title: Distinct and overlapping roles of STAG1 and STAG2 in cohesin localization and gene expression in embryonic stem cells [ChIP-seq]

city: Chapel Hill

first: Jill

last: Dowen

state: NC

address: 250 Bell Tower Drive

institute: University of North Carolina at Chapel Hill

summary:
Purpose: Cohesin is a ring-shaped complex that is critical to genome organization and is an important regulator of gene expression. How cohesin accessory proteins contribute to cohesin function remains unclear. The purpose of this study is to assess the roles of cohesin accessory proteins, STAG1 and STAG2, in cohesin localization and gene expression.

Method: Genome wide binding patterns of the STAGs and cohesin in wildtype cells as well as in STAG CRISPR/Cas9 genome edited knockout cells were assessed via chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq). The redundancy of the STAGs was addressed by using siRNA against STAG1 in a STAG2-knockout background.

overall_design:
ChIP-seq was performed in wildtype mESCs in duplicate for STAG1, STAG2 and RAD21. ChIP-seq was performed in CRISPR-Cas9 genome edited STAG deletion mESCs in duplicate for the reciprocal STAG and RAD21. ChIP-seq was performed in wildtype and Stag2-KO cells after being treated with either siGLO transfection control or siStag1.

Please note that each processed data was generated from both replicates and is linked to the corresponding *Rep1 sample records.

first: Nicole

middle: L

last: Arruda

first: Zachary

last: Carico

first: Megan

last: Justice

first: Ying

middle: F

first: Junjie

last: Zhou

first: Holden

middle: C

last: Stefan


title: Gene expression changes of Planococcus kocurii O516 in various culture conditions (with or without sulfamethoxazole)

status: Public on Aug 08 2020

city: Qingdao

first: Sijia

last: Kong

address: 238 Songling Road

country: China

institute: Ocean University of China

type: Expression profiling by high throughput sequencing

summary: The strain Planococcus kocurii O516 isolated from the marine culture environment was found to be stably and highly effective for the degradation of sulfamethoxazole. We investigated the expression behavior of functional genes related to this degradation ability in P. kocurii O516 under the culture conditions with or without sulfamethoxazole.

overall_design: Five milliliters of bacterial resuspension (in each medium) were inoculated into 50 mL of respective test media and the cultures were shaken at 120 rpm at 30°C. As test media, Muller-Hinton Broth medium with and without sulfamethoxazole were used. After incubation at 24 h, 50 mL of each culture was taken and used for RNA-Seq. A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequence data by HiSeq_4000 were assembled and analyzed using the CLC Genomics Workbench and the statistical software R with various packages (edgeR, and DEGSeq). HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Then, using DEGSeq R package (1.20.0), the gene expression values relative to the two different mediums were calculated to log2 values of fold change (log2FC) with FDR (false discovery rate) values under the four culture conditions.

first: Yangguo

last: Zhao


title: Resolving fate and transcriptome of hematopoietic stem cell clones [LT_ST_HSC]

status: Public on Aug 11 2020

city: Nanjing

first: Xi

last: Wang

address: Longmian Avenue 101, Jiangning District

institute: Nanjing Medical University

summary: Hematopoietic stem cell (HSC) differentiation into mature lineages has been studied under physiological conditions in vivo by genetic barcoding-driven lineage tracing. HSC clones differ in output (differentiation-inactive versus differentiation-active), and in fates (multilineage versus lineage-restricted). Single-cell sequencing data revealed transcriptome diversity of HSC and progenitors, and suggested differentiation pathways. However, molecular hallmarks of functionally distinct HSC clones have not been resolved because existing lineage tracing experiments did not provide transcriptomes, and single cell RNA sequencing lacked information on precursor-product relationships, and hence fate. To close this gap, here we introduce PolyloxExpress, a Cre recombinase-dependent DNA substrate for in situ barcoding in mice that is expressed as mRNA. PolyloxExpress barcoding allows parallel readout of clonal HSC fates (via comparison of barcodes in HSC and mature lineages), and transcriptomes (via single-cell RNA sequencing and barcode matching). Analysing a total of 91 individual HSC clones, we show that differentiation-inactive versus differentiation-active HSC clones reside in different regions of the transcriptional landscape. Inactive HSC clones are closer to the origin of the transcriptional trajectory, yet are proliferatively not more quiescent than active clones. Multilineage versus myelo-erythroid fate-restricted HSC clones show very few transcriptional differences at the HSC stage, yet pronounced fate-specific profiles at the multipotent progenitor stage. Projecting HSC clones with defined fates onto transcriptional landscapes provides a basis for future studies into the molecular determinants for stem cell fate.

overall_design: Phenotypic long-term (LT) (Lin-Kit+Sca1+CD150+CD48-) and short-term (ST) (Lin-Kit+Sca1+CD150-CD48-) HSC were sorted and transcriptomes of each stem cell were profiled using single-cell RNA-sequencing. Their gene expression profiles were then used to characterize every HSC profiled in the PolyloxExpress experiments.

first: Ann-Kathrin

last: Fanti

first: Katrin

last: Busch

first: Hans-Reimer

last: Rodewald


title: Effect of SB100X-mediated integration on the transcriptome of CD34+ cells

status: Public on Aug 07 2020

city: Seattle

first: hongjie

last: wang

state: WA

address: 1959 NE Pacific Street

country: USA

institute: University of washington

summary: We performed RNA-seq on human CD34+ cells that stably expressed mgmt/GFP transgenes after SB100x-mediated integration and O6BG/BCNU selection in vitro

overall_design: CD34+ cells were infected with a HDAd5/35++ vector containing a GFP/mgmt cassette under control of the Ef1a promoter alone or in combination with HDAd-SB. Transduced cells were expanded in erythroid differentation medium for 16 days. Two rounds of O6BG/BCNU selection (50 µM O6BG + 35 µM BCNU) were enriched for GFP-positive cells with integrated transposons. Total RNA were extracted from both samples and subjected to RNA-seq.

first: Andre

last: Lieber

first: Hongjie

first: Aphrodite

last: Georgakopoulou


title: Ribosome profiling of SMN-primed ribosomes and active ribosome profiling from brains of a mouse model of severe SMA

city: New Haven, US & Trento, Italy

first: Toma

last: Tebaldi

state: CT

address: 300 George Street

department: CIBIO & Department of Internal Medicine, Yale Cancer Center

institute: Yale University & University of Trento

summary:
Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality, with an incidence of around 1 in 6,000-10,000 live births. SMA is caused by low levels of full-length survival of motor neuron protein (SMN). We demonstrate that SMN binds to ribosomes and that this interaction is tissue-dependent. We performed ribosome profiling analyses on lysates from P5 wild-type mouse brains exposed to RNase I, from which we isolated SMN-primed ribosomes by immunoprecipitation. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs which are enriched for translational enhancer sequences in the 5’UTR and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Additionally, we analyzed the positioning of active ribosomes using the Active-RiboSeq method based on RiboLace and compared early symptomatic SMA mouse brains to age-matched controls. We found that loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins involved in motor neuron function and stability.
Keywords: motor neuron disease, spinal muscular atrophy, SMA, SMN, SMN1, survival of motor neuron, SMN-primed ribosome profiling, translation, RiboLace, Ribo-Seq, active translation, ribosome heterogeneity

overall_design: We performed ribosome profiling of SMN-primed ribosomes in brain extracts from control mice at P5 (Smn+/-; SMN2tg/0). Experiments were performed in biological triplicate. In parallel, samples from immunoprecipitation with IgG were used as controls for possible unspecific isolation of ribosome protected fragments. Additionally, we performed active ribosome profiling with RiboLace (PMID: 30355487) and ribosome profiling in SMA early symptomatic mouse brains (Smn-/-; SMN2tg/0). Experiments were performed in biological duplicate. Ribosome profiling of age-matched controls were deposited under accessions GSE102354 (RiboLace) and GSE102318 (ribosome profiling).

first: Fabio

last: Lauria

first: Paola

last: Bernabò

first: Massimiliano

last: Clamer

first: Elena

last: Perenthaler

first: Ewout

middle: J

last: Groen

first: Alessandro

last: Quattrone

first: Thomas

middle: H

last: Gillingwater

first: Gabriella

last: Viero


title: The first transcriptome analysis of sugar beet in compatible and incompatible interactions with Heterodera schachtii

city: Ghent

first: Bruno

last: Verstraeten

state: Oost-Vlaanderen

address: Technologiepark 71

country: Belgium

institute: Ghent University

summary: Sugar beet (Beta vulgaris subsp. vulgaris) is an economically important crop and provides nearly one third of the global sugar production annually. The beet cyst nematode (BCN), Heterodera schachtii, causes major yield losses in sugar beet worldwide. The most effective and economic approach to control this nematode is growing tolerant or resistant cultivars. To identify candidate genes involved in susceptibility and resistance, the transcriptome of sugar beet and BCN in compatible and incompatible interactions at two time points, was studied using mRNA-seq. In total, 16 cDNA libraries were constructed and 442 691 707raw reads were obtained. In the compatible interaction, many alterations in phytohormone-related genes were detected. The effect of exogenous application of methyl jasmonate and ethephon was therefore investigated and the results revealed significant reduction of J2s infection and female development rates in treated susceptible plants. Our results revealed candidate genes putatively involved in the Hs1pro1-induced resistance, such as genes related to phenylpropanoid pathway, putative R genes and genes encoding F-box proteins, zinc finger and NAC transcription factors, ABC transporters, BURP and CYSTM proteins. Also, the transcriptome of BCN in the infected root samples was analyzed and several nematode effector genes were found. Our study is the first investigation of the transcriptome profile in the compatible and incompatible interactions between sugar beet and BCN.

overall_design: Two cultivars of sugar beet, a susceptible and a resistant variety, were infected with Heterodera schachtii. On two time points, 4 days after infection and 10 days after infection, plants were sampled. For each combination of sugar beet type and time point a corresponding non-infected control was sampled as well.

first: Razieh

last: Ghaemi

first: Ebrahim

last: Pourjam

first: Naser

last: Safaie

first: Bagher

last: Mahmoudi

first: Rahim

last: Mehrabi

first: Tim

last: De Meyer

first: Tina

last: Kyndt


title: Resolving fate and transcriptome of hematopoietic stem cell clones [multiple cell types]

first: Xi

last: Wang

overall_design:
We devised the PolyloxExpress barcoding system to parallel readout clonal HSC fates via comparison of barcodes in HSC and mature lineages, and transcriptomes via single-cell RNA sequencing and barcode matching.
PolyloxExpress mice were analyzed between 7 and 20 weeks after birth; HSC, MPP, CMP, CLP, lineage progenitors and peripheral cells were sorted and sequenced for barcodes and transcriptomes.

