title: Absence of cGAS-Mediated Type I IFN Responses in HIV-1-Infected T-Cells

status: Public on May 19 2020

city: Bochum

first: Daniel

last: Todt

address: Universitätsstr. 150

department: Molecular & Medical Virology

institute: Ruhr University Bochum

summary: The DNA sensor cGAS senses cytosolic DNA and catalyzes the production of the cyclicdinucleotide cGAMP, resulting in type I IFN responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T-cells. Activated primary CD4+ T-cells expressed cGAS and responded robustly to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T-cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Basal expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T-cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4+ T-cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T-cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1ΔUL41N) resulted in a cGAS dependent type I IFN response. In accordance with our results in primary CD4+ T-cells, plasmid challenge or HSV-1ΔUL41N inoculation of representative T-cell lines provoked an entirely cGAS-dependent type I IFN response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent type I IFN response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T-cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1ΔUL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T-cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS.

overall_design: Human CD4+ T cells of four healthy human donors were purified from whole blood samples by negative selection. Plated cells were inoculated with HIV-1Ba-L or control supernatant and subsequently treated with EFV. Total RNA from CD4+ T cells was extracted after the 3, 8, 48, 144, or 312 h post inoculation and RNAseq was performed.

first: Carina

last: Elsner

first: Aparna

last: Ponnurangam

first: Julia

last: Kazmierski

last: Zillinger

first: Katinka

last: Döhner

first: Shuting

last: Xu

first: Aurélie

last: Ducroux

first: Nils

last: Kriedemann

first: Angelina

last: Malassa

first: Pia-Katharina

last: Larsen

first: Gunther

last: Hartmann

first: Winfried

last: Barchet

first: Eike

last: Steinmann

first: Ulrich

last: Kalinke

first: Beate

last: Sodeik

first: Chrisitine

last: Goffinet


title: SMARCA4 Rescue Profiling [RNA]

status: Public on May 20 2020

city: SEATTLE

first: John

middle: A

last: Stamatoyannopoulos

state: WA

address: 2211 Elliott Avenue, 6th Floor

department: Genome Sciences

country: USA

institute: Altius Institute / University of Washington

type: Expression profiling by high throughput sequencing

summary: Mutations in SMARCA4, a central ATPase of the human BAF/PBAF chromatin remodeling complexes, cause developmental abnormalities and promote cancer development.  The pathogenic effects of SMARCA4 loss are often linked to the de-regulation of a relatively small number of key target genes. Here, to understand how chromatin remodeling by SMARCA4 results in specific transcriptional perturbations, we used genome engineering to correct a homozygous mutation in SMARCA4 in the well-characterized lung adenocarcinoma A549 cell line and profiled changes in SMARCA2/4 occupancy, chromatin accessibility, histone marks and transcription. Restoration of SMARCA4 causes a dramatic increase in chromatin accessibility at low affinity TF binding sites. Despite the widespread increase in chromatin accessibility, we observe comparatively attenuated changes in gene expression. Although there is a marked correlation between the number of local activated DHSs and the transcriptional responsivity of a gene, the influence of distal DHSs appears modified by a gene's promoter architecture and domain-scale chromatin organization. The largest changes in expression occur for genes in isolated, SMARCA4 sensitive chromatin domains that undergo region-wide chromatin remodeling upon reintroduction of SMARCA4. Our results reveal that interactions between distal enhancers, genome organization, and promoter architecture add transcriptional specificity to the global chromatin effects of BAF/PBAF complex perturbation and target the response to key developmental pathways.

overall_design: Regulatory profiling of a lung adenocarcinoma cell line with and without SMARCA4.  The loss of function mutation in SMARC4A in the SMARCA4-null cell line, A549, was corrected and DNaseI-Seq, RNA-Seq, and CUT-&-RUN ChIP-Seq was performed to study the effects of the correction on the chromatin landscape.

last: Lazar

first: Richard

last: Sandstrom


title: SMARCA4 Rescue Profiling [ChIP]

type: Genome binding/occupancy profiling by high throughput sequencing


title: MKRN2 physically interacts with GLE1 to regulate mRNA export and zebrafish retinal development (RNA-Seq)

city: Ipswich

first: Eric

last: Wolf

email: ewolf@neb.com, ericjwolf1@gmail.com

state: MA

address: 240 County Road

department: RNA Division

institute: New England Biolabs

summary: The canonical mammalian mRNA export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through mRNP remodeling. We conducted a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network revealed unexpected interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assessed MKRN2 epistasis with GLE1 in a genetically tractable zebrafish model. Strikingly, morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescued retinal developmental defects seen upon GLE1 depletion.  Next, using iCLIP, we showed that MKRN2 binds preferentially to the 3'UTR of a diverse subset of mRNAs. Next-generation sequencing of fractionated cell extracts revealed that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, these results indicate that MKRN2 selectively interacts with GLE1 to regulate mRNA nuclear export and retinal development.

overall_design: Examination of nucleocytoplasmic transcript distribution upon MKRN2 knockdown in HEK293 cells

middle: J

first: Andrew

last: Emili


title: MKRN2 physically interacts with GLE1 to regulate mRNA export and zebrafish retinal development

overall_design: Examination of the transcriptome-wide RNA-binding profile of EGFP-MKRN2 by individual-nucleotide resolution crosslinking and immuno-precipitation sequencing (iCLIP)


title: RNA seq analysis of primary human macrophages exposed to atheroma plaques homogenates with or without the Liver X receptor (LXR) antagonist GSK 2033

city: Dijon

first: David

last: MASSON

address: Bvd Jeanne D'Arc, UFR des sciences de santé

country: France

institute: INSERM

summary: To identify potential LXR target genes induced in the context of atherosclerosis we performed  a transcriptomic analysis of human macrophages exposed or not to human carotid plaque homogenates in the presence or the absence of the LXR antagonist GSK 2033. We used primary  macrophages from three distincts donors exposed to three differents plaque homogenates.

overall_design: After 6 days of differentiation in the presence of M-CSF, differentiated human macrophages were treated  with whole plaque homogenate (1% v/v in cell culture medium) for 48 hours with or without 1μM GSK2033. For plaque extracts used in cells treatment, lipid rich cores of human carotid plaque samples were carefully dissected under aseptic conditions and were homogenized with 3 volumes of cold NaCl 150 mmol/L and briefly sonicated.

first: D

last: Masson

first: L

last: Menegaut


title: Baseline Frequency of Inflammatory Cxcl9 Expressing Tumor-Associated Macrophages Predicts Response to Avelumab (anti-PDL-1) Treatment

status: Public on May 22 2020

city: san diego

first: Ji

last: Wen

state: CALIFORNIA

address: 10777 science center dr

institute: Pfizer Inc.

summary: scRNA-Sequencing of CD45+ TILs taken from a single tumor of a bilateral pair prior to avelumab treatment

overall_design: CD45+ TILs were FACS purified from CT26 tumors resected 8 days post s.c. implantation. Tumor sampled was a single tumor belonging to a bilateral pair, following resection mice were treated with avelumab to allow correlation of pre-treatment tumor immune cell profiles with treatment response.

first: Graham

last: Thomas


title: Combined targeting of G-protein-coupled receptor and EGF receptor signaling overcomes resistance to PI3K pathway inhibitors in PTEN-null triple negative breast cancer

status: Public on May 18 2020

city: London

first: Davide

last: Zecchin

address: Gower Street

country: United Kingdom

institute: University College London

type: Non-coding RNA profiling by high throughput sequencing

summary: MDA-MB-468 cells were infected with a whole-genome shRNA library, treated with PI3Kbeta inhibitor (AZD8186), pan class I PI3K inhibitor (GDC0941) or AKT inhibitor (MK2206) for 6+6 days. At the end of the treatment genomic DNA was extracted. PCR amplified and shRNAs inserts were quantified by NGS.

overall_design: shRNA screen in one triple-negative breast cancer cell line treated with PI3Kbeta, pan-PI3K or AKT inhibitor for 12 days

first: Stuart

last: Horswell

first: Julian

last: Downward


title: Comparison of Transcriptomes of Wild Type and Paraquat-treated cells by Next Generation RNA Sequencing

city: New York

first: Liza

middle: A.

last: Pon

state: NY

department: Pathology and Cell Biology

institute: Columbia University

summary: We use RNAseq to compare the transcriptomes of wild type and cells treated with 2.5 mM paraquat (PQ) for 8 hr. We find that PQ treatment results in changes in the transcripts for hundreds of genes. A notable subset of PQ-induced transcripts encodes proteins activated by the retrograde response pathway. This pathway is activated by ETC dysfunction and regulates carbohydrate metabolism to increase synthesis of biosynthetic intermediates through pathways including the TCA cycle, glutamine biosynthetic pathway, and isocitrate metabolism. Transcripts encoding oxidative stress response proteins (e.g. amino acid and iron transporters and siderophores) are also more abundant in PQ-treated cells.

overall_design: mRNA profiles of mid-log wild type (WT) yeast cells and cells with 2.5 mM PQ treatment were generated by deep sequencing

first: Pin-Chao

last: Liao


title: RNAseq of B16F10 melanoma CD31+ cells

city: Uppsala

first: L.

last: He

address: Dag Hammarskjölds Väg 20

country: Sweden

institute: Uppsala University

summary: Intravasation, vascular dissemination and metastasis of malignant tumor cells require their passage over the vascular wall which is commonly composed of pericytes (mural cells) and the endothelial cell barrier. We currently decided to investigate the relative contribution of these cell types for B16F10 melanoma metastasis in mice using an experimental model of Shb gene inactivation. RNAseq of tumor endothelial cells from these mice revealed changes in cellular components such as adherens junctions and focal adhesions by gene ontology analysis that were in line with the observed effects on leakage and junction morphology.

overall_design: Endothelial cells from B16F10 melanomas grown on wild type mice (N=4 ) and mice with Shb conditionally inactivated in endothelial cells (N=6) were isolated and subjected to RNAseq comparison.

first: Qi

first: Xiujuan

last: Li

first: Liqun

first: Michael

last: Welsh


title: Single-cell RNA-sequencing of CD45+ cells isolated from mouse dura mater or brain after photothrombotic injury

city: Daejeon

first: Bong Ihn

last: Koh

address: 291 Daehakro

country: South Korea

institute: Korea Advanced Institute of Science and Technology

summary: To further elucidate molecular differences in immune cells and identify potential sources of Vegfa in response to photothrombotic injury, we performed single-cell RNA-sequencing of CD45+ cells isolated from the mouse dura mater or brain after photothrombotic injury.

