genome-wide level and if similar mechanisms are activated by the different IFNs in vascular cells as compared to macrophages and dendritic cells.
Therefore, we compared genome-wide transcriptional responses of mouse VSMCs, macrophages (BMDMs) and dendritic cells (DCs) in response to IFNα, IFNγ or LPS alone, or after combined treatment using RNA-seq. Consequently, commonly up-regulated genes in VSMCs, BMDMs and DCs could be identified after combined treatment with IFNα+LPS, as well as after combined treatment with IFNγ+LPS. Generally, in all three cell-types combined treatment with IFNα+LPS or IFNγ+LPS resulted in a synergistic increase in gene expression as compared to single treatments. Also, significant functional overlap could be observed between commonly upregulated genes in response to IFNα+LPS and IFNγ+LPS. Using ChIP-seq on chromatin from VSMCs, STAT1 and p65 bound IFNα+LPS and IFNγ+LPS up-regulated genes were identified, containing GAF/GAS, ISGF3/ISRE or NF-kB binding motifs located in promoter regions, but also to up- and downstream genomic regions. Comparing IFNα+LPS and IFNγ+LPS commonly upregulated genes identified a substantial overlap of ISRE-containing genes. Interestingly, STAT1 as part of ISGF3 was shown to bind the promoter of these ISRE-containing genes in response to IFNα as well as IFNγ. Moreover, different binding modes of STAT1-p65 co-binding were detected, including GAS-NFκB, ISRE-NFκB or GAS-ISRE-NFκB, shared by IFNα+LPS and IFNγ+LPS up-regulated genes. This cooperative involvement involved a STAT1-dependent role in the nearby recruitment of p65 already upon single IFNα or IFNγ treatment, via closely located GAS-NFĸB or ISRE-NFĸB binding sites in promoter regions. More important, this STAT1-p65 co-binding was significantly increased upon subsequent LPS exposure and resulted in amplified transcriptional activity of IFNα+LPS and IFNγ+LPS upregulated genes.

overall_design: RNA-seq samples were derived from IFN alpha- (8 hours), IFN gamma- (8 hours), LPS- (4hours), IFN alpha+LPS- and IFN gamma+LPS-treated Vascular Smooth Muscle Cells (VSMC), Bone Marrow-Derived Macrophages (BMDM) and Dendritic Cells (DC) at least in triplicates.


title: EOMES-dependent epigenetic reprogramming during CD8 T cell development leads to acquisition of memory traits (ChIP-Seq)

status: Public on Jun 06 2019

city: Gosselies

first: Stanislas

last: Goriely

address: Rue Adrienne Bolland 8

country: Belgium

institute: Université Libre de Bruxelles

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Memory CD8 T cells have a unique ability to provide lifelong immunity against pathogens. Although memory features generally arise after a challenge with foreign antigen, naïve CD8 single positive (SP) thymocytes may already acquire phenotypic and functional characteristics of memory cells in response to cytokines such as interleukin-4. This process is associated with the induction of the T-box transcription factor Eomesodermin (EOMES). However, the underlying molecular mechanisms remain ill-defined. Using epigenomic profiling, we show that these “innate memory” CD8SP cells acquire only a portion of the active enhancer repertoire of conventional memory cells. This reprograming was secondary to EOMES recruitment, mostly to RUNX3-bound enhancers. Furthermore, we identified direct interaction between EOMES and BRG1 and showed that in vivo acquisition of EOMES-dependent program by CD8SP thymocytes was dependent on this chromatin remodeling factor. In conclusion, our results support a strong epigenetic basis for EOMES-driven establishment of CD8 T cell unconventional memory program.

overall_design: CD8SP thymocytes: Innate Memory, Naïve, WT, and Eomes-TG

first: Nicolas

last: Istaces

first: Marion

last: Splittgerber

first: Viviana

middle: L

last: Silva

first: Muriel

last: Nguyen

first: Séverine

last: Thomas

first: Aurore

last: Le

first: Younes

last: Achouri

first: Emilie

last: Calonne

first: Matthieu

last: Defrance

first: François

last: Fuks

first: Abdulkader

last: Azouz


title: Gene expression analysis of micoRNA-221 overexpressing transgenic mice livers with and without metformin intervention upon chronic liver injury induced by carbon tetrachloride

status: Public on Jun 03 2019

city: Ferrara

first: Massimo

last: Negrini

state: Ferrara

address: 70 Via Fossato di Mortara

department: Morphology, Surgery and Experimental Medicine

country: Italy

institute: University of Ferrara

summary:
Metformin is a hypoglycaemic agent used to treat type 2 diabetes mellitus (DM2) patients, with a broad safety profile. Since previous epidemiological studies had shown that incidence of hepatocellular carcinoma (HCC) decreased significantly in metformin treated DM2 patients, we hypothesized that intervention with metformin could reduce the risk of neoplastic transformation of hepatocytes.
HCC is the most common primary liver malignancy and it generally originates in a background of liver fibrosis and cirrhosis. In the present study, we took advantage of a transgenic mouse (TG221) characterized by microRNA-221 overexpression, with cirrhotic liver background induced by chronic administration of carbon tetrachloride (CCl4). This mouse model develops fibrosis, cirrhosis and liver tumors that become visible in 100% of mice at 5-6 months of age.

overall_design: We compared geneset enrichment of differentially expressed genes in livers between metformin+CCl4 and CCl4 treated TG221 mice. Chronic liver injury was induced in all mice with CCl4 for 14 weeks and metformin intervention was started after 3 weeks of initiating CCl4 challenge until the experimental end-point of 24 weeks.


title: Genome-wide collaboration of canonical and non-canonical STAT1 complexes with NF-κB to control signal integration between Interferons and TLR4 in vascular and immune cells [ChIP-seq]

status: Public on Jun 04 2019

overall_design: ChIP-seq samples were derived from IFN alpha- (8 hours), IFN gamma- (8 hours), LPS- (4hours), IFN alpha+LPS- and IFN gamma+LPS-treated Vascular Smooth Muscle Cells (VSMC) in duplicates.


title: Constrained transcriptional polarity in the organization of mammalian Hox gene clusters (Strand specific RNA-seq)

city: Lausanne

first: Fabrice

last: Darbellay

address: Station 19

department: SV/ISREC

country: Switzerland

institute: EPFL

type: Expression profiling by high throughput sequencing

summary: In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, for the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters towards a tighter structure during the evolution of the vertebrate lineage. Here we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster such as to invert one or several transcription unit(s), with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this genetic system, and the distinct problems elicited by these inversion alleles illustrate various aspects of the intricate regulations implemented at these loci. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in-cis.

overall_design: Strand specific RNA-seq generated from wild-type and mutant HoxD alleles

first: Lucille

last: Delisle-Lopez

first: Denis

last: Duboule


title: Genome-wide collaboration of canonical and non-canonical STAT1 complexes with NF-κB to control signal integration between Interferons and TLR4 in vascular and immune cells

first: Anna

last: Piaszyk-Borychowska

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

overall_design: Refer to individual Series


title: Tissue-specific gene expression during productive HPV16 infection of cervical, foreskin and tonsil epithelium.

city: Cambridge

last: Salzberg

state: MA

address: 399 Binney St UNIT 300

country: USA

institute: Rubius Therapeutics

summary: We performed microarray analysis of the effect of productive HPV16 infection in cervical, foreskin and tonsil epithelial trancriptome. Our study for the first time compares and contrast the gene expression changes and differential activation of cellular pathways induced by the virus in the three tissue types.

overall_design: RNA was isolated from 10-day organotypic raft cultures obtained from uninfected and HPV16 infected cervical, foreskin and tonsil keratinocytes. For each tissue type, rafts from the uninfected keratinocytes were used as the control.

first: Craig

last: Meyers

first: Sreejata

last: Chatterjee


title: Histone H2B monoubiquitination regulates heart development via epigenetic control of cilia motility [RNA-Seq]

city: New Haven

first: Martina

last: Brueckner

state: CT

address: 789 Howard Ave

department: Pediatrics

institute: Yale University

summary: Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution from mutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD are unknown. Histone H2B mono-ubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40 and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment=6.01, p=1.67x10-03), some of whom had abnormal laterality associated with cilia dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right asymmetry and impaired cilia motility. Rnf20, Rnf40 and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and non-ciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3.  Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissue-specific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function.

overall_design: Xenopus embryo mRNA profiling in wt (in duplicate) and RNF20 morpholino (in triplicate) at 12 hpf, 13 hpf, 14 hpf, 15 hpf, and 16 hpf

first: Mustafa

last: Khokha


title: The RNA NAD+ cap is widespread in the Arabidopsis transcriptome and probably promotes translation

city: Beijing

first: shaofang

last: Li

state: Beijing

address: No 50, ZhanghuaLu, HAIDIAN

department: Beijing Vegetable Research Center

country: China

institute: Beijing Academy of Agriculture and Forestry Science

summary: As the most common mRNA cap, the m7G cap impacts the fate of an mRNA in eukaryotes. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD+), can be used as an initiating nucleotide in RNA synthesis to result in NAD+-capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD+ cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD+- capped RNAs are widespread in Arabidopsis thaliana. NAD+-capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes but not the chloroplast genome. NAD-capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD+-capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms of translation initiation on NAD+-capped mRNAs. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, and maybe regulated by, mRNA NAD capping.

overall_design: We analyzed the NAD+-capped RNA in Arabidopsis seedling and inflorescence total RNA population, as well as NAD+- capped RNA in seedling polyA mRNA and polysomal RNA fraction. Each sample indcludes at least 2 replicates.

first: Yuan

last: Wang

first: Shaofang

first: Xuemei

last: Chen


title: A Cellular Atlas of Pitx2-Dependent Cardiac Development

first: Matthew

middle: C

last: Hill

address: 75 Aimes Street

institute: Broad Institute of MIT and Harvard

summary: The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation (AF), however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2-null embryos. We uncovered that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2. Our findings offer insight into Pitx2 biology and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease, and arrhythmogenesis.

overall_design: Here, we used single cell transcriptomics to inspect Pitx2 function in cardiac development and left-right cellular specification. Deployment of a high-throughput scRNA-seq platform on cardiac tissue dissected from both control and Pitx2-null embryos at E10.5 and E13.5 was carried out to characterize all deviations in cell composition, cellular state, and differentiation trajectories.