first: Weike

last: Pei

first: Fuwei

last: Shang

first: Thorsten

middle: B

last: Feyerabend

last: Höfer

first: Hans-Reimer

last: Rodewald


title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

status: Public on Aug 06 2020

city: Nijmegen

first: Gert

middle: Jan

last: Veenstra

state: Not US or Canada

address: Geert Grooteplein 28

department: Molecular Developmental Biology

country: Netherlands

institute: Radboud University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Polycomb Repressive Complex 2 (PRC2) plays an essential role in gene repression during development, catalysing H3 lysine 27 trimethylation (H3K27me3). MTF2 in the PRC2.1 sub-complex, and JARID2 in PRC2.2, are central in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). To investigate how PRC2.1 and PRC2.2 cooperate, we combined Polycomb mutant mESCs with chemical inhibition of binding to H3K27me3. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, which are mutually reinforced. Whereas PRC2.1 recruitment is mediated by MTF2 binding to DNA, JARID2-containing PRC2.2 recruitment is more dependent on PRC1. Both recruitment axes are supported by core subunit EED binding to H3K27me3, but EED inhibition exhibits a more pronounced effect in Jarid2 null cells. Finally, we show that PRC1 and PRC2 enhance reciprocal binding. Together, these data disentangle the interdependent interactions that are important for PRC2 recruitment.

overall_design: ChIP-seq for Mtf2, Ezh2, Jarid2, Ring1b, H3K27me3 and input for reference performed in WT mESC or mESC mutant for Mtf2,  Jarid2, EED, Ring1a/b with or without the PRC2 inhibitor EED226 or the PRC1 inhibitor MG32. Validation of quantification with spike-In ChIP for Ezh2 in WT, EED KO and Mtf2 GT lines. Part of the data are re-analized from Perino et al, 2018, Nat. Gen.

first: Matteo

last: Perino

first: Guido

last: van Mierlo

first: Chet

last: Loh

first: Sandra

last: Wardle

first: Dick

last: Zijlmans

first: Hendrik

last: Marks


title: Gut multi-omics profiling in Crohn’s disease: A preliminary study

status: Public on Aug 10 2020

city: Warszawa

first: Filip

last: Ambrozkiewicz

state: Mazowieckie

address: ul.Roentgena 5

department: Genetics

country: Poland

institute: Centrum Onkologii -  Instytut im. Marii Skłodowskiej - Curie

summary: Background: Inflammatory bowel diseases are classic polygenic disorders, with genetic loads that reflect immunopathological processes in response to intestinal microbiota. To assess gut bacterial community, microRNA (miRNA), and short chain fatty acid (SCFA) signatures associated with the activity of Crohn’s disease (CD).  Methods: DNA, miRNA, and metabolites were extracted from stool samples of 15 CD patients, eight with active disease and seven in remission, and nine healthy individuals. Microbial, miRNA and SCFA profiles were assessed using datasets from 16s rRNA sequencing, Nanostring miRNA and GC-MS targeted analysis, respectively.  Results: Pairwise comparisons showed that 11 and 27 taxa differed between controls and CD patients with active and inactive disease, respectively. Seven taxa were common to both comparisons, whereas eight taxa differed in CD patients. α-Diversity was lower in both CD groups than in controls. The levels of 13 miRNAs differed (p-value <0.05; FC >1.5) in CD patients and controls. Comparisons of controls with CD patients having active and inactive disease identified 12 and seven significantly different miRNAs, respectively. Of six SCFAs, the levels of two differed significantly (p-value <0.05, FC >1.5) in CD patients and controls, and the levels of four differed in patients with active and inactive CD. PLS-DA revealed models with high discriminatory powers (AUC >0.9) for bacteria and miRNA. The levels of 14 miRNAs and 39 bacterial taxa correlated with the level of SCFAs.   Conclusion: CD-related gut dysbiosis correlates significantly with miRNA and SCFA profiling, indicating complex relationships among all these factors in response to intestinal inflammation.

overall_design: Fecal miRNA was isolated from stool samples (approximately 200 mg) using mirVana miRNA Isolation Kits, according to the manufacturer’s protocol. miRNA was screened using 100 ng miRNA,  as recommended, and analyzed with nCounter human v2 miRNA expression assay kits, which allow detection of 800 human miRNAs. Hybridization was performed on the nCounter Prep Station, and miRNA was detected with an nCounter Digital Analyzer. Data were processed and analyzed with nSolver™ Analysis Software 4.0.

first: Jakub

last: Karczmarski

first: Maria

last: Kulecka

first: Agnieszka

last: Paziewska

first: Magdalena

last: Niemira

first: Natalia

last: Zeber

first: Edyta

last: Zagorowicz

first: Adam

last: Kretowski

first: Jerzy

last: Ostrowski


title: Modeling human T-cell mediated beta cell destruction

status: Public on Aug 05 2020

city: Cambridge

first: Haiting

last: Ma

state: MA

address: 455 Main Street

institute: Whitehead Institute

summary: Autoimmune destruction of pancreatic β cells underlies type 1 diabetes (T1D). To understand T-cell mediated immune impact on human pancreatic β cells, we combine β cell specific expression of a model antigen CD19 and anti-CD19 chimeric antigen receptor T (CAR-T) cells. Co-culturing CD19-expressing -like cells and CD19 CAR-T cells results in T-cell mediated β-like cell death with release of activated T cell cytokines. Transcriptome analysis of β-like cells and human islets treated with conditioned medium of the immune reaction identifies upregulation of immune reaction genes and the pyroptosis mediator GSDMD as well as its activator CASP4. Caspase-4-mediated cleaved GSDMD is detected in β-like cells under inflammation and endoplasmic reticulum (ER) stress conditions. Among the immune regulatory genes, PDL1 is one of the most upregulated, and PDL1 overexpression partially protects β-like cells in mice transplanted with human β cells. This experimental platform identifies potential mechanisms of β cell destruction and may allow testing therapeutic strategies.

overall_design: Examination of gene expression differences in primary human islets and human pluripostent stem cells differentiated beta-like cells under control or T cell conditioned medium condition.

first: Rudolf

last: Jaenisch


title: Pre- Treatment Mutational and Transcriptomic Landscape of Responding Metastatic Melanoma Patients to Anti-PD1 Immunotherapy

city: Cleveland

first: Jihye

last: Kim

state: Ohio

address: 9500 Euclid Ave.

department: Quantitative Health Sciences

institute: Cleveland Clinic

summary: Immunotherapy, such as anti-PD1, has improved the survival of patients with metastatic melanoma;  However, predicting which patients will respond to immunotherapy is still unknown. In this study we analyzed pre-immunotherapy treated tumors from 52 patients with metastatic melanoma and monitored their response based on RECIST 1.1 criteria.  The responders group contained 21 patients that had a complete or partial response, while the 31 non-responders had stable or progressive disease.  Whole exome sequencing (WES) was used to identify biomarkers of anti-PD1 response from somatic mutations between the two groups.  Variants in codons G34 and G41 in NFKBIE, a negative regulator of NF-kB, were found exclusively in the responders. NKBIE-related genes within the responder group were also enriched compared to the non-responders.  Patients that harbored NFKBIE-related gene mutations also had a higher mutational burden, decreased tumor volume with treatment, and increased progression-free survival.  RNA sequencing on a subsection of tumor samples identified differential expression of the TNFA signaling via NFKB pathway, which includes CD83.  By overexpressing NFKBIEG34E we were able to demonstrate this mutation is related to increased NF-kB activity, including increased CD83 protein expression when compared with the wildtype.  These results suggest that increased NF-kB signaling as a consequence of an NFKBIE mutation may contribute to a favorable anti-PD1 treatment response, including a possible novel role of CD83 in solid tumors.

overall_design: We analyzed pre-immunotherapy treated tumors from 52 patients with metastatic melanoma and monitored their response. The responders group contained 21 patients that had a complete or partial response, while the 31 non-responders had stable or progressive disease. RNA sequencing on a subsection of tumor samples identified differential expression of the TNFA signaling via NFKB pathway, which includes CD83

first: Carol

middle: M

last: Amato

first: Keith

last: Wells

first: Jennifer

middle: D

last: Hintzsche

first: Allison

last: Applegate

first: Nicholas

last: Gordon

first: Victoria

last: Vorwald

first: Richard

middle: P

last: Tobin

first: Kelsey

last: Nassar

first: Yiqun

last: Shellman

first: Theresa

last: Medina

first: Matthew

last: Rioth

first: Karl

last: Lewis

first: Martin

last: McCarter

first: Aik-Choon

last: Tan

first: William

middle: A

last: Robinson


title: Transcriptomic profiling of human effector and regulatory T cell subsets identifies predictive population signatures

first: Daniel

last: Campbell

state: Washington

address: 1201 Ninth Avenue

institute: Benaroya Research Institute

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

overall_design: Refer to individual Series


title: Identification of A Drug Blocking SARS-CoV-2 Infection using Human Pluripotent Stem Cell-derived Colon Organoids

city: New York

first: Shuibing

last: Chen

state: NY

address: A 827B, 1300 York Ave

department: Surgery

institute: Weill Cornell Medical College

summary: The current COVID-19 pandemic is caused by the novel coronavirus SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 virus biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients often present with respiratory symptoms including cough, dyspnea, and respiratory distress but upwards of 25% of respiratory dysfunction, many COVID-19 patients have digestive system indications, including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with more severe COVID-19 outcomes1. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of different colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple types of hESC-derived colon cells, but are highly enriched in hPSC-derivedKRT20+ enterocytes. Distinct cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which is further validated in vivo using a humanized mouse model. Finally, we adapted hPSC-derived COs to a high throughput platform to screen 1280 FDA-approved drugs. Mycophenolic acid was found to block the entry of SARS-Cov-2 pseudo-entry virus in COs, and confirmed to block infection of SARS-CoV-2 virus. In summary, this study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified a drug suitable for rapid clinical testing that blocks SARS-CoV-2 infection.

overall_design: scRNA-seq analysis of human pluripotent stem cell-derived colonic organoids (hPSC-COs) infected with SARS-CoV-2 pseudo-entry virus.

first: Xiaohua

last: Duan

first: Liuliu

last: Yang

first: Yuling

last: Han

first: Benjamin

last: Nilsson

first: Pengfei

last: Wang


title: Parallel scRNA-seq and genetic recording reveals lineage decisions in early mouse embryogenesis

city: Boston

first: Bradley

middle: E

last: Bernstein

address: 185 Cambridge Street

department: Pathology

institute: Massachusetts General Hospital

summary: How cell fate is specified at early stages of development is a fundamental question in biology. We apply deep transcriptomic profiling to map cell lineages at single cell resolution in embryoid bodies (EBs); a spontaneous, manipulatable, in vitro system for embryonic development. We predict the overall cell fate trajectory and define the transcriptomic landscape of cells at each critical branch. We present a dual barcoding system, comprising a static cellular barcode and inducible genetic recorder, and leverage long-read sequencing to obtain both barcodes and transcriptome from each single cell. Using this system, we confirm overall trajectory architecture, and validate key branch points, including the early isolation of the primordial germ cell (PGC) like lineage from preimplantation epiblast-like cells. Our data suggest DNA methylation is a critical determinant of PGC cell fate choice before the postimplantation epiblast cell state is determined. Together, our data provide a high resolution lineage decision map in an organoid model of early embryogenesis, with implications for the developing embryo.

overall_design: Examination of single cell RNA-seq in mouse Embryoid body (EB) differentiation. Examination of single cell RNA-seq and targeted genomic DNA-seq in dual-barcode integrated EBs at day 14. Examination of single cell RNA-seq in EBs treated by 5-azacytidine or DMSO control.