overall_design: 3 days after photothrombotic injury on 8-week-old mice, CD45+ cells of the dura mater or brain were isolated by FACS and subjected to single-cell RNA-sequencing using the 10X Chromium system.

first: Bong

middle: I


title: FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development

city: Paris

first: Emilie

last: Dambroise

address: 24 bd Montparnasse

institute: Institut Imagine

summary: In order to study FGFR3’s role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof1/lof1). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype, we performed single-cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell-cell communication, mineralization, and new osteoblast recruitment.

overall_design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish (fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377), we profiled the transcriptome of nearly 28000 single cells using the Chromium system (10x Genomics). Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types (osteogenic cells, chondrogenic cells, immune cells epidermal cells, endothelial cells). The scRNA-seq libraries were generated with a Chromium Single Cell 3′ Library & Gel Bead Kit v.3 (10x Genomics). The purified libraries were sequenced on a Novaseq 6000 (Illumina) on paired end strands (read 1: 28bp; read 2: 91bp), with a mean read depth of 25000 reads per cell.

first: Ghaith

last: Abdessalem

first: Michaël

last: Menager

first: Alessandro

last: Brombin

first: Laurence

last: Legeai-Mallet


title: RNA-sequencing of dura mater or brain ECs isolated from 8-week-old mice

summary: To further elucidate fundamental molecular differences between dura mater and brain ECs, we performed RNA-sequencing of primary dura mater or brain ECs isolated from adult mice

overall_design: Dura mater or ECs were isolated by FACS from 8-week-old mice and mRNA profiles were generated by deeping sequencing, in duplicate, using Illumina NextSeq 500.

first: Injune

last: Kim


title: Presymptomatic Increase of an Extracellular RNA in Blood Plasma Associates with the Development of Alzheimer’s Disease

city: La Jolla

first: Sheng

last: Zhong

state: California

address: 9500 Gilman Drive MC 0412

department: Bioengineering

institute: University of California, San Diego

summary: The extracellular RNAs (exRNAs) from human biofluid have recently been systematically characterized. However, the correlations of biofluid exRNA levels and human diseases remain largely untested. Here, considering the unmet need for presymptomatic biomarkers of sporadic Alzheimer’s disease (AD), we leveraged the recently developed SILVER-seq (small-input liquid volume extracellular RNA sequencing) technology to generate exRNA profiles from a longitudinal collection of human plasma samples. These 164 plasma samples were collected from research subjects 70 years or older with up to 15 years of clinical follow-up prior to death and whose clinical diagnoses were confirmed by pathological analysis of their post mortem brains. The exRNAs of AD-activated genes and transposons in the brain exhibited a concordant trend of increase in AD plasma in comparison with age-matched control plasma. However, when we required statistical significance with multiple testing adjustments, phosphoglycerate dehydrogenase (PHGDH) was the only gene that exhibited consistent upregulation in AD brain transcriptomes from 3 independent cohorts and an increase in AD plasma as compared to controls. We validated PHGDH’s serum exRNA and brain protein expression increases in AD by using 5 additional published cohorts. Finally, we compared the time-course exRNA trajectories between “converters” and controls. Plasma PHGDH exRNA exhibited presymptomatic increases in each of the 11 converters during their transitions from normal to cognitive impairment but remained stable over the entire follow-up period in 8 out of the 9 control elderly subjects. These data suggest the potential utilities of plasma exRNA levels for screening and longitudinal exRNA changes as a presymptomatic indication of sporadic AD.

overall_design: exRNA profiles of 164 plasma samples from 35 pathology-confirmed participants, including 15 AD participants (donor group AD), 9 age-matched controls (donor group N), and 11 participants who converted (donor group C) from normal to mild cognitive impairment (MCI) or AD (converters) were generated by SILVER-seq.

first: Zixu

last: Zhou

first: Zhangming

last: Yan

first: Qiuyang

last: Wu


title: Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance [RNA-Seq]

city: San Antonio

first: Zhijie

middle: Jason

last: Liu

state: TEXAS

address: 7703 Floyd Curl Drive

department: Department of Molecular Medicine

institute: Universality of Texas Health Science Center at San Antonio

summary: Cellular plasticity has emerged as an important mechanism of therapy resistance in cancers, yet the underlying molecular mechanisms remain unclear. Using an established breast cancer cellular model for endocrine resistance, we show that hormone resistance is associated with enhanced cellular plasticity, indicated by a general downregulation of luminal/epithelial differentiation markers and upregulation of basal/mesenchymal invasive markers. Our extensive omics studies including GRO-seq on enhancer landscapes demonstrate that the global enhancer gain/loss reprogramming driven by the differential interactions between ERα and other oncogenic transcription factors (TFs), predominantly GATA3 and AP1, profoundly alter breast cancer transcriptional programs. Our functional studies in various biological systems support a coordinated role of GATA3 and AP1-mediated enhancer reprogramming in driving cellular plasticity to achieve endocrine resistance or cancer invasive progression. Thus, changes in TF-TF and TF-enhancer interactions can lead to genome-wide enhancer reprogramming, resulting in transcriptional dysregulations that promote plasticity and cancer therapy-resistance progression.

overall_design: Total RNA extraction followed by high throughput sequencing (RNA-seq)

first: Zhao

last: Zhang

first: Mingjun

last: Bi


title: Single Cell RNA-sequencing of murine back skin interfollicular epidermis basal cells

city: Bruxelles

first: Cedric

last: Blanpain

address: 808, route de Lennik, CP 637

country: Belgium

institute: Université Libre de Bruxelles (ULB)

summary: The ability of the skin to grow in response to stretching has, for decades, been exploited in reconstructive surgery. The response of epidermal cells to stretching has been studied in vitro. However, it remains unclear how mechanical forces affect epidermal cell behaviour in vivo. Here, we develop a mouse model in which the consequences of stretching on skin epidermis can be studied. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA-seq, we show that stretching induces skin expansion by a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the mechanisms that control stretch-mediated tissue expansion.

overall_design: The purpose of this study was to define, at the single cell level, the transcriptional profile of murine dorsal epidermal basal cells during homeostasis in adult animals, during tissue stretching at day (D)1 (EXPD1) and D4 (EXPD4) and upon  12-O-Tetradecanoylphorbol-13-acetate (TPA) tretment. Tissue stretching was performed using self-inflating hydrogels purchased from the company Osmed. The analysis was performed on CD1 mice and on basal interfollicular epidermal cells that were FACS sorted for the positive expression of ITGA6 and negative expression of CD34. Additionally, mice in stretching at D2 (EXPD2) and treated with the MEK1/2 inhibitor Trametinib and with the MAL inhibitor CCG203971 were also analysed.

first: Mariaceleste

last: Aragona

first: Milan

last: Malfait

first: Alejandro

last: Sifrim

first: Thierry

last: Voet


title: Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance [ChIP-Seq]

overall_design: Chromatin Immunoprecipitation (ChIP) assay followed by high throughput sequencing (ChIP-seq)


title: Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance [ATAC-Seq]

overall_design: Assay for Transposase-Accessible Chromatin followed by high throughput sequencing (ATAC-seq)


title: Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance [GRO-Seq]

overall_design: Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq)


title: Oligosaccharide-dependent anti-inflammatory role of galectin-1 for macrophages in inflammatory bowel disease

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

country: Japan

institute: Osaka univ.

summary: Our previous study demonstrated that Galectin-1 plays a protective role for colitis by binding with polylactosamine structures in β-1,4-galactosyltransferase I-deficient mice, but precise function of galectin-1 remains unknown. In the present study, we investigated about the anti-inflammatory role of galectin-1 for macrophages to ameliorate inflammatory bowel disease in both animal model and human tissue sample. To know the anti-inflammatory effect of galectin-1 in vivo, transfer of BMDMs cultured with galectin-1 into Recombination activating gene (Rag) 2-/- mice and treatment with galectin-1 in dextran sodium sulfate (DSS) colitis model were performed. Furthermore, RNA sequencing was performed to know the character of macrophages treated with galectin-1. In UC patients, tissue expressions of galectin-1 were decreased in inflamed mucosa compared with those in non-inflamed mucosa. Galectin-1 induced IL-10 production in BMDMs, and the production was abrogated by the addition of lactose, which inhibits the interaction of oligosaccharide-galectin binding. DSS colitis was significantly ameliorated in Rag2-/- mice to which galectin-1-treated BMDMs were transferred, than those transferred with vehicle-treated BMDMs. RNA sequence showed that treatment with Galectin-1 increased the expression of CCAAT/enhancer binding protein β and CD163 but decreased CD80 on BMDMs. Galectin-1 ameliorates murine colitis through the induction of oligosaccharide-dependent anti-inflammatory properties to macrophages.

first: Shuko

last: Iwatani

first: Shinichiro

last: Shinzaki


title: A plant-specific ISWI subunit is involved in fertility by inhibiting the pistil overgrowth

city: Beijing

first: Xin-Jian

last: He

address: No. 7, zhongguancun life science park road, changping district

country: China

institute: National Institute of Biological Sciences

summary: we identified the forkhead-associated domain 2 (FHA2) as a plant-specific subunit of an ISWI chromatin-remodeling complex in Arabidopsis. By in vivo and in vitro analyses, we demonstrated that FHA2 directly binds to RLT1 and RLT2, two redundant subunits of the ISWI complex in Arabidopsis. A reduced-fertility phenotype was observed in both the fha2 and rlt1/2 mutant plants and the phenotype was ascribed to overgrowth of the pistil during the floral development. However, the early-flowering phenotype of the rlt1/2 mutant was not shared by the fha2 mutant. Consistently, our RNA-seq analysis indicated that the fha2 mutant affects a subset of rlt1/2-regulated genes but not genes involved in the regulation of flowering time. This study demonstrates that FHA2 functions as a previously uncharacterized subunit of the Arabidopsis ISWI complex to exclusively regulate the pistil growth during the floral development.