title: IRF2BP2 Modulates the Crosstalk between Glucocorticoid and TNF Signaling

city: Kuopio

first: Einari

middle: A

last: Niskanen

address: Yliopistonranta 8

department: School of Medicine

country: Finland

institute: University of Eastern Finland

summary: Here we have analyzed the role of interferon regulatory factor-2 binding protein-2 (IRF2BP2) in glucocorticoid and tumor necrosis factor alpha (TNF) signaling. We used ChIP-seq to analyze chromatin binding of IRF2BP2 in glucocorticoid (dexamethasone, dex) and vehicle treated HEK293 cells expressing GR (HEK293-GR). Furthermore, we used RNA-seq to analyze how silencing of IRF2BP2 modulates transcriptional responses to dex treatment in HEK293-GR cells, and dex, TNF and co-treatment (dex and TNF, DT) in A549 cells.

overall_design:
For ChIP-seq, HEK293-GR cells were grown on steroid-depleted medium for two days and treated with dex or vehicle for one hour. Cells were fixed with 1% (v/v) formaldehyde, chromatin was fragmented to ~200-400 bp by sonication, chromatin immunoprecipitation was done using anti-IRF2BP2 antibody and libraries were prepared using NEBNext Ultra II kit (New England Biolabs). Two biological replicates of both conditions were sequenced using Illumina HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany).
For RNA-seq, HEK293-GR or A549 cells were cultured in steroid-depleted media and transfected with siRNA against IRF2BP2 (siIRF2BP2, 20nM, Dharmacon, ON-TARGETplus SMARTpool, L-007177-02-0005) or control siRNA (siNON, Dharmacon, non-targeting pool) using Lipofectamine RNAiMAX (Invitrogen). After three days, cells were treated with dexamethasone (dex, 100 nM), Tumor Necrosis Factor alpha (TNF, 10 ng/ml), or vehicle (EtOH, equal amount of ethanol) for six hours. RNA from three biological replicates was extracted using RNeasy Plus Mini kit (Qiagen) and mRNA isolated using NEBNext Poly(a) mRNA Magnetic Isolation Module (New England Biolabs). Libraries were prepared using NEBNext Ultra II kit (New England Biolabs) and sequenced with HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany).

first: A

middle: K

last: Manjur

first: Joanna

last: Lempiäinen

first: Marjo

last: Malinen

first: Jorma

middle: J

last: Palvimo


title: Enhanced DNA repair and genomic stability identify a novel HIV related Diffuse Large B-cell Lymphoma subtype [aCGH]

city: Scottsdale

first: Alanna

last: Maguire

state: Arizona

address: 13400 East Shea Blvd

department: Research

institute: Mayo Clinic

summary:
Transcriptional and genomic profiling study of HIV positive and HIV negative Diffuse Large B-Cell Lymphoma (DLBCL). Tumors were subtyped using the Lymph2Cx assay. Only GCB (germinal center like B-cell) subtypes were then subjected to digitial gene expression profiling and array comparative genomic hybridization (aCGH).
The study used DNA extracted from FFPE DLBCL tumors derived from HIV(+) and HIV(-) patients to assess copy number variation differences between the HIV(+) and HIV(-) cohorts. aCGH data was correlated with gene expression & IHC data.

overall_design:
aCGH was performed on 19 HIV positive and 20 HIV negative GCB DLBCL samples using Agilent SurePrint G3 human CGH arrays.
DLBCL is more agreesive in HIV(+) patients compared to HIV(-) individuals. Study objective was to identify novel markers and potential therapeutic targets that contribute to the enhanced agressive nature of this disease in HIV infected individuals.

first: Xianfeng

first: Lee

last: Wisner

first: Smriti

last: Malasi

first: Colleen

last: Ramsower

first: Samantha

last: Kendrick

first: Michael

middle: T

last: Barrett

first: Betty

last: Glinsmann-Gibson

last: McGrath

first: Lisa

middle: M

last: Rimsza


title: Innate immune activity correlates with suppression of HIV replication and CD4+ T cell-associated HIV-1 DNA decline during treatment with pegylated IFN-α2a in chronic ART-suppressed HIV infection

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: In study NCT00594880, we successfully administered weekly doses of pegylated interferon-α-2a (Peg-IFN-α2a) with antiretroviral therapy (ART) for 5 weeks, before continued Peg-IFN-α2a monotherapy to 12 weeks (primary endpoint) in chronic HIV-infected subjects. Subjects maintaining HIV viral load <400 copies/ml by primary endpoint were defined as responders. We now describe innate immune correlates and transcriptional profiles associated with viral control and decrease in integrated HIV DNA after Peg-IFN-α2a immunotherapy. Peripheral blood samples were obtained prior to Peg-IFN-α2a administration (ART), after 5 weeks of ART+Peg-IFN-α2a dual treatment, and after 12 weeks of Peg-IFN-α2a monotherapy. Cell subset modulation, natural killer cell (NK) function and signaling, as well as inflammatory mediators and gene expression were assessed. Results were analyzed using R.2.5.1 or MATLAB 7.10.0. Five weeks of ART+Peg-IFN-α2a preserved the frequency of immune subsets and NK cytotoxicity, while increased the levels of inflammatory mediators, and decreased cell-responsiveness to in vitro IFN-α re-stimulation. Gene expression analysis showed that induction of host restriction factors after ART+Peg-IFN-α2a was not solely predictive of a virologic response, and revealed a 108 gene signature that identified subjects who did not modulate genes after ART+Peg-IFN-α2a. A 29 gene signature along with higher NK cell activation and IFN-γ-induced protein 10 (IP-10) levels on ART, as well as higher in vitro responses to IFN-γ-induced NK cytotoxicity, and decrease in the frequency of NK bearing inhibitory receptors [i.e. killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail 1 (KIR2DL1) or KIR2DL2/DL3] and C-C chemokine receptor type 7+ (CCR7) myeloid dendritic cells after ART+Peg-IFN-α2a distinguished responders from non-responders. Reductions in integrated HIV DNA after immunotherapy were associated with expression patterns of genes that are associated with activated cell-mediated response and NK cytotoxicity. In summary, innate activation and NK cell cytotoxicity are identified as correlates of HIV control and reduction after Peg-IFN-α2a immunotherapy in ART-suppressed subjects.

overall_design:
Expression from cryo-preserved PBMC for patients on pegylated interferon-α-2a (Peg-IFN-α2a) with antiretroviral therapy (ART) treatment

Please note that [1] the visit 3, 5, and 8 represents;
Visit 3: prior to Peg-IFN-α2a administration (ART)
Visit 5: after 5 weeks of ART+Peg-IFN-α2a dual treatment
Visit 8: after 12 weeks of Peg-IFN-α2a monotherapy

[2] the 'characteristics: response' values represent;
RC: response based on gene expression to 5 weeks of ART+Peg-IFN-α2a treatment, controlled plasma viral load
RN: response based on gene expression to 5 weeks of ART+Peg-IFN-α2a treatment, do not control plasma viral load
NN: no response based on gene expression to 5 weeks of ART+Peg-IFN-α2a treatment, do not control plasma viral load

first: Papasavvas

last: Emmanouil

first: Montaner

last: Luis


title: SIRT7 tethering of L1 elements to the nuclear lamina mediates their transcriptional repression

city: Piscataway

first: Joshua

last: Thackray

state: NEW JERSEY

address: 145 Bevier Road

department: Human Genetics Institute of New Jersey

institute: Rutgers University

summary: Long interspersed elements-1 (LINE-1, L1) are retrotransposons that hold the capacity of selfpropagation in the genome with potential mutagenic outcomes. How somatic cells restrict L1 activity and how this process becomes dysfunctional during aging and in cancer cells is poorly understood. L1s are enriched at lamin associated domains, heterochromatic regions of the nuclear periphery. Whether this association is necessary for their repression has been elusive. Here we show that the sirtuin family member SIRT7 participates in the epigenetic transcriptional repression of L1 genome-wide in both mouse and human cells. SIRT7 depletion leads to increased L1 expression and retrotransposition. Mechanistically, we identify a novel interplay between SIRT7 and Lamin A/C in L1 repression. Our results demonstrate that SIRT7-mediated H3K18 deacetylation regulates L1 expression by promoting L1 association with elements of the nuclear lamina. The failure of such activity might contribute to the observed genome instability and compromised viability in SIRT7 knockout mice. Overall, our results reveal a novel function of SIRT7 on chromatin organization by mediating the anchoring of L1 to the nuclear envelope, and a new mechanism by which the nuclear lamina maintains genome integrity.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Chromatin from 14.5 day WT and SirT7-/- MEFs or the human K562 cell line was subjected to ChIP using antibodies raised against SIRT7 and H3K18Ac and subjected to high-throughput sequencing.

first: Lourdes

last: Serrano


title: Constrained transcriptional polarity in the organization of mammalian Hox gene clusters (ChIP-seq)

first: Fabrice

last: Darbellay

overall_design: ChIP-seq analysis of H3K4me3 and H3K27me3 in two mutant HoxD alleles

first: Lucille

last: Delisle-Lopez

first: Denis

last: Duboule


title: Re-education of tumor-associated macrophages by CXCR2 blockade drives senescence and tumor inhibition in advanced prostate cancer

city: Bellinzona

first: Andrea

last: Rinaldi

state: Ticino

address: via vela 6

institute: IOR - oncology institute of southern switzerland

summary: Tumor-associated macrophages (TAMs) represent a major component of the tumor microenvironment that supports tumorigenesis. Here we found that prostate tumors are strongly infiltrated by TAMs expressing the C-X-C chemokine receptor type 2 (CXCR2). Pharmacological blockade of the CXCR2 receptor by a selective antagonist promoted the re-education of TAMs towards a pro-inflammatory phenotype. In vitro gene expression profiling revealed that CXCL2 promotes a pro-tumorigenic (M2-like) functional state in macrophages.

overall_design: In vitro gene expression profiling of macrophagesand sorted CD45+ negative epithelial cells

first: Diletta

last: Di Mitri

first: Jelena

last: Vasilevska

first: Michela

last: Mirenda

first: Arianna

last: Calcinotto

first: Nicolas

last: Delaleu

first: Ajinkya

last: Revandkar

first: Veronica

last: Gil

first: Gunther

last: Boysen

first: Marco

last: Losa

first: Simone

last: Mosole

first: Emiliano

last: Pasquini

first: Rocco

last: D'antuono

first: Elena

last: Zagato

first: Giovanna

last: Chiorino

first: Paola

last: Ostano

first: Letizia

last: Gnetti

first: Mariona

last: Graupera

first: Ana

middle: R

last: Fonseca

first: David

last: Waugh

first: Simon

last: Barry

first: Johann

last: De Bono

last: Alimonti


title: Constrained transcriptional polarity in the organization of mammalian Hox gene clusters (4C-seq)

first: Fabrice

last: Darbellay

summary: n many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, for the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters towards a tighter structure during the evolution of the vertebrate lineage. Here we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster such as to invert one or several transcription unit(s), with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this genetic system, and the distinct problems elicited by these inversion alleles illustrate various aspects of the intricate regulations implemented at these loci. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in-cis.