first: Ik

middle: S

last: Kim

first: Jingyi

last: Wu


title: Diet-MEF2 Interactions Shape Lipid Droplet Diversification in Muscle to Influence Drosophila Lifespan

city: College Station

first: Jason

last: Karpac

state: Texas

address: Reynolds Medical Building

department: Molecular and Cellular Medicine

institute: Texas A&M University Health Sciences Center

summary: The number, size, and composition of lipid droplets can be influenced by dietary changes that shift energy substrate availability. This diversification of lipid droplets can promote metabolic flexibility and shape cellular stress responses in unique tissues with distinctive metabolic roles. Using Drosophila, we uncovered a role for myocyte enhancer-factor 2 (MEF2) in modulating diet-dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle-specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester-enriched intramuscular lipid droplets in response to high calorie, carbohydrate sufficient diets. The diet-dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up-regulation of Cyclin E is required for diet-and-MEF2 dependent diversification of intramuscular lipid droplets. The integration of MEF2-regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation.

overall_design: We wanted to explore MEF-2 dependent muscle transcriptional networks, putatively influenced by carbohydrate availability, that could modulate lipid droplet diversification and potentially alter organismal lifespan. Utilizing transcriptomics (RNA-seq.), we generated genome-wide expression profiles from dissected thoraces of Act88FGS>UAS-MEF2RNAi (+/- RU486) flies fed a high calorie diet (compared to controls [Act88FGS>w1118 +/- RU486]).

first: Xiao

last: Zhao


title: Visceral adipose tissue macrophage subsets

city: Pittsburgh

first: Partha

last: Dutta

state: PA

address: 200 Lothrop Street

department: Medicine

institute: University of Pittsburgh

summary: We identified two different subsets of macrophages in visceral adipose tissue. These two subsets were transcriptomically different. We observed that C3CR1-low CCR2-low macrophages are transcriptomically different after myocardial infarction (MI).

overall_design: We sorted CX3CR1-high CCR2-high and CX3CR1-low and CCR2-low macrophages from visceral adipose tissue and blood monocytes from mice in the steady state, and CX3CR1-low and CCR2-low macrophages from visceral adipose tissue of mice with MI or sham surgery directly in RNA extraction buffer (Applied Biosystems Pico Pure Kit for mRNA extraction) and performed RNA-seq analysis.


title: Transcriptional analysis of resting T cells from human blood and skin

summary:
After activation, CD4+ T helper (Th) cells differentiate into functionally specialized populations that coordinate distinct immune responses and protect against different types of pathogens.  In humans, these effector and memory Th cell subsets can be readily identified in peripheral blood based on their differential expression of chemokine receptors that govern their homeostatic and inflammatory trafficking.  Foxp3+ regulatory T (Treg) cells can also be divided into subsets that phenotypically mirror each of these effector populations, and share expression of key transcription factors and effector cytokines.  In this study, we performed comprehensive transcriptional profiling of 11 phenotypically distinct Th and Treg cell subsets sorted from peripheral blood of healthy individuals.  Despite their shared phenotypes, we found that mirror Th and Treg subsets were transcriptionally dissimilar, and that Treg cell populations showed limited transcriptional diversity compared to Th cells.
We identified core transcriptional signatures shared across all Th and Treg cell populations, and unique signatures that define each of the Th or Treg populations.  Finally, we applied these signatures to bulk Th and Treg RNA-seq data and found enrichment of specific Th and Treg cell populations in different human tissues.  These results further define the molecular basis for the functional specialization and differentiation of Th and Treg cell populations, and provide a new resource for examining Th and Treg specialization in RNA-seq data.

overall_design:
RNA-seq was performed on CLA+ central and effector memory CD4+ T cells sorted from human blood and skin
CLA+CD4+ T cells were sorted from blood and skin of 6 different donors as central memory (Tcm, CD45RA-CCR7+) and effector memory (CD45-CCR7-) populations for RNA-seq.  14 samples passed quality control metrics and were used in the subsequent analyses.

first: D

first: I

middle: K

last: Gratz

first: B

last: Höllbacher

first: M

last: Klicznik

first: P

last: Morawski

first: S

last: Motley


title: Next generation sequencing of a unique set of glycovariant MC38 mouse colorectal cancer cell lines generated with CRISPR-Cas9 and CRISPR-dCas-VPR technology

city: Amsterdam

first: Anouk

last: Zaal

address: Boelelaan 1108

institute: Amsterdam UMC

summary: To investigate the effect of different tumor-associated glycans, mouse colorectal cancer cells (MC38 cell line) were genetically modified using CRISPR-Cas9 and CRISPR-dCas-VPR technology targeting different genes involved in glycosylation pathways. To study the effect of altered glycosylation at the transcriptional level, next generation sequencing was performed on the entire panel of glycovariant MC38 cell lines. The MC38 knockout cell lines created with the CRISPR-Cas9 technology are: MC38-MOCK#1, MC38-CMAS KO, MC38-COSMC KO, MC38-CMAS+COSMC KO. The MC38 overexpressing cell lines generated with the CRISPR-dCas9-VPR technology are: MC38-MOCK#2, MC38-FUT4, MC38-FUT9. Details on the cell lines can be found in the corresponding manuscript(s).

overall_design: 24 samples were used for next generation sequencing, including triplicates for each MC38 glycovariant cell line. The MC38-MOCK#1 and MC38-MOCK#2 were treated in the same way as the modified cell lines and serve as reference samples for the MC38 knockout and overexpressing cell lines, respectively.

first: Athanasios

last: Blanas

first: Sandra

last: van Vliet


title: Expression data from THP1-wt and THP1-NTR+ cells with LSD1 inhibitor (OG86), Nitroreductase NfsB (NTR) prodrug and negative control

status: Public on Aug 04 2020

city: Freiburg

first: Manfred

last: Jung

address: Albertstr. 25

department: Institut für Pharmazeutische Wissenschaften

country: Germany

institute: University Freiburg

summary:
The Nitroreductase NfsB (NTR) prodrug of an LSD1 inhibitor is inactive in wild-type THP1 cells, but the prodrug gets activated in the LSD1 inhibitor by the NTR in NTR-expressing THP1 cells (THP1-NTR+).
Cell-type specific inhibition of LSD1 in THP1-NTR+ cells with a NTR prodrug is shown with the expression data of the two cell lines, THP1-wt and THP1-NTR+. Data for the LSD1 inhibitor and a negative control with both cell lines are included.

overall_design: Cells were incubated with vehicle, prodrug, inhibitor or negativ control for 24 hours. Cells were harvested, RNA extracted, converted in ss-cDNA and hybridized on Affymetrix human Clariom S microarrays.

first: Mirjam

last: Hau


title: RNA-seq of human CD4+ T cell subsets

first: Daniel

last: Campbell

overall_design:
Transcriptional analysis of 11 different CD4+ T cell populations from 3 separate donors
11 CD4+ T cell populations were sorted from the blood of 3 individual donors as follows: Naïve (CD127+CD25-CD45RA+), Th1 (CD127+CD25-CD45RA-CXCR3+CCR6-), Th2 (CD127+CD25-CD45RA-CXCR3-CCR6-CCR4+), Th17 (CD127+CD25-CD45RA-CXCR3-CCR6+CCR4+CCR10-), Th1/17 (CD127+CD25-CD45RA-CXCR3+CCR6+), Th22 (CD127+CD25-CD45RA-CXCR3-CCR6+CCR4+CCR10+), Treg1 (CD127-CD25+CD45RA-CXCR3+CCR6-), Treg2 (CD127-CD25+CD45RA-CXCR3-CCR6-CCR4+), Treg17 (CD127-CD25+CD45RA-CXCR3-CCR6+CCR4+CCR10-), Treg 1/17 (CD127-CD25+CD45RA-CXCR3+CCR6+), Treg 22 (CD127-CD25+CD45RA-CXCR3-CCR6+CCR4+CCR10+)

first: Barbara

first: Thomas

last: Duhen


title: Distinct Contribution of DNA Methylation and Histone Acetylation to the Genomic Occupancy of Transcription Factors

city: Oxford

first: Skirmantas

last: Kriaucionis

address: Old Road Campus Research Building, Roosevelt Drive, Headington

country: United Kingdom

institute: Ludwig Cancer Research Oxford

type: Genome variation profiling by high throughput sequencing

summary: DNA methylation has been shown to act by directly modulating transcription factor affinity to DNA binding sites and by recruiting methyl-CpG binding proteins, which form complexes with histone deacetylases (HDACs). Both mechanisms can affect gene regulation, but the extent to which HDACs contribute to the function of DNA methylation and transcription factor occupancy is not clear. To address this question we measured gene expression alterations, chromatin accessibility and transcription factor occupancy in wild type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. We observe widespread elevated chromatin accessibility at repeat elements following histone hyper-acetylation, which was magnified by the absence of DNA methylation. Subsets of repeat elements displayed enriched binding by distinct transcription factors and elevated expression. The pronounced additive effect of HDAC inhibition in DNA methylation deficient cells demonstrate that DNA methylation and histone deacetylation act largely independently to supress transcription factor binding and gene expression.

overall_design: 126 samples in total. 1) ChIP-seq with exogenous spike-in (16 samples):  8 H3ac ChIP-seq samples including 4 conditions with 2 biological replicates each. 8 input control samples, one for each of the ChIP-seq sample. 2) ATAC-seq experiment (32 samples): 16 ATAC-seq samples including 4 conditions with 4 biological replicates each. 16 control Tn5 tagmented genomic DNA, one for each of the ATAC-seq samples. 3) ChIP-seq (70 samples): 60 ChIP-seq samples: 5 different antibodies with 4 conditions and 3 biological replicates each. 4 Igg control ChIP-seq samples. 6 Input control samples. 4) RNA-seq (8 samples): 4 conditions with 2 biological replicates each.

first: Martin

last: Cusack


title: Genome-scale mapping reveals complex regulatory activities of RpoN in Yersinia pseudotuberculosis

city: Umea

first: Maria

last: Fallman

address: Petrus Laestadius väg

department: Molecular Biology

country: Sweden

institute: Umea university


title: Alternative enhancer usage and targeted Polycomb marking hallmark promoter choice during T cell differentiation

city: Macclesfield

first: Muhammad

middle: Ahmad

last: Maqbool

address: Alderley Park

department: Cancer Research UK Manchester Institut

institute: University of Manchester

summary: To characterize the regulatory landscape of developing T-cells, we performed a wide epigenetic and transcriptional analysis of mouse primary thymocytes and ex-vivo differentiated T-helper cells. Based on this data, we portrayed extensively the enhancer and promoter landscape used during differentiation. Our investigations shed light to a very dynamic putative enhancer landscape, many of which we could validate genome-wide using CapStarr-seq. Using our data, we could also show that RNA polymerase II occupancy can be used as an excellent proxy for enhancer activity, when combined with H3K4me1 and H3K27ac epigenetic marks. We further show that enhancer dynamicity is in contrast to more modest variations in gene expression and long-distance interactions. However and interestingly, we found that genes using multiple promoters during differentiation display increased enhancer usage, suggesting that apparent ‘enhancer redundancy’ might relate to isoform selection. Furthermore, in the case of the Runx3 locus, we could identify that each of its two active promoters display long-range interactions with specific enhancers. Finally, our analyses reveal that the control of promoter usage is also associated to the PRC2-deposited H3K27me3 signal at the vicinity of the promoter that is apparently repressed, suggesting a novel function for this complex whereby it contributes to regulation of specific isoform expression.

