title: HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence [RNA-seq]

city: Goettingen

first: Argyris

last: Papantonis

address: Robert-Koch-Str 40

department: Department of Pathology

institute: University Medical Center Goettingen

summary: Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome-wide in two primary cell lines. We integrated ChIP-seq and Hi-C with graph theory to uncover clustering of HMGB1-marked topological domains that harbour genes required for paracrine senescence. Using sCLIP and functional tests, we show that HMGB1 is also a bona fide RNA-binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate the availability of SASP-relevant transcripts. Our findings highlight a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell-autonomous and paracrine senescence.

overall_design: Two biological replicates (IMR90 I10 and I79) were treated with NTC or HMGB1 siRNA.of proliferating or senescent IMR90 were used.

first: Natasa

last: Josipovic

first: Konstantinos

last: Sofiadis


title: HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence [Ribo-seq]

status: Public on Apr 10 2021

country: Germany

type: Expression profiling by high throughput sequencing

overall_design: Single IMR90 isolates (I90-10 and -79, passage 5; Coriell Biorepository) were continuously passaged to replicative exhaustion. Three independent replicas of proliferating or senescent IMR90 were used.

first: Audrey

last: Michel

first: James

last: Keane


title: HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence [ChIP-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Two replicates of proliferating/senescent IMR90 and one replicate of proliferating HUVEC cells were used for ChIP-seq experiment for immunoprecipitation with HMGB1 and inputs.


title: Genomic-Wide Transcriptomic Analysis of the Forebrain of postnatal Slc13a4+/- mice

city: Brisbane

first: David

last: Simmons

state: Queensland

address: The Faculty of Medicine

department: School of Biomedical Sciences

country: Australia

institute: The University of Queensland

summary: We performed bulk RNA-seq on Nas2Del +/- Het and wild type postnatal Day 20 mice on samples collected from the mouse forebrain.

overall_design: Bulk genome wide RNA-Seq analysis was performed with 6 biological replicates. Three Nas2Del+/Het mice and three wild type mice at postnatal day 20.

first: Michael

last: Piper

first: Tracey

middle: J

last: Harvey

first: Raul

last: Ayala Davila

first: Diana

last: Vidovic


title: HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence

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

overall_design: Refer to individual Series


title: Therapeutic IL-6 trans-signalling inhibition by olamkicept (sgp130Fc) in patients with active inflammatory bowel disease

city: Kiel

first: Joana

middle: Pimenta

last: Bernardes

address: Am Botanischen Garten 11

department: Systems Immunology

institute: Institute of Clinical Molecular Biology

summary: Background  A large unmet therapeutic need exists in inflammatory bowel diseases (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL-6/IL-6R blockade is limited by profound immunosuppression. Evidence has emerged, that chronic pro-inflammatory activity of IL-6 is mainly mediated by trans-signalling via a complex of IL-6 bound to soluble IL-6R engaging the gp130 receptor without the need of membrane bound IL6R. We have developed a decoy protein, sgp130Fc, which exclusively blocks IL-6 pro-inflammatory trans-signalling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression.  Methods  We present a 12-week, open label, prospective phase IIa trial (FUTURE) in 16 patients with active IBD treated with the trans-signalling inhibitor olamkicept (sgp130Fc) to assess molecular mechanisms, safety and effectiveness of IL-6 trans-signalling blockade in vivo. We performed in-depth molecular profiling at various time points before and after therapy induction to identify the mechanism of action of olamkicept.  Results  Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-TNF (infliximab) or anti-integrin (vedolizumab) therapies was identified.  Conclusion  Our data suggest that blockade of IL-6 trans-signaling holds large promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy of IBD.

overall_design: The trial was conducted over 21 months between 2016 and 2019. 600 mg of olamkicept were administered by intravenous infusion every 2 weeks for 12 weeks (i.e. 7 infusions). Clinical disease activity (including endoscopy) was assessed at baseline and weeks 2, 6 and 14. Additional visits for biosampling (blood, sigmoid mucosal biopsies) were conducted 4 h and 24 h after therapy. Whole blood and sigmoid mucosal biopsy samples (collected from the most inflamed region within 30 cm of colon sigmoideum) were collected as a longitudinal dataset from which the RNA for transcriptome analyses was isolated. The samples were gathered at baseline (0 h) and 4 h, 24 h, 2 weeks, 6 weeks and 14 weeks after the first olamkicept infusion. Whole RNA libraries were prepared using TruSeq RNA Library Prep technology and, in total, 104 blood and 105 biopsy samples were sequenced using HiSeq 4000  (both data sets were of paired data with 2x75bp).

first: Stefan

last: Schreiber

first: Konrad

last: Aden

middle: P

first: Claudio

last: Conrad

first: Florian

last: Tran

first: Hanna

last: Höper

first: Valery

last: Volk

first: Neha

last: Mishra

first: Johanna

middle: I

last: Blase

first: Susanna

last: Nikolaus

first: Johannes

last: Bethge

first: Tanja

last: Kühbacher

first: Christoph

last: Röcken

first: Minhu

last: Chen

first: Ian

last: Cottingham

first: Niclas

last: Petri

first: Birgitte

middle: B

last: Rasmussen

first: Juliane

last: Lokau

first: Lennart

last: Lenk

last: Garbers

first: Friedrich

last: Feuerhake

last: Rose-John

first: Georg

middle: H

last: Waetzig

first: Philip

last: Rosenstiel


title: scRNA-seq of DAC-treated and mock-treated HCT116.

city: Shinagawa-ku

first: Hideyuki

last: Takeshima

state: Tokyo

department: Institute for Advanced Life Sciences

country: Japan

institute: Hoshi University

summary: Epigenetic reprogramming using demethylating drugs is a promising approach for cancer therapy, but its efficacy is highly dependent on the dosing regimen.  Low-dose treatment for a prolonged period shows a high therapeutic efficacy, despite its small demethylating effect.  Here, we aimed to reveal the mechanisms of how such low-dose treatment shows high efficacy by focusing on epigenetic reprograming at the single-cell level.  Single-cell RNA-sequencing of HCT116 cells treated with decitabine (DAC) revealed that up-regulated genes were highly variable at the single-cell level.  To analyze functional consequences at the single-cell level, DAC-treated HCT116 cells were cloned.  While only partial reduction of methylation levels was observed in bulk cells, complete demethylation of specific cancer-related genes was observed, depending upon clones.  For example, p16 was completely demethylated in the H3-32 clone out of 9 clones, and this clone showed slower proliferation than other clones without demethylation.  In addition, in this clone, the fraction of cells with tetraploid became much larger, indicating that cellular senescence was induced.  These results showed that epigenetic reprogramming of specific cancer-related pathways at the single-cell level is likely to underlie the high efficacy of low-dose DNA demethylating therapy.

overall_design: scRNA-seq was conducted for two samples, DAC-treated HCT116 cells and mock-treated HCT116 cells, using the Chromium Controller (10x Genomics ) and Illumina HiSeq platform.

first: Yukie

last: Yoda

first: Satoshi

last: Yamashita

first: Toshikazu

last: Ushijima


title: A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics

status: Public on Apr 09 2021

city: New York

first: Daniel

last: Blanco Melo

state: NY

address: One Gustave L. Levy Place, Box 1124

department: Microbiology

country: USA

institute: Icahn School of Medicine at Mount Sina

summary: The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here, we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production, and the recruitment of circulating immune cells. In chips infected with pseudotyped SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection, but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential.

sample_organism:
Homo sapiens
Mesocricetus auratus

platform_organism:
Homo sapiens
Mesocricetus auratus

overall_design: Independent biological triplicates of transformed lung alveolar (A549) transduced with a vector expressing human ACE2 were pre-treated with amodiaquine (10 μM) for 1hr before SARS-CoV-2 infection (USA-WA1/2020, MOI: 0.1); 24hrs post infection samples were examined by mRNA-sequencing. Additionally, Syrian golden hamsters were treated with amodiaquine (75mg/kg) by oral gavage one day before SARS-CoV-2 infection (100 PFU), on the day of infection, and one and 2 days after infection. The animals were sacrificed and lungs harvested for mRNA-sequencing on day 3 post infection.

first: Benjamin

middle: R

last: tenOever

last: Blanco-Melo


title: The miR-26 family regulates neural differentiation-associated microRNAs and mRNAs by directly targeting REST

city: Würzburg

first: Nina

last: Was

state: Bavaria

address: Zinklesweg 10

department: Institut für medizinische Strahlenkunde und Zellforschung

institute: Universität Würzburg

summary: Our data identify the miR-26 family as a trigger of a self-amplifying system essential   for neural differentiation that acts upstream REST-controlled miRs and initiate a  neuronal gene expression program.

overall_design: Methods:Total RNAs from two independent clones of WT and tKO26b/a1/a2 cell lines and two   biological replicates and NCs (day 15) were isolated using peqGold RNA pure  (PeqLab). rRNA was removed using the Ribo-Zero rRNA Removal Kit (Illumina),  followed by cDNA library preparation using NEB Next Ultra Directional RNA Library  Prep Kit for Illumina (New England Biolabs). Sequence reads were aligned to the   mouse genome (version mm10) using TopHat (Trapnell et al., 2012). Transcripts were   quantified by Cufflinks and Cuffdiff and differential expression was determined  (Trapnell et al., 2012). Small RNA cDNA libraries were generated and deep sequenced  (Hafner et al., 2010). Reads were mapped against murine miR annotation database  (miRBase) using Burrow-Wheeler aligner. Each miR profile was normalized to relative   read frequencies (Farazi et al., 2012).

