title: Glucocorticoid Receptors are Required Effectors of TGFb1-Induced p38 MAPK Signaling to Advanced Cancer Phenotypes in Triple Negative Breast Cancer

status: Public on May 01 2020

city: Minneapolis

first: Carol

last: Lange

state: Minnesota

address: 2231 6th St SE

department: Medicine

institute: University of Minnesota

summary: Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in triple negative breast cancer (TNBC) and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress. We probed GR regulation by factors (cytokines, growth factors) that are rich within the tumor microenvironment (TME). TNBC cells harboring endogenous wild-type (wt) or S134A-GR species were created by CRISPR/Cas knock-in and subjected to transwell migration, invasion, soft-agar colony formation, and tumorsphere assays. RNA-Seq was employed to identify pS134-GR target genes that are regulated both basally (intrinsic) or by TGFβ in the absence of exogenously added GR ligands. Regulation of selected basal and TGFβ-induced pS134-GR target genes was validated by qRT-PCR and chromatin immunoprecipitation assays. Bioinformatics tools were used to probe public data sets for expression of pS134-GR 24-gene signatures. In the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses TGFβ1 and MAPK signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3ζ were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feed-forward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGFβ1 predicts shortened overall survival in breast cancer patients. Phospho-S134-GR is a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic “sensor” of intrinsic stimuli as well as extrinsic factors rich within the TME (TGFβ1) that enables potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC.

overall_design: Examination of the effect of TGFβ1 in TNBC cell line MDA-MB-231 with mutation in the glucocorticoid receptor on Ser134.

first: Carlos

last: Perez Kerkvliet


title: Identification of AflR binding sites in the genome of Aspergillus flavus by ChIP-seq

status: Public on May 02 2020

city: Qingdao

first: Qing

last: Kong

state: Shandong

address: Yushan Road 5

department: Food Science and Engineering

country: China

institute: Ocean University of China

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We report here the AflR binding motif of Aspergillus flavus for the first time with the aid of ChIP-seq analysis. Of the 540 peak sequences associated with AflR binding events, 66.8% were  located within 2 kb upstream (promoter region) of translational start sites. The identified 18-bp  binding motif was a perfect palindromic sequence, 5′-CSSGGGWTCGAWCCCSSG’3′ with S  representing G or C and W representing A or T. On closer examination, we hypothesized that the  18-bp motif sequence identified contained two identical parts (here called motif A and motif B).  Motif A was in positions 8–18 on the upper strand, while motif B was in positions 11-1 on the bottom  strand. The inferred length and sequence of the putative motif identified in A. flavus were similar to  previous findings in A. parasiticus and A. nidulans. Gene ontology analysis indicated that AflR bound  to other genes outside the aflatoxin biosynthetic gene cluster.

overall_design: Examination of AflR binding sites in A. flavus (three replicates)

first: Shihua

last: Shan


title: Global translation during early development depends on the essential transcription factor PRDM10 [RNA-seq]

status: Public on May 06 2020

city: Singapore

first: Brenda

middle: Yuyuan

last: Han

address: 61 Biopolis Drive, Proteos

country: Singapore

institute: Institute of Molecular and Cell Biology, A*STAR

type: Expression profiling by high throughput sequencing

summary: Members of the PR/SET domain-containing (PRDM) family of zinc finger transcriptional regulators play diverse roles in embryonic development and lineage differentiation. PRDM10 is a yet uncharacterized family member, with unknown functions in vivo. Here we report an essential requirement for PRDM10 in the pre-implantation embryo and embryonic stem cells (mESCs), where loss of PRDM10 results in severe cell growth inhibition. Genomic and biochemical analyses reveal that PRDM10 functions as a sequence-specific transcription factor which modulates gene expression by binding to target gene promoters. We identify Eif3b, which encodes a core component of the eukaryotic translation initiation factor 3 (eIF3) complex, as a key downstream target and demonstrate that growth inhibition in PRDM10-deficient mESCs is largely mediated through EIF3B-dependent effects on global translation. This work elucidates the molecular function of PRDM10 in maintaining global translation and establishes its essential role in early embryonic development and mESC homeostasis, offering novel insights into the functional repertoire of PRDMs as well as the transcriptional mechanisms regulating global translation.

overall_design: RNA-seq analysis of gene expression in Prdm10F/F; Rosa26-CreERT2 mouse embryonic stem cells (mESCs) at Day 2 and Day 4 after 4-OHT-induced Prdm10 deletion; 3 replicate samples per condition.

middle: Y

first: Michelle

middle: K

last: Seah

first: Imogen

middle: R

last: Brooks

first: Delia

middle: H

last: Quek

first: Dominic

last: Huxley

first: Chuan-Sheng

last: Foo

first: Li

middle: T

last: Lee

first: Heike

last: Wollmann

first: Huili

last: Guo

first: Daniel

middle: M

last: Messerschmidt

first: Ernesto

last: Guccione


title: Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells

city: PHILADELPHIA

first: Kai

last: Tan

state: PA

address: 3501 CIVIC CENTER BLVD.

country: USA

institute: Children's Hospital of Philadelphia

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

overall_design: Refer to individual Series


title: Genome-wide DNA methylation differences in nucleus accumbens of smokers vs. nonsmokers

city: Research Triangle Park

first: Dana

last: Hancock

state: NC

address: 3040 East Cornwallis Road

institute: RTI International

type: Methylation profiling by genome tiling array

summary: Cigarette smoking remains the leading cause of preventable death worldwide. Cigarette smoking behaviors (e.g., initiation, nicotine dependence, cessation) are heritable, and many genetic risk loci have been identified. However, the neurobiological mechanisms underlying the genetic risk loci along the trajectory of smoking are largely unknown. DNA methylation (DNAm) differences between smokers and nonsmokers can provide insight into mechanisms that predispose to smoking behaviors and consequences of the smoking exposure itself. Differential DNAm by smoking has been found at many CpG sites in blood, but because of the tissue specificity of gene regulation, differential DNAm that can only be detected in brain may have been missed. Here, we provide Illumina HumanMethylationEPIC array data generated in nucleus accumbens, an addiction-relevant brain tissue, from 221 deceased individuals: 53 current cigarette smokers, 168 nonsmokers. From these data, we have conducted an epigenome-wide association study (EWAS) and identified several CpG associations with smoking. A subset of the identified CpGs map to genes that were previously indicated as blood-based smoking DNAm biomarkers, but the other CpGs map to genes that were previously undetected in blood and may reflect brain-specific processes related to smoking.

overall_design: Genome-wide DNAm data were measured in postmortem nucleus accumbens tissue with the HumanMethylationEPIC BeadChip array. The dataset includes 221 deceased individuals: 101 African Americans (23% current cigarette smokers, 77% nonsmokers) and 120 European Americans (26% current cigarette smokers, 74% nonsmokers).


title: Analyses of CED-3 caspase and PMK-1 p38 MAPK in gene regulation in C elegans

city: Dallas

first: Benjamin

last: Weaver

address: 6001 Forest Park Rd

department: Pharmacology

institute: University of Texas Southwestern Medical Center

summary: We identified more than 300 genes inversely regulated by ced-3 and pmk-1 that enriched for epidermal stress response factors in C elegans

overall_design: We analyzed total mRNA and ribosome-associated mRNAs at the L3 stage for wild type, ced-3(n717), and pmk-1(km25) strains with two independent biological replicates

first: B

middle: P

first: Y

middle: W


title: Reducing metastases stiffness by targeting fibroblasts improves therapeutic outcome in colorectal cancer

city: Heidelberg

first: Thomas

last: Schmidt

state: Baden-Württemberg

address: Im Neuenheimer Feld 110

department: Department of General, Visceral and Transplantation Surgery

country: Germany

institute: University Hospital Heidelberg


title: Reducing metastases stiffness by targeting fibroblasts improves therapeutic outcome in colorectal cancer [Cells]

summary: Analysis of primary fibroblasts isolated from human colorectal primary tumors(CAFs) and liver metastases(MAFs) and identify corresponding gene expression signatures

overall_design: 6 CAFs and 6 MAFs cell samples deriving from 12 patients are examined with Next Generation Sequencing (NGS)

first: Ying

last: Shen


title: Expression data from tumor-infiltrating macrophages.

city: St. Louis

first: MALAY

last: HALDAR

state: MO

address: 7751 charing square lane

department: Pathology and Immunology

institute: Washington University in St. Louis

summary:
The microenvironment has profound effect on macrophage phenotype. Here we examine the phenotype of macrophages infiltrating murine undifferentiated pleomorphic sarcomas. We used microarray to examine gene expression profile of tumor-associated macrophages in murine undifferentiated pleomorphic sarcomas.

The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor associated macrophages (TAMs) but not immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we found that the TME induced retinoic acid (RA) production by tumor cells, which polarized intratumoral monocyte differentiation towards TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production by tumor cells or pharmacologic inhibition of RA signaling within TME increased stimulatory monocyte-derived cells, enhanced T cell-dependent anti-tumor immunity and demonstrated striking synergy with immune checkpoint blockade. Further, RA responsive gene signature in human monocytes correlated with an immunosuppressive TME in multiple human tumors. RA has been long considered as an anti-cancer agent, but our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

overall_design: Undifferentiated pleomorphic sarcomas were generated by injecting adenoviral cre recombinase in genetically engineered mice where Trp53 deletion and Kras activation occurs in the presence of Cre recombinase. Tumors were subjected to enzymatic digestion to generate single cell suspension and macrophages (7AAD-CD45+B220-CD11C-F4/80+) isolated by FACsorting. Microarray was performed on isolated macrophages.

first: Samir

last: Devalaraja

first: Tsun

last: To

first: Ian

last: Folkert

first: Ramakrishnan

last: Natesan

first: M

middle: Z

last: Alam

first: Minghong

last: Li

first: Yuma

last: Tada

first: Konstantin

last: Budagyan

first: Mai

last: Dang

last: Zhai

first: Graham

last: Lobel

first: Gabrielle

middle: E

last: Ciotti

first: T

middle: S

last: Eisinger-Mathason

first: Irfan

middle: A

last: Asangani

first: Kristy

last: Weber

middle: C

last: Simon

first: Malay

last: Haldar


title: Expression data from cultured mouse bone-marrow monocytes.

last: HALDAR

summary:
Retinoic acid signaling regulates monocyte differentiation into dendritic cells or macrophages. We used microarrays to uncover gene expression changes associated with retinoic acid exposure in mouse monocytes.

