title: A genome-scale screen for synthetic drivers of T-cell proliferation

first: Neville

last: Sanjana

address: 101 Avenue of the Americas

institute: New York Genome Center

summary: The engineering of patient T-cells for adoptive cell therapies has revolutionised the treatment of several cancer types. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T-cell functions and raise safety concerns due to permanent genome modification. Here we identify positive T-cell regulators via overexpression of ~12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased primary human CD4+ and CD8+ T-cell proliferation, activation, and secretion of key cytokines like interleukin-2 and interferon-gamma. In addition, we developed a single-cell genomics method (OverCITE-seq) for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T-cells. The top-ranked ORF, lymphotoxin beta receptor (LTBR), is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T-cells, LTBR induced profound transcriptional and epigenomic remodelling, increasing T-cell effector functions, as well as resistance to exhaustion in chronic stimulation settings, via constitutive activation of the canonical NF-kappaB pathway. LTBR and other top-ranked genes improved antigen-specific chimeric antigen receptor (CAR) T-cell and gamma delta T-cell responses, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next generation T-cell therapies via induction of new synthetic cell programmes.

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: Screen data: Primary T cells from three donors were transduced with a DNA barcoded ORF library. Best proliferating cells after restimulated were sorted and barcode abundance was compared to unsorted population. Single cell data: T cells overexpressing each ORF were separately transduced and cultured, and pooled only prior to the single cell experiment. Barcoded hashing antibodies were added for sample identification and overloading. Additionally, barcoded anti-NGFR antibody was added. In addition to transcriptome and TCR sequencing, direct ORF capture was performed. RNAseq: CD4 and CD8 T cells were transduced in triplicate with LTBR or tNGFR, and total RNA was extracted from resting or stimulated cells for 3' RNA sequencing in bulk. ATAC-seq: CD8 T cells were transduced in duplicate with LTBR  or tNGFR; T cells were either rested or stimulated with CD3/CD28 for 24 h prior to nuclei isolation and tagmentation.

first: Mateusz

last: Legut

first: Zoran

last: Gajic

first: Maria

last: Guarino

first: Zharko

last: Daniloski

first: Jahan

last: Rahman

first: Xinhe

last: Xue

first: Congyi

last: Lu

first: Lu

first: Eleni

last: Mimitou

first: Stephanie

last: Hao

first: Teresa

last: Davoli

first: Catherine

last: Diefenbach

first: Peter

last: Smibert


title: Effects of DNA topoisomerase inhibitor acriflavine on endothelial gene expression and chromatin accessibility

status: Public on Jan 19 2022

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

country: Germany

institute: MPI for heart and lung research

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes

status: Public on Jan 17 2022

city: Milan

first: Monah

last: Abou Alezz

address: Via Olgettina 60

country: Italy

institute: SR-TIGET

type: Genome variation profiling by high throughput sequencing


title: DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes [DNA-Seq]

summary: We generated iPS cells knock out (KO) for two Aicardi Goutières syndrome genes, TREX1 and RNASEH2b to model the disease in a human neurological context. However, it has been reported that after precise genome engineering human iPSCs may show severe alterations of the p53 gene. With the aim to evaluate the genome engeneering impact on the  p53 pathway in the CRISPR-CAS9 knock out clones we verified integrity of the p53 genome sequence after gene editing.

overall_design: Healthy donor-derived iPS cells were engineered through the CRISPR-Cas9 technology. All editing reagents were delivered transiently through electroporation, followed by limiting dilution of single clones of which we isolated around 30-40 per condition for molecular screening. All clones were analyzed by mismatch-sensitive molecular assay, Sanger sequencing of individual alleles obtained through TOPO TA cloning assay and by Western Blot (WB). One bi-allelic KO clone per gene was selected. A clone obtained from the screen harboring the WT genome was selected as a WT reference for all further experiments. To check if  Trex1 and RNaseH2b knock out iPSCs showed any mutation on p53 gene we isolated them from their murine embrionic fibroblasts feeder cells. Avoiding any murine DNA contamination we isolated the human iPSCs genomic DNA sequencing for p53. We then compared the KO sequences to the WT one. WT samples came from a negative clone.

first: Anna

last: Giordano

first: Sara

last: Valsoni

first: Ivan

last: Merelli

last: Kajaste-Rudnitski


title: Analysis of transcriptome response of rhizobia to aluminium toxicity

city: Jena

first: Clabe

middle: Simiyu

last: Wekesa

address: Dornburgerstr. 159

department: Plant physiology

institute: FSU

summary:
Purpose: High throughput sequencing has revolutionized methods of microbial pathway analysis in response to various envoronmental stimuli. The aim of this study was to determine mechanisms in which Rhizobium phaseoli employ towards tolerance or resistance to aluminium toxicity through RNA sequencing.
Methods: Total RNA was extracted from bacteria treated with aluminium after 48 hours with trizol and sequenced with illumina’s Miseq at Novogene Corporation Inc. U.K. After QC, reads were aligned to the genome with bowtie2 and differential expression performed with DESeq2.
Results: Sequencing resulted to about 6.7-9.0 M reads that aligned to all the four plasmids and one chromosome of R. phaseoli genome. Most enriched genes were found localized in the membrane of the bacteria.
Conclusion: The membrane of R. phaseoli is possibly the target site for toxicity and tolerance against aluminium toxicity. This is the first time such a study is described.

overall_design: Total RNA was isolated from 0.1 mM aluminium treated and untreated cells. The experiment was repeated three times. Together, six samples were analysed.

middle: S

first: Alexandra

middle: C

last: Furch

first: Ralf

last: Oemüller


title: Circulating MicroRNAs From Serum Small Extracellular Vesicles as Potential diagnostic Biomarkers in Pediatric Epilepsy and Drug-Resistant Epilepsy

status: Public on Jan 20 2022

city: Hangzhou

first: Yilong

last: Wang

state: Zhejiang

address: NO.3333 binsheng Road

department: Department of Neurology

country: China

institute: Children’s Hospital, Zhejiang University School of Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: Pediatric epilepsy is a neurological condition that causes repeated and unprovoked  seizures and is more common in 1–5-year-old children. Drug resistance has been  indicated as a key challenge in improving the clinical outcomes of patients with  pediatric epilepsy. In the present study, we aimed to identify serum small extracellular  vesicles (sEVs) derived microRNAs (miRNAs) from the serum samples of children  for predicting the prognosis in patients with epilepsy and drug-resistant epilepsy

overall_design: Expression of circulating miRNAs  from serum sEVs between epileptic children and healthy  children was assessed in this multiphase case-control study.

first: Yeping

first: Yi

last: Chen

last: Hua

last: Xu

first: Mengying

last: Zhu

first: Congying

last: Zhao

first: Weiran

last: Zhang

first: Guoxia

last: Sheng

first: Liu

last: Liu

first: Peifang

last: Jiang

first: Zhefeng

last: Yuan

first: Zhengyan

first: Feng

last: Gao


title: DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes [scRNA-Seq]

first: Monah

last: Abou Alezz

summary: The contribution of the different CNS cell types to Aicardi-Goutiéres Syndrome pathogenesis is still unclear. With the aim to elucidate how oligodendrocytes, neurons and astrocytes impact AGS human phenotype  we interrogate transcriptomes of the different cell populations performing single-cell RNA-Seq (scRNASeq). We compared cells deriving from AGS patient-derived iPSC harboring mutations in TREX1 (AGS1) or RNASEH2B (AGS2) to healthy donor control (WT) cells differentiated into mixed neuronal/glial cell populations.

overall_design: To study AGS pathological mechanisms in a physiologically relevant neural context we differentiated iPS cells from AGS patients harboring mutations in TREX1 (AGS1: compound heterozygous for p.R97H and p.S88fs*22 (Ferraro et al., 2019) or RNASEH2B (AGS2: homozygous for p.A177T (Ferraro et al., 2019) respectively and an healthy donor control (WT) into mixed neuronal/glial cell populations (Frati et al., 2018; Santos et al., 2017). The resulted oligodendrocytes, neurons and astrocyte mixed population were identified by several literatures reported markers and further analyzed for differential expression of the key pathways involved in the disease.

first: Anna

last: Giordano

first: Sara

last: Valsoni

first: Marco

last: Genua

first: Renato

last: Ostuni

last: Merelli


title: Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection

city: Munich

first: Markus

last: Joppich

state: Bavaria

address: Amalienstr. 17

department: Department of Informatics

institute: LMU Munich

summary: The antiviral immune response to SARS-CoV-2 infection can limit viral spread and prevent development of pneumonic COVID-19. However, the protective immunological response associated with successful viral containment in the upper airways remain unclear. Here, we combine a multiomics approach with longitudinal sampling to reveal temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients and associate specific immune trajectories with upper airway viral containment. We see a distinct systemic rather than local immune state associated with viral containment, characterized by interferon stimulated gene (ISG) upregulation across circulating immune cell subsets in non-pneumonic SARS-CoV2 infection. We report reduced cytotoxic potential of Natural Killer (NK) and T cells, and an immune-modulatory monocyte phenotype with protective immunity in COVID-19. Together, we show protective immune trajectories in SARS-CoV2 infection, which have important implications for patient prognosis and the development of immunomodulatory therapies.

overall_design: Single cell RNA Sequencing data of longitudinally sampled PBMCs of non-pneumonic and pneumonic SARS-CoV-2 infected patients and control patients

first: Kami

last: Pekayvaz

first: Alexander

last: Leunig

first: Ralf

last: Zimmer

first: Konstantin

last: Stark

first: Leo

last: Nicolai


title: Effects of DNA topoisomerase inhibitor acriflavine on endothelial gene expression and chromatin accessibility (RNA-Seq)

first: Stefan

last: Günther

summary: RNA-Seq of human endothelial cells treated with ACF revealed massive changes on gene expression. These were non-randomly and strongly conserved to murine lung endothelial cells potentially due to DNA topoisomerase inhibition rather than HIF inhibition. Surprisingly, in contrast to protein-coding genes, RNA-Seq yielded that an exceeding number of lncRNAs is upregulated, e.g. FENDRR, H19, HIF1α-AS1 and FLJ31356, whereas BOLA3-AS1 and MEG3 were strongly downregulated. ATAC-Seq demonstrated that ACF leads to strong changes on chromatin accessibility on lncRNA promoters.

overall_design: Examination of acriflavine treatment in mouse lung endothelial cells and HUVECs.

