title: Fat1 deletion promotes hybrid EMT state with enhanced tumor progression, stemness, and metastasis (human RNA-Seq)

overall_design: Human skin SCC cell line (A388) is FAT1 WT. We perfomed Crispr KO of FAT1 in A388 and perfomed bulk RNA-sequenicng on 2 FAT1KO clones and 2 single-cell derived FAT1 WT clones.


title: Fat1 deletion promotes hybrid EMT state with enhanced tumor progression, stemness, and metastasis (DNA-Seq)

status: Public on Dec 04 2020

city: Bruxelles

first: Yura

last: Song

address: 808, route de Lennik

country: Belgium

institute: Université Libre de Bruxelles

summary: FAT1, a protocadherin, is among the most frequently mutated genes in human cancers. However, the role and the molecular mechanisms by which FAT1 mutations control tumor initiation and progression are poorly understood. Here, using different mouse cancer models including skin squamous cell carcinoma (SCC) and lung tumors we found that Fat1 deletion accelerated tumor initiation and malignant progression and promoted hybrid epithelial to mesenchymal transition (EMT) phenotype. This hybrid EMT state was also found in FAT1 mutated human SCCs. Fat1 deleted skin SCCs presented increased tumor stemness and spontaneous metastasis. Transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies revealed that FAT1 loss of function activates a CAMK2/CD44/SRC axis that promotes YAP/ZEB1 nuclear translocation and stimulates the mesenchymal state, as well as a CAMK2-EZH2 axis that promotes activation of SOX2, which sustains the epithelial state. This comprehensive analysis also identified drug resistance and vulnerabilities in FAT1 deficient tumors with important implications for cancer therapy. Altogether, our studies revealed that Fat1 loss of function promotes tumor initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.

overall_design: We FACS isolated tumor cells from PDX by excluding CD45+, CD31+ and PDGFRb+ (lin-) non-tumoral cells.

first: Ievgenia

last: Pastushenko

first: Federico

last: Mauri

first: Cédric

last: Blanpain


title: Fat1 deletion promotes hybrid EMT state with enhanced tumor progression, stemness, and metastasis (mouse RNA-Seq)

type: Expression profiling by high throughput sequencing

overall_design: FACS isolated Epcam+ and Epcam- tumor cells from DMBA/TPA K14Cre;RYFP and K14Cre;Fat1fl/fl;RYFP skin SCCs, and from Kras;p53;RYFP and Kras;p53;Fat1fl/fl;RYFP lung cancers.


title: A dedicated three component regulatory system (GSP/Ghk/Grr) activates gallocin transcription in Streptococcus gallolyticus UCN34

city: Paris

first: Rachel

last: Legendre

address: 28, rue du docteur Roux

department: Research and Resource Center for Scientific Informatics

country: France

institute: Institut Pasteur

summary: Bacteriocins are natural antimicrobial peptides produced by a bacterium to kill closely related competitors. Streptococcus gallolyticus subsp. gallolyticus (Sgg) UCN34 produces a two-component bacteriocin named gallocin to colonize the gut by killing resident enterococci. In the present work, we investigated how gallocin was regulated by deleting individually three genes present in the locus and encoding potentially an inducing peptide (gsp), a histidine kinase (ghk) and a LytTR-containing response regulator (grr). Comparative transcriptional analyses of these three mutants as compared to the WT UCN34 showed that this 3- component regulatory system induces the transcription of the whole gallocin locus encoding gallocin but also five others putative bacteriocins and genes necessary for bacteriocins biosynthesis and secretion. Beside gallocin locus, only two other pairs of genes were coordinately induced in Sgg genome, encoding an ABC transporter and hypothetical proteins. We conclude that this regulatory system appears highly specialized in bacteriocins induction in Sgg UCN34.

overall_design: Whole transcriptomic analysis of Streptococcus gallolyticus subspecies gallolyticus (Sgg) UCN34 WT, Δgsp, Δghk and Δgrr

first: Alexis

last: Proutière

first: Hugo

last: Varet

first: Odile

last: Sismeiro

first: Shaynoor

last: Dramsi


title: O-linked mucin-type glycosylation regulates the transcriptional programme downstream of EGFR in breast cancer.

city: Naples

first: Gennaro

last: Gambardella

address: Via Campi Flegrei 34,

country: Italy

institute: Telethon Institute of Genetics and Medicine (TIGEM)

summary: Aberrant mucin type O-linked glycosylation is a common occurrence in cancer. This type of O-linked glycosylation can occur on many cell surface glycoproteins where only a small number of sites may be present. EGFR is one such glycoprotein. Upon EGF ligation, EGFR induces a signaling cascade but can also translocate to the nucleus where it can directly regulate gene transcription. Here we show that upon EGF binding, breast cancer cells carrying different O-linked glycans respond by transcribing differential gene expression signatures. This is not a result of changes in signal transduction but due to the differential nuclear translocation of EGFR in the two glyco-phenotypes. This appears to be regulated by the formation of a EGFR/galectin-3/MUC1 complex at the cell surface that is present in cells carrying short core1-based O-glycans characteristic of tumour cells but absent in core 2 O-glycan carrying cells representative of normal mammary epithelial cells.

overall_design: T47D non-treated control cells, T47D cells treated with EGF for 2h, T47D cells treated with EGF for 6h, E2J non-treated control cells, E2J cells treated with EGF for 2h, E2J cells treated with EGF for 6h.3 biological replicates for each condition were used. Technical duplicates were run of T47D non reated control samples and T47D cells treated with EGF for 2h.

first: Virginia

last: Tajadura-Ortega

first: Joy

middle: M

last: Burchell


title: Whole genome transcriptome of in vivo Treg cells and ex vivo TGF-beta induced Treg cells from WT and Aim2 knockout mice

city: Chapel Hill

first: Zengli

last: Guo

state: NC

address: 125 Mason Farm Road

department: Lineberger Comprehensive Cancer Center

country: USA

institute: University of North Carolina at Chapel Hill

summary: To understand the whole genome transcriptome of in vivo Treg cells and ex vivo TGF-beta induced Treg cells from WT and Aim2 knockout mice, the total RNA was extracted from indicated Treg cells using the Direct-zol miniprep kit (Zymo Research, R2060). The RNA samples were firstly enriched by Oligo(dT) magnetic beads  and used to construct BGISEQ-500 libraries. RNA-seq libraries sequenced using the 50bp single-end protocol (in vivo isolated Treg cells) or 100bp paired-end protocol (TGF-β induced Treg cells) via the BGISEQ-500 sequencer per the manufacturer’s protocol. After filtering of adaptors and low quality reads, clean reads (>26 Million reads per sample for in vivo isolated Treg cells and >40 Million reads per sample for TGF-β induced Treg cells) are mapped to mouse reference genome using HISAT /Bowtie2 tool. Mapping results are stored in BAM files using SAMtools.  Total read counts in gene level were summarized using featureCounts function in the Rsubread in R environment, with the R package biomaRt for gene and transcripts mapping. The differential expression (DE) genes were analyzed by DESeq2 package with default setting using total read counts as input, and the adjusted p value (padj) less than 0.05.

overall_design: Examination of differential gene expression of in vivo islolated Treg cells with or without 24-hour TCR stimulation and TGF-beta induced Treg (iTreg) cells at day one from naive CD4 T cells in WT and Aim2 knockout mice.

first: Wei-Chun

last: Chou

first: Xianming

last: Tan

first: Jenny

middle: Y

last: Ting


title: Expanding the spectrum of CNS EWSR1-PATZ1 rearranged tumors: an infantile case with leptomeningeal dissemination

status: Public on Dec 03 2020

city: Rome

first: Sabina

last: Barresi

address: Viale San Paolo 15

institute: Ospedale Pediatrico Bambino Gesu

summary: This case shows similarities with 2 previously reported cases, including intraventricular location, histologic appearance (pushing borders, oligodendrocyte-like morphology, rich vascular network) and immunophenotype with co-expression of OLIG2, GFAP and synaptophysin. The onset in an infant, the presence of a high-grade component and the leptomeningeal dissemination, however, have not been previously reported in EWSR1-PATZ1 rearranged tumors, expanding the clinico-pathological spectrum.

overall_design: One Tumor sample was analyzed

first: Sabrina

last: Rossi


title: Regulation of plant DNA methylation by UV-B light in Arabidopsis

city: St Louis

first: Xuehua

last: Zhong

state: MO

address: One Brookings Drive

department: Biology

institute: Washington University in St. Louis

type: Methylation profiling by high throughput sequencing

summary: This study investigated the effect of UV-B treatment on DNA methylation in A. thaliana.

overall_design: A. thaliana plants were growth on 1/2X MS agar (0.7%) + 1% sucrose throughout treatment.  Treatment began on Day 1 after sowing with 1.5  mE of UV-B (315 nm) and 60 mE white light  and terminated on Day 10 of treatment when the plants were frozen in liquid nitrogen before mRNA or DNA extraction for sequencing.

first: Jianjun

last: Jiang

first: Jie

last: Liu

first: Dean

last: Sanders


title: Transcriptomic Profiling of Fetal Membranes of Mice Deficient in Biglycan and Decorin as a Model of Preterm Birth

city: Tampa

first: Derek

middle: E

last: Wildman

state: Florida

address: 3720 Spectrum Blvd

department: College of Public Health

institute: University of South Florida

summary: Approximately 25% of all preterm births are due to preterm premature rupture of membranes. Mice deficient in proteoglycans biglycan (Bgn) and decorin (Dcn) display abnormal fetal membranes and increased incidence of preterm birth. We conducted RNA-Seq to profile fetal membranes and identify molecular pathways that may lead to preterm birth in double knockout (DKO) mice (Bgn −/−; Dcn −/− ) compared to wild-type (WT) at two different gestational stages, E12 and E18 (n=3 in each group). 3264 transcripts were differentially regulated in E18 DKO vs. WT fetal membranes, and 96 transcripts differentially regulated in E12 DKO vs. WT fetal membranes (FDR<0.05, log 2 FC≥1). Differentially regulated transcripts in E18 DKO fetal membranes were significantly enriched for genes involved in cell cycle regulation, extracellular matrix-receptor interaction, and the complement cascade. 50 transcripts involved in the cell cycle were altered in E18 DKO fetal membranes (40↓, 10↑, FDR<0.05), including p21 and p57 (↑), and Tgfb2, Smad3, CycA, Cdk1, and Cdk2(↓). Thirty-one transcripts involved in the complement cascade were altered (11↓, 20↑, FDR<0.05) in E18 DKO fetal membranes, including C1q, C2, and C3 (↑). Differentially expressed genes in the top three molecular pathways (1) showed evidence of negative or purifying selection, and (2) were significantly enriched (Z-score>10) for transcription factor binding sites for Nr2f1 at E18. We propose that in DKO mice, cell cycle arrest results in lack of cell proliferation in fetal membranes, inability to contain the growing fetus, and preterm birth.

