title: DNA sequence-dependent heterochromatin microdomain formation

city: Colchester

first: Vladimir

last: Teif

address: Wivenhoe Park

institute: University of Essex

summary: Cell-type specific gene expression programs are established by distinct chromatin state patterns that involve thousands of heterochromatin microdomains of ~1-2 kb in size marked by di- and trimethylation of histone H3 at lysine 9 (H3K9me2/me3). However, no theoretical framework exists to predict the location and boundaries of such domains from the DNA sequence. Here, we compare H3K9me2/me3-heterochromatin microdomains in mouse embryonic stem cells (ESCs) that are dependent on the histone methylases SUV39H1/2 and GLP, transcription factor ADNP or chromatin remodeler ATRX. By applying a novel Ising-type chromatin hierarchical lattice (ChromHL) model, we identify two different microdomain types that are distinct with respect to their dependence on DNA sequence motifs or nucleosome interactions. ChromHL is able to predict microdomain location, extension and boundaries based on binding sites of PAX3, PAX9, ADNP, CTCF and repeat sequence motifs, the concentration of heterochromatin protein 1 (HP1) and the strength of nucleosome-nucleosome interactions. Thus, our result provides insight how distinct patterns of silenced heterochromatin states are implemented and regulated.

overall_design: Wild type murine embryonic stem cell (ESC) wt26 and two ATRX knock out cell lines (KO1-40 and KO1-45) were constructed as described previously (Sadic et al., EMBO Rep, 2015). Cells were cultured on 0.2% v/v gelatine (in PBS) in high glucose DMEM (gibco 31053-028) supplemented with 1 mM sodium pyruvate and 4 mM L-Glutamine (PAA M11-006), 15% v/v FCS (Sigma F7524, lot: 091M3398), 1% v/v Penicillin-Streptomycin (PAN Biotech P06-07100), 100 µM β-mercaptoethanol (Sigma 63689), 1% v/v non-essential amino acids and 0.41% v/v LIF (self-made; supernatant from LIF-producing cells; batch: 7/26/14). Chromatin immunoprecipitation. Cells were fixed in 1% formaldehyde (Thermo Scientific 28906) in PBS for 10 minutes and stopped by addition of glycine to a final concentration of 125 mM. Cells were washed twice with PBS/0.5 mM PMSF, dissolved in swelling buffer (25 mM Hepes pH7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, protease inhibitor COMPLETE from Roche) and incubated on ice for 10 minutes. After pelleting they were digested with MNase at 37°C for 15 minutes in a buffer containing 25 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 50 mM Tris/HCl pH7.4 and 1x protease inhibitor from Cell Signalling. Chromatin was fragmented with a Covaris S2 sonicator (parameters: 900 s, burst 200, cycle 20%, intensity 8) in sonication buffer (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na-deoxycholate) and centrifuged at 13000 rpm and 4°C for 15 minutes. The supernatant was mixed with 4 µg normal rabbit IgG (R&D Systems, AB-105-C, lot: ER1212071) and incubated with ChIP-grade protein G magnetic beads (Cell Signalling 9006S, 25 µl/sample) at 4°C for 1:45 h (pre-clearance). 1/20 of the supernatant was used as input sample and the remaining material was used for the IP reaction by adding 4 µg of anti-H3K9me3 antibody (Abcam, ab8898, lot: GR148830-2). The sample was incubated for 2 h at 4°C, protein G magnetic beads were added and incubated at 4°C overnight. On the next day, the IP samples were washed with sonication buffer, high-salt buffer (50 mM Hepes pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), Li buffer (20 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and finally twice with TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Subsequently, the DNA-antibody complexes were eluted by incubation with a buffer containing 50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3 at 37°C for 15 minutes. IP samples and input control were treated with RNase A added to a final concentration of 10 µg/ml for one hour at 37°C. Then Proteinase K (Genaxxon Bioscience M3037) and NaCl were added to final concentrations of 80 µg/ml and 200 mM, respectively and samples were incubated over night at 65°C for protein digestion and reversal of crosslinks. DNA was precipitated and the input samples were used to assess the fragment size by gel electrophoresis on a 2% agarose E-Gel and on a DNA1000 bioanalyzer ChIP. Experiments were conducted for two replicates of each cell line.

first: Graeme

middle: J

last: Thorn

first: Christopher

last: Clarkson

first: Hulkar

last: Mamayusupova

first: Anne

last: Rademacher

first: Gunnar

last: Schotta

first: Karsten

last: Rippe


title: Pervasive transcription-coupled DNA repair in E. coli [RNA-seq 1]

status: Public on Feb 05 2022

city: New York

first: Evgeny

last: Nudler

state: NY

address: 450 E 29th St

country: USA

institute: NYU Medical Center

type: Expression profiling by high throughput sequencing

summary: Global Genomic Repair (GGR) and Transcription-Coupled Repair (TCR) have been viewed, respectively, as major and minor sub-pathways of the nucleotide excision repair (NER) process that removes bulky lesions from the genome. Here we applied a next generation sequencing assay, CPD-seq, in E. coli to measure the levels of cyclobutane pyrimidine dimer (CPD) lesions before, during, and after UV-induced genotoxic stress, and, therefore, to determine the rate of genomic recovery by NER at a single nucleotide resolution. We find that active transcription is necessary for the repair of not only the template strand (TS), but also the non-template strand (NTS), and that the bulk of TCR is independent of Mfd – a DNA translocase that is thought to be necessary and sufficient for TCR in bacteria. We further show that repair of both TS and NTS is enhanced by increased readthrough past Rho-dependent terminators. We demonstrate that UV-induced genotoxic stress promotes global antitermination so that TCR is more accessible to the antisense, intergenic, and other low transcribed regions. Overall, our data suggests that GGR and TCR are essentially the same process required for complete repair of the bacterial genome.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Transcription profiles of WT and mfd(-) E. coli with and without UV damage exposure (2 replicates).

first: Britney

last: Martinez

first: Binod

last: Bharati

first: Vitaly

last: Epshtein


title: AHR roles in regeneration of intestinal epithelium

status: Public on Feb 09 2022

city: London

first: Brigitta

last: Stockinger

address: 1 Midland Road

country: United Kingdom

institute: Francis Crick Insititue

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Pervasive transcription-coupled DNA repair in E. coli [RNA-seq 2]

platform_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Transcription profiles of WT and 2 Rho mutant strains (Rho-L187R and Rho-D210G).


title: Transcriptome alterations of hiPSC-CMs after alcohol treatment, alcohol and losartan co-treatment

city: Beijing

first: Yuanxiu

last: Song

address: 49 Huayuanbei Road, Haidian District, Beijing, China,100191

country: China

institute: Peking University Third Hospital

summary: In order to investigate the changes of hiPSC-CMS transcriptome after alcohol treatment and whether losartan has an effect on the changes of hiPSC-CM transcriptome after alcohol treatment, RNAseq was performed on hiPSC-CMs treated with 100mM alcohol and 100mM alcohol co-treated with1uM losartan.

overall_design: transcriptome alterations of hiPSC-CMs after 100mM alcohol treatment and 100mM alcohol co-culture with 1 μM losartan.

first: Hongxia

last: Li

first: Ming

last: Cui

first: Feng

last: Lan


title: CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity

city: Cambridge

first: Julia

last: Joung

state: MA

address: 75 Ames St

institute: Broad Institute

summary: RNA-seq data for candidate genes identified from CRISPR activation screen for tumor immune evasion

overall_design: Candidate genes were overexpressed in A375 cells for 7 days before RNA-seq

last: Zhang


title: Pervasive transcription-coupled DNA repair in E. coli


summary: ChIP-seq data for candidate gene JUNB identified from CRISPR activation screen for tumor immune evasion

overall_design: N-term and C-term FLAG tagged JUNB was overexpressed in A375 cells. ChIP was performed using FLAG-tagged antibodies.


title: Pervasive transcription-coupled DNA repair in E. coli [CPD-seq]

overall_design: Damage profiles over time in several different conditions.


title: Total RNA sequencing of mouse LSK cells in E14.5 fetal liver

status: Public on Feb 04 2022

city: Chicago

first: Susan

last: Ross

address: 835 South Wolcott Ave

department: Microbiology & Immunology

institute: University of Illinois at Chicago

summary: The study is to obtain a transcriptome-wide map of prenatal murine stem and progenitor cells. VavCre+ mice were used for conditional knockout of Ddx41. Wild-type, Ddx41 heterozygous (Ddx41+/-) and Ddx41 knockout (Ddx41-/-) LSK cells from embryonic 14.5 day fetal liver were harvested (three samples from each group) for RNA. Gene expression analysis and alternative splicing analysis were performed. RNA-seq data were confirmed by RT-qPCR and regular PCR followed by gel quantification. A global change of gene expression and substantial differential splicing events were observed in KO samples compared to WT and het.

overall_design: Total RNA profiling of E14.5 fetal liver LSK cells of WT (Ddx41+/+), het (Ddx41+/-) and KO (Ddx41-/-) mice.

middle: R

first: Jing

last: Ma


title: The PAX5-JAK2 translocation acts as a dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation

city: Vienna

first: Moritz

middle: M

last: Gaidt

address: Campus-Vienna-Biocenter 1

country: Austria

institute: IMP

summary: While PAX5 is an important tumor suppressor in B-ALL, it is also involved in oncogenic translocations coding for PAX5 fusion proteins. PAX5-JAK2 encodes a protein consisting of the PAX5 DNA-binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of PAX5-JAK2 in a mouse model expressing it from the endogenous Pax5 locus. The Pax5Jak2/+ mice rapidly developed an aggressive B-ALL in the absence of another cooperating mutation. The DNA-binding function and kinase activity of Pax5-Jak2, as well as IL-7 signaling, all contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the wild-type Pax5 allele, allowing efficient DNA binding of Pax5-Jak2. While we could not find evidence for a nuclear role of Pax5-Jak2 as an epigenetic regulator, active phosphorylated Stat5 was present at a high level in Pax5Jak2/+ B-ALL tumors, consistent with increased expression of Stat5 target genes. Together, these data identified Pax5-Jak2 as an important nuclear driver of leukemia formation by maintaining phosphorylated Stat5 levels in the nucleus.