first: Sandra

last: Seredinski

middle: P

last: Brandes

first: Matthias

last: Leisegang


title: Single-cell profiling of skeletal muscle reveals a novel senolytic target; CRYAB

city: Novato

first: Simon

last: Melov

state: CA

address: 8001 Redwood Blvd

country: USA

institute: Buck Institute for Research on Aging

summary: Skeletal muscle mass and function decline with aging, a process known as sarcopenia, and is linked to functional alterations of critical cell types within mature muscle, including fibro-adipogenic progenitors (FAPs) and satellite cells (SCs). Senescence is a form of cellular arrest linked with degenerative diseases of aging, and is thought to be a key driver of aging pathophysiology. We utilized single-cell RNA sequencing to identify novel targets in isolated FAPs and SCs which potentiate senescent cell killing - a process termed senolysis. Using molecular and cellular approaches, we identified the small alpha crystalline heat shock protein CRYAB as a novel senolytic target. We targeted CRYAB using chemical inhibitor screening to identify 25-hydroxycholesterol (25HC), an endogenous metabolite of cholesterol biosynthesis as a potent senolytic capable of killing senescent cells. We validated the potential of 25HC as a senolytic in both mouse and human cells, and in vivo in several tissues including muscle and fat. 25HC treatment therefore represents a potential new class of senolytics, which may be useful in combating diseases or physiologies in which cellular senescence is a key driver.

first: Chandani

last: Limbad

first: Serban

last: Ciotlos

first: Kevin

last: Perez


title: The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling

city: Berlin

first: IBRAHIM

middle: AVSAR

last: ILIK

state: Berlin

address: Ihnestraße 63-73

institute: Max-Planck-Institut für molekulare Genetik

summary: The RNA helicase BRR2 (SNRNP200) is one of the key remodeling factors of the spliceosome. Here we show its direct interaction with TSSC4.

overall_design: Identification of TSSC4 RNA targets with FLASH, using GFP as a negative control

first: Ibrahim

last: Ilik

first: Alexandra

last: Bergfort


title: tRNA-Derived Fragments Expression Profile and Their Potential Roles in Lung Adenocarcinoma Tissues

city: Shenyang

first: Le-Tian

last: Huang

state: Liaoning

address: NO.39 Huaxiang Road, Tiexi District

department: Department of Oncology

institute: Shengjing Hospital of China Medical University

summary: Transfer RNA-derived fragments (tRFs) and transfer RNA halves (tiRNAs) have been shown to play crucial roles in gene regulation. This study targets to reveal the expression profile of tRFs and tiRNAs and their possible biological roles in lung adenocarcinoma (LUAD). Five paired clinical lung adenocarcinoma tissues (LAT) and adjacent normal lung tissues (ANLT) were selected to conduct the expression of tRFs and tiRNAs. The sequencing results showed that 109 tRFs and tiRNAs were differentially expressed between LAT and ANLT, out of which 60 were upregulated and 49 were downregulated. Compared with ANLT, lower expression levels of three tRF-1s (tRF-Ser-TGA-010, tRF-Arg-CCT-018 and tRF-Val-CAC-017) in LAT were verified by quantitative real-time polymerase chain reaction. Subsequently, the putative target genes of tRF-1s were analyzed by computational prediction and the top ten significant results of GO and KEGG pathway enrichment analysis were presented. These signaling pathways are involved in the carcinogenesis of LUAD. This study has showed a landscape of tRFs and tiRNAs expression proﬁles in LUAD. Three newly found diﬀerentially expressed down-regulated tRF-1s may be involved in the pathogenesis of LUAD and might serve as potential diagnostic biomarkers.

overall_design: tRFs&tiRNAs profiles of 5 pairs of lung adenocarcinoma tissues and adjacent normal samples by deep sequencing, using Illumina NextSeq 500.

first: Wang

last: Jia-He

first: Huang

last: Le-Tian

last: Yi-Bing


title: Gene expression profiles of human patient derived ectocervical stem cells and 3D organoids with and without Chlamydia trachomatis infection

status: Public on Jan 16 2022

city: Würzburg

first: Cindrilla

last: Chumduri

address: Am Hubland

department: Chair of Microbiology, Biozentrum

institute: Julius-Maximilians-Universität Würzburg

summary: Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modelled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renew capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrate the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression.

overall_design: Patient-derived human ectocervical epithelial stem cells were cultured in 2D and as 3D organoids as described in Chumduri et al. 2021, Nature Cell Biology. The 2D stem cells were genetically manipulated to introduce HPV16 E6E7 oncogenes into their genome to mimic HPV persistence in vivo. The HPV negative and positive 2D stem cells and 3D organoids were either uninfected or infected with Chlamydia trachomatis for 2 days or 5days, respectively as described in this manuscript. Total RNA was extracted from samples and hybridized on Agilent-8x60k Human custom arrays as single-color hybridizations.

first: Stefanie

last: Koster

first: Rajendra

middle: K

last: Gurumurthy

first: Hans-Joachim

last: Mollenkopf

first: Thomas

middle: F

last: Meyer

first: Hilmar

last: Berger


title: Arabidopsis germination and two week growth on ionic vs osmotic stress

city: Observatory

first: Lara

middle: Elizabeth

last: Donaldson

state: Western Cape

address: Werner and Beit South, UCT Medical School, Anzio Road

country: South Africa

institute: International Centre for Genetic Engineering and Biotechnology

summary:
Arabidopsis Col-0 seeds were germinated and grown for two weeks on Arabidopsis thaliana salt media (ATS, control) or ATS media supplemented 50, 75, 100 or 125 mM NaCl that imposes both an ionic and osmotic stress; or ATS media supplemented with iso-osmolar concentrations of  sorbitol (100, 150, 200 or 250 mM) that imposes only an osmotic stress. The aim of the study was to identify genes involved in plant growth and adaptation to ionic stress compared to genes involved in growth and adaptation to osmotic stress conditions. To do this we identified lists of genes that are differentially expressed in plants grown in NaCl (A) and lists of genes differentially expressed in plants grown in sorbitol (B). We then compared these lists to find ionic/salt-specific genes that are only expressed in plants grown in NaCl and not in plants grown in sorbitol; and osmotic genes that are expressed both in plants grown in NaCl and in plants grown in sorbitol.
Associated publication: Cackett et al. (2022) Salt-specific gene expression reveals elevated auxin levels in Arabidopsis thaliana plants grown under saline conditions, DOI: 10.3389/fpls.2022.804716

overall_design: 27 samples including 3 replicates x 9 different treatment conditions. Controls were Arabidopsis thaliana Col-0 plants grown on ATS media. Treatments were plants grown on ATS media supplemented with 50 mM NaCl, 75 mM NaCl, 100 mM NaCl, 125 mM NaCl, 100 mM sorbitol, 150 mM sorbitol, 200 mM sorbitol or 250 mM sorbitol

middle: E

first: Lee

last: Cackett


title: Expression data from murine MPS I fibroblasts under flavonoids treatment

status: Public on Jan 15 2022

city: Gdańsk

first: Marta

middle: Magdalena

last: Moskot

address: Kładki 24

country: Poland

institute: Institute of Biochemistry and Biophysics, Polish Academy of Sciences

summary:
Flavonoids are biological compounds with high potency to modulate molecular processes. The study was designed to estimate genistein, kaempferoland their mixture impact on global gene expression, with special focus on key processes in MPS pathogenesis.
We used microarrays to detail the global program of gene expression underlying flavonoid treatment and identified distinct classes of regulated genes during this process.

overall_design: Murine MPS I fibroblasts were cultivated for 24 hours with 60 and 120 µM genistein, 60 and 120 µM kaempferol and 60 and 120 µM genistein and kaempferol 1:1 mixture.

first: Magdalena

last: Gabig-Cimińska


title: MYC overexpression leads to increased chromatin interactions at superenhancers and MYC binding sites

city: Singapore

first: Melissa

middle: Jane

last: Fullwood

address: 60 Nanyang Drive

country: Singapore

institute: Nanyang Technological University

summary: The MYC oncogene encodes for the MYC protein and is frequently dysregulated across multiple cancer cell types, making it an attractive target for cancer therapy. MYC overexpression leads to MYC binding at active enhancers, resulting in a global transcriptional amplification of active genes. Since superenhancers are frequently dysregulated in cancer, we hypothesized that MYC preferentially invades into superenhancers and alters the cancer genome organization. To that end, we performed ChIP-seq, RNA-seq, 4C-seq and SIQHiC (Spike-in Quantitative Hi-C) on the U2OS osteosarcoma cell line with tetracycline-inducible MYC. MYC overexpression in U2OS cells modulated histone acetylation and increased MYC binding at superenhancers. SIQHiC analysis revealed increased global chromatin contact frequency, particularly at chromatin interactions connecting MYC binding sites at promoters and enhancers. Immunofluorescence staining showed that MYC molecules formed punctate foci at these transcriptionally active domains after MYC overexpression. These results demonstrate the accumulation of overexpressed MYC at promoter-enhancer hubs and suggest that MYC invades into enhancers through spatial proximity. At the same time, the increased protein-protein interactions may strengthen these chromatin interactions to increase chromatin contact frequency. CTCF siRNA knockdown in MYC overexpressed U2OS cells demonstrated that removal of architectural proteins can disperse MYC and abrogate the increase in chromatin contacts. By elucidating the chromatin landscape of MYC driven cancers, we can potentially target MYC associated chromatin interactions for cancer therapy.

overall_design: Spike-In Quantitative Hi-C (SIQHiC), ChIP-Seq, RNA-Seq and 4C-Seq performed on 2 biological replicates of U2OS cells treated with doxycycline or vehicle (H2O) for 30h, with NIH3T3 mouse cells spiked in.

first: Yi Xiang

last: See

first: Kaijing

last: Chen

middle: J


title: STAT6 Blockade Abrogates Aspergillus-Induced Eosinophilic Chronic Rhinosinusitis and Asthma, a Model of Unified Airway Disease

city: Houston

first: John

middle: Morgan

last: Knight

state: TX

address: One Baylor plaza, M917

department: Pathology and Immunology

institute: Baylor College of Medicine

summary: Unified airway disease, including concurrent asthma and chronic rhinosinusitis (CRS), is a common, but poorly understood disorder with no curative treatment options. To establish a murine model of chronic unified eosinophilic airway inflammation, mice were challenged with Aspergillus niger, and sinonasal mucosa and lung tissue were evaluated by immunohistochemistry, flow cytometry and gene expression. Inhalation of A niger conidia resulted in a Th2-biased lung and sinus inflammation that typifies allergic asthma and CRS. Gene network and pathway analysis correlated with human disease with upregulation of the JAK-STAT and helper T cell pathways, but also less expected pathways governing the spliceosome, osteoclast differentiation, and coagulation pathways. Utilizing a specific inhibitor and gene-deficient mice, we demonstrate that STAT6 is required for mycosis induced sinus inflammation. These findings confirm the relevance of this new model and portend future studies that further extend our understanding of the immunopathologic basis of airway mycosis and unified airway disease.

overall_design: Total RNA was quantified from sinus tissue of mice (n = 3/group) challenged every other day with PBS or A. niger (400k live conidia) for 12 weeks

middle: M

first: David

middle: B

last: Corry


title: Reorganization of 3D genome architecture across wild boar and Bama pig adipose tissues

status: Public on Jan 14 2022

city: Chengdu

first: jin

last: long

address: Hui ming

institute: Sichuan Agricultural University

summary: Domestication of pig results in modifications of many traits, including fatness traits, which are important in pig production since they have effect on meat quality, fattening efficiency, reproduction and immunity.In this study, we investigate 3D genome organization and transcriptomic characterization of adipose tissues (ATs) between wild boars and Bama pig, a typical indigenous domestic pig in China, to uncover molecular mechanisms of fatness-phenotypic shifts.