overall_design: This study was performed in a primary and in vitro differentiated mouse T cells. It contains ChIP-seq data for RNA Pol II, H3K4me1, H3K4me3, H3K27ac and H3K27me3 as well as RNAseq, capSTARRseq, and 4C-seq data.

first: Leo

last: Pioger

first: Amal

middle: Z

last: el aabidine

first: Nezih

last: Karazu

first: Anne

last: Molitor

first: Francois

middle: V

last: Laethem

first: Romain

last: Fenouil

first: Frederic

last: Koch

first: Ivo

last: Gut

first: Marta

first: Sebastian

last: Amigorena

first: Olivier

last: Joffre

last: Sexton

first: Salvatore

last: Spicuglia

first: Jean-Christophe

last: Andrau


title: Impaired phagocytic function in CX3CR1+ tissue-resident skeletal muscle macrophages prevents muscle recovery after influenza A virus-induced pneumonia in aged mice

city: Chicago

first: Alexander

last: Misharin

state: Illinois

address: 303 E Superior

institute: Northwestern University

summary: Skeletal muscle dysfunction in survivors of pneumonia is a major cause of lasting morbidity that disproportionately affects older individuals. We found that skeletal muscle recovery was impaired in aged compared with young mice after influenza A virus-induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue-resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in aged mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue-resident skeletal muscle macrophages from aged compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from aged mice failed to downregulate MHCII expression during recovery from influenza A virus induced pneumonia and showed impaired phagocytic function in vitro. Like aged animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1+ tissue-resident skeletal muscle macrophage population in aged mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.

overall_design: Skeletal muscle macrophages were flow cytometry-sorted from 4 and 18 months-old C57BL/6J mice either naive or 60 days post influenza A virus infection. RNA-seq libraries were prepared using SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian kit and sequenced on Illumina NextSeq 500 instrument.

first: Constance

last: Runyan

first: Lynn

last: Welch

first: Emilia

last: Lecuona

first: Masahiko

last: Shigemura

first: Luciano

last: Amarelle

first: Hiam

last: Abdala-Valencia

first: Nikita

last: Joshi

first: Ziyan

last: Lu

first: Nikolay

last: Markov

first: Alexandra

last: McQuattie-Pimentel

first: Raul

last: Piseaux-Aillon

first: Yuliya

last: Politanska

first: Lango

last: Sichizya

first: Satoshi

last: Watanabe

first: Kinola

last: Williams

first: GR

last: Budinger

first: Jacob

middle: I

last: Sznajder


title: Transcriptome of tambaqui Colossoma macropomum during gonad differentiation: different molecular signals leading to sex identity

city: X

first: Igor

last: Lobo

address: X

country: France

institute: X

summary:
Purpose: The gene molecular network involved in teleost fish sex determination and differentiation is highly variable among species and even in some cases among populations of the same species. The objectives of the present study were to identify the period of gonadal sex differentiation in tambaqui juveniles, as well as the genes and pathways potentially involved in this process.
Methods: Histological analysis of juveniles was carried out to establish a timeline of the gonadal differentiation in tambaqui. Based on that knowledge, ten juveniles were selected before the first evidence of histological sex differentiation and total RNA was extracted from their trunks and used for RNA-Sequencing and a subsequent de novo transcriptome assembly. Principal Component Analysis (PCA) of the whole transcriptome data was used to cluster samples into two distinct groups: putative males and putative females. Differential gene expression, functional annotation and gene enrichment were used to identify genes and pathways related to sex  differentiation in tambaqui to which was applied the Mann-Whitney non-parametric t test (p <0.05) confirming the statistical significance of the expression dimorphism between the groups.
Results: The first sign of histological sex differentiation in tambaqui was the formation of the ovarian cavity detected in individuals measuring about 40 mm in total length. Before the differentiation period, components of the Wnt / β-catenin pathway, fox and fst genes (p <0.05) suggest female sex development in the putative females, whereas antagonistic pathways (gsk3b, wt1 and fgfr2), sox9 and genes for androgen synthesis (p <0.05) are indicative of a male-like differentiation.
Conclusions: Tambaqui juveniles prior to the morphological ovarian differentiation present the Wnt / β-catenin pathway exerting putative role on the sex differentiation target, either upregulated in female-like individuals, or antagonized in male-like individuals. Thus, the present work provides a molecular basis for future studies on the application of tambaqui monosex cultivation.

overall_design: Ten juveniles were selected before the first evidence of histological sex differentiation and total RNA was extracted from their trunks and used for RNA-Sequencing.

first: Áquila

middle: R

last: do Nascimento

first: Michel

last: Yamagishi

first: Yann

last: Guiguen

first: Gilvan

middle: F

last: da Silva

first: Dany

last: Severac

first: Aldessandro

last: Amaral

first: Vanessa

last: Reis

first: Fernanda

middle: L

last: de Almeida


title: Synthetic IL-22 signaling revealed biological activity of homodimeric IL-10 receptor 2 and functional cross-talk with the IL-6 receptor gp130

city: Duesseldorf

first: Birgit

last: Knebel

address: Auf'm Hennekamp 65

department: Cinical Biochemistry and Pathobiochemistry

institute: German Diabetes Center

summary: Cytokine signaling is transmitted by cell surface receptors which function as natural biological switches to control among others mainly immune related processes. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of GFP- and mCherry-nanobodies fused to trans-membrane and intracellular domains of cytokine receptors, which phenocopied cytokine signaling induced by non-physiological homo- and heterodimeric GFP-mCherry ligands. Interleukin 22 signals via IL-22Rα1 and the common IL-10R2, belongs to the IL-10 cytokine family and is critically involved in tissue regeneration. IL-22 SyCyRs phenocopied native IL-22 signal transduction as shown by induction of cytokine-dependent cellular proliferation, signal transduction and transcriptome analysis. Whereas homodimeric IL-22Rα1 SyCyRs failed to activate signaling, homodimerization of the second IL-22 signaling chain, SyCyR(IL-10R2), which was considered to not induce signal transduction, lead to induction of signal transduction. Interestingly, the SyCyR(IL-10R2) and SyCyR(IL-22Rα1) were able to form functional heterodimeric receptor signaling complexes with the synthetic IL-6 receptor chain SyCyR(gp130). In summary, we demonstrated that IL-22 signaling can be phenocopied by synthetic cytokine receptors. Further we identified a novel IL-10R2 homodimeric receptor complex and receptor cross-talk with gp130.

overall_design: We used microarrays to detail the overall influence of the constructs to confirm background free action of the  receptor constructs.

first: Sofie

last: Mossner

first: Doreen

last: Floss

first: Jürgen

last: Scheller


title: Effects of TNFR1 deletion in mice in early life

first: Cambrian

middle: Yangshao

last: Liu

state: IL

address: 5841 S Maryland Ave

department: Department of Medicine

institute: The University of Chicago

summary: Athough anti-TNF therapies can be used to treat colitis associated with inflammatory bowel disease, in mice the loss of the TNF receptor TNFR1 (Tnfrsf1a) in the Il10-/- spontaneous colitis background results in acceleration of disease onset. Whereas Il10-/- mice on the Bl/6 background are relatively protected from colitis throughout life, Il10-/- Tnfr1-/- mice develop colitis beginning at 4 wks of age. Their disease results in nearly 50% mortality by 12 wks of age. Strikingly, Tnfr1-/- mice (with functional IL-10) exhibit evidence of mucosal dysfunction at 4 and 12 wks of age. These mucosal abnormalities include loss of barrier integrity, increased epithelial cell proliferation, crypt malformations, and increased immune cell infiltrate. Because of the early onset of mucosal abnormalities in Tnfr1-/- mice, with or without IL-10 expression, we hypothesized that TNFR1 plays important roles in colonic mucosal function in early life, prior to weaning. To test this hypothesis, we profiled, using mRNA-Seq, the colonic transcriptomes from wildtype and Tnfr1-/- mice at 2 wks of age. The results demonstrate that Tnfr1-/- mice have important gene expression changes, including reduced expression of Il1b, a marker of the proinflammatory "weaning reaction" that is required for establishment of mucosal tolerance in later life. TNFR1 therefore has key roles in colonic mucosal homeostasis in early life.

overall_design: A total of 11 samples were analyzed. Each sample is composed of total RNA isolated from the full-thickness colon of a 2-wk-old mice. Five wildtype (WT) and six Tnfr1-/- mice were profiled.

middle: Y


title: A subpopulation of Periostin-expressing fibroblasts is required for cardiac muscle and neuronal maturation after birth

city: Cinicnnati

first: Katherine

last: Yutzey

state: OH

address: 240 Albert Sabin Way

department: Divison of Molecular Cardiovascular Biology

institute: Cinicnnati Children's Hospital Medical Center

summary: During the postnatal period in mammals, the cardiac muscle transitions from hyperplasic to hypertrophic growth, the extracellular matrix (ECM) undergoes remodeling, and the heart loses regenerative capacity. While ECM maturation and crosstalk between cardiac fibroblasts (CFs) and cardiomyocytes (CM) have been implicated in neonatal heart development, not much is known about specialized fibroblast heterogeneity and functions in the early postnatal period. In order to better understand CF functions in heart maturation and postnatal cardiomyocyte cell cycle arrest, we have performed gene expression profiling and ablation of postnatal CF subpopulations. Fibroblast lineages expressing Tcf21 or Periostin were traced in transgenic GFP reporter mice and their biological functions and transitions during the postnatal period were examined in sorted cells using RNAseq. A subpopulation of highly proliferative Periostin (Postn)+ CFs was found from postnatal day (P)1 to P11 but was not detected at P30. This population was less abundant and transcriptionally different from Tcf21+ resident CFs, which persist in the mature heart. The Postn+ subpopulation preferentially expresses genes related to cell proliferation and neuronal development, while Tcf21+ CFs differentially express genes related to ECM maturation at P7 and immune crosstalk at P30.  Ablation of the Postn+ CFs from P0 to P6 led to altered cardiac sympathetic nerve patterning and a reduction in CM binucleation, maturation, and hypertrophic growth. Thus, postnatal CFs are heterogeneous and include a transient proliferative Postn+ subpopulation required for cardiac nerve development and cardiomyocyte maturation soon after birth.

overall_design:
We have characterized  expression and function of different subpopulations of cardiac fibroblasts during postnatal cardiac development.
Use Tcf21MCM and PostnMCM with a tamoxifen-inducible R26eGFP reporter to trace different populations of fibroblast. These populations were GFP FACS sorted for RNA isolation and posterior RNAseq.