overall_design: Examination of the mRNA expression in Arabidopsis


title: SMARCA4 Rescue Profiling [Dnase]

first: John

last: Stamatoyannopoulos

overall_design: Regulatory profiling of a lung adenocarcinoma cell line with and without SMARCA4.  The loss of function mutation in SMARC4A in the SMARCA4-null cell line, A549, was corrected and DNaseI-Seq, RNA-Seq, and CUT-&-RUN ChIP-Seq was performed to study the effects of the correction on the chromatin landscape.

last: Lazar

first: Richard

last: Sandstrom


title: Heat shock stimulates genomic instability and phenotypic variations in yeast

city: Hangzhou

first: Daoqiong

last: Zheng

state: Zhejiang

address: 866 Yuhangtang Road

department: Institute of Microbiology

institute: College of Life Sciences, Zhejiang University

summary: Yeast Saccharomyces cerevisiae has been widely used as a model system for studying genomic instability. In this study, heat-shock-induced genomic alterations were explored in the heterozygous diploid yeast strain JSC25-1. In combination of the whole-genome microarray, the patterns of chromosomal instability induced by heat shock could also be explored at a whole genome level. Using this system, we found heat-shock treatment resulted in hundreds-fold higher rate of genomic alterations, including aneuploidy and loss of heterozygosity (LOH).

overall_design: The JSC25-1 cells were cultured in liquid YPD to an OD600 of 0.25 (initial OD600 was 0.05), then cells at a concentration of 1 OD600 were heat-shocked at 52℃ for 4 min. The treated cells were then plated on YPD plates to allow colonies formation at 30℃ for 3 days. A total of 21 independent colonies (named JP1-JP21) were randomly selected for whole-genome microarray analysis.

first: Dao-Qiong

first: Thomas

middle: D

last: Petes


title: Mechanisms of stretch-mediated skin expansion at single cell resolution [ATAC-seq]

address: route de Lennik, 808

institute: Université Libre De Bruxelles

summary: The ability of the skin to expand in response to stretching has, for decades, been exploited in reconstructive surgery. Several studies have investigated the response of stretching epidermal cells in vitro. However, it remains unclear how mechanical forces affect epidermal stem cell behaviour in vivo. Here, we develop a mouse model in which the temporal consequences of the stretching the skin epidermis can be studied. Using a multidisciplinary approach that combines clonal analysis and mathematical modelling, we show that mechanical force induces skin expansion by promoting the renewal of epidermal stem cells. This occurs through a structured response in which cell fates are coordinated locally by stem cells that switch between states primed for renewal or differentiation. Transcriptional and chromatin profiling identifies the gene regulatory networks modulated by mechanical force. Using a combination of pharmacological inhibition and several conditional gene loss-of-function mouse mutants, we dissect the signalling pathways that control force-mediated tissue expansion.

overall_design: We used ATACseq to profile the chromatin landscape of  basal cells isolated from the interfolliular epidermis during force-mediated tissue expansion. Interfollicolar basal epideramal cells form the back skin were FACS sorted for the positive expression of ITGA6 and negative expression of CD34. One CD1 mouse was subjected to force-mediated tissue expansion and an untreted animal was used as control. For each sample, 10000 basal cells were FACS sorted and used for ATAC preparation.

first: Yura

last: Song


title: Minimum embedding dimension analysis uncovers reduced network complexity during neuronal differentiation in autism spectrum disorder

city: Freiburg im Breisgau

first: Galina

last: Erikson

address: Stübeweg 51

country: Germany

institute: Max Planck Institute of Immunobiology and Epigenetics

summary:
Background: Neuronal activity can be modeled as a nonlinear dynamical system to yield novel measures of neuronal state and dysfunction. We hypothesized that electrical activity during neuronal differentiation would be marked by a reduction in dynamical complexity in autism spectrum disorder (ASD).
Methods: Electrical activity of induced pluripotent stem cell (iPSC)-derived neurons from ASD patients and controls were recorded using a multielectrode array (MEA). Minimum embedding dimension (MED) analysis was performed to characterize the complexity present in the electrical recordings of the neuronal cultures during differentiation. Gene expression changes with respect to the MED were analyzed, and gene ontology analysis and the BrainSpan Atlas were used to identify gene enrichments in specific biological pathways, brain regions, and developmental stages.
Results: We found that the MED showed a statistically significant deficit in the ASD samples, suggesting that, compared to controls, network complexity is reduced in ASD during neuronal differentiation. MED was correlated to clinical endpoints such as nonverbal intelligence and was associated with 53 differentially expressed genes. These differentially expressed genes were overrepresented in ASD gene lists and pathways related to neurodevelopment, cell morphology, and cell migration. Spatiotemporal analysis also showed a prenatal temporal enrichment in cortical and deep brain structures.
Conclusion: Reduced network complexity in ASD was observed by MED analysis during neuronal differentiation, indicating deficits in integrated and synchronized firing. The results present nonlinear dynamical analysis of iPSC-derived neuronal electrical recordings as a new paradigm for the investigation of aberrant brain activity in neurodevelopmental disorders.

overall_design: Examination of gene expression correlates to dynamical electrical properties in autistic and control cell lines

first: Debha

last: Amatya

first: Yeni

last: Kim


title: Xrn1p Acts at Multiple Steps in the Budding-Yeast RNAi Pathway to Enhance the Efficiency of Silencing

city: Cambridge

first: David

middle: P.

last: Bartel

address: 455 Main St.

institute: Whitehead Institute

summary: RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation, and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5′-to-3′ exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand, and degradation of sliced target RNA.

overall_design: The experiments consist of small RNA-seq and RNA-seq experiments from Naumovozyma castellii strains grown to mid-log phase. Two of the strains (WT_selection, xrn1_KO_selection) had hairpin-based silencing constructs against HIS3, URA3, and GFP genes and an extra copy of Argonaute (AGO1) and Dicer (DCR1) inserted into their genomes, which allowed this strain to be used in a genetic selection for components of budding-yeast RNAi pathways. Three other strains (WT_non-selection, xrn1_KO_non-selection, ago1_mut_non-selection) were in a genetic background in which their endogenous Argonaute protein was N-terminally FLAG-tagged (FLAG-tagged).

first: Matthew

last: Getz

middle: P


title: IRE1α-XBP1 signaling in leukocytes controls prostaglandin biosynthesis and pain

status: Public on May 16 2020

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: The IRE1α-XBP1 arm of the unfolded protein response (UPR) maintains endoplasmic reticulum (ER) homeostasis, but also controls UPR-independent processes such as cytokine production and lipid metabolism. Yet, the physiological consequences of IRE1α-XBP1 activation in immune cells remain largely unexplored. Here, we report that leukocyte-intrinsic IRE1α-XBP1 signaling drives prostaglandin biosynthesis and pain. Transcriptomic analyses revealed that induction of prostaglandin-endoperoxide synthase 2 (Ptgs2/Cox-2) and prostaglandin E synthase (Ptges/mPGES-1) was compromised in IRE1α-deficient myeloid cells undergoing ER stress or stimulated via pattern recognition receptors. Inducible biosynthesis of prostaglandins, including PGE2, was markedly decreased in myeloid cells lacking IRE1α or XBP1, but not altered in the absence of the two other ER stress sensors PERK and ATF6. Mechanistically, IRE1α-activated XBP1 bound to and directly induced the expression of human PTGS2 and PTGES to enable PGE2 generation. Mice selectively lacking IRE1α-XBP1 in leukocytes, or treated with pharmacological IRE1α inhibitors, failed to induce PGE2 upon challenge with inflammatory stimuli and demonstrated reduced behavioral pain responses in PGE2-dependent models of pain. Our study uncovers an unexpected role for IRE1α-XBP1 as a key mediator of prostaglandin biosynthesis and indicates that targeting this pathway may represent an alternative approach to control pain.

overall_design: RNA-seq for bone marrow derived dendritic cells from mice untreated and treated with Zymosan and LPS in IRE1α wild type and knockout conditions

first: Sahil

last: Chopra

first: Juan

middle: R

last: Cubillos-Ruiz


title: Molecular Alterations of Extracellular Matrix in the Brain of Newborns with Congenital Zika Syndrome

city: São Paulo

first: Helder

middle: I.

last: Nakaya

address: 438, Av. Prof. Lúcio Martins Rodrigues, 374 - Butantã

country: Brazil

institute: Universidade de São Paulo

summary: Zika virus (ZIKV) infection during pregnancy could cause a set of severe abnormalities in the fetus known as congenital Zika syndrome (CZS). Experiments using animal models and in vitro systems significantly contributed to our understanding of the physiopathology of ZIKV infection. However, the molecular basis of CZS is not yet studied in humans. Here, we used a systems biology approach to integrate transcriptomic, proteomic and genomic data from post-mortem brains of neonates with CZS. We observed that collagen genes were greatly reduced in CZS brains at both the RNA and protein levels and that neonates with CZS have several polymorphisms in collagen genes associated with osteogenesis imperfect and arthrogryposis. These findings were validated using immunohistochemistry and collagen staining of ZIKV infected and non-infected samples. Additionally, it was found that cell adhesion genes that are essential for neurite outgrowth and axon guidance were up-regulated and thereby confirmed the neuronal migration defects observed. This work provided new insights into the underlying mechanisms of CZS and revealed host genes associated with CZS susceptibility.

overall_design: RNA-seq of post-mortem cortex samples from ZIKV infected infants with microcephaly

first: Renato

middle: S

last: Aguiar

first: Fabio

last: Pohl

first: Guilherme

middle: L

last: Morais

middle: C

last: Nogueira

first: Joseane

middle: B

last: Carvalho

first: Letícia

last: Guida

first: Luis

middle: W

last: Arge

first: Adriana

last: Melo

first: Maria

middle: E

last: Moreira

first: Daniela

last: Cunha

first: Leonardo

last: Gomes

first: Elyzabeth

last: Portari

first: Erika

last: Velasquez

first: Rafael

last: Melani

first: Paula

last: Pezzuto

first: Fernanda

last: de Castro

first: Victor

last: Geddes

first: Alexandra

last: Gerber

first: Girlene

last: Azevedo

first: Bruno

last: Schamber-Reis

first: Alessandro

last: Gonçalves

first: Inácio

first: Milton

middle: Y

last: Junior

first: Paulo

last: Ho

first: Alessandra

last: Schanoski

first: Viviane

last: Schuch

first: Amilcar

last: Tanuri

first: Leila

last: Chimelli

first: Zilton

middle: F

last: Vasconcelos

first: Gilberto

last: Domont

first: Ana

middle: T


title: Molecular interplay between dormant bone marrow-resident cells (BMRCs) and CTCs in breast cancer.