overall_design:
6 Bio-ChIP-Seq samples, 2 experiments, 2 replicates per condition:  experiment 1: 29936 + 29938 (wild type)  experiment 2: 38870 + 40031 (experimental), 40030 + 40031 (control)  19 ChIP-Seq samples, 5 experiments:  experiment 1: 100885-100883, 12 samples  experiment 2: 138001 (input), 138002 (experimental), 138003 (control)  experiment 3: 35059 + 35061 (wild type)  experiment 4: 35327 (input)  experiment 5: 8265 (wild type)
21 RNA-Seq samples, 4 experiments, 2-4 replicates each; experiment 1: 36328/36329, 36346-36348 (6 samples); experiment 2: 24394-24397 (4 samples); experiment 3: 38492-38497 (5 samples); experiment 4: 99560-99565 (6 samples)

first: Sabine

last: Jurado

first: Anna

middle: S

last: Fedl

first: Markus

last: Jaritz

first: Daniela

last: Kostanova-Poliakova

first: Stephen

middle: G

last: Malin

first: Charles

last: Mullighan

last: Strehl

first: Maria

last: Fischer

first: Meinrad

last: Busslinger


title: Secretory cells are the primary source of pIgR in small airways

status: Public on Feb 11 2022

city: Nashville

first: Bradley

middle: Winston

last: Richmond

state: Tennessee

address: 1161 21st Avenue South

department: Medicine

institute: Vanderbilt University Medical Center

summary:
Background:  Loss of secretory immunoglobulin A (SIgA) is common in COPD small airways and likely contributes to disease progression.  We hypothesized loss of SIgA results from reduced expression of pIgR, a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium.
Methods:  pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in-situ hybridization, immunostaining, and single-cell RNA sequencing.  Complementary studies in mice utilized immunostaining, primary murine tracheal epithelial cell (MTEC) culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR.  SIgA degradation by human neutrophil elastase or secreted bacterial proteases from non-typeable Haemophilus influenzae (NTHi) was evaluated in vitro.
Results: We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways.  Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression.  Neutrophil elastase and NTHi-secreted proteases are both capable of degrading SIgA in vitro and may also contribute to a deficient SIgA immunobarrier in COPD.
Interpretation:  Loss of the SIgA immunobarrier in small airways of patients severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.

overall_design: We performed single-cell RNA sequencing on distal lung parenchymal samples from the explanted lungs of 11 COPD patients undergoing lung transplantation and 10 control patients.  Raw data from control samples is available under GSE135893.

middle: W

first: Jessica

middle: B

last: Blackburn


title: Reduced RXRa signaling increases dry eye disease inducing gdT17 cells in the conjunctiva

status: Public on Feb 03 2022

city: Houston

first: JEHAN

last: ALAM

state: TEXAS

address: Baylor One Plaza

institute: Baylor College of Medicine


title: Control of hematopoiesis by Cbx2

city: Boston

first: Grant

last: Rowe

address: 1 Blackfan Circle

institute: Boston Children's Hospital

summary: Hematopoiesis changes over life to meet the demands of maturation and aging.  Here, we find that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from gestation into adulthood, a process regulated by the heterochronic Lin28b/let-7 axis.  Native fetal and neonatal HSPCs distribute with a pro-lymphoid/erythroid bias with a shift toward myeloid output in adulthood.  By mining transcriptomic data comparing juvenile and adult HSPCs and reconstructing coordinately activated gene regulatory networks, we uncover the Polycomb repressor complex 1 (PRC1) component Cbx2 as an effector of Lin28b/let-7?s control of hematopoietic maturation.  We find that juvenile Cbx2-/- hematopoietic tissues show impairment of B-lymphopoiesis, a precocious adult-like myeloid bias, and that Cbx2/PRC1 regulates developmental timing of expression of key hematopoietic transcription factors.  These findings define a novel mechanism of epigenetic regulation of HSPC output as a function of age with potential impact on age-biased pediatric and adult blood disorders.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
CUT&RUN analysis of histone H2A lysine 119 monoubiquitylation in mouse and human hematopoietic stem and progenitor cells
Please note that raw data for the human HUDEP1 and K562 cell lines will be submitted to dbGAP.


title: Reduced RXRa signaling increases dry eye disease inducing gdT17 cells in the conjunctiva [Sequencing]

summary: Cultured monocytes were divided into three groups; 1) a control group 2) monocytes treated with LPS (0.5 ug/ml) for 6 hours 3) monocytes pre-treated with 100nM 9CisRA for 1 hour followed by LPS treatment for 6 hours.

overall_design: Examination of open and close promotor regions in monocytes in response to LPS and 9CisRA treatment

first: Jehan

last: Alam

first: Steven

last: Pflugfelder


title: Smarca4 ChIP-seq in XX and XY  mESCs

status: Public on Feb 02 2022

city: Royal Parade

first: Andrew

last: Keniry

state: Victoria

department: Molecular Medicine

country: Australia

institute: The Waler and Eliza Hall Institute of Medical Research

summary: This experiment is designed to test if Smarca4 acts directly on the establishing inactive X chromosome

overall_design: n=1. ChIP-seq for Smarca4 in male or female mESCs at day 4 of differentiation, with either depletion of Smarcc1 or nontargeting control

first: Marnie

last: Blewitt


title: RIP-Seq of Eif4a2 in mouse embryonic stem cell

first: Martin

last: Leeb

address: Dr.-Bohr-Gasse 9/3

department: Max Perutz Labs

institute: University of Vienna

summary: RNA-immunoprecipitation can be used to identify transcripts bound by RNA binding proteins.  Here we identified transcripts bound by the translation initiation factor Eif4a2 in ESCs. We found that several pluripotency transcription factor encoding mRNAs and factors regulating ESC differentiation show binding to Eif4a2 stronger than expected by transcript abundance.

overall_design: Identification of transcripts bound by Eif4a2

first: Elena

last: Galimberti

first: Robert

last: Sehlke

first: Michelle

last: Huth

first: Marius

last: Garmhausen

first: Merrit

last: Romeike

first: Julia

last: Ramesmayer

first: Sarah

last: Stummer

first: Fabian

last: Titz-Teixeira

first: Veronika

last: Herzog

first: Anastasia

last: Chugunova

first: Katrin

middle: F

last: Leesch

first: Laurenz

last: Holcik

first: Klara

last: Weipoltshammer

first: Laura

last: Santini

first: Arndt

last: von Haeseler

first: Christa

last: Bücker

first: Andrea

last: Pauli

first: Christian

last: Schoefer

first: Stefan

middle: L

last: Ameres

first: Austin

last: Smith

first: Andreas

last: Beyer


title: AI-guided use of balanced PPARα/g dual agonist finetunes macrophage responses in inflammatory bowel disease

city: La Jolla

first: Debashis

last: Sahoo

state: California

address: 9500 Gillman Drive

department: Pediatrics

institute: UCSD

summary: A computational platform, the Boolean network explorer (BoNE), has recently been developed to infuse AI-enhanced precision into drug discovery; it enables querying and navigating invariant Boolean Implication Networks of disease maps for prioritizing high-value targets. Here we used BoNE to query an Inflammatory Bowel Disease (IBD)-map and prioritize two nuclear receptors, PPARa/g. Balanced agonism of PPARa/g was predicted to impact macrophage processes, ameliorate colitis in network-prioritized animal models, ‘reset’ the gene expression network from disease to health, and achieve a favorable therapeutic index that tracked other FDA-approved targets. Predictions were validated using a balanced and potent PPARa/g-dual agonist (PAR5359) in two pre-clinical murine models, i.e., Citrobacter rodentium-induced infectious colitis and DSS-induced colitis. Mechanistically, we show that such balanced dual agonism promotes bacterial clearance more efficiently than individual agonists both in vivo and in vitro; PPARa is required and its agonism is sufficient to induce the pro-inflammatory cytokines and cellular ROS, which are essential for bacterial clearance and immunity, whereas PPARg-agonism blunts these responses, delays microbial clearance and induces the anti-inflammatory cytokine, IL10. Balanced agonism achieved controlled inflammation while protecting the gut barrier and ‘reversal’ of the transcriptomic network. Furthermore, dual agonism effectively reversed the defective bacterial clearance observed in PBMCs derived from IBD patients. These findings not only deliver a macrophage modulator for use as barrier-protective therapy in IBD, but also highlight the potential of BoNE to accelerate and enhance the precision of drug discovery.

overall_design: Mice were orally challenged with C. rodentium at 5 x 108 CFU in 0.1 ml of PBS. Distal colon samples were collected to assess histology and levels of mRNA (by qPCR and RNA Seq). PPARa agonist (GW7647, 20 ug/kg body weight/day), PPARg agonist (Pioglitazone, 20mg/kg body weight/day), PPARa/g dual agonist (PAR5359, 1 mg/kg body weight/day) were administered via intraperitoneal route in 200 ul total volume (DMSO less than 4%). Total RNA was isolated using TRIzol reagent (Life Technologies) and Quick-RNA MiniPrep Kit (Zymo Research, USA). Sequencing was performed.

first: Soumita

last: Das

first: Pradipta

last: Ghosh


city: Columbus

first: Erin

last: Gloag

state: Ohio

address: 460 W. 12th Ave

department: Microbial  Infection and Immunity

institute: The Ohio State Univerisity

summary:
Purpose: Use RNA-seq to investigate the transcriptional response of Pseudomonas aeruginosa when adhering to different surfaces.
Methods: Coupons of silicone, glass, and polycarbonate plastic were purchased from Biosurfaces Technologies (Catalog numbers: RD128-Si, RD128-GL, RD128-PC). Coupons were placed into a 24-well plate and conditioned in LB for at least 10 min prior to bacterial addition. LB was removed and replaced with 2mL of logarithmic phase bacterial culture in LB (OD600 0.7). At the indicated time point, the coupon was rinsed twice in PBS and placed into 1 mL of Trizol. Trizol was collected and stored at -80°C for RNA isolation. HiSeq 4000 sequencing was performed, generating approximately 300 million total paired end 300 bp reads. Reads were aligned to the reference PAO1 genome using Rockhopper.
Results: A total of 833 genes were differentially expressed across the three surfaces. Most genes were reduced (522 genes) rather than elevated (269 genes), with 42 genes showing both elevated and reduced RNA levels across two or more surfaces.
Conclusion: Bacteria had a nearly unique transcriptional response on each surface, with the vast majority regulated on only one surface

overall_design: RNA-Seq analysis of wild type Pseudomonas aeruginosa adhered to three different medically relevant surfaces; silicone, glass, and polycarbonate plastic. Each time point was analyzed in triplicate

first: Christopher

last: Jones

first: Nikolas

last: Grotewold

first: Daniel

last: Wozniak


title: Global mRNA half-lives measurement in nonsense-mediated mRNA decay deficient ESCs

first: Martin

last: Leeb

summary: Nonsense-mediated mRNA decay (NMD) pathway promotes the degradation of several mRNA species, including transcripts containing a premature termination codon. Here,we measured global mRNA half-lives in NMD deficient and wild-type (WT) embryonic stem cells (ESCs). This allowed us to discriminate which transcripts are affected by NMD. Several transcripts had an impaired half-life in Smg5 and Smg6 KO ESCs compared to WT. They belong to different pathways and cellular processing indicating that NMD regulates multiple processes in the cells. Finally, majority of the transcripts had an impairment on the half-life in both Smg5 and Smg6 KO ESCs indicating that endo- and exonucleolityc branches of the NMD pathway target the same mRNAs.