overall_design: Comparison of mouse fetal membranes following double knockout of biglycan and decorin versus wild-type at two embryonic days E12 and E18

first: Priyadarshini

last: Pantham

first: Don

middle: L

last: Armstrong

first: Jonathan

last: Bodnariuc

first: Owen

last: Haupt

first: Amy

last: Wagoner Johnson

first: Lori

last: Underhill

first: Renato

middle: V

last: Iozzo

first: Beatrice

last: Lechner


title: Analysis of gene expression changes in the spinal cord during chronic pain.

city: Chuo-ku

first: Takashi

last: Hozumi

state: Chiba

address: 1-8-1, Inohana

country: Japan

institute: Chiba University

summary: Purpose: The goal of this study is to examine the DEGs in the spinal cord of schiatic nerve cuff model mouse using RNA-seq compared with control (sham).

overall_design: Examination of DEGs in schiatic nerve cuff mouse model at day 28 post surgery compared with control (sham).

first: Atsushi

last: Yamaguchi


title: COVID-19 cytokines and the hyperactive immune response: Synergism of TNF-α and IFN-γ in triggering inflammation, tissue damage, and death

city: Memphis

first: Geoffrey

last: Neale

state: TN

address: 262 Danny Thomas Place

department: Hartwell Center

institute: St Jude Childrens Research Hospital

summary: COVID-19 has rapidly circulated around the globe and caused significant morbidity and mortality. The disease is characterized by excessive production of pro-inflammatory cytokines and acute lung damage and patient mortality. Although initial cytokine cascades may be beneficial to the host for clearing the virus, enhanced production of pro-inflammatory cytokines and increasing levels in the systemic circulation, referred to as cytokine storm, can promote tissue damage by inducing inflammatory cell death in both infected and bystander cells. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combination of TNF-α and IFN-γ specifically induced cell death characterized by GSDME¬–mediated pyroptosis, caspase-8–mediated apoptosis, and MLKL–mediated necroptosis. Cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. However, deletion of pyroptosis, apoptosis, or necroptosis individually was not sufficient to protect against cell death. Mechanistically, the STAT1/IRF1 axis activated by TNF-α and IFN-γ co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of inflammatory cell death protected mice from TNF-α and IFN-γ–induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. To determine the physiological relevance of protection, we neutralized both TNF-α and IFN-γ in multiple disease models associated with cytokine storm and found that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models. Collectively, our findings reveal that blocking the COVID-19 cytokine-mediated inflammatory cell death signaling pathway identified in this study may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancer where TNF-α and IFN-γ synergism play key pathological roles.

overall_design: We used microarrays to compare the transcription profiles of primary mouse bone marrow-derived macrophages (BMDMs) from Irf1-/- and WT mice that had been stimulated in culture with TNF-a, IFN-g, or both cytokines simultaneously.

first: Rajendra

last: Karki

first: Bhesh

middle: R

last: Sharma

first: Shraddha

last: Tuladhar

first: Balaji

last: Banoth

first: Parimal

last: Samir

first: Thirumala-Devi

last: Kannaganti


title: Immune-stimulating antibody conjugates elicit robust myeloid activation and durable anti-tumor immunity II

city: Redwood City

first: Lisa

middle: K

last: Blum

email: lblum@boltbio.com, info@boltbio.com

state: CA

address: 640 Galveston Dr

institute: Bolt Biotherapeutics

type:
Expression profiling by array
Other

summary: Innate  pattern recognition  receptors,  including  toll-like  receptors  (TLRs),  can  alter  the  tumor microenvironment and prime adaptive anti-tumor immunity. However, TLR agonists have not been well-tolerated due to toxicities associated with widespread immune activation following systemic administration. To design a therapeutic that is suitable for systemic delivery and capable of eliciting a tumor-targeted response, we developed a novel class of immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 agonist conjugated to tumor-targeting antibodies. Systemically administered anti-HER2 ISACs were well-tolerated in vivo and elicited robust activation of intratumoral myeloid cells, resulting in tumor clearance and subsequent immunological memory. In vitro potency and in vivo efficacy required tandem activity driven by intact Fc functionality and TLR agonism to enable phagocytosis and T cell-mediated anti-tumor immunity. ISAC-mediated immunological memory was not limited to HER2, as ISAC-treated mice were protected from rechallenge with a HER2-negative tumor. These results provide strong rationale for  the  clinical  development  of ISACs and  show  that  synergy  between  FcgR  and  TLR7/8 signaling contributes to the mechanism of ISAC-mediated anti-tumor immunity.

overall_design: We measured mouse gene expression in syngeneic MMC tumors from 20 mice (n = 5 per group) one day following treatment with anti-rHER2, anti-rHER2 T785-ISAC, Isotype Control, or Isotype T785-ISAC.

first: Michael

middle: N

last: Alonso

first: Shelley

last: Ackerman


title: Synthetic lethality by TET-dioxygenase inhibition

city: Cleveland

first: Babal

last: Jha

state: Ohio

address: 9500 Euclid Ave, NE6-253

department: Translational Hematology and Oncology

institute: Cleveland Clinic

summary: To characterize target specificity of TETi76, we performed global gene expression analyses of K562TET2+/+ and TET2-/- control cells; TETi76 mimicked expression signatures generated by the loss of TET2 in K562. The addition of Ascorbic Acid (AA), known to enhance TET-dioxygenase activity, counteracted the changes induced by TETi76. In order to study the molecular pathway of synthetic lethality by TET-dioxygenase inhibition, natural TET2-/- mutant cell line SIGM5 was treated with TET inhibitor TETi76 and global gene expression analysis was performed by RNAseq. Result demostrate a significant upregulation of TNT-α signaling and the down regulation of Interferon-α signaling. Interestingly, we also observed significant up-modulation of oxidative stress response pathway genes consistent with the inhibition of dioxygenases. In particular, TETi76 treatment induces 8-fold increase of oxidative stress sensor NQO1 a NRF2 target gene that has been shown earlier to induce pro-apoptotic cell death in cancer cells.

overall_design: SIGM5 and isogenic K562 (TET2+/+ and TET2-/-) cells mRNA profile of TETi76 treatment for 24 hours

first: Jaroslaw

middle: P

last: Maciejewski

first: Yihong

last: Guan


title: Regulatory proteins control a transcriptional network in response to antibiotic stress

city: Brookings

first: Nicholas

middle: C.

last: Butzin

state: SD

address: 1224 Medary Ave.

institute: South Dakota State University

summary: In a given bacterial population, antibiotic treatment kills a large portion of the population, while a small, tolerant subpopulation survives. Tolerant cells disrupt the efficacy of antibiotic treatment and increase the likelihood that a population gains antibiotic resistance. Antibiotic tolerance is different from resistance because tolerant cells cannot grow and replicate in the presence of the antibiotic, but when the antibiotic is removed, they begin to propagate. When a population becomes resistant, the antibiotic becomes ineffective, which is a major health concern. Since antibiotic tolerance often leads to antibiotic resistance, we have taken a systems biology approach to examine how regulatory networks respond to antibiotic stress so that cells can survive and recover after antibiotic treatment. We have compared gene expression with and without ampicillin in E. coli.

overall_design: RNA-sequencing from three different E. coli populations: (1) stationary phase cells and two populations incubated for three hours in fresh media (2) without ampicillin (untreated) and (3) with ampicillin at bactericidal concentrations (treated).

first: Heather

middle: S

last: Deter

middle: C


title: Protein Arginine Methyltransferase 3 Fine-Tunes the Assembly/Disassembly of Pre-ribosomes to Repress Nucleolar Stress by Interacting with RPS2B in Arabidopsis

city: Beijing

first: Jing

last: Sun

address: No. 1 West Beichen Road

department: IGDB

country: China

institute: Chinese Academy of Sciences

summary: Ribosome biogenesis, which takes place mainly in the nucleolus, involves coordinated expression of pre-ribosomal RNAs (pre-rRNAs) and ribosomal proteins, pre-rRNA processing, and subunit assembly with the aid of numerous assembly factors. Our previous study showed that the Arabidopsis thaliana protein arginine methyltransferase AtPRMT3 regulates pre-rRNA processing; however, the underlying molecular mechanism remains unknown. Here, we report that AtPRMT3 interacts with Ribosomal Protein S2 (RPS2), facilitating processing of the 90S/Small Subunit (SSU) processome and repressing nucleolar stress. We isolated an intragenic suppressor of atprmt3-2, which rescues the developmental defects of atprmt3-2 while produces a putative truncated AtPRMT3 protein bearing the entire N-terminus but lacking an intact enzymatic activity domain. We further identified RPS2 as an interacting partner of AtPRMT3, and found that loss-of-function rps2a2b mutants were phenotypically reminiscent of atprmt3, showing pleiotropic developmental defects and aberrant pre-rRNA processing. RPS2B binds directly to pre-rRNAs in the nucleus, and such binding is enhanced in atprmt3-2. Consistently, multiple components of the 90S/SSU processome were more enriched by RPS2B in atprmt3-2, which accounts for early pre-rRNA processing defects and results in nucleolar stress. Collectively, our study uncovered a novel mechanism by which AtPRMT3 cooperates with RPS2B to facilitate the dynamic assembly/disassembly of the 90S/SSU processome during ribosome biogenesis and repress nucleolar stress.

overall_design: All wildtype and mutant seeds were grown on MS plate (with 1% sucrose) at 22°C under long day condition (LD, 16 hours light, 8 hours dark). 12 day after germination (12DAG), whole seedling were harvested for RNA-seq.