status: Public on May 15 2020

city: Albuquerque

first: Dario

last: Marchetti

state: NM

address: 1 University of New Mexico

department: Internal Medicine

institute: University of New Mexico

summary:
Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. To overcome these hurdles, we developed a novel experimental model of tumor dormancy employing circulating tumor cells (CTCs) derived from metastatic breast cancer patients (de novo CTCs), transplanted them in immunocompromised mice, and re-isolated these cells from xenografted mice bone marrow (ex vivo BMRCs) and blood (ex vivo CTCs) to perform downstream transcriptomic analyses.
Here we report that the balance between mTORC1 vs mTORC2 signaling regulates BMRC mitotic and/or dormancy characteristics.

overall_design: Blood samples were collected from 6 (2 each of ER/PR+, HER2+ and triple negative) metastatic breast cancer patients in accordance to a protocol approved by Institutional Ethical Review Board at Houston Methodist Research Institute. After red blood cell lysis, the remaining buffy coat layer was subjected to FACS for enrichment of CTC populations by (i) doublet discrimination and dead cell elimination (DAPI-), followed by (ii) depletion of cells normally present in the peripheral blood using lineage-specific (CD45-/CD34-/CD105-/CD90-/CD73-) antibodies. Next, we implanted the residual CTC-enriched/lineage-negative cells into NOD scid-gamma (NSG) mice by intra-cardiac injection. Mice were euthanized after 4-8 months of in vivo selection, and bone marrow and blood were subjected to FACS to isolate ex vivo BMRCs and CTCs. The ex vivo BMRC/CTCs were defined as live (DAPI-), HLA-ABC+ (confirming human origin), PanCK+ (epithelial phenotype) or CD44+/CD24- (stem-like phenotype). Sorted cells were collected directly into a pre-chilled tube maintained at 4oC containing RNA lysis buffer and total RNA was collected, followed by RNA and cDNA amplifications, quality controls and gene expression arrays using the Affymetrix HTA 2.0 array. Only mice that were confirmed to be free of overt metastasis, were used for this study. Ex vivo BMRC/CTC gene expression was compared to whole gene expression data from CTCs isolated from 10 metastatic breast cancer patients (de novo CTCs) publicly available at GSE99394. Comparison between ex vivo BMRC/CTCs and de novo CTCs highlighted the transcriptomic landscape of dormant breast cancer BMRC/CTCs and identified mTORC2 signaling as a key regulator of metastatic dormancy.

first: Debasish

last: Boral


title: Genes expression in glioma stem-like cells

city: Madrid

first: Massimo

last: Squatrito

address: Calle Melchor Fernandez Almagro 3

country: Spain

institute: Spanish National Research Center (CNIO)

summary: Expression profiling by high throughput sequencing of human GBM tumorspheres

overall_design: 6 independent  glioma stem-like cellines


title: Phloem and non-phloem associated translatomes during plum pox virus infection in Prunus domestica L. II

city: College Park

first: James

middle: N

last: Culver

state: MD

address: 4291 Fieldhouse Dr.

department: Plant Science and Landscape Architecture

institute: University of Maryland, College Park

summary: In this study we used a translating ribosome affinity purification strategy to identify phloem and non-phloem associated translatomes in Prunus domesitca L during PPV infection. Three different promoter:His6FLAG-RPL18 lines were used. These included two phloem specific promoters (pSUC2 and pSULTR2;2) as well as the more ubiquitously expressed cauliflower mosaic virus 35S promoter (p35S). Immunopurification of ribosome-mRNA complexes was accomplished by the method described in Reynoso et al. (Plant Functional Genomics: Methods and Protocols, 185-207; 2015). The dataset includes samples from plum leaves taken at 2, 4, 6, and 12 weeks post cold induced dormancy.

overall_design: 100 samples, 4 time points (2, 4, 6, and 12 weeks), 3 promoter lines, PPV infected and healthy controls, 2-6 biological replicates

first: J

first: T

last: Collum

first: C

last: Dardick

first: Y

last: Tamukong


title: VDR ChIP-seq in human monocytic THP-1 cell line

city: Debrecen

first: Laszlo

last: Nagy

address: Egyetem ter 1.

country: Hungary

institute: University of Debrecen

summary:
Treatment with calcitriol, a specific VDR agonist, augmented the heterodimerization between VDR and RXR. We also wanted to investigate its impact on VDR binding to genomic regions. ChIP-seq experiments were carried out with the human monocytic THP-1 cell line under either vehicle (1:1 DMSO-ethanol) or calcitriol (100 nM) treatment conditions.
Our finding is that calcitriol may have both a stimulatory or an inhibitory effect on VDR binding depending on the presence of its specific response element (VDRE), a less specific nuclear receptor (NR) half-site or absence thereof ("None"). Upon calcitriol activation, VDRE-containing genomic regions showed considerably higher occupancies on average than in the control, vehicle-treated sample. A similar induction, but to a much lesser extent, was detected for the NR half-site-containing regions. In contrast, genomic regions not containing any of these specific response elements did not show any induction upon calcitriol treatment; they rather showed a decrease of binding in promoter regions. We can conclude that ligand-induced heterodimerization and binding of the NR to its response elements are correlated events.

overall_design: ChIP-seq experiment was carried out in human monocytic THP-1 cell line under either vehicle, 1:1 DMSO-ethanol, or calcitriol (100 nM) treatment conditions.

first: Dora

last: Bojcsuk

first: Gyorgy

last: Vamosi


title: Expression profiles of in vitro cultured murine undifferentiated and differentiating spermatogonia

city: East lansing

first: Yuan

last: Wang

state: MI

address: 474 S Shaw Ln

institute: Michigan State University

summary: Total RNA from in vitro cultured murine undifferentiated and differentiating spermatogonia (by retinoic acid induction) were collected for RNA-seq

overall_design: In vitro cultured murine undifferentiated  permatogonia were induced by 100 µm retinoic acid for 36 hours to differentiate into  Kit+ differentiating spermatogonia (>80%). Duplicates  from undifferetniated and differentiating spermatognia were prepared for total RNA extraction which were then processed for RNA-seq.

first: Wei

last: Chen

first: Haihui

last: Fu

first: Suxu

last: Tan


title: The CCR4-NOT complex component NOT1 regulates RNA-directed DNA methylation and transcriptional silencing by facilitating Pol IV-dependent siRNA production [Bisulfite-seq]

first: Xin-Jian

last: He

type: Methylation profiling by high throughput sequencing

summary: Small interfering RNAs (siRNAs) are required for establishment and maintenance of DNA methylation through RNA-directed DNA methylation (RdDM) pathway in plants. While the siRNA biogenesis is well known, relatively little is known about how the process is regulated. By a forward genetic screen in Arabidopsis thaliana, we identified a mutant defective in NOT1 and demonstrated that NOT1 is required for transcriptional silencing at RdDM target genomic loci. Furthermore, we revealed that NOT1 is required for Pol IV-dependent siRNA accumulation and DNA methylation at a subset of RdDM target loci. By immunoprecipitation combined with mass spectrometry, we showed that NOT1 is a constituent of a multi-subunit CCR4-NOT deadenylase complex. We demonstrated that the CCR4-NOT components can function as a whole to mediate chromatin silencing. Our work thus establishes that the CCR4-NOT complex regulates the production of Pol IV-dependent siRNAs and thereby facilitates DNA methylation and transcriptional silencing in Arabidopsis.

overall_design: Examination of the DNA methylation level in Arabidopsis

first: Xin-jian


title: Spatiotemporal and cell-type dependent phasiRNA biogenesis in maize male reproduction (RNA-seq2)

city: St. Louis

first: Blake

middle: C.

last: Meyers

state: MO

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

summary: Maize anthers, the male reproductive floral organs, express two classes of phased, small interfering RNAs (phasiRNAs). RNA profiling from ten sequential cohorts of staged maize anthers plus mature pollen revealed that 21-nt phased siRNAs (21-phasiRNAs) from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, while 24-nt phased siRNAs (24-phasiRNAs) from 176 loci coordinately accumulate during meiosis and persist as haploid gametophytes differentiate into pollen. RNA sequencing of anther developmental mutants, together with in situ RNA hybridization detection of phasiRNA biogenesis factors, demonstrated that 21-phasiRNAs and 24-phasiRNAs are independently regulated. Furthermore, 21-phasiRNAs require epidermal cells while 24-phasiRNAs require functional tapetal cells. Maize phasiRNAs and mammalian PIWI-interacting RNAs (piRNAs) illustrate convergent evolution of small RNAs to support male reproduction.

overall_design:
Examination of maize phasiRNAs by high throughput sequencing for RNA-seq.
This series is an extension of the data previously submitted in GSE52290.

first: Jixian

last: Zhai

first: Han

last: Zhang

first: Arikit

last: Siwaret

first: Kun

last: Huang

first: Virginia

last: Walbot


title: Stem cell dynamic and signalling controlling mechanical force mediated skin expansion

first: Cedric

last: Blanpain

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

overall_design: Refer to individual Series


title: Comparative tRNA-seq for rapid profiling of tRNA modifications in a non-model organism

city: Boston

first: Satoshi

last: Kimura

state: Massachusetts

address: 181 Longwood Avenue

institute: Brigham and Women's Hospital

summary: tRNAs are heavily decorated with post-transcriptional modifications (tRNA modifications). Profile of tRNA modifications in non-model organisms are largely uncharacterized. Here using high-throughput sequencing, sites and frequency of tRNA modifications are predicted in Vibrio cholerae and Escherichia coli. During cDNA synthesis, some modifications cause misincorporation of a wrong base or termination of reverse transcription (RT). Using these RT-derived signatures we aim to explore organism-specific modifications and to track changes in modification frequency among different cellular conditions.

sample_organism:
Escherichia coli
Vibrio cholerae

platform_organism:
Escherichia coli
Vibrio cholerae

overall_design: High throughput sequencing of tRNA fractions from Escherichia coli (log and stationary phase) and Vibrio cholerae (log phase, stationary phase, and cecal fluid from infant rabbit) was performed using Illumina miSeq.