overall_design: We analyzed chromatin conformation capture sequencing (Hi-C) data and RNA sequencing (RNA-seq) data of two adipose tissues ( upper layer of backfat tissue and greater omentum fat tissue)of wild boar and Bama pig with 2 replicates for inter-breed comparison

first: Jiaman

last: Zhang

first: Pengliang

last: Liu

first: Diyan

last: Li

first: Mingzhou


title: Comparison of intestinal epithelial transcriptomes of WT and FACI-/- mice

city: Hong Kong

first: Yun

last: Cheng

state: Hong Kong

address: 21 Sassoon Road, Pokfulam

institute: The University of Hong Kong

summary: CREB-H is a key transcription factor governing lipid metabolism. Faci (C11orf86) is a novel CREB-H-induced gene preferentially expressed in enterocyte. To explore its physiological function, we compared intestinal epithelial transcriptomes of WT and Faci-/- mice.

overall_design: The total RNA from the intestinal epithelium of 4-month-old WT and Faci-/- mice (n = 3 per group) was extracted by RNAiso Plus reagent (Takara) and sent to BGI Shenzhen Genomics Institute of for RNA sequencing.

first: Xiao-Zhuo

last: Kang


title: DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes [RNA-Seq]

first: Monah

last: Abou Alezz

summary: We generated iPS cells knock out (KO) for two Aicardi Goutières syndrome genes, TREX1 and RNASEH2b to model the disease in a human neurological context. Their transcriptomes were analyzed at several differentiation stages: iPSC-derived Neural Stem Cells, iPSC-derived proinflammatory astrocytes and iPSC-derived neurons. We then evaluated when and to which extent the AGS-related transcriptional alterations arised in all KO and WT cells.

overall_design: To study AGS pathology in a physiologically relevant neural context we generated isogenic iPSCs knock-out (KO) for TREX1 or RNASEH2B. Healthy donor-derived iPS cells were engineered through the CRISPR-Cas9 technology. All editing reagents were delivered transiently through electroporation, followed by limiting dilution of single clones of which we isolated around 30-40 per condition for molecular screening. All clones were analyzed by mismatch-sensitive molecular assay, Sanger sequencing of individual alleles obtained through TOPO TA cloning assay and by Western Blot (WB). One bi-allelic KO clone per gene was selected. A clone obtained from the screen harboring the WT genome was selected as a WT reference for all further experiments. At first, WT, T-/- and R-/- clones were differentiated into induced neural stem/progenitor cells (iPSC-NSCs) (Chambers et al., 2009), Later they were differentiated in more committed cells such inflammation-responsive astrocytes as previously reported (Santos et al., 2017) and towards neurons following a commercial differentiation protocol. Pluripotent stem cells and more committed derived cells were analyzed by RNA Seq to characterize the transcriptional changes at different stages of differentiations.

first: Anna

last: Giordano

first: Sara

last: Valsoni

first: Ivan

last: Merelli

last: Kajaste-Rudnitski


title: Cell type-specific mechanism of Setd1a heterozygosity in schizophrenia pathogenesis [single-cell RNA-seq]

status: Public on Jan 12 2022

city: Doha

first: Mohamed Nadhir

last: Djekidel

state: Doha

address: Gharafa

department: Translational Medicine Department

country: Qatar

institute: Sidra Medicine

summary: Schizophrenia (SCZ) is a chronic, serious mental disorder with severe burden on patients’ families and society. Although over 100 genes have been linked to SCZ pathogenies, the underlying molecular and cellular mechanisms remain largely unknown. Here, we generated a Setd1a haploinsufficiency mouse model to understand how this SCZ-associated epigenetic factor affects gene expression programs in cells of brain regions highly relevant to SCZ. By comparing single-cell RNA-seq results from wild type and Setd1a+/- mice, we found Setd1a heterozygosity causes highly diverse transcriptional adaptations across different cell types in prefrontal cortex and striatum, suggesting brain region- and cell type-specific mechanisms contribute to pathophysiology of SCZ. Interestingly, we found the Foxp2+ neurons exhibit the most prominent gene expression changes among the different neuron subtypes in PFC, which correlate with the H3K4me3 changes. Importantly, many of the genes dysregulated in heterozygous Setd1a mice are linked to neuron morphogenesis and synaptic function. Consistently, heterozygous Setd1a mice exhibit certain behavioral features of SCZ patients. Collectively, our study establishes Setd1a heterozygous mice as a model for understanding SCZ and uncovers a complex brain region- and cell type-specific changes that potentially underlying SCZ pathogenesis.

overall_design: To mimic Setd1a loss-of-function mutations in a mouse model, we used CRISPR-Cas9 technique and targeted the 15th exon of Setd1a , which is right before the SET domain encoded by exons 16-18.  we dissected PFC tissues from acute coronal sections of Setd1a+/- and WT mouse brains and dissociated the tissues to single cells, which were processed with the 10x Chromium platform (10X Genomics, CA).  In additional to PFC, we performed similar scRNA-seq to analyze the transcriptional effect of Setd1a heterozygosity in striatum (including both dorsal and ventral striatum) of two WT and two Setd1a+/- mice.

first: Renchao

first: Yiqiong

first: Mohamed

middle: N

first: Wenqiang

first: Aritra

last: Bhattacherjee

first: Zhiyuan

first: Ed

last: Scolnick

first: Yi


title: Cell type-specific mechanism of Setd1a heterozygosity in schizophrenia pathogenesis [CUT&RUN]

overall_design: To study epigenetic effect of Setd1a depletion, we performed CUT&RUN on Setd1 and H3K4me3 from PFC nuclei or with sorted GFP+ nuclei from Foxp2-Cre/Setd1a+/+ and Foxp2-Cre/Setd1a+/- mice.

last: Chen


title: Direct regulation of shikimate, early phenylpropanoid and stilbenoid pathways by Subgroup 2 R2R3-MYBs in grapevine

status: Public on Jan 10 2022

city: New York

first: Shao-shan

middle: Carol

last: Huang

state: NY

address: 12 Waverly Pl

department: Biology

institute: New York University

summary: The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis vinifera L.). A few transcription factors (TFs) have been identified as regulators of this pathway, but the extent of this control has not been deeply studied. Here we demonstrate how DNA affinity purification sequencing (DAP-Seq) allows for genome-wide TF binding site interrogation in grape. We obtained 5,190 and 4,443 binding events assigned to 4,041 and 3,626 genes for MYB14 and MYB15, respectively (around 40% of peaks located within -10kb of transcription start sites). DAP-Seq of MYB14/MYB15 was combined with aggregate gene co-expression networks (GCNs) built from more than 1,400 transcriptomic datasets from leaves, fruits and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15 and MYB13, a third uncharacterized member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 out of 47 STS family genes. Moreover, all three MYBs bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene candidates amongst which ROMT2-3 were validated enzymatically. A high proportion of DAP-Seq bound genes was induced in the activated transcriptomes of transient MYB15-overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3-MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP-Seq and reciprocal GCNs offers a rapid framework for gene function characterization using genome-wide approaches in the context of non-model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialized metabolism.

overall_design: Identification of binding sites for three transcription factors in Vitis vinifera by direct sequencing of affinity purified genomic DNA fragments.

first: José

middle: T

last: Matus

first: Dario

last: Cantu


title: Cell type-specific mechanism of Setd1a heterozygosity in schizophrenia pathogenesis [bulk RNA-seq]

overall_design: Bulk RNA-seq samples were generated from the PFC in WT and Setd+/- to study the global transcriptional changes induced by Setd1 loss. Fox2p+ PFC were FACS sorted and their transcriptome sequenced.

last: Zhang


title: Nanostring Human v3 miRNA Assay on CD4+ T naïve, central memory and effector memory total RNA

first: Francesco

last: Gualdrini

address: Via Adamello, 16

department: Department of Experimental Oncology

institute: European Institute of Oncology

summary: Buffy coats from four independent human donors were enriched for CD4+ cells using MACS CD4 beads and LS columns. Cells were subsequently sorted into Naïve, Central Memory and Effector Memory using following markers: Naive: CD4+CD25–CD45RA+CCR7+; Central Memory: CD4+CD25–CD45RA–CCR7+; Effector Memory: CD4+CD25-CD45RA-CCR7-. Total RNA from these cell subsets was extracted and 100ng was used for Nanostring SPRINT run according to manufacturer's instructions. Overall aim of the experiment is assessing the expression level of human T helper cell miRNome.

overall_design: Total RNA from resting CD4+ human T cells. No treatment.

first: Michele

last: Chirichella

first: Niccolò

last: Bianchi


title: An RFX/miR-150 axis regulates proliferation of primary human T lymphocytes

summary: ATAC-seq was performed to identify the cis-regulatory genomic elements in in primary human naïve and memory T cells untreated or activated with anti-CD3 and anti-CD28 antibodies for one or three days.

overall_design: Primary Naïve and memory T cells from two independent donors were sorted based on the expression of the following surface markers: Naive: CD4+CD25–CD45RA+CCR7+; Memory: CD4+CD25–CD45RA–CCR7+/-and activated for 48hrs on anti-CD3 and anti-CD28 antibodies. From each subset 10^5 cells were taken at day 0 (ex vivo, immediately after sorting), at day1 (24h after activation) and at day3 and lysed. Nuclei were than isolated by centrifugation and treated with Tn5 transposase (made in-house) for 1hr at 37°C. Chromatin tagmentation was inactivated by incubating the samples for 30min at 40°C with a cleanup buffer containing EDTA. Tagmented DNA was isolated using 2× AMPure XP beads and amplified by PCR with barcoded primers using 14 cycles of PCR. Finally, fragments smaller than 500 bp were purified with 0,65x AMPure XP beads and primers removed by purification with 1,8x AMPure XP beads. Libraries were sequenced paired-end on an Illumina NOVAseq.