The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor associated macrophages (TAMs) but not immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we found that the TME induced retinoic acid (RA) production by tumor cells, which polarized intratumoral monocyte differentiation towards TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production by tumor cells or pharmacologic inhibition of RA signaling within TME increased stimulatory monocyte-derived cells, enhanced T cell-dependent anti-tumor immunity and demonstrated striking synergy with immune checkpoint blockade. Further, RA responsive gene signature in human monocytes correlated with an immunosuppressive TME in multiple human tumors. RA has been long considered as an anti-cancer agent, but our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

overall_design: To identify retinoic acid-induced gene expression changes in monocytes, we performed microarray-based gene expression profiling of mouse Ly6c-high bone marrow monocytes cultured under conditions that promote dendritic cell differentiation in the presence or absence of all-trans retinoic acid.

last: Devalaraja

first: Li


title: Reducing metastases stiffness by targeting fibroblasts improves therapeutic outcome in colorectal cancer [Tissue]

first: Thomas

last: Schmidt

summary: Analysis of colorectal primary tumors (pTU) and liver metastases (LM) tissue samples from patients and identify corresponding gene expression signatures

overall_design: 12 pTU and 10 LM tissue samples deriving from 22 patients are examinated with Next Generation Sequencing(NGS)

first: Ying

last: Shen


title: Understanding Heterogeneity of Fetal Hemoglobin Induction through Comparative Analysis of F- and A-erythroblasts

city: Philadelphia

first: Gerd

last: Blobel

address: 3615 Civic Center Blvd, ARC 316

department: Division of Hematology

summary: Here we set out to understand the heterogeneity of fetal hemoglobin (HbF) activation by developing techniques to purify and profile differentiation stage-matched late erythroblast F-cells and non-F cells (A-cells) from the human HUDEP2 erythroid cell line and primary CD34 cell erythroid cultures. Transcriptional and proteomic profiling of these cells demonstrated very few differences between F- and A-cells at the RNA level either under baseline conditions of following treatment with HbF inducers hydroxyurea or pomalidomide. Surprisingly, we did not find RNA or protein level differences in any known HbF regulator such as BCL11A or LRF that would account for HbF activation. Our analysis shows that F-erythroblasts are not significantly different from non HbF-expressing cells and that the primary differences likely occur at the transcriptional level at the b-globin locus.

overall_design: RNA-seq analysis of sorted stage-matched HbF high and low erythroblasts from HUDEP2 cells and from primary CD34-derived erythroid cultures

first: Eugene

last: Khandros


title: Next Generation Sequencing Facilitates Quantitative Analysis of feeding and fasting coilia nasus liver Transcriptomes

city: wuxi

first: denghua

last: yin

state: jiangsu

address: shanshui east road 9

institute: Freshwater Fisheries Research Center

summary:
Purpose: To gain a comprehensive understanding of differential gene expression in anadromous C. nasus during feeding and fasting conditions.
Methods: we characterized transcriptomics of C. nasus livers between feeding and fasting using RNA-seq
Results:A total of 23,159 mRNA moleculeswere selected to identify the mechanisms of feeding and fasting. Our results provide insight into the activation of protein synthesis and energy metabolism, identification of key genes involved in food intake regulation, motivation of the fatty acid biosynthesis, glycolysis, TCA cycle and oxidative phosphorylation and decrease of amino acids and linoleic acid metabolism in feeding conditions compared to fasting.
Conclusions:This is the first study describing differential gene expression and metabolic changes accompanying feeding and fasting in this interesting but endangered group. Our findings will be useful for future research on feeding characteristics, energy utilization and survival strategies of C. nasus.

overall_design: liver mRNA profiles of natural feeding and fasting  C. nasus were generated by deep sequencing, using Illumina HiSeq 4000.

first: Denghua

last: Yin

first: Kai

last: Liu


title: Gene Expression Data from Influenza A Virus-Infected and Mock-infected Mouse Eosinophils

city: Memphis

first: Robert

last: Rooney

state: TN

address: 50 N. Dunlap, Room 366R, LeBonheur CFRC

department: Pediatrics

institute: University of Tennessee Health Science Center

summary:
Eosinophils are terminally differentiated granulocytes that recently have been shown to become infected with Influenza A Virus (IAV).  Here, we investigate transcriptional changes that occur to bone marrow-derived eosinophils following exposure to IAV.
Global changes in the transcriptome of bone marrow derived mouse eosiniphils were assessed following mock- and Influenza A virus-infection using Affymetrix mouse gene 2.0 ST microarrays.

overall_design: Bone marrow-derived eosinophils were infected with MOI 0.1 Influenza A Virus strain PR8 for 1h and incubated at 37°C/5% CO2 for a further 23h at a density of 1 million cells/mL.

first: Kim

last: LeMessurier

first: Amali

last: Samarasinghe


title: Expression data

first: MALAY

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Cardiac mesenchymal stromal cells from right and left ventricles display different biological properties.

city: Milano

first: Gualtiero

middle: Ivanoe

last: Colombo

address: Via Carlo Parea, 4

country: Italy

institute: Centro Cardiologico Monzino IRCCS

summary:
Aims. We sought to analyze the physiological differences of human C-MSC between the left ventricle (LV) versus the  right ventricle (RV) .
Methods and results. Autoptic cardiac specimens from LV and RV were obtained from healthy donors. Part was used for tissue sections; part was processed to obtain C-MSC. Isogenic LV and RV C-MSC were compared as for surface markers expression, cell proliferation/death ratio, differentiation capabilities and transcriptomics. Our results indicated a greater presence of C-MSC in RV vs. LV total tissue. Moreover, more cells can be obtained from RV than from LV after isolation procedures. LV and RV C-MSC are characterized by the expression of the same surface markers. Functional studies revealed RV-LV C-MSC comparable groth curves but higher apoptosis rate within the LV cells. Moreover, higher propensity of the RV to to collagen deposition was obseved. Transcriptome analysis confirmed differences in the expression of genes related to apoptosis regulation and extracellular matrix organization. Alongside, we unveiled further pathways  specifically associated to LV or RV C-MSC.
Conclusions. These results contribute to understand the role of  RV and LV C-MSC in physiological condition. Moreover, the obtained information can unravel their differential contribution to RV or LV- specific pathologic scenarios.  RNA-Seq allowed identifying novel differentially expressed genes in RV vs. LV cells, which might uncover unappreciated mechanisms underlying ventricle-specific C-MSC regulation.

overall_design: The poly(A)+ RNA profiles of 6 paired C-MSC samples obtained from the RV and 6 from the LV of the same human tissue were generated through RNA-Seq using the Ion GeneStudio S5 Prime System .

first: Ilaria

last: Stadiotti

first: Luca

last: Piacentini

first: Chiara

last: Vavassori

first: Mattia

last: Chiesa

first: Barbara

last: Micheli

first: Anna

last: Guarino

first: Gianluca

last: Polvani

middle: I

first: Giulio

last: Pompilio

first: Elena

last: Sommariva


title: Expression data from ileum samples from FADD IEC-KO (intestinal epithelial cell-knock out) and CASP8 IEC-KO mice in combination with different genetic mutations (Zbp1 -/-, Mlkl -/-, Ripk3 IEC-KO)

city: Cologne

first: Manolis

last: Pasparakis

state: NRW

address: Joseph Stelzmann Strasse, 26

department: Institute for Genetics

institute: University of Cologne

summary:
FADD-IEC KO and CASP8 IEC-KO mice spontaneously develop chronic ileitis charcterized by inflammatory gene expression. We characterized the role of MLKL, RIPK3, ZBP1, in the upregulation of inlflammatory genes in these mice.
We used microarray to study the effect of MLKL, RIPK3 and ZBP1 on the gene expression profile in FADD IEC-KO and/or CASP8 IEC-KO mice and detect effects on specific inflammatory genes.

overall_design: ileum samples were collected from 10-12 week old mice for RNA extraction and subsequent microarray analysis.

first: Robin

last: Schwarzer


title: Genome-wide RNA-seq of Bacteroides thetaiotaomicron VPI-5482 with Bacteroides phage challenge

city: Ann Arbor

first: Jaime

middle: Joel

last: Fuentes

state: MI

address: 1150 West Medical Center Drive, 6751 Medical Science BLDG II

department: Microbiology and Immunology

institute: University of Michigan

summary:
Purpose: Examining the transcriptome of Bacteroides thetaiotaomicron VPI-5482 challenged with Bacteroides phage to assess surface molecule expression changes
Methods: Bacteroides thetaiotaomicron was grown in BPRM in vitro or Germ-Free mice were monocolonized with Bacteroides thetaiotaomicron and gavaged with ARB25 phage. Fold change was calculated as live phage versus heat-killed phage treated samples with n=3 biological replicates.  Once cells reached an optical density corresponding to mid-log phase growth (absorbance between 0.4-0.5), RNA was isolated and rRNA depleted. Samples were multiplexed for sequencing on the Illumina HiSeq platform at the University of Michigan Sequencing Core. Data was analyzed using Arraystar software (DNASTAR, Inc.) using DEseq2 normalization with default parameters. Genes with significant up- or down-regulation were determined by the following criteria: genes with an average fold-change >5-fold and with at least 2/3 biological replicates with a normalized expression level >1% of the overall average, and a p-value < 0.05 (t test with Benjamini-Hochberg correction)
Results: Specific capsule expression was increased in wild-type B. thetaiotaomicron during phage infection in vitro and in vivo. Many corresponding in vivo genes were upregulated as well as other surface layer proteins.

overall_design: rRNA-depleted, mRNA profiles of Bacteroides thetaiotaomicron VPI-5482 grown in vitro or isolated from Germ-Free mice challenged with Bacteroides phage using Heat killed phage as a reference in biological triplicate run on Illumina NextSeq platform.

middle: J

first: Eric

last: Martens

first: Nathan

last: Porter


title: RNA-Seq of GPR56+/- populations

city: Montreal

first: Guy

last: Sauvageau

state: QC

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute for Research in Immunology and Cancer

summary: Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). The lack of clone-specific surface markers, however, precludes prospective isolation and functional characterisation of distinct clones. Here, we performed RNA-Sequencing and assessed LSC frequencies for 56 primary AML samples in xenograft assays. We found that G-protein coupled receptor 56 (GPR56) identified the engrafting fraction in CD34positive samples and was associated with most high-risk mutations and poor outcome. Furthermore, variant allele frequency analysis revealed different clonal composition of engrafting GPR56+ versus non-engrafting GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low leukemia initiating capacity. Our data suggest that the NSG-model rather than reproducing the full clonal diversity of the human disease selects for leukemic sub-clones based on their distinct functional properties.

overall_design: RNA-Seq of GPR56+/- populations

first: Caroline

last: Pabst

first: Anne

last: Bergeron

first: Vincent

last: Lavallée

first: Jonathan

last: Yeh

first: Patrick

last: Gendron

first: Gudmundur

middle: L

last: Norddahl

first: Jana

last: Krosl

first: Isabel

last: Boivin

last: Deneault

first: Jessica

last: Simard

first: Suzan

last: Imren

first: Geneviève

last: Boucher

first: Kolja

last: Eppert

first: Tobias

last: Herold

first: Stefan

last: Bohlander

first: Keith

last: Humphries

first: Sébastien

last: Lemieux

first: Josée

last: Hébert

first: Frédéric

last: Barabé


title: Expression data from cultured human monocytes

first: MALAY

last: HALDAR

summary:
Retinoic acid signaling regulates monocyte differentiation into dendritic cells or macrophages. We used microarrays to uncover gene expression changes associated with retinoic acid exposure in human monocytes.