first: Peter

last: Dedon

first: Matthew

middle: K

last: Waldor


title: Mechanisms of stretch-mediated skin expansion at single cell resolution [array]

summary:
The ability of the skin to expand in response to stretching has, for decades, been exploited in reconstructive surgery. Several studies have investigated the response of stretching epidermal cells in vitro. However, it remains unclear how mechanical forces affect epidermal stem cell behaviour in vivo. Here, we develop a mouse model in which the temporal consequences of the stretching the skin epidermis can be studied. Using a multidisciplinary approach that combines clonal analysis and mathematical modelling, we show that mechanical force induces skin expansion by promoting the renewal of epidermal stem cells. This occurs through a structured response in which cell fates are coordinated locally by stem cells that switch between states primed for renewal or differentiation. Transcriptional and chromatin profiling identifies the gene regulatory networks modulated by mechanical force. Using a combination of pharmacological inhibition and several conditional gene loss-of-function mouse mutants, we dissect the signalling pathways that control force-mediated tissue expansion.
We used microarray to molecularly profile basal cells isolated from the interfolliular epidermis during force-mediated tissue expansion and after 12-O-Tetradecanoylphorbol-13-acetate (TPA) tretment.

overall_design: Interfollicolar basal epideramal cells form the back skin were FACS sorted for the positive expression of ITGA6 and negative expression of CD34. The mice were subjected to force-mediated tissue expansion or 12-O-Tetradecanoylphorbol-13-acetate (TPA) tretment. Untreted animals were used as control. The three replicates samples for control and expanded condition at day4 (EXPD4) were from 3 different  CD1 mice. The two replicates for the TPA treatment were from 2 different CD1 mice.  For each sample, 300 basal cells were FACS sorted and used for RNA extraction and hybridization on Affymetrix microarrays.

first: Mariaceleste

last: Aragona


title: High-throughput propagation of human prostate tissue from induced-pluripotent stem cells.

city: Belfast

first: Rebecca

middle: Elizabeth

last: Steele

address: 97 Lisburn Road

department: CCRCB

institute: Queen's University Belfast

summary: We demonstrate a new high-throughput model where rapidly proliferating and easily handled induced pluripotent stem cells enable generation of human prostate tissue in vivo and, for the first time, in vitro. Using a co-culture technique with urogenital sinus mesenchyme, we recapitulated the in situ prostate histology, including the stromal compartment and the full spectrum of epithelial differentiation.

overall_design: Examination of transcripts in IPSC, Definitive Endoderm and Prostate Pro Organoids at 12 Weeks through RNA-seq analysis

first: A

last: Hepburn

first: E

last: Curry

first: M

last: Moad

first: R

first: O

last: Franco

first: L

last: Wilson

first: P

last: Singh

first: S

last: Crawford

first: I

middle: G

last: Mills

last: Hayward

first: C

last: Robson

last: Heer


title: Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance

city: San Antonio

first: Zhijie

middle: Jason

last: Liu

address: 7703 Floyd Curl Drive

institute: Universality of Texas Health Science Center at San Antonio


title: 7-Hydroxycoumarin attenuates colistin-induced kidney injury in mice through the inhibition of HDAC1 and the activation of Nrf2 signaling

status: Public on May 14 2020

city: Harbin

first: Jian

last: Wang

address: 600 Changjiang Road

institute: Northeast Agricultural University

summary: RNA sequencing of kidney tissues was performed to investigate the  mechanism of 7-Hydroxycoumarin attenuates colistin-induced kidney injury in mice

overall_design: This study aimed to investigates the renoprotective effect of 7-Hydroxycoumarin (7-HC) against colisitn-induced kidney injury.  Mice were randomly divided into four groups: saline solution (con group); 7-HC administered at 50 mg/kg of body weight/day (7-HC group); colistin at 15 mg/kg/day (col group); colistin at 15 mg/kg/day plus 7-HC at 50mg/kg/day (col+7-HC group). All mice were treated for 7 consecutive days, kidney tissued were collected and used to RNA sequencing.


title: Epigenetically  bistable regions across neuron-specific genes govern neuron  eligibility to a coding ensemble in the hippocampus

city: New York

first: Miklos

last: Toth

address: 1300 York Ave, W506

department: Pharmacology

institute: Weill Cornell Medicine

summary: Sensory inputs activate sparse ensembles of neurons in the dentate gyrus of the hippocampus, but how eligibility of individual neurons to recruitment is determined remains elusive. We identified thousands of largely bistable (CpG methylated or unmethylated) regions within neuronal gene bodies, established during mouse dentate gyrus development. Reducing DNA methylation and the proportion of the methylated epialleles at bistable regions compromised novel context-induced neuronal activation. Conversely, increasing methylation and the frequency of the methylated epialleles at bistable regions enhanced intrinsic excitability. Single-nuclei profiling revealed enrichment of specific epialleles from a subset of bistable regions in activated neurons. Genes displaying both differential methylation and expression in activated neurons defined a network of proteins regulating neuronal excitability and structural plasticity. We propose a model in which bistable regions create neuron heterogeneity, and constellations of exonic epialleles dictate, via modulating gene expression and neuronal excitability, eligibility to a coding ensemble

overall_design: Compared tissue and cells from dorsal dentate gyrus C57BL6 male animals that were either injected with an AAV-DJ-SYN-DNMT3A virus, control animals, or animals exposed to a novel environment for 15 minutes.  Bulk RRBS DNA methylation, single nuclei DNA methylation, and single nuclei RNA expression profiles were collected.

first: Shannon

last: Odell

first: Faten

last: Taki

first: Anika

last: Nabila


title: Reciprocal fine-tuning of progesterone and prolactin-regulated gene expression in breast cancer cells

city: Chuo-ku, Kobe

first: Bakhtiyor

last: Nosirov

state: Hyogo

address: Minatojima-minamimachi, 2-2-3

institute: BDR RIKEN, Japan

summary: To determine how progesterone and prolactin effect the transcription activity of each individual hormone in breast cancer cells

overall_design: T47D breast cancer cells were treated with vehicle (water for prolactin and EtOH for R5020), 100 ng/ml prolactin, 10nM R5020, or both, for 6 hrs. RNA was isolated immediately following treatment.

first: Christy

middle: R

last: Hagan

first: Sean

middle: M

last: Holloran


title: Macrophage-Tumor Cell Interaction Promotes ATRT Progression and Chemoresistance (GEMM)

city: Münster

first: Kornelius

last: Kerl

address: Albert-Schweitzer-Campus 1

department: Department of Pediatric Hematology and Oncology

institute: University Children's Hospital Münster

summary: Atypical teratoid/rhabdoid tumors (ATRT) are known for their heterogeneity concerning pathophysiology and outcome. However, predictive factors within distinct subgroups still need to be uncovered. Using multiplex immunofluorescent staining and singe-cell RNA sequencing we unraveled distinct compositions of the immunological tumor microenvironment (TME) across ATRT subgroups. CD68+ cells predominantly infiltrate ATRT-SHH and ATRT-MYC and are a negative prognostic factor for patients’ survival. Within the murine ATRT-MYC and ATRT-SHH TME, Cd68+ macrophages are core to intercellular communication with tumor cells. In ATRT-MYC distinct tumor cell phenotypes express macrophage marker genes. These cells are involved in the acquisition of chemotherapy resistance in our relapse xenograft mouse model. In conclusion, the tumor cell-macrophage interaction contributes to ATRT-MYC heterogeneity and tumor recurrence.

overall_design: We used a mouse model, Rosa26-creERT2::Smarcb1fl/fl , to profile the transcriptome of SHH and MYC-ATRT by single cell RNA-sequencing (scRNA-seq). We crossed the Smarcb1 fl/fl strain with a mouse line harboring the CreERT2 coding region under the control of ubiquitous (Rosa26) promoter. Pregnant females were induced with a single intraperitoneal dose (50 mg/kg) of Tamoxifen at 6.5 days post-coitum (post-coital plug observation was considered as day 0.5). All mice were intensively monitored until neurological symptoms indicating tumor growth were observed. Tumors were dispersed into single cells and further processed for scRNA-seq. Quality-filtered sequence data were merged and subjected to dimensionality reduction and cell cycle regression to mitigate the effects of cell cycle heterogeneity. This resulted in a total of 24,199 cells, which represent tumor cells as well as tumor-associated non-tumor cells including immune cells.

first: Viktoria

last: Melcher

first: Monika

last: Graf

first: Marta

last: Interlandi


title: Macrophage-Tumor Cell Interaction Promotes ATRT Progression and Chemoresistance (NODSCID)

overall_design: We used 7-9 weeks old immune-compromised NOD/SCID mice (Jackson Laboratory, www.jax.org) for xenograft experiments. Ten thousand single sphere-forming cells of the BT-16 cell line (ATRT-MYC) were diluted in 5 µl sterile PBS and injected into the striatum (anteroposterior: 0 mm; lateral: 2.5 mm; dorso-ventral: 3 mm). Orthotopically transplanted NOD/SCID mice were monitored regularly for signs of tumor formation and examined by small animal Magnetic Resonance Imaging (MRI) for the assessment of tumor engraftment and growth. After detection of tumor presence, mice were treated in adaptation to the European Rhabdoid Registry (EU-RHAB) to assess potential therapy-resistant ATRT.  Tumors from chemotreated and untreated mice were isolated, dispersed into single cells and further processed for scRNA-seq. Quality-filtered sequence data were merged and subjected to dimensionality reduction and cell cycle regression to mitigate the effects of cell cycle heterogeneity. This resulted in a total of 9,637 cells, which represent tumor cells as well as tumor-associated non-tumor cells including immune cells.


title: Enarodustat, a hypoxia-inducible factor stabilizer, counteracts the diabetic renal energy metabolism alterations in the early stages of diabetic kidney disease

city: Tokyo

first: Sho

last: Hasegawa

address: 7-3-1, Hongo, Bunkyo-ku

department: Division of Nephrology and Endocrinology

institute: University of Tokyo

summary: We analyzed the effects of enarodustat (JTZ-951; HIF stabilizer) on renal energy metabolism in alloxan-induced diabetic mouse model. Transcriptome analysis of renal tissue revealed that genes related to fatty acid metabolism were upregulated in diabetes, whereas genes related to glucose metabolism were upregulated by enarodustat. In addition, genes related to amino acid metabolism were upregulated by diabetes and downregulated by enarodustat. Thus, HIF stabilization counteracts the renal energy metabolism alterations occurring in the early stages of diabetic kidney disease.