first: Luis

last: Hortells

first: Iñigo

last: Valiente


title: Effects of TNFR1 deletion on Il10-/- mouse colitis in early life

summary: The loss of TNFR1 (Tnfrsf1a) in the Il10-/- spontaneous mouse colitis background results in acceleration of disease onset. Whereas Il10-/- mice on the Bl/6 background are relatively protected from colitis throughout life, Il10-/- Tnfr1-/- mice develop colitis beginning at 4 wks of age. Their disease results in nearly 50% mortality by 12 wks of age. We hypothesized that this early-onset colitis was due to dysregulation of immune signals in early life, defining a key period known as the "weaning reaction" in which proinflammatory signals help induce mucosal tolerance of the microbiome. To test this hypothesis, we profiled, using mRNA-Seq, the colonic transcriptomes from Il10-/- and Il10-/- Tnfr1-/- mice at 2 wks of age. The results presented here show that Il10-/- Tnfr1-/- mice have reduced expression of cytokines at this early age, suggesting that they cannot amount an appropriate immune response. Thus, TNFR1 plays an important role in the weaning reaction and the acquisition of tolerance.

overall_design: A total of 12 samples were analyzed. Each sample is composed of total RNA isolated from the full-thickness colon of a 2-wk-old mice. Six Il10-/- and six Il10-/- Tnfr1-/- mice were profiled.

first: Sharon

last: Tam

first: Ying

last: Huang

first: David

middle: B

last: Polk


title: Genome-scale mapping reveals complex regulatory activities of RpoN in Yersinia pseudotuberculosis [RNA-seq]

first: Maria

last: Fallman

summary:
We combined phenotypic and genomic assays to provide insight into its role in this pathogen. RpoN was essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility.
Mapping of genome-wide associations of Y. pseudotuberculosis RpoN identified an RpoN-binding motif at 103 inter- and intragenic sites located on both the sense and anti-sense strands
Transcriptomic analysis of bacteria lacking rpoN showed a large impact on gene expression, including down-regulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing
There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with peak binding locations implicated around 130 genes or operons potentially activated or repressed by RpoN

overall_design: RNA-seq of WT and RponN mutant in stationary and induction conditions.

first: A

last: Firoj Mahmud

first: Kristina

last: Nilsson

first: Rajdeep

last: Choudhury

last: Fållman


title: Genome-scale mapping reveals complex regulatory activities of RpoN in Yersinia pseudotuberculosis [ChIP-seq]

summary: We combined phenotypic and genomic assays to provide insight into its role in this pathogen. RpoN was essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping of genome-wide associations of Y. pseudotuberculosis RpoN identified an RpoN-binding motif at 103 inter- and intragenic sites located on both the sense and anti-sense strands. Transcriptomic analysis of bacteria lacking rpoN showed a large impact on gene expression, including down-regulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with peak binding locations implicated around 130 genes or operons potentially activated or repressed by RpoN

overall_design: Examination of RpoN binding genome wide during different conditions

last: Nillsson


title: Genome-wide screens reveal that resveratrol induces replicative stress in human cells

city: Montreal

first: Lea

last: Harrington

state: Quebec

address: 2950 chemin de Polytechnique, Pavillon Marcelle-Coutu

country: Canada

institute: University of Montreal

summary: Using genome-wide CRISPR-Cas9 screens and other cell-based assays, we show that the pro-longevity agent resveratrol and its analogue pterostilbene induce a genetic signature of replicative stress that is similar to hydroxyurea

overall_design: Genome-wide CRISPR-Cas9 screens were conducted in two cell lines: NALM-6 cells treated with resveratrol (16 µM), pterostilbene (16 µM), or hydroxyurea (100 µM), and Jurkat cells treated with resveratrol (25 µM), compared to their respective DMSO-treated controls.

first: Yahya

last: Benslimane

first: Thierry

last: Bertomeu

first: Jasmin

last: Coulombe-Huntington

first: Mary

last: McQuaid

first: María

last: Sánchez-Osuna

last: Papadopoli

first: Daina

last: Avizonis

first: Mariana

last: Russo

first: Caroline

last: Huard

first: Ivan

last: Topisirovic

first: Hugo

last: Wurtele

first: Mike

last: Tyers


title: Fc receptor-like 4 (FCLR4) and Fc receptor-like receptor 5 (FCRL5) define and control human atypical memory B cells

city: Silver Spring

first: Frederick

middle: C

last: Mills

state: MD

address: Building 52, Room 2108

department: CDER, OPQ, OBP, Division IV

institute: FDA

type:
Expression profiling by array
Other

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

overall_design: Refer to individual Series


title: Fc receptor-like 4 (FCLR4) and Fc receptor-like receptor 5 (FCRL5) define and control human atypical memory B cells [RNA-Seq]

summary: We performed NGS on Ig heavy chain V sequences amplified from RNA obtained from human tonsillar B cell subsets. From these NGS data, we reconstructed ongoing B cell responses as lineage trees, providing crucial in vivo developmental context. Each memory subset typically maintained its lineage, denoting mechanisms enforcing their phenotypes. FCRL4-FCRL5+ cells were the most represented memory subset in lineage trees, indicating robust participation in ongoing responses. Affinity maturation of both FCRL4-FCRL5+ and FCRL4+FCRL5+ atypical memory cells was stalled in chronic streptococcus infection, conceivably due to negative feedback from IgG immune complexes. We propose that FCRL4 and FCRL5 function as sensors of immune complexes, thus controlling atypical memory B cell responses.

overall_design: B cells were isolated from human tonsil cells and FACS-sorted into subsets on the basis of CD19-, IgD CD38, FCRL4, and FCRL5 surface expression. CD38-IgD- memory cells were sorted into three subsets: FCRL4- FCRL4+ (F5), FCRL4- FCRL5- (DN) and FCRL4+ FCRL5+ (DP). In addition, IgD+CD38- naïve/IgD+ memory cells(Naive), IgD-D38+ GC cells(GC) , and IgD-CD38++ plasmablasts (PB) subsets were also sorted . IgM, IgA or IgG HC variable regions were separately amplified from each sorted subset and these amplified samples were subjected to deep sequencing.

first: Mate

last: Tolnay


title: TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T cell numbers

city: Shanghai

first: Feizhen

last: Wu

state: Shanghai

address: Zhangheng Road 825, Pudong

department: Intelligent Medicine Institute

institute: Fudan Univ, Shanghai, China

type: Methylation profiling by high throughput sequencing

summary: Host immune response is a fundamental mechanism for attenuating cancer progression. Here we report a pivotal role of the DNA demethylase and tumor suppressor TET2 played in host antitumor immunity. Deletion of Tet2 dramatically elevated the IL-6 level in Tet2-/- mice upon tumor challenge. The elevated IL-6 greatly stimulated the immunosuppressive granulocytic myeloid-derived suppressor cells (G-MDSCs), which in turn significantly reduced CD8+ T cells upon tumor challenge. Consequently, systematic knockout of the Tet2 in mice led to accelerated syngeneic tumor growth, which was constrained by anti-PD-1 blockade. Removal of G-MDSCs by the anti-mouse Ly6g Ab restored the numbers of CD8+ T cells in Tet2-/- mice and rebooted their anti-tumor activity. Importantly, anti-IL-6 Ab treatment blocked the expansion of G-MDSCs and inhibited syngeneic tumor growth. Collectively, these findings reveal a TET2-mediated IL-6/G-MDSC/CD8+ T immune response cascade that safeguards host adaptive anti-tumor immunity, offering a cell non-autonomous mechanism of TET2 for tumor suppression.

overall_design:
Identifying the differential expression genes between Tet2-KO and WT in syngeneic tumors and gMDSCs, and examining the 5mC and 5hmC profile of Tet2-KO and WT gMDSC in tumor bearing mice.

Please note that the *mr.txt.gz processed data was generated from each BS and OxBS pair sample and is linked to the corresponding *-BS sample records.

first: Shuangqi

last: Li

first: Jiuxing

last: Feng


title: Fc receptor-like 4 (FCLR4) and Fc receptor-like receptor 5 (FCRL5) define and control human atypical memory B cells [array]

summary: We performed gene array analysis of three memnory cell subsets sorted from three tonsil donors with the following patterns of surface expression: FCRL4-, FCRL5+ (designated F5), FCRL4 -, FCRL 5- (designated DN) ,  and FCRL4+, FCRL5+ (designated DP). The three memory subsets were clearly segregated by principal component analysis and cluster analysis as distinct,  and hundreds of genes were found differentially expressed. between any two subsets

overall_design: B cells were isolated from human tonsils, stained with antibodies for CD19, IgD, CD38, FCRL4, and FCRL5, and sorted to obtain F5, DN, and DP populations.   RNA was prepared from sorted cells, labeled, hybridized to Agilent  human 8X60K v2 microarrays, and scanned .  Microarray data were normalized  and analyzed for enrichment of gene expression and gene pathways between the three B cell subsets


title: Molecular effects of neonicotinoid thiacloprid in honey bees (Apis mellifera)

city: Muttenz

first: Fent

last: Karl

address: Langackerstrasse 30

department: School of Life Sciences

country: Switzerland

institute: University of Applied Sciences and Arts Northwestern Switzerland

summary: The low and high concentrations led to significant alteration of 5 and 71 genes, respectively, of which more were up- than down-regulated. Genes belonging to diverse pathways were affected. KEGG analysis showed that in the high exposure concentration ribosome proteins, mitochondrial oxidative phosphorylation and pyrimidin metabolism were among the three most prominent altered pathways.

overall_design: 1) Solvent control (5 replicates) 2) Treatment with 25 ng Thiacloprid/bee (5 replicates) 3) Treatment with 250 ng Thiacloprid/bee (5 replicates)

first: Michael

last: Schmid

first: Karl

last: Fent


title: Gene expressions of Tfap2a/b heterozygous knockout in mouse brain

city: Goettingen

first: Yang

last: Hu

institute: MPIBPC

summary: The aim of this study is to compare the transcriptome profiles between wild type (WT, B6N in this case), with mutant mice (Tfap2a+/- or Tfap2b+/- respectively)

overall_design: Brain mRNA profiles of adult WT, Tfap2a+/-, Tfap2b+/- male mice

first: Henrik

last: Bringmann


title: A bifunctional nucleosome binding protein mediates specialized mSWI/SNF complex targeting and activity (RNA-seq dataset)

city: Boston

first: Cigall

last: Kadoch

state: Massachusetts

address: 450 Brookline Avenue

department: Pediatric Oncology

institute: Dana Farber Cancer Institute

summary: Here we identify and characterize a minimal domain within the SS18-SSX fusion oncoprotein hallmark to synovial sarcoma, that exhibits a high-affinity interaction with the nucleosome acidic patch. We find that this region is necessary and sufficient for nucleosome binding, mSWI/SNF (BAF) chromatin remodeling complex targeting, catalytic activity, and activation of cancer-specific gene expression. Moreover, we identify PRC1-mediated H2AUb119 as a further rheostat governing further stability and binding. Together, our results define an unexpected direct bifunctional histone detector on SSX that is responsible for disease-specific chromatin recruitment of a major family of chromatin regulatory complexes.

