title: The transcription factor Pdr802 regulates Titan cell production and Cryptococcus neoformans pathogenicity (RNA-Seq)

city: St Louis

first: Daniel

last: Paiva Agustinho

state: MO

address: 660 S Euclid Ave

department: Molecular Microbiology

institute: Washington University in St. Louis

summary: We report on our study the role of C. neoformans transcription factor Pdr802, whose expression is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. We found that direct targets of Pdr802 include the calcineurin targets Had1 and Pmc1, which are important for C. neoformans virulence, the transcription factor Bzp4, which promotes cryptococcal melanization and capsule thickness, and 35 transmembrane transporters. Notably, a strain engineered to lack Pdr802 showed a dramatically increased population of Titan cells. Since Titan cells are not phagocyted and do not disseminate via the Trojan horse mechanism or via penetration of biological barriers, this likely explains the reduced dissemination of pdr802 cells to the central nervous system and consequently reduced pathogenicity of this strain. The role of Pdr802 as a negative regulator of titanisation is thus critical for cryptococcal virulence.

overall_design: We studied the target of the C. neoformans transcription factor Pdr802

first: Julia

middle: V

last: Reuwsaat

middle: P

last: Agustinho

first: Heryk

last: Motta

first: Holly

last: Brown

first: Andrew

last: Chang

first: Livia

last: Kmetzsch

first: Tamara

last: Doering


title: Single cell- Cluster-RNA seq and Spatial Transcriptomics from the trunk of mouse embryos of different genotypes (wild type, Pax2-GFP, Pax2-GFP; Gata3 knockout) and developmental stages (E8.75, E9.5, E11.5) [Spatial Transcriptomics]

status: Public on Feb 16 2021

city: Montreal

first: Alain

last: Pacis

state: QC

address: 740 Dr Penfield Ave

country: Canada

institute: Canadian Centre for Computational Genomics (C3G)

type: Expression profiling by high throughput sequencing

summary: To study cell heterogeneity and state transitions in the developing urinary tract we performed spatial transcriptomics from the trunk of E9.5 Pax2GFP mouse embryos using the 10X Genomics Visium Spatial Gene Expression protocol

overall_design: Identify cell heterogenity and state transitions in the developing urinary tract

first: Oraly

last: Sanchez-Ferras

first: Maria

last: Sotiropoulou

first: Yuhong

last: Zhang

first: Yu

middle: C

last: Wang

first: Mathieu

last: Bourgey

first: Guillaume

last: Bourque

first: Ioannis

last: Ragoussis

first: Maxime

last: Bouchard


title: A single cell atlas of human teeth (Periodontium scRNA-seq)

city: Basel

first: Andreas

last: Moor

address: Mattenstrasse 26

department: Department of Biosystems Science and Engineering

country: Switzerland

institute: ETH

summary: Teeth exert fundamental functions related to mastication and speech. Despite their big biomedical interest, an overall picture of their cellular and molecular composition is still lacking. We here mapped the transcriptional landscape of the various stem cell populations and their microenvironments in human teeth at single-cell resolution. Our analysis identified significant cellular heterogeneity between the various dental tissues. Unexpectedly, we found that the molecular signatures of the stem cell populations were very similar in vivo, and that their distinctive behavior was due to substantial differences between their microenvironments. Furthermore, we showed that the evolutionarily conserved Notch signaling pathway is important for the interactions between dental stem cells and the diverse cell types composing their niches. Our findings reveal that the microenvironmental specificity is responsible for the major functional differences of the stem cells located in the various tooth compartments and open new perspectives towards dental cell-based therapeutic approaches.

overall_design: Single-cell RNA sequencing analysis (10x) of human dental periodontium. For each tissue, the analysis was performed on biopsies obtained from 5 distinct patients. Healthy dental periodontium were selected

first: Pierfrancesco

last: Pagella

first: Laura

last: de Vargas Roditi

first: Bernd

last: Stadlinger

middle: E

first: Thimios

middle: A

last: Mitsiadis


title: Single cell- Cluster-RNA seq and Spatial Transcriptomics from the trunk of mouse embryos of different genotypes (wild type, Pax2-GFP, Pax2-GFP; Gata3 knockout) and developmental stages (E8.75, E9.5, E11.5) [single-cell RNA-seq]

summary: To study cell heterogeneity and state transitions in the developing urinary tract we isolated renal cells (single cells or clusters) by Fluorescence-activated cell sorting (FACS) from the trunk of E8.75, E9.5 and E11.5  mouse embros and performed 10X Genomics single cell and Cluster RNA sequencing


title: Expression profiling of mouse melanomas with or without UVB-induced neoantigens and after combination immunotherapy

city: Charlestown

first: Jennifer

last: Lo

state: MA

address: 149 13th Street

department: Cutaneous Biology Research Center

country: USA

institute: Massachusetts General Hospital/Harvard Medical School

summary: Syngeneic grafts of the D4M.3A.3 (parental) mouse melanoma cell line (derived from a Tyr::CreER;BrafCA;Ptenlox/lox mouse) in C56BL/6 mice model poorly immunogenic, low neoantigen human melanomas. The D3UV2 (UV2) cell line was derived by serial UVB irradiation and single cell cloning. The addition of UVB-induced putative neoantigens sensitizes UV2 syngeneic melanoma grafts to immune checkpoint inhibitors and triggers epitope skewing to tumor-lineage self-antigens, a phenomenon that can be successfully mimicked in parental melanomas through treatment combinations such as anti-PD-1 with ablative fractional photothermolysis and imiquimod. Our mouse models were used to characterize gene expression changes between neoantigen rich and neoantigen poor melanomas, and with immunotherapy.

overall_design: RNA-sequencing was performed to study gene expression changes in bulk tumors after addition of UVB-induced putative neoantigens (parental vs. UV2 melanomas) or treatment with anti-PD-1 or combination immunotherapy (anti-PD-1 + ablative fractional photothermolysis + imiquimod).

first: David

middle: J

last: Lieb

first: Marcelo

last: Pereira da Silva

last: Fisher


title: The transcription factor Pdr802 regulates Titan cell production and Cryptococcus neoformans pathogenicity

first: Daniel

last: Paiva Agustinho

type: Genome binding/occupancy profiling by high throughput sequencing

first: Julia

last: Reuwsaat

last: Agustinho

first: Heryk

last: Motta

first: Holly

last: Brown

first: Andrew

middle: L

last: Chang

first: Livia

last: Kmetzsch

first: Tamara

last: Doering


title: A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation

city: Los Angeles

first: Colette

last: Picard

state: California

address: 610 Charles E Young Dr East, Room 4045

department: Molecular, Cell and Developmental Biology

institute: University of California - Los Angeles

type: Methylation profiling by high throughput sequencing

summary: CRISPR-based epigenome editing was recently used to activate gene expression through direct transcriptional activation or site-specific DNA demethylation. Viral delivery of guide RNAs for these purposes remains to be developed. Furthermore, currently available viral delivery tools for genome editing show meager rates of heritability. Here, we have developed a tobacco rattle virus (TRV)-based guide RNA delivery system for both transcriptional activation and targeted DNA demethylation. To promote heritable epigenome editing specifically within plant meristems and the germline, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome, thus facilitating cell-to-cell movement of the RNA in plants. We achieved up to ~8% heritability of the induced phenotype in the progeny of virus-inoculated plants and 25% in the following generation, indicating high rates of heritability for targeted DNA demethylation. Thus, TRV delivery, in combination with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.

overall_design: BS-seq and BS-PCR followed by sequencing were used to evaluate DNA methylation in plants expressing SunTag-TET1, with virally-supplied guide RNAs (+gRNAs) or no guide RNA sequence  (-gRNAs; controls). Three different gRNAs (g4, g10, g18) were designed against several regions in the 5' region of the FWA locus and its promoter. Two Tobacco rattle virus vectors encoding a single guide RNA (g4) and multiple guide RNAs (g4, g10, and g18)  were used to deliver the guide RNAs. BS-PCR was used to evaluate methylation at several regions of the FWA promoter in two pairs of +gRNA and matched -gRNA control lines, one at three days post-infection (3 dpi) and one at six dpi. BS-seq was later used to assess methylation in first- and second-generation descendants of these treated lines (2 +gRNAs and 2 -gRNA lines per generation), to evaluate changes both locally at FWA, and genome-wide.

first: Basudev

last: Ghoshal

first: Brandon

last: Vong

first: Suhua

last: Feng

first: Janet

middle: M

last: Tam

first: Steven

last: Jacobsen


title: Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro [RNA-seq]

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

country: Japan

institute: Kyoto University, Graduate school of medicine

summary: Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ∼20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (∼50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.

overall_design: Transcriptome analysis of mouse PGCLC culture suppoted by Cyclosporin A and FGF signaling.

first: Hiroshi

last: Ohta

first: Kazuki

last: Kurimoto

first: Tomonori

last: Nakamura

first: Yusuke

last: Murase

first: Takuya

last: Yamamoto

first: Mitinori

last: Saitou


title: Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro [bis-seq]


title: Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro.