first: Runlai

last: Hang

first: Zhen

last: wang

first: Chunyan

last: Liu

first: Xiaofeng

last: Cao


title: Immune-stimulating antibody conjugates elicit robust myeloid activation and durable anti-tumor immunity I

summary: Innate  pattern recognition  receptors,  including  toll-like  receptors  (TLRs),  can  alter  the  tumor microenvironment and prime adaptive anti-tumor immunity. However, TLR agonists have not been well-tolerated due to toxicities associated with widespreadimmune activation following systemic administration. To design a therapeutic that is suitable for systemic delivery and capable of eliciting a tumor-targeted response, we developed a novel class of immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 agonist conjugated to tumor-targeting antibodies. Systemically administered anti-HER2 ISACs were well-tolerated in vivo and elicited robust activation of intratumoral myeloid cells, resulting in tumor clearance and subsequent immunological memory. In vitro potency and in vivo efficacy required tandem activity driven by intact Fc functionality and TLR agonism to enable phagocytosis and T cell-mediated anti-tumor immunity. ISAC-mediated immunological memory was not limited to HER2, as ISAC-treated mice were protected from rechallenge with a HER2-negative tumor. These results provide strong rationale for  the  clinical  development  of ISACs and  show  that  synergy  between  FcgR  and  TLR7/8 signaling contributes to the mechanism of ISAC-mediated anti-tumor immunity.

overall_design: We measured mouse gene expression in HCC1954 tumor xenografts from 20 mice (n = 5 per group) one day following treatment with Trastuzumab, Trastuzumab T785-ISAC, Rituximab, or Rituximab T785-ISAC.


title: Colibactin causes genomic instability and promotes Wnt independence in primary colon epithelial cells

city: Berlin

first: Thomas

middle: F

last: Meyer

address: Chariteplatz 1

department: Molecular Biology

country: Germany

institute: Max Planck Institute for Infection Biology

summary: Colorectal cancer is driven by a sequential cascade of mutations known as the adenoma-carcinoma sequence. Recent studies have revealed that specific bacterial species present in the colonic microbiota can induce mutations and contribute to this malignancy. Specifically, genotoxic colibactin-producing pks+ Escherichia coli strains can induce DNA double strand breaks (DSBs) and promote tumor development in mouse models of colorectal cancer. Here, we investigated the transformation potential of colibactin by using organoids and polarized monolayers derived from primary murine colon epithelial cells and reveal striking phenotypic changes upon short-term infection.  This study demonstrates the direct pro-oncogenic potential of pks+ E. coli, as such transformations in vivo could facilitate colitis-associated colorectal carcinogenesis.

overall_design: RNAseq of mouse colorectal epithelium (Wnt independent organoids derived from pks+ infected cells and uninfected control organids)

first: Amina

last: Iftekhar

last: Sigal

first: Francesco

last: Boccellato

first: Hilmar

last: Berger


title: Immune-stimulating antibody conjugates elicit robust myeloid activation and durable anti-tumor immunity

first: Lisa

last: Blum

type:
Other
Expression profiling by array

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

overall_design: Refer to individual Series


title: Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation

city: Versailles

first: Filipe

last: Borges

address: Route de St-Cyr (RD10)

institute: Institut Jean-Pierre Bourgin - INRAE

summary: 5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, while mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, while mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small-interfering RNAs and H3K9me2, without gaining CHH methylation. This suggests that spontaneous epialelles that arise in plant cell cultures are stably maintained by small RNA independently of the canonical RNA-directed DNA methylation pathway. In contrast, loci that fail to produce siRNA are targeted for demethylation when cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones.

overall_design: Whole-genome bisulfite sequencing of Arabidopsis cell cultures

first: Robert

middle: A

last: Martienssen


title: Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons

status: Public on Dec 02 2020

city: Baltimore

first: Dong Won

middle: Thomas

last: Kim

state: Maryland

address: 733 N.Broadway, MRB building (Rm 332)

department: Neuroscience

institute: Johns Hopkins University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Purpose: highthroughput sequencing (bulk RNA-Seq, bulk ATAC-Seq, and scRNA-Seq) to study hypothalamic Lhx6 neurons

overall_design: Bulk RNA-Seq, bulk ATAC-Seq, scRNA-Seq on flow-sorted Lhx6 neurons from the cortex and hypothalamus using Lhx6-eGFP and Lhx6-CreER;Ai9 lines

first: Dong

middle: W

first: Kai

first: Zoe

middle: Q

last: Wang

first: Yi

last: Zhang

first: Abhijith

last: Bathini

first: Matthew

last: Brown

first: Sonia

middle: H

last: Lin

first: Parris

last: Washington

first: Changyu

first: Susan

last: Lindtner

first: Bora

last: Lee

first: Hong

first: Tomomi

last: Shimogori

first: John

last: Rubenstein

first: Seth

last: Blackshaw


title: Combined BRAF, MEK and CDK4/6 inhibition depletes intratumoral immune-potentiating myeloid populations in melanoma

city: Melbourne

first: Emily

middle: Jane

last: Lelliott

state: VIC

address: 305 Grattan Street

department: Research

country: Australia

institute: Peter MacCallum Cancer Centre

summary: Combined inhibition of BRAF, MEK and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on anti-tumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA-seq analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted pro-inflammatory macrophages and cross-priming CD103+ dendritic cells (DCs), the absence of which correlated with poor overall survival and clinical responses to ICB in melanoma patients. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo. While combined BRAF, MEK and CDK4/6 inhibition demonstrates favourable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact on anti-tumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

overall_design: Single-cell RNA-seq and paired TCR-seq on the CD45.2+ immune cells from YOVAL1.1 melanoma tumors treated for 7 days with vehicle or triple (one sample per group representative of 10 tumors pooled)

first: Karen

last: Sheppard

first: Jane

last: Oliaro

first: Grant

last: McArthur

middle: J

first: Stefano

last: Mangiola


title: Genetic analysis of the role of the conserved inner membrane protein CvpA in EHEC resistance to deoxycholate

status: Public on Dec 01 2020

city: Boston

last: Waldor

state: MA

address: 181 Longwood Avenue

department: Microbiology

institute: Harvard Medical School

summary: The function of cvpA, a bacterial gene predicted to encode an inner membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpA’s genetic interactions and identify common pathways that rescue the sensitivity of a ∆cvpA EHEC mutant to DOC. These screens demonstrated that mutations predicted to activate the σE-mediated extracytoplasmic stress response bypass the ∆cvpA mutant’s susceptibility to DOC. Consistent with this idea, we found that deletions in rseA and msbB and direct overexpression of rpoE restored DOC resistance to the ∆cvpA mutant. Analysis of the distribution of CvpA homologs revealed that this inner membrane protein is conserved across diverse bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA plays a role in cell envelope homeostasis in response to DOC and similar stress stimuli in diverse bacteria.

overall_design: We performed transposon-insertion sequencing (Tn-Seq) on transposon mutagenized libraries in wild-type (WT) enterohemorrhagic E. coli EDL933 and a strain in which cvpA gene is deleted (∆cvpA).

first: Alyson

last: Warr

first: Rachel

middle: T

last: Giorgio


title: Single-cell RNAsequencing of Drosophila PI Circadian Output Cells

city: Chicago

first: Daniel

last: Cavanaugh

state: IL

address: 1050 W Sheridan Rd., Life Sciences Building Room 215

institute: Loyola University Chicago

summary: The circadian system produces ~24-hr oscillations in behavioral and physiological processes to ensure that they occur at optimal times of day and in the correct temporal order. At its core, the circadian system is composed of dedicated central clock neurons that keep time through a cell-autonomous molecular clock. To produce rhythmic behaviors, time-of-day information generated by clock neurons must be transmitted across output pathways to regulate the downstream neuronal populations that control the relevant behaviors. An understanding of the manner through which the circadian system enacts behavioral rhythms therefore requires the identification of the cells and molecules that make up the output pathways. To that end, we recently characterized the Drosophila pars intercerebralis (PI) as a major circadian output center that lies downstream of central clock neurons in a circuit controlling rest:activity rhythms. We have conducted single-cell RNA sequencing (scRNAseq) to identify potential circadian output genes expressed by PI cells, and used cell-specific RNA interference (RNAi) to knock down expression of ~40 of these candidate genes selectively within subsets of PI cells. We demonstrate that knockdown of the slowpoke (slo) potassium channel in PI cells reliably decreases circadian rest:activity rhythm strength. Interestingly, slo mutants have previously been shown to have aberrant rest:activity rhythms, in part due to a necessary function of slo within central clock cells. However, rescue of slo in all clock cells does not fully reestablish behavioral rhythms, indicating that expression in non-clock neurons is also necessary. Our results demonstrate that slo exerts its effects in multiple components of the circadian circuit, including PI output cells in addition to clock neurons, and we hypothesize that it does so by contributing to the generation of daily neuronal activity rhythms that allow for the propagation of circadian information throughout output circuits.

overall_design: 5 single GFP-labeled Drosophila pars intercerebralis (PI) cells were extracted by pipet aspiration from 4 different animals. Antisense RNA was subjected to 3 rounds of amplification. Single-cell RNA sequencing was performed to determine expressed genes.


title: Bulk RNA sequencing of SARS-CoV-2 infected alveolar type I- and type II like cells

status: Public on Dec 07 2020

city: Utrecht

first: Jelte

last: van der Vaart

address: Universiteitsweg 100

department: Center for Molecular Medicine

country: Netherlands

institute: University Medical Center Utrecht

summary:
Bulk RNA sequencing was performed on control and SARS-CoV-2 infected 2D bronchioalveolar-like and small airway cultures

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), may result in acute respiratory distress syndrome (ARDS), multi-organ failure and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interphase culture system which was characterized by confocal-, electron-microscopy and mRNA expression analysis. In this model, alveolar type I- (ATI-L) and type II-like (ATII-L) cells, but also basal cells, neuroendocrine cells and club cells, are grown from 3D self-renewing lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with both ATI-L and ATII-L cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of a type I/III interferon response program. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.

overall_design: RNA was extracted from 2D pulmonary cultures that were either grown in expansion conditions (EXP) or differentiation conditions (DIF) and were not treated or SARS-CoV-2 for 72 hours. After this, RNA was collected.