The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor associated macrophages (TAMs) but not immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we found that the TME induced retinoic acid (RA) production by tumor cells, which polarized intratumoral monocyte differentiation towards TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production by tumor cells or pharmacologic inhibition of RA signaling within TME increased stimulatory monocyte-derived cells, enhanced T cell-dependent anti-tumor immunity and demonstrated striking synergy with immune checkpoint blockade. Further, RA responsive gene signature in human monocytes correlated with an immunosuppressive TME in multiple human tumors. RA has been long considered as an anti-cancer agent, but our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

overall_design: To identify retinoic acid-induced gene expression changes in monocytes, we performed microarray-based gene expression profiling of human monocytes cultured under conditions that promote dendritic cell differentiation in the presence or absence of all-trans retinoic acid.

first: Samir

last: Devalaraja

first: Tsun

middle: K

last: To

first: Ian

last: Folkert

first: Ramakrishnan

last: Natesan

first: M

last: Alam

first: Minghong

last: Li

first: Yuma

last: Tada

first: Konstantin

last: Budagyan

first: Mai

last: Dang

first: Li

last: Zhai

first: Graham

last: Lobel

first: Gabrielle

last: Ciotti

first: T

last: Eisinger-Mathason

first: Irfan

last: Asangani

first: Kristy

last: Weber

last: Simon

first: Malay

last: Haldar


title: small RNA-seq in HepG2 cells

city: Montpellier

first: Rosemary

last: Kiernan

address: 141 rue de la cardonille

country: France

institute: UMR9002 CNRS-UM

type: Non-coding RNA profiling by high throughput sequencing

summary: The effect of NF90 on the abundance of miRNAs was measured using small RNA-seq.

overall_design: HepG2 cells were transfected with a non-targeting control siRNA (siScr) or a siRNA targeting NF90 for 7 days. RNA was extracted and analyzed by deep sequencing, in triplicate, using BGISEQ-500, SE50

first: Giuseppa

last: Grasso

first: Jerome

last: Barbier


title: Paired-end RNA-seq in HepG2 cells

summary: The effect of NF90 on transcript abundance was measured using RNA-seq.

overall_design: HepG2 cells were transfected with a non-targeting control siRNA (siScr) or a siRNA targeting NF90 for 7 days. RNA was extracted and analyzed by deep sequencing, in triplicate, using BGISEQ-500, PE100


title: small RNA-seq in HEK-293t cells

overall_design: HEK-293t cells were transfected with a non-targeting control siRNA (siScr) or a siRNA targeting NF90 for 10 days. RNA was extracted and analyzed by deep sequencing, in triplicate, using Standard Library Preparation (protocol small RNA illumina), NextSeq, Single-Reads, 50bps.

first: Gabriel

last: Sanchez


title: NAD+ regeneration rescues lifespan but not ataxia in a mouse model of brain mitochondrial complex I dysfunction

status: Public on Apr 30 2020

city: Chicago

first: Navdeep

last: Chandel

state: IL

address: 303 E Superior St, SQ5

department: Pulmonary and Critical Care

institute: Northwestern University

summary: Mitochondrial complex I regenerates NAD+ and proton pumps for TCA cycle function and ATP production, respectively. Mitochondrial complex I dysfunction has been implicated in many brain pathologies including Leigh Syndrome and Parkinson’s disease. We sought to determine whether NAD+ regeneration or proton pumping is the dominant function of mitochondrial complex I in protection from brain pathology. We generated a mouse that conditionally expresses the yeast NDI1 protein, a single enzyme that can replace the NAD+ regeneration capability of the 45-subunit mammalian mitochondrial complex I without proton pumping. NDI1 expression was sufficient to dramatically prolong lifespan without significantly improving motor function in a mouse model of Leigh Syndrome. Therefore, mitochondrial complex I activity in the brain supports organismal survival through its NAD+ regeneration capacity, while optimal motor control requires the bioenergetic function of mitochondrial complex I.

overall_design: RNA sequencing of mouse cerebellum tissue, 24 total samples, 3 male and 3 female biological replicates for 4 different genotype groups. Cre control mice, NDI1 expressing control mice, cKO mice, and cKO+NDI1 mice.

first: Gregory

last: McElroy

first: Colleen

middle: R

last: Reczek

first: Paul

last: Reyfman

first: Divakar

last: Mithal

first: Craig

last: Horbinski


title: NF90 Modulates Processing of a Subset of Human Pri-miRNAs

summary:
This SuperSeries is composed of the SubSeries listed below.
MicroRNAs are predicted to regulate the expression of more than 60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The dsRNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of pri-miRNAs. Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 25 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to ac  quisition of NF90 association, as determined by RNA EMSA. NF90-bound and modulated pri-miRNAs are embedded in introns of host genes and expression of several is concomitantly modulated, including an oncogene implicated in metastasis of hepatocellular carcinoma, TIAM2. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

overall_design: Refer to individual Series


title: ND1 ex vivo gene therapy modulates the transcription of genes relevant to AD pathogenesis

city: Lund

first: Jeovanis

last: Gil Valdés

state: Skane

address: Sölvegatan 19, BMC D13

department: Department of Biomedical Engineering

country: Sweden

institute: Lund University

summary:
Transcriptome analysis of human iPSC-derived hippocampal neurons overexpresing NEUROD1
We employed Affymetrix GeneChip microarray (Human Clariom D) to examine early transcriptomic changes in APP variant cells compared to control, upon viral delivery of ND1.

overall_design: Human iPSC-derived hippocampal neurons from healthy donor and Alzheimer's disease patient carrying APP mutation were transfected with GFP or NEUROD1. Three biological replicates were performed. Summarized probe set signals in log2 scale were calculated by using the GCCN-SST-RMA algorithm with the Affymetrix GeneChip Expression Console v1.4 Software.

first: Yuriy

last: Pomeshchi

first: Oxana

last: Klementieva

last: Gil

first: Isak

last: Martinsson

first: Marita

middle: G

last: Hansen

first: Tessa

last: de Vries

first: Anna

last: Sancho-Balsells

first: Kaspar

last: Russ

first: Ekaterina

last: Savchenko

last: Collin

first: Ana

last: Vaz

first: Silvia

last: Bagnoli

first: Benedetta

last: Nacmias

first: Claire

last: Rampon

first: Sandro

last: Sorbi

first: Dora

last: Brites

first: György

last: Marko-Varga

first: Zaal

last: Kokaia

first: Melinda

last: Rezeli

first: Gunnar

last: Gouras

first: Laurent

last: Roybon


title: Multi-omics analysis reveals divergent epigenetic regulation of gene expression and drivers of esophageal squamous cell carcinoma

city: San Francisco

first: Wei

last: Wu

state: CA

address: 600 16th street

department: Department of Medicine

institute: University of California, San Franciso

type: Methylation profiling by high throughput sequencing

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


title: RNA-seq of iPSC-derived astrocytes that are capable of exerting pro-maturational effect on synaptic networks

city: Cardiff

first: Jimena

last: Monzón-Sandoval

address: Maindy Road

country: United Kingdom

institute: UK Dementia Research Institute at Cardiff University

summary: Astrocytes interact closely with neurons and facilitate neuronal maturation and function by providing trophic support, regulating the extracellular environment, and engaging in a variety of inter-cellular signalling mechanisms. We have described the generation of human astrocytes and neurons from a common cortical progenitor pool, thereby recapitulating aspects of in vivo development. Firstly, we show that the iPSC-derived astrocytes exhibit many of the key molecular and functional hallmarks predicted of astrocytes. Secondly, we provide functional and transcriptomic analyses of the astrocytes’ capacity to interact with neurons both through rapid regulation of ongoing synaptic transmission as well as exerting pro-maturational effects on the synaptic networks. Transcriptional analysis included the comparison of the present RNA-seq of iPSC-derived astrocytes to other iPSC-derived astrocyte datasets as well as fetal and adult human astrocytes. We investigated differences in gene expression between our maturation-enhancing iPSC-astrocytes and iPSC-astrocytes previously shown to be unable to promote synaptic network maturation. By integrating transcriptomic data, functional genomic annotations and protein-protein interaction resources we support that astrocytic extracellular signalling is important for the ability of astrocytes to mediate functional neuronal maturation. This work provides a foundation for future investigations into astrocyte-neuron interactions in human health and disease.

overall_design: Bulk RNA-seq for 9 samples of human induced pluripotent stem cell (iPSC) derived astrocytes from 3 different donors.

first: Anne

last: Hedegaard

first: Sarah

last: Newey

first: Emma

last: Whiteley

first: Caleb

last: Webber

first: Colin

last: Akerman


title: DNA sequencing coverage in wild type and RNase H deficient Bacillus subtilis cells [1]

first: Taylor

last: Nye

address: 1105 N University Ave

department: MCDB

summary: RNA-DNA hybrids form throughout the chromosome during normal cell growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, increase mutagenesis, and reduce gene expression. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion of RNA-DNA hybrids. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore resulting in SOS-dependent inhibition of cell division. We conclude that B. subtilis NCIB 3610 encodes functional RNase HI, HII, and HIII, and pBS32-encoded RNase HI contributes to replication fork progression and chromosome stability while RNase HIII is important for chromosome stability and plasmid hyper-replication.

sample_organism: Bacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10

overall_design: Assessment of sequencing coverage across the chromosome and plasmid pBS32

first: Daniel

middle: B

last: Kearns

first: Lyle

last: Simmons


title: RNA-seq of dendritic cell subsets

first: Cindy

last: Audiger

state: Québec

address: 2900, boul. Édouard-Montpetit

institute: Univeristy of Montreal

summary:
Comparison of cDC1, cDC2, mcDC transcriptome
Conventional dendritic cells (cDCs) are classically subdivided in two subsets, named cDC1 and cDC2. We demonstrated that mcDC are a new subset of cDCs. To understand their transcriptomic relatedness, we perform a RNA sequencing on cDC1, cDC2 and mcDCs sorted from mouse spleens. We demonstrate that cDC1, cDC2 and mcDCs all cluster independently in a PCA analysis and an unbiased hierarchical cluster analysis reveals unique gene profile for all three cDC subsets. As mcDC cluster slightly more towards cDC1 than cDC2, we next aimed to more closely examine the transcriptomic relatedness between mcDC and cDC2. We compared the DC subsets gene expression signatures based on a selected gene set known to define cDC2. Both the PCA and hierarchical cluster analysis show that mcDC exhibit a different signature to cDC2. Altogether, we demonstrate that mcDC are a distinct subsets of cDCs with a specific transcriptomic signature.