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

overall_design: Refer to individual Series


title: Epigenetic therapy strengthens immune synapses to potentiate susceptibility of lung cancer cells to adoptive γδ T cell therapy

city: Taipei

first: Rueyhung

middle: Roc

last: Weng

address: No. 1, Roosevelt Rd., Sec. 4

department: Internal Medicine

country: Taiwan

institute: National Taiwan University Hospital

type: Methylation profiling by genome tiling array


title: Single cell- and Clumps-RNA seq from the trunk of E9.5 Pax2-GFP and Pax2-GFP; Gata3 knockout embryos

first: Alain

last: Pacis

summary: To study cell heterogeneity and state transitions in the developing urinary tract we isolated Pax2-GFP positive single cells or clumps of cells by Fluorescence-activated cell sorting (FACS) from the trunk of E9.5 Pax2-GFP BAC transgenic embryos and performed 10X Genomics single cell and Clumps RNA sequencing

first: Oraly

last: Sanchez-Ferras

first: Yuhong

last: Zhang

first: Yu

last: Wang

first: Mathieu

last: Bourgey

first: Guillaume

last: Bourque

first: Ioannis

last: Ragoussis

first: Maxime

last: Bouchard


title: Epigenetic therapy strengthens immune synapses to potentiate susceptibility of lung cancer cells to adoptive γδ T cell therapy [seq]

summary:
Purpose: Epigenetic drug DNA methyltransferase inhibitors (DNMTis) are immunomodulating agents that may sensitize cancer cells to immune cell attack. However, it is still unclear how DNMTi affects cancer cells to the killing by immune cells. We therefore try to uncover the alternations of genomic DNA methylation, chromatin accessibility and transcriptomes after DNMTi treatment of cancer cells.
Methods: Six human lung cancer cell lines (A549, H1299, CL1-0, CL1-5, H1792, PC9) treated without (Mock) or with 100 nM decitabine (DAC) for 72 hours followed by growing in drug-free medium for three days were subjected to mRNA-seq and Omni-ATAC-seq analyses.
Results: mRNA-seq and ATAC-seq analyses reveal upregulation of actin and intermediate filament, and downregulation of microtubule modules by decitabine in human lung cancer cell lines.
Conclusion: Decitabine primes lung cancer cells to γδ T cell-mediated killing through regulating cytoskeleton associated genes potentially involved in immune synapse formation.

overall_design: mRNA-seq and ATAC-seq analyses of control (Mock) and decitabine (DAC) treated human lung cancer cell lines.

middle: R

first: Hsuan-Hsuan

last: Lu

first: Chong-Jen

last: Yu

first: Hsing-Chen

last: Tsai


title: DNA methylome analysis of DNA methyltransferase inhibitor reagent-treated lung cancer cells

summary:
Purpose: Epigenetic drug DNA methyltransferase inhibitors (DNMTis) are immunomodulating agents that may sensitize cancer cells to immune cell attack. However, it is still unclear how DNMTi affects cancer cells to the killing by immune cells. We therefore try to uncover the alternations of genomic DNA methylation after DNMTi treatment of cancer cells.
Methods: Six human lung cancer cell lines (A549, H1299, CL1-0, CL1-5, H1792, PC9) before (Mock) and after (DAC) treatement with 100 nM decitabine (DAC) for 72 hours followed by growing in drug-free medium for three days were subjected to Illumina MethylationEPIC microarray analysis.
Results: DNA methylome analysis reveals DNMTi-induced DNA demethylation at transcription start site (TSS)-flanking regions of several actin, intermediate filament and microtubule molecules in human lung cancer cell lines.
Conclusion: Decitabine primes lung cancer cells to γδ T cell-mediated killing through regulating cytoskeleton associated genes potentially involved in immune synapse formation.

overall_design: Methylome altenation analysis between control (Mock) and decitabine (DAC) treated human lung cancer cell lines (A549, H1299, CL1-0, CL1-5, H1792, PC9).


title: CRISPRi reveals genes modulating yeast growth in lignocellulose hydrolysate

status: Public on Feb 15 2021

city: Heidelberg

first: Cosimo

last: Jann

address: Meyerhofstr. 1

department: Genome Biology

country: Germany

institute: EMBL Heidelberg

summary: CRISPR interference screening of 129 protein kinases and 161 transcription factors in S. cerevisiae. Repression effects on yeast growth in oxygen-limited conditions were quantified in synthetic complete media (SCM), SCM supplemented with 10% lignocellulose hydrolysate and SCM supplemented with 45% of a mixture of growth-inhibiting lignocellulosic compounds. The aim of this project was to determine the reproducibility of CRISPRi effects across studies and to characterize CRISPRi for screening of phenotypes relevant for industrial biotechnology. We identify gene functions in general growth in oxygen-limited conditions, and specific for cellular fitness in lignocellulose hydrolysate. A further screen with a cocktail of lignocellulosic compounds enables us to explain hydrolysate-specific gene functions with roles in toxicity.

overall_design: CRISPRi screens in bulk yeast populations on three selection conditions: competitive growth profiling over 25 generations days in SCM, 10% spruce hydrolysate and 45% lignocellulosic inhibitor cocktail.

first: Friederike

last: Gutmann

first: Filipa

last: Pereira

first: Andreas

last: Johansson

first: Lars

last: Steinmetz

first: Kiran

last: Patil


title: Dual β-catenin and γ-catenin loss in hepatocytes impacts their polarity through altered TGFβ and HNF4⍺ signaling

status: Public on Feb 14 2021

city: Pittsburgh

first: Shuchang

last: Liu

state: PA

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Previously, we have shown that loss of β-catenin at adherens junctions (AJs) is compensated by β-catenin and dual loss of both catenins in dual knockouts (DKO) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Here, we identify simultaneous loss of β- and γ-catenin in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defect in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli.  Loss of polarity in DKO livers manifested as epithelial-to-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of TGFβ signaling and repression of Hnf4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may be playing a pathogenic role in subsets of cholangiopathies. Our findings also support a previously unknown role β- and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β and γ-catenin could potentially benefit development of new therapies for cholestasis.

overall_design: Control and hepatocyte-specific β- and γ-catenin double knockout (DKO) mouse model. Three replicates per group.

first: Tirthadipa

last: Pradhan-Sundd

first: Satdarshan

last: Pal Monga

first: Silvia


title: Quantitative evaluation of chromosomal rearrangements in primary gene-edited human stem cells by preclinical CAST-Seq

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

summary: Genome editing with programmable nucleases has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by chromosomal translocations or the insertion of mutations at off-target sites. Here, we describe CAST-Seq, an innovative assay to identify and quantify chromosomal aberrations derived from on- and off-target activities of CRISPR-Cas nucleases or TALENs. CAST-Seq also detected novel types of chromosomal rearrangements, including homology-mediated translocations that are mediated by homologous recombination. Depending on the employed designer nuclease, translocations occurred in 0–0.5% of gene-edited human stem cells and some 20% of target loci harbored gross aberrations. In conclusion, CAST-Seq analyses are particularly relevant for therapeutic editing of stem cells to enable a thorough risk assessment before clinical application of gene editing products.

overall_design: CD34 positive HSPC were treated with CRISPR/CAS9 or TALEN nucleases. In total 30 samples treated with nulceases were analysed together with their respective untreated controls (one control per sample). The same non-treated sample was used whenever we analysed the Wild-Type or the High Fidelity (HiFi) nuclease. Several days post treatment were considered: 1, 4 and 14.

first: Giandomenico

last: Turchiano

first: Julia

last: Klermund

first: Melanie

last: Boerries

first: Toni

last: Cathomen


title: Comparison of Transcriptional Profiles of T cells after Activation by TCR and Chimeric Antigen Receptors

city: Beijing

first: Xin

last: Lin

address: Shuangqing Street, Haidian District

department: School of Medicine

country: China

institute: Tsinghua University

summary: Chimeric antigen receptor T (CAR-T) cell therapies for B cell malignancies demonstrate high response rate and durable disease control. However, in the case of solid tumors, CAR-T cells have shown dysfunction ascribed to some intrinsic defects in CAR signaling. Here, we construct a multi-chain chimeric receptor, termed as Synthetic T Cell Receptor and Antigen Receptor (STAR), which incorporates antigen-recognition domain of antibody and engages entire CD3 signaling machinery of T cell receptor (TCR). In multiple solid tumor models, STAR-T cells prominently outperform CAR-T cells without notable toxicity. STAR triggers strong and sensitive TCR-like signaling upon antigen stimulation. We compared the transcriptional profiles of STAR/CAR/TCR-T cells after stimulation for different time points (0, 6, 24, 72 hours), in order to figure out whether signaling difference of these receptors led to distinct gene expression. Our results showd that STAR activation phencopied TCR, while CAR drove  a different program, displayed as various pathways related to effector function, cytokine response and cell survival were altered.

overall_design: For sample 1-36, we compare the cetuximab-derived anti-EGFR STAR-T cells, 28zCAR-T cells with 1G4-TCR which recognized NY-ESO-1 peptide in the context of HLA-A*02:01. EGFR+ A549 cells were over-expressed HLA-A*02:01 and NY-ESO-1 protein, so that it could be used to stimulate STAR, CAR and TCR. Transduced RFP+ T cells from three PBMC donors were sorted by FACS for CD8 positive and conducted coculture with target cells. T cells were retrieved from coculture system using CD3 positive selection kit and subjected to RNA preparation. For sample 37-39, we compared the cetuximab-derived anti-EGFR STAR-T cells, 28zCAR-T cells and BBzCAR-T cells (from a single PBMC donor) after stimulation by immobilized EGFR protein for 72 hours.

first: Yue

first: Daosheng

last: Huang

first: Zhixiao

last: Zhou


title: MicroRNA-29 is an essential regulator of non-CG methylation during brain maturation [Bisulfite-seq]

city: Dallas

first: Hume

last: Stroud

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern

summary: While embryonic brain development and neurodegeneration have been examined extensively, the events that govern postnatal brain maturation are less understood. During this period, widespread changes in synaptic connectivity are evident1,2 and, perturbations of synaptic development or maturation can have severe neurobehavioral consequences as seen in autism spectrum disorders (ASD), epilepsy and schizophrenia3-6. Recent studies have led to the unexpected discovery that marked increases in non-canonical (non-CG) DNA methylation occur during postnatal brain maturation7-14. This de novo CH methylation (where H= A, C, T), which occurs 1-4 weeks postnatally in mice and 1-15 years in humans7, is important for experience-dependent fine tuning of gene expression15 . The critical period of CH methylation appears to be mediated by Dnmt3a, the DNA methyltransferase that catalyzes CH methylation8,9. During brain maturation, Dnmt3a levels are elevated and then sharply decline7,16, and a strict control of CH methylation seems important as mutations or variants of Dnmt3a are associated with ASD, epilepsy and intellectual disability17-19. Here we identify the microRNA miR-29 as an essential regulator of Dnmt3a and CH methylation during brain maturation. Mice deficient in miR-29 or mice in which miR-29 can no longer target Dnmt3a exhibit enhanced levels of Dnmt3a, global CH hypermethylation, and repression of neuronal genes specifically associated with synaptic activity and neurotransmission. Importantly, these mice appear normal at birth, but subsequently exhibit severe neurological deficits including susceptibility to seizures and premature lethality. Together, these results identify an essential function of miR-29 in defining the critical period of DNA methylation during brain maturation, the absence of which results in neurobehavioral deficits.