first: Mark

last: Sauer

first: Markus

last: Hafner

first: Matthias

last: Becker

first: Thomas

last: Ziegenhals

first: Xiantao

last: Wang

first: Utz

last: Fischer


title: Contextual cues from cancer cells govern CAF heterogeneity [scRNA-seq]

city: Houston

first: Chenghang

last: Zong

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: Cancer cells function as primary architects of the tumor microenvironment. Yet, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.

overall_design: Single cell RNA-seq on tCAFs from KrasLA1 mice

first: Jonathan

middle: M

last: Kurie

first: Neus

last: Bota-Rabassedas

first: Yichi

last: Niu

first: Jiayi

last: Luo


title: Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos [ES cell RIP-seq]

city: Shanghai

first: Chuan

last: Chen

address: 1239 Siping Road

country: China

institute: Tongji University

summary: N6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the embryonic stem cell (ESC) self-renewal and differentiation potency, which highly depends on its m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on LINE1 scaffold-regulated regions, including 2-cell retrotransposons, is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

overall_design: rRNA-free total RNA-seq was performed for Ythdc1 f/f (control), Ythdc1 cKO, W378A-YTHDC1 rescue, WT-YTHDC1 rescue, WT (control for Mettl3 KO), Mettl3 KO, control shRNA-expressed and Kap1 shRNA-expressed mouse ES cell lines with 2-3 replicates for each line. Nuclear RIP-seq for YTHDC1 was performed using control ES cells with 2 replicates for IP and 1 reaction for input. Cross-Linked ChIP-seq of KAP1 and H3K9me3 was perfomed for Ythdc1 f/f , Ythdc1 cKO, W378A-YTHDC1 rescue and WT-YTHDC1 rescue ES cells with 2 replicates for each IP and 1 reaction for each input. Low-input RNA-seq was performed for control and Ythdc1 KO ICM cells with 2 replicates each. Ultra-low-input native ChIP-seq of H3K9me3 was performed for control and Ythdc1 KO ICM cells with 2 replicates for each IP and 1 reaction for each input.

first: Wenqiang

last: Liu

first: Bin

last: Shen

first: Yawei

last: Gao

first: Shaorong


title: Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos [ICM RNA-seq]


title: Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos [ES cell RNA-seq]


title: Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos [ES cell ChIP-seq]


title: Pharmacological inhibition of tyrosine kinase 2 preserves interferon-λ-mediated protection

city: London

first: Andreas

last: Wack

address: 1 Midland Road

country: United Kingdom

institute: The Francis Crick Institute


title: Pharmacological inhibition of tyrosine kinase 2 preserves interferon-λ-mediated protection (airway epithelial cells)

summary: Treating inflammatory diseases with Janus kinase 1/2 (JAK1/2) inhibitors bears the risk that patients acquire viral infections due to unwanted immune suppression. Tyrosine kinase 2 (TYK2), a JAK family member, is required for type I interferon (IFN-α/β) signaling, but its role in type III IFN (IFN-λ) signaling is still under debate. We found that the selective TYK2 inhibitor BMS-986165 blocked potentially noxious type I IFN signaling without altering IFN-λ-mediated gene expression. We show that epithelial cells do not require TYK2 for IFN-λ-mediated signaling or antiviral protection. Lack of TYK2 diminished IFN-α-induced protection against lethal influenza virus infection of mice, but did not impair IFN-λ-mediated antiviral protection. Our findings suggest that selective TYK2 inhibitors likely represent a superior treatment option for type I interferonopathies than broadly acting JAK1/2 inhibitors, as selective TYK2 inhibitors may counteract inflammatory responses without abolishing the beneficial antiviral effects of IFN-λ.

overall_design: WT or TYK2 KO airway epithelial cultures were  mock treated or treated with either IFNab (3 ng/ml or 0.3 ng/ml) or IFNL (0.3ng/ml). Biological triplicates were run for every treatment group.

first: Daniel

last: Schnepf

first: Peter

last: Staeheli


title: Contextual cues from cancer cells govern CAF heterogeneity [Bulk RNA-seq]

overall_design: RNA-seq on tCAFs co-cultured with cancer cells


title: Pharmacological inhibition of tyrosine kinase 2 preserves interferon-λ-mediated protection (neutrophils)

overall_design: WT or TYK2 KO neutropihls were  mock treated or treated with either IFNab (0.3ng/ml) or IFNL (0.3/ml). Biological triplicates were run for every treatment group.


title: RNA from mouse lung conditioned medium

status: Public on Apr 11 2021

city: Matsumoto

first: Sachie

last: Hiratsuka

address: 3-1-1 Asahi

department: Medicine

institute: Shinshu University

summary:
RNA fragments were isolated from mouse lung conditioned medium and analyzed by a microarray
This analysis surveys RNAs, probably complexed with RNA-binding protein, secreted in the culture media

overall_design: RNAs were directly collected from culture medium, which was used to culture diced mouse lungs for 24 hours


title: Efficient conversion of human induced pluripotent stem cells into microglia by defined transcription factors

status: Public on Apr 08 2021

city: Taipei

first: Yu-Hui

last: Wong

address: No.155, Sec.2, Linong Street

country: Taiwan

institute: National Yang-Ming University

summary: Microglia, the immune cells of the central nervous system (CNS), play critical roles in brain physiology and pathology.  We report a novel approach that produces within ten days the differentiation of human induced pluripotent stem cells (hiPSCs) into microglia (iMG) by forced expression of both SPI1 and CEBPA.  High-level expression of the main microglial markers and the purity of the iMG cells were confirmed by RT-qPCR, immunostaining and flow cytometry analyses.  Whole transcriptome analysis demonstrated that these iMGs resemble human fetal/adult microglia but not human monocytes.  Moreover, these iMGs exhibited appropriate physiological functions, including various inflammatory responses, ADP/ATP-evoked migration and phagocytic ability.  When co-cultured with hiPSC-derived neurons (iNs), the iMGs respond and migrate toward injured neurons.  This study has established a protocol for the rapid conversion of hiPSCs into functional iMGs, which should facilitate functional studies of human microglia using different disease models and also help with drug discovery.

overall_design: Transcriptomal profile of iMGs from two hiPS cell lines in independent triplication, comparing with RNA-seq data of human adult microglia, human fetal primary microglia, CD14+ monocytes and hiPSC-derived microglia obtained using other differentiation protocols downloaded from NCBI GEO (GSE89189 and GSE110952)

first: Shih-Wei

first: Yu-Sheng

last: Hung

first: Jong-Ling

last: Fuh

first: Nien-Jung

first: Yeh-Shiu

last: Chu

first: Shu-Cian

first: Ming-Ji

last: Fann


title: Organoid phenotypic testing identifies IFN-beta as neuroprotective compound against ZIKA virus (ZIKV)

city: Vienna

first: Thomas

middle: Rainer

last: Burkard

address: Dr. Bohrgasse 7

country: Austria

institute: IMBA

summary: Administration of recombinant interferons to 3D brain organoid cultures infected with ZIKV identifies IFN-beta as compound that alleviates organoid damage and inhibits ZIKV

overall_design: Comparison of transcriptomes from ZIKV infected or MOCK organoids treated with IFN-alpha or IFN-beta or left untreated (6 conditions) at two time points

first: Veronica

last: Krenn

first: Juergen

middle: A

last: Knoblich


title: shRNA Knockdown of ILF3 in Monocyte-Derived Dendritic Cells (MDDCs)

city: Seattle

first: Aderem

last: Lab

state: WA

address: 307 Westlake Avenue North, Suite 500

institute: Seattle Children's Research Institute

summary: Antigen presenting cells such as myeloid dendritic cells (DCs) are key sentinels of the innate immune system. In response to pathogen recognition and innate immune stimulation, DCs transition from an immature, inactive state to a mature, active state that is characterized by widespread changes in host gene expression. Several key transcription factors are known to drive these gene expression changes, but the mechanisms that negatively regulate DC maturation are less well understood. Here, we have identified that the transcription factor Interleukin Enhancer Binding Factor 3 (ILF3) is a negative regulator of innate immune responses and DC maturation. Depletion of ILF3 in primary human monocyte-derived DCs (MDDCs) using shRNA knockdown led to increased expression of maturation markers and potentiated innate responses at baseline.

overall_design: DCs were derived from the peripheral blood from three deidentified human donors and transduced with lentiviral control or two individual shRNAs targeting ILF3 in the presence of Vpx. RNA expression was measured at day 5 post-transduction.

first: Rodolfo

last: Nazitto

first: Lynn

last: Amon

first: Alan

last: Aderem


title: Overexpression of NF110b and NF110bΔDZF in Monocyte-Derived Dendritic Cells

summary: We have found through previous experiments that ILF3 acts as a negative regulator of myeloid dendritic cell (DC) maturation. Mutagenesis experiments targeting various domains of both major isoforms of ILF3 (NF90b and NF110b) revealed that domain-associated with zinc finger (DZF) of NF110b was required for suppression of DC maturation. To determine which innate immune pathways may underlie the mechanism behind ILF3's suppression of DC maturation, we used lentiviral vectors expressing control (LKO), wildtpe NF110b, or  an NF110b DZF-deletion mutant in 4 individual donors. On day 5 post-transdiction, RNA was isolated and sent for total RNA sequencing. Differential gene expression between the wildtype and DZF mutant, relative to LKO controls, revealed enrichment for Cholesterol Homeostatis and Oxidative Phosphorylation pathways implicating involvement of these pathways in supression of DC maturaturation.