first: Amy

last: Kenyon

first: Gioacchino

last: Natoli

first: Silvia

last: Monticelli


title: Genomic variants affecting homoeologous gene expression dosage contribute to agronomic trait variation in allopolyploid wheat

city: Manhattan

first: Eduard

last: Akhunov

state: Kansas

address: 1712 Claflin Road

department: Plant Pathology

institute: Kansas State University

summary: We have collected RNA-seq data from the total RNA isolated from the 2-week seedlings of 198 diverse wheat accessions. These accessions were selected among nearly 3,000 lines to represent the broad geographic and genetic diversity of wheat populations. On average, 65.7 million paired-end Illumina reads (2 x 100 bp) were collected for each sample, and after quality trimming were mapped to the wheat reference genome RefSeq v.1.0. The proportion of reads unambiguously mapped to the individual wheat genomes was 81%, with the accuracy of correct read mapping estimated by simulation achieving 98%. The expression levels measured as Transcripts Per Million (TPM) were estimated for high confidence (HC) gene models in the wheat reference genome, with 82,092 gene models (66,333 genes) showing TPM > 0.5 in at least two wheat lines (PRJNA670223)

overall_design: 204 Samples

first: Fei

last: He

first: Wei

last: Wang

first: William

last: Rutter

first: Katherine

middle: W

last: Jordan

first: Jie

last: Ren

first: Alina

last: Akhunova

first: Les

last: Szabo

first: Matthew

last: Rouse


title: DNA methylation analysis of breast cancer patient-derived xenografts and organoids

status: Public on Jan 09 2022

city: Salt Lake CIty

first: Jason

last: Gertz

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: University of Utah

type: Methylation profiling by high throughput sequencing

summary: To understand if the generation of xenograft and organoid models of breast cancer alters DNA methylation, we compared the genome-wide methylation profile of matching patient tumors, patient derived xenografts, and organoid cultures derived from xenografts.

overall_design: Reduced Representation Bisulfite Sequencing (RRBS) or Reduced Representation Enzymatic Methylation Sequencing (RREMS) was performed on DNA extracted from 11 sets of matching patient tumor samples, patient derived xenografts, and organoid culture derived from the xenograft tumors.

last: Varley

first: Bryan

last: Welm

first: Alana

middle: L


title: Glucagon-like peptide-1 suppresses sympathetic activation via peripheral chemosensory inhibition

status: Public on Jan 11 2022

city: Bristol

first: Audrys

middle: G

last: Pauza

state: Somerset

address: University of Bristol, The Dorothy Hodgkin Building, Whitson Street

department: Bristol Medical School (THS)

country: United Kingdom

institute: University of Bristol

summary: In order to investigate the molecular basis of carotid body chemoreceptor sensitisation in the Spontaneously Hypertensive Rat (SHR) we have sequenced the transcriptomes of bilateral carotid body samples from aged-matched, male SHR and control Wistar-Kyoto rats.

overall_design: Bilateral (right and left) carotid body samples of n=6 individual SHR/NHsd and WKY/NHsd (male, 12-14 weeks old) rats were microdissected from common carotid artery bifurcations following intracardial perfusion with ice-cold PBS. Total RNA was isolated using phenol-chloroform phase seperation method and Zymo Directzol RNA Microprep kit. Average RNA Integrity Number (RIN) of samples processed for sequencing (n=24) was 7.67. Poly-A enriched sequencing libraries were constructed using TruSeq® Stranded mRNA library preparation kit (20020594, Illumina). Libraries were pooled and sequenced on Illumina NextSeq500 platform using NextSeq 500/550 High Output v2.5 (150 Cycles) Kit (20024907; Illumina) on a paired-end multiplex mode.

first: David

last: Murphy

first: Julian

last: Paton


title: The role of Hippo pathway in ER positive breast cancer

city: La Jolla

first: Shenghong

last: Ma

address: 2880 TORREY PINES SCENIC DR, Sanford Consortium

institute: UCSD

summary: We report the transcriptional difference of LATS1/2 gene knock out T47D cells with wild type control using RNA-seq technology. We further generated the chromatin binding of multiple proteins, including YAP, TEAD2, Estrogen receptor alpha, VGLL3 and several histone marks, as well as ATAC profiling between LATS1/2 gene knock out MCF7 cells with wild type control.

overall_design: mRNA profiling between WT and LATS1/2 dKO T47D cell. Examination of multiple histone modifications, PolII, pPolII-S2, CTCF, YAP, TEAD2, ER-alpha, VGLL3 and ATAC chromatin signal between WT and LATS1/2 dKO MCF7 cells


title: miR-146a Pulldown-sequencing data

city: Milan

first: Francesco

last: Gualdrini

summary: Human CD4+ Total memory cells (CD4+CD25-CD45RA-CCR7+/-) were activated with anti-CD3/anti-CD28 antibodies and transfected with biotinylated miR-146a mimic or biotinylated mimic negative control by Exiqon. After 24h cells were lysed and miRNA:mRNA complexes pulled-down with streptavidin agarose O/N at 4°C. RNA was then purified using TRI-reagent and RNA zymospin columns. Four independent donors were used for this experiment. Total RNA was sequenced in 2 lanes, paired ends. This experiment is assessing direct targets bound by miR-146a in human T cells.

overall_design: RNA from biotinylated miR-146a pulldown sequencing. Total memory cells, human, CD4+.

first: N

last: Bianchi

first: M

last: Chirichella


title: Local and global crosstalk among heterochromatin marks drives epigenome patterning in Arabidopsis

city: Bunkyo-ku

first: Taiko

middle: Kim

last: To

state: Tokyo

address: 7-3-1 Hongo

department: Biological Science

country: Japan

institute: University of Tokyo

summary: Transposable elements (TEs) are robustly silenced by multiple epigenetic marks, but dynamics of crosstalk among these marks remains enigmatic. In Arabidopsis, TEs are silenced by cytosine methylation in both CpG and non-CpG contexts (mCG and mCH) and histone H3 lysine 9 methylation (H3K9me). While mCH and H3K9me are mutually dependent for their maintenance, mCG and mCH/H3K9me are independently maintained. Here we show that establishment, rather than maintenance, of mCH depends on mCG, accounting for the synergistic colocalization of these silent marks in TEs. When mCG is lost, establishment of mCH is abolished in TEs. mCG also guides mCH in active genes, although  the resulting mCH/H3K9me is removed thereafter. Unexpectedly, targeting efficiency of mCH depends on relative, rather than absolute, levels of mCG within the genome, suggesting underlying global negative controls. We propose that local positive feedback in heterochromatin dynamics, together with global negative feedback, drive robust and balanced DNA methylome patterning.

overall_design: Examination of DNA methylation (WGBS) and transcription (RNA-seq) in Arabidopsis WT, several DNA methylase and histone modification mutants, and their genetic hybrids.

first: Chikae

last: Yamasaki

first: Shoko

last: Oda

first: Sayaka

last: Tominaga

first: Akie

last: Kobayashi

first: Yoshiaki

last: Tarutani

first: Tetsuji

last: Kakutani


title: Mouse Global gene expression profiles of mouse embryonic day3.75 inner cell mass cells

city: Cambridge

first: Giuliano

middle: Giuseppe

last: Stirparo

state: Cambridgeshire

address: 418 Cambridge Science Park Milton Rd

institute: Cerevance ltd

summary: The goal of this study is elucidating the mechanisms of the primitive endoderm (PrE, founder of the yolk sac) segregates from the epiblast (EPI, founder of the foetus). Within the uterus and ex vivo, precursors of the EPI and PrE emerge in an unsorted manner in inner cell mass (ICM). Coincident with identity acquisition, the cells physically sort, resulting in PrE establishing a single layer of cells covering the cavity-facing surface of the ICM with the EPI enclosed between the PrE and polar trophectoderm. The mechanism that determines this spatial segregation between EPI and PrE is still poorly understood. To achieve this goal, we have compared mouse embryonic day (E) 3.75 ICM cells’ transcriptome profiling to other existing mouse E3.5 and E4.5 ICM cells’ RNA-seq data. We have identified the timing of EPI and PrE lineages specification and maturation and each stage- and lineage-specific genes expression profiles.

overall_design: Single cells of mouse PDGFRa::H2B-GFP/+ E3.75 ICMs mRNA profiles were generated using Smart-seq2.

first: G

first: A

last: Yanagida


title: miR-150 Pulldown-sequencing data

summary: Human CD4+ Total memory cells (CD4+CD25-CD45RA-CCR7+/-) were activated for 5d with anti-CD3/anti-CD28 antibodies and transfected with biotinylated miR-150 mimic or biotinylated mimic negative control by Exiqon. After 24h cells were lysed and miRNA:mRNA complexes pulled-down with streptavidin agarose O/N at 4°C. RNA was then purified using TRI-reagent and RNA zymospin columns. Three independent donors were used for this experiment. Inputs were also sequenced. Total RNA was sequenced in single end mode. This experiment is assessing direct targets bound by miR-150-5p in human T cells.

overall_design: RNA from biotinylated miR-150 pulldown sequencing. Total memory cells, human, CD4+.


title: The secreted factor TSK maintains slow-twitch myofiber integrity and exercise endurance and contributes to muscle regeneration

status: Public on Jan 08 2022

city: Ann Arbor

first: Jiandie

last: Lin

state: MI

address: 210 Washtenaw Ave

department: Cell & Developmental Biology, Life Science Institute

institute: University of Michigan

summary: Mammalian skeletal muscle contains heterogenous myofibers with different contractile and metabolic properties that sustain muscle mass and endurance capacity. The transcriptional regulators that govern these myofiber gene programs have been elucidated. However, the hormonal cues that direct the specification of myofiber types and muscle endurance remain largely unknown. Here we uncover the secreted factor Tsukushi (TSK) as an extracellular signal that is required for maintaining muscle mass, strength, and endurance capacity, and contributes to muscle regeneration. Mice lacking TSK exhibited reduced grip strength and impaired exercise capacity. Muscle transcriptomic analysis revealed that TSK deficiency results in a remarkably selective impairment in the expression of myofibrillar genes characteristic of slow-twitch muscle fibers that is associated with abnormal neuromuscular junction formation. AAV-mediated overexpression of TSK failed to rescue these myofiber defects in adult mice, suggesting that the effects of TSK on myofibers are likely restricted to certain developmental stages. Finally, mice lacking TSK exhibited diminished muscle regeneration following cardiotoxin-induced muscle injury. These findings support a crucial role of TSK as a hormonal cue in the regulation of contractile gene expression, endurance capacity, and muscle regeneration.

overall_design: RNA-seq on WT vs TSK KO mouse quadriceps

first: Tongyu

last: Liu

first: Qiuyu

last: Wang


title: ΔNp63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes

city: Rome

first: Stefano

last: Scalera

address: Via Elio Chianesi 53

institute: IRCCS Regina Elena National Cancer Institute

summary: ΔNp63, a master regulator of epithelial biology, is involved in regulating epithelial stem cell function, maintaining the integrity of stratified epithelial cells and committing epidermal cells to the differentiation program. To this end, ΔNp63 exploits several direct mechanisms. Here, we elucidated a novel mechanism whereby ΔNp63 efficiently sustains the expression of epidermal differentiation genes. We show that ΔNp63 interacts with Senataxin (SETX), an RNA/DNA helicase able to resolve the R-loop intermediates over the GC-rich termination sites of coding genes. Notably, we found that SETX and ΔNp63 co-regulate a subset of genes involved in the early step of the keratinocyte differentiation program. At the molecular level, SETX physically binds the p63 DNA binding motifs present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3’ ends and thus promoting efficient transcriptional termination and gene expression. Remarkably, SETX loss affects the activation of the proper epidermal differentiation program in vitro and impacts epidermal layer stratification in organotypic human skin. Furthermore, we found that SETX is mutated or downmodulated in SCC and SETX gene mutation is a negative prognostic factor for cutaneous SCC patient survival. Collectively, our results unveil SETX as a novel molecular player of skin homeostasis, potentially involved in hyperproliferative skin disorders.