overall_design: BS-seq on cortical tissue


title: MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells

city: Bronx

first: Paul

last: Frenette

state: NY

address: 1301 Morris Park Avenue , Room 101

department: Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research

institute: Albert Einstein College of Medicine

summary: Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island1, is a membrane-associated E3 ubiquitin ligase essential for HSC maintenance and lymphoid commitment. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and function and leads to a lethal myeloproliferative syndrome. By contrast, MAEA expression is essential for the development of acute myeloid leukaemia (AML) and up-regulated in human and mouse AML. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in absence of Maea, thereby prolonging their intracellular signalling. Additionally, the autophagy flux in HSCs, but not in mature haematopoietic cells, is dramatically impaired. Administration of autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. These results thus suggest that MAEA is a pivotal E3 ubiquitin ligase guarding HSC function via autophagy.

overall_design: mRNA profiles were generated from bone marrow hematopoietic stem cells of 8wk old control and MaeaCsf1r-Cre littermates in triplicates by Illumina NextSeq500 sequencing

first: Qiaozhi

last: Wei


title: Chicken dermal papilla complex in dorsal growing feather

first: Wei-Ling

last: Chang

address: 2011 Zonal Ave HMR 313B

institute: Keck School of Medicine

summary: Comparison of mRNA profiles in anterior and posterior of chicken dorsal growing feather

overall_design: RNA-seq was performed to study the gene expression profiling between the anterior of the dermal papilla and the posterior of the dermal papilla. There are 2 replicates for each sample group.

first: C

last: Chuong

first: WeiLing


title: Interspecies Chimeric Conditions Affect the Developmental Rate of Human Pluripotent Stem Cells

city: Boston

first: Jared

last: Brown

address: Mailtop CLS11007, 450 Brookline Ave

department: Data Science

institute: Dana Farber Cancer Institute

summary: We performed RNA sequencing experiments on chimeric samples of mixed human and mouse pluripotent stem cells over time courses of neural differentiation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Mixed mouse and human pluripotent stem cells were differentiated in vitro and periodically sampled. Submission 399 series has four sets of samples, each sampling a 33 day timecourse. Submissions 441 and 464 sample every day for the first eight days, and every two days thereafter out to forty-two days. Sequenced transcripts were aligned to a combined human-mouse transcriptome.

first: Christopher

last: Barry

first: Matthew

middle: T

last: Schmitz

first: Cara

last: Argus

first: Jennifer

last: Bolin

first: Michael

last: Schwartz

first: Amy

last: Van Aartsen

first: John

last: Steill

first: Scott

last: Swanson

first: Ron

last: Stewart

first: James

last: Thomson

first: Christina

last: Kendziorski


title: A chemical screen identifies compounds limiting the toxicity of C9ORF72 dipeptide repeats

city: Madrid

first: Oscar

last: Fernandez-Capetillo

address: MFA 3

department: Genomic Instability Group

country: Spain

institute: CNIO

summary:
Purpose: Compare parental NSC34 cells with NSC34 PR20-resistant clones
Methods: NSC-34 cell cultures were treated with PR20, a peptide that contains 20 copies of Proline-Arginine aminoacids, for two weeks. mRNA profiles of resistant clones were generated by RNAseq. Sequencing reads were aligned and analyzed for differential gene expression.
Results: NSC34 clones that gained spontaneous resistance to PR20 present a higher levels of translation than parental cells. Gene Set Enrichment Analysis (GSEA) of this dataset revealed an overall upregulation of factors from the “mTORC1 signaling” hallmark

overall_design: mRNA profiles of NSC34 PR20-resistant clones and untreated cells were generated by deep sequencing. Differential gene expression pathway analyses were performed.

first: Lafarga

last: Vanesa

first: Fernandez-Capetillo

last: Óscar


title: RNA-Seq of in vitro generated purified cDC1 from c-Jun/JunB fl/fl and  c-Jun/JunB Δ/Δ Itgax-Cre mice

city: Vienna

first: Philipp

last: Novoszel

address: Borschkegasse 8A

country: Austria

institute: Institute of Cancer Research

summary: We used RNA-Seq to define genes that control cDC1 indentity in the absence of the transcription factors c-Jun and JunB.

overall_design: FLT3 Ligand-derived cDC1 were generated from murine bone marrow in vitro, sort-purified for RNA extraction and sequenced by RNA-Seq to compare the transcriptional profile between c-Jun/JunB fl/fl and c-Jun/JunB Δ/Δ Itgax-Cre cDC1.

first: P

first: M

last: Sibilia


title: Prevention of MECP2 phosphorylation at S423 changes global transcription in primary cortical neurons co-cultured with BV2 cells upon LPS/IFNγ-induced activation.

status: Public on Feb 13 2021

city: Kuopio

first: Mikko

last: Hiltunen

address: Yliopistonranta 1E

department: Institute of Biomedicine

country: Finland

institute: University of Eastern Finland

summary: Methyl-CpG-binding protein 2 (MECP2) is a transcriptional regulator critical for synaptic function. Dysfunction of synapses, in turn, as well as microglia-mediated neuroinflammation belong to the earliest pathological events in Alzheimer’s disease (AD). Functional versatility of MECP2, including its affinity for different binding partners, transcriptional regulation of different gene sets, and effects on neuronal plasticity, are modulated by post-translational modifications, such as phosphorylation. To characterize expression changes through blocking of MECP2 S80 and S423 phosphorylation in neuron-BV2 co-cultures upon induction of neuroinflammation, mouse primary cortical neurons were transduced with MECP2-WT and MECP2-S80A and MECP2-S423A phopsho-variants. Neuroinflammation was induced with LPS/IFNγ and the samples were subjected to RNA sequencing. In neurons co-cultured with BV2 cells, blocking of MECP2 S423 phosphorylation increased the expression of several genes important for neuron and synapse maintenance and protection upon inflammatory stress conditions. Blocking of S80 phosphorylation did not lead to major global expression changes.The results suggest that MECP2 S423 phosphorylation might play a role in activation of neuronal gene expression conveying neuroprotection under neuroinflammation related stress conditions.

overall_design: Cortical DIV4 neurons were transduced with lentivirus vectors encoding MECP2-WT-Myc-IRES-ZsGreen (MECP2 reference sequece: NMNM_004992), MECP2-S80A-Myc-IRES-ZsGreen, MECP-S423A-Myc-IRES-ZsGreen or ZsGreen only (Control). BV2 cells were added 48 h after transduction and inflammation was induced with LPS (200 ng/ml) and IFNγ (20 ng/ml). Four replicates of primary cortical neurons were transduced independently of each other in each of the four sample groups. 16 RNA samples were analyzed in total.

first: Rebekka

last: Wittrahm

first: Mari

last: Takalo

first: Mikael

last: Marttinen

first: Teemu

last: Kuulasmaa

first: Petra

last: Mäkinen

last: Natunen

first: Annakaisa

last: Haapasalo


title: RNAseq of sorted a4b7+CCR9+ CD4+ Teff cells from human PBMC cultured in 10uM CITCO or DMSO control for 24 hours

city: Jupiter

first: Mark

last: Sundrud

state: FL

address: 120 Scripps Way

department: Immunology and Microbiology

institute: The Scripps Research Institute

summary: Bile acids (BAs) are a fundamental class of lipid emulsifying metabolites that are synthesized by hepatocytes, important for fat-soluble vitamin absorption, and maintained in vivo through enterohepatic circulation between the liver and ileum1. However, BAs are also lipophilic detergents and can be cytotoxic in enterohepatic tissues2. Whereas several nuclear receptors are known to prevent BA-driven toxicity in hepatocytes and intestinal enterocytes3, the mechanisms by which mucosal immune cells protect themselves from BAs in the ileum remains poorly understood. We recently reported that CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter, MDR1, in the ileum to protect against BA toxicity and suppress Crohn’s disease-like small bowel inflammation4. Here, we identify the BA- and xenobiotic-sensing nuclear receptor, constitutive androstane receptor (CAR/NR1I3), as a key regulator of MDR1 expression in mucosal Teff cells. CAR promotes large-scale transcriptional reprogramming of Teff cells in the small intestine lamina propria (siLP), which shares similarities with CAR-dependent gene expression in hepatocytes and involves the induction of both detoxifying enzymes and transporters, as well as anti-inflammatory cytokines, such as Il10. Accordingly, loss of CAR in Teff cells exacerbates, whereas pharmacologic CAR activation suppresses, BA-driven ileitis in T cell-reconstituted Rag1-/- mice. Further, CAR transcriptional activity is evident in human CD4+ T cells, but only in 4+7+CCR9+ Teff cells that are licensed for small bowel homing. In these cells, CAR activation also promotes cyto-protective and anti-inflammatory gene expression, including a core set of transcriptional targets that are similarly regulated by CAR in mouse mucosal Teff cells and hepatocytes. Together, these results suggest that mucosal T cells rely on CAR to activate a hepatocyte-like transcriptional response in the ileum that detoxifies BAs and safeguards immune homeostasis. Pharmacologic activation of this program may offer an unexpected strategy to treat small bowel Crohn’s disease.

overall_design: 6 total samples, triplicates of 2 conditions; Sorted a4b7+CCR9+ CD4+ Teff cells from human PBMC cultured in 50uM TC or DMSO control for 24 hours

last: Chen

middle: S


title: RNAseq of approximately 500 sorted congenic wild type or CAR deficient CD4+ T cells isolated from small intestine lamina propria, colonic lamina propria, or spleen from Rag deficient hosts at 3 weeks post transfer

overall_design: 18 total samples, triplicates of 6 populations


title: CAR directs T cell adaptation to bile acids in the small intestine


title: Corticosterone inhibits Gas6 to govern hair follicle stem cell quiescence

city: Cambridge

first: Sekyu

last: Choi

state: Massachusetts

address: 7 Divinity Ave, Sherman-fairchild 312

institute: Harvard University

summary: Chronic, sustained exposure to stressors can profoundly impact tissue homeostasis, although the mechanisms by which these changes occur are largely unknown. Here, we report the adrenal gland-derived stress hormone corticosterone (the rodent equivalent of cortisol) regulates hair follicle stem cell (HFSC) quiescence and hair growth in mice. Without systemic corticosterone, HFSCs enter substantially more rounds of the regeneration cycle throughout life. Conversely, under chronic stress, elevated corticosterone levels prolong HFSC quiescence and keep hair follicles in an extended resting phase. Mechanistically, corticosterone acts on dermal papilla (DP) to suppress the expression of a secreted factor, Growth Arrest Specific 6 (Gas6). Restoring Gas6 expression overcomes stress-induced inhibition of HFSC activation and hair growth. Our work identifies corticosterone as a systemic inhibitor of HFSC activity via its impact on the niche, and demonstrates that removal of such inhibition drives HFSCs into frequent regeneration cycles with no observable defects long-term.