first: Mart

last: Lamers

first: Kèvin

last: Knoops

first: Sandra

last: Riesebosch

first: Tim

last: Breugem

first: Anna

last: Mykytyn

first: Joep

last: Beumer

first: Debby

last: Schipper

first: Karel

last: Bezstarosti

first: Raimond

last: Ravelli

first: Hans

last: Duimel

first: Jeroen

last: Demmers

first: Georges

last: Verjans

first: Marion

last: Koopmans

first: Peter

last: Peters

last: Clevers

first: Bart

last: Haagmans


title: Arginine methylation of the intrinsically disordered C-terminus of METTL14 controls N6-methyladenosine function in DNA repair

city: Philadelphia

first: Lan

last: Lin

state: PA

address: 3501 Civic Center Boulevard, Office 9052

department: Pathology and Laboratory Medicine

institute: The Children’s Hospital of Philadelphia

summary: The N6-methyladenosine (m6A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m6A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m6A methyltransferase complex, as a novel pathway that controls the function of m6A in DNA repair. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with and methylates the intrinsically disordered C-terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNAPII. Mouse embryonic stem cells (mESCs) expressing arginine methylation-deficient METTL14 exhibit dramatically reduced global m6A levels. Transcriptome-wide m6A analysis reveals that arginine methylation-dependent m6A enhances the translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation-deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m6A regulation that could have broad implications in normal development and in diseases such as cancer.

overall_design: Using a modified m6A-seq protocol (Dominissini et al., 2013), we profiled the genome-wide m6A methylomes of WT, Mettl14 KO, KO+WT, KO+5RK, and KO+13RK mESCs. For each sample, we analyzed three experimental replicates using both RIP-seq with an m6A-specific antibody and standard RNA-seq of the input control.

first: Samir

last: Adhikari

first: Zhicheng

last: Pan


title: In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes

city: Cambridge

first: Joshua

last: Levin

address: 75 Ames St

department: Stanley Center

institute: Broad Institute

summary: We developed and applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes (see https://www.biorxiv.org/content/10.1101/791525v1 for complete list). Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA sequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes.

overall_design: All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Harvard University and of the Broad Institute of MIT and Harvard. In utero lentiviral injection into the lateral ventricles was performed at E12.5 in Cas9 transgenic mice (4-6 month old, Jax #026179), and each single-cell library was made by combining the BFP+ cells from 1-3 litters (4-20 animals) of P7 animals harvested on the same day. The FACS-purified cells were sorted into cold Hibernate A/B27 medium and subjected to single-cell RNA sequencing library preparation. Our analysis comprises 18 independent libraries of Perturb-Seq cells (17 after filtering). Single-cell RNA sequencing libraries were created using the Chromium Single Cell 3' Solution v2 kit (10x Genomics) following the manufacturer’s protocol. Each library was sequenced with Illumina NextSeq high-output 75-cycle kit with sequencing saturation above 70%. Dial-out PCR was performed to extract the perturbation barcode in each cell.

first: Xin

last: Jin

first: Sean

last: Simmons

first: Aviv

last: Regev

first: Paola

last: Arlotta

first: Feng

last: Zhang


title: MetR is a global regulator controlling phytochrome-dependent light responses in Aspergillus nidulans

city: Warszawa

first: Marek

last: Skoneczny

email: kicia@ibb.waw.pl, mrksknc@gmail.com

address: Pawińskiego 5A

country: Poland

institute: Institute of Biochemistry and Biophysics PAS

summary: Phytochrome-dependent light signaling has been studied in several fungi. In Aspergillus nidulans light-stimulated phytochrome activates the HOG signaling pathway and thereby controls the expression of a large number of genes, many of which are related to stress responses. In a genome-wide expression analysis in A. nidulans we found that phytochrome, fphA, is under strict expression control of the central regulator of the sulfur-starvation response, MetR. This transcriptional regulator is required for the expression of genes involved in inorganic sulfur assimilation. In the presence of organic sulfur, MetR is probably ubiquitinated and possibly degraded and the transcription of sulfur-assimilation genes, e.g. sulfate permease, is turned off. The expression analysis described here revealed, however, that MetR additionally controls the expression of hundreds of genes, many of which are required for secondary metabolite production. We also show that metR mutation phenocopies fphA deletion, and five other histidine-hybrid kinases are down-regulated in the metR1 mutant. Furthermore, we show that light and phytochrome regulate the expression of at least three carbon-sulfur hydrolases. This work is a further step towards understanding the interplay between light sensing and metabolic pathways.

overall_design: Comparison of transcriptomes of the filamentous fungus Aspergillus nidulans two congenic strains, W1 (yA2; pyroA4; veA1) and RM114 (yA2; metR1; pyroA4; veA1). Two biological replicates of W1 vs metR1  were performed. Each biological replicate had two technical replicates with dye swapping.

first: Marzena

last: Sieńko

first: Jerzy

last: Brzywczy


title: Ash1 and Tup1 Dependent Repression of the Saccharomyces cerevisiae HO promoter Requires Activator-Dependent Nucleosome Eviction

city: Salt Lake City

first: David

last: Stillman

state: Utah

address: 15 N Medical Drive East, EEJMRB

department: Pathology

institute: University of Utah

summary: Transcriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.

overall_design: Genomic localization of Ash1-V5, Tup1-V5 and Rpd3-V5 was determined by chromatin immunoprecipitation from cross-linked S. serevisiae cells, followed by Illumina paired-end sequencing. Three replicate ChIPs and a single input control are included for each strain.

first: Emily

last: Parnell

first: Timothy


title: The SARS-CoV-2 RNA-protein interactome in infected human cells

city: New York

first: Caleb

last: Lareau

state: New York

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

institute: Memorial Sloan Kettering

summary: Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry (RAP-MS), we identify up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrate the SARS-CoV-2 RNA interactome with proteome abundance changes induced by viral infection and link interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrate by genetic perturbation that CNBP and LARP1, two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduces viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.

overall_design: 1. We mapped direct protein-RNA interaction sites using enhanced crosslinking and immunoprecipitation (eCLIP). 2. We identified RNA sequences crosslinked to proteins purified by RNA antisense purification (RAP-MS) by following the procedure described in the protocols section.

first: Mathias

last: Munschauer


title: Sperm DNA Methylation Epimutation Biomarker for Paternal Offspring Autism Susceptibility

city: Pullman

first: Michael

last: Skinner

state: WA

address: Abelson 507

department: SBS

institute: WSU

summary: Autism spectrum disorder (ASD) has increased over ten-fold over the past several decades, and appears predominantly associated with paternal transmission. Although genetics is anticipated to be a component of ASD etiology, environmental epigenetics is now thought to be an important factor. Epigenetic alterations, such as DNA methylation have been correlated with ASD. The current study was designed to identify a DNA methylation signature in sperm as a potential biomarker to identify paternal offspring autism susceptibility. Sperm samples were obtained from fathers, many undergoing in vitro fertilization (IVF) procedures, that have children with or without autism, and the sperm then assessed for alterations in DNA methylation. Differential DNA methylation regions (DMRs) were identified in the sperm of fathers with autistic children in comparison to those without ASD children. An MeDIP-seq procedure was used to identify DMRs. The genomic features and genes associated with the DMRs were identified. The potential sperm DMR biomarker was validated with a blinded test set of individuals. Observations demonstrate a significant set of DMRs in sperm can potentially act as a biomarker for paternal offspring autism susceptibility.

overall_design: Paternal males with children affected by autism (case) or without (control) were recruited and paternal sperm samples were collected at the Andrology Laboratory of IVIRMA Clinic in Valencia, Spain. The sperm sample was collected upon enrollment. Thirty-six patients were enrolled, which included thirteen in the control group, thirteen in the autism case group, and ten for the blinded test group. The DNA was extracted from the sperm then fragmented for a methylated DNA immunoprecipitation (MeDIP) analysis in order to identify differential DNA methylated regions (DMRs) between the case and control samples.

first: Nicolás

last: Garrido

first: Fabio

last: Cruz

first: Rocio

last: Egea

first: Carlos

last: Simon

first: Ingrid

last: Sadler-Riggleman

first: Daniel

last: Beck

first: Eric

last: Nilsson

first: Millissia

middle: B

last: Maamar


title: Expression data from reconstructed human epidermis

city: Isnes Gembloux

first: Roland

last: Hubaux

state: Namur

address: rue Jean Sonet 10

department: R&D

institute: StratiCELL

summary:
Gene expression profiles of reconstructed human epidermis following exposure to interleukins 4, 13, and 25 for 48 hours
We used microarrays to study the changes in gene expression of reconstructed human epidermis after 48 hours of exposure to interleukins 4, 13, and 25

overall_design: Reconstructed human epidermal tissue samples at 14 days of culture at the air-liquid interface were treated with Th2 interleukins 4, 13, and 25, during 48 hours. At the end of the experiments, the total RNA of each sample were extracted using the Qiagen RNeasy kit and processed for hybridization on GeneChip Human Gene 2.0 ST arrays (Affymetrix, 902112)

first: Michel

last: Salmon


title: miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling (genomic Variation dataset)

city: Ann Arbor

first: Analisa

last: DiFeo

state: MI

address: 1600 Huron Parkway

institute: The University of Michigan

summary:
Genomic instability predisposes cells to malignant transformation, however the molecular mechanisms that allow for the propagation of cells with a high-degree of genomic instability remains unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells- the precursor cell type for the majority of high-grade serous ovarian cancers- through the inhibition of RB1 and simultaneously drives a cell protective inhibition of the stimulator-of-interferon-genes (STING) in order to maintain a microenvironment conducive to the propagation of cells with a high-degree of genomic instability. We found that miR-181a inhibition of RB1 leads to profound nuclear defects, genomic instability, and nuclear rupture resulting in a persistence of genomic material in the cytoplasm. While normally, this persistence of genomic material in the cytoplasm induces interferon response through STING to drive cell death, miR-181a directly downregulates STING and prevents apoptosis. The most common mechanism by which oncogenic miRNAs promote tumorigenesis is through the direct inhibition of tumor suppressor genes, however our studies highlight a new mechanism of oncomiR transformation through the combination of tumor suppressor gene inhibition and abrogation of immune surveillance that initiates and propagates tumor cell survival. Importantly, we found that miR-181a induction in ovarian patient tumors is tightly associated with decreased IFNg response and downregulation of lymphocyte infiltration amd leukocyte fraction. To date, DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation thus, our findings are the first to identify a genetic factor, miR-181a, that can downregulate STING expression, suppress activation of the immunosurveillance machinery, and impair signaling in cancer cells creating a survival advantage. Our studies support the notion that the induction of STING-mediated signaling in cancer cells could lead directly to cancer cell death however these effects are abrogated by miR-181a. Given the recent interest in the development of STING agonists as a strategy to harness the immune system to treat cancer, this study introduces novel patient selective biomarker as well as potent therapeutic target for development of the most effective combination treatments.
Infinium CytoSNP-850K  v1.2 BeadChip (Illumina)  was performed to assess genomic variation