overall_design: splenic sorted cDC1, cDC2 and mcDC

first: Edith

last: Janssen

first: Sylvie

last: Lesage


title: Expression data from colon samples from FADD IEC-KO (intestinal epithelial cell-knock out) and CASP8 IEC-KO mice in combination with different genetic mutations (Zbp1 -/-, Mlkl -/-, Ripk3 IEC-KO)

first: Manolis

last: Pasparakis

summary:
FADD-IEC KO and CASP8 IEC-KO mice spontaneously develop chronic colitis charcterized by inflammatory gene expression. We characterized the role of MLKL, RIPK3, ZBP1, in the upregulation of inlflammatory genes in these mice.
We used microarray to study the effect of MLKL, RIPK3 and ZBP1 on the gene expression profile in FADD IEC-KO and/or CASP8 IEC-KO mice and detect effects on specific inflammatory genes.

overall_design: Colon samples were collected from 10-12 week old mice for RNA extraction and subsequent microarray analysis.

first: Robin

last: Schwarzer


title: Single-cell RNA-sequencing for dissecting the immunological network in autoimmune myocarditis

city: Beijing

first: Jiangping

last: Song

address: 167A Beilishi Road

department: State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases

institute: Chinese Academy of Medical Sciences and Peking Union Medical College

summary: We report the scRNA-seq for cardiac  immune cells in the different phases of experimental autoimmune myocarditis (EAM).

overall_design: scRNA-seq for CD45+ cell isolated from hearts of different phases of EAM including Day 0, Day 14, Day 21 and Day 60


title: Multi-omics analysis reveals divergent epigenetic regulation of gene expression and drivers of esophageal squamous cell carcinoma (RNA-Seq)

summary: Purpose: Epigenetic landscapes can shape physiologic and disease phenotypes. We used whole genome bisulfite sequencing, RNAseq, and ChIP-seq technologies to understand the methylome profiling, gene expression and regulation via epigenetic alterations on esophageal sequamouse carcinoma comparing to adjacent non-tumor tissues.

overall_design: Methods:  Frozen surgical removed specimens of ten matched normal and tumor samples were used for DNA and RNA extraction.

last: Cao


title: RNA-seq of subcutaneous and visceral adipose tissue from sheep prenatally exposed to BPA

first: Kelly

last: Bakulski

address: 1415 Washington Heights

department: Department of Epidemiology

summary: Subcutaneous and visceral adipose tissue from sheep prenatally exposed to BPA

overall_design: 16 total samples: 4 subcutaneous adipose controls, 4 subcutaneous adipose BPA exposed samples, 4 visceral adipose controls, 4 visceral adipose BPA exposed samples

first: John

middle: F

last: Dou

first: Muraly

last: Puttabyatappa

first: Vasantha

last: Padmanabhan

middle: M


title: LIN28B underlies the pathogenesis of a subclass of Ewing sarcoma

city: Torino

first: Paolo

last: Provero

address: Via Nizza 52

department: Molecular Biotechnology and Health Sciences

institute: University of Turin

summary: Ewing sarcoma (EwS) is associated with poor prognosis despite current multimodal therapy. Targeting of EWS-FLI-1, the fusion protein responsible for its pathogenesis, and its principal downstream targets has not yet produced satisfactory therapeutic options, fuelling the search for alternative approaches. Here, we show that the oncofoetal RNA-binding protein Lin28B regulates the stability of EWS-FLI-1 mRNA in about 10% of EwS. Lin28B depletion in these tumours leads to a decrease in expression of EWS-FLI-1 and its direct transcriptional network, abrogating EwS cell self-renewal and tumorigenicity. Moreover, pharmacological inhibition of Lin28B mimics the effect of Lin28B depletion, suggesting that Lin28B sustains the emergence of a subset of EwS in which it also serves as an effective therapeutic target.

overall_design: Four samples of spherogenic cultures from primary Ewing sarcomas

first: Matteo

last: Torsello

first: Gaylor

last: Boulay

first: Sandrine

last: Cornaz-Buros

first: Arnaud

last: Bakaric

first: Tugba

last: Keskin

first: Patricia

last: Waszyk

first: Thibaud

last: Geiser

last: Martin

first: Angela

last: Volorio

first: Sowmya

last: Iyer

first: Anupriya

last: Kulkarni

first: Igor

last: Letovanec

first: Stéphane

last: Cherix

first: Gregory

last: Cote

first: Edwin

last: Choy

first: Antonia

last: Digklia

first: Michael

last: Montemurro

first: Ivan

last: Chebib

first: Petur

last: Nielsen

first: Angel

last: Carcaboso

first: Jaume

last: Mora

first: Raffaele

last: Renella

first: Mario

last: Suvà

first: Carlo

last: Fusco

first: Miguel

middle: N

last: Rivera

first: Nicolò

last: Riggi

last: Stamenkovic


title: Time-course analysis of transcriptomic changes of mesothelial cells after stress exposure

city: Biberach

first: Meshal

last: Ansari

address: Birkendorfer Straße 65

institute: Boehringer Ingelheim

summary: To determine the transcriptional program that imparts adhesion capacity to healthy mesothelial cells, we analyzed subtle gene expression changes by performing highly parallel single-cell RNA-seq genome-wide expression profiling of individual mesothelial cells exposed to stress (Extended Figure 6A) using the Drop-seq workflow (Pubmed ID 26000488). We individually sequenced >16,000 cells from Met-5A mesothelial cells at various time-points after exposure to a 15 min desiccation shock, as well as under control unstressed conditions.

overall_design: Single-cell transcriptomics time-course experiment after desiccation shock

first: Adrian

last: Fischer

first: Tim

last: Koopmans

first: Yuval

last: Rinkevich


title: Transcriptional analysis of glnA deletion mutant in Haloferax mediterranei

city: San Vicente del Raspeig

first: Julia

last: Esclapez

state: Alicante

address: Crta San Vicente del Raspeig s/n

department: Biochemistry

country: Spain

institute: University of Alicante

summary: Transcriptional profiling of  Haloferax mediterranei DglnA and HM26 strains  in two different culture media: a) cells were grown exponentially on complex media + 40 mM Gln, b) cells were shifted to nitrogen starvation conditions (72h).  In the Nsta(∆glnA)-Cx and Nsta-Cx contrasts, glnA2 was up-regulated, and glnA3 did not show any difference. Therefore, glnA2 and glnA3 were unable to compensate lack of glnA because either they do not properly encode GS or are not expressed under the same conditions.

overall_design: Four-condtions experiment with four biological replicates, combined in a loop design.

first: V

last: Rodríguez-Herrero

first: G

last: Payá

last: Bautista

first: A

last: Vegara

first: M

last: Cortés

last: Camacho

first: J

last: Bonete


title: CG14906 (mettl4) mediates m6A methylation of U2 snRNA in Drosophila

city: Bad Nauheim

first: Lei

last: Gu

state: MASSACHUSETTS

address: parkstreet 1

institute: Max Planck Institute for heart and lung research

summary: We identified U2 as the substrate of mettl4 in fly

overall_design: Investigating gene experession in mettl4 wt and ko fly cells


title: Association between progesterone concentration in early diestrus and endometrial gene expression in grazing dairy cows

city: Utrecht

first: Maria

middle: Belen

last: Rabaglino

address: Yalelaan 7

department: Population Health Science

country: Netherlands

institute: Utrecht University

summary: The objective of this study was to evaluate the association between progesterone concentration (P4) at day 4 of the estrous cycle (beginning of diestrus) and endometrial global gene expression at day 9 (middle diestrus) in lactating grazing dairy cows. Blood samples were obtained on days 0, 4 and 9 for P4 measurement by chemiluminescence. Cows were asignated to one of the following groups (n=3/group): cows with low physiological P4 at day 4 (PLP4), cows in anestrous (ANE), cows with high physiological P4 at day 4 (HPP4) and superovulated cows (SO). Endometrial biopsy samples were obtained on day 9 for RNA sequencing. Quality control and determination of differentially expressed genes (DEG; FDR<0.05) were determined using the edgeR package for R. The number of DEG between groups were: HPP4 vs LPP4: 453; HPP4 vs ANE: 623; SO vs LPP4: 603; SO vs ANE: 1102; LPP4 vs ANE: 261 and SO vs HPP4: 0. Functional analysis using the DAVID database revealed that endometrial genes upregulated by low P4 are enriched in the immune system and inflammatory response. Conversely, genes upregulated by high P4 are enriched inmodulation of uterine relaxation-contraction, GnRH signaling pathway, focal adhesion and EGF-like related terms. In conclusion, our results support the concept of changes in P4 at the beginning of the luteal phase could be associated with the endometrial expression of critical genes involved in the preparation of the uterine environment for embryo implantation.

overall_design: 12 endometrial samples were analyzed, with 3 replicates per group (4 groups).

first: Laura

middle: V

last: Madoz

last: BRabaglino

first: Ana

last: Migliorisi

first: María

last: Jaureguiberry

first: Santiago

middle: P

last: Wallace

first: Nicolás

last: Lorenti

first: Germán

last: Domínguez

first: Mauricio

last: Giuliodori

first: Rodolfo

last: de la Sota


title: LIN28B underlies the pathogenesis and therapeutic sensitivity of a subclass of Ewing sarcoma

first: Paolo

last: Provero

summary: Ewing sarcoma (EwS) is a pediatric bone malignancy associated with high mortality despite aggressive multimodal therapy. Targeting EWS-FLI-1, the fusion protein responsible for EwS pathogenesis, or its principal downstream target gene products have not produced effective therapeutic options, fuelling the quest for alternative approaches. Here, we show that the oncofoetal RNA-binding protein Lin28B is expressed in a subset of EwS in which it directly binds and safeguards EWS-FLI-1 transcripts. Depletion and pharmacologic inhibition of Lin28B in primary patient-derived EwS cultures led not only to a decrease in EWS-FLI-1 expression but also to the deconstruction of its direct transcriptional network, abrogating EwS cell self-renewal and tumorigenicity. Our observations demonstrate that Lin28B provides indispensable support for EWS-FLI-1 to sustain the emergence of a EwS subset in which it also constitutes a promising therapeutic target.