overall_design: Cell type: FACS purified dermal papilla cells (DP) from the mouse skin; Conditions: Sham (Control) vs ADX (adrenalectomized mice); GR KO (Pdgfra-CreER; GRfl/fl) vs control; 2 replicate for each condition. FACS purified hair follicle stem cells (HFSC) from the mouse skin; Conditions: Sham vs ADX vs ADX+CORT (corticosterone); GR KO vs control; 2 replicate for each condition.

first: Bing

last: Zhang

first: Sai

last: Ma

first: Ya-Chieh

last: Hsu


title: High-throughput and automated acoustic trapping of extracellular vesicles to identify microRNAs with diagnostic potential for prostate cancer

city: Lund

first: Yvonne

last: Ceder

address: Scheelevägen 2

country: Sweden

institute: Lund University

type: Non-coding RNA profiling by high throughput sequencing

summary: We performed small RNA sequencing on 173 urinary extracellular vesicles derived from patients with either positive or negative prostate cancer biopsy in order to assess its potential as biomarkers.

overall_design: Comparison of the microRNA profile from urinary extracellular vesicles of patients with Gleason Grade Group ≥ 4 to patients with Gleason Grade Group ≤ 3, including patients with undetectable prostate cancer on biopsy.

first: Jacob

last: Fredsøe

first: Karina

middle: D

last: Sørensen

first: Michael

last: Borre

last: Evander

first: Thomas

last: Laurell

first: Hans

last: Lilja


title: The immune suppressive tumor microenvironment affects efficacy of radio-immunotherapy in breast-to-brain metastasis

status: Public on Feb 12 2021

last: Schulz

address: Breisacher Straße 64

department: Institute for Neuropathology

institute: University Hospital Freiburg

summary: The tumor microenvironment in brain metastases is characterized by high myeloid cell content with immune-suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T cell-directed therapies fail to elicit effective anti-tumor immune responses. Here we seek to evaluate the applicability of radio-immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy-induced immune modulation resulted in an increase in cytotoxic T cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experimental breast-to-brain metastases. However, long-term efficacy was not observed. Recurrent brain metastases showed accumulation of blood-borne PD-L1+ myeloid cells after radio-immunotherapy indicating the establishment of an immune-suppressive environment to counteract re-activated T cell responses. This finding was further supported by transcriptional analyses indicating a crucial role for monocyte-derived macrophages in mediating immune-suppression and regulating T cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.

overall_design: RNA-Seq of FACS purified myeloid and lymphoid cells out of a syngeneic breast-to-brain metastasis (infl. Monocytes, monocyte-derived macrophages, microglia, CD4+, CD8+, B220+ cells) and their normal cellular counterparts (healthy microglia, peripheral blood monocytes, blood CD4, CD8, and B220 cells)

first: K

last: Niesel

first: M

first: L

last: Sevenich


title: Cytoplasmic accumulation of FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and defects in inhibitory synapses

city: Strasbourg

first: Salim

last: Megat

state: Alsace

address: 1 rue Eugène Boeckel

country: France

institute: Center for Research in Biomedicine of Strasbourg (CRBS)

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived frontal cortex transcriptome profiling (RNA-seq) from control and a mouse of ALS-FUS.
Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a profound increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo. Importantly, RNAseq analysis suggested involvement of defects in inhibitory neurons, that was confirmed by ultrastructural and morphological defects of inhibitory synapses and increased synaptosomal levels of mRNAs involved in inhibitory neurotransmission. Thus, cytoplasmic FUS triggers inhibitory synaptic deficits, leading to increased neuronal activity and behavioral phenotypes. FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, but also in other neurodegenerative diseases with FUS mislocalization.

overall_design: Frontal cortex mRNA from 6- and 22-month-old WT and FUS-NLS male mice were generated by deep sequencing using Illumina Hiseq 4000.

first: Luc

last: Dupuis


title: Expression data from Eomes+ and Eomes- CD8 T cells following aOX40/aCTLA-4 treatment

status: Public on Feb 11 2021

city: Portland

first: Dana

middle: Alexander

last: Emerson

state: OR

address: 3181 SW Sam Jackson Park Road L220

department: Molecular Microbiology & Immunology

institute: OHSU

summary:
Development, maturation, and function of CD8 T cells is regulated by transcription factor expression. Expression of the transcription factor Eomesodermin was found to be significantly increased in CD8 T cells following aOX40/aCTLA-4 immunotherapy in mice.
We used microarray analysis to detail gene expression in Eomes+ and Eomes- CD8 T cells following aOX40/aCTLA-4 treatment to determin functional differences between these two populations.

overall_design: MCA-205 tumor bearing male Eomes-GFP C/57BL6 mice were treated with aOX40 and aCTLA-4 when tumors reached a size of 50-80mm2. Tumors were harvested seven days after the start of treatment and sorted for GFP+ and GFP- CD8 T cells by Fluorescence-activated cell sorting for gene array analysis by 3 independent experiments.

first: William

last: Redmond


title: RNAseq analysis of integrin β3-mediated responses to in vitro docetaxel in 4T1 breast cancer cells

city: St. Louis

first: Katherine

middle: N.

last: Weilbaecher

address: 660 S. Euclid Ave., Box 8069

institute: Washington University School of Medicine

summary: We sought to transcriptomically characterize the effect of integrin β3 expression on breast cancer cell responses to the microtubule-inhibiting chemotherapeutic agent docetaxel. Experiments were performed in 4T1 murine breast cancer cells with or without CRISPR mediated deletion of the Itgb3 gene.

overall_design: mRNA profiles from β3WT and β3KO 4T1 murine breast cancer cells after 24 hours of treatment with either DMSO or 10nM docetaxel and 48 hours of rest in fresh media. 3 biological replicates per group.

first: Gregory

last: Fox

first: Kristin

last: Kwakwa

first: Ha

middle: X

last: Dang

first: Christopher

last: Maher

middle: N


title: Synthetic lethality-mediated precision oncology via the tumor transcriptome

city: Gang-nam gu

first: Sehoon

last: Lee

state: Seoun

address: Ilwon

department: Division of Hematology-Oncology, Department of Medicine

country: South Korea

institute: SAMSUNG MEDICAL CENTER

summary: Precision oncology has made significant advances in the last few years, mainly by targeting actionable mutations in cancer driver genes. However, the proportion of patients whose tumors can be targeted therapeutically remains limited. Recent studies have begun to explore the benefit of analyzing tumor transcriptomics data to guide patient treatment, raising the need for new approaches for systematically accomplishing that. Here we show that computationally derived genetic interactions can successfully predict patient response.

overall_design: Assembling a broad repertoire of 32 datasets spanning more than 1,500 patients and including both tumor transcriptomics and response data

first: Joo

first: Se-Hoon


title: mRNA transcriptome analysis of bone in a mouse model of implant-associated Staphylococcus aureus osteomyelitis

city: Guangzhou

first: Yihuang

last: Lin

state: Guangdong

address: No.1838 North of Guangzhou Avenue

department: Orthopaedics

institute: Nanfang Hospital, Southern Medical University

summary:
Objective: To investigate the molecular pathogenesis of bone with implant-associated osteomyelitis (IAOM) in early and chonic stages.
Methods: After a 2 mm sterile stainless pin (0.3 mm in diameter) was inserted into the bone marrow cavity, 2 μl S. aureus solution at 1 × 10e6 CFU/ml was slowly injected into the bone marrow cavity using a micro-syringe to create IAOM mice while only an equal volume of sterile PBS to create controls. Transcriptomic sequencing was performed on day 3 and 14 post-operation, respectively. Differential expression analyses of IAOM_3D vs Ctrl_3D and of IAOM _14D vs Ctrl_14D were performed using the DESeq2 R package (1.16.1); P-values were adjusted using the Benjamini and Hochberg’s approach to control the false discovery rate. The genes with an adjusted P-value < 0.05 and |log2 (Fold Change)| > 1 were recognized as differentially expressed genes (DEGs). GO, KEGG and PPI analysis of DEGs were performed to identify key genes.
Results: Differential expression analyses detected a total of 101 DEGs, among which 96 were up-regulated and 5 down-regulated in the femur, when the IAOM and control groups were compared by day 3 post-infection. The number of DEGs significantly increased up to 1,702 by day 14 post-operation, with 1,065 up-regulated and 637 down-regulated in the IAOM femurs compared with controls. Further GO, KEGG and PPI analyses of DEGs suggested that IL-6, CXCL10, IFN-γ and CXCL9 were associated with early development of IAOM.
Conclusions: Our study has revealed transcriptomic characteristics of IAOM and suggested that IL-6, CXCL10, IFN-γ and CXCL9 might play an important role in the early stage of IAOM.

overall_design: 3 mice in the IAOM group and the control group were performed total RNA sequencing on the days 3 and 14 post-operation, respectively.

first: Jianwen

last: Su

first: Yutian

last: Wang

first: Daorong

last: Xu

first: Xianrong

last: Zhang

first: Bin

last: Yu


title: CRISPR perturbations at many coronary artery disease loci impair vascular endothelial cell functions [RNA-seq]

city: Montreal

first: Florian

last: Wuennemann

address: 5000 Rue Bélanger

institute: Montreal Heart Institute

summary: Genome-wide association studies have identified 161 genetic variants associated with coronary artery disease (CAD), but the causal genes and biological pathways remain unknown at most loci. This knowledge is critical to optimize precision medicine strategies and develop new drugs for CAD. Here, we performed multiple pooled CRISPR screens to test the impact of CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect by flow cytometry on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signaling). At a false discovery rate ≤10%, we identified 42 significant variants located within 26 CAD loci. Although a few of these loci include genes previously characterized in endothelial cells (e.g. MIA3/AIDA, FURIN, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed cellular and molecular characterization of the RNA helicase DHX38, as well as CRISPR-mediated over-expression experiments at the FURIN/FES, CCDC92/ZNF664 and CNNM2 loci, revealed a strong effect on vascular endothelial cell senescence. Our findings implicate many CAD-associated loci in the regulation of atherosclerosis-related endothelial functions and offer new insights into how modulating these functions could help prevent or treat CAD.