overall_design: 4 samples were processed in duplicate


title: Genomic alterations during the in situ to invasive ductal breast carcinoma transition shaped by the immune system

first: Kornelia

last: Polyak

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

overall_design: Bulk RNA-Seq: 6 patient samples following macrodissection of FFPE slides

first: Anne

last: Trinh

middle: R

last: Del Alcazar

first: Sachet

last: Shukla

first: Koei

last: Chin

first: Jennifer

last: Eng

first: Young

last: Chen

first: Bojana

last: Jovanovic

first: Marcelo

last: Aldaz

first: So

middle: Y

last: Park

first: Joon

last: Jong

first: Catherine

last: Wu

first: Joey

last: Gray


title: F-box protein CFK1 interacts with and destabilizes de novo DNA methyltransfease in Arabidopsis

city: St Louis

first: Xuehua

last: Zhong

state: MO

address: One Brookings Drive

institute: Washington University in St. Louis

summary: This study investigated the regulation of  de novo DNA methyltransfease DRM2 by CFK1 to control DNA methylation homeostasis.

overall_design: Whole-genome bisulfite sequencing of drm2, and CFK1 overexpression plants to determine genome wide CHH hypo DMRs.

first: Jiani

first: Jie

last: Liu


title: Transcription factor POU3F2 regulates TRIM8 expression contributing to cellular functions implicated in schizophrenia

city: SYRACUSE

first: Mira

last: Krendel

address: 505 Irving Ave

institute: Upstate Medical University

summary: Schizophrenia (SCZ) is a neuropsychiatric disorder with aberrant expression of multiple genes. However, identifying its exact causal genes remains a considerable challenge. The brain-specific transcription factor POU3F2 (POU domain, class 3, transcription factor 2) has been recognized as a risk factor for SCZ, but our understanding of its target genes and pathogenic mechanisms are still limited. Here we report that 42 SCZ-related genes are regulated by POU3F2 in knockdown and RNA sequencing experiments of human neural progenitor cells (NPCs). Among those SCZ-related genes, TRIM8 (Tripartite motif containing 8) is located in SCZ-associated genetic locus and aberrantly expressed in patients with SCZ. Luciferase reporter and electrophoretic mobility shift assays (EMSA) showed that POU3F2 induces TRIM8 expression by binding to the SCZ-associated SNP (single nucleotide polymorphism) rs5011218, which impacts POU3F2 binding efficiency at the promoter region of TRIM8. We investigated the cellular functions of POU3F2 and TRIM8 as they co-regulate several pathways related to neural development and synaptic function. Knocking down either POU3F2 or TRIM8 promoted the proliferation of NPCs, inhibited their neuronal differentiation, and impaired the excitatory synaptic transmission of NPC-derived neurons. These results indicate that POU3F2 regulates TRIM8 expression through the SCZ-associated SNP rs5011218, and both genes may be involved in the etiology of SCZ by regulating neural development and synaptic function.

overall_design: A total of 12 samples were analyzed (three POU3F2 knockdown and three POU3F2 control; three TRIM8 knockdown and three TRIM8 control)

first: Chunyu

first: Chunling

last: Zhang

first: Chaodong

last: Ding

first: Shishi

last: Min


title: RNA Sequencing - Zebrafish FASD Model

first: Olivia

last: Weeks

address: 77 Ave Louis Pasteur, New Research Building #458

institute: Brigham and Women's Hospital

summary: Purpose: The transcriptional alterations underlying physological changes in fetal alcohol spectrum disorder are largely unidentified. Here we perform RNA sequencing on control and embryonic alcohol exposed (EAE/FAE) zebrafish at larval (13 dpf) and adult stages in order to identify significant transcriptional alterations under normal and HFHC diet conditions.

overall_design: All fish were treated with 0% or 1% EtOH from 12 hpf - 5 dpf and removed from EtOH at 5 dpf. For larval experiments, 3 biological samples, each containing a group of pooled whole embryos, were included in the study. For adult experiments, 5 biological samples/individual livers were included per condition. Each sample represents an individual adult liver after 8 weeks of normal or HFHC diet challenge.

middle: G

first: Wolfram

last: Goessling

first: Isaac

last: Oderberg

first: Yariv

last: Houvras


title: Epigenetic gene-expression links heart failure to memory impairment

city: Goettingen

first: Md Rezaul

last: Islam

address: von siebold str. 3a

institute: DZNE

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Functional microRNA screening for dietary vitamin E regulation of abdominal fat deposition in broilers

city: Longyan

first: Yanfa

last: Sun

state: Fujian

address: Dongxiao North Road No. 1, Xinluo District

department: College of Life Science

institute: Longyan University

summary: Functional microRNA (miRNA) screening for abdominal fat tissue with different dietary vitamin E (VE) levels was performed  in broilers.

overall_design: A total of 240 1-d-old healthy female chicks were randomly allocated into five dietary treatments with 0, 20, 50, 75 and 100 IU DL-α-tocopherol acetate. The sequencing of miRNAs of abdominal fat tissues was performed between control and 50 IU VE treatment.

first: Min


title: Loss of Laminin α3 induces cell invasion and macrophage infiltration in  cutaneous squamous cell carcinoma

city: London

first: Matthew

middle: Peter

last: Caley

state: london

address: Centre for Cutaneous Research, BLizard Building, Newark Street

country: United Kingdom

institute: QMUL

summary: Cutaneous squamous cell carcinoma (cSCC) is a common cancer which invades into dermis through the basement membrane.  Laminins are basement membrane proteins which are copiously secreted by SCC keratinocytes.   Here we provide evidence using in vitro and in vivo 3D models as well as human tumors that the loss of the laminin a3 chain observed in poorly differentiated cSCC increases tumor invasion with an amoeboid phenotype.  There is an increase in recruitment and differentiation of TAMs around invading tumor with a shift towards M2 TAMs.  These data provide evidence that loss of the laminin a3 chain in cSCC has an effect on both the epithelial and immune components of cSCC resulting in an aggressive tumor microenvironment.

overall_design: RNASeq Analysis of SCCIC1 cell line with shRNA mediated knockdown of LAMA3, LAMB3, LMC2 and SHc control


title: SO018 muscle gene expression

city: clermont ferrand

first: frederic

last: capel

address: 28 place henri dunant

department: alimentation humaine

institute: INRA

summary: identification of the effect of transgenerationnal supplementation with omega 3 fatty acids on the resistance to a diet induced obesity challenge

overall_design: 3 groups of mice were included n=8 per group, a control low fat diet group, a high fat-control lineage group and a high fat-omega 3 lineage group

first: alexandre

last: pinel

first: Claire

last: Naylies

first: Franck

last: Soubès

first: Yannick

last: Lippi


title: Inhaled multi-walled carbon nanotubes-induced gene expression profile in rat lung (NM-403)

city: Vandoeuvre les Nancy

first: Carole

last: Seidel

address: Rue du Morvan

institute: INRS

summary: In this work we study the pulmonary toxicological properties of carbon nanotubes using molecular toxicological approache. For this, we exposed Sprague Dawley rats by nose-only inhalation 6 hours/day, 5 days/week for 4 weeks.  Lung samples have been collected up to 180 days after the end of exposure and transcriptomics analysis were performed.

overall_design: 13 week old female Sprague Dawley rats were exposed by nose-only inhalation  6 hours/day, 5 days/week for 4 weeks to two doses of carbone nanotubes (NM-403). Carbon nanotube-induced gene expression profile in rat lung was performed 3, 30, 90 and 180 days post exposure (4 time points). Each time point is compsed of 6 control,  6 exposed rats to a low dose and 6 exposed rats to a high dose. A total of 72 samples (microarrays) was used.

first: Laëtitia

last: Chézeau

first: Sarah

last: Valentino

first: Sylvie

last: Sébillaud

first: Mylène

last: Lorcin

first: Christian

last: Darne

first: Sébastien

last: Bau

first: Stéphane

last: Grossmann

last: Viton

first: Hervé

last: Nunge

first: Laurine

last: Douteau

last: Michaux

first: Frédéric

last: Cosnier

first: Laurent

last: Gaté


title: Cadherin 11 promotes immunosuppression and extracellular matrix deposition to promote growth of pancreatic tumors and resistance to gemcitabine in mice

city: Washington

first: Sonia

last: de Assis

state: DC

address: 3970 Reservoir Rd. NW, New Research Building E408

department: Oncology

institute: Georgetown University

summary:
Background & Aims: Pancreatic ductal adenocarcinomas (PDAC) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule cadherin 11 (CDH11), which has been associated with other fibrotic disorders and is expressed by activated fibroblasts.
Methods: We compared levels of CDH11 mRNA in human pancreatitis and pancreatic cancer tissues and cells, compared with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11+/+ and Cdh11–/– mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored.
Results: Levels of CDH11 mRNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11+/– and KPC/Cdh11–/– mice survived significantly longer than KPC/Cdh11+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/– and KPC/Cdh11–/– mice, whose lesions also contained fewer FOXP3+ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11+/+ mice, tumors of KPC/Cdh1+/– mice had increased markers of antigen processing and presentation; more lymphocytes and cytokines associated with lymphocyte infiltration; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival only of KPC/Cdh11+/– and KPC/Cdh11–/– mice or reduced subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice.
Conclusions: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