overall_design: Four samples of spherogenic cultures from primary Ewing sarcomas, plus six samples of the same after various shRNA, overexpression and drug treatments

first: Matteo

last: Torsello

first: Gaylor

last: Boulay

first: Sandrine

last: Cornaz-Buros

first: Arnaud

last: Bakaric

first: Tugba

last: Keskin

first: Patricia

last: Martin

first: Angela

last: Volorio

first: Sowmya

last: Iyer

first: Anupriya

last: Kulkarni

first: Igor

last: Letovanec

first: Stéphane

last: Cherix

first: Gregory

last: Cote

first: Edwin

last: Choy

first: Antonia

last: Digklia

first: Michael

last: Montemurro

first: Ivan

last: Chebib

first: Petur

last: Nielsen

first: Angel

last: Carcaboso

first: Jaume

last: Mora

first: Raffaele

last: Renella

first: Mario

last: Suvà

first: Miguel

last: Rivera

first: Nicolò

last: Riggi

last: Stamenkovic


title: Uterine transpriptome of pre-pubertal WT and ERa knockout (Ex3aERKO) mice

status: Public on Apr 29 2020

city: RTP

first: NIEHS

last: Microarray Core

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

summary:
Pre-pubertal (21 days old) WT or ERa knockout
we compared RNA from 21-day old WT and Ex3aERKO uteri that were treated with saline vehicle (V)  estradiol (E2) or IGF1 for 2 hours or 24 hours

overall_design: 3 replicates of each treatment

first: Sylvia

last: Hewitt

first: Kenneth

middle: S

last: Korach


title: Genome-wide maps of chromatin state in CD8 Naïve T cells upon differentiation and subsequent reversion to Naïve-like phenotype

city: Birmingham

first: Paul

last: Moss

state: West Midlands

address: Vincent Drive

department: Immunology and Immunotherapy

institute: University of Birmingham

summary: We have employed whole genome ATAC-seq to profile the chromatin accssibility landscape of naive CD8+ T cells (TN) and these same cells following activation and differentiation to CM/EM and following incubation with homeostatic cytokines, a reverted naive-like phenotype (TNrev). Four umbilical cord blood samples were used to enrich for TN and to produce the respective EM CM and TNrev phenotypes.

overall_design: Chromatin accessibility profile was measured by ATAC-seq of TN cells from 4 human cord blood samples and in the same cells after activation with anti CD3 for 6 days, followed by incubation with IL-7 25 ng/ml for further 13 days.

first: G

last: Frumento

first: K

last: Verma

first: W

last: Croft


title: Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells  [ChIP-Seq]

city: PHILADELPHIA

first: Kai

last: Tan

address: 3501 CIVIC CENTER BLVD.

institute: Children's Hospital of Philadelphia

overall_design: Examination of genome-wide of 4 histone modifications during the ontogeny of hematopoietic stem cells

first: Changya

last: Chen

first: Peng

last: Gao


title: Peri- and post-pubertal estrogen exposures of female mice optimize uterine responses later in life

summary:
At birth, all female mice, including those that either lack estrogen receptor α (ERα-knockout) or that express mutated forms of ERα (AF2ERKI), have a hypoplastic uterus. However, uterine growth and development that normally accompanies pubertal maturation does not occur in ERα-knockout or AF2ERKI mice, indicating ERα mediated estrogen signaling is essential for this process. Mice that lack Cyp19 (aromatase, ArKO mice), an enzyme critical for estrogen (E2) synthesis, are unable to make E2, and lack pubertal uterine development. A single injection of E2 into ovariectomized adult (10 weeks old) females normally results in uterine epithelial cell proliferation, however, we observe that, although ERα is present in the ArKO uterine cells, no proliferative response is seen. We assessed the impact of exposing ArKO mice to E2 during pubertal and post-pubertal windows and observed that E2 exposed ArKO mice acquired growth responsiveness. Analysis of differential gene expression between unexposed ArKO samples and samples from animals exhibiting the ability to mount an E2-induced uterine growth response (WT or E2 exposed ArKO) revealed activation of EZH2 and HAND2 signaling and inhibition of GLI1 responses. EZH2 and HAND2 are known inhibit uterine growth, and GLI1 is involved in IHH signaling, which is a positive mediator of uterine response. Finally, we show that exposure of ArKO females to dietary phytoestrogens results in their acquisition of uterine growth competence. Altogether our findings suggest that pubertal levels of endogenous and exogenous estrogens impact biological function of uterine cells later in life via ERα-dependent mechanisms.
We compared uterine RNA from ovariectomized adult aromatase knockout mice (ARKO) mice that were untreated to WT mice and to ARKO that were administered estradiol benzoate (EB) to induce uterine epithelial cell growth competence

overall_design: RNA samples include 3 replicates each of WT untreated or EB treated, and ARKO untreated or EB treated

middle: C

first: Marleny

last: Carmona

last: Foley

first: Lauran

last: Donoghue

first: Sydney

last: Lierz

first: Wipawee

last: Winuthayanon


title: Transcriptome analyses of the nervous system of Familial Dysautonomia-model mice identify novel cellular pathways dependent on Ikbkap/Elp1 (CNS)

city: Baltimore

first: Supriyo

last: De

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

institute: NIA-IRP, NIH

summary: Familial Dysautonomia (FD; OMIM #223900) is both a developmental and progressive autosomal recessive neurodegenerative disorder that results from a nervous-system reduction in the IKAP/ELP1 protein due to a mutation in a splice acceptor site of the IKBKAP/ELP1 gene. The function of this gene in the nervous system is unresolved. To obviate the embryonic lethality of mice completely null for Ikbkap, we generated conditional knock out (CKO) mouse models for FD that recapitulate hallmarks of the human disease. To derive insight into potential intracellular functions for Ikbkap, we conducted a genome-wide transcriptome analysis of both the peripheral and central nervous systems from Ikbkap CKO mice, and identify over 100 shared misregulated genes that reveal roles for IKAP in several metabolic and signaling pathways in addition to synaptic transmission. Importantly, our data are the first to demonstrate that in the absence of IKAP, neurons undergo intracellular stress that is marked by transcriptional elevations in ATF5, p53, and several CREB target genes, as well as an increase in reactive oxygen species. These data will aid in the identification of common upstream and downstream targets for therapeutics for preventing the progressive demise of neurons in FD and potentially other neuropathies.

overall_design: Examining the major genetic pathways that require IKAP are hampered by that Ikbkap null mice are embryonic lethal (E10), but can circumvented by genome-wide transcriptome analysis in the brains in adult Tuba1a-Cre;IkbkapLoxP/LoxP mice, which live on average 6 months. These lines were obtained by crossing mice homozygous for a floxed allele of Ikbkap (exon 4) mice to mice that are heterozygous for this floxed allele, and a Tuba1a-Cre transgene. For this study, brain tissue (cerebellum and brainstem) was removed from 2-5 month old adult Tuba1a-cre;IkbkapLoxP/LoxP  mice and stored at -80 until RNA was isolated. Four wildtype and four conditional knock-out animals were analyzed for this experiment.

first: Frances

last: Lefcort

first: Yongqing

last: Zhang

first: Elin

last: Lehrmann

first: Lynn

last: George

first: Marta

last: Chaverra

first: Julian

last: Thorne

first: Sarah

last: Ohlen

first: Yumi

last: Ueki

first: Dani

last: Bergey

first: Kevin

last: Becker


title: Single Cell RNA-Sequencing of Human Limb Skeletal Muscle across Development and Myogenic Culture from Pluripotent Stem Cells

city: Los Angeles

first: Haibin

last: Xi

address: 615 Charles E Young Dr South, BSRB 257

department: MIMG

institute: UCLA

summary: The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. To address this, we employed single cell RNA-sequencing to profile human skeletal muscle tissues from embryonic, fetal and postnatal stages. In silico, we identified myogenic as well as other cell types and constructed a “roadmap” of human skeletal muscle ontogeny across development. In a similar fashion, we also profiled the heterogeneous cell cultures generated from multiple human pluripotent stem cell (hPSC) myogenic differentiation protocols, and mapped hPSC-derived myogenic progenitors to an embryonic-to-fetal transition period. Additionally, we found differentially enriched biological processes and discovered co-regulated gene networks and transcription factors present at distinct myogenic stages. In summary, this work serves as a resource for advancing our knowledge of human myogenesis. It also provides a tool for better characterization and understanding of hPSC-derived myogenic progenitors for translational applications in skeletal muscle based regenerative medicine.

overall_design: Whole hindlimbs or hindlimb skeletal muscle tissues were obtained from human prenatal development up to 18 weeks as well as juveniles and adults. Dissociated cells were subjected to single cell RNA-sequencing (scRNA-seq) via the Drop-seq method (Macosko et al., Cell, 2015) without or with minimal enrichment. Similarly, myogenic cultures from three human pluripotent stem cell (hPSC) differentiation protocols (Xi et al., Cell Rep, 2017; Chal et al., Nat Biotechnol, 2015; Shelton et al., Stem Cell Rep, 2014) were also subjected to scRNA-seq/Drop-seq. Single cells from the whole tissues/cultures were analyzed using the Seurat package in R to explore the broad cell types present. The skeletal myogenic populations were subset and reanalyzed to identify myogenic subpopulations. Skeletal muscle stem and progenitor cells from in vivo tissues were computationally purified and assembled to construct a trajectory of human myogenesis, and progenitor cells derived from hPSCs in vitro were aligned to the trajectory to determine their developmental identities.

first: April

middle: D

last: Pyle


title: Next generation sequencing facilitates transcriptome analysis of wild type and Kap114-knockout Saccharomyces cerevisiae under high salt conditions

city: Taipei

first: Kuo-Chiang

last: Hsia

address: 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan

department: Institute of Molecular Biology

country: Taiwan

institute: Academia Sinica

summary: RNA-Seq-based transcriptomic analysis were carried out to investigate the global gene exprssion changes of Saccharomyces cerevisiae in the presence of 1.5 NaCl.

overall_design: mRNA profiles of wild type and Kap114-knockout Saccharomyces cerevisiae under high salt conidtions (1.5 M NaCl) were generated by deep sequencing, in triplicate.

first: Chung-Chi

last: Liao


title: Multidimensional gene regulatory landscape of a bacterial pathogen in plants

city: Cologne

first: Tatsuya

last: Nobori

address: Carl-von-Linne Weg 10, Max-Planck Institute for Plant Breeding Research

summary: Understanding the gene regulation of plant pathogens is crucial for pest control and thus global food security. An integrated understanding of bacterial gene regulation in the host is dependent on multi-omic datasets, but these are largely lacking. Here, we simultaneously characterized the transcriptome and proteome of a bacterial pathogen in plants. We found a number of bacterial processes affected by plant immunity at the transcriptome and proteome levels. For instance, the salicylic acid-mediated plant immunity suppressed the accumulation of proteins comprising the tip component of bacterial type III secretion system. Interestingly, there were instances of concordant and discordant regulation of bacterial mRNAs and proteins. Gene co-expression analysis uncovered previously unknown gene regulatory modules underlying virulence. This study provides molecular insights into the multiple layers of gene regulation that contribute to bacterial growth in planta and elucidate the role of plant immunity in affecting pathogen responses.