overall_design: Vehicle (n = 6; group A [Sham]) or 90 mg/kg of alloxan were intravenously administered into mice 3 days before grouping. We selected the mice for group B and C (n = 9, for each group) by matching blood glucose and body weight. Group A (Sham) and B (DKD) were given normal feed, while group C (DKD+enarodustat) was given feed mixed with 0.01% (w/w) of enarodustat. Kidney samples were collected 16 days after grouping. Total RNAs from renal tissues were analyzed.

first: Tetsuhiro

last: Tanaka

first: Tomoyuki

last: Saito

first: Kenji

last: Fukui

first: Takeshi

last: Wakashima

first: Masaomi

last: Nangaku


title: Endocrine-exocrine signaling in obesity-driven pancreatic ductal adenocarcinoma

city: Cambridge

first: A

last: Bhutkar

address: 77 Massachusetts Avenue

institute: MIT

summary: Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how and when obesity contributes to PDAC progression is not well understood. Leveraging an autochthonous mouse model, we demonstrate a causal and reversible role for obesity in early PDAC progression, showing that obesity markedly enhances tumorigenesis, while genetic or dietary induction of weight loss intercepts cancer development. Bulk and single cell molecular analyses of human and murine samples define microenvironmental consequences of obesity that promote tumor development rather than new driver gene mutations. We observe increased inflammation and fibrosis and also provide evidence for significant pancreatic islet cell adaptation in obesity-associated tumors. Specifically, we identify aberrant beta islet cell expression of the peptide hormone cholecystokinin (CCK) in tumors as an adaptive response to obesity. Furthermore, islet CCK expression promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Our studies argue that PDAC progression is driven by local obesity-associated changes in the tumor microenvironment – rather than systemic effects – and implicate endocrine-exocrine signaling beyond insulin in PDAC development. Furthermore, our demonstration that these obesity-associated adaptations are reversible supports the use of anti-obesity strategies to intercept PDAC early during progression

overall_design: Tumor-bearing pancreata from KCO (Tumor_ob), KC (Tumor_Non_ob), KPC (Tumor_p53). Biologic replicates include 15 KCO, 8 KC, and 9 KPC pancreata.

first: Katherine

last: Chung

first: Jaffarguriqbal

last: Singh

first: Lauren

last: Lawres

first: Kimberly

middle: J

last: Dorans

first: Cathy

last: Garcia

first: Daniel

last: Burkhardt

first: Rebecca

last: Robbins

first: Arjun

last: Cardone

first: Xiaojian

last: Zhao

first: Ana

last: Babic

first: Sara

middle: A

last: Vayrynen

first: Andressa

middle: D

last: Costa

first: Jonathan

last: Nowak

last: Chang

first: Richard

last: Dunne

first: Aram

last: Hezel

first: Albert

last: Koong

first: Joshua

last: Wilhelm

first: Melena

last: Bellin

first: Vibe

last: Nylander

first: Anna

last: Michael

first: Mark

middle: I

last: McCarthy

last: Kibbey

first: Smita

last: Krishnaswamy

first: Brian

last: Wolpin

first: Tyler

last: Jacks

first: Charles

middle: S

last: Fuchs

first: Mandar

last: Muzumdar


title: Enarodustat, hypoxia-inducible factor stabilizer, counteracts the diabetic renal energy metabolism alterations in streptozotocin-induced diabetic rats

first: Sho

last: Hasegawa

summary: We analyzed the effects of enarodustat (JTZ-951; HIF stabilizer) on renal energy metabolism in streptozotocin-induced diabetic rat model. Transcriptome analysis of renal cortex revealed that  genes related to fatty acid metabolism and amino acid metabolism were upregulated in diabetes and downregulated by enarodustat, whereas genes related to glucose metabolism were upregulated by enarodustat. Thus, HIF stabilization counteracts the renal energy metabolism alterations occurring in the early stages of diabetic kidney disease.

overall_design: Vehicle (n = 5; group A [Sham]) or 65 mg/kg of streptozotocin (STZ) (n = 19) were intravenously administered into rats 7 days before grouping. We selected the rats for group B and C (n = 7, for each group) by matching blood glucose and body weight. Group A (Sham) and B (DKD) were given normal feed, while group C (DKD+enarodustat) was given feed mixed with 0.01% (w/w) of enarodustat. Kidney samples were collected 14 days after grouping. Total RNAs from renal cortical tissues were analyzed.

first: Tetsuhiro

last: Tanaka

first: Tomoyuki

last: Saito

first: Kenji

last: Fukui

first: Takeshi

last: Wakashima

first: Masaomi

last: Nangaku


title: The Role of Endothelial Autocrine NRG1/ERBB4 Signaling in Cardiac Remodeling

city: Antwerp

first: Ligia

middle: Monica

last: Mateiu

address: Universiteitsplein 1

department: Center for Molecular Neurology

institute: VIB

summary: Neuregulin-1 (NRG1) is a paracrine growth factor, secreted by cardiac endothelial cells (Ecs) in conditions of cardiac overload/injury. The current concept is that the cardiac effects of NRG1 are mediated by activation of ERBB4/ERBB2 receptors on cardiomyocytes. However, recent studies have shown that paracrine effects of NRG1 on fibroblasts and macrophages are equally important. Here, we hypothesize that NRG1 autocrine signaling plays a role in cardiac remodeling.  We generated EC–specific Erbb4 knockout mice to eliminate endothelial autocrine ERBB4 signaling without affecting paracrine NRG1/ERBB4 signaling in the heart. We first observed no basal cardiac phenotype in these mice up to 32 weeks. We next studied these mice following transverse aortic constriction (TAC), exposure to angiotensin II (Ang II) or myocardial infarction in terms of cardiac performance, myocardial hypertrophy, myocardial fibrosis and capillary density. In general, no major differences between EC–specific Erbb4 knockout mice and control littermates were observed. However, 8 weeks following TAC both myocardial hypertrophy and fibrosis were attenuated by EC–specific Erbb4 deletion, albeit these responses were normalized after 20 weeks. Similarly, 4 weeks after Ang II treatment myocardial fibrosis was less pronounced compared to control littermates. These observations were supported by RNA-sequencing experiments on cultured endothelial cells showing that NRG1 controls the expression of various hypertrophic and fibrotic pathways.   Overall, this study shows a role of endothelial autocrine NRG1/ERBB4 signaling in the modulation of hypertrophic and fibrotic responses during early cardiac remodeling. This study contributes to understanding the spatio-temporal heterogeneity of myocardial autocrine and paracrine responses following cardiac injury.

overall_design: PBS-treated samples: L.01-L.04, L.17-L.20; 8 h NRG1-treated samples: L.05-L.08, L21-L.24; 24 h NRG1-treated samples: L.09-L.12, L.25-L.28

first: Lindsey

last: Dugaucquier

first: Eline

last: Feyen

first: Tine

last: Bruyns

first: Gilles

last: De Keulenaer

first: Vincent

last: Segers


title: Inhibition of HDAC1/2 along with TRAP1 causes Synthetic Lethality in Glioblastoma Model Systems

status: Public on May 13 2020

first: Markus

last: Siegelin

state: New York

institute: Columbia University

summary: Pan and selective HDAC inhibition is synthetically lethal with TRAP1 inhibition in various model systems of glioblastoma, including patient derived xenograft (PDX) cells. Mechanistically, this occurs through several mechanisms, including the induction of metabolic stress by interference with tumor cell energy metabolism accompanied by modulation of pro- and anti-apoptotic Bcl-2 family proteins and the induction of a cell death with apoptotic features.

overall_design: Compared OXPHOs complex (SHDA and SDHB) level between U87 and U87PbR

first: Trang

last: Nguyen


title: Multiplexed imaging of nucleome architectures in single cells of mammalian tissue

city: New Haven

first: Siyuan

last: Wang

state: CT

address: 333 Cedar Street

department: Department of Genetics and Department of Cell Biology

institute: Yale University

summary: The three-dimensional architecture of the genome affects genomic functions. Multiple genome architectures at different length scales, including chromatin loops, domains, compartments, and regions associated with nuclear lamina and nucleoli, have been discovered. However, how these structures are arranged in the same cell and how they are correlated with each other in different cell types in mammalian tissue are largely unknown. Here, we developed Multiplexed Imaging of Nucleome Architectures that measures multiscale chromatin folding, copy numbers of numerous RNA species, and associations of numerous genomic regions with nuclear lamina, nucleoli and surface of chromosomes in the same, single cells. We applied this method in mouse fetal liver, and identified de novo cell-type-specific chromatin architectures associated with gene expression, as well as chromatin organization principles independent of cell type. Polymer simulation showed that both intra-chromosomal self-associating interactions and extra-chromosomal interactions are necessary to establish the observed organization. Our experiments and modeling provide a multiscale and multi-faceted picture of chromatin folding and nucleome architectures in mammalian tissue and illustrate physical principles for maintaining chromatin organization. Here we submit our bulk RNA-sequencing data on E14.5 mouse fetal liver, used in the study to validate image-based RNA profiling results.

overall_design: Bulk mRNA sequencing from mouse embryonic day 14.5 fetal liver tissue

first: Miao

last: Liu

first: Yanfang

last: Lu


title: DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

city: Baltimore

first: Marina

last: Bellani

state: Maryland

address: 251 Bayview Blvd

department: LMB

institute: NIA

summary: Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of “stressed” replisome. However, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. CHIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late

overall_design:
FANCM and GFP(-DONSON) Chip-seq analysis in asynchronous Hela cells stably expressing GFP-DOSNON

Third-party reanalysis: Hela_eigenvector_HiC.hg38.bedgraph was calculated on Hi-C data for Hela cells downloaded from NCBI, dbGaP phs000640.v8.p1. See README files for data processing details.

first: Jing

last: Zhang

first: Michael

last: Seidman


title: Transcriptomic changes resulting from STK32B overexpression identifies pathways potentially relevant to essential tremor

city: Montreal

first: Cal

last: Liao

state: QC

address: 1033 Avenues des Pins

country: Canada

institute: Mcgill University

summary: We report RNA sequencing of overexpressed STK32B in DAOY cells compared to matched controls, with 200M reads per sample. We identified approximately 4000 differentially expressed genes and potential pathways relevant to essential tremor.

overall_design: Case-control of overexpressed STK32B cell lines compared to controls

first: Calwing

first: Guy

last: Rouleau


title: A memory of eS25 loss drives resistance phenotypes

city: Stanford

first: Julien

last: Couthouis

state: CA

address: 279 Campus Dr

department: Genetics

institute: Stanford University

summary:
In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.