overall_design: TeloHAEC cell lines stably expressing Cas9 or dCas9-VP64 (CRISPRa) were infected with lentiviruses containing sgRNA targeting coronary artery disease loci or a safe-harbor locus as control, at a multiplicity of infection of 0.3. Following viral infection, cells were selected using zeocin (teloHAEC-CRISPRa or G418 (teloHAEC-Cas9) for five (teloHAEC-CRISPRa) or 7 days (teloHAEC-Cas9) in vascular cell basal medium (ATCC PCS-100-030) to remove any cells that did not incorporate a vector. After selection, cells expressing Cas9 were stimulated using TNFɑ (10ng/ul]) for four hours to induce a pro-inflammatory response.

last: Wünnemann

first: Guillaume

last: Lettre

first: Melissa

last: Beaudoin


title: Chromosome-scale assembly of the Symbiodinium microadriaticum genome

city: Worcester

first: Job

last: Dekker

address: 368 Plantation Street

institute: University of Massachusetts Medical School

summary: Hi-C assisted genome assembly of the Symbiodinium microadriaticum genome

overall_design: Hi-C

first: Ankita

last: Nand

first: Ye

last: Zhan


title: Illumina EPIC array on pooled DNA samples from peripheral blood

status: Public on Feb 10 2021

city: Cologne

first: Richard

middle: James

last: Acton

state: North Rhine-Westphalia

address: CECAD Cluster of Excellence at the University of Cologne, Joseph-Stelzmann-Straße 26

department: Bioinformatics Core

institute: CECAD

summary: Genome-wide DNA methylation profiling using the Illumina EPIC 850k DNA methylation BeadChip array on 8 pools of human genomic DNA from whole blood for 190 individuals age matched at 4 time points; ~4, ~28, ~63, & ~78 years.

overall_design: 8 pools of DNA: Pools 3-8 are comprised of DNA from 25 individuals each, all female; pools 1 and 2 have only 20 individuals each, with pool 1 being male and pool 2 female.

middle: G

last: Bell


title: Nicotinamide for the treatment of heart failure with preserved ejection fraction

city: Graz

first: Andreas

last: Prokesch

state: Styria

address: Harrachgasse 21/7

department: Institute of Cell Biology, Histology, and Embryology

institute: Medical University Graz

summary: Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD+). Elevating NAD+ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats) or cardiometabolic syndrome (in ZSF1 obese rats). Mechanistically, this effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics, as evidenced by cardiac trasncriptome and metabolome analyses. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium ATPase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD+ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.

overall_design: The aim of this study was to investigate the role of NAD+ in diastolic dysfunction and HFpEF. We quantified cardiac NAD+ in clinical and experimental HFpEF and evaluated the association between dietary intake of naturally occurring NAD+ precursors and cardiac mortality in a long-term human cohort. To examine the effect of increasing cellular NAD+ abundance on diastolic dysfunction in vivo, the NAD+ precursor NAM was orally supplemented to ZSF1 obese rats, which model key risk factors for HFpEF, namely hypertension, obesity and metabolic syndrome. We applied a multitude of in vivo and in vitro assays, including invasive hemodynamics, echocardiography, exercise tolerance testing with indirect calorimetry, RNA sequencing, metabolome profiling, and myocardial bioenergetics. For RNA sequencing, 4 myocardial rat samples per group were used, including lean ZSF1 rats as non-failing controls.

first: Mahmoud

last: Abdellatif

first: Jorge

last: Alegre-Cebollada

first: Simon

last: Sedej


title: PDK1 is required for maintenance of CD4+ FoxP3+ regulatory T cell function [Hemizygous]

city: Jersey City

first: Thomas

last: Postler

state: New Jersey

address: 95 Greene Street 5-018

department: Discovery Science

institute: International AIDS Vaccine Initiative

summary: The goal of this study was to determine the effect of Treg-specific Pdpk1 (PDK1) deletion on the transcriptome of Treg cells. CD4+ YFP+ Treg cells from spleens and peripheral lymph nodes of hemizygous Foxp3 YFP-Cre/0 Pdpk1 +/+ (WT) and Foxp3 YFP-Cre/0 Pdpk1 FL/FL (KO) were  sorted, RNA was isolated and RNA-seq libraries were constructed and sequenced by Hiseq2000. Note companion data set using Pdpk1-deleted Treg from Cre-heterozygous mice, which do not develop spontaneous inflammation.

overall_design: RNA-seq with mRNA isolated from CD4+ YFP+ (Treg) cells of Foxp3 YFP-Cre/0 Pdpk1 FL/FL or Foxp3 YFP-Cre/0 Pdpk1 +/+ mice.

first: Hyunju

last: Oh

first: Jingyao

last: Zhao

first: Yenkel

last: Grinberg-Bleyer

first: Sankar

last: Ghosh


title: CRISPR perturbations at many coronary artery disease loci impair vascular endothelial cell functions

last: Wuennemann

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

overall_design: Refer to individual Series


title: PDK1 is required for maintenance of CD4+ FoxP3+ regulatory T cell function


title: Single cell RNA-seq of Sox10-Cre::GFP+ population

city: Stockholm

first: Marek

last: Bartosovic

address: Solnavagen 9

institute: Karolinska Institute

summary: We performed single-cell RNA-seq on GFP+ sorted population from the brain from the Sox10-Cre::GFP transgenic mouse at postnatal day P21

overall_design: Brain was dissociated using Neural tissue dissociation kit (P) (Miltenyi Biotec), and debris was removed using Debris removal solution (Miltenyi Biotec). GFP+ cells were sorted on FACS Aria III and single-cell RNA seq was performed using chromium single cell 3' library preparation kit (v3)

first: Mukund

last: Kabbe

first: Goncalo

last: Castelo-Branco


title: Single-cell profiling of histone modifications in the mouse brain

summary: H3K27ac-directed HiChIP in cultured primary mOPCs

overall_design: HiChIP in cultured primary mOPCs

first: Gonçalo


title: PDK1 is required for maintenance of CD4+ FoxP3+ regulatory T cell function [heterozygous]

summary: The goal of this study was to determine the effect of Treg-specific Pdpk1 (PDK1) deletion on the transcriptome of Treg cells in the absence of inflammatory conditions. CD4+ YFP+ Treg cells from spleens and peripheral lymph nodes of Cre-heterozygous Foxp3 YFP-Cre/+ Pdpk1 +/+ (WT) and Foxp3 YFP-Cre/+ Pdpk1 FL/FL (KO) were  sorted, RNA was isolated and RNA-seq libraries were constructed and sequenced by Hiseq2500. Note companion data set using hemizygous Cre (induces spontaneous inflammation).

overall_design: RNA-seq with mRNA isolated from CD4+ YFP+ (Treg) cells of Foxp3 YFP-Cre/+ Pdpk1 FL/FL or Foxp3 YFP-Cre/+ Pdpk1 +/+ mice.


title: Integrative analysis of liver-specific noncoding regulatory variants associated with the risk of coronary artery disease

first: Minna

middle: U

last: Kaikkonen

address: Neulaniementie 2

institute: A.I.Virtanen Institute for Molecular Sciences

summary: We performed promoter Capture Hi-C in HepG2 to investigate interactions between gene promoters and distal elements as a transcription-regulating mechanism contributing to these phenotypes. We also performed ChIP-Seq at 2h, 8h and 23h timepoints in HepG2 for Il1B treatment.

overall_design: Promoter Capture Hi-C and Chip-Seq library was produced from HepG2

first: Ilakya

last: Selvarajan

first: Kristina

last: Garske

first: Oscar

middle: H

last: Liu

first: Päivi

last: Pajukanta


title: CRISPR perturbations at many coronary artery disease loci impair vascular endothelial cell functions [pooled CRISPR screen]

first: Florian

overall_design: Human, immortalized teloHAEC cell lines stably expressing different Cas9 variants (Cas9, dCas9-KRAB (CRISPRi), dCas9-VP64 (CRISPRa)) were infected with lentiviruses containing sgRNA targeting candidate loci and LD proxies for coronary artery disease (CAD) in the human genome. Cells were selected via anibiotic selection to remove cells not infected with an sgRNA. TeloHAEC expressing Cas9 or dCas9-KRAB (CRISPRi) were stimulated with TNFa to induce a proinflammatory response, whereas teloHAEC expressing CRISPRa were not stimulated. After 5 (CRISPRa) or 7 (Cas9 or dCas9-KRAB (CRISPRi)) days respectively, cells were stained and sorted for endothelial adhesion molecules (E-SELECTIN, ICAM1, VCAM1) or physiological readouts (NO, ROS, Ca2+). The top and bottom 10% of cells for each FACS marker were collected, gDNA extracted, sgRNA sequences amplified via PCR and sequenced.

last: Wünnemann

first: Guillaume

last: Lettre

first: Melissa

last: Beaudoin


title: Cytokine-sensing primary enhancer cluster launches secondary enhancers to activate multi-gene locus [RNA-seq]

city: Bethesda

first: Michaela

last: Willi

address: 9000 Rockville Pike

department: NIDDK

institute: NIH

summary: Enhancer clusters are platforms occupied at high-density by the transcription machinery to activate lineage-specific genes. However, it is not clear yet whether they are merely a collection of conventional enhancers or represent unique and new types of regulatory elements. Specifically, the question remains whether they activate additional enhancer programs in multigene loci. We addressed this issue in the mouse mammary gland, where genes are highly activated during pregnancy. We identify a 300kb multi-gene locus with twenty-one regulatory elements, including a cytokine-dependent primary enhancer cluster (PEC). Using genome editing in combination with ChIP-seq, RNA-seq and 3C-technologies, we show that the PEC initiates the activation of lineage-specific genes through contacting and activating secondary enhancers. This new framework of enhancer clusters indicates a hitherto unrecognized hierarchy of regulation by which a PEC activates genes via regulating secondary enhancers.