overall_design: Measurement of global mRNA half-lives in WT and NMD deficient ESCs by SLAM-Seq. We generated stable Smg5 KO and Smg6 KO ESCs. We then measured mRNAs half-lives in Smg5 and Smg6 KO and in WT ESCs.

first: Elena

last: Galimberti

first: Robert

last: Sehlke

first: Michelle

last: Huth

first: Marius

last: Garmhausen

first: Merrit

last: Romeike

first: Julia

last: Ramesmayer

first: Sarah

last: Stummer

first: Fabian

last: Titz-Teixeira

first: Veronika

last: Herzog

first: Anastasia

last: Chugunova

first: Katrin

last: Leesch

first: Laurenz

last: Holcik


title: Expression profiling of tracheobronchial basal cells derived from patients with Tracheobronchopathia Osteochondroplastica (TO), before and after in vitro differentiation

city: Haikou

first: Yue

last: Hong

address: 58 Renmin Road

department: School of Life and Health Sciences

institute: Hainan University

summary: Next-generation sequencing of mRNA extracted from immature tracheobronchial basal cells and their differentiated structures from air-liquid interface (ALI) was performed to quantitatively analyze TO-associated changes at transcriptional levels. RNA-Seq data revealed distinct expression profiles and divergent fate commitments between normal tissue-derived and TO lesion region-derived basal cells. In particular, there were 339 genes up-regulated at stem cell state and 1266 up-regulated upon differentiation (cutoff at fold change>2, p<0.05) in TO, whilst 173 TO-associated down-regulated genes and 1646 genes with reduced expression (cutoff at fold change<-2, p<0.05) were detected before and after differentiation respectively, compared to normal controls. Our results show that TO-derived basal cells, although exhibit high similarities on clone morphology and key marker staining, are transcriptionally different from normal tissue-derived counterpart and possess altered multipotency evidenced by transcriptome characterization. To further evaluate airway basal cell conditions in lesion-free regions of TO patients, RNA-Seq data was generated on cultured TBBCs derived from patient-matched (PM) relatively normal regions, and their ALI differentiated structures. Compared to stem cell state, there were 3314 genes induced in expression whilst 1627 down-regulated in the process of PM-TBBCs differentiation. Distinct differentiation-induced gene expression patterns were detected across TO patients, indicating a change of basal cell condition from normal mucociliary-committed status to a more disease-prone, inflammatory state during TO progression, and such basal cell alteration appeared prior to nodule formation.

overall_design: RNA-Seq data was generated on normal tissue-derived （donors n=7）, TO PM-derived  and TO lesion region-derived basal cell clones (donors n=6); for differentiated state, data was generated on normal cell-derived and TO cell-derived ALIs, corresponding to sample cases of which basal cell clones were subjected to RNA-Seq.

first: Tao

last: Ren


title: Constant inhibition of local BMP signaling contributes to recovery of mucociliary epithelium from Tracheobronchopathia Osteochondroplastica (TO)-associated pathophysiology via basal cell restoration

summary: Tracheobronchial basal cells have been found to undergo persistent functional changes, via acting as a local repository for BMP signaling elements, thereby play a driven role in TO pathogenesis. Although it would be unlikely to fundamentally correct cell fate alteration, because of epigenetic changes occurred, partial restoration of epithelial physiology may be achieved by braking self-amplifying BMP signaling loop. To investigate the effect of BMP inhibition on TO-derived basal cells, ALI structures derived from routine differentiation without Noggin treatment and modified differentiation with constant BMP inhibition were subjected to RNA-sequencing. While a differentiation pattern of 3538 genes up-regulated and 2109 genes down-regulated (>2-fold or <-2-fold, p<0.05) upon routine differentiation of a typical TO-derived pedigree, differentiation in the presence of BMP inhibition resulted in an up-regulation of 4101 genes and down-regulation of 2337 genes (equivalent cutoff as above). Among those 1554 genes with significantly increased expression on ALI under BMP inhibition, a major enrichment shown up in categories related to cilium assembly and function, and an up-turned Smoothened signaling were detected. In contrast, 482 genes with decreased expression under BMP inhibition were mainly fallen into categories related to biomineral tissue development. These data suggest that constant repression of local BMP signaling has a favorable effect, to some extent, on functional rescue of pathological basal cells.

overall_design: Samples at stem cell state, differentiated without BMP inhibitor treatment and differentiated along BMP inhibition were analyzed by RNA-Seq, in singleton.


title: Global mRNA half-lives measurement and RIP-Seq of Eif4a2 in mouse embryonic stem cell

overall_design: Refer to individual Series


title: RNA-seq in differentiating female FVBxCAST F1 ES cells with Smarca4 gene knockdown [Female Smarca4 RNA-seq]

first: Andrew

last: Keniry

summary:
Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation.
This experiment is designed to test if X chromosome inactivation is altered upon Smarca4 gene knockdown.

overall_design: n=1 Smarca4 knockdown for two different hairpins, n=1 for matched nonsilencing controls. Samples taken at day 0 and 6 of FVBxCAST F1 ES cell differentation

first: Marnie

last: Blewitt


title: Transcriptional and epigenetic profiling of tracheobronchial basal cells derived from patients with Tracheobronchopathia Osteochondroplastica (TO)

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


title: Neuronal and cardiac toxicity of pharmacological compounds identified through transcriptomic analysis of human pluripotent stem cell-derived embryoid bodies

status: Public on Jan 31 2022

city: Bangalore

first: Rajarshi

last: Pal

address: CCAMP, NCBS

country: India

institute: Eyestem research

summary: Concurrent with the '3R' principle, the embryonic stem cell test (EST) using mouse embryonic stem cells, developed in 2000, remains the solely accepted in vitro method for embryotoxicity testing. However, the scope and implementation of EST for embryotoxicity screening, compliant with regulatory requirements, are limited. This is due to its technical complexity, long testing period, labor-intensive methodology, and limited endpoint data, leading to misclassification of embryotoxic potential. In this study, we used human induced pluripotent stem cell (hiPSC)-derived embryoid bodies (EB) as an in vitro model to investigate the embryotoxic effects of a carefully selected set of pharmacological compounds. Morphology, viability, and differentiation potential were investigated after exposing EBs to folic acid, all-trans-retinoic acid, dexamethasone, and valproic acid for 15 days. The results showed that the compounds differentially repressed cell growth, compromised morphology, and triggered apoptosis in the EBs. Further, transcriptomics was employed to compare subtle temporal changes between treated and untreated cultures. Gene ontology and pathway analysis revealed that dysregulation of a large number of genes strongly correlated with impaired neuroectoderm and cardiac mesoderm formation. This aberrant gene expression pattern was associated with several disorders of the brain like mental retardation, multiple sclerosis, stroke and of the heart like dilated cardiomyopathy, ventricular tachycardia, and ventricular arrhythmia. Lastly, these in vitro findings were validated using in ovo chick embryo model. Taken together, pharmacological compound or drug-induced defective EB development from hiPSCs could potentially be used as a suitable in vitro platform for embryotoxicity screening.

overall_design: We tested low/ medium/ heavy toxicity imparting compounds using in vitro platform for embryotoxicity screening with pluripotent stem cell derived embryoid bodies


title: NOMe-seq in differentiating FVBxCAST F1 ES cells with Smarcc1 gene knockdown [NOMe-Seq]

type: Methylation profiling by high throughput sequencing

summary:
Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation.
This experiment is designed to test if nucleosome remodelling during X chromosome inactivation is altered upon Smarcc1 gene knockdown.

overall_design: n=1 Smarcc knockdown, n=1 Nonsilencing control. Samples taken at days 0, 4, 5, 6 and 7 of FVBxCAST F1 ES cell differentation


title: RNA-seq in differentiating Xmas cells ES cells [Xmas Differentiation RNA-seq]

summary:
Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation.
This experiment is designed to test if Xmas ES cells are transcriptionally similar to published ES cell lines during differentiation.

overall_design: n=4. Samples taken daily for 9 days of Xmas ES cell differentation


title: PM01183 and Cisplatin on SCLC cell lines

city: Colmenar Viejo

first: Javier

middle: Díez

last: Pérez

state: Madrid

address: Avda. De los Reyes, 1

department: I+D

country: Spain

institute: Pharma Mar

summary: Small Cell Lung Cancer (SCLC) is an aggressive neuroendocrine malignancy with a poor prognosis. Here, we focus on the neuroendocrine SCLC subtypes SCLC-A and SCLC-N, whose transcription addiction was driven by ASCL1 and NEUROD1 transcription factors which target E-box motifs to activate up to 40% of total genes, the promoters of which are maintained in a steadily open chromatin environment according to ATAC and H3K27Ac signatures. This leverage is used by the marine agent lurbinectedin, which preferentially targets the CpG islands located downstream of the transcription start site, thus arresting elongating RNAPII and promoting its degradation. This abrogates the expression of ASCL1 and NEUROD1 and of their dependent genes such as BCL2, INSM1, MYC and AURKA, which are responsible for relevant SCLC tumorigenic properties such as inhibition of apoptosis and cell survival, as well as for a part of its neuroendocrine features. In summary, we show how the transcription addiction of these cells becomes their Achilles' heel, and how this is effectively exploited by lurbinectedin as a novel and highly specific SCLC therapeutic endeavor.

overall_design: detect the impact of lurbinectedin vs cisplatin on transciption factor binding and replication

first: Federico

last: Costanzo

middle: D


title: Comparison of genome architecture at two stages of male germline cell differentiation in Drosophila

status: Public on Feb 01 2022

city: Moscow

first: Anna

last: Kononkova

address: Nobel st., Building 1

department: Skoltech Center of Life Sciences

country: Russia

institute: Skolkovo Institute of Science and Technology

summary: Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs), some of them being attached to the nuclear lamina (NL) in lamina-associated domains (LADs). Here, we identified LADs and TADs at two consecutive stages of Drosophila spermatogenesis – in spermatogonia (SpG) and spermatocytes (SpCs). We found that launching of SpC-specific transcription frequently correlates with promoters’ detachment from the NL accompanied by spatial insulation of adjacent regions. We also revealed an increased contact frequency between SpC-specific genes implying their de novo gathering into “transcription factories”. In SpG and SpCs, single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.