RNA-seq of polyadenylated mRNAs as the first step to acquire a snapshot of the transcriptional state of wild-type, RPS25 KOs, and eS25-HA AB HAP1 cells

overall_design: HAP1 cell lines were seeded into the wells of 6-well plates with equal live cell counts (250,000 cells/well), grown under standard conditions, and harvested by trypsinization, pelleting and washing cells with PBS 48 hrs post-seeding. To normalize growth conditions prior to harvest, media was removed 6 hrs prior to harvest and replenished with fresh media. RNA was purified from cells pellets using the PureLink RNA mini kit (ThermoFisher cat.#12183025). Prior to library preparation, RNA concentration and sample quality by RNA integrity number (RIN) were checked using Tapestation RNA ScreenTape reagents (Agilent cat.# 5067-5576). cDNA libraries were prepared with the SureSelect Strand-Specific RNA Library Prep Kit (Agilent cat.#G9691B) on an Agilent Bravo Automated Liquid Handling Platform accordingly to the protocol (Version E0, March 2017, G9691-90030). Library concentration and integrity were checked using Tapestation D1000 ScreenTape reagents (Agilent cat.#5067-5582) and Qubit dsDNA BR Assay Kit (Invitrogen cat.#Q32850). Sequencing was performed on an Illumina HiSeq 4000 with 2x101 base pair reads and Illumina Single Index. Reads were aligned to the hg38 reference genome using STAR v2.5.3a (Dobin et al., 2013) and differential expression between samples was computed using R v3.4.0 and the DESeq2 package (Love et al., 2014) (detailed pipeline and options available on https://github.com/emc2cube/Bioinformatics/). In the rare case that fold change P-values were too small for R to calculate and estimated as 0, these were imputed at 10-300 to facilitate logarithmic conversion.

first: Alex

last: Johnson

first: Shizuka

middle: B

last: Yamada

first: Aaron

last: Gitler

first: Joseph

last: Puglisi


title: Complementation can maintain a quasispecies of drug sensitive and resistant HIV

city: Durban

last: Sigal

state: -- SELECT --

address: 719 Umbilo Road, Congella

country: South Africa

institute: Africa Health Research Institute (AHRI)

type: Genome variation profiling by high throughput sequencing

summary:
The goal of this study was to determine how an HIV quasispecies is maintained in the face of selection. We deep sequenced the HIV provirus from cell populations as well as single cells at different time points from in vitro evolution experiments and found that when a less fit and more fit infect the same cell, they share components (complmentation) and therefore allow the less fit to perpetuate.
We reproduced a quasispecies to an HIV reverse transcriptase inhibitor. The drug resistant genotype never completely supplanted the drug sensitive genotype, which stabilized at about 20% of viral sequences. Single-cell sequencing showed that resistant genotype frequency plateaued when cells were co-infected with sensitive and resistant genotypes, suggesting a sharing of viral proteins in co-infected cells (complementation), masking genotypic differences. To test if complementation can confer phenotypic drug resistance, we co-transfected fluorescently labelled molecular clones of sensitive and resistant HIV and observed drug resistance in genotypically sensitive virus from co-transfected cells. Resistant virus preferentially co-infected cells with drug sensitive HIV, explaining initiation of co-infections. Modelling showed that a stable quasispecies could form at the experimental multiplicities of infection.
Conclusions: Complementation can lead to a quasispecies in infection environments where multiple infections per cell are common

overall_design: Sequencing of Reverse transcriptase region of HIV proviral DNA from infected cell populations (3 independent experiments) as well as single cells (either 30 or 60 cells from each time point)

first: Laurelle

last: Jackson

first: Sandile

last: Cele

first: Gila

last: Lustig

first: Jennifer

last: Giandhari

first: Tulio

last: de Oliveira

first: Richard

last: Neher


title: Novel enhancer emerge with high interindividual variability and less frequently associate with disease

city: Utrecht

first: Menno

middle: P

last: Creyghton

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: Mutations in non-coding regulatory DNA such as enhancers underlie a wide variety of diseases including developmental disorders and cancer. As enhancers rapidly evolve, understanding their function and configuration in non-human disease models can have important clinical applications. Here, we analyze enhancer configurations in tissues isolated from the common marmoset, a widely used primate model for human disease. Integrating these data with human and mouse data, we find that enhancers containing trait-associated variants are preferentially conserved. In contrast, most human-specific enhancers are highly variable between individuals, with a subset failing to contact promoters. These are located further away from genes and more often reside in inactive B-compartments. Our data show that enhancers typically emerge as instable elements with minimal biological impact prior to their integration in a transcriptional program. Furthermore, our data provide insight into which trait variations in enhancers can be faithfully modeled using the common marmoset.

overall_design: ChIP-sequencing for H3K27ac and H3K4me3 of marmoset tissue samples

first: B

last: Castelijns

first: M

last: Baak

first: G

last: Geeven

middle: W

last: Vermunt

first: C

last: Wiggers

first: I

last: Timpanaro

last: Kondova

first: W

last: de Laat


title: TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7–9

city: Vienna

first: Vitaly

last: Sedlyarov

address: Lazarettgasse 14, AKH BT 25.3

country: Austria

institute: Research Center for Molecular Medicine

summary: Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses. Here we show that a previously uncharacterized protein encoded by CXorf21—a gene that is associated with systemic lupus erythematosus—interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease. Loss of this type-I-interferon-inducible protein, which we refer to as ‘TLR adaptor interacting with SLC15A4 on the lysosome’ (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus.

overall_design: 3 biological replicates, 2 independent sgRNAs per gene

first: Leonhard

middle: X

last: Heinz

first: JangEun

last: Lee

first: Felix

last: Kartnig

first: Konstantinos

last: Papakostas

first: Adrian

last: César-Razquin

first: Patrick

last: Essletzbichler

first: Ulrich

last: Goldmann

first: Adrijana

last: Stefanovic

first: Johannes

last: Bigenzahn

first: Stefania

last: Scorzoni

first: Peter

last: Májek

first: André

middle: C

last: Müller

first: F

last: King

first: Jun

last: Li

first: Enrico

last: Girardi

middle: L

last: Mbow

first: Utkarsh

last: Kapoor

first: Mattia

last: Pizzagalli

first: Ariel

last: Bensimon

first: Charles

middle: E

last: Whitehurst

first: Manuele

last: Rebsamen

first: Giulio

last: Superti-Furga


title: Human Naive Pluripotency Attained by Transient Inhibition of Mtor

city: Buffalo

first: Jian

last: Feng

state: NY

address: 955 Main Street, Room 3102

department: Department of Physiology and Biophysics

institute: State University of New York at Buffalo

summary: The ability to support blastocyst chimera formation is the most critical property of naïve pluripotent stem cells (PSCs). Transient inhibition of mTOR converts human PSCs from primed to naive state. Naïve human PSCs cultured in 2iLI robustly incorporate into mouse embryonic development after injected to mouse blastocysts.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Quantification of human cells in E17.5 chimeric mouse embryos using next generation squencing of amplicons covering the human and mouse V3 region of 18S rDNA.

first: Hanqin

first: Zhixing

last: Hu

first: Houbo

last: Jiang

first: Yong

last: Ren

first: Xinyang

last: Yu

first: Jingxin

last: Qiu

first: Aimee

last: Stablewski

first: Boyang

last: Zhang

first: Michael

last: Buck


title: Human Naive Pluripotency Attained by Transient Inhibition of mTor

summary: Mouse embryonic stem cells (mESCs) are in naive pluripotency that represents the ground state of development, from which all cells in the mouse embryo are derived. In contrast, human embryonic stem cells (hESCs) are in a primed state of pluripotency with many different properties. Despite intense efforts to generate naive human pluripotent stem cells (hPSCs), it has not been possible to derive naive hPSCs without relying on transgene overexpression or chemicals. Here, we show that a transient treatment with Torin1, a selective inhibitor of mTOR, converted hPSCs from primed to naive pluripotency. The naive hPSCs were maintained in the same condition as mESCs in defined media with 2iLI (MEK inhibitor, GSK3b inhibitor, LIF and Insulin). Like mESCs, they exhibited high clonal efficiency, rapid cell proliferation, active mitochondrial respiration, X chromosome activation, DNA hypomethylation, and transcriptomes similar to those of human blastocysts than primed hESCs. Most importantly, the naive hPSCs significantly contributed to mouse embryos when transferred to mouse blastocysts. mTor inhibition induced nuclear translocation of TFE3, a critical transcription factor at the interplay of autophagy and pluripotency. TFE3 with mutated nuclear localization signal blocked the conversion from primed to naive pluripotency. It appears that by mimicking diapause at the cellular level, naive pluripotency in human can be readily attained from primed hPSCs, thus establishing the unified ground state of pluripotency in mammals.

overall_design: We compared the differences in gene expression between primed state and naive state human pluripotent stem cells. RNAseq was performed on primed H9 (PH9), naive H9 (NH9), primed RUES2 (PRUES2), and naive RUES2 (NRUES2) with 3 biological replicates for each line. For each sample, colonies of hPSCs were manually picked and homologized in 1ml TRIzol reagent (Thermo Fisher Scientific). RNeasy Mini Kit (QIAGEN) was used for RNA extraction. Quality of purified RNA was monitored by agarose gel electrophoresis. PolyA+ RNA enrichment, cDNA library preparation, sequencing and RPKM calculation were performed at our core facility with standard procedure.