overall_design: Expression constructs of V5-tagged SS18-SSX fusion or specific fusion mutations were overexpressed in CRL7250 human fibroblast cells to determine the role of specific amino acids in directing the chromatin targeting of this fusion. Alternatively, shRNA hairpins targeting the SS18-SSX fusion in synovial sarcoma cell lines were expressed to see how the chromatin targeting of mSWI/SNF complexes or polycomb complexes and the histone landscape is altered upon loss of the fusion.

first: Matthew

last: McBride

first: Andrew

last: D'Avno


title: Virus-Host Interactome Reveals the Unique Blockage of Host RNAi Machinery by the Zika Virus Capsid

city: Los Angeles

first: Zhifei

last: Luo

state: CA

address: 1450 Biggy Street, NRT 6514

institute: University of Southern California

type: Non-coding RNA profiling by high throughput sequencing

summary: The recent outbreaks of Zika virus (ZIKV) and its association with birth defects known as Congenital Zika Syndrome warrant investigation on the molecular processes related to its infection and pathogenesis. Among the flavivirus family, only ZIKV is linked to microcephaly as announced by World Health Organization, suggesting uniqueness of ZIKV infection compared to other members. By analyzing the ZIKV-host interactome, we found that the key microRNA (miRNA) processing enzyme Dicer was a leading target of ZIKV capsid protein in neural stem cells (NSCs), and its deficiency facilitated ZIKV infection. Mechanistically, ZIKV capsid can directly interact with Dicer and block its ribonuclease activity, dampening the production of host miRNAs that are essential for neurogenesis. Interestingly, this capsid-mediated immune evasion is specific to ZIKV because capsid proteins from other close flaviviruses, e.g., dengue, yellow fever and West Nile viruses, cannot bind to Dicer or inhibit its function. By molecular mapping, we defined a ZIKV capsid H41R mutant with loss of interaction to Dicer and no longer affecting its activity. More importantly, ZIKV H41R mutant exerted almost no impact on neurogenesis in vitro when expressed in NSCs compared to wild type capsid, and in utero infection of recombinant ZIKV-H41R mutant virus resulted in less inhibition on corticogenesis than wild-type ZIKV in mouse embryos. Interestingly, the epidemic ZIKV strain reinforces the capsid-Dicer interaction by two amino acid substitution compared to ancient Africa strain. Thus, our study demonstrated that capsid-dependent suppression of Dicer function is a unique determinant of ZIKV immune evasion and pathogenesis, which may unveil a new mechanism for ZIKV-mediated microcephaly.

overall_design: miRNA-seq of neural stem cells (NSCs) infected with ZKIV or ZKIV H41R or without infection

first: Jianxiong

last: Zeng

first: Shupeng

last: Dong

first: Bishi

last: Fu

first: Ping

first: Xiaochun

last: Xie

first: Xing

last: Yang

first: Yujie

last: Chen

first: Xin

last: Wang

first: Chengrong

last: Liu

first: Zhenshan

first: Jing

first: Jian

last: Zhang

first: Gang

last: Long

first: Shou-Wei

last: Ding

first: Shitao

first: Zhen

last: Zhao

first: Qiming

last: Liang


title: A bifunctional nucleosome binding protein mediates specialized mSWI/SNF complex targeting and activity (ChIP-seq dataset)

first: Cigall

last: Kadoch

first: Matthew

first: Andrew


title: Comparison of tumor-associated YAP1 fusions identifies a recurrent set of functions critical for oncogenesis

city: Seattle

first: Sonali

last: Arora

state: WA

address: 1100 Fairview Ave N,

institute: FHCRC

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Distinct Cdk9-phosphatase switches act on elongation factor Spt5 at the beginning and end of the RNA polymerase II transcription cycle

city: Bronx

first: PABITRA

last: PARUA

address: 1300 Morris Park Avenue, Forchheimer 236, Molecular Pharmacology

department: Molecular Pharmacology

institute: Albert Einstein College of Medicine

summary: The Pol II transcription cycle is ordered by CDKs and phosphatases. In fission yeast, Cdk9 phosphorylates carboxy-terminal repeats (CTRs) of Spt5 while inhibiting PP1 during elongation. Transcription past the cleavage and polyadenylation signal (CPS) coincides with PP1-dependent Spt5 dephosphorylation and leads to Pol II pausing with phosphorylated CTD-Ser2 (pSer2). Here we show this switch is conserved in humans: Cdk9 inhibition decreases phosphorylation of both PP1g and Spt5-Thr806 (pThr806), and induces pSer2 upstream of the CPS, whereas PP1 depletion increases pThr806. Moreover, in unperturbed cells, pThr806 is diminished in 3’-paused complexes where pSer2 is maximal. Cdk9 also phosphorylates Spt5 on Ser666, a PP1-refractory site between conserved KOW4 and KOW5 motifs; pSer666 increases upon promoter-proximal pause release and, in contrast to pThr806, persists beyond the CPS. We identify PP4—another target of inhibitory phosphorylation by Cdk9—as a pSer666 phosphatase. PP4 and PP1g are enriched at 5’ and 3’ ends of genes, respectively, consistent with pSer666 and pThr806 distributions. Therefore, distinct Cdk9-phosphatase switches operate on Spt5 at different steps in transcription.

overall_design: ChIP-seq: Data represent nineteen different ChIP-seq experiments in human colon carcinoma (HCT116) cells, performed in biological replicates. There are raw and processed files (bigWig) for all biological replicates.

first: Pabitra

last: Parua

first: Bradley

last: Benjamin


title: High Resolution Genome Wide Expression Analysis of Single Myofibers Using SMART-Seq

city: Ottawa

first: Theodore

last: Perkins

state: Ontario

address: 501 Smyth Rd.

department: Regenerative Medicine Program

institute: Ottawa Hospital Research Institute

summary: Skeletal muscle is a heterogeneous tissue. Individual myofibers that make up the contractile muscle tissue exhibit variation in their metabolic and contractile properties. Although there are biochemical and histological assays to study myofiber heterogeneity, methods to analyze the transcriptomes of individual myofibers are lacking.  We have developed single myofiber RNA-Seq (smfRNA-Seq) to analyze the whole transcriptome of an individual myofiber by combining single fiber isolation with Switching Mechanisms at 5’ end of RNA Template (SMART) technology. Our method provides high-resolution genome wide expression profiles of single myofibers. This method will be useful to study developmental and age-related dynamics in the composition of skeletal muscle.

overall_design: Gene expression analysis of single myofibers, groups of 5 or 20 myofibers, and whole muscle, each in triplicate

middle: J

first: Vahab

middle: D

last: Soleimani


title: HUVEC gene expression in response to monocytes

status: Public on Aug 03 2020

city: Hannover

first: Sibylle

last: von Vietinghoff

address: Carl Neuberg Strasse 1

department: Internal Medicine

institute: Hannover Medical School

summary: Monocytes are central for atherosclerotic vascular inflammation. The human non-classical, patrolling subtype, which expresses high levels of CD16 and fractalkine receptor CX3CR1, strongly associates with cardiovascular events. Compared to classical CD14+ monocytes or transwell cocultures, CD16+ monocytes enhanced endothelial STAT1 and NFκB p65 phosphorylation, upregulated expression of CX3CL1 and IL-1β, numerous CCL and CXCL chemokines and molecules promoting leukocyte patrolling and adhesion such as ICAM1 and VCAM1.

overall_design: RNA of HUVECs from two donors was isolated after 24h either direct or transwell coculture with either CD14+ or CD16+ monocytes from two different donors and pooled for gene expression analysis.

first: Alexandra

last: Helmke

first: Eva

last: Roy-Chowdhury


title: Metabolic conditioning of CD8+ effector T cells for adoptive cell therapy

city: Baltimore

first: Immunometabolism

last: Department

state: Maryland

address: 1650 Orleans Street

department: Immunometabolism

institute: Johns Hopkins University

summary: Effector CD8+ T cell (TE) proliferation and cytokine production depends on enhanced glucose metabolism. However, circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE metabolically primes effector functions and enhances tumour clearance in mice. Tumor-specific TGR CD8+ TE co-cultured with tumor spheroids in replete conditions display enhanced effector molecule expression, and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumor clearance. Mechanistically, TGR TE undergo metabolic remodelling that upon glucose re-exposure supports enhanced glucose uptake, increased carbon allocation to the pentose phosphate pathway (PPP), and a cellular redox shift toward a more reduced state, all indicators of a more anabolic program to support their enhanced functionality. Thus, metabolic conditioning could be utilized to promote efficiency of T cell products for adoptive cellular therapy.

overall_design: CD8+ T cells were obtained from spleens and lymph nodes isolated from 8-12-week-old OT-I mice. Single cells were sequenced using the 10x Chromium controller. One sample per treatment was used.

first: Ramon

last: Klein Geltink

first: Joy

last: Edwards-Hicks

first: Petya

last: Apostolova

first: David

last: O’Sullivan

last: Sanin

first: Annette

last: Patterson

first: Daniel

last: Puleston

first: Nina

middle: A

last: Ligthart

first: Joerg

middle: M

last: Buesscher

first: Katarzyna

last: Grzes

first: Agnieska

last: Kabat

first: Michal

last: Stanczak

first: Jonathan

last: Curtis

first: Fabian

last: Haessler

first: Franziska

last: Uhl

first: Mario

last: Fabri

first: Robert

last: Zeiser

first: Edward

last: Pearce

first: Erika


title: Inflammatory Signals induce AT2 Cell-Derived Damage-Associated Transient Progenitors that Mediate Alveolar Regeneration

city: Cambridge

first: Joo-Hyeon

last: Lee

address: Puddicombe Way, Cambridge Biomedical Campus

institute: Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge

summary: Tissue regeneration is a multi-step process mediated by diverse cellular hierarchies and states that are also implicated in tissue dysfunction and pathogenesis.  Here, we leveraged single-cell RNA sequencing in combination with in vivo lineage tracing and organoid models to finely map the trajectories of alveolar lineage cells during injury repair and lung regeneration. We identified a distinct AT2-lineage population, Damage-Associated Transient Progenitors (DATPs), that arises during alveolar regeneration. We found that interstitial macrophage-derived IL-1β primes a subset of AT2 cells expressing Il1r1 for conversion into DATPs via a HIF1α-mediated glycolysis pathway, which is required for mature AT1 cell differentiation. Importantly, chronic inflammation mediated by IL-1β prevents AT1 differentiation, leading to aberrant accumulation of DATPs and impaired alveolar regeneration.  Together, this step-wise mapping to cell fate transitions shows how an inflammatory niche impairs alveolar regeneration by controlling stem cell fate and behavior.

overall_design: For scRNA-seq, lung organoids derived from AT2 cells and lung tissues from control or Bleomycin-treated SPC-CreERT2;R26RTomato mice were collected and sorted by FACS, and the expression levels were quantified using the Chromium Controller (10X Genomics). For ATAC-seq of bulk (control) and Il1r1+ AT2, Tomato+ AT2 cells were isolated by FACS.