overall_design: Hi-C analysis of chromatin spatial structure in Drosophila male germline cells at two consequitive stages of differentiation.


title: RNA sequencing of 53 early stage lung adenocarcinoma and AMG900-treated LUAD cells

first: Seungyeul

last: Yoo

address: 1425 Madison Avenue

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai


city: Stockholm

first: Artem

last: Ilin

address: Svante Arrhenius väg 20C

department: MBW

country: Sweden

institute: Stockholm University

summary: Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). Here, we identified LADs and TADs at two stages of Drosophila spermatogenesis – in bam∆86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). We found that initiation of SpC-specific transcription correlates with promoters’ detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. We also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, we uncovered the specific X chromosome organization in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.

overall_design: Amplified PCR fragments from DamID procedure were sequenced on Illumina HiSeq 2000, cDNA libraries were sequenced on Illumina Novaseq 6000

middle: A

last: Ilyin


title: RNA sequencing of 53 early stage lung adenocarcinoma

city: New York

summary: Lung adenocarcinoma is the most common histological subtype of lung cancer. Although early-stage LUAD (esLUAD) patients have much better prognosis than patients with advanced disease, among early stage patients treated primarily with surgery, some of early stage patients will develop metastasis with overall 5 year survival for stage 1 and 2 non-small cell lung cancer of 70% and 35%, respectively. Within lung adenocarcinoma, histology is heterogenous and associated with tumor invasion and clinical outcomes. Invasiveness is one of cancer hallmarks and is directly related with metastatic potential and clinical outcomes of the tumor. In this study, we characterize invasiveness mechanisms in esLUAD by analyzing gene expression of a novel cohort of 53 histologically heterogenous esLUAD samples.

overall_design: Tumor specimens were acquired from 53 patients of resected lung adenocarcinoma in situ and minimally invasive adenocarcinoma. Tumors tissue were collected from 2000~2010 and retrieved via an IRB approved protocol. All cases of AIS, MIA and LPA for which frozen tissue was collected were obtained. In addition, small invasive adenocarcinomas (prioritized as <2.0 cm, but all<3.0 cm) with node metastasis were identified (10 cases) and matched to 10 cases of the same T stage size grouping and with the same predominant invasive pattern but lacking node metastasis.

first: Abhilasha

last: Sinha

first: Jun

last: Zhu

first: Charles

last: Powell


title: Signatures of protein and small RNA cargoes in extracellular vesicles of mouse testis

city: New Territories

first: Ellis

last: Fok

address: Shatin

department: School of Biomedical Sciences

country: Hong Kong

institute: Chinese University of Hong Kong

type: Non-coding RNA profiling by high throughput sequencing

summary: We report the small RNA transcriptome of testicular extracellular vesicles in mouse testis. We established a testis dissociation protocol to isolate testicular extracellular vesicles. After treatment with proteinase K and RNase A, the RNA inside the extracellular vesicles was extracted and sequenced by small RNA-seq.

overall_design: Investigate the RNA species in extracellular vesicles of mouse testis. Two sets of sequencing were performed

middle: K


title: H3K27ac ChIP upon NeuroD1 overexpression

city: Belfast

first: Aditi

last: Singh

state: Please Select State

address: 97 Lisburn Road, Belfast

department: The Wellcome-Wolfson Institute for Experimental Medicine, SMDBS

institute: Queen's University Belfast

summary: Cell-fate specification relies on a close cooperation of distinct transcription factors and their crosstalk with the epigenetic landscape during embryonic development. NeuroD1 is known to be essential for eliciting neurogenesis and is able to reprogram other cell types into neurons. Despite progress, its global impact on the epigenome and cooperativity with other transcription factors during neurogenesis remains unknown. Here we show that despite NeuroD1 binding and a gain of active chromatin, only sites that show high local density of adjacent bHLH motifs, higher minor groove width and propeller twist as well as enrichment of another novel bHLH motif were able to get transcriptionally induced. Transcription factor activity analysis on the epigenome timecourse following NeuroD1 induction further identified this bHLH factor among top factors that gained activity during this process. Importantly, this bHLH factor was induced simultaneously to NeuroD1 as cells transit from an apical to a basal progenitor state during cortical development. Moreover, this bHLH factor interacts with NeuroD1 and co-occupies NeuroD1 target distinct genomic regions. We further show that the loss of this bHLH factor during cortical development severely impairs neurogenesis. Our study not only reveals the mechanistic basis underlying a productive transcriptional response of NeuroD1 during neurogenesis but also identified a novel bHLH transcription factor that partners with NeuroD1 and plays a critical role during cortical development.

overall_design: 1. Ectopic NeuroD1 was induced for 48 hours (+Dox) in ES cells for checking initiation of neuronal transcriptional program in comparison to uninduced condition (-Dox); 2. H3K27ac ChIP-seq was performed after 0hours, 12 hours and 48 hours of NeuroD1 induction in ES cells.

first: Arun

last: Mahesh

first: Florian

last: Noack

last: Calegari

first: Vijay

last: Tiwari


title: RNA-seq in differentiating male C57/BL6 ES cells with Smarcc1 or Smarca4 gene knockdown [Male Smarc RNA-seq]

last: Keniry

summary: This experiment is designed to test if differentiation is altered upon depletion of either Smarcc1 or Smarca4

overall_design: n=1 Smarcc1 knockdown for two different hairpins, n=1 Smarca4 knockdown for two different hairpins, n=2 for matched nonsilencing controls. Samples taken at day 4, 5, 6 and 7 of  ES cell differentation

last: Blewitt


title: Pseudo-temporal analysis of Single-cell RNA sequencing reveals trans-differentiation potential of Greater Epithelial Ridge cells into hair cells during postnatal development of cochlea in Rats

status: Public on Jan 29 2022

city: Shanghai

first: Jianyong

last: Chen

state: Yangpu

address: 1665 Kongjiang Rd

department: Otorhinolaryngology Head and Neck Surgery

institute: Xinhua Hospital, Shanghai Jiaotong University School of Medicine

summary: The hair cells of the cochlea play a decisive role in the process of hearing damage and recovery, yet knowledge of their regeneration process is still limited. Greater epithelial ridge (GER) cells, a type of cell present during cochlear development that has the characteristics of a precursor sensory cell, disappear at the time of maturation of hearing development. Its development and evolution remain mysterious for many years. Here, we performed single-cell RNA sequencing to profile the gene expression landscapes of rats' cochlear duct from P1, P7, and P14 and identified eight major subtypes of GER cells. Furthermore, single-cell trajectory analysis for GER cells and hair cells indicated that among the different subtypes of GER, four subtypes had transient cell proliferation after birth and could transdifferentiate into inner and outer hair cells, and two of them mainly transdifferentiated into inner hair cells. The other two subtypes eventually transdifferentiate into outer hair cells. Our study lays the groundwork for elucidating the mechanisms of the key regulatory genes and signaling pathways in the trans-differentiation of GER cell subtypes into hair cells and provides potential clues to understand hair cell regeneration.

overall_design: The cochlear duct of postnatal rats at day 1 (P1) , day 7 (P7) and day 14 (P14) to identify subsets of greater epithelial ridge cell and hair cell, and pseudo-temporal analysis of single-cell RNA sequencing to reveal the trans-differentiation potential of Greater Epithelial Ridge cells into hair cells.

first: Dekun

last: Ga

first: Junmin

last: Yang


title: Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

status: Public on Jan 30 2022

first: Aixia

last: Song

address: Dong'an Road

institute: Fudan University

summary: Pleiotropic transcription regulator RNA polymerase II (Pol II)-associated factor 1 (PAF1) governs multiple transcriptional steps and the deposition of several epigenetic marks. However, it remains unclear how ultimate transcriptional outcome is determined by PAF1 and whether it relates to PAF1-controlled epigenetic marks. We utilize rapid degradation systems and reveal direct PAF1 functions in governing pausing partially by recruiting Integrator-PP2A (INTAC), in addition to ensuring elongation. Following acute PAF1 degradation, destabilized polymerase undergoes early termination or effective release, which presumably relies on skewed balance between INTAC and P-TEFb resulting in hyperphosphorylated substrates including SPT5. Impaired Pol II progression during elongation, along with altered pause release frequency, determines the final transcriptional outputs. Moreover, PAF1 degradation causes a cumulative decline in histone modifications. These epigenetic alterations in chromatin likely further influence the production of transcripts from PAF1 target genes.

overall_design: We performed PRO-seq, ChIP-seq, or TT-seq to investigate the role of PAF1 in regulating transcription. In this study, samples for each condition were collected in biological duplicates. Cells were treated with dTAG13 for 3,or 24 hours, the corresponding control cells were treated for the same duration with vehicle only (DMSO) at the same vol/vol dilution.

first: Zhenning

last: Wang

first: Fei

middle: X


title: PM01183 on SCLC cell lines [ChIP-Seq, DMS-53]

city: Bellinzona

first: Federico

last: Costanzo

address: Via Vela 6

country: Switzerland

institute: Institute of Oncology Research IOR

summary: detect the impact of lurbinectedin on transciption factor binding

overall_design: DMS-53 cell line

first: Juan

last: Ignacio Diaz Hernandez


title: Characterization of genome-wide chromatin accessibility in airway basal cells derived from patients with Tracheobronchopathia Osteochondroplastica (TO)

first: Yue

last: Hong

summary: The assay for transposase-accessible chromatin using sequencing (ATAC-Seq) was applied to investigate the TO-associated chromatin alteration(s) appeared in tracheobronchial basal cells (TBBC). ATAC-Seq data was generated on cultured TBBCs derived from lesion regions and patient-matched lesion-free regions of two patients with Stage I and Stage III disease respectively, in parallel with control TBBCs from two normal donors. Six samples were spontaneously divided into two main groups (TO and non-TO) based on both differentiation phenotype and clustering results. The relationship between chromatin accessibility and future gene expression during basal stem cell differentiation was analyzed in combination with RNA-Seq. We identified 222914 increased chromatin accessible regions whilst 6406 decreased chromatin accessible regions in TO cells compared to non-TO cells. Among 641 loci with TO-related increased accessibility at the promoter region, 22 were associated with up-regulated gene expression (cutoff at fold change>2, p<0.05), and out of which, 18 exhibited enhanced transcriptional induction during basal cell differentiation. On the other hand, there were 459 loci with decreased accessibility at the promoter region in TO, among which, 107 were associated with down-regulated expression (cutoff at fold change<-2, p<0.05) and 58/107 exhibited transcriptional suppression during differentiation. Our study suggests that chromatin remodeling may provide an explanation for the sustained basal cell malfunction in TO pathogenesis.