overall_design: Transcriptome profiling of KPC/CDH11+/+ and KPC/CDH11 +/- mice with and without gemcitabine treatment; pancreatic cancer tissue from 3 mice per group was analyzed

first: Stephen

last: Byers

first: Ivana

last: Peran

first: Matthew

middle: D

last: McCoy


title: Post-transcriptional gene regulation by microRNA-194 promotes neuroendocrine transdifferentiation in prostate cancer [ChIP-Seq]

status: Public on Nov 30 2020

city: Bedford Park

first: Luke

last: Selth

state: SA

address: Flinders Drive

department: College of Medicine and Public Health

institute: Flinders University

summary: Potent therapeutic inhibition of the androgen receptor (AR) in prostate adenocarcinoma can lead to the emergence of neuroendocrine prostate cancer (NEPC), a phenomenon associated with enhanced cell plasticity. Here, we show that microRNA-194 (miR-194) is a regulator of epithelial-neuroendocrine transdifferentiation. In clinical prostate cancer samples, miR-194 expression and activity were elevated in NEPC and inversely correlated with AR signalling. Over-expression of miR-194 facilitated the emergence of neuroendocrine features in prostate cancer cells, a process mediated by its ability to directly target a suite of genes involved in cell plasticity. One such target gene was FOXA1, which encodes a transcription factor with a vital role in maintaining the prostate epithelial lineage. Importantly, a miR-194 inhibitor blocked epithelial-neuroendocrine transdifferentiation and inhibited the growth of cell lines and patient-derived organoids possessing neuroendocrine features. Overall, our study reveals a post-transcriptional mechanism regulating the plasticity of prostate cancer cells and provides a rationale for targeting miR-194 in NEPC.

overall_design: ChIP-seq for FOXA1 in LNCaP cells transfected with a miR-194 mimic or a negative control

first: Rayzel

last: Fernandes


title: Galectin-9 bridges human B cells to vascular endothelium while programming regulatory pathways.

city: Miami

first: Charles

last: Dimitroff

state: Florida

address: AHC2, Rm 678

department: Translational Medicine

institute: Herbert Wertheim College of Medicine, Florida International University

summary: Humoral immunity is reliant on efficient recruitment of circulating naive B cells from blood into peripheral lymph nodes (LN) and timely transition of naive B cells to high affinity antibody (Ab)-producing cells.  Current understanding of factor(s) coordinating B cell adhesion, activation and differentiation within LN, however, is incomplete.  Prior studies on naïve B cells reveal remarkably strong binding to putative immunoregulator, galectin (Gal)-9, that attenuates BCR activation and signaling, implicating Gal-9 as a negative regulator in B cell biology.  Here, we investigated Gal-9 localization in human tonsils and LNs and unearthed conspicuously high expression of Gal-9 on high endothelial and post-capillary venules.  Adhesion analyses showed that Gal-9 can bridge human circulating and naïve B cells to vascular endothelial cells (EC), while decelerating transendothelial migration.  Moreover, Gal-9 interactions with naïve B cells induced global transcription of gene families related to regulation of cell signaling and membrane/cytoskeletal dynamics.  Signaling lymphocytic activation molecule F7 (SLAMF7) was among key immunoregulators elevated by Gal-9-binding, while SLAMF7’s cytosolic adapter EAT-2, which is required for cell activation, was eliminated.  Gal-9 also activated phosphorylation of pro-survival factor, ERK. Together, these data suggest that Gal-9 promotes B cell – EC interactions while delivering anergic signals to control B cell reactivity.

overall_design: In this report, we analyzed the effects of galectin-9 (Gal-9) on (2) donors A and B.  The folllowing groups were analyzed by RNA isolation, cDNA synthesis and RNAseq: 1.) A2 and B2 - Buffer alone; 2.) A3 and B3 - Gal-9; 3.) A4 and B4 - Gal-9 + Lactose; and 4.) A5 and B5 - Lactose alone.

first: Asmi

last: Chakraborty

first: Caleb

last: Staudinger

first: Sandra

middle: L

last: King


title: Identification of stable markers of  EGFR inhibitor resistance in breast cancer

city: West Lafayette

first: Michael

last: Wendt

state: IN

address: 201 S. University St.

department: MCMP

institute: Purdue University

summary: EGFR expression in primary mammary tumors  predicts for decrease patient surival, but targeted EGFR inhibitors have failed to improve patient outcomes. To identifiy mediators of resistance to EGFR inhibition in breast cancer, normal murine mammary gland (NMuMG) cells specifcally transformed by overexpression of EGFR (NME)  were cultured under 3D conditions in the presence of the EGFR inhibitor AG1478.  Using this approach we were able to isolate a cell conlony that demonstrated sponaneous resistance to inhibition of EGFR.  These AG1478 resistant (AGR) cells were expanded in vitro and analysed by microarray.

overall_design: Each of the three groups were analyzed in triplicate, three control NME samples and three AGR samples.

middle: K


title: Inhaled multi-walled carbon nanotubes-induced gene expression profile in rat lung (NM-401)

first: Carole

last: Seidel

overall_design: 13 week old female Sprague Dawley rats were exposed by nose-only inhalation  6 hours/day, 5 days/week for 4 weeks to two doses of carbone nanotubes (NM-401). Carbon nanotube-induced gene expression profile in rat lung was performed 3, 30, 90 and 180 days post exposure (4 time points). Each time point is compsed of 6 control,  6 exposed rats to a low dose and 6 exposed rats to a high dose. A total of 72 samples (microarrays) was used.

first: Laëtitia

last: Chézeau

first: Sarah

last: Valentino

first: Sylvie

last: Sébillaud

first: Mylène

last: Lorcin

first: Christian

last: Darne

first: Sébastien

last: Bau

last: Grossmann

last: Viton


title: Inhibitory signaling sustains a distinct early memory CD8+ T cell precursor that is resistant to DNA damage

city: Philadelphia

first: E

middle: JOHN

last: WHERRY

address: 421 Curie Blvd,354 BRB II/III

department: Systems Pharmacology       and Translational Therapeutics,Institute for Immunology

institute: University of Pennsylvania

summary:
The developmental origins of memory T cells remain controversial. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8+ T cells that possessed unique characteristics including expression of CD62L, TCF-1 and Eomes; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1+ pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8+ T cells, coupled with elevated expression of multiple inhibitory receptors including PD-1, LAG-3, CTLA-4, CD5, and CD160.  Indeed, genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62Lhi TCF-1+ subset and subsequent CD8+ T cell memory. Although central memory phenotype CD8+ T cells (TCM) were formed in the absence of these cells, subsequent memory CD8+ T cell recall responses were compromised. Together, these results suggest a parsimonious model wherein there is more than a single developmental origin of long-term memory CD8+ T cells, and point to novel links between genome integrity, regulation of signal intensity during priming, inhibitory receptors and generation of long-term CD8+ T cell memory.
To gain deeper insight into the biology of this CD62Lhi subset, we performed RNAseq on purified CD62Lhi, IL-7Rαhi and KLRG1hi subsets on day 8 post LCMV Arm infection and included naive P14 CD8+ T cells from uninfected mice as a control.

overall_design: CD62Lhi, IL-7Rαhi and KLRG1hi P14 CD8+ T cell subsets were sorted from d8 LCMV Arm infected mice on day 8 pi and naive P14 CD8+ T cells were sorted from uninfected P14 mice. Three biological replicates were analyzed for each of the defined CD8+ T cell subsets.

first: Jonathan

last: Johnnidis

first: Yuki

last: Muroyama

last: Wherry


title: The effect of IFNAR1 blockade on Tfh-cell production of IL-21 and IFN-γ loci

city: Newark

first: Jason

last: Weinstein

state: NJ

address: New Jersey Medical School Cancer Center 205 South Orange Ave

department: Center for Immunity and Inflmmation

institute: Rutgers New Jersey Medical School

summary: To determine if the effect of IFNAR1 blockade on Tfh-cell production of IL-21 and IFN-γ was a consequence of altered chromatin accessibility, we used ATAC-seq to characterize their gene loci.

overall_design: Sorted Tfh cells (CD4+CD44+GITR-PSGL-1loCXCR5hiPD-1hi) from 5-month old B6/Sle1.Yaa mice treated twice a week for 4 weeks with either anti-IFNA1 or anti-rat IgG

first: Alex

last: Lemenze


title: Target identification of an antimalarial oxaborole identifies AN13762 as an alternative chemotype for targeting CPSF3 in apicomplexan parasites.

city: La Tronche

first: Alexandre

last: Bougdour

state: Isère

address: Domaine de la Merci, Campus santé

department: Prevention and Therapy of Chronic diseases

institute: Centre de Recherche Inserm U1209 / CNRS UMR5309 / UGA

summary: Purpose: To shed light on the parasiticidal mechanisms of AN13762, we have adapted an original workflow which combines a forward genetic approach based on transcriptome sequencing, computational mutation discovery and CRISPR/Cas9 genome editing in Toxoplasma gondii. Drug-resistant parasites were generated by chemical random mutagenesis. Multiple independent resistant lines were isolated. Single nucleotide variations (SNVs) were identified based on NGS transcriptomic analysis. By focusing on mutations present in coding sequences, we identified a single gene, TgCPSF3, that harbored SNVs leading to amino acid substitutions in each of the 7 drug-resistant lines that were not present in the parental strain. Finally, using CRISPR/Cas9 genome editing we confirmed that the mutations identified confer resistance against AN13762.

sample_organism:
Toxoplasma gondii
Homo sapiens

platform_organism:
Toxoplasma gondii
Homo sapiens

overall_design: Methods: RNA profiles were generated from HFF cells infected by RH wild-type or AN13762-resistant T. gondii strains. Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). The RNA quality was assessed using TapeStation System (Agilent Technologies, Palo Alto, California, USA).  Illumina TruSEQ RNA library prep of selected polyA RNAs and sequencing reagents were used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2 × 125 bp) on the Illumina Hiseq platform and generated at least 40 million reads for each sample.