first: Jinwook

last: Choi

first: Jong-Eun

last: Park

first: Georgia

last: Tsagkogeorga

first: Motoko

last: Yanagita

first: Bon-Kyoung

last: Koo

first: Namshik

last: Han


title: A High Docosahexaenoic Acid Diet Alters the Lung Inflammatory Response to Acute Dust Exposure

city: Fort Collins

first: Tara

last: Nordgren

state: CO

address: Colorado State University, Physiology 133

department: Environmental and Radiological Health Sciences

institute: Colorado State University

summary: Agricultural workers are at risk for the development of acute and chronic lung diseases due to their exposure to organic agricultural dusts. A diet intervention using the omega-3 fatty acid docosahexaenoic acid (DHA) has been shown to be an effective therapeutic approach for alleviating a dust-induced inflammatory response. We thus hypothesized a high-DHA diet would alter the dust-induced inflammatory response through the increased production of specialized pro-resolving mediators (SPMs). Mice were pre-treated with a DHA-rich diet 4 week  s before being intranasally challenged with a single dose of an extract made from dust collected from a concentrated swine feeding operation (HDE  ). This omega-3-fatty-acid-rich diet led to reduced arachidonic acid levels in the blood, enhanced macrophage recruitment, and increased the production of the DHA-derived SPM Resolvin D1 (RvD1) in the lung following HDE exposure. An assessment of transcript-level changes in the immune response demonstrated significant differences in immune pathway activation and alterations of numerous macrophage-associated genes among HDE-challenged mice fed a high DHA diet. Our data indicate that consuming a DHA-rich diet leads to the enhanced production of SPMs during an acute inflammatory challenge to dust, supporting a role for dietary DHA supplementation as a potential therapeutic strategy for reducing dust-induced lung inflammation.

overall_design: Randomly selected left lung tissues (n = 24) out of the possible 40 mice, representing tissues collected across three unique experimental trials, were homogenized and RNA was extracted using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, California, USA). RNA sample quality was quantified using the NanoDrop ND-100 (NanoDrop Technologies, Inc, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (UC Riverside Core Facilities, Agilent Technologies, Santa Clara, CA, USA). The assessment of transcript-level gene expression changes was performed using the NanoString mouse Immunology Panel (NanoString Technologies, Seattle, WA, USA), a codeset designed to target 561 genes related to inflammation and the immune response. Fifty to one hundred nanograms of total RNA was mixed with the codeset and reporter probes and hybridized for 16 h to form a purified target–probe complex that was then imaged and quantified with a nCounter Sprint profiler. Gene expression data analysis was performed using the nCounter Analysis System, nSolver 4.0 software. The expression data were normalized by using the geometric mean of 4 housekeeping genes: OAZ1, PPIA, RPL19, and EEF1G. Following normalization, 20 of the samples passed normalization and were viable to use for the subsequent analyses, including 4 control diet + saline, 6 control diet + HDE, 3 DHA diet + saline, and 7 DHA + HDE samples.

last: Dominguez

first: Art

last: Heires

first: Jacqueline

last: Pavlik

first: Tricia

last: Larsen

first: Stephanie

last: Guardado

first: Joseph

middle: H

last: Sisson

first: Michelle

last: Baack

first: Debra

last: Romberger


overall_design:
For ATAC-seq of bulk (control) and Il1r1+ AT2, Tomato+ AT2 cells were isolated by FACS.
Please note that each processed .bw data was generated from both replicates and is linked to the corresponding *_1 records.


title: Effect of HDAC inhibitor TSA on haploid embryogenesis [dataset 1]

city: Wageningen

first: Anneke

last: Horstman

state: Nederland

address: Droevendaalsesteeg,

department: Plant Science Group

institute: Wageningen University and Research

summary: Haploid embryos can be induced from cultured immature pollen following a stress treatment. In Brassica napus, application of the histone/lysine deacetylase (HDAC/KDAC) inhibitor trichostatin A (TSA) to pollen cultures enhances the production of differentiated embryos and embryogenic callus when applied together with heat stress (Li et al., 2014). To identify genes associated with the induction of B. napus haploid embryogenesis, we compared the transcriptomes of untreated pollen cultures and pollen cultures treated with either heat-stress or heat-stress plus TSA.

overall_design: 17 samples were analyzed. For each condition, two to three biological replicates were analyzed (1 genotype, 3 treatments).

first: Charlotte

last: Siemons

first: Patricia

last: Martinez

first: Kim

last: Boutilier


title: Gene expression analysis of wildtype AML12 versus VWA8-null AML12 cells

city: Tucson

first: Dawn

middle: K

last: Coletta

state: AZ

address: 1501 N. Campbell Ave

department: Medicine

institute: UNIVERSITY OF ARIZONA

summary: To obtain a more complete picture of gene expression changes induced by deletion of VWA8 null cells, Affymetrix microarray analysis of gene expression was carried out using RNA isolated from wildtype and VWA8-null AML12 cells.

overall_design: To determine changes in gene expression in AML12 liver cells associated with deletion of VWA8, separate cultures of wildtype  and knockout cells were differentiated (n=4 each). Total RNA was isolated from cells using RNAStat solution (Tel-Test Inc., Friendswood, TX) and the total RNA was purified with RNeasy and DNase I treatment (Qiagen, Valencia, CA).  RNA was prepared for hybridization to Affymetrix Mouse 2.0 arrays according to manufacturer’s instructions

first: Moulun

last: Luo

first: Lawrence

last: Mandarino


title: Epigenetic basis for monocyte dysfunction in acute-on-chronic liver failure and alcoholic hepatitis [RNA-seq]

status: Public on Aug 02 2020

city: Gosselies

first: Stanislas

last: Goriely

address: Rue Adrienne Bolland 8

institute: Université Libre de Bruxelles

summary:
BACKGROUND/ AIMS:
Severe forms of alcoholic liver disease are associated with increased susceptibility to infections largely due to immune dysfunction. In this study, we analyze the phenotypic, molecular and functional profiles of the mononuclear phagocyte system of patients with cirrhosis, severe alcoholic hepatitis (sAH) or acute-on-chronic liver failure (ACLF).
RESULTS: sAH and ACLF patients display a severe impairment of the TH1-promoting IL-12p70/IFNgamma/CXCL10 axis. Furthermore, the CD14+ monocytes and classical dendritic cells of these patients are unable to mount an appropriate pro-inflammatory response after stimulation with PAMPs associated with gram-negative bacteria or fungal pathogens. These cells also have reduced polyfunctionality, which is associated with a higher risk of subsequent infection. Finally, we show that CD14+ monocytes of sAH and ACLF patients display a similar transcriptional profile characterized by immunosuppressive features. This was accompanied with changes in chromatin accessibility in enhancer regions of key immune and metabolic genes.
CONCLUSIONS: Altered transcriptional program and functional properties of monocytes from sAH and ACLF patients has strong epigenetic determinants.

overall_design: We recruited 39 patients with alcoholic cirrhosis (including 11 patients with sAH and 12 with ACLF), 12 healthy controls and 11 patients with chronic alcohol consumption. We performed whole blood phenotypic analysis of surface markers and stimulation with various pathogen-associated molecular patterns (PAMPs). We also performed RNAseq and ATACseq on CD14+ monocytes from 6 HC, 4 sHA and 4 ACLF patients.

first: Laura

last: Weichselbaum

first: Abdulkader

last: Azouz

first: Kinga

last: Smolen

first: Jishnu

last: Das

first: Marion

last: Splittgerber

first: Frederick

last: Libert

first: Thierry

last: Gustot


title: Single-cell profiling reveals Colonic CD8 Topography in IBD -hIL26 Mouse RNA-Seq

city: Oxford

first: Alison

last: Simmons

address: John Radcliffe Hospital/Headley Way

department: The MRC Weatherall Institute of Molecular Medicine

institute: University of Oxford

summary:
Aim: To assess the effect of IL-26 on mouse colonic tissue at steady state and in DSS-induced colitis through next-generation RNA sequencing of bulk RNA from colonic tissue.
Results: We demonstrate an anti-inflammatory effect of IL-26 at both steady-state and after induction of DSS-colitis, through suppression of both cell recruitment and activation pathways.

overall_design: Methods: Mice do not express IL-26, but do express the putative IL-26 receptor (IL10RB/IL20RA). The study therefore utilized WT (C57BL/6 B6) or TG mice (C57BL/6 expressing human IL26 BAC transgene) littermate mice housed in pathogen-free conditions with regularized day-night cycles and ad-libitum access to sterile food and water. These mice were either assessed at age 20-24 weeks (steady-state,for any spontaneous inflammation) or exposed to 2.5% DSS at age 10-14 weeks for 6 days, with and without exposure to anti-IL26 antibody or isotype control antibody. Bulk RNA was extracted from the colonic tissue of these mice, enriched for mRNA with strand-specific information, and analyzed with next-generation RNA sequencing.

first: Daniele

last: Corridoni

first: Agne

last: Antanaviciute

first: Dai

last: Ishikawa

first: Ryo

last: Hatano

first: Chikao

last: Morimoti

first: Marta

last: Jagielowicz


title: Epigenetic basis for monocyte dysfunction in acute-on-chronic liver failure and alcoholic hepatitis [ATAC-seq]


title: Transcriptom analysis of normal and Slug overexpressing human endothelial cells in a 3D fibrin-gel bead angiogenesis assay

status: Public on Aug 01 2020

city: Irvine

first: Christopher

last: Hughes

address: 2400 Biological Sciences III, Hughes Lab

institute: University of California, Irvine

summary:
Purpose:To identify transcriptional changes in human endothelial cells (HUVEC) both during normal sprouting angiogenesis (different time points) and with Slug overexpression (different Slug expression), in a 3D fibrin-gel bead angiogenesis
Methods: Angiogenic endothelial cell with different level of Slug expression (normal and overexpressing) at different stage (early/sprouting and late/lumen formation) were harvested from a 3D fibrin-gel bead angiogenesis assay. Their transcriptom profile was generated by Next Generation RNA-sequencing as single reads, performed on an Illumina HiSeq 2500 platform, using three donor HUVEC lines.At least 30 million reads per sample were sequenced. Quality control was performed on data using FASTQC(v. 0.11.2), and reads were trimmed using Trimmomatic(v.0.32) with Illumina TruSeq adapter sequences using a PHRED quality score 15 and minimum length 20 bases. Trimmed reads were then aligned to the human hg19 reference genome using the Ensembl GRCh37 annotations and post processed using Tophat2(v.2.0.12), Bowtie2(v.2.2.3) and Samtools(v.0.1.19). Expression levels were quantified both with FPKM (Fragment per kilobase per million mapped reads) using Cufflinks(v. 2.1.1) and with raw counts using HTSeq(v.0.6.1p1.). Differential analysis was done using DESeq2 and significant genes were considered (p-value < 0.05, 2 fold change). K-means clustering was performed to extract dynamic gene clusters based on differential expression between GFP and SLUG knockdown time-points. Gene set enrichment analysis (GSEA) was performed on expressed genes to identify significant gene sets across sample comparisons. Additional gene ontology analysis was performed for each cluster and reported using Metascape. Clustering was performed in R and heatmaps were generated using JavaTree.
Results: Using several different gene expression analysis platforms, we found consistent and significant changes, during both normal angiogenesis and with Slug overexpression, in genes related to proliferation, migraion, cell morphology/polarity and junction regulations, all of which are consistent with a partial-EMT program. We also found changes in Notch signaling components in Slug overexpressing cells. Changes in specific genes related to EMT and Notch signaling were validated with both qPCR and WB.
Conclusion: This is the first comprehensive transcriptome analysis in human angiogenic EC during normal and excessive sprouting induced by Slug expression in an in vitro 3D angiogenesis assay system. We conclude that a partial-EndoMT is necessary for normal angiogenesis and that endothelial Slug expresion level regulates Notch signaling to determines at least partly the extent of EC's commitment towards a mesenchymal identity.