overall_design: 4C-seq analysis of Hoxd9 and lacZ interactions in E13.5 metanephros in two HoxD mutant alleles.

first: Lucille

last: Delisle-Lopez

first: Denis

last: Duboule


title: RNA-seq of DV90 cells with REG4 siRNA

status: Public on Jun 01 2019

city: Shanghai

first: Shenglin

last: Huang

address: 270 Dongan

institute: Fudan University

summary: DV90 cells with REG4 siRNAs were subjected to RNA isolation and RNA-seq analyses were generated by deep sequencing using Illumina Hiseq.

overall_design: RNA-seq analyses of DV90 cells with REG4 siRNA by deep sequencing using Illumina Hiseq.

first: Hui

last: Yu


title: Enhanced DNA repair and genomic stability identify a novel HIV related Diffuse Large B-cell Lymphoma subtype [nanostring]

first: Alanna

last: Maguire

summary:
Transcriptional and genomic profiling study of HIV positive and HIV negative Diffuse Large B-Cell Lymphoma (DLBCL). Tumors were subtyped using the Lymph2Cx assay. Only GCB (germinal center like B-cell) subtypes were then subjected to digitial gene expression profiling and array comparative genomic hybridization (aCGH).
The study used total RNA extracted from FFPE DLBCL tumors derived from HIV(+) and HIV(-) patients to assess differential expression of known cancer genes. Gene expression was correlated with IHC and array CGH data.

overall_design:
Digital gene expression profiling of 739 cancer related genes was performed on 19 HIV positive and 21 HIV negative GCB-DLBCL samples using the PanCancer Pathways panel and nCounter Technology from NanoString with customized spike-ins.
DLBCL is more agressive in HIV(+) patients compared to HIV(-) individuals. Study objective was to identify novel markers and potential therapeutic targets that contribute to the enhanced agressive nature of this disease in HIV infected individuals.

first: Xianfeng

last: Chen

first: Lee

last: Wisner

first: Smriti

last: Malasi

first: Colleen

last: Ramsower

first: Samantha

last: Kendrick

first: Michael

last: Barrett

first: Betty

last: Glinsmann-Gibson

last: McGrath

first: Lisa

last: Rimsza


title: miR-100 maintains phenotype of tumor associated macrophages by targeting mTOR to promote tumor metastasis via Stat5a/IL-1ra pathway in mouse breast cancer

first: Wei

last: Wang

address: No.5 Dongdansantiao

institute: PUMC

type: Non-coding RNA profiling by high throughput sequencing

summary: Tumor-associated macrophages (TAMs)，the main part of immune cells in tumor microenvironment (TME)，play a potent role in promoting tumorigenesis through mechanisms such as stimulating angiogenesis, enhancing tumor migration and suppressing antitumor immunity. MicroRNAs (miRNAs) are considered as crucial regulators in multiple biological processes. The relationship between miRNAs and macrophages function has been extensively reported, but the roles that miRNAs play in regulating TAMs phenotype remains unclear. In this study, we screened highly-expressed microRNAs in TAMs, and first identified that miR-100 represented a TAMs-high expression pattern and maintained TAMs phenotype by targeting mTOR signaling pathway. Moreover, miR-100 expression level in TAMs was positively related to IL-1ra secretion, a traditional immune-suppressive cytokine, which was determined to promote tumor cells stemness via stimulating Hedgehog pathway. Mechanism study suggested that mTOR/Stat5a pathway was involved in IL-1ra transcriptional regulation process mediated by miR-100. More importantly, tumor metastasis and invasion capacity were significantly decreased in a 4T1 mouse breast cancer model injected intratumorally with miR-100 antagomir, and combination therapy with cisplatin showed much better benefit. In this study, we confirmed that highly expressed miR-100 maintains the phenotype of TAMs and promotes tumor metastasis via enhancing IL-1ra secretion. Interfering miR-100 expression of TAMs in mouse breast cancer model could inhibit TAMs pro-tumor function and reduce tumor metastasis, which suggested that miR-100 could serve as a potential therapy target to remodel tumor microenvironment in breast cancer.

overall_design: Tumor associated macrophages isolated from mouse breast tumor tissues were performed microRNA Solexa sequencing,using IlluminaGAIIX , macrophages from naïve healthy mice spleen were viewed as control.

first: Yan

last: Liu


title: Secreted Protein Acidic and Rich in Cysteine Improves Glucose Tolerance via AMP-activated Protein Kinase Activation

city: Kyoto

first: Wataru

last: Aoi

address: 1-5 Hangicho Shimogamo, Sakyo-ku

country: Japan

institute: Kyoto Prefectural University

summary:
Metabolic dysfunction of skeletal muscle is often prevalent at an early stage in the development of several non-communicable diseases. Here, we investigated the effect of a myokine, secreted protein acidic and rich in cysteine (SPARC), on glucose tolerance in human and mouse skeletal muscles.
SPARC knockout mice showed marked decreases in parameters for whole-body glucose metabolism, along with reduced phosphorylation of AMPK and Akt in skeletal muscle tissues compared with wild-type mice. Furthermore, mice injected with SPARC showed improved glucose tolerance concomitant with AMPK activation. Exogenous SPARC treatment accelerated glucose uptake in muscle tissues isolated from wild-type mice but not from AMPKγ3 knockout mice. In muscle cells, SPARC increased glucose uptake concomitant with AMPK activation, mediated by a calcium-dependent signal. Chronic treatment of SPARC restored metabolic functions in diet-induced obese mice. These findings suggest that SPARC improves glucose metabolism via AMPK activation in skeletal muscle, providing mechanistic insights on exercise-induced metabolic benefits and physical inactivity-induced glucose intolerance.

overall_design: We performed a microarray analysis to compare the metabolic gene expression profiles in the skeletal muscle from each mouse.

first: Nariyuki

last: Hirano

first: David

middle: G

last: Lassiter

first: Marie

last: Björnholm

first: Alexander

middle: V

last: Chibalin

first: Kunihiro

last: Sakuma

first: Yuko

last: Tanimura

first: Katsura

last: Mizushima

first: Tomohisa

last: Takagi

first: Yuji

last: Naito

first: Juleen

last: Zierath

first: Anna

last: Krook


title: Constrained transcriptional polarity in the organization of mammalian Hox gene clusters (ChIPmentation-seq)

first: Fabrice

last: Darbellay

overall_design: ChIPmentation-seq analysis of CTCF and RAD21 in wild-type and mutant HoxD alleles

first: Lucille

last: Delisle-Lopez

first: Denis

last: Duboule


title: Transcriptional profile of pDyn-lineage spinal interneurons in the developing mouse

city: Cincinnati

first: Mark

last: Baccei

state: OH

address: 231 Albert Sabin Way

department: Anesthesiology

institute: University of Cincinnati

summary:
Purpose: Mounting evidence suggests that the spinal dorsal horn (SDH) contains multiple subpopulations of inhibitory interneurons that play distinct roles in somatosensory processing, as exemplified by the importance of spinal dynorphin-expressing neurons for the suppression of mechanical pain and chemical itch.  While it is clear that GABAergic transmission in the SDH undergoes significant alterations during early postnatal development, little is known about the maturation of discrete inhibitory “microcircuits” within the region.  As a result, the goal of the present study was to elucidate the gene expression profile of spinal dynorphin (pDyn)-lineage neurons throughout life.
Methods: We isolated nuclear RNA specifically from pDyn-lineage SDH interneurons at postnatal days 7, 21, and 80 using the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique in conjunction with Fluorescence-activated Nuclei Sorting (FANS), followed by  RNAseq analysis.
Results: Over 650 genes were ≥2-fold enriched in adult pDyn nuclei compared to non-pDyn spinal cord nuclei, including targets with known relevance to pain such as galanin (Gal), prepronociceptin (Pnoc), and nitric oxide synthase 1 (Nos1).  In addition, the gene encoding a membrane-bound guanylate cyclase, Gucy2d, was identified as a novel and highly selective marker of the pDyn population within the SDH.  Differential gene expression analysis comparing pDyn nuclei across the three ages revealed sets of genes that were significantly upregulated (such as Cartpt encoding cocaine- and amphetamine-regulated transcript peptide) or downregulated (including Npbwr1 encoding the receptor for neuropeptides B/W) during postnatal development.
Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: Transcriptional profiles of pDyn-lineage spinal cord neurons were generated from nuclear RNA and compared to non-pDyn lineage spinal cord nuclei at postnatal ages P7, P21, and P80.  Additionally, the transcriptional profiles of pDyn-lineage nuclei were compared between the three postnatal ages to elucidate age-dependent changes in gene expression.

first: Elizabeth

last: Serafin

last: Chamessian

first: Jie

last: Li

first: Xiang

last: Zhang

first: Amanda

last: McGann

first: Chelsie

last: Brewer

first: Temugin

last: Berta


title: Genome wide expression profiling of PRMT double deletion strains

city: Innsbruck

first: Ingo

last: Bauer

state: Tirol

address: Innrain 80-82

department: Institute of Molecular Biology

country: Austria

institute: Medical University of Innsbruck

summary: In filamentous fungi, arginine methylation has been implicated in morphogenesis, mycotoxin biosynthesis, pathogenicity, and stress response although the exact role of this posttranslational modification in these processes remains obscure. Here, we present the first genome-wide transcriptome analysis in filamentous fungi that compared expression levels of genes regulated by type I and type II protein arginine methyltransferases (PRMTs).

overall_design: Three strains  (wild type, rmtA/rmtB deletion, and rmtB/rmtC deletion) grown for 24 h  were analyzed in triplicates.

first: Angelo

last: Pidroni

first: Gerald

last: Brosch

first: Stefan

last: Graessle


title: Cell-type-specific differential methylation in rheumatoid arthritis peripheral blood samples

city: San Francisco

first: Lindsey

middle: A.

last: Criswell

state: CA

address: 513 Parnassus Avenue

institute: UCSF

summary: We measured 371 genome‐wide DNA methylation profiles of sorted peripheral blood samples from 63 patients with rheumatoid arthritis and 31 unaffected control subjects. Methylation profiles were measured for cell subsets of CD14+ monocytes, CD19+ B cells, CD4+ memory T cells, and CD4+ naive T cells.