overall_design: RNA profiles of the human head and neck squamous cell carcinoma cell line A253 and the immortalized human keratinocytes (Ker-CT) transfected with siRNA targeting p63 (sip63), SETX (siSETX) or a nonrelevant mRNA (scramble, SCR) were generated by deep sequencing using Illumina Novaseq. We analyzed three biological replicates for each conditions. qRT–PCR validation was performed using SYBR Green assays.

first: Angelo

last: Peschiaroli

first: Francesca

last: De Nicola

first: Veronica

last: Gatti

first: Claudia

last: Fierro


title: Differential Expression of miRNAs in Bovine Skeletal Muscle that Display Varying Degrees of the Dark-cutting Phenotype.

status: Public on Jan 07 2022

city: College Station

first: Dustin

middle: A

last: Therrien

state: Texas

address: 474 Olsen Blvd

department: Animal Science

institute: Texas A&M University

summary: Identify specific genetic regulatory and signaling mechanisms that result in occurrence of “dark cutting meat” from beef carcasses in order to develop novel strategies for reducing incidence

overall_design: RNA-Seq was performed on total RNA that was extracted from muscle biopsies taken from a group of steers that expressed varying degrees of dark cutting in order to detect and profile miRNAs that were potentially associated with the dark-cutting phenotype.

first: Penny

last: Riggs

first: H

middle: R

last: Cross


title: Time-course transcriptome analyses identify salt stress responding mechanisms in Chlamydomonas reinhardtii strain GY-D55

city: Jinan

first: Luoyan

last: Zhang

address: wenhuadong 88

institute: shandong normal university

summary: It is highly necessary to understand the molecular mechanism underlying the salt stress response in green algae, which may contribute to finding the evolutionary cues of abiotic stress response in plants.In the present study, we examined the gene expression pattern in Chlamydomonas reinhardtii GY-D55 cells at different time points, from early stage (2-24 h) to late stage (up to 96 h).

overall_design: mRNA profiles of Chlamydomonas reinhardtii GY-D55 cells at control and NaCl treated different time points: 2,4,8,12,24,48,72 and 96 h.

first: Shoujin

last: Fan


title: Cell type-specific mechanism of Setd1a heterozygosity in schizophrenia pathogenesis

first: Mohamed Nadhir

last: Djekidel

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

overall_design: Refer to individual Series


title: YAP1 Drives Lineage Specification and Patterning in hESC-derived Gastruloids

city: San Diego

first: April

middle: Elizabeth

last: Williams

state: California

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute for Biological Studies

summary: The gastrulation process is controlled by the interplay between morphogenetic signals from BMP, WNT and NODAL pathways. Increasing evidences support an emerging role of the Hippo-YAP signaling in the cell-fate decisions that guide lineage specification in mouse and human Embryonic Stem Cells (hESCs).  However, the contribution of YAP to the process of gastrulation in hESCs remains unknown. Here, we show that YAP1 regulates the specification, size and patterning of the three-germ layers.  Using hESC-derived 2D-micropatterned gastruloids and directed differentiation approaches we show that YAP maintains a semi-active NODAL signaling during gastrulation essential to regulate the exit of pluripotency. In absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei, impeding the exit of pluripotency and the acquisition of the ectodermal gene program. Accordingly, the inhibition of NODAL signaling is sufficient to rescue the gastrulation-defective phenotype of the YAP1 KO hESCs. Our work revealed that Hippo-YAP1 signaling is an important component of the developmental network that coordinate hESC pluripotency and gastrulation.

overall_design: We used a directed differentiation protocol (StemCell Technologies #05230) to induce human embryonic stem cells  (hESC) differentiation towards ectoderm, mesoderm and endoderm lineages in WT and YAP1 KO and performed gene expression profiling using RNA-Seq. We also included controls of undifferentiated hESC. In addition, single-nuclei ATAC-seq  (snATAC) was used to examine the genome-wide chromatin accessibility profile in WT and YAP1 KO hESCs.

first: Servando

last: Giraldez

first: Eleonora

last: Stronati

first: Hui-Ting

last: Hsu

first: Ling

last: Huang

first: Elizabeth

last: Abraham

first: Kathy

last: Jones

first: Conchi

last: Estaras


title: The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells II

city: Montpellier

first: Dominique

last: Helmlinger

address: 1919 route de Mende

department: CRBM

country: France

institute: CNRS

summary: Transcription is essential for cells to respond to signaling cues and involves factors with multiple distinct activities. One such factor, TRRAP, functions as part of two large complexes, SAGA and TIP60, which have essential roles during transcription activation. Structurally, TRRAP belongs to the family PIKKs but is the only member classified as a pseudokinase. Recent studies established that a dedicated HSP90 co-chaperone, the TTT complex, is essential for PIKK maturation and activity. Here we used endogenous auxin-inducible degron alleles to show that the TTT subunit TELO2 promotes TRRAP assembly into SAGA and TIP60 in human colorectal cancer cells (CRC). Transcriptomic analysis revealed that TELO2 contribute to TRRAP regulatory roles in CRC cells, most notably of MYC target genes. Surprisingly, TELO2 and TRRAP depletion also induced the expression of type I interferon genes. Using a combination of nascent RNA, antibody-targeted chromatin profiling (CUT&RUN) and kinetic analyses, we show that TRRAP directly represses the expression of IRF9, which is a master regulator of interferon stimulated genes. We have therefore uncovered a new, unexpected transcriptional repressor role for TRRAP, suggesting a previously unidentified mechanism by which TRRAP may contribute to tumorigenesis.

overall_design: TRRAP occupancy profiling from HCT116 cells edited using CRISPR-Cas9 and treated with either NaOH or auxin were generated by CUT&RUN experiment followed by deep sequencing. Two replicates and an IgG control experiment, to determine experimntal background, were sequenced in one lane of an Illumina HiSeq 3000/4000, with 150bp paired-end reads.

first: Dom

first: Dylane

last: Detilleux

first: Peggy

last: Raynaud


title: Lycopene supplementation in serum-free maturation medium improves bovine in vitro embryo development, quality, and modulates embryonic transcriptomic profile

status: Public on Jan 06 2022

city: Merelbeke

first: Osvaldo

last: Bogado Pascottini

state: Flanders

address: Salisburylaan 133

country: Belgium

institute: Ghent University

summary: Bovine embryos are typically cultured at reduced oxygen tension to lower the impact of oxidative stress on embryo development. However, oocyte in vitro maturation (IVM) is performed at atmospheric oxygen tension since low oxygen during maturation has a negative impact on oocyte developmental competence. Lycopene, a carotenoid, acts as a powerful antioxidant and may protect the oocyte against oxidative stress during maturation at atmospheric oxygen conditions. Here, we assessed the effect of adding 0.2 μM lycopene (antioxidant), 5 μM menadione (pro-oxidant), and their combination on reactive oxygen species (ROS) generation in mature oocytes and subsequent blastocyst development, quality, and transcriptome in a bovine in vitro model. ROS fluorescent intensity in mature oocytes was significantly lower in the lycopene group, and resulting embryos showed a significantly higher blastocyst rate on day 8 and lower apoptotic cell ratio than all other groups. Transcriptomic analysis disclosed a total of 296 differentially expressed genes (Benjamin-Hochberg adjusted P < 0.05 and ≥ 1 log2 fold change) between lycopene and control groups with pathways associated with cellular function, metabolism, DNA repair, and anti-apoptosis upregulated in lycopene. Lycopene supplementation in serum-free maturation medium neutralizes excess ROS during maturation, enhances blastocyst development and quality, and modulates the transcriptomic landscape.

overall_design: We hypothesized that supplementation of antioxidants in a serum-free maturation medium would be effective in obtaining optimal ROS neutralization and maintenance of equilibrium during IVM and embryo development. This study aimed to assess if lycopene supplementation in serum-free maturation medium reduces ROS generation in matured oocytes and improves subsequent embryo development, quality, transcriptomic profile, and whether lycopene supplementation during maturation exerted extended protective actions on vitrified-warmed embryos.

first: Shehu

last: Sidi

first: Daniel

last: Angel-Velez

first: Nima

last: Azari-Dolatabad

first: Krishna

last: Chaitanya Pavani

first: Gretania

last: Residiwati

first: Tim

last: Meese

first: Filip

last: Van Nieuwerburgh

first: Elias

last: Kambai Bawa

first: Ambrose

last: Alikidon Voh

first: Joseph

last: Olusegun Ayo

first: Ann

last: Van Soom


title: The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells

overall_design: mRNA profiles from HCT116 cells edited using CRISPR-Cas9 and treated with either NaOH or auxin were generated by deep sequencing, in triplicate, in one lane of either an Illumina HiSeq 2500 (TELO2-AID) or 4000 (AID-TRRAP), with 1x 50bp single reads.


title: Preparation of the working viruses of B.1.1, Delta and Omicron SARS-CoV-2

city: Minato-ku

first: Kei

last: Sato

address: 4-6-1 Shiroganedai

department: Department of Microbiology and Immunology

institute: The Institute of Medical Science, The University of Tokyo

summary: To investigate the virological properties of SARS-CoV-2 variants, we amplified the clinical isolates of an early pandemic D614G-bearing isolate (B.1.1 lineage, strain TKYE610670; GISAID ID: EPI_ISL_479681), a Delta isolate (B.1.617.2 lineage, strain TKYTK1734; GISAID ID: EPI_ISL_2378732) and an Omicron isolate (BA.1 lineage, strain TY38-873; GISAID ID: EPI_ISL_7418017) and prepared the working viruses.