overall_design: The landscape of altered chromatin accessibility in TO-derived tracheobronchial basal cells was explored by ATAC-Seq, in biological triplicate, compared to Non-TO basal cells derived from normal airway tissues.

first: Tao

last: Ren


title: PM01183 on SCLC cell lines (ChIP-Seq)

overall_design: PM01183 on SCLC cell lines

middle: I

last: Diaz Hernandez


title: Tissue extracellular matrix hydrogels as alternative to Matrigel for culturing gastrointestinal organoids

city: Seoul

first: Suran

last: Kim

address: Yonsei-ro 50

department: Department of Biotechnology

country: South Korea

institute: Yonsei University

summary: To determine the performance of the decellularized GI tissue ECM hydrogel, we analyzed differences in mRNA expression levels in native gastric tissues, gastric organoids cultured in Matrigel and those cultured in decellularized gastric ECM hydrogel. We also compared mRNA expression levels of native small intestinal tissues, small intestinal organoids grown in Matrigel and those grown in decellularized intestinal ECM hydrogel.

overall_design: Examination of mRNA expression levels in 6 different groups

first: Sungjin

last: Min

first: Yi

last: Choi

first: Sung-Hyun

last: Jo

first: Jae

last: Jung

first: Kyusun

last: Han

first: Jin

first: Yong

last: Ji

first: Yun-Gon

first: Seung-Woo

last: Cho


title: Aryl hydrocarbon receptor-dependent and -independent pathways mediate curcumin anti-aging effects

city: Düsseldorf

first: Vanessa

last: Brinkmann

state: NRW

address: Auf'm Hennekamp 50

country: Germany

institute: IUF- Leibniz Research Institute for Environmental Medicine

summary:
In this study we investigated if curcumin has AhR-dependent effects on aging.
This transcriptomic analysis should help shed light on the AhR-dependent and AhR-independent mechanisms of curcumin.

overall_design: transcriptomic analysis in wild-type and ahr-1(ju145) either treated with DMSO or curcumin

first: Natascia

last: Ventura

first: Alfonso

last: Schiavi


title: RNA-seq in differentiating female FVBxCAST F1 ES cells with Smarcc1 gene knockdown [Female Smarcc1 RNA-seq]

first: Andrew

last: Keniry

institute: The Waler and Eliza Hall Institute of Medical Research

summary:
Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation.
This experiment is designed to test if X chromosome inactivation is altered upon Smarcc1 gene knockdown.

overall_design: n=1 Smarcc1 knockdown for two different hairpins, n=1 for matched nonsilencing controls. Samples taken at day 0, 4, 5, 6 and 7 of FVBxCAST F1 ES cell differentation

first: Marnie

last: Blewitt


title: RNA sequencing of AMG900-treated LUAD cells

first: Seungyeul

last: Yoo

summary: From RNAseq analysis of 53 esLUAD, we identified a gene signature distinguishing invasive and indolent tumors. Regulatory network analysis identified aurora kinases as master regulators of the gene signature. So we applied an aurora kinase pan inhibitor, AMG900, using A549 and H1792 cells to confirm whether the expression of pro-invasive genes were impacted by aurora kinase activity suppression.

overall_design: For RNA sequencing of A549 and H1792 cells treated with DMSO or 0.1 uM AMG900 for 48 hours. Total RNA was purified from cells using RNeasy kit (Qiagen) for sequencing performed on NextSeq 500 (Illumina).

first: Abhilasha

last: Sinha

first: Jun

last: Zhu

first: Charles

last: Powell


title: LNCaP prostate cancer cells

status: Public on Jan 28 2022

city: Philadelphia

first: Peter

state: PA

address: 3501 Civic Center Blvd, CTRB 4400

department: Pathology and Laboratory Medicine

institute: The Children's Hospital of Philadelphia

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

overall_design: Refer to individual Series


title: PM01183 on SCLC cell lines Chem-seq

first: Federico

last: Costanzo

summary: Chem seq to compare the binding activity of biotynilated drugs.Lurbinectedin and cisplatin comparison

overall_design: 6 samples

first: Juan

last: Hernandez


title: RNA-sequencing in LNCaP prostate cancer cells

last: Choi

summary: We used RNA-Seq to profile gene expression in LNCaP cells with and without PRMT1 knockdown.

overall_design: RNA-Seq of LNCaP cells infected with doxycycline-inducible PRMT1 shRNA and were either untreated (control) or treated with doxycycline to induce PRMT1 knockdown. Three replicates for each condition.

first: Stephen

last: Tang

first: Srinivas

last: Viswanathan


title: Single cell immune response to the mRNA SARS-CoV-2 vaccine and yellow fever in the lymph nodes of mice

city: Stanford

first: Madeleine

last: Scott

state: CA

address: 729 Lagunita Dr

institute: Stanford

summary:
Purpose: to understand the mechanisms of vaccines in the lymph nodes of mice
Methods: mice were treated with mRNA SARS-CoV-2 vaccine or the yellow fever vaccine. The draining inguinal or illiac lymph nodes were harvested 1, 3, or 7 days after treatment and analyzed by scRNA-seq
Results: TBD
Conclusions: TBD

overall_design: Mice treated with either yellow fever vaccine (YFV) or Pfizer BioNTech mRNA COVID vaccine (mRNA). Samples were taken from the draining inguinal lymph node (LN) or the illiac lymph node (LC). Lymph nodes were harvested one day (D1), three days (D3) and seven days (D7) after treatment.

first: Chunfeng

last: Li

first: Prabhu

last: Arunachalam


title: Phase I clinical trial evaluating the safety and anti-tumor activity of ADP-A2M10 SPEAR T-cells in patients with MAGE-A10+ head and neck, melanoma, or urothelial tumors

city: Houston

first: David

last: Hong

state: Texas

address: Room FC8.3050, 1400 Holcombe Blvd.

department: Department of Investigational Cancer Therapeutics

institute: University of Texas M.D. Anderson Cancer Center

summary: Expression of several gene groups was compared between pre-treatment tumour samples from patients with either head&neck, melanoma or urothelial cancers

overall_design: Pre-treatment (screening or baseline) biopsies from primary or metastatic tumours (3 each: head&neck, melanoma and urothelial cancers) were compared.

first: Marcus

last: Butler

first: Russell

last: Pachynski

first: Ryan

last: Sullivan

first: Partow

last: Kebriaei

first: Sarah

last: Boross-Harmer

first: Armin

last: Ghobadi

first: Matthew

last: Frigault

first: Ecaterina

middle: E

last: Dumbrava

first: Amy

last: Sauer

first: Francine

last: Brophy

first: Jean-Marc

last: Navenot

first: Svetlana

last: Fayngerts

first: Zohar

last: Wolchinsky

first: Robyn

last: Broad

first: Dzmitry

last: Batrakou

first: Ruoxi

last: Wang

first: Luisa

last: Soto

first: Dzifa

middle: Y

last: Duose

first: Joseph

middle: P

last: Sanderson

last: Gerry

first: Diane

last: Marks

first: Jane

last: Bai

first: Elliot

last: Norry

first: Paula

last: Fracasso


title: PM01183 on SCLC cell lines (ATAC-Seq)

first: Federico

last: Costanzo

first: Juan

last: Diaz Hernandez


title: The tubulin code during cardiac hypertrophy and failure [III]

first: Benjamin

last: Prosser

address: 700 CRB, 415 Curie Blvd

department: Physiology

institute: University of Pennsylvania

summary: A proliferated and post-translationally modified microtubule network underlies cellular growth in cardiac hypertrophy and contributes to contractile dysfunction in heart failure. Yet how the heart achieves this modified network is poorly understood. Determining how the “tubulin code” – the permutations of tubulin isoforms and post-translational modifications - is rewritten upon cardiac stress may provide new targets to modulate cardiac remodeling. Further, while tubulin can autoregulate its own expression, it is unknown if autoregulation is operant in the heart or tuned in response to stress. Here we use heart failure patient samples and murine models of cardiac remodeling to interrogate transcriptional, autoregulatory, and post-translational mechanisms that contribute to microtubule network remodeling at different stages of heart disease.  We find that autoregulation is operant across tubulin isoforms in the heart and leads to an apparent disconnect in tubulin mRNA and protein levels in heart failure. We also find that within 4 hours of a hypertrophic stimulus and prior to cardiac growth, microtubule detyrosination is rapidly induced to help stabilize the network. This occurs concomitant with rapid transcriptional and autoregulatory activation of specific tubulin isoforms and microtubule motors. Upon continued hypertrophic stimulation, there is an increase in post-translationally modified microtubule tracks and anterograde motors to support cardiac growth, while total tubulin content increases through progressive transcriptional and autoregulatory induction of tubulin isoforms. Our work provides a new model for how the tubulin code is rapidly rewritten to establish a proliferated, stable microtubule network that drives cardiac remodeling, and provides the first evidence of tunable tubulin autoregulation during pathological progression.

overall_design: For animal experiments: 8wks old C57/Bl6 mice were used. total RNA was isolated from mouse heart tissue harvested after 4 hours or 4 days (day 0 and day 2 injection) post 10mg/kg phenylephrine or 5mg/kg isoproterenol injections. Isolated cardiomyocytes treated with 6h of DMSO or 10uM colchicine or 10uM tax were used as positive control in the tubulin autoregulation panel. (Replicates: 4h Ctrl, 4h PE, 4h Iso = 6 and 4d Ctrl = 7, 4d PE = 7, 4d Iso = 6) For human experiment: total RNA was isolated from post-mortem heart tissue. (Replicates: non-failing = 12, failing = 23) The total RNA were analyzed using custom Nanostring nCounter panels.

first: Sai

last: Phyo


title: RNAsequencing of DOT1L inhibitor EPZ5676 treated ALDH1+ and ALDH1- cell populations

city: Miami

first: Derek

last: Van Booven

state: FL

address: 1501 NW 10th Ave

institute: University of Miami

summary: Global gene expression profiles obtained after treating cells with DOT1L inhibitor, EPZ5676

overall_design: MDA-MB-468 cells were first treated with DOT1L inhibitor, EPZ5676 for 10 days and then FACS isolated into ALDH1+ and ALDH1- cell populations. Global gene expression profiles that change after treatment with DOT1L inhibitor are reported.