overall_design: Examination of differentially expressed genes by wildetype NF110b and NF110b DZF deletion mutant (NF110bΔDZF)

last: Diercks


title: Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos [ICM ChIP-seq]

first: Chuan

last: Chen

first: Wenqiang

last: Liu

first: Bin

last: Shen

first: Yawei

last: Gao

first: Shaorong


title: Optimization of MethylRAD sequencing to detect changes in DNA methylation at enhancer elements in differentiating embryonic stem cells

city: West Lafayette

first: Nadia

middle: Marie

last: Atallah

state: IN

address: 201 S. University St

department: Center for Cancer Research

institute: Purdue University

type: Methylation profiling by high throughput sequencing

summary: Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given DNA methylation across most of the mammalian genome is maintained, use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome.  Here we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed at PpGe with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation at regulatory regions including with low/intermediate CpG content, thus asserting it use as a method of choice for diagnostic purpose.

overall_design: methylRAD was used to identify methylated CCGG and CCWGG sites in undifferentiated, differentiated, and differentiated cells treated with PG

first: Allison

last: Norvil

first: Debapriya

last: Saha

last: A Lanman

first: Humaira

last: Gowher


title: Effect of type I interferons against HSV infection in cerebral organoids

address: Dr. Bohr gasse 3

institute: Institute for Molecular Biotechnology (IMBA)

summary: Administration of recombinant interferons to 3D brain organoid cultures infected with HSV reveals a strong antiviral effect of IFN-alpha against HSV

overall_design: Comparison of transcriptomes from HSV infected or MOCK organoids treated with IFN-alpha or IFN-beta or left untreated (6 conditions) at two time points

last: Burkard


title: Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior  [TRAP II]

city: Tallahassee

first: Michelle

middle: N

last: Arbeitman

state: Florida

address: 1115 W. Call Street

department: Department of Biomedical Sciences

institute: Florida State University

summary: Chromatin modifications have essential roles in directing nervous system development and behavior. Drosophila courtship is an ideal model for understanding the relationships between chromatin modifications, gene expression, sexual dimorphisms in neuroanatomy, and reproductive behaviors. These behaviors are largely innate and are under the control of sex determination hierarchy genes doublesex (dsx) and fruitless (fru) that encode sex-specific transcription factors. Transcripts from the fru P1 promoter are sex-specifically spliced, resulting in male-specific proteins, FruM, which are required for male courtship behaviors. FruM has been shown to form a complex with Bonus/TIF1, a transcriptional cofactor, and interact with chromatin modifying proteins. This indicates that sex-differential modification of the chromatin landscape contributes to creating and maintaining the potential for sexually dimorphic behavior. To examine chromatin modifications genome-wide in fru P1-expressing neurons, we have developed a method to purify chromatin in a cell-type-specific manner using a tagged histone H2B under UAS control, “Chromatag”, paired with sequential ChIP-seq. Here, we evaluate five chromatin modifications: H3K27ac, H3K4me3, H3K36me3, H3K9me3, and H3K27me3 to investigate fru P1-expressing neurons at three developmental stages in both sexes. These modifications have been well characterized for their roles in transcriptional activation or repression. We observe greater changes in chromatin modification profiles across developmental stages than between sexes. In addition, we have generated cell-type specific RNA-seq data sets, using Translating Ribosome Affinity Purification (TRAP), to examine gene expression in fru P1-expressing neurons of both sexes. Informed by these data sets, we have identified several genes with roles in neuronal function and examined their expression in fru P1-expressing neurons, revealing sexually dimorphic expression patterns. Altogether, this work offers insights into stage and sex-differences in chromatin modifications and gene expression in fru P1-expressing neurons.

overall_design: Cell-type-specific RNA-seq libraries for fru P1-expressing neurons were generated using Translating Ribosome Affinity Purification (TRAP). fru-P1 TRAP was performed in 10-12 day old adult heads and coincides with the 10-12 day adult chromatin modification data in our study. TRAP (IP) and total mRNA (input) samples were prepared from whole heads from both males and females with 4 biological replicates per sex.

first: Colleen

last: Palmateer

first: Surjyendu

last: Ray

first: Shawn

last: Moseley


title: Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior [ChIP]

overall_design: Cell-type-specific examination of 5 histone modifications and histone H3 in Drosophila fru P1-expressing and elav expressing neurons for males and females at three time-points (life stages): 48hr after puparium formation (48hAPF), 16-24hr adults (1-day adults), and 10-12 day adults. Libraries were prepared from 3-4 independent biological replicates per modification, time-point, and sex. Chromatin samples were obtained from whole pupae for 48hr APF or whole head tissue for adult stages. Sex and stage specific input chromatin libraries were generated for each stage and sex examined in our study.


title: Cytosine base editors induce prevalent unwanted out-of-protospacer editing and target-strand editing [Targeted amplicon sequencing]

city: Beijing

first: Howard

last: MENG

state: Beijing

address: No.5 Yiheyuan Road Haidian District

department: School of Life Sciences

institute: Peking University

summary: The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells.     Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level.     Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the  FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates.     Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities.

overall_design: Detect-seq utilized a dU-mapping strategy to profile on-target and off-target editing events by CBE at the whole-genome level. We applied Detect-seq to off-target evaluation in HEK293T and MCF7 cells for BE4max with several frequently used sgRNAs: “VEGFA site 2”, “HEK293 site 4”, “EMX1”, and “RNF2”.

first: Zhixin

last: Lei

first: Meng

last: Haowei

first: Lv

last: Zhicong

first: Yi

last: Chengqi


title: RNAseq expression analysis of FACS-sorted macrophages from cxcr3.2 mutant and wt zebrafish larvae

first: Vincenzo

last: Torraca

state: London

address: Keppel street

institute: LSHTM

summary: The zebrafish Cxcr3.2 is a functional homolog of the human chemokine receptor CXCR3. Zebrafish macrophages lacking this receptor have impaired motility and a rounded shape compared to their wildtype counterparts. To investigate the effects of cxcr3.2 mutation on the transcriptional profile of macrophages, we sorted macrophages from zebrafish larvae lacking a functional cxcr3.2 and compared their transcriptome to that of macrophages from wildtype larvae. Mutant and wildtype macrophages could be clearly distinguished based on the overall differential expression profiles. Classification of genes by compartment showed that peroxisomal, lysosomal and Golgi-related genes were most frequently up-regulated. Moreover, lysosomal and Golgi-related terms were significantly differentially represented in Gene Ontology and KEGG enrichment analysis. Of note, several lysosomal markers  (including acidic hydrolases and voltage ATPases) were consistently upregulated in cxcr3.2 mutant macrophages, indicating that cxcr3.2-mediated chemokine signaling is tightly connected to the regulation of lysosomal function.

overall_design: 3 independent groups of 150-200 5dpf Tg (mpeg1: mCherryF cxcr3.2-/- and cxcr3.2+/+) larvae were dissociated and red fluorescent macrophages were FACS sorted. RNA was extracted, retrotranscribed into cDNA and amplified using the SMARTer® Universal Low Input RNA Kit for Sequencing (Clontech), prior Illumina sequencing.

first: Frida

last: Sommer

first: Eveline

last: in 't Veld

first: Joost

last: Willemse

first: Annemarie

last: Meijer


title: Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior [TRAP I]

overall_design: Cell-type-specific RNA-seq libraries for fru P1-expressing neurons were generated using Translating Ribosome Affinity Purification (TRAP). fru-P1 TRAP was performed at 48hr APF when fruM is at its peak expression in males and to coincide with the 48hr APF chromatin modification data in our study. TRAP (IP) and total mRNA (input) samples were prepared from whole 48hr APF pupae from both males and females with 4-5 biological replicates per sex.


title: A Transcriptional Switch Governs Fibroblast Activation in Heart Disease

city: San Francisco

first: Pawel

last: Przytycki

state: California

address: 1650 Owens Street

department: GIDB

institute: Gladstone Institutes

summary: In diseased organs, stress-activated signaling cascades alter chromatin, triggering maladaptive cell state transitions. Fibroblast activation is a common tissue stress response that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains obscure. Pharmacologic BET inhibition alleviates cardiac dysfunction, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here, we leverage single-cell epigenomic interrogation of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch underlying fibroblast activation. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed novel DNA elements whose accessibility dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer regulating the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program, and was required for TGFβ-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFβ-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction, and also demonstrate its upregulation upon activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide new trans- and cis- targets for treating fibrotic disease.

overall_design:
2 replicates for each sample were run for both single cell RNA and ATAC seq (2x Sham, 2x TAC, 2x TAC JQ1, 2xTAC JQ1 withdrawn).
2 replicates for each sample were run for PRO seq (2x Unstimulated, 2x TGFb, 2x TGFb + siRNA Ctrl, 2x TGFb + siRNA Meox1).
1 replicate for each sample was ran for 4C (1x Unstimulated, 1x TGFb).
2 replicates for MEOX1 ChIPseq.
3 replicates for each sample for bulkRNAseq (3x Sham, 3x TAC, 3x TAC JQ1).
8 PROseq samples (2x Unstimulated, 2x TGFb, 2x TGFb + siRNA Ctrl, 2x TGFb + siRNA Meox1).
6 MEOX1 ChIPseq samples (3x for Unstim and 3x for TGFb-treated conditions).
4 H3K27ac ChIPseq samples (3x for Unstim and 3x for TGFb-treated conditions).

first: Michael

last: Alexanian

middle: F


title: Experience induces rapid nucleus-scale movements of chromatin.

first: Thomas

last: Burkard

summary: The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. Dynamic chromatin movements at the scale of the nucleus are believed to support transcriptional alterations. Applying in vivo two-photon imaging in a novel transgenic mouse model allowing photolabeling of histones, we show that chromatin organization of neurons can undergo movements within minutes. Pharmacological manipulation of neuronal activity induces chromatin movement in an in vitro brain slice model. Furthermore, we show that a behavioral learning paradigm prompting changes in gene expression patterns in auditory cortex, induces chromatin movement in neurons in vivo. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional plasticity at the time scales that could not be realized by passive diffusion. Together, our findings open a framework to chronically study chromatin dynamics at the nucleus-scale in a physiologically relevant context.