sample_organism: Severe acute respiratory syndrome coronavirus 2

overall_design:
To investigate the virological properties of SARS-CoV-2 variants, we amplified the clinical isolates of an early pandemic D614G-bearing isolate (B.1.1 lineage, strain TKYE610670; GISAID ID: EPI_ISL_479681), a Delta isolate (B.1.617.2 lineage, strain TKYTK1734; GISAID ID: EPI_ISL_2378732) and an Omicron isolate (BA.1 lineage, strain TY38-873; GISAID ID: EPI_ISL_7418017) using VeroE6/TMPRSS2 cells and prepared the working viruses. At 3 days postinfection, the culture media were harvested and centrifuged, and the supernatants were collected as the working virus. The genome sequences of the working viruses were verified using a viral RNA-sequencing analysis. Viral RNA was extracted using QIAamp viral RNA mini kit (Qiagen, Cat# 52906). The sequencing library for total RNA-sequencing was prepared using NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolabs, MA, Cat# E7530). Paired-end, 76-bp sequencing was performed using MiSeq (Illumina, San Diego, CA) with MiSeq Reagent Kit v3 (Illumina, Cat# MS-102-3001).
contributor: The Genotype to Phenotype Japan (G2P-Japan) Consortium


title: A molecular architecture of the vagal interoceptive system

city: New Haven

first: Le

last: Zhang

state: CT

address: 300 George Street

department: Neurology

institute: Yale University

summary: Interoception, the ability to timely and precisely sense changes inside the body, is critical for survival. Vagal sensory neurons (VSNs) form an important body-to-brain axis, navigating along body’s rostral-caudal axis to their target organs and dive across organ’s surface-lumen axis into appropriate tissue layers to sense stimuli with various physical properties. The brain can discriminate numerous body signals through VSNs, yet the underlying molecular coding strategy remains poorly understood. Here we show that VSNs code visceral organ, tissue layer, and stimulus modality, three key features of an interoceptive signal, in different dimensions. Large-scale single-cell profiling of VSNs from seven major organs, including the lung, heart, stomach, esophagus, duodenum, pancreas, and transverse colon, using multiplexed projection-barcodes reveals a ‘visceral organ’ dimension composed of differentially expressed gene modules coding VSN target organs along body’s rostral-caudal axis. Surprisingly, we discover another ‘tissue layer’ dimension with gene modules coding VSN ending locations along organ’s surface-lumen axis. The three independent feature-coding dimensions together specify many parallel VSN pathways in a combinatorial fashion and facilitate complex VSN projection in the brainstem. Our study thus highlights a novel multidimensional coding architecture of the mammalian vagal interoceptive system for effective signal communication.

overall_design: For each sample of Projection-seq, 10 age and gender-matched wildtype mice were first injected with different SNAP-seq AAVs into seven thoracic and abdominal organs, including the lung, heart, stomach, esophagus, duodenum, pancreas, and transverse colon. Vagal ganglia were harvested and acutely isolated 7 days later. Single cell libraries were prepared using 10x Genomics Chromium Single Cell 3′ Reagent Kit v3 chemistry and sequenced on an Illumina NovaSeq6000 S4 sequencer.

first: Qiancheng

last: Zhao

first: Rui

last: Chang


title: SLAM-seq ML-792

city: Oslo

first: Pierre

last: Chymkowitch

address: Blindernveien 31

department: Biosciences

country: Norway

institute: University of Oslo

summary: Tight control of gene expression networks involved in adipose tissue formation and plasticity is required to adapt to energy needs and environmental cues. However, little is known about the mechanisms that orchestrate the dramatic transcriptional changes leading to adipocyte differentiation. We investigated the regulation of nascent transcription by SUMO during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that SUMO has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of chromatin modifiers and specific transcription factors like PPARg/RXR promotes the transcription of adipogenic genes. Our data demonstrate that the sumoylation pathway coordinates the rewiring of transcriptional networks required for formation of functional adipocytes.

overall_design: Analysis of the nascant transcriptome of differentiating 3T3-L1 cells

first: Xu

first: Tao

last: Ye


title: Non-canonical base modifications of bacterial origin in a eukaryotic genome

city: Woods Hole

first: Irina

last: Arkhipova

state: MA

address: 7 MBL St

department: Josephine Bay Paul Center

institute: Marine Biological Laboratory


title: Non-canonical base modifications of bacterial origin in a eukaryotic genome [MeDIP-seq]

summary: Methylation of cytosines at the 5th carbon position of the aromatic ring has long been regarded as the predominant type of DNA base modification in eukaryotes. Often called “the fifth base”, C5-methylcytosine (5mC) plays an important role in genome defense against mobile genetic elements, and is mostly associated with transcriptional silencing, establishment of the closed chromatin configuration, and repressive histone modifications. Recently, another type of DNA modification, N6-methyladenine (6mA), has been added to the repertoire of modified bases in eukaryotic DNA, and was mostly linked to elevated transcription levels. In prokaryotes, 5mC and 6mA typically constitute components of restriction-modification (R-M) systems, along with N4-methylcytosine (4mC), which so far has been confined to bacteria. Here we report the first case of 4mC occurrence in eukaryotic DNA. We find that bdelloid rotifers, small freshwater invertebrates known for their ability to reproduce clonally and to acquire genes from non-metazoan sources, lack the canonical eukaryotic C5-methyltransferases but instead encode an amino-methyltransferase of bacterial origin, N4CMT, which is present in all bdelloid families separated by tens of millions of years of evolution. The recombinant N4CMT introduces 4mC into genomic DNA in vivo and in vitro. Using SMRT-seq (PRJNA558051), MeDIP-seq, ChIP-seq, and RNA-seq, we examined genome-wide distribution of non-canonical base modifications over annotated genomic features and observed an excess of 4mC in silenced transposable elements and certain tandem repeats, while 6mA tends to associate with transcribed genes and active chromatin. The presence of the chromodomain in N4CMT explains its affinity for repressive histone marks, H3K9me3 and especially H3K27me3. Our results expand the known repertoire of eukaryotic base modifications, shed light on the process of recruitment of methyl groups as epigenetic marks in DNA, and highlight the role of horizontal gene transfer as an important driver of evolutionary innovation in eukaryotes.

overall_design: Examination of 4mC and 6mA DNA methylation with MeDIP-seq, ChIP-seq, and RNA-seq in two Adineta vaga strains (reference and L1).

first: Fernando

last: Rodriguez

last: Yushenova

first: Daniel

last: DiCorpo


title: Non-canonical base modifications of bacterial origin in a eukaryotic genome [ChIP-seq]


title: The role of HK2 in breast cancer metastasis

status: Public on Jan 05 2022

city: Chicago

first: Nissim

last: Hay

state: IL

address: 900 South Ashland Ave

department: BMG

institute: University of Illinois Chicago

summary: Here we study the effect on systemic HK2 deletion in breast cancer

overall_design: Investigating role of HK2 in breast cancer using single cell RNA-seq

first: Catherine

last: Blaha

first: Maxim

middle: V

last: Frolov

first: Alexandre

middle: F

last: Aissa

first: Abul

last: Islam


title: Gene expression changes caused by treatment of A375 cells with A-366 versus DMSO (vehicle)

status: Public on Jan 02 2022

city: New York

first: Yariv

last: Houvras

address: 1300 York Ave

department: Surgery

institute: Weill Cornell Medical College

summary: We use RNAseq to examine gene expression changes in A375 cells after treatment with A-366 versus DMSO (vehicle) of mScarlet tagged H3.3(wildtype) vs. H3.3(K9M)

overall_design: 4 Samples of each treatment

first: Raul

last: Martinez-McFaline


title: The impact of whole glucan particles (WGP) treatment on pancreatic myeloid cells

status: Public on Jan 04 2022

city: Louisville

first: Eric

middle: Christian

last: Rouchka

state: Kentucky

address: 522 East Gray Street

department: Biochemistry and Molecular Genetics

institute: University of Louisville

summary: Despite the remarkable success of immunotherapy in many types of cancer, pancreatic cancer has yet to benefit. Innate immune cells are critical players in antitumor immunosurveillance. Recent studies have revealed that innate immune populations may possess a form of memory, termed trained immunity through transcriptomic, epigenetic, and metabolic reprograming. 1Here, we show that intraperitoneal administration of yeast-derived particulate β-glucan results in 90% of that dose trafficking to the pancreas. This trafficking leads to a robust influx of newly characterized innate immune cells with an inflammatory monocyte/macrophage phenotype into the pancreas in a CCR2 dependent manner. These cells also exhibit a trained immunity phenotype upon exposure to tumor-derived factors and show enhanced phagocytosis and ROS-mediated cytotoxicity against pancreatic tumors. In orthotopic models of pancreatic cancer, mice trained with β-glucan show significantly reduced tumor burden and prolonged survival, which is further enhanced when combined with anti-PD-L1 immunotherapy. Cumulatively, these findings highlight a novel trafficking mechanism of yeast-derived particulate β-glucan that incites a phenotype of trained immunity specifically in the pancreas that can be utilized as an innovative strategy to treat pancreatic cancer.

overall_design: mouse pancreatic myeloid cells in triplicate treated with whole glucan particles (WGP) and untreated mouse pancreatic myeloid cells in triplicate

first: Anne

middle: E

last: Geller

first: Rejeena

last: Shrestha

first: Chuanlin

last: Ding

first: Hixun

last: Guo

first: Kalina

last: Andreeva

first: Julia

middle: H

last: Chariker

first: Matthew

last: Woeste

first: Xiaolong

last: Hu

first: Mingqqian

last: Zhou

first: David

last: Tieri

first: Corey

last: Watson

first: Robert

last: Mitchell

first: Huang-ge

first: Yan

last: Li

middle: C

last: Martin II

first: Jun

last: Yan


title: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

city: Vellore

first: Nithin Sam

last: R

state: TamilNadu

address: Bagayam

department: Center for Stem Cell Research

country: India

institute: Christian Medical College

summary: CRISPR-Cas base editors are preferred tool for genome editing as they generate desired editing without any double strand break in the genome, as double stand break is detrimental to the cells. In our study we have demonstrated the significance of base editors in editing the highly homologous HBG promoter (HBG1 and HBG2) region to introduce novel HPFH-like mutation to elevate HbF for therapeutical applications. Previous studies revealed that the base editors can cause unintended Cas-independent edits at transcriptome level. To validate off-target at RNA level, we performed a transcriptome wide analysis. The frequency of unintended edits in the HUDEP-2 stable cell lines expressing the base editors with the gRNA were not significant compared to the control. We determined the ABE mediated A to I conversion and CBE mediated C to U conversion across the base edited samples. The RNA off-target analysis was carried out with the help of REDItools v 2 tool. This data suggests that despite high on-target editing in DNA, the Cas-independent RNA off-target were not at detectable range compared to control. The differential expression of 34 selected genes which necessitate globin regulation were compared between the unedited HUDEP WT, CBE control, ABE control, and edited ABE (with gRNA 2/11) and edited CBE (with gRNA 2/11). We observed that there is no significant differential gene expression between the edited and control cells except the gamma and delta globin genes. These results suggest that base editors are preferred tools to edit highly homologous HBG promoter region to created HPFH-like mutations inducing HbF levels without causing double strand breaks, larger deletions and no significant RNA off-targets which are detrimental to the gene edited cells.