first: Valeria

last: Bellini

first: Christopher

last: Swale

first: Marie-Pierre

last: Brenier-Pinchart

first: Tiffany

last: Pezier

first: Sonia

last: Georgeault

first: Fabrice

last: Laurent

first: Mohamed-Ali

last: Hakimi


title: RNA sequencing analysis of the CAL-27 cell response to over-expressed ZNF750 gene

city: Liaocheng

first: Haiying

last: Chen

state: Shandong

address: Dongchang West Road 67#

institute: Liaocheng People's Hospital

summary: Background: Zinc-finger protein 750 (ZNF750) is a potential tumor suppressor in oral squamous cell carcinoma (OSCC). However, the molecular mechanisms underlying its anti-tumor effect remain elusive in OSCC. We report the application of RNA sequencing to identify differentially expressed genes (DEGs) between vector groups and ZNF750 groups (over-expressed ZNF750 in CAL-27 cell), and to elucidate the genes and pathways involved in tumor suppression following the ZNF750 over-expression in OSCC cell line CAL-27 cell. Methods: The RNA sequence libraries were constructed, and the data were analyzed to identify DEGs between vector groups and ZNF750 groups. QPCR and western-blot was used to validate differential expression of candidate genes with cell cycle regulation. The cell cycle distribution was analyzed by BrdU staining. Results: By RNA sequencing profiling, 7,131 genes were differentially expressed in ZNF750 groups. Among the DEGs, 3,285 genes were upregulated, 3,846 genes were downregulated and 4,507 genes were identified in three main categories (cellular_component, biological process and molecular function) based on the gene ontology (GO) classification. The Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis defined the DEGs could be categorized into 280 pathways and identified the top two most significant pathways involved in spliceosome and cell cycle. Functional categorization and enrichment analysis revealed that most of DEGs involved in binding and catalytic activity, and the cell cycle associated genes was significantly enriched in response to ZNF750 over-expression. ZNF750 induced cell cycle arrest in G0/G1 phase of the cell cycle. Conclusion: Data from this study revealed that the cell cycle pathway was a key factor involved in the anti-tumor effect of ZNF750 in CAL-27 cells.

overall_design: RNA sequence profiles of CAL-27 cell transduced with or without ZNF750 lentivirus were generated by deep sequencing, in duplicate, using Illumina Hiseq 2500 platform.


title: Inhibition of G-protein signaling in cardiac dysfunction of Intellectual Developmental Disorder with Cardiac Arrhythmia (IDDCA) syndrome.

city: Lausanne

first: Sylvain

last: Pradervand

address: Genopode

department: CIG

country: Switzerland

institute: UNI Lausanne

summary: Background: Pathogenic variants of GNB5 encoding the 5 subunit of the guanine nucleotide-binding protein cause IDDCA syndrome, an autosomal recessive neurodevelopmental disorder associated with cognitive disability and cardiac arrhythmia, particularly severe bradycardia. Methods: We used echocardiography and telemetric ECG recordings to investigate consequences of Gnb5 loss in mouse. Results: We delineated a key role of Gnb5 in heart sinus conduction and showed that Gnb5-inhibitory signaling is essential for parasympathetic control of heart rate and maintenance of the sympatho-vagal balance. Gnb5-/- mice were smaller and had a smaller heart than Gnb5+/+ and Gnb5+/-, but exhibited better cardiac function. Lower autonomic nervous system modulation through diminished parasympathetic control and greater sympathetic regulation, resulted in a higher baseline heart rate in Gnb5-/- mice. In contrast, Gnb5-/- mice exhibited profound bradycardia upon treatment with Carbachol, while sympathetic modulation of the cardiac stimulation was not altered. Concordantly, transcriptome study pinpointed altered expression of genes involved in cardiac muscle contractility in atria and ventricles of knocked-out mice. Homozygous Gnb5 loss resulted in significantly higher frequencies of sinus arrhythmias. Moreover, we described thirteen affected individuals, increasing the IDDCA cohort to 44 patients. Conclusions: Our data demonstrate that loss of negative regulation of the inhibitory G-protein signaling causes heart rate perturbations in Gnb5-/- mice, an effect mainly driven by impaired parasympathetic activity. We anticipate that unraveling the mechanism of Gnb5-signaling in the autonomic control of the heart will pave the way for future drug screening.

overall_design: Six biological replicates of knock-out, heterozygous and control mouse atria and ventricles; six biological replicates of knock-out and control mouse hippocampi, cerebellum and cerebral cortex

first: Pasquelena

last: De Nittis

last: Reymond


title: Temporal transcriptomic analysis of Tfh cells in lupus

summary: Spleens were harvested from B6.Sle1.Yaa at 2, 4, and 7 months of age or from LCMV infected mice 8 days post infection. RNA-seq was employed to assess transcriptional changes in these cells throughout disease progression.

overall_design: Sorted Tfh cells (CD4+CD44+GITR-PSGL-1loCXCR5hiPD-1hi) from 2, 4- and 7-month-old mice compared to naive CD4 T cells from B6.Sle1.Yaa mice


title: NELF-A controls Drosophila healthspan by regulating heat-shock protein-mediated cellular protection and heterochromatin maintenance [RNA-seq]

city: Singapore

first: Chin Tong

last: Ong

address: 1 Research Link, National University of Singapore

country: Singapore

institute: Temasek Life Sciences Laboratory

summary: Comparison of differentially expressed genes in Day 50 adult heterozygous NELF-A*/+ mutant (BX) and control sibling (Sib) fly heads (2 replicates).

overall_design: Read quality check was performed using FASTQC and adaptors were trimmed using Trim Galore. Reads were aligned to Drosophila reference genome (BDGP6.22.98) using STAR (v2.5). HTSeq-count was used to identify read counts per gene. Differentially expressed genes (DEGs) were determined using DESeq2 (v1.22.1) on Galaxy platform. The data was visualised by Heatmap2 (v3.0.1). Gene Ontology analysis was performed using DAVID Functional Annotation Bioinformatics (v6.8).

first: Ong

last: Chin-Tong

first: Ngian

last: Zhen-Kai

first: Lin

last: Wei-Qi


title: NELF-A controls Drosophila healthspan by regulating heat-shock protein-mediated cellular protection and heterochromatin maintenance

summary: Epigenetic alteration is a hallmark of aging, but it remains unclear if perturbation of transcription machineries may affect longevity. NELF-mediated pausing of RNA polymerase II (RNAPII) constitutes an important step in transcription regulation. We found that halving NELF-A level in heterozygous mutants or via neuronal-specific depletion of NELF-A improves locomotor activity, stress resistance and lifespan of Drosophila significantly. Mechanistically, lower NELF-A releases paused RNAPII to facilitate productive transcription of the heat-shock protein (Hsp) genes. Elevated HSPs attenuate the level of protein aggregation, reactive oxidative species, DNA damage and systemic inflammation in the brains of NELF-A depleted flies. This protection is conserved in human cells where NELF-A depletion confers improved resistance against oxidative stress. Reduced oxidative DNA damage allows the maintenance of H3K9me2-enriched heterochromatin, which represses the activation of specific retrotransposons during aging. Therefore, by regulating transcription of Hsp genes in the brains, NELF-A controls multiple aspects of aging in Drosophila.

overall_design: Refer to individual Series


title: Metabolic programming drives proximal tubule differentiation and kidney dysfunction

city: Pleasanton

first: Juan

middle: Pablo

last: Romero

state: CA

address: 6230 Stoneridge Mall Rd

department: Computational Biology

institute: 10x Genomics

summary: Renal disease development is poorly understood, mostly because the kidney is made up of more than 20 different cell types, each of them with specific function and complex interaction. Here, we used single cell RNA sequencing to resolve cell-type specific and cell fraction changes

overall_design: Single-cell RNA-seq of engineered human kidney organoids

first: Jihwan

last: Park

first: Poonam

last: Dhillon

first: Carmen

last: Hurtado

first: Lingzhi

last: Li

first: Shizheng

last: Huang

first: Hyun

last: Kang

first: Rojesh

last: Shrestra

first: Shatakshee

last: Chatterjee

first: Patricia

last: Prado

first: SeungYub

last: Han

first: Hongbo

last: Liu

first: Sheng

last: Xin

first: Kirill

last: Bartmanov

middle: P

first: Felipe

last: Prosper

first: Mingyao

first: Liming

last: Pei

first: Junhyong

last: Kim

first: Nuria

last: Montserrat

first: Katalin

last: Susztak


title: RNA-seq data of total RNA treated with different E. coli AlkB variants.

city: Chicago

first: Tao

last: Pan

state: Illinois

address: 929 E. 57th Street

department: Biochemistry and Molecular Biology

institute: University of Chicago

summary: We compared several E. coli AlkB variants for their applications in facilitating sequencing of tRNA species in total RNA. These proteins are wild type AlkB, mutant D135S and mutant D135T.

overall_design: Total RNA isolated from HEK 293T cells were treated with different combinations of enymes: (1) wild type alone, (2) wild type + D135S, (3) wild type + D135T, (4) no enzyme. The treated samples were then subjected to library construction and illumina sequencing.

first: Yuru

last: Wang

first: Christopher

last: Katanski

last: Watkins

first: Jessica

middle: N

first: Qing

last: Dai

first: Zhuoxun

last: Jiang


title: IL-10 inhibition of miR-155 in inflammation

city: Dublin 2

first: Claire

middle: E

last: McCoy

address: 123 St Stephen's Green

country: Ireland

institute: Royal College of Surgeons in Ireland

summary:
Inflammatory disorders including arthritis, inflammatory bowel disease, psoriasis and multiple sclerosis are characterised by the excessive production of pro-inflammatory cytokines resulting in inflammation, tissue damage and chronic pain.
In a healthy immune system, the host has developed mechanisms to protect itself from excessive inflammation. One such mechanism is mediated by the anti-inflammatory cytokine IL-10, which primarily acts to limit the production of pro-inflammatory cytokines and antigen presenting capabilities of cells in the innate immune system. Since the discovery of IL-10, much emphasis has been placed on extrapolating its exact mechanism of inhibition with the aim of potentiating its use therapeutically. Significantly, the systemic administration of IL-10 has had success in many animal models of inflammation as well as clinical success in human disorders such as psoriasis and Crohn’s disease. Despite this, major gaps in our knowledge remain which have weakened the expected therapeutic effect for this anti-inflammatory cytokine.
Our early studies discovered that in response to innate immune stimulation, IL-10 can potently inhibit miR-155 (McCoy et al., 2010), a miRNA characterised as a promoter of inflammation and found over-expressed in autoimmune pathologies. This finding suggests that the IL-10/miR-155 axis is a novel mechanism utilised by IL-10 to administer its function. In this study, an Affymetrix array was employed to characterise genes regulated by the IL-10/miR-155 axis in bone-marrow derived macrophages.
This project proposal aims to characterise the mechanism of IL-10 inhibition on miR-155, as well as understand the functional outcome and significance of this effect by characterising genes regulated by the IL-10/miR-155 axis.