overall_design: Total RNA-seq was done for mammary tissues at pregnancy day 6 and 18 for wild-type and mutant mice carrying enhancer deletions within the Casein locus. One mouse lines carries a deletion of the enhancer cluster identified next to the primordial Odam within (Csn_S1-4). The other two mouse lines carry deletions of single enhancer elements: delCsn3-PE - deletion of the proximal enhancer of Csn3 and delCsn1s2b-E - a deletion of the enhancer of Csn1s2b.

first: Hye

middle: K

last: Lee


title: Bulk Cut&run and Cut&tag

summary: We generated bulk Cut&run data with antibodies agains H3K27me3 in primary mouse OPCs and OPC model cell line Oli-neu, and with antibodies agains H3K27ac in Oli-neu cell line with addition of BSA at various steps of the protocol. These datasets were used as benchmarks for the scCUT&Tag data of the mouse brain.

overall_design: Bulk Cut&run and Cut&Tag in primary mouse OPCs and OPC model cell line Oli-neu


title: Molecular reprogramming and phenotype switching in Staphylococcus aureus lead to high antibiotic persistence and affect therapy success

city: Zurich

first: Markus

last: Huemer

state: Zurich

address: Sternwartstrasse 14, Lab F39

department: Division of Infectious Diseases and Hospital Epidemiology

institute: University Hospital Zurich

summary: The goal of this experiment was to analyse transcriptomic changes upon acidic pH-stress in different S. aureus strains, including a high persister clincal isolate CI-1149. In addition we aimed to identify differentally regulated pathways that can be linked to an increased persister phenotype as seen in our clinical strain.

overall_design: S. aureus strains USA300_JE2, Cowan I and our clinical isolate CI-1149 were grown in DMEM pH 5.5 and pH 7.4 for three days. RNA transcripts were extracted in biological triplicates for each strain and condition. Samples were sequenced in Illumina NextSeq 500 system using 75 bp.

first: Srikanth

last: Mairpady Shambat


title: DAZAP2 controls cancer cell chemosensitivity by orchestrating the p53 response upon DNA damage

city: Heidelberg

first: Guenter

last: Raddatz

address: Im Neuenheimer Feld 580

institute: German Center for Cancer Research

summary: The tumor suppressors p53 and HIPK2 play a central role in cell fate decision-making upon DNA damage and regulate cancer cell chemosensitivity. Here we identify the adaptor protein Deleted in Azoospermia-associated protein 2 (DAZAP2) as a novel player in the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 potentiates cancer cell chemosensitivity in vitro and in vivo. In unstressed cells, DAZAP2 stimulates HIPK2 ubiquitination and degradation through interaction with HIPK2 and the ubiquitin ligase SIAH1. After chemotherapeutic drug-induced DNA damage, DAZAP2 accumulates in the cell nucleus, which is regulated through site-specific phosphorylation by HIPK2. Nuclear DAZAP2 interacts with p53 response elements and specifies p53 target gene expression by modulating a select subset of p53 target genes. Mechanistically, DAZAP2 forms a complex with p53 and modulates target gene expression in association with HDAC1. Our findings identify DAZAP2 as a novel regulator of the p53 response and cancer cell chemosensitivity.

overall_design: Whole transcriptome analysis to identify DAZAP2-regulated genes under physiological conditions or upon DNA damage. Briefly, HCT116 cells were either transfected with a siRNA targeting DAZAP2 or a control siRNA followed by treatment with either DMSO or 50 µM 5-FU for 24 h. 3 biological replicates per group were analyzed.

first: F

last: Lyko

first: T

last: Hofmann


title: Single cell Cut&Tag in the mouse brain

first: Marek

last: Bartosovic

summary: We performed single cell Cut&Tag using 10x genomics chromium platform with antibodies agains H3K4me3, H3K27ac, H3K27me3, H3K36me3, Rad21 and Olig2 on nuclei isolated from the mouse brain. The cells were sorted based on GFP fluorescence, from Sox10-Cre/GFP whole mouse brain at postnatal day P21-25. Additionaly, H3K4m3 and H3K27me3 scC&T includes both Sox10-Cre/GFP positive and negative cells sorted from the whole mouse brain at postnatal day P15.

overall_design: Cut&Tag profiles were generated by antibody directed tagmentation, coupled to standart 10x chromium ATAC-seq protocol. Single cell libraries were sequenced on NovaSeq platform and aligned and processed using cellranger-atac 1.2.0. Data was aggregated into cell by bin matrices and analyzed with R packages Seurat/Signac.

first: Mukund

last: Kabbe

first: Goncalo


title: Single cell profiling of histone modifications and transcription factors in the mouse brain


title: Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets [Bisulfite-Seq]

city: Memphis

first: Chunxu

last: Qu

state: TN

address: 262 Danny Thomas Pl

department: Pathology

institute: St Jude Children's Research Institute

summary: Acute erythroid leukemia (AEL) is a high-risk leukemia with a distinct mutational spectrum. Here, we examined the cooperativity of loss of function alterations in leukemogenesis and tested the therapeutic tractability of each model of AEL. Using CRISPR/Cas9 genome editing, we generated six pools of lentiviral vectors with different combinations of guide RNAs and transduced Cas9-eGFP-mouse mouse hematopoietic stem and progenitor cells (HSPCs) that were used in transplant assays. Induced tumors were characterized by phenotype, genomic/epigenomic analysis and tested for sensitivity to 222 therapeutic agents. Tumor genotype determined tumor phenotype with Bcor and Tp53 driving erythroid leukemogenesis.  Tumors showed acquisition of additional somatic mutations during serial passaging and by transcriptomic analysis AEL models had overexpression of transcription factors required for differentiation of megakaryocyte-erythroid progenitors and recapitulated human AEL.  Dnmt3a-mutated models exhibited global hypomethylation, but hypermethylation when co-occurred with Tet2 mutations.   Drug response was associated with genotype. Tp53 wild-type but Dnmt3a/Tet2 double mutant cells were the most chemo-sensitive models. ViceversaTp53-mutated AEL subtypes were chemo-resistant to several conventional chemotherapeutics but highly sensitivity to PARP inhibitors, including talazoparib. Sensitivity to talazoparib was confirmed in vivo when combined with decitabine. In conclusion, combinations of recurrently mutated genes are critical determinants of leukemia phenotype, with central roles of Tp53 and Bcor mutations in driving the erythroid leukemogenesis. Using these models as preclinical tools, we have shown the potential for bromodomain and PARP inhibition as new therapeutic strategies in AEL.

overall_design: Methyl-seq on mouse AEL models.

first: Ilaria

last: Iacobucci

first: Charles

last: Mullighan


title: Transcriptional profiling of STAT1 gain-of-function reveals common and mutation-specific fingerprints

status: Public on Feb 09 2021

city: Leuven

first: Rik

last: Schrijvers

address: Herestraat 49 bus 1023

department: Department of Microbiology, Immunology and Transplantation, Allergy and Clinical Immunology

country: Belgium

institute: KU Leuven

summary: STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical GAS sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion: our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism towards transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.

overall_design: mRNA sequencing on U3A cells complemented with STAT1 WT or GOF mutants, before and after IFNγ stimulation.

first: Simone

last: Giovannozzi

first: Jonas

last: Demeulemeester

last: Gijsbers


title: A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation

city: Nijmegen

first: Hendrik

last: Marks

address: Geert Grooteplein 26/28

department: Molecular Biology

country: Netherlands

institute: Radboud University Nijmegen, RIMLS

summary: Epigenetic profiling by chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-seq profiling of low-input samples, we constructed microfluidic PDMS-based plates capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time and 4.5 hours machine-running time. These disposable plates can be conveniently loaded into a widely available controller for pneumatics and thermocycling. In light of the Plug and Play (PnP) ChIP plates and workflow, we named our procedure PnP-ChIP-seq. We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for all six post-translational histone modifications that are included in the International Human Epigenome Consortium set of reference epigenomes. PnP-ChIP-seq robustly detects epigenetic differences on promoters and enhancers between naïve and more primed mouse embryonic stem cells (mESCs). Furthermore, we used our platform to generate epigenetic profiles of rare subpopulations of mESCs that resemble the 2-cell stage of embryonic development. PnP-ChIP-seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.

overall_design: Automated low-cell ChIP-seq on various epigenetic marks by the use of novel microfluidic technologies

first: René

middle: A

last: Dirks

first: Peter

middle: C

last: Thomas

first: Haoyu

last: Wu

first: Robert

last: Jones

last: Stunnenberg


title: Integrative genomic analysis of gemcitabine resistance in pancreatic cancer by patient-derived xenograft models

city: Beijing

first: Wenbo

last: Guo

state: Beijing

address: Haidian

institute: Tsinghua University


title: Loss of KDM4B Exacerbates Bone-Fat Imbalance and Mesenchymal Stem Cell Exhaustion in Skeletal Aging [ChIP-seq]

city: Los Angeles

first: Cunyu

last: Wang

state: CA

address: 10833 Le Conte Ave

department: School of Dentistry

institute: university of California, Los Angeles

summary: Mesenchymal stem/stromal cells (MSCs) are multipotent progenitor cells with multilineage differentiation potentials. We previously reported histone demethylases KDM4B promoted osteogenesis and inhibited adipogenesis of human MSCs. To identify genome-wide enrichments of KDM4B and the changes in H3K9me3 marks when loss of KDM4B, Primary MSCs were isolated from Prx1Cre; Kdm4bf/f mice and control mice. ChIP-seq assay was performed using KDM4B, H3K9me3 and H3 antibody, respectively.

overall_design:
Identification of genome-wide enrichments of KDM4B and H3K9me3 marks in mouse bone marrow-derived MSCs.