overall_design: Examination of DNA methylation from cell subsets in peripheral blood using the Illumina HumanMethylation450 BeadChip.

first: Lisa

middle: F

last: Barcellos


title: Profiling of protrusion-enriched RNAs from human breast cancer cell line MDA-MB-231

city: Houston

first: Pengchao

last: zhang

state: TX

address: 6670 Bertner Ave

department: Nanomedicine

institute: Houston Methodist Research Institute

summary: The goals of this study are to compare the gene expression of cell protrusions and corresponding cell bodies by isolating cell protrusions from cultured MDA-MB-231 cells in a novel microfluidic device.

overall_design: mRNA profiles of cell protrusions and corresponding cell bodies of MDA-MB-231 cells were gnertated by deep sequencing, in five replicates, using Illumina Hiseq 4000.

first: Lidong

last: Qin


title: Functional dissection of regulatory landscapes reveals non-essential and instructive roles of TADs in regulating gene expression

city: Berlin

first: Daniel

middle: Murad

last: Ibrahim

state: Berlin

address: Augustenburger Platz 1

department: Center for Regenerative Therapies

country: Germany

institute: Berlin Institute of Health

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

overall_design: Refer to individual Series


title: Functional dissection of regulatory landscapes reveals non-essential and instructive roles of TADs in regulating gene expression [ChIP-Seq]

summary: The genome is organized in megabasesized three-dimensional units, called Topologically Associated Domains (TADs), that are separated by boundaries. TADs bring distant cis-regulatory elements into proximity, facilitated by the cooperative action of cohesin and the DNA binding factor CTCF. However, how TADs and their boundaries impinge on enhancer function remains an open question. Here, we investigate TAD function in vivo in mice at the Sox9/Kcnj locus. We find that TADs are formed by a redundant system of CTCF sites requiring the removal of all major sites within the TAD and at the boundary for two neighboring TADs to fuse. TAD fusion resulted in a low degree of regulatory spread from the Sox9 to the Kcnj TAD, but no major changes in gene expression, indicating that TAD structures provide robustness and precision, but are not essential for developmental gene regulation. Gene misexpression and consecutive disease phenotypes, however, were attained by re-directing regulatory activity through inversions and/or the re-positioning of boundaries. Thus, efficient re-wiring of enhancer promoter interaction and aberrant disease causing gene activation is not induced by a mere loss of insulation but requires the re-direction of contacts.

overall_design: We used ChIP-seq to identify the binding profiles of H3K27ac and CTCF in the limb buds of E12.5 BL6 mice

first: Alexandra

last: Despang

first: Ivana

last: Jerkovic


title: PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression

city: Mexico City

first: David

last: Valle-Garcia

address: Av. Insurgentes Sur 3877, La Fama, Tlalpan

country: Mexico

institute: National Institute of Neurology and Neurosurgery


title: E11.5 Hand1 myocardial knockout RNA-Seq

city: Indianapolis

first: Anthony

last: Firulli

state: IN

address: 1044 W Walnut R4 351

department: Pediatrics - Wells Center

institute: Indiana University School of Medicine

summary:
Aims: To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium.
Methods and Results: Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5-13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or a-myosin heavy chain Cre (aMhc-Cre) driver. Interrogation of transcriptome data via Ingenuity Pathway Analysis (IPA) reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects (VSDs), and abnormal LV papillary muscles (PM). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure.
Conclusions: Collectively, these data reveal that Hand1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicate a role for Hand1 conduction system and papillary morphogenesis.

overall_design:
The goal is to understand changes in gene expression in loss-of-function Hand1 hearts
RNA-seq profiling of Hand1 flox mice and Nkx2.5^Cre mice control Nkx2.5^Cre; Hand1^f/f mice ventricles.

middle: B

first: Beth

first: Hongyu

last: Gao


title: Comprehensive Proteogenomic Analysis of Human Embryonic and Induced Pluripotent Stem Cells

status: Public on Jun 02 2019

city: Munich

first: Gianluca

last: Santamaria

state: Germany

address: Ismaninger Strasse 22

department: Department of Medicine I

institute: Klinikum rechts der Isar – Technical University Munich

summary: Although the concepts of reprogramming and human induced pluripotent stem cells (hiPSCs) generation have undergone to several analysis to strengthen the usefulness of these cells in research and clinic, it still remains controversial whether the hiPSCs are really equivalent to human embryonic stem cells, pointing to the need of further characterizations in order to gain a more comprehensive understanding of human stem cells pluripotency. Most of the experimental evidence comes from the transcriptome analysis, while a little information is available on protein data, and even less is known about the post-translational modifications. Here we report a combined strategy of nLC-MS/MS and gene expression profiling for proteogenomic analysis of two distinct stem cell lines, the commercially available hESCs H9 and the hiPSCs, obtained in our laboratory by reprogramming of peripheral blood T-lymphocytes with the Sendai virus-driven Yamanaka’s 4-transcription factors cocktail. The analysis was further enriched by the study of the phosphoproteomic profile of the two PSC lines. The results obtained through this integrated, multi -“omics” approach, indicate that a small number of distinct pathways is enriched in reprogrammed versus naïve pluripotent stem cells, supporting the view that pluripotency is an extremely complex, multifaceted phenomenon, with peculiarities that are characteristic of each cell type.

overall_design: human induced pluripotent stem cells (hiPSCs) and H9 cells; triplicates

first: Elvira

last: Parrotta


title: m6Am-Exo-Seq mapping of MEL624 control and PCIF1 KO cells

summary: We analyzed m6Am distribution by m6Am-Exo-Seq in order to determine the mRNAs marked by m6Am in human melanoma cells

overall_design:
The experiment was divided in two stages. In the first stage, mRNA from one clonal PCIF1 KO cell line and one control cell line was treated using the m6Am-Exo-Seq protocol and immunoprecipitated with 8 different m6A antibodies. RNA-Seq libraries for Illumina sequencing from Input and immunoprecipitated mRNA were prepared and sequenced in a NextSeq witn an average coverage of 15 million reads per sample. In the second stage, mRNA from three biological replicates from control and PCIF1 KOs was treated with the m6A-Exo-Seq protocol and immunoprecipitated with a single m6A antibody. RNA-seq libraries were prepared from input and immunoprecipitated mRNA and sequenced in a NextSeq with an average coverage of 35 Million reads for Inputs and 18 Million reads for IPs

Here is the antibody information;
Ab1: Monoclonal Rabbit antibody from the EpiMark N6-Methyladenosine Enrichment Kit
Ab2: Mouse monoclonal, clone 80C9, gift of Synaptic Systems
Ab3: Mouse monoclonal, clone 345E11, Synaptic Systems cat # 202 011
Ab4: Mouse monoclonal, clone 212B11, Synaptic Systems cat # 202 111
Ab5: Rabbit polyclonal, affinity purified, batch #8699, gift of Synaptic Systems
Ab6: Rabbit polyclonal, affinity purified, batch #8700, gift of Synaptic Systems
Ab7: Rabbit polyclonal, affinity purified, batch #8701, gift of Synaptic Systems
Ab8: Rabbit polyclonal, affinity purified, batch #8702, gift of Synaptic Systems
For the samples where antibody number is not specified, antibody 4 (Synaptic Systems cat # 202 111) was used.

first: Yang

last: Shi


title: Gene expression atlas of a developing tissue by single cell expression correlation analysis

city: Heidelberg

first: Erica

last: Valentini

address: Im Neuenheimer Feld 580

institute: DKFZ

summary: The Drosophila wing disc has been a fundamental model system for the discovery of key signaling pathways and for our understanding of developmental processes. However, a complete map of gene expression in this tissue is lacking. To obtain a complete gene expression atlas in the wing disc, we employed single-cell sequencing (scRNA-seq) and developed a new method for analyzing scRNA-seq data based on gene expression correlations rather than cell mappings. This enables us to discover 824 genes with spatially restricted expression patterns, and to compute expression maps for all genes in the wing disc. This approach identifies both known and new clusters of genes with similar expression patterns and functional relevance. As proof of concept, we characterize the previously unstudied gene CG5151 and show it regulates Wnt signaling. This novel method will enable the leveraging of scRNA-seq data for generating expression atlases of undifferentiated tissues during development.

overall_design: Single cell transcriptome experiments from female wandering 3rd instar wing discs  were generated: two samples using Drop-seq and one sample using the 10x genomics platform. Bulk polyA-RNA-seq experiment from the same tissue was conducted for comparison.

first: Josephine

last: Bageritz

first: Philipp

last: Willnow

first: Svenja

last: Leible

first: Michael

last: Boutros

first: Aurelio

last: Teleman


title: Genome-wide characterisation of replication and transcription changed induced by G4-stabilisation with PhenDC3

city: Montpellier

first: Marcel

last: Mechali

address: 141, Rue de la Cardonille

department: Replication and Genome Dynamics

country: France

institute: Institute of Human Genetics, CNRS

summary: Genome-wide characterisation of replication and transcription programs upon G4-stabilisation by PhenDC3

overall_design: Genome-wide maps of replication origins in PhenDC3 treated cells

first: Benoit

last: Ballester


title: m6A MeRIP-Seq of MEL624 control and PCIF1 KO cells

summary: We analyzed m6A distribution by m6A MeRIP in otder to determine the effect of PCIF1 KO over m6A distribution in human melanoma cells

overall_design:
mRNA from two independent clonal PCIF1 KO cell lines and one control cell line was immunoprecipitated using an antibody against m6A. RNA-Seq libraries were generated from three biological replicates for Input (RNAseq) and m6A-immunoprecipitated mRNA. Libraries were sequenced in a HiSeq with an average coverage of 20 million reads per sample.