overall_design: 14 samples

first: Nithin

last: Ravi

first: Stacia

middle: K

last: Wyman

first: Kumarasamypet

middle: M

last: Mohankumar


title: Non-canonical base modifications of bacterial origin in a eukaryotic genome [RNA-seq]

first: Irina

last: Arkhipova

first: Fernando

last: Rodriguez

last: Yushenova

first: Daniel

last: DiCorpo


title: Single Cell sequencing analysis of Bone Marrow samples

city: Houston

first: Ganiraju

last: Manyam

state: TX

address: 1515 Holcombe Blvd

institute: UT MD Anderson Cancer Center

summary: Study of hematopoietic stem and progenitor cell (HSPC) hierarchies in bone marrow cells from myelodysplastic syndromes through the transcriptional analysis of single cells in distinct differentiation states

overall_design:
Lin-CD34+ HSPCs were FACS-purified from magnetically CD34-enriched mononuclear cells isolated by density gradient from bone marrow aspirates. A total of 3000 cells were sorted and input onto the Chromium Single Cell A Chip Kit (10x Genomics, Pleasanton, CA), in which single cells were lysed and barcoded for reverse-transcription. The pooled single-stranded, barcoded cDNA was amplified and fragmented for library preparation. During library preparation, appropriate sequence primer sites and adapters were added for sequencing on a NextSeq 500 sequencer (Illumina).
------------------------------------------------
**The submitter declares that the "Raw data to be uploaded to dbGaP" due to patient privacy concerns.**

first: Irene

last: Ganan-Gomez

first: Ma

last: Feiyang

first: Simona

last: Colla


title: Genome-wide epigenetic changes of leukemia cell lines in reponse to byrostatin treatment

city: Seattle

first: Rimas

last: Orentas

state: Washington

address: 1100 Olive Way NE

department: BTCCCR

institute: Seattle Children's Research Institute

summary: We report the application of cut&tag technology for high-throughput profiling of histone modifications in leuemia cells in reponse to bryostatin treatment. We generated genome-wide chromatin-state landscape of NALM6, REH and Raji cells.  Dual- and tri-methylation of lysine 4 on histone H3 protein indicate genes that are actively expressed/regulated. Tri-methylation of lysine 27 on histone H3 protein marks genes that are negatively regulated or suppressed for expressiom. We compared differential peaks for cells treated with or without bryostatin to demonstrate genes responding to bryostatin treatment on epigenetic level.

overall_design: Examination of histone modifications (H3K4me2,  H3K4me3 and H3K27me2)  in NALM6, REH and Raji cells treated with or without Bryostatin, using cut&tag for epigentic landscape profiling

first: Yue

last: Zhang


title: Next Generation Sequencing analysis to compare expression patterns of myeloid progenitors in healthy donors and MDS patients

summary: Transcriptional changes occurring in the CMPs and GMPs of untreated "CMP pattern" and "GMP pattern" MDS patients were compared with the transcriptional profile of CMPs and GMPs from healthy donors

overall_design:
Lin-CD34+CD38+CD123+CD45RA- CMPs and Lin-CD34+CD38+CD123+CD45RA+ GMPs were FACS-purified from mononuclear cells previoulsy isolated by density gradient from fresh bone marrow aspirates and magnetically-enriched in CD34+ cells . Total RNA was purified using the PicoPure RNA Isolation Kit (Thermo Fisher Scientific). Quality checks were performed in a 2100 Expert bioanalyzer (Agilent). cDNA libraries were prepared using the Ovation Single Cell RNA-Seq System (NuGEN Technologies). RNA-seq was performed with the HiSeq 4000 System (Illumina), generating 76-base pair reads.
***Submitter states that raw data will be submitted to dbGaP for controlled access***

first: Jing

last: Wang


title: Waves of sumoylation support transcription dynamics during adipocyte differentiation [ChIP-seq]

first: Pierre

last: Chymkowitch

summary: Tight control of gene expression networks involved in adipose tissue formation and plasticity is required to adapt to energy needs and environmental cues. However, little is known about the mechanisms that orchestrate the dramatic transcriptional changes leading to adipocyte differentiation. We investigated the regulation of nascent transcription by SUMO during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that SUMO has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing sumoylome dynamics in differentiating adipocytes by mass spectrometry we found that SUMOylation of specific transcription factors like PPAR/RXR and chromatin modifiers promotes the transcription of adipogenic genes. Our data demonstrate that the sumoylation pathway helps coordinates the rewiring of transcriptional networks required for formation of functional adipocytes.

overall_design: Analysis of Sumo-2/3 on the chromatin of differentiating adipocytes

first: Xu

last: Zhao


title: Epigenetic changes of Raji Epstein–Barr viral genome in reponse to byrostatin treatment

summary: We report the application of cut&tag technology for high-throughput profiling of histone modifications Raji Epstein–Barr viral genome in reponse to bryostatin treatment. Dual- and tri-methylation of lysine 4 on histone H3 protein indicate genes that are actively expressed/regulated. Tri-methylation of lysine 27 on histone H3 protein marks genes that are negatively regulated or suppressed for expression.

overall_design: Examination of histone modifications (H3K4me2,  H3K4me3 and H3K27me2) of Epstein–Barr viral genome in Raji cells treated with or without Bryostatin, using cut&tag for epigentic landscape profiling


title: Waves of sumoylation support transcription dynamics during adipocyte differentiation

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

overall_design: Refer to individual Series


title: Comparison of changes in mRNA expression profiles in arteries and heart valves in rabbit models of dietary lipid-triggered cardiovascular diseases

city: Liege

first: Zheshen

last: Jiang

state: Liege

address: Avenue de l'hopital, 1

department: GIGA

institute: University of Liege

summary: We report RNA-seq data obtained on dissected aorta and four heart valves from rabbits fed with chow diet (n=7), cholesterol-rich diet (n=6) or lard-rich diet (n=7). Rabbits that received cardiac irradiation combined with the cholesterol-rich diet were also included (n=7).

overall_design: mRNA expression was analyzed in 5 tissues for all rabbit groups.

first: Nathalie

last: Donis

first: Cécile

last: Oury


title: Non-redundancy of IL-1α and IL-1β in infection is defined by distinct functions and spatially restricted expression

city: Wien

first: Kevin

last: Eislmayr

address: Dr.-Bohr-Gasse 9/4

country: Austria

institute: University of Vienna

summary: Analysis of the spleen and liver transcriptom of Il1a-/-, Il1b-/- and WT mice 24 h after infection with Streptococcus pyogenes or from untreated animals. The experiment revealed a spatial restriction and non-redundant functions of Il1a and Il1b.

overall_design: RNA was isolated 24 h after infection or PBS treatment from the liver and spleen of l1a-/-, Il1b-/- and WT mice (4 per genotype). Sequencing was performed in two rounds. In the first one, RNA from the liver and spleen of for 24 h infected mice were processed and multiplexed with the QuantSeq 3’ mRNA-Seq Library Prep Kit and the mixed multiplexed samples were sequenced on two lanes on an Illumina HiSeqV4 SR50 platform with a read length of 50 bases. For the second one, only the RNA from the liver of uninfected Il1a-/-, Il1b-/- and WT mice (4 per genotype) were used and multiplexed samples were sequeneced on one lane.

first: Annika

last: Bestehorn

first: Luisa

last: Morelli

first: Martina

last: Borroni

first: Pavel

last: Kovarik


title: RNAseq analysis of skeletal muscle transcriptomes from wild-type and LONP1 skeletal muscle specific knockout (LONP1-MKO) mice

city: Nanjing

first: Zhenji

last: Gan

address: 12 Xuefu Road

department: Model Animal Research Center of Nanjing University

institute: Nanjing University

summary: Mitochondria serve diverse functions and are essential organelles that require continuous surveillance to maintaintheir integrity and function. LONP1 is an evolutionarily conservedserine peptidase that safeguards mitochondrial protein quality from yeast to human.To investigatethe physiological role of LONP1-mediated mitochondrial quality-control in skeletal musclein vivo, we generated skeletal muscle-specificLonp1-knockout mice (referred to as LONP1 MKO). We performedtranscriptome analysis by whole-genome gene expression profiling experiments in gastrocnemius (GC) muscle from both wild-type (WT) and LONP1-MKO mice. Knockout of LONP1 in skeletal muscle resulted in deregulation of 457 genes in 2-week-old mice and of 1922 genes in 6-week-old mice. Gene ontology analysis revealed that LONP1 deficiency triggers unfolded protein response (UPR) in skeletal muscle.Moreover, GSEA analysis of transcriptomic datain 6-week-old micefurther revealed that genes deregulated by LONP1 deficiency were significantly enriched during aging.Together, the transcriptional profiling results suggest a critical role of LONP1 in regulating skeletal muscle metabolism and health.

overall_design: Gastrocnemius muscle mRNA profiles of 2-week-old and 6-week-old WT and LONP1-MKO mice were generated by deep sequencing, in duplicate, using Illumina HiSeq 4000.

first: Zhisheng

last: Xu

first: Qiqi

last: Guo


title: The Induction of Trained Immunity in the Pancreas Incites Anti-tumor Activity to Control Pancreatic Cancer Progression

first: Eric

last: Rouchka

summary: Following treatment with WGP, we have observed an influx of immune cell populations into the pancreas over time. These cells have increased anti-tumor activities, which results in protection against pancreatic cancer growth. The study objective is to better understand the cell types involved in this process and the temporal nature of this influx. It is known that the immune influx peaks at day 7, so we are interested in the changes in the immune populations of the pancreas 3 days and 7 days following WGP treatment. To gain a more in depth analysis of the cell populations present in the pancreas after WGP treatment, a single cell RNA sequencing (scRNA-Seq) was performed on sorted CD45+ cells from PBS treated mice and mice treated with WGP 3 and 7 days prior.

overall_design: Mice were treated with PBS or WGP 7 days prior and 3 days prior to euthanization. The pancreas of these mice were harvested, digested, and stained with Viability dye and an anti-CD45 fluorescent antibody. Cells were then run through a cell sorter, where only live CD45+ cells were acquired. Cells were washed with 400ug/mL BSA 2x and then counted.

first: Anne

last: Geller

first: Rejeena

last: Shrestha

first: Matthew

last: Woeste

first: Xiaolong

last: Hu

first: Chuanlin

last: Ding

first: Hixun

first: Kalina

last: Andreeva

first: Julia

last: Chariker

first: Mingqqian

last: Zhou

first: David

last: Tieri

first: Corey

middle: T

last: Watson

first: Robert

last: Mitchell

first: Huang-ge

first: Yan

last: Li

last: Martin II

first: Jun

last: Yan


title: Single cell RNAseq analysis in embryonic spinal cords to profile transcriptomes in control and Jmjd3cKO motor neurons.

city: Buffalo

first: Soo-Kyung

last: Lee

state: NY

address: 109 Cooke Hall

department: Department of Biological Sciences

institute: University at Buffalo, The State University of New York

summary: To characterize Jmjd3 function in motor neuron development, we isolated GFP+ cells derived from Olig2 expressing progenitor cells that included motor neuron population and then employed single cell RNAseq. We found some novel motor neuron subtypes in addition to well-known motor columns, and specific genes in each subtype by unbiased and comprehensive analysis. Moreover, by comparing control and Jmjd3cKO motor neuron lineage cells, we found many downregulated and upregulated genes affected by Jmjd3 loss of function. Single cell RNAseq provided very powerful tool to analyze different regulated genes in each subtype rather than overall motor neuron groups.