overall_design: Examination of incubation of acute brain slices in a pharmacological cocktail

first: Manuel

last: Peter

first: Dominik

last: Aschauer

first: Florian

last: Grössl

first: Wulf

last: Haubensak

first: Simon

last: Rumpel


title: Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior

first: Michelle

last: Arbeitman

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

overall_design: Refer to individual Series


title: Low-dose DNA demethylating therapy induces reprogramming of diverse cancer-related pathways at the single-cell level

status: Public on Apr 07 2021

first: Hideyuki

last: Takeshima

type: Methylation profiling by genome tiling array


title: Astrocytic chromatin remodeling in response to neuroactive compounds [ATAC-seq]

first: Brittney

last: Lozzi

department: Cell & Gene Therapy

summary: The goal of the study is to profile chromatin landscape of astrocytes in response to neuroactive chemicals. We performed ATAC-seq on mES_astrocytes induced for 30 minutes in presence of neuroactive chemicals (glutamate, gamma-aminobutyric acid, acetylcholine, noradrenaline, and serotonin) to understand how induction from different types of neuronal signaling remodels chromatin in astrocytes.

overall_design: mES_astrocytes were collected in triplicate after induction for 30 minutes with each neuroactive chemical at day 35 for profiling using ATAC-Seq.

first: Debosmita

last: Sardar

first: Benjamin

last: Deneen


title: Direct Immunodetection of Global A-to-I RNA Editing Activity with a Chemiluminescent Bioassay

city: Atlanta

first: Steven

middle: Douglas

last: Knutson

state: GA

address: 1515 Dickey Drive

department: Chemistry

institute: Emory University

summary: We report the development of an ELISA-based chemiluminescent assay to measure global levels in A-to-I editing. We directly compare this performance to RNA-seq analysis using the Alu Editing Index. These attached datasets represent sequenced mRNA isolated from HEK293T cells transfected with increasing amounts of ADAR1 to induce a global increase in A-to-I RNA editing activity.

overall_design: Measurement of global A-to-I editing activity in cellular mRNA.

first: Steve

middle: D

first: Robert

last: Arthur

first: H

last: Johnston

first: Jennifer

last: Heemstra


title: Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients [III]

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Epstein-Barr virus (EBV) is a human gamma-herpesvirus that is causally associated with various lymphomas and carcinomas.  Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from multiple myeloma patients.  Here, we analyzed two EBV+ MM-patient derived cell lines IM9 and ARH77 and found defective viral genomes and atypical viral gene expression patterns. We performed RNA-seq transcriptomics to characterize the viral and cellular properties of the two EBV+ cell lines compared to canonical MM cell line 8226. Principal component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226.  ImmGen analysis designate these cells as stem-cell and bone marrow derived.  IM9 and ARH77 displayed atypical viral gene expression, including a leaky lytic cycle gene expression with absence of lytic DNA amplification. Genome sequencing revealed that EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 have numerous EBV genome heterogeneity suggestive of cell harboring multiple and variant viral genomes.  Among the lytic genes, we identified atypical high-level expression of BLRF2 and BLRF1.  BRLF2 encodes a tegument protein previously shown to bind BNRF1 and to also interact with cGAS, similar to KSHV ORF52, also known as the inhibitor of cGAS (KicGAS).  We demonstrate the shRNA depletion of BLRF2 alters viral and host gene expression, including a reduction in lytic gene activation and DNA amplification.  These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B-cells derived from MM tumors, and suggest that EBV may contribute to etiology of MM.

overall_design: Whole genome sequencing in EBV positive multiple myeloma cell lines

first: Paul

last: Lieberman

first: Fang

last: Lu


title: A cellular and spatial map of the choroid plexus across brain ventricles and ages

city: Cambridge

first: Rebecca

last: Herbst

state: MA

address: 415 Main Street

department: Klarman Cell Observatory

institute: Broad Institute of MIT and Harvard

summary: The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. We apply single cell and single nuclei sequencing to identify region and age specific shifts in gene expression of the constituent cell types of the choroid plexus. First, we sequenced whole cells (15,620) from each ventricle (lateral, third and fourth) of the embryonic mouse brain (Embryonic day (E) 16.5). Our analyses and validation of gene (smFISH) and protein (immunohistochemistry) expression combined with spatial mapping (confocal imaging) revealed the identity and location of major cell types, subtypes, proliferating cells, progenitor populations and regionalized gene expression, across each ventricle in the developing brain. Next, to track age-dependent shifts in the choroid plexus properties, we performed single nuclei sequencing (83,040) across each ventricle of the embryonic (E16.5), adult (4 months) and aging (20 months) mouse brain. Epithelial and mesenchymal cells showed regionalized gene expression patterns by ventricle, starting at embryonic stages and persisting with age. Dramatic transcriptional shifts were found with maturation (embryonic to adult) and a smaller shift within each aged cell type. With aging, epithelial cells upregulated host defense programs and resident macrophages enhanced expression of IL-1b signaling genes. Our atlas revealed ChP brain barrier cellular diversity, architecture and signaling across ventricles during development, maturation and aging.

overall_design: We sequenced whole cells isolated from lateral, third and fourth ventricle of embryonic brains (E16.5, CD-1) in triplicate. We sequenced whole nuclei isolated from each ventricle of embryonic (E16.5), adult (4 months post natal) and aging (20 months post natal) mouse (C57BL/6J) brains.

first: Aviv

last: Regev

first: Maria

last: Lehtinen

first: Naomi

last: Habib


title: Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients [I]

overall_design: RNA-seq in EBV positive and negative multiple myeloma cell lines


title: Comparative metabolic and transcriptomic analysis of macrozoids and palmelloid cells of Haematococcus pluvialis related to astaxanthin accumulation

city: Seville

first: Ana

middle: Belén

last: Romero-Losada

state: Seville

address: St. Américo Vespucio, 49

department: Institute for plant biochemistry and photosynthesis

country: Spain

institute: University of Seville

summary: In this work, we propose a metabolomic and transcriptomic comparative study between a vegetative state and intermediate reddish palmelloid state growing in continuous culture conditions, giving rise to a profound understanding of the first steps of the adaptation of Haematococcus pluvialis to nitrogen limitation and the correlation with astaxanthin biosynthesis. In addition, other related metabolic pathways as fatty acid biosynthesis, starch accumulation, citric acid cycle, and amino acid biosynthesis have been analyzed. Understanding the molecular basis of astaxanthin accumulation will be useful for the optimization of astaxanthin production.

overall_design: Haematococcus lacustris (strain CCAP 34/8) was grown photoautotrophically in six jacketed bubble columns (0.06 m diameter, 0.50 m height), containing 1.8 L of cell suspension. Photobioreactors were continuously sparged with air (6.6 L (per L culture)/h). Temperature was maintained at 25ºC by flowing thermostated water through the jacked of the photobioreactors. The pH was controlled by on demand injection of pure CO2 into the air stream entering the culture maintained to 7.0. Each photobioreactor was illuminated by six surrounding Phillips Master TL5HO 24W/840 white-light lamps in a solar cycle simulated (12h/12h) with maximum impinging irradiance on the reactor surface of 1500 uE m-2 s-1. Experiments were carried out in continuous mode, at dilution rate of 0.8/day. The basal inorganic culture medium used to feed the reactor was previously described (Del Río et al., 2015). The medium was supplied with sodium nitrate, as to reach the final nitrate concentration indicated (15 or 2 mM).Escribe un mensaje.

first: Francisco

last: Romero-Campero

first: Mercedes

last: García-González


title: Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients [II]

overall_design: RNA-seq in control and shBLRF2 conditions in ARH77 and IM9 cell lines


status: Public on Apr 06 2021

city: Nanjing

first: Rong

last: Yin

address: Baiziting 42

department: Thoracic Surgery

institute: Nanjing Medical University Affiliated Jiangsu Cancer Hospital

summary:
LncRNA LINC00525 suppresses p21 expression via mRNA decay and triplex-mediated changes in chromatin structure in lung adenocarcinoma
Our findings demonstrate that LINC00525 promote lung adenocarcinoma progression by reducing the transcription and stability of p21 mRNA in concert with EZH2 and RBMS2, which highlights the importance of LINC00525 acting as a potential therapeutic target of LUAD.

overall_design: In human lung adenocarcinoma A549 cells, we downregulated the expression of LINC00525 utilizing small interference RNA (siRNA) and used scrumble as negative control. Then we used RNA-Seq to reveal the change of mRNA profiles after the silence of LINC00525.

first: Tongyan

last: Liu


title: The ETS inhibitor YK-4-279 suppresses thyroid cancer progression independent of TERT promoter mutations

city: Guangzhou

first: Haipeng

last: Xiao

address: 58 Zhangshan er Road

institute: The First Affiliated Hospital of Sun yat-sen University

summary: Hotspot mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been well established to associate with aggressive clinical characteristics, radioiodine refractory, tumor recurrence and mortality in thyroid cancer. Several E-twenty-six (ETS) transcription factors were reported to selectively bound to the mutant TERT promoter and activated TERT expression. In this study we aimed to investigate whether TERT promoter mutations confer sensitivity to ETS inhibitor YK-4-279 in thyroid cancer cells and whether this inhibitor could be served as a potential therapeutic agent for thyroid cancer. In vitro assays showed that YK-4-279 treatment sharply suppressed cell viability, colony formation, migration and invasion, as well as induced cell cycle arrest and apoptosis in a panel of thyroid cancer cells. The cell viability after YK-4-279 treatment were similar between cell lines harboring mutant and wild-type TERT promoter. Furthermore, YK-4-279 treatment reduced both luciferase activity and mRNA expression of TERT independent of TERT promoter mutation status. Data from RNA-seq further revealed that YK-4-279 significantly affected biological processes including DNA replication and cell cycle. Reduced DNA helicase activity and decreased expression of several helicase genes were observed after YK-4-279 treatment. Moreover, YK-4-279 significantly inhibited tumor growth and induced apoptosis in a xenograft mice model. Thus, ETS inhibitor YK-4-279 suppressed TERT expression and conferred anti-tumor activity in a TERT promoter mutation-independent manner, and it could be a potential agent for the treatment of advanced thyroid cancers.