Please note that the m6A rabbit monoclonal antibody from the EpiMark N6-Methyladenosine Enrichment Kit was used. This antibody is produced by Cell Signaling and distributed by NEB in the kit, but is not sold independently and doesn't have a catalog number.

first: Wanqiang

last: Sheng


title: Functional dissection of regulatory landscapes reveals non-essential and instructive roles of TADs in regulating gene expression [Capture Hi-C-seq]

first: Daniel

last: Ibrahim

overall_design: We used Capture Hi-C enriching a 6 Mb region at the Sox9locus in Mus musculus to study the effects of CRISPR/Cas9-induced CTCF-site deletions, insertions, and inversions on the genomic landscape. We analysed wildtype and homozygous mutant limb buds (from 9 mutants) as singleton (limb buds of 3-8 embryos for each condition), as it was done in similar studies (See Franke et la., Nature, 2016 or Bianco et al., Nat. Gen. 2018). Embryonic E12.5 limb tissue was used.

first: Alexandra

last: Despang

first: Robert

last: Schöpflin


title: Pro-Seq of MEL624 control and PCIF1 KO cells

first: David

last: Valle-Garcia

summary: We analyzed human melanoma cells by PRO-Seq in order to determine the effect of PCIF1 KO in Pol II dynamics

overall_design: PRO-seq libraries from 3 independent biological replicates were generated for Control MEL624 cells, PCIF1-KO clone 1 and PCIF1-KO clone 2, using 1 million cells per sample. Samples were spiked with 40,000 Drosophila S2 cells as a normalization spike-in control. Samples were sequenced on the NextSeq using a High Output 75-cycle kit, generating paired end 42 nt reads. Libraries were sequenced to an average read depth of 50 million mappable reads per sample.

first: Telmo

last: Henriques

first: Karen

last: Adelman


title: Identification and functional analysis of yap1b: a new Yap family member in teleosts that evolved a divergent transcriptional activation domain.

first: Javier

last: Vázquez-Marín

state: Baden-Württemberg

address: Im Neuenheimer Feld 230, 5th floor

department: Centre for Organismal Studies (COS)

institute: Heidelberg University

summary: The vertebrate paralogs Yap1 and Taz are well known Hippo pathway effectors playing a key regulatory process in growth, morphogenesis, cell survival and cancer progression. Upon translocation to the nucleus Yap/Taz interact with Tead proteins and regulate transcription through their conserved C-terminal activation domain. Here we describe yap1b a new teleost paralog of the Yap1/Taz family that evolved specifically in Euteleostei -the largest clade within vertebrates- by duplication and subsequent adaptation of yap1. In contrast, taz paralogs were progressively lost in the Euteleostei lineage. We show that yap1b displays a unique C-terminal transcriptional activation domain that cannot be phosphorylated by kinases of the Src/Yes family. Using DamID-seq, we obtained comparative maps of yap1 and yap1b occupancy in the medaka genome during early embryogenesis; as well as of yap1 and taz in the zebrafish embryo. Our analyses show that both medaka paralogs target a largely overlapping set of genes. To further understand yap1b role, we mutated it by CRISPR-Cas9 in medaka. Yap1b mutants do not display any overt phenotype during embryogenesis or adulthood. However, yap1b mutation strongly enhances the early embryonic malformations described for medaka yap1 mutants. Thus yap1-/- ; yap1b-/- double mutants display more severe body flattening, eye misshaping, and increased apoptosis than yap1-/- single mutants. This negative epistatic interaction indicates overlapping gene functions between yap1 and yap1b. Taken together, our results indicate that, despite its divergent C-terminal transactivation domain, the newly evolved paralog yap1b cooperates with yap1 in regulating cell survival and tissue morphogenesis during early development.

sample_organism:
Danio rerio
Oryzias latipes

platform_organism:
Danio rerio
Oryzias latipes

overall_design: iDamID-seq data from zebrafish and medaka late gastrula embryos, injected with DAM-zYAP1/DAM-zTAZ and DAM-mYAP1/DAM-mYAP1B mRNA, respectively, including their corresponding DAM-GFP control peaks.

first: Juan

last: Martínez-Morales


title: Defective expression of mitochondrial, histone and vesicle genes in a C. elegans SMA model

city: Changsha

first: Long

last: Ma

address: 110 Xiangya Road, Kaifu District

institute: Central South University

summary: Spinal muscular atrophy (SMA) is a severe congenital motor neuron degenerative disease caused by loss-of-function mutations in the conserved survival motor neuron gene SMN1. SMN is required for the assembly of small nuclear ribonucleoprotein complexes, the core components of the spliceosome and also affects other molecular processes. It is hypothesized that defective gene expression and pre-mRNA splicing underlie SMA. However the immediate target genes of SMN are unclear. We used the nematode Caenorhabditis elegans as a model to study the function of SMN. We analyzed the transcriptome of a C. elegans smn-1 deletion mutant and identified that multiple biological processes were affected. Specifically the expression of numerous mitochondrial genes and vacuolar H+ -ATPase was significantly reduced while the expression of multiple histone genes was significantly increased. We also found that the U2AF large subunit mutation n4588 could ameliorate the defective expression of genes in the mitochondrial and V-ATPase pathways caused by the smn-1 mutation, which is consistent with our previous finding that uaf-1(n4588) could improve the behavior and lifespan of smn-1 mutants. Our study identified key molecular processes affected by SMN and suggests that mitochondrial and V-ATPase dysfunctions might contribute to SMA.

overall_design: Detect expression differences across N2, smn-1(ok355), uaf-1(n4588) and smn-1(ok355); uaf-1(n4588) on day 2 post the L1 larval stage. At least two replicates were used for each genotype.

first: Xiaoyang

last: Gao


title: Linking YAP to Müller glia quiescence exit in the degenerative retina

city: Saclay

first: Jerome

middle: Eric

last: Roger

state: Please select

address: 151 Route de la Rotonde, Campus CEA Saclay - Bat 151

department: CERTO

institute: CNRS-Universite Paris Sud

summary: Contrasting with fish or amphibian, retinal regeneration from Müller glial cells is largely limited in mammals. In our quest towards the identification of molecular cues that may boost their stemness potential, we investigated the involvement of the Hippo pathway effector YAP, which we previously found to be upregulated in Müller cells following retinal injury. We report that conditional Yap deletion in Müller cells prevents the upregulation of cell cycle genes that normally accompanies reactive gliosis upon photoreceptor cell death. This occurs as a consequence of defective EGFR signaling. Consistent with a function of YAP in triggering Müller glia cell cycle re-entry, we further show that in Xenopus, a species endowed with efficient regenerative capacity, YAP is required for their injury-dependent proliferative response. Finally, and noteworthy, we reveal that YAP overactivation in mouse Müller cells is sufficient to induce their reprogramming into highly proliferative cells. Overall, we unravel a pivotal role for YAP in tuning Müller cell response to injury and highlight a novel YAP-EGFR axis by which Müller cells exit their quiescence state, a critical step towards regeneration.

overall_design: Retinal samples were harvested from Yapflox/flox;  Rax-CreERT2 mouse line allowing for Cre-mediated conditional gene ablation specifically in Müller cells. It is named Yap CKO while “control” refers to Yapflox/flox mice. Yap deletion was induced in fully differentiated Müller cells, through 4-hydroxytamoxifen (4-OHT) intraperitoneal injection at P10. All animals were injected with 4-OHT.  Each sample included 1 frozen retina and experiments were performed in triplicate. RNA-seq transcriptome libraries were constructed from 1 ug of total RNA.

first: Annaïg

last: Hamon

first: Divya

last: Ail

first: Diana

last: García-García

first: Juliette

last: Bitard

first: Deniz

last: Dalkara

first: Morgane

last: Locker

first: Jérôme

middle: E

first: Muriel

last: Perron


title: Functional dissection of regulatory landscapes reveals non-essential and instructive roles of TADs in regulating gene expression [ATAC-Seq]

first: Daniel

last: Ibrahim

overall_design: We used ATAC-seq to compare chromatin accessibility in 3 different tissues and to compare the accessibility of the enhancer in WT and mutant animals.

first: Christina

last: Paliou


title: The synarcual of the little skate: Novel development among the vertebrates

city: Newcastle upon Tyne

first: Katherine

last: James

address: Urban Sciences Building

department: School of Computing

country: United Kingdom

institute: Newcastle University

summary: The synarcual is a specialized adaptation of the anterior axial skeleton comprising a putatively fused array of vertebral elements characteristic of jawed vertebrate (gnathostome) clades such as batoid and chimaeroid chondrichthyans, as well as a fossil group known as the placoderms. Placoderms represent the phylogenetically most basal jawed vertebrates and the presence of a synarcual in these and chondrichthyans may suggest a conserved vertebral type for jawed vertebrates, predating the divergence of stem and crown gnathostomes. Alternatively, synarcuals may have evolved independently in these lineages, exhibiting a remarkable case of morphological convergence. We investigated the early development of the cervicothoracic synarcual of an emerging model chondrichthyan, the Little skate Leucoraja erinacea, by combining x-ray computed tomography, classical histology, and a de novo transcriptome assembly for two developmental stages of the skate synarcual and post-synarcual axial skeletal elements.

overall_design: We produced a de novo transcriptome assembly and expression analysis from two developmental stages (28 and 33) of the skate,  Leucoraja erinacea, synarcual (Syn) and post-synarcual (Psyn) axial skeletal elements.

first: Zerina

last: Johanson

first: Gareth

last: Fraser

first: Kyle

last: Martin


title: S. coelicolor, control (untreated) vs ciprofloxacin-treated

city: Powai, Mumbai

first: Sarika

last: Mehra

state: MH

address: IIT Bombay

department: Chemical Engineering

country: India

institute: IIT Bombay

summary:
Transcriptional profiling of S. coelicolor cells treated with sub-inhibitory or inhibitory concentrations of ciprofloxacin in comparision to untreated control when cultured in R5 media.
Two-condition experiment, Control Vs CIP treatment.

overall_design: 10 samples and seven biological replicates to study the response of S. coelicolor cells to ciprofloxacin

first: Minal

last: Patkari


title: Transcriptomic comparison of Listeria monocytogenes EGDe and its Δlmo0599 mutant.

status: Public on May 31 2019

city: Wernigerode

first: Samuel

last: Hauf

address: Burgstr. 37

department: Division of Enteropathogenic Bacteria and Legionella (FG11)

institute: Robert Koch-Institute

summary: Using RNA-Seq we show that the PadR-type transcriptional repressor Lmo0599 (LltR) mainly regulates expression of the lmo0599-0600-0601 operon.

overall_design: Comparison of wildtype and mutant transcriptomes. Harvest of cells in mid exponential phase (OD600 = 0.5). Three replicates were analyzed.

first: Sven

last: Halbedel


title: Transcriptomic comparison of Listeria monocytogenes EGDe and its Δlmo0422 mutant.

summary: Using RNA-Seq we describe the regulon of the PadR-type transcriptional repressor Lmo0422 (LstR). The main regulatory target of LstR is the lmo0423-0422-0421 operon.


title: Longevity is determined by ETS transcription factors in multiple tissues and diverse species

city: London

first: Adam

last: Dobson

address: Gower Street

department: GEE

institute: University College London

overall_design: Refer to individual Series


title: Spaceflight causes changes in gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina

city: Loma Linda

first: Xiao

middle: Wen

last: Mao

address: 11175 Campus St

department: Basic Sciences

institute: Loma Linda University

summary:
Purpose:The purpose of this study was to determine whether the spaceflight environment induces oxidative damage on ocular structure and how gene expression profiles change during spaceflight.