overall_design: Wild-type and miR-155 knockout bone-marrow derived macrophages were stimulated with LPS and LPS+IL-10 in biological duplicate. N=8 total; WT_BMDM LPS (n=2), WT_BMDM LPS_IL10 (n=2), miR155KO_BMDM LPS (n=2), miR55KO_BMDM LPS_IL10 (n=2)

first: Linden

last: Gearing


title: Histone H3.3G34-mutant interneuron progenitors co-opt PDGFRA for gliomagenesis

city: Montreal

first: Carol

last: Chen

state: QC

address: 1001 Decarie Boulevard, Rm E01.4380

department: Human Genetics

country: Canada

institute: Research Institute of McGill University Hospital Centre

summary: Histone H3.3 glycine 34 to arginine/valine (H3.3G34R/V) mutations occur in deadly hemispheric high-grade gliomas. These tumors show exquisite regional and temporal specificity, suggesting a developmental context permissive to the effects of G34R/V mutations. Here we present the molecular landscape of G34R/V gliomas (n = 83) and show that ~50% bear activating mutations in PDGFRA, with strong selection pressure for PDGFRAMUT clones at recurrence. We show that G34R/V tumors arise in interneuron progenitors of the foetal ventral forebrain, expressing GSX2 and the DLX family of homeobox transcription factors, where terminal neuronal differentiation is impaired through aberrant G34R/V-mediated H3K27me3. Frequent co-occurrence of G34R/V & PDGFRAMUT is facilitated in this interneuron lineage-of-origin as PDGFRA forms an aberrant chromatin loop with the adjacent GSX2, hijacking its active chromatin conformation. At the single-cell level, G34R/V tumors entirely lack oligodendroglial transcriptional programs prominent in other glioma entities, and instead harbour dual neuronal and astroglial compartments. CRISPR-removal of H3.3G34R/V does not impact tumorigenicity suggesting this mutation becomes dispensable, while PDGFRAMUT are potently oncogenic regardless of G34 mutation. Collectively, our results suggest that G34R/V gliomas arise in foetal interneuron progenitors unable to terminally differentiate, enabling co-option of PDGFRA through inappropriate expression and activating mutations to promote gliogenesis and oncogenicity. Reliance on PDGFRA for oncogenesis may be of therapeutic opportunity in G34R/V glioma.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: ChIP-seq, Hi-C and RNA-seq were performed on CRISPR-edited and parental G34R/V glioma-derived cell lines and neural progenitor cells (NPCs) derived from in-utero electroporated mice.

middle: C

first: Shriya

last: Deshmukh

first: Véronique

last: Lisi

first: Selin

last: Jessa

first: Djihad

last: Hadjadj

first: Augusto

last: Faria Andrade

first: Damien

last: Faury

first: Hiromichi

last: Suzuki

first: Manav

last: Pathania

first: A

last: Deli

first: Frank

last: Dubois

first: Eleanor

last: Woodward

first: Steven

last: Hébert

first: Marie

last: Coutelier

first: Jason

last: Karamchandani

first: Steffen

last: Albrecht

first: Sebastian

last: Brandner

first: Nicolas

last: De Jay

first: Tenzin

last: Gayden

first: Andrea

last: Bajic

first: Ashot

middle: S

last: Harutyunyan

first: Dylan

middle: M

last: Marchione

first: Leonie

last: Mikael

first: Nikoleta

last: Juretic

first: Michele

last: Zeinieh

first: Caterina

last: Russo

first: Nicola

last: Maestro

first: Angelia

middle: V

last: Bassenden

first: Peter

last: Hauser

first: Almos

last: Klekner

first: Michal

last: Zapotocky

first: Ales

last: Vicha

first: Lenka

last: Krskova

first: Katerina

last: Vanova

first: Josef

last: Zamecnik

first: David

last: Sumerauer

first: Paul

last: Ekert

last: Ziegler

first: Benjamin

last: Ellezam

first: Mathieu

last: Blanchette

first: Jordan

last: Hansford


title: NELF-A controls Drosophila healthspan by regulating heat-shock protein-mediated cellular protection and heterochromatin maintenance [ChIP-seq]

first: Chin Tong

last: Ong

summary: Comparison of differential H3K9me2 enrichment in Day 50 adult heterozygous NELF-A*/+ mutant (BX) and control sibling (Sib) fly heads (2 biological replicates).

overall_design: Read quality check was performed using FASTQC and adaptors were trimmed. Reads were mapped to Drosophila reference genome (BDGP6.22.98) using STAR (v2.5) with following parameters: (--alignEndsType EndToEnd --alignIntronMax 1). Downstream analysis was performed using Galaxy platform. Peak calling was performed using MACS2 (v2.1.1), using an effective genome size of 120000000 --no model --extsize 200 --broad --keep-dup auto --bdg. Differential peaks between NELF-A*/+ (BX) and control siblings (Sib) was performed using MACS2 bdgdiff with following parameters: -g 100 -l 200. Alignment files (bam format) were transformed into read coverage files (bigwig format) using DeepTools: bamCompare (v3.3.2), normalized by readcount, scores representing ChIP minus Input.

first: Ong

last: Chin-Tong

first: Ngian

last: Zhen-Kai

first: Lin

last: Wei-Qi


title: Molecular classification of zebrafish retinal ganglion cells links genes to behavior

city: Seattle

first: Joshua

middle: William

last: Hahn

address: 3747 W Stevens Wy NE

department: Department of Neurobiology & Biophysics

institute: University of Washington

summary: Retinal ganglion cells (RGCs) form an array of feature detectors, which transmit visual information through segregated channels to central brain regions. Characterizing RGC diversity is required to understand the logic of this functional parcellation. Using single- cell transcriptomics, we systematically classified RGCs in adult and larval zebrafish, thereby identifying marker genes for >30 stable and several transient cell types, cell type-specific markers, and their maturational changes. We used this catalog to engineer transgenic driverreporter lines, enabling experimental genetic access to specific RGC types. Strikingly, expression of a few transcription factors often predicts dendrite morphological features, in particular and axonal projections to specific tectal layers and extratectal targets. Moreover, iIn vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuningphysiological properties. Finally, chemogenetic ablation of eomesa+ RGCs, which comprise three melanopsin-expressing types with a specific projections to thalamus, pretectum and one deep tectal layerpattern, selectively impaired a visual behavior, phototaxis. Our study establishes a framework and provides a resource for systematically studying the functional architecture of the visual system.

overall_design: RGCs were labeled using transgenic Tg(isl2b:tagRFP) zebrafish that express RFP in all RGCs. Retinas from larval (5 dpf) and adult (4-6 months) fish were dissected and sorted using FACS. For the larval dataset, 200 manually dissected retinas were dissociated in one experiment and single cell profiles were collected across 3 replicates. For the adult dataset, about 20 retinas per batch were dissected and dissociated and droplet RNA sequencing was performed collecting a total of 15 replicates across 5 experiments. The cDNA libraries were sequenced on the Illumina HiSeq 2500 to a depth of ~30,000 reads per cell.

first: Yvonne

last: Kolsch

first: Anna

last: Sappington

first: Manuel

last: Stemmer

first: António

last: Fernandes

first: Thomas

middle: O

last: Helmbrecht

first: Shriya

last: Lele

first: Salwan

last: Butrus

first: Eva

last: Laurel

first: Irene

last: Arnold-Ammer

first: Karthik

last: Shekhar

last: Sanes

first: Herwig

last: Baier


title: Ustilago maydis effector Jsi1 interacts with Topless corepressor, hijacking plant JA/ET signaling

city: Vienna

first: Martin

middle: Alejandro

last: Darino

address: Dr. Bohr-Gasse 3

country: Austria

institute: Gregor Mendel Institute of Molecular Plant Biology

summary:
•	Ustilago maydis (U. maydis) is the causal agent of maize smut disease. During the colonization process, the fungus secretes effector proteins which suppress immune responses and redirect the host metabolism in favor of the pathogen. As effectors play a critical role during plant colonization, their identification and functional characterization is essential to understanding biotrophy and disease.
•	Using biochemical, molecular, and transcriptomic techniques, we performed a functional characterization of the U. maydis effector Jasmonate/Ethylene signaling inducer 1 (Jsi1).
•	Jsi1 interacts with several members of the plant co‐repressor family Topless/Topless related (TPL/TPR). Jsi1 expression in Zea mays (Z. mays) and Arabidopsis thaliana (A. thaliana) leads to transcriptional induction of the ethylene response factor (ERF) branch of the jasmonate/ethylene (JA/ET) signaling pathway. In A. thaliana, activation of the ERF‐branch leads to biotrophic susceptibility. Jsi1 likely activates the ERF‐branch via an EAR motif, which resembles EAR motifs from plant ERF transcription factors, that interacts with TPL/TPR proteins.
•	EAR motif‐containing effector candidates were identified from different fungal species including Magnaporthe oryzae, Sporisorium scitamineum, and Sporisorium reilianum. Interaction between plant TPL proteins and these effector candidates from biotrophic and hemibiotrophic fungi indicates the convergent evolution of effectors modulating the TPL/TPR co‐repressor hub.

overall_design: We analyzed two independent Arabidopsis lines expressing Jsi1 fused to mCherry (Jsi1-mCherry) under the control of the β-estradiol (XVE) inducible promoter. As control we used an Arabidosis lines expressing mCherry under the XVE promoter. We treated lines for six hours with β-estradiol and we took three independent replicates from each line. In addition, for the control line we also included three replicates without β-estradiol treatment. In total we have 12 samples.

middle: A

first: Luz

last: Soto Jimenez

first: Armin

last: Djamei