last: Li


title: Hierarchical transcriptional regulation of quorum sensing genes in Vibrio harveyi

city: Bloomington

first: Julia

last: van Kessel

state: IN

address: 1001 E 3rd Street

department: Biology

institute: Indiana University

summary: Quorum sensing controls hundreds of genes in vibrios required for cell density-specific behaviors, including bioluminescence, biofilm formation, competence, secretion, and motility. The central regulator in the quorum sensing pathway in vibrios is LuxR/HapR, which directly regulates >100 genes in the 625-gene regulon of Vibrio harveyi. Among these directly regulated genes are 15 transcription factors, which we predicted would comprise the second tier in the hierarchy of the quorum sensing regulon. To better study the mechanism of regulation of the quorum sensing network, we mapped all transcriptional start sites in V. harveyi using dRNA-seq. From these data, we determined the relative position of LuxR binding sites in the promoters of genes directly regulated by LuxR. We confirmed that LuxR directly binds to the promoters of the genes encoding transcription factors and quantified the extent of LuxR activation or repression of transcript levels. Finally, we determined the individual regulons for a subset of transcription factors that have not been previously studied. For regulators such as LysR- or AsnC/Lrp-type transcription factors, the regulons contained >100 genes that contained both unique and overlapping genes with the LuxR regulon. These data support a model in which LuxR directly regulates other transcription factors, which act to further alter the second tier of the gene expression cascade producing cell density behaviors in V. harveyi.

overall_design: RNA-seq of cultures grown to OD600=1.0

first: Ryan

last: Chaparian

first: Alyssa

last: Ball


title: BAMscale: quantification of DNA sequencing peaks and generation of scaled coverage tracks

status: Public on May 17 2020

city: Bethesda

first: Lorinc

last: Pongor

address: 37 Convent Dr,

department: DTB

institute: NIH

type:
Other
Third-party reanalysis

summary: We present a tool for processing NGS DNA-seq BAM files to quantify peaks and create coverage tracks from ChIP-seq, NS-seq, ATAC-seq, OK-seq, END-seq and replication timing data

overall_design:
BAMscale is a one-step tool that processes DNA sequencing datasets from chromatin binding (ChIP-seq) and chromatin state changes (ATAC-seq, END-seq) experiments to DNA replication data (OK-seq, NS-seq and replication timing). The outputs include normalized peak scores in text format and scaled coverage tracks (BigWig) which are directly accessible to data visualization programs. BAMscale (available at https://github.com/ncbi/BAMscale) effectively processes large sequencing datasets (~100Gb size) in minutes, outperforming currently available tools.

This series includes re-analyzed data from: GSE101512 (ATAC-seq on peripheral blood), GSE116321 (Mouse_Okseq_ENDseq_RepliSeq on Activated primary B cells), ERR2760956 (OK-seq on K562) and GSE122489 (RNA-seq).

first: Jacob

last: Gross

first: Christophe

last: Redon

first: Mirit

last: Aladjem

first: Yves

last: Pommier


title: Single-cell RNA Sequencing (scRNA-seq) of Laryngeal Squamous Cell Carcinoma (LSCC) Tissues

status: Public on May 12 2020

city: Harbin

first: Lianhao

last: Song

address: Baojian road

institute: Harbin Medical University

summary: Background: Laryngeal squamous cell carcinoma (LSCC) is a highly malignant tumor originated from respiratory system. Although there have been many improvements in therapy until now, reducing the high mortality is still difficult. Understanding the cellular heterogeneity of LSCC could contribute to improve this problem.  Methods: Single-cell RNA sequencing (scRNA-seq) was applied to dissect the cell composition and molecular characteristics of LSCC tissues. Immunohistochemistry (IHC) staining of the LSCC tissues was performed to identify the spatial location of tumor cells. Survival analysis of marker genes was executed in TCGA to verify the correlation between each cell clusters and patients' prognosis.   Results: The LSCC tissue cells were finely grouped into various clusters, including tumor cells, immune cells, epithelial cells, fibroblasts and endothelial cells. Notably, in tumor cells, keratinocyte-like cells were located in the core of tumor while malignant proliferating cells were located at the tumor edge. The malignant proliferating cells were correlated with poor prognosis.  Conclusion: In summary, this is the first study to delineate a landscape of the LSCC intra-tumor heterogeneity. Our work might help researchers have a better understanding for tumor progression.

overall_design: Laryngeal squamous cell carcinoma (LSCC) tissues of a 66-year-old male patient and a 64-year-old male patient.

first: Siwei

first: Siyang

first: Lanlan

last: Wei


title: Transcriptome analysis of Senecavirus A-infected cells: type I interferon is a critical anti-viral factor

city: Yangzhou

first: jing

last: wang

address: 12 Wen-hui East Road

institute: Yangzhou university

summary:
Purpose: to study the changes in various genes in cells during the early stages of SVA infection. Differentially expressed genes were screened from them, and GO enrichment analysis and KEGG pathway enrichment analysis were performed. This study provides some help for better prevention and control of SVA infection.
Methods: Deep sequencing of SVA-infected cells at 6hpi and 12hpi and mock-infected cells were performed, in triplicate. The raw data from the sequencing is filtered using ngqc software and compared to the reference sequence. Differential and enrichment analyses were then performed.   PK:mock-infeted groups   TP:SVA-infected groups
Results: Using RNA-seq technology, more than 46 billion raw reads of each sample were generated, and the proportion of clean reads was >94%. The ratios of clean reads successfully mapped to the swine reference genome in all samples ranged from 90.41% to 95.92%, and the ratios of the uniquely mapped reads were >91.48%. The R2 values of the samples in the same group were >0.99. 1584 genes had a significant difference at 6hpi between SVA- and mock-infected groups and 9785 DEGs were screened at 12hpi, 28 of these DEGs were validated with RT–qPCR. Altered expression of 28 genes was confirmed with RT–qPCR, demonstrating the high degree of sensitivity of the RNA-seq method. At the same time, we selected three important DEGs for WB experiments and analyzed their protein-level alterations. The results were consistent with RNA-seq data, Further confirmation of the accuracy of RNA-SEQ technology
Conclusion: Our experiments analyzed in detail the changes in various genes in cells after SVA infection. A series of cytokines, such as interleukins (IL6, TNF-α), chemokines (CCL4, CCL5), and immune-related factors (IFN-α, IFN-β, RSAD2, MX1), are involved in the battle between SVA and the host. We postulated that this innate immune response is the main mean used by the host for the initial response to SVA infection. This study evaluated SVA-induced immune responses and provides information that can be used to investigate the molecular mechanisms of SVA-host interactions.

overall_design: expressed genes of 2 different hours after SVA infection; TP represents the SVA-infected groups and PK represents the mock-infected groups.

first: Zhenhai

last: Chen

first: Jing

last: Wang


title: Characterizing transcriptomic profiles with human SORLA overexpression in mice

city: La Jolla

first: Timothy

last: Huang

address: 10901 N Torrey Pines Rd.

institute: Sanford Burnham Prebys Medical Discovery Institute

summary: The purpose of this study is to determine global changes in mRNA expression with transgenic expression of human SORLA in mouse hippocampus

overall_design: Hippocampus mRNA profiles from 3-month-old male mice (n=2 WT, n=3 SORLA TG) were generated by RNAseq.

first: LuLin

last: Jiang


title: Drosophila Histone Locus Body assembly and function involves multiple interactions

city: Carrboro

first: Ashlesha

last: Chaubal

state: NC

address: 3342 Genome Sciences Building

department: Integrative Biological and Genome Sciences

institute: University of North Carolina at Chapel Hill

summary: The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome has a single locus comprised of ~100 copies of a tandemly arrayed 5 kB repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the ~200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters (12xPR) does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression. ChIP-qPCR and ChIP-Seq experiments were done on homozygous histone locus (HisC) deletion embryos rescued by the 12 copy promoter replacement (PR) or designer wild type (DWT) arrays on the third chromosome (VK33) to determine CLAMP's interaction with the locus

overall_design: ChIP was performed according to (Blythe and Wieschaus, 2015) with the following particulars. 2-4 hours old embryos (~200 embryos for each of three biological replicates) from HisC/HisC; 12xDWT/12xDWT or 12xPR/12xPR strains were collected, fixed and stored at −80°C until further processing. Immunoprecipitation of chromatin preparations was performed using rabbit anti-CLAMP antibody (Rieder et al., 2017) and a polyclonal rabbit IgG (Millipore-Sigma, 12-370). qPCR was performed as described in (Urban et al., 2017). Three biological replicates and two technical replicates were used for each genotype. We synthesized libraries using 4ul of inputs and 36ul of each ChIP sample (CLAMP or IgG) according to Blythe and Wieschaus (2015). We made libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs), pooled 12 samples per lane, and sequenced libraries on an Illumina HiSeq 2500 in 2×150-bp mode (Genewiz).

first: Leila

last: Rieder

first: Katy

last: Koreski


title: Neutrophil Extracellular Traps (NETs) promote macrophage inflammation and impair atherosclerosis resolution in mice with diabetes

city: New York

first: Emily

last: Brown

address: 435 E 30th St

department: Medicine

institute: NYU Langone

summary: Neutrophil extracellular traps (NETs) promote inflammation and atherosclerosis progression. In diabetes they are increased and impair wound healing, during which inflammation normally resolves. Atherosclerosis regression, a process resembling wound healing, is also impaired in diabetes. Thus, we hypothesized that NETs impede atherosclerosis regression in diabetes through unresolved inflammation. Objective: To investigate in diabetes the effect of NETs on plaque macrophage inflammation and whether NETs reduction improves atherosclerosis regression. Findings: Transcriptomic profiling of plaque macrophages from NET positive and negative areas in Ldlr-/- mice revealed inflammasome and glycolysis pathway upregulation, indicating a pro-inflammatory phenotype. During atherosclerosis regression in non-diabetic mice, plaque NET content decreased. In contrast, in diabetic mouse plaques NETs were enriched and persisted after lipid-lowering. DNase1 treatment (to degrade NETs) of diabetic mice reduced plaque NETs and macrophage inflammation and improved atherosclerosis regression after lipid-lowering. Conclusions: NETs decline during atherosclerosis regression in non-diabetic mice, but persist in diabetes and impair regression by exacerbating macrophage inflammation. DNase1 reduced diabetic plaque NETs and macrophage inflammation, and restored atherosclerosis resolution after lipid-lowering, despite ongoing hyperglycemia. Given that humans with diabetes also exhibit impaired atherosclerosis resolution with lipid-lowering, these data suggest that NETs contribute to the increased CVD risk in this population.

overall_design: Transcriptomic profiiling of atherosclerotic macrophages in NET positive versus NET negative areas

first: Tatjana

last: Josefs

first: Tessa

last: Barrett

first: Alexandra

last: Quezada

first: Xiaoyun

last: We

first: Jaume

last: Amengual

first: Edward

last: Fisher