Primary mMSCs were isolated from Prx1Cre; Kdm4bf/f mice (KO) and Prx1Cre; Kdm4bw/w mice (WT).
ChIP assay was performed using KDM4B, H3K9me3 and H3 antibody (rabbit anti-KDM4B (1:400, Bethyl Laboratories, Cat No: A301-478A, Lot No.: 2); rabbit anti-KDM4B (1:400, Abcam, Cat No: ab191434, Lot No.: EPR18603-2); rabbit anti-H3K9me3 (1:500, Abcam, Cat No: ab8898, Lot No.: GR3176466-6) and rabbit anti-H3 (1:200, Cell Signaling Technology, Cat#2650, Lot No.: 20)), , respectively. Yeast chromatin was added as a spike-in control for H3K9me3 ChIP-seq. Libraries were prepared using KAPA Hyper Prep Kit (Kapa Biosystems, KK8502) per the manufacturer’s protocol. The sequencing was completed using HiSeq 3000 system (Illumina) or Nextseq 500 at the Technology Center for Genomics & Bioinformatics (TCGB) of UCLA.

first: Peng

last: Deng

first: Trent

last: Su

first: Jing


title: Loss of KDM4B Exacerbates Bone-Fat Imbalance and Mesenchymal Stem Cell Exhaustion in Skeletal Aging [RNA-seq]

summary: Primary mMSCs were cultured in control medium (Con), containing DMEM and 15% FBS, or were subjeucted to osteogenic induction (OI) for 2 days. RNA was isolated using RNeasy Mini Kit (Qiagen, Cat#74104) from three biological replicates of primary MSCs from Prx1Cre;Kdm4bf/f mice and control mice. Total RNA was then purified with Dynabeads™ mRNA Purification Kit (Invitrogen, Cat#61006). RNA-seq libraries were constructed using Stranded RNA-Seq Library Preparation Kit (Kapa Biosystems, Cat#KK8400), and sequenced using an illumina HiSeq 3000 sequencer at the Technology Center for Genomics & Bioinformatics (TCGB) core at UCLA.

overall_design: Identification of gene expression profiling in Kdm4b-/- mBMSCs.


title: DNA methylation data of PAC PDX models after Gemcitabine treatment

summary:
Purpose: Gemcitabine is most commonly used for pancreatic cancer (PC). However, the molecular features and mechanisms of the frequently occurred resistance remain unclear. This work aims at exploring the molecular features of gemcitabine resistance and identifying candidate biomarkers and combinatorial targets for the treatment.
Experimental Design: In present study, we established 66 patient-derived xenografts (PDXs) based on clinical PC specimens and treated them with gemcitabine. We generated multi-omics data (including whole exome-seq, RNA-seq, miRNA-seq and DNA methylation array) of 15 drug sensitive and 13 resistant PDXs before and after the gemcitabine treatment. We performed integrative computational analysis to identify the molecular networks related to gemcitabine intrinsic and required resistance. Then, shRNA-based high-content screening was implemented to validate the function of the de-regulated genes.
Results: The comprehensive multi-omics analysis and functional experiment revealed that MRPS5 and GSPT1 had strong effects on cell proliferation, and CD55 and DHTKD1 contributed to gemcitabine resistance in PC cells. Moreover, we found miR-135a-5p was significantly associated with the prognosis of PC patients and could be a candidate biomarker to predict gemcitabine response. Comparing the molecular features before and after the treatment, we found that PI3K-Akt, p53, HIF-1 pathways were significantly altered in multiple patients, providing candidate target pathways for reducing the acquired resistance.
Conclusions: This integrative genomic study systematically investigated the predictive markers and molecular mechanisms of chemoresistance in pancreatic cancer and provide potential therapy targets for overcoming gemcitabine resistance.

overall_design: xenografts derived from 28 pacreatic cancer patients after gemcitabine treatment

first: Gang

last: Yang

first: Wenfang

last: Guan

first: Zhe

last: Cao

first: Guangbing

last: Xiong

first: Fangyu

last: Zhao

first: Mengyu

last: Feng

first: Jiangdong

last: Qiu

first: Yueze

last: Liu

first: Michael

middle: Q

last: Zhang

first: Lei

last: You

first: Taiping

first: Yupei

first: Jin

last: Gu


title: DNA methylation data of PAC PDX models before Gemcitabine treatment

first: Wenbo

overall_design: xenografts derived from 28 pacreatic cancer patients before gemcitabine treatment


title: Clinical Application of a Lung Tumoroid Culture System

city: Cincinnati

first: Minzhe

state: OH

address: 3333 BURNET AVENUE

department: Pulmonary Biology

institute: Cincinnati Children's Hospital Medical Center

summary: Our goal and objective of this study is to identify genetic alterations using RNA-seq in a lung cancer organoid (tumoroid) line that we generated in our hospital.

overall_design: We extracted RNA from the lung tumoroid line that was generated from a lymph node metastasis originating from lung adenocarcinoma.  We analyzed it using RNA-seq.

first: Etsuko

last: Yokota

first: Takuya

last: Fukazawa

first: Yutaka

last: Maeda

first: Tomoki

last: Yanmatsuji


title: Diverse CGG binding proteins across eukaryotes produced by independent domestications of hAT transposons

city: Toronto

first: Isaac

last: Yellan

state: Ontario

address: 160 College St.

department: Molecular Genetics

institute: University of Toronto

summary: The human transcription factor (TF) CGGBP1 (“CGG Binding Protein”) is conserved only in amniotes, and is believed to derive from the zf-BED and Hermes transposase DNA-binding domains (DBDs) of a hAT DNA transposon. Here, we examine the DNA binding preferences of a wide variety of metazoan CGGBP1-like TFs with this bipartite domain using PBM experiments. The derived motifs are available at ...

sample_organism:
Locusta migratoria
Acyrthosiphon pisum
Aedes aegypti
Culex quinquefasciatus
Petromyzon marinus
Latimeria chalumnae
Acipenser ruthenus
Cyprinus carpio
Salmo salar
Fundulus heteroclitus
Sparus aurata
Chrysemys picta
Crocodylus porosus
Gallus gallus
Homo sapiens
Rhodnius prolixus
Erpetoichthys calabaricus
Rhipicephalus appendiculatus
Kryptolebias marmoratus
Nematostella vectensis
Austrofundulus limnaeus
Hucho hucho
Anabas testudineus
Acanthochromis polyacanthus
Amphiprion ocellaris
Labeo rohita
Mola mola
Hippocampus comes
Amphiprion percula
Salarias fasciatus
Sphaeramia orbicularis
Hofstenia miamia
Anabarilius grahami
Rhizophagus irregularis
Paramormyrops kingsleyae

overall_design: Protein binding microarray (PBM) experiments were performed for 106 CGGBP1-like proteins and related hAT transposons. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006.

first: Ally


title: Arterial stiffness and cardiac dysfunction in Hutchinson-Gilford Progeria Syndrome corrected by inhibition of Lysyl Oxidase

city: Philadelphia

first: Ryan

last: Von Kleeck

state: PA

address: 421 Curie Blvd

institute: University of Pennsylvania

summary:
Purpose: Arterial stiffening is a hallmark of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), but the molecular regulators remain unknown. Here, we show that the LMNAG609G mouse model of HGPS recapitulates the premature arterial stiffening seen in human HGPS. To gain a better understanding of potential stiffness-regulators in LMNAG609G mice, we performed RNA-sequencing analysis on cleaned descending aortas from 2- and 24-month WT and 2-month LMNAG609G mice on a C57BL6 background.
Methods: Descending aortas containing the intimal, medial and adventitial layers were isolated from 2- and 24-month male WT and 2-month HGPS mice, and RNA was extracted using the RNeasy Plus Micro kit (Qiagen 74034). The high-throughput library was prepared using the TruSeq stranded total RNA (ribo-Zero) kit (Illumina 20037135). Paired-end sequencing was performed on a HiSeq4000 Sequencing System (Illumina) and generated 14-30 million reads/sample.

overall_design: Aortic mRNA profiles of young and old wild type (WT) and young LMNAG609G mice.

first: Richard

last: Assoian

first: John

middle: W

last: Tobias


title: Tissue-specific reprogramming of host tRNA transcriptome by the microbiome

city: Guangzhou

first: Xiaoyun

last: Wang

address: 190 Kaiyuan Avenue, Huangpu

department: Infection and Immunity

institute: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

summary: The role of gut microbiota in modulating host tRNA modifications and expression has not yet been studied. In this experiment, we applied DM-tRNA-seq and normal tRNA-seq to obtain and infer the cytosolic and mitochondrial tRNA modifications and expression of four tissues (brain, intestine, kidney, and liver) in the specific pathogen-free (SPF) and germ-free (GF) mice, respectively. In conclusion, we confirmed that the modifications and expression of both cytosolic and mitochondrial tRNAs were influenced by not only tissue-specific but also microbiota-dependent manners. The present study facilitates comprehensive insights and better understanding into the interactions between the gut microbiota and the tRNA modifications and expression among host tissues

overall_design: We apply the DM-tRNA-seq and normal tRNA-seq to a total of 48 samples (SPF/GF mice, four tissues (brain, intestine, kidney, and liver), demethylase-treated/untreated, and three replicates). tRNA modifications and expression of their sequencing data are further analyzed in this study

first: Jia

last: Huang

first: Wenjun

last: Chen

first: Tao

last: Pan


title: Alternative RNA Stuctures Formed During Transcription Depend on Elongation Rate and Modify RNA processing

city: Aurora

first: David

last: Bentley

state: Colorado

department: BMG

institute: UCD AMC

summary: Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing, and determined how the “nascent RNA structureome” relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing.  Introns that become more structured at their 3’ splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary the plasticity of nascent transcripts has widespread effects on RNA processing.

overall_design: RNA pol II immunoprecipiation in isogenic HEK293 cells expressing a slow pol II mutant or wild type pol II was combinded with either in vivo chemical RNA structure probing using DMS or enzymatic probing using structure specific nuleases to identify cotranscriptional RNA structures

first: Ben

last: Erickson


title: Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition

city: Seattle

first: Jacob

middle: Tyler

last: Polaski

state: Washington

address: 1100 Fairview Ave. N.

department: Public Health Sciences/Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary: Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles for the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections between minor introns, hematopoiesis, and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNAs converged on LZTR1, a regulator of Ras-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing, and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as cancer drivers, and links between ZRSR2 mutations, LZTR1 regulation, and leukemias.

overall_design: RNA-seq of hematopoietic stem/progenitor cells (DAPI- lineage-negative c-Kit+ cells) harvested from Mx1-Cre-Zrsr2 conditional knockout mice.