overall_design: KHM-5M cells were treated with DMSO and YK-4-279 at different dosage (0.3 μM and 1 μM) with three repllication.

first: Rengyun

first: Junyu

last: Xue

first: Shiyong

last: Li

first: Mengke

last: Chen

first: Shuang

last: Yu

first: Shubing

last: Hong

first: Peijie

last: Shi


title: Epigenetic aging of the prefrontal cortex and cerebellum in humans, chimpanzees, and rhesus macaques

city: Durham

first: Elaine

last: Guevara

state: North Carolina

address: 130 Science Drive

department: Evolutionary Anthropology

institute: Duke University

summary: Genome-wide prefrontal cortex and cerebellum DNA methylation profiles of younger and older adult humans, captive chimpanzees, and captive rhesus macaques

sample_organism:
Macaca mulatta
Pan troglodytes
Homo sapiens

overall_design: Bisulfite convrted DNA for 22 individuals of three species was profiled by the Illumina Infinium EPIC array


title: Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients

first: Priyankara

last: Wickramasinghe


title: Cell-intrinsic glial pathology is conserved across human and murine models of Huntington Disease

city: Rochester

first: John

last: Mariani

address: 601 Elmwood Avenue

department: Neurology

institute: University of Rochester

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Arcuate organoid cells

first: Hongjun

last: Song

address: 415 Curie Boulevard

institute: University of Pennsylvania

summary: We report the application of bulk and single-cell RNAseq profiling to arcuate organoid cells

overall_design: Bulk and single-cell RNAseq profiling of arcuate organoid cells

first: Samuel

last: Wong


title: Circulating miRNAs in serum as biomarkers for early diagnosis of non-small cell lung cancer

first: Xiaoxia

last: Zhu

state: Guangdong

address: No. 253 Gongye Street

department: Radiation Oncology Department

institute: Zhujiang Hospital, Southern Medical University

summary: The aim of this study was to explore novel miRNA biomarkers in the serum of lung cancer patients based on miRNA profiling. A miRCURY™ LNA Array was used to identify differentially altered miRNAs in the serum of lung cancer patients (n = 6) and healthy controls (n = 6). Total RNA was isolated using TRI reagent BD (MRCgene, TB-126) according to manufacturer’s instructions. After having passed RNA quantity measurement using the NanoDrop 1000, the samples were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.18.0). Following the washing steps the slides were scanned using the Axon GenePix 4000B microarray scanner.Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs were identified through Volcano Plot filtering. Finally, hierarchical clustering was performed to show distinguishable miRNA expression profiling among samples.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: miRNA profiling in serum samples were analyzed by the 7th generation of miRCURYTM LNA Array (v.18.0, Exiqon). The 7th generation of miRNA array contains 3100 capture probes, covering all human, mouse and rat microRNAs annotated in miRBase 18.0, as well as all viral microRNAs related to these species. In addition, this array contains capture probes for 25 miRPlus™ human microRNAs. These are proprietary microRNAs not found in miRBase.


title: Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

city: Boulder

first: Evan

last: Lester

state: CO

address: JSC Biotech bldg

department: Biochemistry

institute: University of Colorado

summary:
We report the isolation and sequencing of tau aggregates from
[1] HEK293 cells expressing Tau-RD-P301S-CFP/YFP that have been seeded with preformed fibrils from the brain of P301S mice (B6-Tg(Thy1-MAPT*P301S)2541; referred to as Tg2541 mice). Tau aggregates were isolated by differential centrifugation followed by fluorescence automated particle sorting using a BD FACSaraia. We found that these tau aggregates were enriched for particular small non-coding RNAs, including snoRNAs and snRNAs.

[2] the following mice: FvBB6F1-Tg(Camk2a-tTa),(tetO-MAPT*wt)21221 (referred to as rTg21221 or WT tau mice in the paper) and FvBB6F1-Tg(Camk2a-tTA)1Mmay, (tet)-tdTomato-Syp/EGFP)1.1Luo/J,(tetO-MAPT*P301L)4510 (referred to as rTg4510 or P301L mice in the paper). Briefly, tau aggregates were isolated by 1% sarkosyl extraction (to enrich for insoluble proteins) followed by immunoprecipitation of tau using the tau-12 antibody (see Methods section of associated paper for further details). We found that these tau aggregates were enriched for particular small non-coding RNAs, including snRNAs and some snoRNAs.

[3] Sequencing of HEK293 tau biosensor cells with and without tau aggregates reveals evidence of significant splicing alterations. Specifically we observed an increase in intron retention events in cells that contain tau aggregates relative to cells without tau aggregates.

overall_design: Examination of the RNA composition of tau aggregates in HEK293 cells [GSM4477465-GSM4477468] and from the brains of P301L mice (rTg4510) [GSM4484210-GSM4484221]. Examination of total RNA in HEK293 tau biosensor cells with and without tau aggregates [GSM5227473-GSM5227478].

first: Felicia

middle: K

last: Ooi

first: Nadine

last: Bakkar

first: Jacob

last: Ayers

first: Amanda

middle: L

last: Woerman

first: Joshua

last: Wheeler

first: Robert

last: Bowser

first: George

last: Carlson

first: Stanley

last: Prusiner

first: Roy

last: Parker


title: Regnase-1 suppresses TCF-1+ precursor exhausted T cell formation to limit CAR T cell responses against ALL

city: Memphis

first: Hongbo

last: Chi

state: TN

address: 262 Danny Thomas Place

department: Immunology

institute: St Jude Children's Research Hospital


title: Neonatal mouse exposed to hyperoxia

city: Fukushima

first: Hayato

last: Go

state: Fukushima

address: Hikarigaoka 1

department: Pediatrics

institute: Fukushima Medical University

summary: We performed miRNA array analysis from 2 groups (neonatal lung control, neonatal lung after hyperoxia).   We used pools of every 100ng of total RNA of three samples for each groups.

overall_design: Examination of miRNA levels in hyperoxia

first: Phyllis

last: Dennery


title: Extracellular vesicle miRNA-21 is a potential biomarker for predicting chronic lung disease in premature infants


title: Human hypothalamus

summary: We report the application of single-cell RNAseq profiling of human hypothalamus cells

overall_design: Single-cell RNAseq profiling of human hypothalamus cells


title: Transcriptional and imprinting complexity in Arabidopsis seeds at single-nucleus resolution

city: Los Angeles

first: Colette

last: Picard

address: 610 Charles E Young Dr East, Room 4045

department: Molecular, Cell and Developmental Biology

institute: University of California - Los Angeles

summary: Seeds are the basis of agriculture, yet their full transcriptional complexity has remained unknown. Here, we employ single-nucleus RNA-sequencing to characterize developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the site of gene imprinting in plants, mediates the relationship between the maternal parent and embryo. We identify new cell types in the chalazal endosperm region, which interfaces with maternal tissue for nutrient unloading. We further demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. These data indicate imprinting in endosperm is heterogeneous and suggest that parental conflict, which is proposed to drive the evolution of imprinting, is fiercest at the boundary between filial and maternal tissues.

overall_design:
To identify cell/nuclei types and investigate imprinting dynamics within endosperm, single nucleus RNA-seq was performed using the Smart-seq2 method on seed nuclei, targeting either the 3C or 6C FANS peak in order to enrich for triploid endosperm (other seed tissues are diploid). A total of 1,664 libraries were sequenced, of which 64 were negative controls (no DNA in library prep or no cell sorted), 51 were made from two nuclei to test single-nuclei sorting accuracy, 112 failed QC checks (< 1,500 genes detected and/or <1,000 detected with 5+ reads), and the remaining 1,437 were high-quality single-nuclei libraries used in final analysis. The final dataset consists primarily of seeds derived from Col, Cvi reciprocal crosses at 4 days after pollination (DAP), although some nuclei were instead obtained from Col, Ler crosses and/or other timepoints.