Methods: RNA sequencing, immunofluorescence, and microCT assays were performed on two groups of mice to assess the effects of spaceflight on the mammalian retina. Ten week old adult C57BL/6 mice was flown aboard the ISS for 35 days and returned to Earth alive. Ground control mice were maintained on Earth in identical flight hardware. Within 38 (+/-4) hours of splashdown, mice were euthanized and ocular tissues were collected for analysis.

Results:RNA sequencing detected 600 differentially expressed genes in spaceflight retina. This set of differentially expressed genes is enriched for genes related to visual perception, the phototransduction pathway, and numerous retina and photoreceptor phenotype categories. Eleven of the differentially expressed genes are associated with the disease retinitis pigmentosa, a retinal disease characterized by dystrophy of the rods and cones of the photoreceptor layer. Analysis of differentially expressed transcription factors indicate changes in chromatin structure, offering clues to the causes of the phenotypic changes observed. Additionally, immunofluorescence assays showed degradation of cone photoreceptors and increased oxidative stress in the retina. Retinal thickness, as well as thickness of the retinal pigment epithelium and choroid layers, were significantly reduced after spaceflight.

Conclusions: These results indicate that retinal performance may decrease over extended periods of spaceflight and cause visual impairment.

overall_design: Retinal RNA-Seq libraries of ten week old adult C57BL/6 mice were constructed (Ovation® Mouse RNA-Seq System; Nugen) and sequencing performed on  Illumina HiSeq 4000

middle: W

first: Eliah

last: Overbey


title: Longevity is determined by ETS transcription factors in multiple tissues and diverse species [PntRNAi]

summary: Ageing populations pose one of the main public health crises of our time. Reprogramming gene expression by altering the activities of sequence-specific transcription factors (TF) can ameliorate deleterious effects of age. Here we explore how a circuit of TFs coordinates pro-longevity transcriptional outcomes, which reveals a multi-tissue and multi-species role for an entire protein family: the E-twenty-six (ETS) TFs. In Drosophila, reduced insulin/IGF signalling (IIS) extends lifespan by coordinating activation of Aop, an ETS transcriptional repressor, and Foxo, a Forkhead transcriptional activator. Aop and Foxo bind the same genomic loci, and we show that, individually, they effect similar transcriptional programmes in vivo. In combination, Aop can both moderate or synergise with Foxo, dependent on promoter context. Moreover, Foxo and Aop oppose the activities of Pnt, an ETS transcriptional activator, effecting a transcriptomic programme that correlates lifespan outcomes. Directly limiting Pnt extended lifespan, suggesting this is how Aop and Foxo promote longevity. The lifespan-limiting role of Pnt appears to be balanced by a requirement for metabolic regulation in young flies, in which the Aop-Pnt-Foxo circuit determines nutrient storage, and Pnt regulates lipolysis and responses to nutrient stress. Molecular functions are conserved amongst ETS TFs, suggesting others may also affect ageing. We show that Ets21C limits lifespan, functioning in the same genetic network as Foxo and IIS. Other ETS TFs appear to play roles in fly ageing in multiple contexts, since inhibiting the majority of the family in intestine, adipose or neurons extended lifespan. We expand the repertoire of lifespan-limiting ETS TFs in C. elegans, confirming their conserved function in ageing. Altogether this study reveals that roles of ETS TFs in physiology and lifespan are conserved throughout the family, both within and between species.

overall_design: PntRNAi in S106 D. melanogaster, polyA RNAseq.

first: A

first: N

last: Alic


title: Autophagy inhibition elicits emergence from metastatic dormancy by inducing and stabilizing Pfkfb3

status: Public on May 30 2019

city: Cleveland

first: William

last: Schiemann

state: Ohio

address: 2103 Cornell Road

institute: Case Western Reserve University

summary: Transcriptome analysis of formalin fixed paraffin embedded murine lung tumors

overall_design: We analyzed the transcriptomes of FFPE tumor tissues obtained using laser capture microdissection. Both primed-3D and unprimed-2D D2.A1 and D2.OR cells (murine breast cancer cell lines) were injected into the lateral tail veins of BALB/c mice. Following lung harvest, processing/embedding, laser capture microdissection, and RNA extraction, RNA derived from these tumors was analyzed using Affymetrix GeneChip Mouse 2.0 ST microarrays.

first: Alyssa

middle: L

last: Flynn

first: Benjamin

middle: C

last: Calhoun

first: Arishya

last: Sharma

first: Jenny

last: Chang

first: Alexandru

last: Almasan

middle: P


title: An RNA binding region in CTCF regulates chromatin looping and CTCF nuclear organization

city: Berkeley

first: Robert

last: Tjian

address: University of California, Berkeley 450 Li Ka Shing #3370

department: MCB

institute: UC Berkeley

summary: The study investigates CTCF/cohesin binding and chromatin looping by ChIP-seq and Micro-C. By ChIP-seq, we determined the genome-wide binding profiles of CTCF and Smc1a cohesin subunit in a knock-in mouse ES cell line (wt-CTCF; clone C59) with endogenously tagged wild type CTCF (FLAG-Halo-mCTCF) and Rad21 (mRad21-SNAPf-V5), and compared them to the same ES line expressing a mutant CTCF (ΔRBR-CTCF; clone C59D2), where we replaced a previously described RNA binding region with a short linker (GDGAGLINS) followed by a 3xHA tag (N576_D611del::3xHA). By Micro-C, we compared nucleosome-resolution chromosome folding maps of the same ES cell lines C59 and C59D2 described above, to determine the effect of deleting CTCF RNA binding region on chromatin looping.

overall_design: 1) ChIP-Seq of CTCF and Smc1a cohesin subunit in mouse ES cells homozygous for FLAG-Halo-mCTCF and mRad21-SNAPf-V5 (C59: wt-CTCF) and in the same ES cell line where we deleted CTCF RNA binding region (C59D2: ΔRBR-CTCF). ChIP-Seq libraries were prepared independently from two ChIP biological replicates, using normal serum IgGs as a control, and including total inputs as reference datasets.   2) Three replicates of Micro-C data were generated in the same cell lines as ChIP-seq. Two replicates (named as “QC”) were initially generated for evaluating library quality and improving experimental protocol. These samples qualitatively and quantitatively reproduce the results by the optimized protocol, regardless a higher percentage of PCR duplicates. High quality and coverage of Micro-C data (named as “highCov”) were generated with an optimized protocol producing over 650M unique pairs per sample. 3) RNA-Seq of total RNA (ribosomal RNA depleted) in mouse ES cells homozygous for FLAG-Halo-mCTCF and mRad21-SNAPf-V5 (C59: wt-CTCF) and in the same ES cell line where we deleted CTCF RNA binding region (C59D2: ΔRBR-CTCF). RNA-Seq libraries were prepared from two biological replicates.

first: Claudia

last: Cattoglio

first: Tsung-Han

middle: S

last: Hsieh

first: Anders

last: Hansen

first: Xavier

last: Darzacq


title: Longevity is determined by ETS transcription factors in multiple tissues and diverse species [FxAxP]

overall_design: foxo, aopACT and pntP1 overexpression in S106 D. melanogaster, polyA RNAseq.


title: Transcriptomic comparison of Listeria monocytogenes EGDe and its Δlmo1408 mutant.

first: Samuel

last: Hauf

summary: Using RNA-Seq we confirmed that the PadR-type transcriptional repressor Lmo1408 (LadR) regulates expression of lmo1409 (mdrL), lmo1617 (mdrM) and lmo1618.

overall_design: Comparison of wildtype and mutant cell transcriptomes. Harvest of cells in mid exponential phase (OD600 = 0.5). Three replicates were analyzed.

first: Sven


title: Bioaccumulation, cellular and molecular effects in adult zebrafish after exposure to cadmium sulphide quantum dots and ionic cadmium

city: Leioa

first: José María

last: Lacave

state: Bizkaia

address: Sarriena s/n

department: Zoology and Animal Cell Biology

country: Spain

institute: University of the Basque Country

summary: Few works have addressed the effects provoked by the exposure to cadmium containing nanoparticles (NPs) on adult zebrafish (Danio rerio). We studied the effects of CdS NPs (5 nm) or ionic cadmium (10 µg Cd/L) after 3 and 21 d of exposure and at 6 months post-exposure (mpe). Acute toxicity was recorded after exposure to both forms of cadmium. Significant cadmium accumulation was measured in the whole fish after both treatments and autometallography showed a higher accumulation of metal in the intestine than that in the liver. Histopathological alterations, such as inflammation in gills and vacuolization in the liver, were detected after the exposure to both cadmium forms and, in a lower extent, at 6 mpe. X-ray analysis proved the presence of CdS NPs in these organs. The hepatic transcriptome analysis revealed that gene ontology terms such as “immune response” or “actin binding” were over-represented after 21 d of exposure to ionic cadmium respect to CdS NPs treatment. Exposure to CdS NPs caused a significant effect on pathways involved in the immune response and oxidative stress, while the exposure to ionic cadmium affected significantly pathways involved in DNA damage and repair and in the energetic metabolism. Oxidative damage to liver proteins was detected after the exposure to ionic cadmium, while a stronger destabilization of the hepatocyte lysosomal membrane was recorded under exposure to CdS NPs. In summary, although ionic cadmium provoked stronger effects than CdS NPs, both cadmium forms exerted an array of lethal and sublethal effects to zebrafish.