overall_design: Angiogenic HUVEC mRNA profiles at early and late stage sprouting angiogenesis with normal and Slug overexpression

last: Hultgren

first: Ricardo

last: Ramirez

first: Katrina

last: Welch-Reardon

first: Chritopher

first: Jenny

last: Wu

first: Seung-Ah

last: Chung

first: Melanie

last: Oakes


title: Phagocytosis-related NADPH oxidase 2 subunit gp91phox contributes to neurodegeneration after repeated systemic challenge with lipopolysaccharides

city: Bonn

first: Harald

last: Neumann

address: Venusberg-Campus 1

institute: University Hospital of Bonn

summary: Here, we investigated the role of mononuclear phagocytes associated nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX-2) in inflammatory neurodegeneration. Cybb-deficient mice NOX-2 knock-out (KO) and control wild type (WT) mice were treated intraperitoneally daily over four consecutive days with 1 µg/gbw/day LPS. Repeated LPS injections led to inflammation and neuronal loss in the brains of WT mice, which was attenuated after knockout of NOX-2. Transcriptome analysis by RNA-seq of total brain tissue indicated increased LPS-induced upregulation of genes belonging to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) production, complement and lysosome activation as well as apoptosis and necroptosis in WT compared to NOX-2 KO mice.

overall_design: Examination of brain mRNA profiles after repeated LPS or PBS (vehicle control) application of 3-month-old WT and NOX-2 KO mice by RNA-Seq (6 animals per group).

first: Anahita

last: Shahraz

first: Jannis

last: Wißfeld

first: Aurélien

last: Ginolhac

first: Mona

last: Mathews

first: Lasse

last: Sinkkonen


title: Microarray-based transcriptome profiling after H19 KO

first: Christian

last: Bär

address: Carl-Neuberg-Str. 1

department: IMTTS

summary: Treatment of pathological cardiac remodeling and subsequent heart failure represents an unmet clinical need. The well conserved lncRNA H19 shows as powerful therapeutic potential in the treatment of pathological cardiac hypertrophy. H19 is strongly repressed in failing hearts from mice, pigs and humans. Gene therapy using murine but also human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. Using microarray , GSEA and ChIP-Seq we identified a link between H19 and NFAT signalling. H19 physically interacts with PRC2 to epigenetically induced Tescalcin repression which in turn leads to reduced NFAT expression and activity.

overall_design: RNA was isolated from the cardiomyocyte fractions of H19 knockout mice and corresponding wt littermates generated 6 weeks after TAC (transverse aortic constriction) surgery. RNA quality and quantity was assessed before microarray-based transcriptome profiling. Deregulated mRNAs due to the absence of H19 were analysed by Gene Set Enrichment Analysis GSEA.

first: Anne

last: Bührke

first: Janika

last: Viereck

first: Thomas

last: Thum


title: Chromatin ImmunoPrecipitation DNA-Sequencing after H19 OE and KD

overall_design: HL-1 cells were transfected with siRNA for H19 KD (50nM: siH19, siCtrl) to induce H19KD or transduced with lentivirus for stable H19 OE (pLV+H19, pLV+empty). Chromatin was isolated and 4 ug of antibody against H3K27me3 were applied for the isolation of genomic DNA regions of interest. ChIP DNA was purified.


title: A bifunctional nucleosome binding protein mediates specialized mSWI/SNF complex targeting and activity

first: Cigall

last: Kadoch

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

overall_design: Refer to individual Series


title: The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation

city: SHANGHAI

first: HONGHUI

last: MA

address: Jimo Road 150

institute: Tongji University School of Medicine

summary: Input mRNAs and ribosome protected fragments (RPFs) from wildtype and Mettl5 knockout (KO) mESC cells were analyzed by TruSeq Ribo Profile (Illumina) library preparation and high throughput sequencing

overall_design: The mESCs were cultured in 2i/lif medium. The 2i/lif medium contained N2B27 medium with 2i (PD0325901:1µM, Selleck; CHIR99021: 3µM, Selleck) and leukemia inhibitory factor lif (1,000U/ml, Millipore). N2B27 medium consisted of 50% DMEM/F12(GIBCO, #10565-018),50%Neurobasal medium (GIBCO, #21103-049) supplemented with 1xN2 (GIBCO, #17502048), 1xB27 (GIBCO, #17504044), 1xGlutamax (GIBCO, #35050061),1mM NEAA (GIBCO, #11140050),1xSodium Pyruvate (GIBCO, #11360070) and 0.1mM 2-mercaptoethanol (Sigma-Aldrich, #M3148). Input mRNAs and RPFs were isolated, ribosomal RNAs were depleted and cDNA libraries were prepared according to the TruSeq Ribo Profile pipeline (Illumina)

first: Ming

last: Xing

first: Hao

last: Chen

first: Qi

last: Liu

first: Honghui

last: Ma


title: Whole genome bisulfite sequencing of Picocholorum soloecismus across several days of its growth cycle

city: Los Alamos

first: Christina

middle: Rene

last: Steadman

state: NM

address: MS888 PO Box 1663

department: Biosciences

institute: Los Alamos National Laboratory

summary: Epigenetic modifications have not been well characterized in most microalgae species, particularly those used for biofuel production. We report the first analysis of DNA methylation of the species Picocholorum soloecismus across its growth cycle using whole-genome bisulfite sequencing. DNA methylation occurs in all three cytosine contexts (CpG, CHH, CHG) in P. soloecismus. While global DNA methylation is low overall (~1.15% of the 15 MB genome), methylation occurs appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution. The P. soloecismus genome becomes hypomethylated during the growth cycle (across several days in culture). 5-aza-2’-deoxycytidine (5AZA) treatment was used to alter DNA methylation patterns, which resulted in altered algal physiology and site specific changes in CpG DNA methylation.

overall_design: whole genome bisulfite sequencing on days 4, 5, 6, 7, and 10 in culture in control and 20 uM 5-aza-2'deoxycytidine treated cultures (biological triplicates) for a total of 30 sequencing runs


title: Single-cell analysis RNA sequencing of prenatal and neonatal gonads/ovaries at E11.5, E12.5, E14.5, E16.5, E18.5, P1 and P5

first: Allan

last: Spradling

address: 3520 San Martin Drive

institute: Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution for Science

summary: We sequenced more than 52,500 single cells from E11.5 to P5 gonads to analyze primordial follicles and wave 1 medullar follicles during mouse fetal and perinatal oogenesis. Germ cells clustered into six meiotic substages as well as dying/nurse cells. We also define genes expressed by epithelial progenitors, and clarify the similar but distinct genetic programs of bipotential-derived and epithelial-derived pre-granulosa cell progenitors. Their differentially expressed genes are candidates to control the distinctive developmental programs of wave 1 and wave 2 follicles. These observations provide a strong basis for further studies of the development, physiology, and evolutionary conservation of mammalian ovarian follicle formation.

overall_design: We performed single cell RNA-seq on developing prenatal gonads/ovaries atE11.5 (sex determination), E12.5 (cycle mitotically to form germline cysts), E14.5 (transition to meiosis), E16.5 (progress to pachytene),E18.5 and P1 (organelle transfer, cyst breakdown, oocyte differentiation and germ cell turnover), and P5 (primordial follicle formation).  The selected time points span key events during perinatal ovarian development. After a meticulous dissection and trypsin incubation, ovaries were dissociated into single-cell suspensions. 52,542 dissociated cells were subsequently captured, loaded onto oil droplets, and used for cDNA library construction, deep sequencing, and cluster analysis.  Expression information on an average of 2,700 different genes was recovered from each cell.

first: Wanbao

last: Niu


title: DOT1L-mediated Murine Neuronal Differentiation associates with H3K79me2 Accumulation and preserves SOX2-Enhancer Accessibility

city: Freiburg

last: Manke

address: Stuebeweg 51

institute: Max Planck Institute of Immunobiology and Epigenetics

summary: To study whether acute inhibition of DOT1L induces global chromatin states alterations, we profile and compare the transcriptome, chromatin accessibility and epigenome (H3K4me3, H3K4me1, H3K27ac, H3K27me3, H3K36me3, H3K9me3, H3K79me2) of mESC and ES-derived NPC, treated with DMSO or EPZ5676.

overall_design: full epigenome (duplicates), transcriptome (triplicates) and chromatin acceccibility profiling (duplicates) of mouse embryonic stem cells and in-vitro differentiated neurons, treated with DMSO and EPZ5676

first: Francesco

last: Ferrari

first: Laura

last: Arrigoni

first: Eirini

last: Trompouki

first: Tanja

last: Vogel


title: Extracellular matrix remodelling is associated with muscle force increase in overloaded mouse plantaris muscle.

city: Berlin

first: Markus

last: Schuelke

state: Berlin

address: Augustenburger Platz 1

department: Neuropediatrics

institute: Charite

summary:
Aims: Transforming growth factor-β (TGF-β) signalling is thought to contribute to the remodelling of extracellular matrix (ECM) of skeletal muscle and to functional decline in patients with muscular dystrophies. We wanted to determine the role of TGF-β-induced ECM remodelling in dystrophic muscle.
Methods: We experimentally induced the pathological hallmarks of severe muscular dystrophy by mechanically overloading the plantaris muscle in mice. Furthermore, we determined the role of TGF-β signalling on dystrophic tissue modulation and on muscle function by (i) overloading myostatin knockout (Mstn-/- ) mice and (ii) by additional pharmacological TGF-β inhibition via halofuginone.
Results: Transcriptome analysis of overloaded muscles revealed upregulation predominantly of genes associated with ECM, inflammation and metalloproteinase activity. Histology revealed in wild-type mice signs of severe muscular dystrophy including myofibres with large variation in size and internalized myonuclei, as well as increased ECM deposition. At the same time, muscle weight had increased by 208% and muscle force by 234%. Myostatin deficiency blunted the effect of overload on muscle mass (59% increase) and force (76% increase), while having no effect on ECM deposition. Concomitant treatment with halofuginone blunted overload-induced muscle hypertrophy and muscle force increase, while reducing ECM deposition and increasing myofibre size.
Conclusions: ECM remodelling is associated with an increase in muscle mass and force in overload-modelled dystrophic muscle. Lack of myostatin is not advantageous and inhibition of ECM deposition by halofuginone is disadvantageous for muscle plasticity in response to stimuli that induce dystrophic muscle.

overall_design: Case-control design, using wildtype and Myostatin knockout (Mstn-/-) mice. In the cases, overload of the plantaris muscle was achieved by removal of the soleus and part of the gastrocnemius muscle