first: Hetakshi

last: Kurani

first: Joyce

last: Slingerland


title: PM01183 on SCLC cell lines


title: Expression data from the visceral adipose tissues of Lgr4 mutant (KO) and wild-type (WT) mice

first: Jiqiu

address: No. 197, Ruijin 2nd Road

department: Endocrinology and Metabolism

institute: Ruijin Hospital

summary:
Lgr4 acts as a key factor to regulate energy expenditure and body weight. Visceral fat (epidydimal white adipose tissue, eWAT) show a significant reduction in Lgr4 mutant mice than littermate controls, especially in high-fat diet (HFD) condition. A white-to-brown fat switch of eWAT in Lgr4 mice was reported by our group. However, the whole-genome expression profile of eWAT has not been comphrehensively studied.
We used microarrays to reveal the potential novel factors underlying browning process.

overall_design: eWAT tissues from both Lgr4 mutant and WT mice under chow diet and HFD diet were sampled for RNA extraction and hybridization on Affymetrix microarrays.


title: Profile of plasma exosome-derived circrnas from acute ischemic stroke patients compared with controls

city: Chengdu

first: Kejie

last: Chen

state: Sichuan

address: 783#, Xindu Avenue

institute: Chengdu Medical College

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: Stroke is one of the major causes of death and long-term disability, of which acute ischemic stroke (AIS) is the most common type. Although circRNA expression profiles of AIS patients have been reported to be significantly altered in blood and peripheral blood mononuclear cells, the role of exosome-contained circRNAs after AIS is still unknown. Plasma exosomes from ten AIS patients and ten controls were isolated, and through microarray and bioinformatics analysis, profile and putative function of circRNAs in the plasma exosomes were studied. A total of 198 circRNAs were differentially quantified (|log2 FoldChange|≥1.00, P<0.05) between AIS patients and controls.The functions of host genes of differentially quantified circRNAs, including RNA and protein process, focal adhesion and leukocyte transendothelial migration, were associated with the development of AIS. As miRNA sponge, differentially quantified circRNAs had the potential to regulate pathways related to AIS, like PI3K-Akt, AMPK and chemokine pathways. Of 198 differentially quantified circRNAs, 96 circRNAs possessing strong translational ability could affect cellular structure and activity, like focal adhesion, tight junction and endocytosis. Most differentially quantified circRNAs were predicted to bind to EIF4A3 and AGO2- two RNA binding proteins (RBPs)- and play a role in AIS. In conclusion, plasma exosome-derived circRNAs were significantly differentially quantified between AIS patients and controls, and participated in the occurrence and progression of AIS by sponging miRNA/RBPs or translating into proteins, indicating that circRNAs from plasma exosomes could be crucial molecules in the pathogenesis of AIS and promising candidates as diagnostic biomarkers and therapeutic targets for the condition.

overall_design: Plasma exosomes from ten AIS patients ten controls were collected.


title: A new female embryonic stem cell system reveals the requirement to open promoter chromatin to establish X chromosome inactivation.

city: Royal Parade

first: Andrew

last: Keniry

department: Molecular Medicine


title: The tubulin code during cardiac hypertrophy and failure [II]


title: Circulating miR-499a-5p is a potential biomarker of MYH7-associated hypertrophic cardiomyopathy

first: Maria

last: Pisklova

address: 3rd Cherepkovskaya, 15a

institute: NATIONAL MEDICAL RESEARCH CENTER OF CARDIOLOGY

summary: Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disease with significant genetic and phenotypic heterogeneity. To search for novel biomarkers, which could increase the accuracy of HCM diagnosis and possibly improve understanding of its phenotype formation, we analyzed the levels of circulating microRNAs (miRNAs) − stable non-coding regulatory RNAs. Performed high throughput sequencing of miRNAs in plasma of HCM patients and controls pinpointed miR-499a-5p as one of 35 miRNAs dysregulated in HCM.

overall_design: We performed miRNA profiling in plasma of peripheral blood of 9 patients with hypertrophic cardiomyopathy (HCM) and 6 healthy controls (HC).

first: Natalia

last: Baulina

first: Ivan

last: Kiselev


title: The tubulin code during cardiac hypertrophy and failure [I]


title: PM01183 on SCLC cell lines (RNA-Seq)


title: Analysis of BTLA regulated transcripts in human B and CD4+ T cells

city: San Diego

first: John

last: Sedy

address: 10770 Wateridge Cir

institute: AnaptysBio

summary: Purified B or CD4+ T cells were analyzed for transcriptional changes induced by the BTLA inhibitory receptor. BTLA receptors were activated using anti-human BTLA coupled to microbeads. Gene expression was analyzed using Nanostring

overall_design: Transcriptional analysis was measured in three separate donors for T cells and B cells. Cells from each donor were treated as control or with anti-BTLA. To determine differential expression paired analysis was performed on analyzed data.

middle: R


title: PM01183 on SCLC cell lines BrdU

summary: detect the impact of lurbinectedin on transciption factor binding

overall_design: 4 Samples

last: Hernandez


city: Columbus

first: Erin

last: Gloag

address: 460 W. 12th Ave

institute: The Ohio State Univerisity

summary:
Purpose: Use RNA-seq to investigate the kinetics of bacterial response during the first hour of surface attachment
Methods: Logarithmic phase bacterial culture in LB with optical density 600 (OD600) of 0.5 was inoculated in an Ibidi μ-Slide. At the indicated time from 5 - 60 min post-attachment, the channel was rinsed twice in PBS to remove non-adherent cells and 200μL of Trizol was added to the channel. Trizol was collected and stored at -80°C for RNA isolation. HiSeq 4000 sequencing was performed, generating approximately 300 million total paired end 300 bp reads from the 15 total samples, with a mean quality score of 37.4. Reads were aligned to the reference PAO1 genome using Rockhopper.
Results:In total, 453 genes were differentially regulated between 5 min post-attachment, and at least one of the later time points. Expression of most of the regulated genes was elevated (390 genes) rather than reduced (63 genes) upon surface attachment
Conclusion: This rapid response indicates that biofilm phenotypes may occur more rapidily than previously proposed, and that the phenotype may be triggered by surface contact

overall_design: RNA-Seq analysis of wild type Pseudomonas aeruginosa adhered to a surface for 5, 10, 15, 30, 45, or 60min. Each time point was analyzed in triplicate

first: Christopher

last: Jones

first: Nikolas

last: Grotewold

first: Daniel

last: Wozniak


title: Primordial germ cells identified as one potential cell of origin of MYC rhabdoid tumors

city: Münster

first: Kornelius

last: Kerl

address: Albert-Schweitzer-Campus 1

department: Department of Pediatric Hematology and Oncology

institute: University Children's Hospital Münster

summary: Rhabdoid tumors (RT) are rare and highly aggressive pediatric neoplasms. Their epigenetically-driven intertumoral heterogeneity is well described; however, the cellular origin of RT remains an enigma. Here, we established and characterized different genetically engineered mouse models driven under the control of distinct promoters and being active in early progenitor cell types with diverse embryonic onsets. From all models only Sox2-positive progenitor cells give rise to murine RT. Using single-cell analyses, we identified distinct cells of origin for the SHH and MYC subgroups of RT, rooting in early stages of embryogenesis. Intra- and extracranial MYC tumors harbor common genetic programs and potentially originate from fetal primordial germ cells (PGCs). Using PGC specific Smarcb1 knockout mouse models we validated that MYC RT originate from these progenitor cells. We uncovered an epigenetic imbalance in MYC tumors compared to PGCs being sustained by epigenetically-driven subpopulations. Importantly, treatments with the DNA demethylating agent decitabine successfully impaired tumor growth in vitro and in vivo.

overall_design: We utilized gene expression profiling (bulk RNA-seq) of tumors derived from the Dppa3cre:: Smarcb1 knockout models. For this, we crossed the Smarcb1 fl/fl strain with the mouse line harboring the Cre coding region under the control of PGC cell specific (Dppa3) promoter. Mice were intensively monitored until neurological symptoms or macroscopical abnormalities indicating tumor growth were observed. Total RNA from the tumor sample was extracted and submitted to RNA-Sequencing.

last: Moreno

first: Marta

last: Interlandi

first: Monika

last: Graf


title: Tumor eQTM screening reveals distinct CpG panels for deconvolving cancer immune signatures

city: Tampa

first: Xuefeng

last: Wang

address: 12902 Magnolia Dr

institute: H. Lee Moffitt Cancer Center and Research Institute

type: Methylation profiling by genome tiling array

summary: Genome-wide DNA methylation profiling of melanoma samples. The Illumina Infinium Human DNA methylation EPIC Beadchip Kit was used to obtain DNA methylation profiles across approximately 850,000 CpGs.

overall_design: Bisulphite-converted DNA from the 30 melanoma samples were hybridised to the Illumina Infinium Human DNA Methylation Beadchip.


overall_design: We used a mouse model, Rosa26-creERT2::Smarcb1fl/fl , to profile the transcriptome of  rhabdoid tumors of the MYC subgroup, by single cell RNA-sequencing (scRNA-seq). We crossed the Smarcb1 fl/fl strain with a mouse line harboring the CreERT2 coding region under the control of ubiquitous (Rosa26) promoter. Pregnant females were induced with a single intraperitoneal dose (50 mg/kg) of Tamoxifen at 6.5 days post-coitum (post-coital plug observation was considered as day 0.5). All mice were intensively monitored until neurological symptoms indicating tumor growth were observed. Tumors were dispersed into single cells and further processed for scRNA-seq (10X Genomics Platform).


title: Molecular phenotyping of breast cancer cell lines at the single-cell level for automated tumour diagnosis and prediction of drug sensitivity

city: Naples

first: Gennaro

last: Gambardella

address: Via Campi Flegrei 34,

country: Italy

institute: Telethon Institute of Genetics and Medicine (TIGEM)

summary: Breast Cancer (BC) patient stratification is mainly driven by tumour receptor status and histological grading and subtyping, with about twenty percent of patients for which absence of any actionable biomarkers results in no clear therapeutic intervention to apply. Here, we evaluated the potential of single-cell transcriptomics for automated diagnosis and drug treatment of breast cancer. We transcriptionally profiled 35,276 individual cells from 32 BC cell-lines covering all main BC subtypes to yield a Breast Cancer Single Cell Atlas. We show that single cell transcriptomics can successfully detect clinically relevant BC biomarkers and that atlas can be used to automatically predict cancer subtype and composition from a patient’s tumour biopsy. We found that BC cell lines harbour a high degree of heterogeneity in the expression of clinically relevant BC biomarkers and that such heterogeneity enables cells with differential drug sensitivity to co-exist even within a genomically stable isogenic cell line. Finally, we developed a novel bioinformatics approach named DREEP (Drug Response Estimation from Expression Profiles) to automatically predict responses to more than 450 anticancer agents starting from single-cell transcriptional profiles. We validated DREEP both in-silico and in-vitro by selectively inhibiting the growth of the HER2-deficient subpopulation in the MDAMB361 cell line.  Our work shows that transcriptional heterogeneity is common, dynamic and that its plasticity plays a relevant role in determining drug sensitivity. Moreover, our Breast Cancer Single Cell Atlas and DREEP approach are a unique resource for the BC research community and to advance the use of single-cell sequencing in the clinic.