sample_organism: Pseudomonas syringae pv. tomato str. DC3000

overall_design: Transcriptomes of P. syringae pv. tomato DC3000 during infection to four Arabidopsis thaliana genotypes. Bacterial suspension was syringe infiltrated to plant leaves at OD600=0.005. At 48 h after infection, the infected leaves were harvested and bacterial cells were isolated, followed by RNA extraction. Ribosomal RNA of bacteria and plants were removed with a commercial kit to enrich bacterial mRNA. Then, cDNA libraries were generated and RNA-Seq was performed. For each condition, three independent replicates were taken.

first: Kenichi

last: Tsuda


title: Kaposi’s Sarcoma-associated herpesvirus fine-tunes the temporal expression of its genes manipulating a specific host RNA quality control pathway

status: Public on Apr 28 2020

city: Dallas

first: Nicholas

last: Conrad

state: TX

address: 6000 Harry Hines Blvd

department: Microbiology

institute: UT Southwestern Medical Center

summary: Kaposi’s Sarcoma-associated herpesvirus (KSHV) transcripts are subject to degradation by at least two host-mediated nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) and an ARS2-dependent decay pathway. KSHV expresses the multifunctional protein ORF57, which increases viral transcripts stability by protecting them preferentially from ARS2-dependent decay. However, a subset of viral transcripts succumb to PPD even in the presence of ORF57, but the role of PPD during KSHV infection is not completely understood. We inactivated both decay pathways using siRNA and monitored their contribution to viral gene expression in the presence of ORF57 by RNA-Seq at 24 hours post induction (hpi). Inactivation of PPD, but not ARS2-mediated decay, results in aberrant accumulation of late transcripts, which are typically expressed at 48 hpi. PPD exerts its functions post-transcriptionally as the upregulation of late genes is independent of viral transactivation factors needed for their expression. Remarkably, at their proper time of expression, PPD inactivation has no effect on late transcripts. We further show that PPD evasion by late transcripts requires the host factor NRDE2. In conclusion, our studies show that KSHV uses PPD to fine-tune the temporal expression of its genes by preventing their premature accumulation.

overall_design: RNA-seq experiment to compare the contribution of the host-mediated RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) and an ARS2-dependent decay pathway to KSHV gene expression. PPD and ARS2-dependent decay pathways were inactivated by depleting iSLK WT cells of PAPα/γ or ARS2, respectively, using siRNAs.

first: Julio

last: Ruiz


title: Genome-wide maps of histone modification and chromatin remodeler binding in mouse calvaria derived stromal cell line (OP9)

city: Kolkata

first: Amitava

last: Sengupta

state: West Bengal

address: CN 6, Sector V, Salt Lake

department: Cancer Biology & Inflammatory Disorder Division

country: India

institute: CSIR - Indian Institute of Chemical Biology (Translational Research Unit of Excellence)

summary: We have used chromatin immunoprecipitation followed by high throughput sequencing to map regions of chromatin remodeler and  histone modification state. We have used this data to understand the global distribution in stromal cell and probable mechanism of transcriptional regulation.

overall_design: Binding profiles of two unique and one core  subunit of  ATP-ase dependent  chromatin remodeler PBAF along with histone modification mark H3K27ac , in duplicate, in shCtrl and shPbrm1 stable cell line has been studied.


title: DHH1/DDX6-like RNA helicases maintain ephemeral half-lives of stress-response mRNAs

city: Riverside

first: Maureen

last: Hummel

state: California

address: 900 University Avenue

department: Botany and Plant Sciences

institute: University of California

summary: Gene transcription is counterbalanced by mRNA decay processes that regulate transcript quality and quantity. We show here that the evolutionarily conserved DHH1/DDX6-like RNA HELICASEs of Arabidopsis thaliana control the ephemerality of a subset of cellular mRNAs. These RNA helicases co-localize with key markers of processing bodies and stress granules and contribute to their subcellular dynamics. They function to limit the precocious accumulation and translation of stress-responsive mRNAs associated with autoimmunity and growth inhibition under non-stress conditions. Given the conservation of this RH subfamily, they may control basal levels of conditionally-regulated mRNAs in diverse eukaryotes, accelerating responses without penalty.

overall_design: 42 samples in total - 3 replicates, 2 genotypes, 2 RNA populations, no treatment and treatments

first: Thanin

last: Chantarachot

first: Reed

last: Sorensen

first: Julia

last: Bailey-Serres


title: In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states

city: Nijmegen

first: Hendrik

last: Marks

address: Geert Grooteplein 26/28

department: Molecular Biology

institute: Radboud University Nijmegen, RIMLS

summary: Upon implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked, and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state, defined by co-expression of naive factors with transcription factor OTX2. Downregulation of blastocyst WNT signals drives transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells (RSCs), a self-renewing naive-primed intermediate. RSCs erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate where control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.

overall_design: Examination of H3K27me3 and H3K4me3 in various mouse pluripotent states. Comparison with previously published 2i ESCs and EpiSCs.

first: Rene

middle: A

last: Dirks

first: Guido

last: van Mierlo

first: Alex

last: Neagu

first: Emiel

last: van Genderen

first: Irene

last: Escudero

first: Lucas

last: Verwegen

first: Dorota

last: Kurek

first: Johannes

last: Lehmann

first: Jente

last: Stel

last: Maas

first: Cindy

last: Eleveld

first: Yang

last: Ge

last: den Dekker

first: Rutger

middle: W

last: Brouwer

first: Wilfred

last: van Ijcken

first: Miha

last: Modic

first: Micha

last: Drukker

first: Joop

middle: H

last: Jansen

first: Nicolaas

last: Rivron

first: Esther

last: Baart

first: Derk

last: ten Berge


title: ERa-binding super enhancers drive key mediators that convey uterine hormone responses

city: Research Triangle Park

first: Sylvia

last: Hewitt

state: NC

address: 111 Alexander Dr

department: RDBL

summary: Estrogen receptor α (ERα) modulates gene expression through interactions with enhancer regions of chromatin that are frequently distal from the promoters of estrogen regulated genes. Active chromatin enriched “super-enhancer” (SE) regions, mainly described in in vitro culture systems, often control production of key cell type determining transcription factors.  Here, we define ERα binding super-enhancers in vivo within hormone responsive uterine tissue. SE are already formed prior to estrogen exposure at the onset of puberty. The SE encode critical developmental factors including Rara and Hoxd.  Using chromosome conformation capture with high throughput sequencing (Hi-C) we demonstrate that most ERα-binding SE are located at a chromatin loop end and most uterine genes in loop ends associated with ERα-binding SEs are estrogen regulated. Although SE are formed prior to puberty, SE-associated genes acquire optimal ERα dependent expression after reproductive maturity, indicating estrogen impacts enhancer function subsequent to assembly. ERα-binding SE-associated genes impact key uterine functions mediated by estrogen, including TGFβ and LIF signaling pathways. This is the first identification of ERα-binding SE interactions underlying hormonal regulation of genes in uterine tissue and optimal development of estrogen response

overall_design: Hi-C of ovariectomized adult WT uterine tissue after treatment for 1 hour with saline vehicle , estradiol, or progesterone or ERa knockout uterine tissue after treatment with saline vehicle for 1 hour

first: Sara

last: Grimm

first: San-Pin

last: Wu

first: Francesco

middle: J

last: DeMayo

first: Kenneth

last: Korach


title: Integrated analyses of translatome and proteome identify the rules of translation selectivity in RPS14-deficient

city: Paris

first: Ismael

last: Boussaid

address: 22 Rue Mechain

institute: Inserm

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

overall_design: Refer to individual Series


title: In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states (II, single-cell RNA-Seq data)

city: Rotterdam

address: Wytemaweg 80

department: Cell Biology

institute: ErasmusMC

summary: Naive pluripotent cells in the implanting mouse blastocyst generate a rosette structure before undergoing lumenogenesis to form the egg cylinder. Simultaneously, they acquire primed pluripotency, the ability to differentiate into the primary germ layers. The existence of discrete intermediate pluripotent states during this transition has not been demonstrated. We identify here a distinct rosette pluripotent state, defined by co-expression of naive factors with transcription factor OTX2. Downregulation of WNT signals in the blastocyst drives transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells (RSCs), a self-renewing naive-primed intermediate. RSCs gain a unique epigenome that includes erasure of constitutive heterochromatin and bivalent marking of primed pluripotency genes. Notwithstanding this primed chromatin landscape, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate where control over pluripotency progression and morphogenesis pivots from WNT to MEK signals.

overall_design: Mouse embryonic stem cells were differentiated towards rosette- and primed pluripotency and sorted into 384-well plates. Afterwards, single-cell transcriptome profiles were obtained using SORT-seq (Muraro, M.J. et al. A Single-Cell Transcriptome Atlas of the Human Pancreas. Cell Syst 3, 385-394 e383 (2016)).

first: Mauro

last: Muraro

first: Emiel

last: van Genderen

first: Nicolas


title: mRNA length and 3’UTR structure govern the translation selectivity in RPS14-haploinsufficient erythroblasts [translatome]

summary:
5q- syndrome is a somatic ribosomopathy linked to the monoallelic deletion of the RPS14 gene and characterized by a proeminent erythroid phenotype. The mechanism of anemia involves an impaired differentiation and increased apoptosis of erythroblasts. Here we show that GATA1 protein expression is low in line with a defect in the representation of its mRNA at the ribosome. A global analysis of transcripts on polysomes indicates that translation is selective with a decreased representation of the transcripts with a short coding sequence and UTRs and a highly structured 3’UTR, a subset of transcripts that includes GATA1. Our whole proteome analysis confirms that post-transcriptionally downregulated proteins were encoded by transcripts with a short length and structured 3’UTR. We identified a subset of post-translationally downregulated proteins including ribosomal proteins and translation elongation factors encoded by 5’TOP mRNAs that were enriched on the ribosome. Our results indicate that the thermodynamic characteristics of 3’UTR and in a lesser extend 5’UTR and the transcript length are the determinants of translation selectivity under RPS14 haploinsufficiency conditions and that a post-translational regulation of ribosomal proteins accounts for their decreased content in the cell.
We performed transcriptome and translatome expression profiling of cells infected with shRPS14 or shSCR

overall_design: The purpose of the dataset is to profile  analyze the effect of silencing RPS14

first: Michaela

last: Fontenay


title: Somatic tissue engineering in mouse models reveals an actionable role for WNT pathway alterations in prostate cancer metastasis