Please note that the counts.txt files for the P17_10A, P21_10A, and P22_10A samples are identical as they are from negative controls that should not have any reads mapping to A. thaliana.
There are  ~50 negative controls in the dataset, and most have at least one or two reads falling in a gene somewhere and so the processed data files (read counts per gene) aren't exactly the same, but the read counts in all three files are actually zero for all genes, so all three files are identical.

first: Rebecca

last: Povilus

first: Ben

last: Williams

first: Mary

last: Gehring


title: Small molecule inhibitor of PRC1 E3 ligase activity induces differentiation of leukemia initiating cells

city: Ann Arbor

first: Tomasz

last: Cierpicki

state: MI

address: 1150 W Medical Center Dr

department: Pathology

institute: University Of Michigan

overall_design: Refer to individual Series


title: Transcriptome analyses of Arabidopsis ldl1ldl2 (lysine-specific demethylase1 [LSD1]-like1 [ldl1]) double mutants as well as wild-type Columbia-0 plants via next-generation sequencing (NGS)

status: Public on Apr 04 2021

city: Busan

first: Ho Won

last: Jung

address: 37 Nakdong-Daero 550beon-gil, Saha-gu

department: Molecular Genetics

country: South Korea

institute: Dong-A University

summary:
Purpose: The goals of this study are to compare transcriptome of ldl1ldl2 double mutants with that of wild-type Columbia-0 plants and to identify genes whose expression are tightly regualted by LDL1/LDL2 prtoeins in Arabidopsis.
Methods: mRNA profiles of 3-week-old wild-type Col-0 and ldl1ldl2 mutant plants were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with a method : TopHat followed by Cufflinks.
Results: Using an optimized data analysis workflow, we mapped about 27~31 million sequence reads per sample to the Arabidopsis genome (TAIR10).
Conclusions:  Our results show that LDL1 and LDL2 protein had  significant effects on transcription of genes important for plant immune reponses against Pseudomonas infection.

first: Sung

middle: W

last: Noh

first: Ri

last: Seo

first: Ho


title: Small molecule inhibitor of PRC1 E3 ligase activity induces differentiation of leukemia initiating cells [RNA-Seq]

summary: We have developed small molecule inhibitors of PRC1. These inhibitors target PRC1, decrease H2A ubiquitination and differentiation of TEX cells.

overall_design: TEX cells treated with DMSO or 25uM WY332 compound for 6 days or 10 days and gene expression was profiled using RNA sequencing.

first: Jolanta

last: Grembecka

first: Jingya

last: Wang


title: Transcriptomic changes of tumor endothelial cells in spontaneous breast tumour model and implanted breast tumour model

status: Public on Apr 05 2021

city: Daejeon

first: Myung Jin

last: Yang

institute: Institute for Basic Science

summary: To investigate the trancriptomic changes of tumor endothelial cells (ECs) in response to STING activation, tumor ECs from MMTV-PyMT mouse model or implanted breast tumour model were either treated by PBS or cGAMP were sorted by FACS and single-cell RNA seq was performed.

overall_design: Tumor endothelial cells treated with PBS or cGAMP were sorted and single-cell RNA libraries were made.

first: Gou

last: Koh

first: Seung-hwan

last: Jeong

first: Myung

middle: J


title: Mitohormesis reprograms macrophage metabolism to enforce tolerance.

city: Berkeley

first: Greg

last: Timblin

state: CA

department: MCB

institute: UC Berkeley

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


title: Serum extracelluar vesicles miRNA in premature neonates with chronic lung disease (CLD) and non CLD

first: Hayato

summary:
Serum were obtained from 4 groups, day0 and day28 neonates with CLD and non-CLD.  Serum samples (250ul) from cord blood at birth and venous blood of neonates at 28 DAYS were transferred to sterile tubes containing 63 μl of ExoQuick precipitation solution (System Bioscience, Mountain View, CA, USA), and mixed.  The mixtures were incubated for 30 min at 4℃, and centrifuged at 1500×g for 30 min.  Then, the supernatants were removed, the pellets (extracellular vesicles) were diluted to 60 μl with phosphosaline buffer, and then thawed on dry ice for total RNA extraction with a miRvana isolation kit (Ambion). 3pM synthetic ath-miR159a was spiked into each sample to correct for technical differences.
Using a human miRNA array, we identified 62 miRNAs that were universally expressed in CLD patients and non-CLD patients (Normalized Template).

overall_design: Examination of miRNA levels in extracelluar vesicles miRNA in premature neonates with CLD and non CLD


title: Mitohormesis reprograms macrophage metabolism to enforce tolerance [BMDM_tolerance_RNAseq]

summary: Macrophages generate mitochondrial reactive oxygen and electrophilic species (mtROS, mtRES) as antimicrobials during Toll-like receptor (TLR)-dependent inflammatory responses. Whether mitochondrial stress caused by these molecules impacts macrophage function is unknown. Here, we demonstrate that both pharmacologically- and lipopolysaccharide (LPS)-driven mitochondrial stress in macrophages triggers a stress response called mitohormesis. LPS-driven mitohormetic stress adaptations occur as macrophages transition from an LPS-responsive to LPS-tolerant state where stimulus-induced proinflammatory gene transcription is impaired, suggesting tolerance is a product of mitohormesis. Indeed, like LPS, hydroxyestrogen-triggered mitohormesis suppresses mitochondrial oxidative metabolism and acetyl-CoA production needed for histone acetylation and proinflammatory gene transcription, and is sufficient to enforce an LPS-tolerant state. Thus, mtROS and mtRES are TLR-dependent signaling molecules that trigger mitohormesis as a negative feedback mechanism to restrain inflammation via tolerance. Moreover, bypassing TLR signaling and pharmacologically triggering mitohormesis represents a novel anti-inflammatory strategy that co-opts this stress response to impair epigenetic support of proinflammatory gene transcription by mitochondria.

overall_design: RNA-seq of primary bone marrow-derived macrophages (BMDMs) treated with LPS, 4-hydroxyestrone (4-OHE1), or both.


title: Mitohormesis reprograms macrophage metabolism to enforce tolerance [BMDM_OHestrogen_RNAseq]

overall_design: RNA-seq of primary bone marrow-derived macrophages (BMDMs) treated with controls or hydroxyestrogens.


title: Small molecule inhibitor of PRC1 E3 ligase activity induces differentiation of leukemia initiating cells [ChIP-seq]

overall_design: TEX cells treated with DMSO or 25uM WY332 compound for 6 days followed by immunoprecipitation with anti-H2Aub antibody. ChIP sequencing was performed and analyzed by ActiveMotif.


title: Mitohormesis reprograms macrophage metabolism to enforce tolerance [vWAT_mac_RNAseq]

overall_design: RNA-seq of visceral white adipose tissue (vWAT) macrophages from high fat diet-fed mice treated with estrogens.


title: Arid1a regulates cell cycle exit of transit-amplifying cells through inhibiting the Aurka-Cdk1 axis in mouse incisor

city: Shanghai

first: Jiahui

last: Du

address: Zhizaoju Road 639

institute: Shanghai Jiao Tong University

summary: We show that Arid1a maintains tissue homeostasis through promoting cell cycle exit and differentiation of TACs by inhibiting the Aurka-Cdk1 axis.

overall_design: We detected the incisor homeostasis  in Gli1-CreER;Arid1afl/fl mice conditional knockout mouse models

first: Yang

last: Chai


title: Mitohormesis reprograms macrophage metabolism to enforce tolerance [BMDM_ChIPseq]

overall_design: ChIP-seq for p65 and H3K27ac in control or hydroxyestrogen-pretreated, LPS-stimulated bone marrow-derived macrophages (BMDMs).


title: A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma [ChIP-seq]

city: Hong Kong

first: Alfred Sze-Lok

last: Cheng

address: Rm 405A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, CUHK

department: School of Biomedical Sciences

country: Hong Kong

institute: The Chinese University of Hong Kong

summary: Insufficient T cell infiltration into non-inflamed tumors, such as hepatocellular carcinoma (HCC), restricts the effectiveness of immune-checkpoint blockade (ICB) for a subset of patients. Epigenetic therapy provides further opportunities to rewire cancer-associated transcriptional programs, but whether and how selective epigenetic inhibition counteracts the immune-excluded phenotype remain incompletely defined. Here, we showed that pharmacological inhibition of histone deacetylase 8 (HDAC8), a histone H3 lysine 27 (H3K27)-specific isozyme over-expressed in a variety of human cancers, thwarts HCC tumorigenicity in a T cell-dependent manner. The tumor-suppressive effect of selective HDAC8 inhibition was abrogated by CD8+ T cell depletion or regulatory T cell adoptive transfer. Chromatin profiling of human HDAC8-expressing HCCs revealed genome-wide H3K27 deacetylation in 1,251 silenced enhancer-target gene pairs that are enriched in metabolic and immune regulators. Mechanistically, down-regulation of HDAC8 increased global and enhancer acetylation of H3K27 to reactivate production of T cell-trafficking chemokines by HCC cells, thus relieving T cell exclusion in both immunodeficient and humanized mouse models. In an HCC preclinical model, selective HDAC8 inhibition increased tumor-infiltrating CD8+ T cells and potentiated eradication of established hepatomas by anti-PD-L1 therapy without evidence of toxicity. Importantly, mice treated with HDAC8 and PD-L1 co-blockade were protected against subsequent tumor re-challenge as a result of the induction of memory T cells and remained tumor-free for greater than 15 months. Collectively, our study demonstrates that selective HDAC8 inhibition elicits effective and durable responses to ICB by co-opting adaptive immunity via enhancer reprogramming.

overall_design:
ChIP-Seq (H3K27ac) of non-alcoholic fatty liver disease (NAFLD)-associated HCC tumor (n=10) and adjacent non-tumor liver tissues (n=10); **Raw data not provided due to patient privacy concerns**

*** Raw data are not provided due to patient privacy concerns ***

first: Weiqin

first: Yu

last: Feng

first: Jingying

last: Zhou

first: Otto

last: Cheung

first: Feng

last: Wu

first: Alfred

middle: S


title: A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma


title: A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma [RNA-seq]

overall_design:
RNA-Seq of non-alcoholic fatty liver disease (NAFLD)-associated HCC tumor (n=10) and adjacent non-tumor liver tissues (n=10); **Raw data not provided due to patient privacy concerns**
Note: Raw data are not provided due to patient privacy concerns


title: MondoA Drives Malignancy in B-ALL through Enhanced Adaptation to Metabolic Stress [ChIP-seq]

city: Munich

first: Maxim

last: Barenboim

address: Koelner Platz 1

department: Department of Pediatrics

institute: TECHNICAL UNIVERSITY OF MUNICH

summary: MondoA (also known as MLXIP), a member of the MYC interactome, has been described as an example of a metabolic sensor. By assessing patient data sets we found that MondoA overexpression is associated with a worse survival in pediatric common acute lymphoblastic leukemia (B-ALL). Using CRISPR/Cas9 and RNA interference approaches, we observed that MondoA depletion reduces transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid (TCA) cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced PDH activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. CHIPseq revealed a modest but highly significant redistribution of MYC towards binding sites shared with MondoA upon loss of MondoA.