overall_design: Single-cell sequencing of 32 breast cancer cell-lines

first: Gaetano

last: Viscido

first: Diego

last: di Bernardo


title: The tubulin code during cardiac hypertrophy and failure [IV]

first: Benjamin

last: Prosser

first: Sai

last: Phyo


overall_design: We utilized gene expression profiling (Affymetrix GeneChip Mouse Gene 2.0 ST Array) of tumors derived from the Sox2-cre, Sox2-creERT2 and Rosa26-creERT2 Smarcb1 knockout models generating SHH and MYC RT. For this, we crossed the Smarcb1 fl/fl strain with the mouse line harboring the Cre or CreERT2 coding region under the control of ubiquitous (Rosa26) or cell specific (Sox2) promoter. When required (CreERT2), pregnant females were induced with a single intraperitoneal dose (50 mg/kg) of Tamoxifen at 6.5 days post-coitum (post-coital plug observation was considered as day 0.5). All mice were intensively monitored until neurological symptoms or macroscopical abnormalities indicating tumor growth were observed. Total RNA from the tumor sample was extracted for a total number of 41 samples, processed into 3 separate batches.


title: The tubulin code during cardiac hypertrophy and failure

overall_design: Refer to individual Series


title: RNA sequencing data of microglia, meningeal macrophages (mMΦ) and perivascular macrophages (pvMΦ) isolated from brains of Cx3cr1-GFP mice at different age.

status: Public on Jan 27 2022

city: Freiburg

first: Gianni

last: Monaco

address: Breisacher Straße

institute: Freiburg Medical Center

summary: All tissue resident macrophages of the central nervous system (CNS), including parenchymal microglia as well as CNS-associated macrophages (CAMs) such as meningeal (mMF) and perivascular macrophages (pvMF) are part of the CNS endogenous innate immune system that acts as the first line of defense during infections or trauma. It has been suggested that microglia and all CAM subsets are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAMs subsets in situ are poorly understood. Using novel fate mapping systems, single-cell profiling and cell-specific mutants, we show that only mMF and microglia share a common prenatal progenitor. In contrast, pvMF originate from perinatal mMF only after birth in an integrin-dependent manner. Furthermore, the establishment of pvMFcritically requires the presence of vascular smooth muscle cells. In summary, our data reveal a novel precisely timed process in distinct anatomical niches for the establishment of CNS macrophage subsets.

overall_design: Microglia, mMΦ and pvMΦ  were isolated from brain tissue of 3-5 individual mice per age and used for the generation of bulk transcriptomics data. RNA expression levels between the different groups and ages were compared.

first: Marco

last: Prinz

first: Takahiro

last: Masuda

first: Lukas

last: Amann


title: A primed immune transcriptional program is activated in oligodendroglia in multiple sclerosis [Cut&Run EZH2i]

city: Stockholm

first: Eneritz

last: Agirre

address: Solnavägen 9

department: MBB

institute: Karolinska Institutet

summary: Multiple sclerosis (MS) is a disease characterized by a targeted immune attack on myelin in the central nervous system (CNS). We have previously shown that oligodendrocytes (OLs), myelin producing cells in the CNS, and their precursors (OPCs), acquire disease-specific transcriptional states in MS. To understand how these alternative transcriptional programs are activated in disease, we performed single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) on the OL lineage in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We identified regulatory regions with increased accessibility in oligodendroglia (OLG) in EAE, some of which in the proximity of immune genes. A similar remodeling of chromatin accessibility was observed upon treatment of postnatal OPCs with interferon-gamma (IFNg), but not with dexamethasone. These changes in accessibility were not exclusive to distal enhancers, but also occurred at promoter regions, suggesting a role for promoters in mediating cell-state transitions. Notably, we found that a subset of immune genes already exhibited chromatin accessibility in OPCs ex vivo and in vivo, suggesting a primed chromatin state in OLG compatible with rapid transitions to an immune-competent state. Several primed genes presented bivalency of H3K4me3 and H3K27me3 at promoters in OPCs, with loss of H3K27me3 upon IFNg treatment. Inhibition of JMJD3/KDM6B, mediating removal of H3K27me3, led to the inability to activate these genes upon IFNg treatment. Importantly, OLGs from the adult human brain showed chromatin accessibility at immune gene loci, particularly at MHC-I pathway genes. A subset of single-nucleotide polymorphisms (SNPs) associated with MS susceptibility overlapped with these primed regulatory regions in OLG from both mouse and human CNS. Our data suggest that susceptibility for MS may involve activation of immune gene programs in OLG. These programs are under tight control at the chromatin level in OLG and may therefore constitute novel targets for immunological-based therapies for MS.

overall_design: We have performed bulk CUT&RUN for H3K27ac, H3K27me3 and  H3K4me3 from FACS sorted Sox10GFP+ oligodendrocyte precursor cells treated with EZH2 inhibitor.  After 4 days IFN-gamma was added  together with new EZH2 inhibitor.

first: Mandy

last: Meijer

first: Mukund

last: Kabbe

first: Cassandra

last: van Tuijn

first: Abeer

last: Heskol

first: Ana

middle: M

last: Falcao

first: M

last: Corces

first: Thomas

middle: J

last: Montine

first: Xingqi

last: Chen

first: Howard

last: Chang

first: Gonçalo

last: Castelo-Branco


title: SARS-CoV-2 Infection Induces Ferroptosis of Human Pluripotent Stem Cell-Derived Sinoatrial Nodal like Pacemaker Cells [single-cell RNA-seq]

status: Public on Jan 26 2022

city: New York

first: Shuibing

address: A 827B, 1300 York Ave

department: Surgery

institute: Weill Cornell Medical College

summary: We established an efficient strategy to derive functional human SAN like pacemaker cells for disease modeling and drug screening. We used a dual-reporter cell line(SHOX2-GFP+MYH6-mcherry) to indicate the sinoatrial nodal like pacemaker cells. We sorted out the double positive cell population after Day20 and prepared the sample for single cell RNA seq analysis.

overall_design: 1 total sample

first: Yuling

last: Han

first: Jiajun

last: Zhu


title: Kinetics of Tbet+ B cells during LCMV infection

city: Newark

first: Jason

last: Weinstein

state: NJ

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

department: Center for Immunity and Inflmmation

institute: Rutgers New Jersey Medical School

summary: Inflammatory (B220+ CD19+ CD44+ CD11c+ Tbet-AmCyanhi) B cells were sorted from female Tbet-AmCyan reporter mice at day 8, 10, and 15 post infection with LCMV-Armstrong. RNA-seq was employed to assess transcriptional changes in inflammatory B cells throughout the infection.

overall_design: Mice were infected i.p. with 2 × 10^5 pfu LCMV Armstrong and inflammatory B cells were sorted from Tbet-AmCyan reporter mice at various day post LCMV infection

first: Alex

last: Lemenze

first: Wenzhi

last: Song


title: Gpr125 is a Unifying Hallmark of Multiple Mammary Progenitors coupled with Tumor Latency

first: Ziyan

last: Lin

state: NY

department: Applied Bioinformatics Laboratories

institute: NYU Grossman School of Medicine

summary: Several markers have been used to identify mammary stem cell subpopulations. However, a specific unifying hallmark of progenitors has not yet been identified yet.  Here we show the orphan adhesion G-protein coupled receptor, Gpr125, identifies and locates long-lived progenitors at multiple sites and stages of mammary development. In particular, Gpr125 is expressed in disparate cell populations with documented regenerative capacity in the nipple-proximal and in the TEB-distal end of the mammary gland. To compare these two major Gpr125 cell populations we used whole RNAseq genome to characterize their gene expression profile. Our data revealed Gpr125+ cells at the nipple-proximal end of the gland are engaged in chemorepulsion and the formation of cell surface structures associated with cell movement. In contrast Gpr125+ cells at the TEB-distal tips exhibit a hybrid epithelial-mesenchymal phenotype and are equipped to bind chemokine and growth factors and secrete a promigratory matrix.

overall_design: We performed whole genome RNAseq on Gpr125+ (FDG+/CD49hi) and Gpr125- (FDG-/ CD49hi) cell population isolated from TEB-distal and nipple-proximal mammary regions of pubertal mice Adgra3-Lz/+ mice. Mammary glands were harvested at puberty. Both abdominal mammary glands from 5 mice were pooled together per each sample used in the study.     The lymph nodes were removed and their position was used to cut the mammary gland into distal and nipple proximal halves. The mammary glands were mechanically dissociated and digested using a mix consisting of 1 mL Collagenase/Hyaluronidase and 9 mL Epicult-B Basal Medium and incubated for 6-8 hours at 37° C. Cells that were positive for CD31, CD45 or Ter119 were first excluded.  Cells were sorted based on their incorporation of fluorescein di-V-galactoside (FDG) and surface expression of CD49fhi.

first: Elena

last: Spina

first: Pamela

last: Cowin


title: Gene Expression of mid-gestation human placenta

city: New Haven

first: Liza

last: Konnikova

state: CT

address: 375 Congress Ave LSOG 407

department: Pediatrics

institute: Yale University

summary: The goal of this study was to transciprtionally profile the three layers of the human placenta (decidua, fetal membrane and placental villi) from the mid-gestation  healthy human placenta.

overall_design: Bulk mRNA extracted from three tissues isolated from matched placentas.

first: Jessica

last: Toothaker


title: Amino acids and IGF1 regulation of fish muscle growth revealed by transcriptome and microRNAome integrative analyses of pacu (Piaractus mesopotamicus) myotubes [miRNA-seq]

city: Jatai

first: Bruna

middle: Tereza Thomazini

last: Zanella

state: Goias

country: Brazil

institute: Federal University of Jatai (UFJ)

summary: Amino acids (AA) and IGF1 have demonstrated to play essential roles in protein synthesis and fish muscle growth. The myoblast cell culture is useful to study the muscle regulation, and omics data have contributed enormously to understand its molecular biology. However, to our knowledge, no study has performed large-scale sequencing of fish cultured muscle cells stimulated with pro-growth signals. In this work, we obtained the transcriptome and microRNAome of pacu (Piaractus mesopotamicus) cultured myotubes treated with AA or IGF1. We identified 1228 and 534 genes differentially expressed by AA and IGF1. Enrichment analysis showed that AA treatment induced chromosomal changes, mitosis, and muscle differentiation, while IGF1 modulated IGF/PI3K signalling, metabolic alteration, and matrix structure. In addition, potential molecular markers were similarly modulated by both treatments. Muscle-miRNAs (miR-1, -133, -206 and -499) were up-regulated especially in AA samples, and we identified molecular networks with omics integration. Two pairs of genes and miRNAs showed high level of relationship, and involvement in myogenesis and muscle growth: marcksb and miR-29b in AA, and mmp14b and miR-338-5p in IGF1. Our work helps to elucidate fish muscle physiology and metabolism, highlights potential molecular markers, and creates perspective for improvements in aquaculture and in vitro meat production.