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: To study genetic factors that influence the progression and therapeutic response of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late stage human disease, can produce tumors that are metastatic and castration resistant.  Unexpectedly, a subset of particularly metastatic tumors acquired WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Harnessing features linked to the EPO-GEMM approach, we validate the WNT pathway as a key event in driving metastatic disease, a finding that we confirm in an orthogonal approach using mouse prostate organoids. Moreover, we show that tumors harboring WNT pathway alterations are sensitive to pharmacological WNT pathway inhibition. Thus, by leveraging the power of EPO-GEMMs, our studies reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as an actionable therapeutic target in metastatic prostate cancer.

overall_design: For RNA-seq analysis of the transcriptional profiles of MPten and MP EPO-GEMM prostate tumors, as well as normal anterior lobes of prostates of wild-type (WT) C57BL/6, total RNA was extracted from bulk tissue using the RNeasy Mini Kit (Qiagen). Purified polyA mRNA was subsequently fragmented, and first and second strand cDNA synthesis performed using standard Illumina mRNA TruSeq library preparation protocols. Double stranded cDNA was subsequently processed for TruSeq dual-index Illumina library generation.

first: Josef

last: Leibold

first: Marcus

last: Ruscetti

first: Zhen

last: Cao

first: Timour

last: Baslan

first: Wassim

last: Abida

first: Min

last: Zhou

first: Judith

last: Feucht

first: Teng

last: Han

first: Francisco

last: Barriga

first: Kaloyan

last: Tsanov

first: Leah

last: Zamechek

first: Amanda

last: Kulick

first: Corina

last: Amor

first: Sha

last: Tian

first: Nelson

last: Salgado

last: Sanchez-Rivera

first: Elisa

last: de Stanchina

first: John

middle: E

last: Wilkson

first: Cory

last: Abate-Shen

first: Philip

last: Watson

first: Lukas

last: Dow

first: Charles

middle: L

last: Sawyers

first: Scott

last: Lowe


title: mRNA length and 3’UTR structure govern the translation selectivity in RPS14-haploinsufficient erythroblasts [transcriptome]

first: Ismael

last: Boussaid

first: Michaela

last: Fontenay


title: Rules governing translation selectivity under limiting ribosome availability

summary:
Ribosomopathies are cell-type-specific pathologies related to a ribosomal protein (RP) gene insult. The 5q- syndrome is a somatic ribosomopathy linked to RPS14 gene haploinsufficiency and characterized by a prominent erythroid hypoplasia. Using quantitative proteomic, we show that GATA1 protein expression is low in shRPS14 cells in which ribosome quantities are diminished.   Here, we investigated the cause of low GATA1 protein expression in limiting ribosome availability. A global analysis of translation in RPs deficiencies highlights the rules that drive translation selectivity. We demonstrate that in addition of the transcript length, a high codon adaptation index (CAI) and a highly structured 3’UTR are the key characteristics for a selective translation. An integrated analysis of transcriptome and proteome confirms that the post-transcriptional regulations of gene expression are directly linked to the criteria governing the translational selectivity. In particular, these criteria explain GATA1 translation default with unprecedented precision. More generally, the proteins that accumulate along normal erythropoiesis share the determinants of translation selectivity revealed by the conditions of limiting ribosome availability.
We performed translatome expression profiling of cells infected with shRPS14 or shSCR


title: Targeted determination of pri-miRNA processing

city: Barcelona

first: Benjamin

last: Lang

address: Carrer del Dr. Aiguader, 88

institute: Centre for Genomic Regulation (CRG)

summary: Targeted sequencing at Microprocessor cleavage sites in pri-miRNAs to determine processing efficiency of several pri-miRNAs in vivo in one experiment

overall_design:
Pri-miRNAs are enriched with targeted probes from Illumina RNA sequencing libraries and sequenced with low depth (high coverage) to focus sequencing reads on pri-miRNA processing
Hepatocellular carcinoma A1-C12 (40 samples); Normal Liver D1-D10 (9 samples)

first: Ulf

last: Andersson Vang Ørom


title: Genome-wide Analysis of Brassinosteroid Responsive small RNAs in Arabidopsis thaliana

status: Public on Apr 27 2020

city: Chuncheon

first: Dong-Hoon

last: Jeong

state: Gangwon-do

address: Room #8306, Department of Life Science, 1 Hallymdaehak-gil

department: Dept. of Life Science

country: South Korea

institute: Hallym University

summary: Brassinosteroids (BRs) are a class of class of phytohormones with important roles in regulating physiological and developmental processes. Small RNAs, including small interfering RNAs and microRNAs (miRNAs), are non-protein coding RNAs that regulate gene expression at the transcriptional and post-transcriptional levels. However, the roles of small RNAs in BR response have not been studied well. In this study, we aimed to identify BR-responsive small RNA clusters and miRNAs in Arabidopsis. In addition, the effect of BR-responsive small RNAs on their transcripts and target genes were examined. Small RNA libraries were constructed from control and epibrassinolide-treated seedlings. After sequencing the small RNA libraries, differentially expressed small RNA clusters were identified by examining the expression levels of small RNAs in 100-nt bins of Arabidopsis genome. To identify the BR-responsive miRNAs, the expression levels of all the annotated mature miRNAs, registered in miRBase, were analyzed. Previously published RNA-seq data were utilized to monitor the BR-responsive expression patterns of differentially expressed small RNA clusters and miRNA target genes. In results, 38 BR-responsive small RNA clusters, including 30 down-regulated and eight up-regulated clusters, were identified. These differentially expressed small RNA clusters were from miRNA loci, transposons, protein-coding genes, pseudo genes and others. Of these, a transgene, BRI1, accumulates small RNAs, which are not found in the wild type. Small RNAs in this transgene are up-regulated by BRs while BRI1 mRNA is down-regulated by BRs. By analyzing the expression patterns of mature miRNAs, we have identified BR-repressed miR398a-5p and BR-induced miR156g. Although miR398a-5p is down-regulated by BRs, its predicted targets were not responsive to BRs. However, SPL3, a target of BR-inducible miR156g, is down-regulated by BRs. BR-responsive small RNAs and miRNAs identified in this study will provide an insight into the role of small RNAs in BR responses in plants. Especially, we suggest that miR156g/SPL3 module might play a role in BR-mediated growth and development in Arabidopsis.

overall_design: Examination of brassinosteroid-responsive small RNAs in Arabidopsis

first: Man-Ho

last: Oh

first: So Young

last: Park


title: HACE1 prevents lung carcinogenesis via inhibition of RAC-family GTPases

city: Vienna

first: Melanie

last: Floderer

address: Dr.-Bohr-Gasse 3

country: Austria

institute: IMBA

summary: HACE1 is an E3 ubiquitin ligase with important roles in tumor biology and tissue homeostasis. Loss or mutation of HACE1 has been associated with the occurrence of a variety of neoplasms, but the underlying mechanisms have not been defined yet. Here we report that HACE1 is frequently mutated in human lung cancer. In mice, loss of Hace1 led to enhanced progression of KRasG12D-driven lung tumors. Additional ablation of the oncogenic GTPase Rac1 partially reduced progression of Hace1-/- lung tumors. RAC2, a novel ubiquitylation target of HACE1, could compensate for the absence of its homolog RAC1 in Hace1-deficient, but not in HACE1-sufficient tumors. Accordingly, ablation of both Rac1 and Rac2 fully averted the increased progression of KRasG12D-driven lung tumors in Hace1-/- mice. In lung cancer patients, increased expression of HACE1 correlated with reduced levels of RAC1 and RAC2 and prolonged survival, while elevated expression of RAC1 and RAC2 was associated with poor prognosis. This work defines HACE1 as a crucial regulator of the oncogenic activity of RAC-family GTPases in lung cancer development.

overall_design: Analysis of gene expression in cultured pneumocytes from different groups

first: Luigi

last: Tortola

last: Kogler


title: Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution

first: Diego

middle: Rivera

last: Gelsinger

state: MD

address: 910 Light Street, Apartment 2

department: Biology

institute: The Johns Hopkins Univeristy

summary: Members of the third domain of life, the Archaea, are ubiquitous in all environments on Earth but remain understudied in many aspects including regulatory processes of the central dogma. Archaea present unique biology because they utilize a mosaic of molecular features from both Bacteria and Eukarya, along with unique features. The advent of a high-throughput view of the translation landscape via ribosome profiling in the Bacteria and the Eukarya has illuminated the complexity and previously underappreciated regulation of translation (i.e. translation efficiency, ribosome surveillance, etc.) that potentially has large scale effects on cellular functionality. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii and elucidated the translational landscape for the first time in the third domain of life. We coupled ribosome profiling with translation inhibitors to: (a) determine the size of the archaeal ribosome footprint, (b) systematically assign translation states of the ribosome to footprint lengths in a mostly leaderless transcriptome, (c) experimentally induce ribosome pauses and clarify the pausing landscape comprehensively, (d) identify putative novel proteins, including small open reading frames (smORFs), and (e) provide evidence that many genes initiate on putative alternative translation start sites (paTSS) around and within open reading frames (ORFs), demonstrating how a microorganism with a gene dense genome can produce proteins with distinct functions using the same gene.