overall_design: 20 samples per group, 5 replicates (4 liver per replicate), 3 groups x 2 times, control, ionic silver and Ag NPs, 3 and 21 days of exposure

first: Jose

first: Eider

last: Bilbao

first: Douglas

last: Gilliland

first: Francesco

last: Mura

first: Luciana

last: Dini

first: Miren

last: Cajaraville

first: Amaia

last: Orbea


title: Genome-scale DNA methylation analysis for blood-based colorectal cancer detection

city: Chengdu

first: xiaomeng

last: liu

address: No.55,Section 4,South Renmin Road

country: Colombia

institute: Sichuan Cancer Hospital & Institute, Sichuan Cancer Center

type: Methylation profiling by high throughput sequencing

summary: Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-scale DNA hypermethylation analysis technique using plasma is attractive for the noninvasive early detection of CRC. The methylated CpG tandems amplification and sequencing (MCTA-Seq) method was applied to plasma samples from 147 CRC patients and 134 controls in which 5 from the CRC patients and 2 from the control subjects failed to pass a quality control value and were excluded from further analysis. The capacity of the method for discriminating CRC and hepatocellular carcinoma (HCC) was assessed. We identified many DNA methylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5 and CHST11) CpG islands for detecting CRC in blood. A panel of 80 markers significantly discriminated early stage CRC and controls with a sensitivity of 78% and a specificity of 90%. This method can efficiently distinguish between early stage CRC and HCC. MCTA-Seq is a promising method for the noninvasive detection of CRC that also shows great potential for identifying the tissue of origin of cancer.

overall_design: The methylated CpG tandems amplification and sequencing (MCTA-Seq) method was applied to 33 tumor tissues, 33 adjacent tumor tissues and plasma samples from 147 CRC patients and 134 controls in which 5 from the CRC patients and 2 from the control subjects failed to pass a quality control value and were excluded from further analysis.

first: Jingyi

last: Li

first: Xin

last: Zhou

first: Xiaomeng

last: Liu

first: Jie

last: Ren

first: Jilian

last: Wang

first: Bingyan

first: Yuxuan

last: Zheng

first: Tao

last: Sun

first: Anding

last: Kang

first: Fuchou

last: Tang

first: Lu

last: Wen

first: Wei

last: Fu


title: Expression of IDO1-2, iNOS and interferon-inducible GTPases in Chlamydia muridarum infected BALB/c mouse lung tissues

status: Public on May 24 2019

city: Szeged

first: Dezső

middle: Peter

last: Virok

address: Dóm tér 10.

department: Department of Medical Microbiology and Immunobiology

country: Hungary

institute: University of Szeged

summary: We wanted to screen the expression of known and putative antichlamydial genes in vivo. As an open approach, we performed RNA sequencing of Chlamydia muriadrum infected mouse lung tissues. RNA seq revealed that a diverse set of antichlamydial  genes were induced by the infection, including previously described unique human and murine genes. We further characterized the source and activity of indolamine 2,3-dioxygenase genes, previously described as key human effectors.

overall_design: Comparison of Chlamydia muridarum infected and uninfected BALB/c mouse lung tissues

first: Katalin

last: Burián


title: Establishment and characterization of a new intrahepatic cholangiocarcinoma cell line resistant to gemcitabine

city: Biella

first: Paola

last: Ostano

address: via Malta 3

institute: Fondazione Edo ed Elvo Tempia

summary: Intrahepatic cholangiocarcinoma (ICC) is one of the most lethal liver cancers. The late diagnosis as well as the resistance to chemotherapy concurred to the scarce outfit of this malignancy. Even the improvement of the knowledge and, as consequence, of the broad spectrum of alternative therapies available (i.e. targeted, immune checkpoint inhibitors), the survival and the quality of life remain poor. The phenomenon of resistance is not still clear. Here, we established one gemcitabine, GEM, (the standard therapy in ICC) resistant cell line, named MTCHC01R1.5, obtained by a gradual exposure to GEM for 9 months (up to 1.5 µM) of the sensible counterpart MT-CHC01. We biologically and molecularly characterized this cell line comparing its behavior with its parental cell line. The drug resistance is irreversible, even if cells are treated with high doses of GEM. They grew slowly, both in vitro and in vivo, and no differences are highlighted in terms of migration and invasion. GEM resistance promoted a multi drug resistance development, but the drug prediction analysis showed that paclitaxel and doxycycline could overcome GEM resistance, as confirmed by cell viability assay.  There is a global enrichment of up-regulated genes involved in the regulation of cell cycle and in DNA related process. The down-regulated genes are involved in response to stimuli, and xenobiotic metabolism, and angiogenesis. A deregulation of more than 20 genes related to drug resistance and epithelial to mesenchymal transition (EMT) was found. The trend in expression of 9 genes related to resistance is confirmed in a primary cell culture. In conclusion, we characterized a new Italian GEM resistance ICC model which could be exploited either to study the mechanism of resistance or to explore new therapies.

overall_design: Comparison between the parental cell line MT-CHC01 and its resistant counterpart MT-CHC01R1.5

first: Chiara

last: Varamo

first: Caterina

last: Peraldo-Neia

first: Marco

last: Basiricò

last: Raggi

last: Bernabei

first: Tiziana

last: Venesio

first: Enrico

last: Berrino

first: Massimo

last: Aglietta

first: Francesco

last: Leone

first: Giuliana

last: Cavalloni


title: Transcriptome analysis of deletion of protein kinase A, catalytic subunit 1 in T. reesei upon growth on cellulose in light and darkness

status: Public on May 18 2019

city: Vienna

first: Monika

last: Schmoll

address: Djerassiplatz 1

department: Centre of Microbiology and Environmental Systems Science

institute: University of Vienna

summary:
The aim of this study was to evaluate changes in the transcriptome due to the lack of PKAc1 in light or darkness. As the cAMP pathway represents an output pathway of heterotrimeric G-protein signaling
PKAc1 is likely to be important for regulation of enzymes in response to different nutrient conditions. Also, a light dependent effect was found earlier.

overall_design: Strains of this study were grown on Mandels Andreotti Minimal Medium with 1 % w/v microcrystalline cellulose as sole carbon source in constant light or constant darkness for 72 hours; 2 biological replicates were included

first: Andre

last: Schuster


title: Chronic hypoxia changes gene expression profile of primary rat carotid body cells: consequences on the expression of NOS isoforms and ET-1 receptors

status: Public on May 22 2019

first: Matias

last: Mosqueira

state: Baden-Wuttenberg

address: Im Neuenheimer Feld, 326 R414

department: Institute of Physiology and Pathop

institute: Heidelberg University Hospital

summary: Chronic hypoxia (CH) produces changes not fully understood in morphology and function of the carotid body (CB). To characterize the effect of CH, primary rat CB cells were exposed to 7 days of CH, total RNA was extracted, cDNA-32P synthesized and hybridized with 1185 genes printed on a nylon membrane. Out of 324 differentially expressed genes, 184 were up-regulated and 140 were down-regulated. Since data analyses showed that nitric oxide synthases (NOS) and endothelin-1 (ET-1) pathways were enriched, we studied the effect of CH at protein levels of NOS isoforms and ET-1 receptors. CH induced an increase in all NOS and in ET-1 receptor B (ETB). Combining CH and SNAP, iNOS and ETB were significantly up-regulated, whereas the ET-1 receptor A (ETA) was down-regulated, while Tezosentan up-regulated iNOS and ETA and L-NAME induced up-regulation of all NOS. These results described the changes of CH on the CB gene expression profile, affecting a possible interaction in between NOS and ET-1 receptors, as part of the adaptive CB response to CH.

overall_design: Total RNA from CB cultured primary rat cells was isolated by adding 1.0 ml of Trizol (Invitrogen) following manufacturer's instructions. The RNA with an absorbance ratio A260/A280 > 1.6 and a total RNA concentration above 0.3 microgram/ml were used and 1.0 microgram of RNA were amplified using RiboAmp (Arcturus) following the manufacturer's protocol. The amplified RNA with an absorbance relation over A260/A280 > 1.9 and concentration over 1.0 microgram/ml were used to synthesize the cDNA probes. Radiolabeled cDNA probes were synthesized from 6 μg of RNA amplified in the presence of dATP, dTTP, dGTP, [α-32P] dCTP (3000 Ci/mmol), poly T, Superscript II Reverse transcriptase (Invitrogen) and the purified (Atlas NucleoSpin Extraction Spin Columns, BD Biosciences Clontech). Atlas Rat 1.2 Array nylon membranes with 1185 genes (Atlas cDNA Expression Arrays Cat Nº 7854-1, BD Biosciences Clontech) were used according to manufacturer’s procedure. Briefly, the membrane was prehybridized in ExpressHyb hybridization buffer, containing the sheared salmon testes DNA for 30 min at 68 ºC in a rotating hybridization oven. The heat-denatured probe (95 ºC for 5 min) was hybridized overnight at 68 ºC with continuous agitation followed by three washing steps [twice at room temperature for 5 min with Sodium Chloride/ Sodium Citrate solution (1 X SSC = 150 mM NaCl/15 mM sodium citrate, pH 7.0), twice with 2 X SSC plus 1% SDS at 68 ºC for 30 min, and twice with 0.1 X SSC plus 0.5% SDS at room temperature for 30 min]. The membranes were exposed on phosphoimager up to 3 hours at room temperature. Image acquisition and quantification of the average of three membranes under Nx and three under CH were performed using the software AtlasImage V2.0 (Clontech). The normalization was performed to each experiment using a coefficient of normalization (adding the averaging of the signal of all genes to the signal values for all the genes that are above the background signal). The parameters used for the data analysis were the ratio threshold (TR) and the difference threshold (TD).

first: Rodrigo

last: Iturriaga


title: Tissue specific comprehensive gene analysis in a murine lens-induced myopia model

status: Public on May 29 2019

city: Takayama-cho Ikoma-shi, Nara

first: Yasuhisa

last: Tanaka

state: Japan

institute: Santen Pharmaceutical CO., LTD

summary: To identify to identify target tissues and molecules involved with refractive myopic shift and axial length elongation in a murine lens-induced myopia model, we performed comprehensive analysis by microRNA array. Negative 30diptor (-30D) lens was fixed on right eye (-30D) of C57BL/6J mice (3weeks old, N=3) for 3 weeks, the refraction and the axial length were measured using a refractometer and a SD-OCT system in all eyes. Eye balls were enucleated and separated to cornea, iris, lens, retina, choroid and sclera. Total RNA was extracted from individual ocular components. MicroRNA expression analysis was carried out using Agilent Mouse miRNA Microarray (8×60K) miRBase21.0 (Agilent). Expression ratio calculation and miRNA varying expression were extracted by GeneSpring GX 14.5 (Agilent). After 3weeks of lens fixing, a refractive change and an axial length elongation change were observed (Normal vs -30D: 0.95 ± 1.85D vs -18.42 ± 3.98D, 0.155 mm ± 0.015mm vs 0.273 ± 0.009 mm), respectively. MiRNA expression changes that induced only by -30D lens fixing was confirmed in each part of the eyeball. By expression ratio calculation and miRNA varying expression analysis, upregulated miRNA (56 in cornea, 13 in iris, 6 in lens, 0 in retina, 29 in choroid and 30 in sclera) and downregulated miRNA (7 in cornea, 28 in iris, 17 in lens, 9 in retina, 7 in choroid and 40 in sclera) were observed. Overlapping miRNAs were also found while each eye tissues. In this study, miRNA varying expression were observed in each ocular part of the murine lens-induced myopia model. These miRNAs dysregulation may be functionally involved with the refractive myopia shift and the axial length elongation.