overall_design: To obtain the microRNAome profile of pacu (Piaractus mesopotamicus) muscle cells treated with amino acids (AA), IGF1 or cultived in nutrient restriction medium

first: Bruno

middle: O

last: Duran

middle: T

first: Erika

middle: S

last: Perez

first: Edson

last: Mareco

first: Maeli

last: Dal-Pai-Silva

first: Daniel

middle: G

last: de la serrana


title: Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis. [RNA-Seq]

city: Fukuoka city

first: Yuki

last: Naitou

state: Japan

address: 3-1-1, Maidashi, Higashi-ku, Fukuoka city

country: Japan

institute: Kyushu university

summary: In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that Ovol2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) driving gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and consequently induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation.

overall_design: Transcriptomes of embryonic stem (ES) cells, epiblast-like cells (EpiLCs), and primordial germ cell like cells (PGCLCs) at day2 in Ovol family genes KO cells. Refer to individual Series.

first: Go

last: Nagamatsu

first: Nobuhiko

last: Hamazaki

first: Kenjiro

last: Shirane

first: Masafumi

last: Hayashi

first: Makoto

first: Satoru

last: Kobayashi

first: Katsuhiko


title: Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis. [ChIP-Seq]

overall_design: ChIP-seq of primordial germ cell like cells derived from Ovol2a-Tg, Ovol2b-Tg ES cells.


title: Rixosomal RNA degradation contributes to silencing of Polycomb target genes

city: boston

first: Danesh

last: Moazed

state: MA

address: 240 longwood avenue

department: cell biology

institute: Harvard medical school

summary: Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1-3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA degradation complex called the rixosome or RIX1 complex4,5. Whether RNA degradation also plays a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Importantly, depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb-target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5’ mono-phosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosome-mediated degradation of nascent RNA is conserved from fission yeast to human, although in human cells the rixosomal RNAe degradation activity is primarily associated withes RNA in facultative rather than constitutive heterochromatin.

overall_design: RNA-seq analysis of HeLa cells

first: Haining

last: zhou


title: High Resolution Slide-seqV2 Spatial Transcriptomics Enables Discovery of Disease-Specific Cell Neighborhoods and Pathways

city: Cambridge

first: Jamie

middle: L

last: Marshall

address: 75 Ames Street, Office 5013C

department: Kidney Disease Initiative

institute: Broad Institute

summary: High resolution spatial transcriptomics is a transformative technology that enables mapping of RNA expression directly from intact tissue sections; however, its utility for the elucidation of disease processes and therapeutically actionable pathways remain largely unexplored. Here we applied Slide-seqV2 to mouse and human kidneys, in healthy and in distinct disease paradigms. First, we established the feasibility of Slide-seqV2 in human kidney by analyzing tissue from 9 distinct donors, which revealed a cell neighborhood centered around a population of LYVE1+ macrophages. Second, in a mouse model of diabetic kidney disease, we detected changes in the cellular organization of the spatially-restricted kidney filter and blood flow regulating apparatus. Third, in a mouse model of a toxic proteinopathy, we identified previously unknown, disease-specific cell neighborhoods centered around macrophages. In a spatially-restricted subpopulation of epithelial cells, we also found perturbations in 77 genes associated with the unfolded protein response (UPR), including Tmed9. Treatment with a TMED9-targeting compound showed efficient removal of toxic mutant proteins and reversal of the UPR. Our studies illustrate and experimentally validate the utility of Slide-seqV2 for the discovery of disease-specific cell neighborhoods and actionable targets.

overall_design: For the BTBR experiments, the BTBR-wt/wt are controls and BTBR-ob/ob are diabetic samples. Seven arrays per mouse and 4 mice of each gentoptype were processed. For the UMOD experiments, UMOD-WT are controls and UMOD-KI are ADTKD samples. Five arrays per mouse and 3 WT mice and 5 KI mice were processed. There are 3 separate files types: BAMs, raw data which is unmapped spatial data, and processed data which contains QCed and mapped kidney cell types.

first: Teia

last: Noel

first: Qingbo

last: Wang

first: Anna

last: Greka


summary: Polycomb Repressive Complex (PRC) 1 and 2 are histone-modifying and chromatin-binding complexes that are required for silencing of developmental regulatory and other genes. Their silencing functions are thought to involve chromatin compaction and condensate formation but whether other mechanisms contribute to silencing is unknown. Here we show that the rixosome, a conserved RNA degradation complex with roles in ribosomal RNA processing and heterochromatic RNA degradation in fission yeast, associates with human PRC complexes, is recruited to promoters of Polycomb target genes in differentiated cell lines and embryonic stem cells, and is required for efficient silencing of Polycomb target genes. These findings reveal an unanticipated role for RNA degradation in Polycomb-mediated gene silencing.

overall_design: ChIP-seq analysis of histone modifications and chromatin associated proteins in HeLa cells


overall_design: ChIP-seq analysis of histone modifications and chromatin associated proteins inHEK293FT cells


title: Inactivation of the LKB1 substrate AMPK accelerates lung adenocarcinoma and leads to immune evasion.

city: Helsinki

first: Tomi

last: Makela

address: Biomedicum 1, Haartmaninkatu 8

country: Finland

institute: University of Helsinki

summary:
Purpose: Mutations in STK11 (LKB1) occur in 17% of lung adenocarcinoma (LUAD) and drive a suppressive (cold) tumor immune microenvironment (TME) and resistance to immunotherapy. The mechanisms underpin the establishment and maintenance of a cold TME in LKB1 mutant LUAD remain poorly understood and the identification of downstream effectors is critical to inform therapeutic strategies to invigorate antitumor immunity. In this study, we investigated the association between inactivation of AMPK, one of the downstream substrates of LKB1, and immune evasion in human LUAD as well as the causal role of AMPK on TME in genetically engineered mouse model (GEMM) of LUAD.
Experimental Design: We modeled AMPK inactivation in vivo using GEMM by deleting both AMPKα1 (Prkaa1) and AMPKα2 (Prkaa2) in KrasG12D-driven LUAD (KAA). Furthermore, we investigated the impact of AMPK inactivation on immune cell infiltration and global gene-expression programs of murine LUAD. Gene expression signature from AMPK-deficient murine LUAD was further compared to that of Lkb1 deficient murineLUAD as well as human LUAD with either LKB1 mutation or attenuated AMPK phosphorylation.
Results: Inactivation of both AMPKα1 and AMPKα2 accelerates KrasG12D-driven LUAD. AMPK-deficient tumors are characterized with reduced infiltration of CD8+/CD4+ T cells and gene signatures associated with compromised immune microenvironment, which resembles LKB1-deficient murine and human LUAD (KL) as well as LKB1-wildtype LUAD with reduced AMPK phosphorylation. Attenuation of the MHC Class 1 component ß2 microglobulin (ß2M) was identified as a shared feature in KL and KAA murine LUAD as well as in human LUAD with either LKB1 mutations or reduced AMPK phosphorylation. Furthermore, reactivation of AMPK by expression of constitutive active form of AMPKα1 leads to restoration of ß2M level in LKB1 mutant LUAD cells.
Conclusions: The results identify attenuation of the LKB1 substrate AMPK as an important mechanism for decreased MHC Class 1 expression and immune evasion in LKB1 mutant LUAD.
Translational relevance: LKB1 loss-of-function mutations rank as the third most common genetic alterations found in lung adenocarcinoma and associated with immune evasion. In the current study, on the basis of GEMM of LUAD we established the causal relationship between inactivation of the LKB1 substrate AMPK and immune evasion. Furthermore we find that the status of AMPK phosphorylation is more sensitive than LKB1 mutation in predicting impaired tumor immune microenvironment and likely also for the resistance to immunotherapy. The results also suggest that restoration of AMPK activity could increase the number of patients benefiting from immunotherapy.

overall_design: mRNA profiling of buik tumors from murine lung with 1) Kras activation,  2) Kras activation and depletion of LKB1, 3) Kras activation and depletion of AMPKa1 (Prkaa1) and AMPKa2 (Prkaa2)

first: Yan

last: Yan

first: Yajing

last: Gao

first: Sushil

last: Tripathi

first: Pekka

last: Päivinen

first: Iris

last: Wong

first: Kari

last: Vaahtomeri

first: Marc

last: Foretz

first: Benoit

last: Viollet

last: Mäkelä


title: H3K56 deacetylation and H2A.Z deposition are required for aberrant heterochromatin spreading

status: Public on Jan 25 2022

city: KunMing

first: Lu

last: Zhang

state: YunNan

address: WuJiaYing Street

institute: YunNan University

summary: Crucial mechanisms are required to restrict self-propagating heterochromatin spreading within defined boundaries and prevent euchromatic gene silencing. In the filamentous fungus Neurospora crassa, the JmjC domain protein DNA METHYLATION MODULATOR-1 (DMM-1) prevents aberrant spreading of heterochromatin, but the molecular details remain unknown. Here, we revealed that DMM-1 is highly enriched in a well-defined 5-kb heterochromatin domain and constrained its aberrant spreading. Interestingly, aberrant spreading of the 5-kb heterochromatin domain observed in the dmm-1KO strain is accompanied by the sharp deposition of histone variant H2A.Z, and deletion of H2A.Z abolishes aberrant spreading of the 5-kb heterochromatin domain into adjacent euchromatin. Furthermore, lysine 56 of histone H3 is deacetylated at the expanded heterochromatin regions, and mimicking H3K56 acetylation with an H3K56Q mutation effectively blocks H2A.Z-mediated aberrant spreading of the 5-kb heterochromatin domain. More importantly, genome-wide analyses demonstrated the general roles of H3K56 deacetylation and H2A.Z deposition in aberrant spreading of heterochromatin.  Altogether, our results illustrate a previously unappreciated regulatory process that mediates aberrant heterochromatin spreading.

overall_design: Dissectingthe molecular mechanisms of ectopic heterochromatin spreading in N. crassa.

first: Chengcheng

first: Shuang

last: Song

first: Yunkun

last: Dang

first: Qun

last: He