overall_design:
H. volcanii H98 single colonies were picked and grown overnight at 42°C with shaking at 220 rpm in Hv-YPC medium supplemented with thymidine (50 μg/mL final concentration) until saturation (OD600 > 1.0). These cultures were diluted to OD600 0.02 in fresh media, grown to  OD600 0.4 and split evenly into two flasks; one flask was used as a no treatment control and the other was treated with either 1 mg/mL homo-harringtonine (CAS Number 26833-87-4), 20 mM serine hydroxamate (CAS Number 55779-32-3), or 100 μg/mL anisomycin, final concentrations. Cells were harvested by centrifugation or direct freezing of the culture in liquid nitrogen. For cells harvested by centrifugation, cultures were immediately centrifuged at 8,000 rpm for 3 minutes at room temperature, the supernatant removed, and the pellets flashed frozen in liquid nitrogen. For cell lysis, the frozen pellets were resuspended in 1 mL of 1x lysis buffer (3.4 M KCl, 500 mM MgCl2, 50 mM CaCl2, 1 M Tris pH 7.5) with an additional 100 μg/mL anisomycin, transferred to a cryomill, and pulverized with 5 cycles (1 minute of grinding at 5 Hz, 1 minute of cooling). Lysates were then thawed at room temperature, centrifuged at 10,000 x g for 5 minutes, and transferred to a new tube on ice. For direct freezing of cultures in liquid nitrogen, 100 mL of culture sprayed directly into liquid nitrogen using a serological pipette. The frozen culture form small pellets which are collected and 50g of pellets were weighed in order to add 1x lysis buffer (3.4 M KCl, 500 mM MgCl2, 50 mM CaCl2, 1 M Tris pH 7.5) and 100 μg/mL anisomycin to prevent ribosome elongation when thawed later on. The pellets were pulverized in a cryomill at 10 cycles due to the larger volume of input (1 minute of grinding at 10 Hz, 1 minute of cooling). The lysates were thawed at room temperature and ribosomes were pelleted over a 60% sucrose cushion (sucrose dissolved in lysis buffer) in an ultracentrifuge with a Ti-70 rotor at 60,000 rpm for 2 hours at 4°C. Ribosome pellets were resuspended in 200 μL lysis buffer. Cell lysates (from cells harvested by centrifugation or direct freezing) were processed by first treating 20 AU of lysate RNA (Nanodrop) with 12,000 units of MNase (Nuclease S7, Roche) for 1 hour at 25°C. After nuclease digestion, samples were loaded onto 10-50% sucrose gradients and RNA was isolated from monosome fractions as described above. Library preparation was performed as previously described (Muhammed, et al 2019). Briefly, 10 µg of RNA fragments was used to purify 15-45 nt RNA fragments by gel electrophoresis (15% TBE Urea gel); RNA fragments were treated with T4 polynucleotide kinase (NEB), then ligated to the linker (NEB Universal miRNA Cloning Linker) using T4 RNA ligase (NEB), and gel extracted from a 10% TBE Urea gel. Lastly, rRNA fragments were subtracted using the Ribo-Zero rRNA removal kit for bacteria (Illumina), then fragments reverse transcribed with SuperScript III (Invitrogen) using custom primers previously described (Muhammed, et al 2019), template RNA was degraded using NaOH and high heat, gel extracted from a 10% TBE Urea gel, circularized using CircLigase (Epicentre) and PCR amplified (8-12 cycles) with Phusion polymerase (NEB) using custom primers (Muhammed, et al 2019), and PCR products gel extracted from a 10% TBE gels. PCR products were analyzed for size and concentration using a BioAnalyzer high sensitivity DNA kit (standard protocol) before sequencing on an Illumina HiSeq 2500 at the Johns Hopkins Sequencing Core Facility (Baltimore, MD).

Adapter to trim: CTGTAGGCACCATCAATAGATCGGAAGAGCACACGTCTGAACTCCAGTCA

middle: R

first: Emma

last: Dallon

first: Rahul

last: Reddy

first: Fuad

last: Muhammed

first: Allen

last: Buskirk

first: Jocelyne

last: DiRuggiero


title: Genomic and physiological resilience in extreme environments are associated with a secure attachment style

status: Public on Apr 26 2020

city: Roma

first: Ivan

last: Arisi

address: viale Regina Elena 295

department: Bioinformatics Facility

institute: European Brain Research Institute

summary: Understanding individual capability to adjust to protracted confinement and isolation may inform adaptive plasticity and disease vulnerability/resilience, and may have long-term implications for operations requiring prolonged presence in distant and restricted environments. Individual coping depends on many different factors encompassing psychological dispositional traits, endocrine reactivity and their underlying molecular mechanisms (e.g. gene expression). A positive view of self and others (secure attachment style) has been proposed to promote individual resilience under extreme environmental conditions. Here, we tested this hypothesis and investigated the underlying molecular mechanisms in 13 healthy volunteers confined and isolated for 12 months in a research station located 1670 km away from the south geographic pole on the Antarctic Plateau at 3233 m above sea level. Study participants, stratified for attachment style, were characterised longitudinally (before, during and after confinement) for their psychological appraisal of the stressful nature of the expedition, diurnal fluctuations in endocrine stress reactivity, and gene expression profiling (Agilent microarray transcriptomics). Predictably, a secure attachment style was associated with reduced psychological distress and endocrine vulnerability to stress. In addition, while prolonged confinement and isolation remarkably altered overall patterns of gene expression, such alteration was largely reduced in individuals characterized by a secure attachment style. Furthermore, increased resilience was associated with a reduced expression of genes involved in energy metabolism (mitochondrial function and oxidative phosphorylation). Ultimately, our data indicate that a secure attachment style may favour individual resilience in extreme environments and that such resilience can be mapped onto identifiable molecular substrates.

overall_design: The mission crew was composed by 13 members. A total of n=49 peripheral blood samples were processed. Except few cases, 4 samples from each person were collected at 4 different time points: PRE (before mission), Day30 (30 days after mission start), Day150 (150 days after mission start), POST (after mission end). In 3 cases only it was not possible to collect all the samples, for technical reasons: in total 11,13,12,13 samples were collected at the PRE, D30, D150, POST time points respectively.

first: Viviana

last: Caputo

first: Maria

middle: G

last: Pacilli

first: Tommaso

last: Mazza

first: Rossella

last: Brandi

first: Alice

last: Traversa

first: Giampietro

last: Casasanta

first: Edoardo

last: Pisa

first: Michele

last: Sonnessa

first: Beth

last: Healey

first: Lorenzo

last: Moggio

first: Mara

last: D’Onofrio

first: Enrico

last: Alleva

first: Simone

last: Macrì


title: MYC ChIP-seq in human pancreatic ductal adenocarcinoma cells

city: Portland

last: Sherman

state: OR

address: 2730 SW Moody Avenue CL6C

institute: Oregon Health & Science University

summary: To assess modulation of global MYC chromatin occupancy by stromal cues, we exposed human pancreatic cancer cells to secreted factors from cancer-associated fibroblasts and performed ChIP-seq in stroma-stimulated cells compared to controls.

overall_design: MIAPaCa2 human PDAC cells were serum-starved for 48 hours, then treated with fresh serum-free media or with serum-free conditioned media from primary cancer-associated fibroblasts. After 3 hours, cells were fixed and ChIP for MYC performed.

first: Wes

last: Horton

first: Sohinee

last: Bhattacharyya


title: Generation of pancreatic β cells from CD177+ anterior definitive endoderm

city: Neuherberg

first: Heiko

last: Lickert

address: Ingolstaedter Landstraße 1

department: Institute of Diabetes and Regeneration Research

institute: Helmholtz Zentrum München German Research Center for Environmental Health

summary: Methods for differentiating human pluripotent stem cells to pancreatic and liver lineages in vitro have been limited by the inability to identify and isolate distinct endodermal subpopulations specific to these two organs. Here we report that pancreatic and hepatic progenitors can be isolated using the surface markers CD177/NB1 glycoprotein and inducible T-cell costimulatory ligand CD275/ICOSL, respectively, from seemingly homogeneous definitive endoderm derived from human pluripotent stem cells. Anterior definitive endoderm (ADE) subpopulations identified by CD177 and CD275 show inverse activation of canonical and noncanonical WNT signaling. CD177+ ADE expresses and synthesizes the secreted WNT, NODAL and BMP antagonist CERBERUS1 and is specified toward the pancreatic fate. CD275+ ADE receives canonical Wnt signaling and is specified toward the liver fate. Isolated CD177+ ADE differentiates more homogeneously into pancreatic progenitors and into more functionally mature and glucose-responsive β-like cells in vitro compared with cells from unsorted differentiation cultures.

overall_design: We performed Affymetrix microarray analysis on flow cytomerty sorted cell populations marked by CXCR4, CD2715 or CD177

first: Pallavi

middle: U

last: Mahaddalkar

first: Katharina

last: Scheibner

first: Sandra

last: Pfluger

first: Ansarullah

last: Ansarullah

first: Michael

last: Sterr

first: Julia

last: Beckenbauer

first: Martin

last: Irmler

first: Johannes

last: Beckers

first: Sebastian

last: Knöbel


title: Ribo-seq on K562 and HepG2 cells

city: Berlin

last: Calviello

address: Robert-Rössle-Straße 10, MDC, Haus 89

department: Berlin Institute for Medical Systems Biology

institute: Max Delbrueck Center for Molecular Medicine

summary: Deep sequencing methods have matured to comprehensively detect the full set of transcribed loci, but there is a gap to determine the function of the resulting highly complex transcriptomes. To this end, we have developed a new approach named ORFquant to annotate and quantify translation at the single open reading frame (ORF) level using Ribo-seq data.

overall_design: After RNase I footprinting in CHX-containing lysis buffer, RNA fragments around 29nt were isolated and subjected to rRNA depletion using RiboZero.

first: Antje

last: Hirsekorn

first: Uwe

last: Ohler


title: Clinical characteristics and exploratory genomic analyses of germline BRCA1 or BRCA2 mutations in breast cancer

status: Public on Apr 25 2020

city: Seoul

first: Yeon Hee

last: Park

address: 81 Ilwon-ro, Gangnam-gu

institute: Samsung Medical Center

summary: gBRCA1/2 mutations increase the incidence of breast cancer (BC) by interrupting the homologous recombination repair (HRR) pathway. Although gBRCA1 and gBRCA2 BC have similar clinical profiles, different molecular characteristics have been observed. In this study, we conducted comprehensive genomic analyses and compared gBRCA1/2 BC. Sanger sequencing to identify gBRCA1/2 mutations was conducted in 2,720 patients, and gBRCA1 (n=128) and gBRCA2 (n=126) mutations were analyzed. Within that population, deep target sequencing (TS) and matched whole transcriptome sequencing (WTS) results were available for 46 and 34 patients, respectively. An internal database of breast-cancer patients with wildtype gBRCA was used to compile a TS (n=195) and WTS (n=137) reference dataset. Three specific mutation sites, p.Y130X (n=14) and p.1210Afs (n=13) in gBRCA1 and p.R294X (n=22) in gBRCA2, were comparably frequent. Immunohistochemistry subtyping determined that the incidence of triple negative BC was higher among those with a gBRCA1 mutation (71.9%), and estrogen receptor (ER)-positive BC was dominant in those with a gBRCA2 mutation (76.2%). gBRCA1/2 mutations were mutually exclusive with PIK3CA somatic mutations (P<0.05), and gBRCA1 frequently co-occurred with TP53 somatic mutations (P<0.05). The median tumor mutation burden was 6.53 per megabase (MB) in gBRCA1 and 6.44 per MB in gBRCA2. The expression of AR, ESR1, and PGR was significantly upregulated with gBRCA2 mutation compared with gBRCA1 mutation. gBRCA1 and gBRCA2 BC have similar clinical characteristics, but they have different molecular subtypes, co-altered somatic mutations, and gene expression patterns. Implications: Even though gBRCA1 and gBRCA2 mutations both alter HRR pathways, our results suggest that they generate different molecular characteristics and different mechanisms of carcinogenesis.

overall_design: whole transcriptome sequencing of 34 breast cancer patients who have germline BRCA mutations by Illumina Hiseq 2500

first: Yeon