first: Daichi

last: Inoue

first: Justin

last: Taylor

first: Robert

last: Bradley

first: Omar

last: Abdel-Wahab


title: RNAseq analysis of a patient with heterozygous frame-shift mutation in TRMT1 gene vs a patient with the mutation in homozygosis

status: Public on Feb 08 2021

city: Barcelona

first: Eva Maria

last: Novoa

address: Dr. Aiguader 88

institute: Center for Genomic Regulation

summary: Analysis of long RNASeq (>200bp)  of RNA obtained from Limphoblastoid cell lines derived from one heterozygous and one homozygous patient for TRMT1 frame-shift mutation

overall_design: long RNASeq for 2 conditions (MUT and WT), with biological duplicates.

first: Eva

middle: M

first: Sonia

last: Cruciani

first: Nicky

last: Jonkhout


title: White matter aging drives microglial diversity

city: Munich

first: Moritz

middle: J

last: Rossner

address: Nussbaumstr. 7

department: Psychiatry

overall_design: Grey and white matter  (corpus callosum) were dissected from the brain of 4 months and 21 months old wild type mice, single cell suspension was isolated using Neural Tissue Dissection kit (myelin was removed on percoll gradient) then microglia cells were isolated using CD11b microbeads by Magnetic separation with MS columns (MACS technology).The cell pellets are resuspended in the RLT buffer (100ul-150ul), snap frozen in liquid nitrogen and stored in -80 freezer. RNA was extracted and transcriptome libraries were generated

first: Shima

last: Safaiyan

first: Nirmal

last: Kannaiyan

first: Mikael

last: Simons


title: UTX constrains antiviral CD8+ T cell responses during chronic virus infection

city: Chapel Hill

first: Joshua

last: Starmer

state: NORTH CAROLINA

address: 120 Mason Farm Road

department: Genetics

institute: University of North Carolina at Chapel Hill

summary: Persistent virus infections can cause pathogenesis that is debilitating or lethal. During these infections, virus-specific T cells fail to protect due to weakened antiviral activity or failure to persist. These outcomes are governed epigenetically, though it is unknown which enzymes contribute to T cell loss or impaired function over time. Here, we show that T cell receptor-stimulated CD8+ T cells increase their expression of UTX (Ubiquitously transcribed Tetratricopeptide repeat, X-chromosome), an enzyme involved in demethylating histone-3 lysine-27 (H3K27), and reduce genome-wide H3K27-me3. UTX limits the frequency and durability of virus-specific CD8+ T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice by promoting inhibitory receptor expression and apoptosis. UTX also increases effector gene expression to improve CTL-mediated protection, a process corresponding to reduced H3K27me3 at effector gene bodies. Thus, UTX guides gene expression patterns in CD8+ T cells, promoting antiviral activity while reducing CD8+ T cell durability.

overall_design: RNA-seq, H3K27me3 CUT&RUN, and UTX CUT&Tag comparision of mouse WT P14 CD8+ T cells to Utx-/- P14 CD8+ T cells following adoptive transfer and LCMV-A22 infection. For RNA-seq, there were 3 WT replicates and 4 Utx -/- replicates 21 days post LCMV-A22 infection. For H3K27me3 CUT&RUN, there were 4 replicates for each group 15 days post LCMV-A22 infection. For UTX CUT&Tag, there were 4 WT replicates and 2 Utx -/- replicates 15 days post LCMV-A22 infection as well as 2 WT replicates prior to infection (day 0).

first: Joseph

last: Mitchell

first: Makayla

last: Lund

first: Karl

last: Shpargel

first: Terry

last: Magnuson

first: Jason

last: Whimire


title: Ferroptotic cell death triggered by conjugated linolenic acids is mediated by ACSL1

first: Jeffrey

middle: R

last: Peterson

address: 333 Cottman Avenue

department: Cancer Biology

institute: Fox Chase Cancer Center

summary: Ferroptosis is associated with lipid hydroperoxides generated by oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. We identified conjugated linoleates including α-eleostearic acid (αESA) as novel ferroptosis inducers. αESA did not alter GPX4 activity but was incorporated into cellular lipids and promoted lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death was mediated by acyl-CoA synthetase long-chain isoform 1, which promoted αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppressed ferroptosis triggered by αESA but not by GPX4 inhibition. Orally administered tung oil, naturally rich in αESA, limited tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a novel approach to ferroptosis, complementary to GPX4 inhibition, with therapeutic potential.

overall_design: Three independent replicates of MDA-MB-231 cells incubated for 5 hours with ML162 (125 nM) or ESA (100  µM) and 2 replicates for the control incubated with the vehicle for ESA (methanol) were sequenced.

first: Yan

last: Zhou

first: Alexander

last: Beatty


title: RNAseq analysis of integrin β3-mediated responses to in vitro docetaxel in PyMT-BO1 breast cancer cells

city: St. Louis

first: Katherine

last: Weilbaecher

state: MO

address: 660 S. Euclid Ave., Box 8069

institute: Washington University School of Medicine

summary: We sought to transcriptomically characterize the effect of integrin β3 expression on breast cancer cell responses to the microtubule-inhibiting chemotherapeutic agent docetaxel. Experiments were performed in PyMT-BO1 murine breast cancer cells with CRISPR mediated deletion of the Itgb3 gene. Cells were subsequently retrovirally rescued using either an empty vector construct (pMx), a functional human integrin β3 construct (hβ3), or a signaling-deficient integrin β3 mutant.

overall_design: mRNA profiles from β3KO PyMT-BO1 murine breast cancer cells rescued with empty vector (pMx), functional integrin β3 (hβ3), or signaling-deficient mutant integrin β3 (Δβ3) and receiving 24 hours of treatment with either DMSO or 10nM docetaxel. 3 biological replicates per treatment group.

first: Gregory

last: Fox

first: Kristin

last: Kwakwa

first: Ha

last: Dang

first: Christopher

last: Maher


title: small RNA sequencing analysis of TRMT1L KO vs WT brain

first: Eva Maria

last: Novoa

summary: TRMT1L is a paralog of the tRNA m22G-methyltransferase TRMT1 and, similarly to TRMT1, it is involved in cognitive functioning. To functionally dissect the biological role of TRMT1L, here we have employed a combined strategy of LC-MS/MS and next-generation sequencing of both long and short RNAs, to compare the levels and locations of m2,2G in brain tissues from TRMT1L knock-out mice, relative to wild-type.

overall_design: small RNASeq for 2 conditions (KO and WT), with biological triplicates.

first: Eva

first: Sonia

last: Cruciani

first: Nicky

last: Jonkhout


title: Single-cell RNA-sequencing of KPPF;Col1smaKO mouse models with pancreatic tumors

status: Public on Feb 07 2021

city: Houston

first: Yang

last: Chen

state: Texas

address: 1881 East Road

institute: UT-MD Anderson Cancer Center

summary: We employed dual-recombinase genetic mouse models of spontaneous PDAC mice (KPPF;Col1smaKO) to delete Col1 (type I collagen) specifically in myofibroblasts, in comparison with control KPPF mice. Single-cell RNA-sequencing analyses were performed on unfractionated live cell mixtures from pancreatic tumors of KPPF mice and KPPF;Col1smaKO mice, to investigate the impact of myofibroblast-specific Col1 deletion on tumor microenvironment.

overall_design: Unfractionated live cell mixtures from pancreatic tumors of KPPF control mice and KPPF;Col1smaKO mice were analyzed by 10x single-cell RNA-sequencing.

first: Raghu

last: Kalluri


title: Integrating multimodal data sets into a mathematical framework to describe and predict therapeutic resistance in cancer

city: Austin

first: Amy

last: Brock

state: TX

address: 107 West Dean Keeton St.

institute: University of Texas at Austin

summary: A significant challenge in the field of biomedicine is the development of methods to integrate the multitude of dispersed data sets into comprehensive frameworks to be used to generate optimal clinical decisions. Recent technological advances in single cell analysis allow for high-dimensional molecular characterization of cells and populations, but to date, few mathematical models have attempted to integrate measurements from the single cell scale with other data types. Here, we present a framework that actionizes static outputs from a machine learning model and leverages these as measurements of state variables in a dynamic mechanistic model of treatment response. We apply this framework to breast cancer cells to integrate single cell transcriptomic data with longitudinal population-size data. We demonstrate that the explicit inclusion of the transcriptomic information in the parameter estimation is critical for identification of the model parameters and enables accurate prediction of new treatment regimens. Inclusion of the transcriptomic data improves predictive accuracy in new treatment response dynamics with a concordance correlation coefficient (CCC) of 0.89 compared to a prediction accuracy of CCC = 0.79 without integration of the single cell RNA sequencing (scRNA-seq) data directly into the model calibration. To the best our knowledge, this is the first work that explicitly integrates single cell clonally-resolved transcriptome datasets with longitudinal treatment response data into a mechanistic mathematical model of drug resistance dynamics. We anticipate this approach to be a first step that demonstrates the feasibility of incorporating multimodal data sets into identifiable mathematical models to develop optimized treatment regimens from data.

overall_design: Single cell RNA-seq of MDA-MB-231 cell line with chemotherapy treatment

first: Kaitlyn

last: Johnson

first: Grant

last: Howard

first: Daylin

last: Morgan

first: Eric

last: Brenner

first: Andrea

middle: L

last: Gardner

first: Russell

middle: E

last: Durrett

first: William

last: Mo

first: Aziz

last: Al’Khafaji

first: Eduardo

middle: D

last: Sontag

first: Angela

last: Jarrett

first: Thomas

last: Yankeelov


title: Genome-wide maps of chromatin state in mTECs

city: Shanghai

first: haohao

last: zhang

state: Shanghai

address: 320 Yueyang Road

institute: Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences

summary: We performed ChIP-Seq against histones H3K4me3 and H3K27me3 in medullary thymic epithelial cells (mTECs) of wild-type mice to analysis bivalent epigenetic modifications.

overall_design: Examination of 2 different histone modifications in mTECs.

first: Zhi

last: Liu

first: Haohao

last: Zhang

first: Yiming

last: Hu

first: Xiaoren


title: Jmjd3 is required for tissue-restricted antigens expression in mTECs

summary: To understand the role of Jmjd3 in medullary thymic epithelial cells (mTECs) function and development, Jmjd3 was conditionally deleted in thymic epithelial cells. At the age of 4 weeks, mTECs were sorted by flow cytometry and RNA-seq was performed to identify the genes targeted by Jmjd3 for repression.

overall_design: RNAseq on control or Jmjd3-deficient murine medullary thymic epithelial cells.