overall_design: Eye balls were enucleated and separated to cornea, iris, lens, retina, choroid and sclera. About Each eye tisses, varying miRNA were comfirmed in control group vs lens induced group (Corne control vs cornea lens induced).


title: Next Generation Sequencing analysis using splenic cells freshly isolated from Wild Type-OT-I (WT) and OTUB1-CD4Cre-OT-I (KO) mice

status: Public on May 16 2019

city: Houston

first: Ganiraju

last: Manyam

address: 1515 Holcombe Blvd

institute: UT MD Anderson Cancer Center

summary: CD8 T cells play a central role in immune responses against pathogens and cancer; however, CD8 T cells are typically rendered hypofunctional in tumor microenvironment due to tolerance to cancer cells and functional exhaustion. How to overcome CD8 T cell tolerance and stimulate strong antitumor responses has become an important issue for cancer immunotherapy 1,2. Here we identified the atypical deubiquitinase Otub1 as a pivotal regulator of CD8 T cell self-tolerance and antigen-stimulated responses. T cell-specific deletion of Otub1 in mice breaks CD8 T cell self-tolerance and promotes the metabolic reprograming and effector functions of activated CD8 T cells, resulting in profoundly stronger immunity against infections and tumorigenesis. The Otub1 deletion also synergizes with an immune checkpoint inhibitor in inducing tumor rejection and greatly increases the efficiency of an adoptive T cell transfer model of cancer immunotherapy. In line with these findings, the expression level of Otub1 is inversely associated with the abundance of CD8 effector T cell signature gene expression and patient survival in human melanoma. Mechanistically, Otub1 negatively regulates activation of AKT, a kinase that integrates the T cell receptor (TCR) and cytokine signals and mediates CD8 T cell metabolism and effector functions. Otub1 deficiency greatly promotes AKT activation by both the TCR signal and the immunostimulatory cytokine IL-15. Otub1 inhibits AKT ubiquitination and, thereby, suppresses its interaction with the membrane lipid phosphoinositol 3,4,5 triphosphate required for membrane translocation. These results demonstrate a ubiquitin-dependent mechanism that controls CD8 T cell responses and implicate Otub1 as a potential therapeutic target for cancer immunotherapy.

overall_design: Fresh splenic naïve CD8 T cells or fresh splenic naïve CD8 T cells cultured with plate-bound anti-CD3 (1ug/ml) plus anti-CD28 (1ug/ml) for 24 hours.  mRNA profiles of 6 to 8-week old wild type -OT-I (WT) and OTUB1-CD4Cre-OT-I (KO)  mice were generated by deep sequencing, in triplicate

first: Li

last: Zhang


title: Clonal replacement of tumor-specific T cells following PD-1 blockade [single cells]

city: Stanford

first: Howard

middle: Y

last: Chang

address: CCSR 2130, 269 Campus Drive

department: Center for Personal Dynamic Regulomes

institute: Stanford University

summary: Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients. However, the clonal origin of tumor-specific T cells following checkpoint blockade in patients remains unclear. Here, we performed paired single-cell RNA- and T cell receptor (TCR)- sequencing on site-matched tumors from patients with basal cell carcinoma (BCC) pre- and post-anti-PD-1 therapy. Tracking TCR clonotypes and transcriptome phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction: the response to treatment was accompanied by a clonal expansion of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this response was not accompanied by an expansion of pre-existing tumor-specific T cell clonotypes; rather, expanded T cell clones post-therapy comprised novel clonotypes, which were not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in tumor-specific exhausted CD8+ T cells, compared to other distinct T cell phenotypes, and was more evident in patients who exhibited a clinical response to treatment. These results, enabled by single-cell multi-omic profiling of clinical samples, demonstrate that the chronic activation of pre-existing tumor-infiltrating T cells may limit their re-invigoration following checkpoint blockade, and that a successful response relies on the expansion of a distinct repertoire of tumor-specific T cell clones.

overall_design: Dissociated tumor samples were sorted as either CD45+ CD3+ tumor-infiltrating T cells, other CD45+ CD3- tumor-infiltrating lymphocytes and CD45- CD3- tumor/stromal cells. Sorted cells were subjected to paired single cell RNA- and TCR-sequencing on the droplet based 10X Genomics platform.

first: Kathryn

last: Yost

first: Ansuman

middle: T

last: Satpathy

first: Daniel

middle: K

last: Wells

first: Yanyan

last: Qi

first: Robin

last: Kageyama

first: Chunlin

last: Wang

first: Kavita

last: Sarin

first: Ryanne

middle: A

last: Brown

first: Samantha

last: Bucktrout

first: Mark

middle: M

last: Davis

first: Anne


title: Alpha-ketoglutarate links p53 to cell fate during tumor suppression

status: Public on May 10 2019

city: New York

first: Yu-Jui

last: Ho

state: New York

address: 417 E 68th St

department: Cancer Biology & Genetics Program

institute: Memorial Sloan Kettering Cancer Center

summary: The tumor suppressor TP53 is mutated in the majority of human cancers, including over 70% of pancreatic ductal adenocarcinoma (PDAC). Wild-type p53 accumulates in response to cellular stress and regulates the expression of genes that alter cell fate and constrain tumorigenesis. p53 also modulates several cellular metabolic pathways, though it remains unclear whether particular p53-regulated metabolites contribute to tumor suppression or whether metabolic alterations driven by p53 mutation sustain cancer progression. Here, we show that restoring endogenous p53 function in cancer cells derived from a murine PDAC model driven by oncogenic Kras and a regulatable p53 short hairpin RNA (shRNA) rewires glucose and glutamine metabolism leading to the accumulation of α-ketoglutarate (αKG), an obligate substrate for several chromatin modifying enzymes. p53 induces transcriptional programs characteristic of premalignant differentiation, an effect that can be partially recapitulated by addition of cell permeable αKG. Similarly, enforcing αKG accumulation in p53-deficient PDAC cells though the inhibition of oxoglutarate (αKG) dehydrogenase (Ogdh), the enzyme that consumes αKG in the tricarboxylic acid cycle, reduces tumor-initiating capacity and promotes tumor cell differentiation. Decreases in 5-hydroxymethylcytosine (5hmC), an αKG-dependent chromatin modification, are associated with the appearance of p53 mutations in the transition from premalignant to de-differentiated malignant lesions, whereas increases in 5hmC accompany tumor cell differentiation triggered by either p53 restoration or Ogdh depletion. Together these data nominate αKG as an effector of p53-mediated tumor suppression whose accumulation in p53-deficient tumors can drive tumor cell differentiation and antagonize malignant progression.

overall_design: 6 samples were analyzed in duplicates of 3 conditions.  1. Control, KPsh cells grown on dox, treated with vehicle DMSO for 72 hours. 2. KPsh cells grown on dox, treated with 4mM cell permeable dimethyl-alpha ketoglutarate for 72 hours.  3. KPsh cells grown off dox for 8 days, treated with DMSO vehicle for 72 hours.

first: Lydia

last: Finley

first: Scott

last: Lowe

first: John

last: Morris IV


title: Copy number variation data from iPS cells with PTCHD1-AS deletions

city: Toronto

first: James

last: Ellis

state: ON

address: 686 Bay St

department: Developmental & Stem Cell Biology

country: Canada

institute: The Hospital for Sick Children

summary: iPS cell lines were generated from a male with ASD (proband or prb) and his unaffected mother (control or ctrl). Both individuals carry X-linked 167kb microdeletions that disrupt both PTCHD1 and PTCHD1-AS. We found that cells PTCHD1/PTCHD1-AS-null cells tended to have abnormal karyotypes. Copy number variation analyses were performed to examine genomic stability in control and proband iPS cell lines.

overall_design: Genomic DNA from fibroblasts and from two iPS cell lines from each individual were hybridized to the Affymetrix CytoScanHD SNP array

first: P

middle: J

last: Ross

first: E

last: Deneault


title: RNASeq differential expression analysis from the lung tissue of IL-33 transgenic mice

status: Public on May 14 2019

city: Rochester

middle: Rothschild

last: OBrien

state: MN

address: 200 First St. SW

institute: The Mayo Clinic

summary: To identify the up & down regulated genes in IL-33 transgenic mice

overall_design: An RNASeq experiment and differential expression analysis was performed on lung epithelial cells of IL-33 transgenic mice and control mice

first: Hirohito

last: Kita

first: Li

last: Drake


title: Molecular characterization of the prp40c mutant transcriptome

city: Buenos Aires

first: Mariano

last: Garcia-Hourquet

address: Av Patricias Argentinas 435

country: Argentina

institute: Fundacion Instituto Leloir

summary: The goal of this study was to compare the transcriptional profile (RNA-seq) of prp40c Arabidopsis mutants with their respective wild type plants grown under continous light conditions

overall_design: prp40c mutant plants were grown together with their respective wild type (Col-0) plants for two weeks under continuous white light (50 μmol·m−2·s−1 ) at 22 degrees centigrades and whole seedling RNA was extracted for transcriptional profiling using RNA-seq. Three biological samples of each genotype were analysed.

first: M

last: Yanovsky

first: C

last: Hernando


title: Elevated Exogenous Pyruvate Potentiates Mesodermal Differentiation through Metabolic Modulation and AMPK/mTOR Pathway in Human Embryonic Stem Cells

status: Public on May 06 2019

city: Macau

first: Ren

last: Zhili

address: Avenida da Universidade

institute: University of Macau

summary: Pyruvate is a key metabolite in glycolysis and tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance towards oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and then modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism, but also modulates signaling pathways in hESC differentiation.

overall_design: Total RNA obtained from H1 human embryonic stem cell (hESC) culture in E8 medium (E8_2 Days), E8 medium with Pyruvate 16 mM treatment  (Pyr 16mM_2 Days);  HESCs were treated with BMP4 for 2 or 6 days for mesoderm differentiation in E8 medium, BMP4 treated for 2 days (BMP4_2 Days), BMP4 treatment with Pyruvate 16mM for 2 days (BMP4+Pyr 16mM_2 Days) or for 6 days (BMP4+Pyr 16mM_6 Days).

first: Guokai

last: Chen