overall_design: ChIP-seq of MYC and MondoA TF in MondoA-WT and MondoA-KO Nalm6 cells

first: Julia

last: Hofstetter

first: Elmar

last: Wolf

first: Stefan

last: Burdach

first: Alexandra

last: Sipol

first: Rupert

last: Oellinger

first: Busheng

last: Xue

first: Roland

last: Rad


title: Genome-wide mapping of OsHOX24-binding sites in rice

city: New Delhi

first: Mukesh

last: Jain

state: New Delhi

address: New Mehrauli Road

country: India

institute: Jawaharlal Nehru University

summary: In this study, we provide a global overview of genome-wide OsHOX24 binding sites in rice under control and desiccation stress conditions in wild-type and OsHOx24 overexpressing rice plants (H49 line) via chromatin immunoprecipitation sequencing (ChIP-sequencing) approach. We identified numerous downstream targets of OsHOX24 under desiccation stress and control by analyzing the comprehensive binding site map of OsHOX24 at whole genome level in rice.

overall_design: We collected three-weeks-old seedlings of wild-type and OsHOX24 over-expression transgenic (H49 line) rice subjected to control (kept in water) and dessication (kept between folds of tissue paper) treatments for 3h. DNA  was isolated from these tissue samples by chromatin immunoprecipitation and subjected to Illumina sequencing. The sequence data was then filtered using NGS QC Toolkit to obtain high-quality reads. The filtered reads were mapped to rice genome sequence (MSU v7) using Bowtie2 software. MACS2 was used for peak calling and confident peaks were identified using Bedops pipeline. Several downstream targets of OsHOX24 were identified in rice under desiccation stress and control conditions.

first: Annapurna

last: Bhattacharjee

first: Prabhakar

last: Srivastava

first: Onkar

last: Tiwari


title: Type 2 immunity is maintained during cancer associated adipose tissue wasting

status: Public on Apr 03 2021

city: Boston

first: Patrick

last: Lenehan

address: 1 Jimmy Fund Way

institute: Dana Farber Cancer Institute

summary:
Purpose: Cachexia is a metabolic phenotype affecting many cancer patients. The immune environment in adipose tissue during the development of cancer associated cachexia has not been characterized. To help address this gap, we isolated visceral adipose tissue from cancer-bearing and sham-operated mice and performed bulk RNA-sequencing.
Methods: mRNA expression was evaluated by RNA-sequencing in visceral adipose tissue (VAT) of 10-week old mice which had been subjected to sham surgery (sham) or had received orthotopic pancreatic tumors four weeks prior to harvest (KPC). Paired-end 150 bp single-index sequencing was performed using an Illumina HiSeq system. Transcript quantification was performed with Salmon, using quasi-mapping mode with selective alignment (validateMappings). We set the rangeFactorizationBins to 4 and passed both the seqBias and gcBias flags to correct for sequence-specific bias and fragment-level GC bias, respectively. Transcript quantifications were collapsed to gene-level quantifications using the tximport package and the Ensembl based annotation package EnsDb.Mmusculus.v79. Differential expression analysis was then performed using DESeq2 with fold change shrinkage by ashr. For downstream analysis and visualization, only genes with a mean TPM (transcripts per million) > 2 in VAT from both sham-operated and tumor-bearing mice were considered.
Results: We identified over 11,000 genes which were expressed at an average of at least 2 TPM (transcripts per million) in both the sham and KPC adipose tissue samples. Differential expression analysis with DESeq revealed 161 upregulated genes (p < 0.05) and 27 downregulated genes (p < 0.05) in the adipose tissue of cancer bearing mice compared to that of sham operated mice. Gene set enrichment analysis suggested an increase in inflammatory pathways, including type 1 and type 2 interferon pathways, in the adipose tissue of cancer bearing mice.
Conclusions: This study represents a comprehensive characterization of adipose tissue in the healthy vs. cachexia setting. The RNA-seq data suggests increased immune infiltration into adipose tissue in the setting of a distant pancreatic tumor. Together with flow cytometry and cytokine abundance analyses, we conclude that reguatory Th2 immunity is maintained in the adipose tissue during this model of cancer associated cachexia.

overall_design: Adipose tissue mRNA profiles of 10 week old mice that were subjected to sham surgery (sham) or inoculated with orthotopic pancreatic tumors (KPC)

first: Assunta

last: Cirella

first: Amiko

last: Uchida

first: Stephanie

last: Crowley

first: Tatyana

last: Sharova

first: Genevieve

last: Boland

first: Michael

last: Dougan

first: Max

last: Heckler


title: The connectome of neural crest enhancers reveals regulatory features of signaling systems

city: Ithaca

first: Marcos

last: Simoes-Costa

state: New York

address: 526 Campus Rd

department: Molecular Biology and Genetics

institute: Cornell University

summary: The neural crest is a migratory stem cell population that gives rise to a wide array of cell types. The formation and differentiation of neural crest cells are controlled by a complex transcriptional network, which endows these cells with unique features such as multipotency and stemness. During migration, signaling systems modulate subcircuits within this network to ensure that neural crest cells differentiate at the right time and place. The wingless (Wnt) signaling pathway, in particular, plays an important role in defining developmental potential of these cells but, surprisingly few direct Wnt-target genes have been identified in the neural crest transcriptional network. To address this, we generated a high-resolution contact map of active enhancers in the neural crest and identified the direct targets of Wnt signaling in the neural crest genome via CUT&RUN for its nuclear effectors LEF1 and CTNNB1 (bCat).

overall_design: CUT&RUN data for Wnt signaling nuclear factors LEF1 and CTNNB1, and CTCF in chick neural crest cells at specification stage (HH9)    CUT&RUN data for LEF1 in chick neural crest cells at specification stage (HH9) upon Wnt knockdown    RNA-seq on neural crest cells upon Wnt knockdown.    H2K27ac HiChIP for dissected neural folds and whole embryos at HH9

first: Ana

middle: P

last: Azambuja


title: A dual role for H2A.Z.1 in modulating Initiating and Elongating RNAPII dynamics

city: Cambridge

first: Laurie

last: Boyer

address: 77 Massachusetts Avenue

department: Biology

institute: Massachusetts Institute of Technology

summary: RNA Polymerase II (RNAPII) pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step for the induction of most metazoan genes. During pause-release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells (ESCs). Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts Negative Elongation Factor (NELF) dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the Pre-Initiation Complex (PIC) components TFIIB and TBP. Together, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs by regulating RNAPII dynamics and pause release.

overall_design: ChIP-seq, RNA-seq, NET-seq, and ChIP-nexus following H2A.Z.1 depletion

first: Constantine

last: Mylonas

first: Choongman

last: Lee

first: Alexander

last: Auld

first: Ibrahim

last: Cissé


title: Identification of clinical combination therapies to induce durable responses in kidney cancers

status: Public on Apr 02 2021

city: Portland

first: Laura

last: Heiser

state: OR

address: 2730 S Moody Ave

department: Biomedical Engineering

institute: Oregon Health and Science University

summary: The lack of effective treatment optionsfor advanced non-clear cell renal cell carcinoma (NCCRCC) is a critical unmet clinical need. Applying a high throughput drug screen to multiple human kidney cancer cells, weidentified the combination of the VEGFR-MET inhibitor cabozantinib and the SRC inhibitor dasatinib acted synergistically in cells to markedly reduce cell viability. Importantly, the combination was well tolerated and caused tumor regression in vivo. Transcriptional and phosphoproteomic profilingrevealed that the combination converged to downregulate the MAPK-ERK signaling pathway, a result not predicted by single agent analysis alone. Correspondingly, the addition of a MEK inhibitor synergized with either dasatinib or cabozantinibto increase its efficacy. This study, by employing approved, clinically relevant drugs provides the rationale for the design of effective combination treatments in NCCRCC that can be rapidly translated to the clinic.

overall_design: Gene expression profiles of triplicate ACHN cells treated for 24 hours with dasatinib, cabozantinib, and dasatinib + cabozantinib co-treatment.

first: George

middle: V

last: Thomas

first: Daniel

last: Derrick


title: Impact of 6-mercaptopurine on LPS treated primary cultures of enteric nervous system

city: Kiel

first: Francois

last: Cossais

address: Otto-Hahn-Platz 8

department: Institute of Anatomy

institute: Kiel University

summary:
Enteric nervous system is involved in the regulation of intestinal inflammation. We developped mouse primary cultures of enteric nervous system to study impact of LPS, as pro-inflammatory mediator, and of the pro-drug 6-mercaptopurine on enteric inflammatory pathways
We used microarrays to detail the global programme of gene expression underlying enteric neuro-inflammation and identified classes of up-regulated genes during this process.

overall_design: Mouse ENS primary cultures were treated with LPS in presence or absence of 6-mercaptopurine. Genome-wide expression analysis was performed using Clariom S microarray

first: François

first: Meike

last: Kähler


title: TnSeq of Enterococcus faecalis OG1RF Tn mutants in biofilm and planktonic samples

city: Minneapolis

first: Julia

last: Willett

state: MN

address: 689 23rd Avenue SE, 4-149 Micro Research Facility

institute: University of Minnesota

summary: Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections.  Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions.  Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical.  Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants.

overall_design: Comparison of Enterococcus faecalis OG1RF Tn mutant abundance in biofilm and planktonic samples using TnSeq

first: Gary

last: Dunny


title: Chromosome Compartments on the Inactive X Guide TAD Formation Independently of Transcription during X-Reactivation

city: Scarborough

first: Guillaume

last: Filion

state: Ontario

address: 1265 Military Trail

department: Biological Sciences

country: Canada

institute: UTSC

summary: To investigate the dynamics of X-chromosome reactivation, we differentiated mouse embryonic stem cells (ESCs) into neural precursor cells (NPCs) and later reprogrammed them into induced pluripotent stem cells (iPSCs). During the conversions of NPCs into iPSC, we assessed the kinetics of X-chromosome reactivation. We generated allele-specific RNA-seq datasets to assess gene reactivation dynamics, employed ATAC-seq to investigate chromatin opening dynamics and performed in situ Hi-C to explore dynamics of chromosome structure.