overall_design: We used Olig2-Cre and mTmG mouse lines to label Olig2-expressing lineage cells which mainly include motor neurons at E12.5 stage and then employed single cell RNAseq of GFP+ cells to profile transcriptomes in control and Jmjd3cKO mice.

first: Wenxian

last: Wang

first: Hyeyoung

last: Cho


title: Accelerated flexor tendon repair in superhealer mice reflects alterations in TGFB1 regulated expression programs linked to inflammatory, fibrosis and cell cycle control

city: Rochester

first: Hani

last: Awad

address: 601 Elmwood Avenue

department: Biomedical Engineering

institute: University of Rochester

summary: Purpose: The goal of this study is to compare transcriptional profiles of flexor tendon healing in wild-type (WT, C57Bl/6J) to superhealer (MRL/MpJ) miceto gain insights in the biological drivers of the tendon injury response between the C57 and MRL mice. Methods: RNA was isolated from patially lacerated or uninjured flexor tendon 7 days post-injury. Results: Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons.  Conclusions: There is altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.

overall_design: Tendon mRNA profiles of 7 days post-injury with C57Bl/6J and MRL/MpJ mice

first: Jacob

last: Kallenbach

first: Margaret

last: Freeberg

last: Abplanalp

first: Jacquelyn

last: Myers

first: John

last: Ashton

first: Alayna

last: Loiselle

first: Mark

last: Buckley

first: Andre

middle: J

last: van Wijnen


title: Single-cell transcriptomics of adult gd T-cell lineage commitment highlights its TCR-instructive nature I

status: Public on Jan 01 2022

city: Marseille

first: Delphine

last: Potier

address: 27 boulevard Lei Roure

institute: CRCM

summary: Single cell RNA-seq study of T lymphocyte differentiation WT and LAT deficient mice in order to deciphy gd T-cell lineage commitment.

overall_design:
Single-cell RNA-seq analysis of the T cell stages straddling the developmental bifurcation leading to αβ and γδ T cells in adult thymus. Thymocyte subsets encompassing such bifurcation were isolated from 6- to 8-week-old mice using established cell surface markers and subjected to single cell RNA sequencing. Double negative cells (DN, i.e. CD8– and CD4–) cells were sorted into 5 subsets corresponding to DN3a bi-potent αβ/γδ precursors which rearrange the genes coding for the TCRγ, TCRδ, and TCRβ chains, post-selected αβ precursors (DN3b TCRγδ– and DN4 TCRγδ–; in short DN3b γδ– and DN4 γδ–), post-selected γδ precursors (DN3b TCRγδ+; in short DN3b γδ+), and immature γδ T cells in the process of functional diversification (DN4 TCRγδ+; in short DN4 γδ+). In addition, to precise the role of γδ TCR signals in the earliest phases of γδ lineage commitment and specification two subsets of DN3 cells expressing signaling-defective γδ TCR from TCRd-H2BEGFP Lat-deficient mice were isolated : DN3 γδint and DN3 γδ–.
Each subset was spike-in using 500 splenic B cells in order to check dataset merging quality.

first: Sara

last: Scaramuzzino

first: Robin

last: Ordioni

first: Pierre

last: Grenot

first: Dominique

last: Payet-Bornet

first: Hervé

last: Luche

first: Bernard

last: Malissen


title: Development and Validation of a Prognostic and Predictive 32-Gene Signature for Gastric Cancer

city: Jacksonville

first: Tae Hyun

last: Hwang

state: FL

address: 4500 San Pablo Rd S.

institute: Mayo Clinic

summary: Genomic profiling can provide prognostic and predictive information to guide clinical care. Biomarkers that reliably predict patient response to chemotherapy and immune checkpoint inhibition in gastric cancer are lacking. In this retrospective analysis, we use our machine learning algorithm NTriPath [Park, Sunho et al. “An integrative somatic mutation analysis to identify pathways linked with survival outcomes across 19 cancer types.” Bioinformatics (2016): 1643-51. doi:10.1093/bioinformatics/btv692] to identify a gastric-cancer specific 32-gene signature. Using unsupervised clustering on expression levels of these 32 genes in tumors from 567 patients, we identify four molecular subtypes that are prognostic for survival. We then built a support vector machine with linear kernel to generate a risk score that is prognostic for five-year overall survival and validate the risk score using three independent datasets. We also find that the molecular subtypes predict response to adjuvant 5-fluorouracil and platinum therapy after gastrectomy and to immune checkpoint inhibitors in patients with metastatic or recurrent disease. In sum, we show that the 32-gene signature is a promising prognostic and predictive biomarker to guide the clinical care of gastric cancer patients and should be validated in a prospective manner.

overall_design: We generated microarray-based mRNA expression profiles from pre-treatment tumor samples from 567 patients who underwent resection at Yonsei University. This series includes a subset of the dataset (135 samples) and the rest of the dataset has been available in series GSE84437 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84437). For more detailed information, please refer to the individual samples.

first: Jae-Ho

last: Cheong

first: Sam

last: Wang

first: Sunho

last: Park

first: Matthew

last: Porembka

first: Alana

middle: L

last: Christie

first: Hyunki

last: Kim

first: Hong

last: Zhu

first: Woo

last: Hyung

first: Sung

last: Noh

first: Bo

last: Hu

first: Changjin

last: Hong

middle: D

last: Karalis

first: In-Ho

last: Lee

first: Tae


title: Nuclear export restricts Gdown1 to a mitotic function

status: Public on Jan 03 2022

city: Iowa City

first: David

last: Price

state: IA

address: 431 Newton Road

department: Department of Biochemistry and Molecular Biology

institute: University of Iowa

summary: Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit called Gdown1, encoded by the POLR2M gene. Previous in vitro and structural studies have established that Gdown1 potently inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in multiple human cell lines. Remarkably, acute depletion of Gdown1 led to minimal direct effects on gene expression and transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is directly regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis prior to complete breakdown of the nuclear envelope, whereupon association with Pol II becomes possible. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with a growth defect and evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 association with Pol II elongation complexes in vitro modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF, and P-TEFb. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for genome integrity.

overall_design: Multiple different HeLa cells enabling the study of loss of function of Gdown1 were developed as described in the methods. To address changes in gene expression linked to Gdown1 loss, total RNA parental and Gdown1 KO #1 and 2 HeLa cell lines was isolated and prepared for sequencing as described in the paper. Samples were prepared in biological triplicate. To assay the effects of Gdown1 loss on nascent transcription, nuclei were isolated from asynchronous and mitotic Parental HeLa cells and Gdown1 KO #3 HeLa cells, and PRO-Seq was performed as described in the methods. PRO-Seq libraries were prepared in biological duplicate. To assay the direct effects of Gdown1 depletion on transcription, nuclei were isolated from Parental HeLa cells and HA-FKBP-Gdown1 HeLa cells treated for 4 h with DMSO or 400 nM dTAGV-1. Libraries were prepared in biological duplicate. To assay Gdown1 occupancy of chromatin, ChIP-Seq for Gdown1 was carried out as described in the text from HA-FKBP-Gdown1 HeLa cells, using an HA antibody. As a background control, the HA antibody was used for ChIP from unmodified Parental HeLa cells. ChIP-Seq was performed in biological duplicate. Gdown1 ChIP signals were compared to a single Pol II ChIP-Seq dataset, performed as described in the text with an antibody identifying POLR2A N-terminus. ChIP-Seq datasets for H3K4me3 and H3K4me1, which were utilized for the desgination of enhancers, were carried out as described. Previously published HeLa NasCap data were utilized in this study, and are accessible at NCBI GEO ####, SRA ####. Processed data are provided as normalized bw files and scaling factors are accessible in the supplemental data file.

first: Christopher

middle: B

last: Ball

first: Mrutyunjaya

last: Parida


title: A b-catenin-TCF-sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

city: Philadelphia

first: Scott

last: Waldman

state: PA

address: 1020 locus st

institute: Thomas Jefferson University

summary:
Background & Aims. Sporadic colorectal cancers arise from mutations in APC, producing oncogenic β-catenin/TCF-dependent transcriptional reprogramming. The tumor suppressor axis regulated by the intestinal epithelial receptor, GUCY2C, is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by β-catenin/TCF signaling.
Methods. We performed RNA-seq analysis of four unique conditional human colon cancer cell models of β-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, ChIP-seq, CRISPR Cas9 knockout, and CRISPR epigenome editing, which were cross-validated with human tissue ChIP-seq datasets, to identify functional gene enhancers mediating GUCY2C ligand loss.
Results. RNA-seq analyses reveal the GUCY2C hormones as two of the most sensitive targets of β-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant β-catenin/TCF signaling.
Conclusions. These studies reveal novel DNA elements regulating co-repression of GUCY2C ligand transcription by β-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could re-establish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUYC2C axis and oppose transformation.

overall_design: Overall Design: Samples are from four human colorectal cancer cell lines expressing stable transgenes enabling inducible silencing of Wnt signaling. RNAseq was performed on all cell lines, with or without the inducing agent, in triplicate (24 samples). RNA Polymerase II- and H3K27ac-ChIPseq was performed on one cell line, with or without the inducing agent, in duplicate (8 experimental samples and 2 input samples).

first: Jeffrey

last: Rappaport

first: Adam

last: Ertel

first: Paolo

last: Fortina


title: Distinct catalytic-dependent or -independent activities of DNMT1 in mouse embryonic stem cells

city: Toyonaka

first: Takamasa

last: Ito

state: Osaka

address: 1-1 Machikaneyama

department: Biological Science

institute: Osaka University

summary: DNMT1 maintains DNA methylation profile and regulates gene expression. Although DNMT1 interacts with other proteins that regulate gene expressions, it remains unclear how the protein-protein interaction or the catalytic activity of DNMT1 itself affect gene expression and DNA methylation pattern. In this study, immunofluorescence staining  showed abnormal DNA methylation pattern  in chromosome arms of DNMT1-deficient (1KO) mouse embryonic stem cells (ESCs), and we hypothesized that DNMT1 helps methylation activity of DNMT3. To validate this hypothesis, we generate transgenic 1KO ESCs expressing catalytic inactive DNMT1 (1KO+1CI).  Here, we used RNA-seq to analyze the effect of catalytic-dependent or -independent DNMT1 activity on gene expression in ESCs or epiblast-like cells (EpiLCs) induction.

overall_design: RNA-seq was performed on WT and 1KO, 1KO+1CI  ESCs in their undifferentiated state (d0) and during the induction of EpiLCs differentiation (d2 and d4).

first: T

first: L

last: Lefebvre

first: M

last: Tada


title: Impact of NFE2L1 knockout on gene expression

city: Stanford

first: Giovanni

middle: Charles

last: Forcina

address: 327 Campus Drive

department: Biology

institute: Stanford University

summary: These files represent DMSO and bortezomib (200 nM, 4 h) treated Control (B2) and NFE2L1 KO1 (B4) and NFE2L1 KO2 (B6) A549 cells.

overall_design: Two replicates of samples treated with DMSO or bortezomib (btz) were collected

last: Dixon