overall_design: Allele-specific total RNA-seq, ATAC-seq and in situ Hi-C in ESC, NPCs derived from ESCs and multiple iPSC samples at different stages of X-chromosome reactivation.

first: Bernhard

last: Payer

first: Moritz

last: Bauer

first: Enrique

last: Vidal


title: Immune dysregulation and autoreactivity correlate with disease severity in SARS-CoV-2-associated multisystem inflammatory syndrome in children

city: New Haven

first: Carrie

last: Lucas

address: 300 Cedar St., S625D

institute: Yale University

summary: Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV2 infection in otherwise healthy children. Here, we define immune abnormalities in MIS-C compared to adult COVID-19 and pediatric/adult healthy controls using single-cell RNA sequencing, antigen receptor repertoire analysis, unbiased serum proteomics, and in vitro assays. Despite no evidence of active infection, we uncover elevated S100A-family alarmins in myeloid cells and marked enrichment of serum proteins that map to myeloid cells and pathways including cytokines, complement/coagulation, and fluid shear stress in MIS-C patients. Moreover, NK and CD8 T cell cytotoxicity genes are elevated, and plasmablasts harboring IgG1 and IgG3 are expanded. Consistently, we detect elevated binding of serum IgG from severe MIS-C patients to activated human cardiac microvascular endothelial cells in culture. Thus, we define immunopathology features of MIS-C with implications for predicting and managing this SARS-CoV2-induced critical illness in children.

overall_design: Single-cell RNA sequencing of PBMCs from 7 patients with MIS-C (severe and moderate, with 2 samples from recovered patients) and 6 pediatric healthy donors.

first: Anjali

last: Ramaswamy


title: Exhaustive drug repurposing strategies support the pursuit of new chemical entities for the development of effective COVID-19 interventions

city: La Jolla

first: Debashis

last: Sahoo

state: California

address: 9500 Gillman Drive

institute: UCSD

summary: The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. Two high-throughput, high-content imaging infection assays (one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells) were developed and used to screen ReFRAME, a best-in-class drug repurposing library. Among the promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 had most favorable in vitro activity, pharmacokinetic and human safety profiles, and both reduced SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. However, only MK-4482 effectively blocked SARS-CoV-2 infection in a hamster model, likely due to inadequate plasma exposure of nelfinavir.

overall_design: Hamster lung from uninfected (U, n=2), vehicle treated (V, n=4) and MK-4482 (EIDD-2801) treated (T, n=4) samples were analyzed using RNASeq platform.

first: Soumita

last: Das

first: Pradipta

last: Ghosh

first: Thomas

last: Rogers

first: Malina

last: Bakowski

first: Nathan

last: Beutler


title: High proliferation and delamination during skin epidermal stratification

city: Cologne

first: Hisham

last: Bazzi

state: NRW

address: Joseph-Stelzmann-Straße 26

department: CECAD & Dermatology

institute: University Hospital Cologne

summary: The development of complex stratified epithelial barriers in mammals is initiated from single-layered epithelia. How stratification is initiated and fueled are still open questions. Previous studies on skin epidermal stratification suggested a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors. Here, we use centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetically ablating centrosomes from the developing epidermis leads to the activation of the p53-, 53BP1- and USP28-dependent mitotic surveillance pathway causing a thinner epidermis and hair follicle arrest. The centrosome/p53-double mutant keratinocyte progenitors significantly alter their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggest that the first and major phase of epidermal development is boosted by high proliferation rates in both basal and suprabasally-committed keratinocytes as well as cell delamination, whereas the second phase maybe uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

overall_design: RNA-Seq analysis from mRNA of primary keratinocytes at P0 or skin epidermis at E13.5 with K14-Cre-specific depletion of Sas-4 and controls. Four replicates for each condition were used.

first: Mareike

last: Damen


title: MondoA Drives Malignancy in B-ALL through Enhanced Adaptation to Metabolic Stress [array]

first: Maxim

last: Barenboim

overall_design: MLXIP-specific sgRNA or scramble was introduced to cALL leukemic cell lines Nalm6 by CRISPR/Cas9. RNA from treated cells was isolated with Trizol and subjected to microarray analysis onto human Gene 1.0 ST arrays following the Affymetrix protocol.

first: S

last: Burdach

first: A

last: Sipol

first: G

middle: H

last: Richter


title: Differentiation of Human induced Pluripotent Stem Cells to Authentic Macrophages using Defined, Serum Free, Open Source Media

city: Oxford

first: Alun

last: Vaughan-Jackson

address: South Parks Road

department: Sir William Dunn School of Pathology

institute: University of Oxford

summary: Human iPSC and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modelling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. We describe here a defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages. This medium is equally capable of maintaining these cells compared to commercial alternatives. The macrophages differentiated in this medium display improved terminally differentiated cell characteristics, reduced basal expression of induced anti-viral response genes, and improved polarisation capacity. We conclude that cells cultured in this medium are an appropriate and malleable model for tissue resident macrophages, on which future differentiation techniques can be built.

overall_design: We generated human iPSC-macrophages from the SFC840-03-03 cell line in two different media conditions. Macrophage precursor cells were harvested after 8, 10, and 12 weeks of differentiation and cultured for a further 7 days in terminal differentiation medium to make authentic macrophages. At this point cells were lysed for collection of RNA for RNA-Seq.

first: Szymon

last: Stodolak

first: Kourosh

last: Ebrahimi

first: Cathy

last: Browne

first: Paul

last: Reardon

first: Elisabete

last: Pires

first: Javier

last: Gilbert-Jaramillo

first: Sally

last: Cowley

first: William

last: James


title: The SvFUL2 transcription factor is required for inflorescence determinacy and timely flowering in Setaria viridis

city: St. Louis

first: Andrea

last: Eveland

state: Missouri

address: 975 North Warson Road

institute: Donald Danforth Plant Science Center

summary: Inflorescence architecture in cereal crops directly impacts yield potential through regulation of seed number and harvesting ability. Extensive architectural diversity found in inflorescences of grass species is due to spatial and temporal activity and determinacy of meristems, which control the number and arrangement of branches and flowers, and underlie plasticity. Timing of the floral transition is also intimately associated with inflorescence development and architecture. Here, we show that a single mutation in a gene encoding an AP1 A-class MADS-box transcription factor significantly delays flowering time and disrupts multiple levels of meristem determinacy in panicles of the C4 model panicoid grass, Setaria viridis.

overall_design: Poly-A+ RNA-seq libraries were generated from pools of hand-dissected inflorescence primordia from  wild-type and ful2 mutant seedlings. Wild-type primordia were sampled at 8, 11, and 17 DAS  while, accounting for the mutant’s developmental progression, ful2 primordia were sampled at 9, 15, and 21 DAS. For each developmental stage, four biological replicates were collected, for a total of 24 data points.  In addition to these samples, we included previously described wild-type S. viridis inflorescence primordia samples (Zhu et al., 2018): 23 additional data points from six inflorescence stages (10,  12,  14,  15,  16,  and  18 DAS).   Form these data We built  a signed gene regulatory network using the WGCNA R package (Langfelder and Horvath, 2008) and integrated it with information derived from regulatory interactions among TFs and their putative targets based on the GENIE3 algorithm (Huynh-Thu et al., 2010). This complementary approach helped us to resolve the directionality and connectivity of important hub genes within the GRN.

first: Jiani

last: Yang

first: Edoardo

last: Bertolini

first: Max

last: Braud

first: Jesus

last: Preciado

first: Adriana

last: Chepote

first: Hui

last: Jiang


title: RNAseq Analysis Investigates Transcriptional Similarities and Differences Between Cryopreserved Parental and Engineered iPSC Derived Macrophages

status: Public on Apr 01 2021

city: Madison

first: Deepika

last: Rajesh

state: WI

address: 525 Science Dr

department: Life Sciences

institute: Fujifilm Cellular Dynamics Inc.

summary: RNA-seq analysis was used to investigate the transcriptional profile to reflect the changes in the parental vs engineered iPSC-derived macrophages. The RNA seq analysis provided an insight to uncover underlying mechanisms that might be triggered by perturbations in SNCA, Progranulin, and MeCP2 genes in iPSC derived macrophages. RNA seq data revealed an upregulation of 431 genes and concomitant downregulation of 276 in SNCA A53T Macrophages, an upregulation of 549 genes and concomitant downregulation of 356 in GRN R493X macrophages, an upregulation of 466 genes and concomitant downregulation of 406 genes in MeCP2HM macrophages when compared to the parental unengineered macrophages.  These genes affected pathways regulating cell metabolism, exocytosis, transport, degranulation, immune activation, vascular circulation and neural differentiation and provided a comprehensive analysis of all cellular pathways perturbed by genetic engineering in SNCA, Progranulin, and MeCP2 genes.

overall_design:
Investigation of perturbations resulting from genetic engineering mimicking neurological diseases; Parkinson's disease (SNCA A53T), Neuronal Ceroid Lipofuscinosis (GRN R493X), and Rett Syndrome (MECP2).

>>> Raw data have not been provided for this study.  Submitter indicates the risk of re-identifying the donor is against FCDI privacy policy. <<<<

first: Christie

last: Munn

first: Sarah

last: Burton

last: Dickerson

first: Anne

last: Strouse

first: Kiranmayee

last: Bakshy