title: RNA-seq on primary human kidney epithelial cells with PPFIBP1 or PLEKHA1 knockdown

city: New Haven

first: Haikuo

last: Li

state: CT

address: 55 Prospect St

institute: Washington University in St. Louis

summary: Here we investigated the transcriptomic changes induced by PPFIBP1 or PLEKHA1 siRNA treatments on human Primary Renal Proximal Tubule Epithelial Cells (RPTECs) with RNA-seq.

overall_design: There are a total of 9 samples derived from 3 groups with 3 replicates per group. sint: RPTECs treated with non-targeting control siRNA (NTsiRNA). sipp: RPTECs treated with PPFIBP1 siRNA. sipl: RPTECs treated with PLEKHA1 siRNA. Libraries were generated with the ribosomal RNA removal method and sequenced with the NovaSeq 6000 S4 platform (2×150 bp) at a target of 30 million reads per library.

first: Benjamin

middle: D

last: Humphreys


title: Multimodal stimulation screens reveal unique and shared regulators limiting T-cell fitness

status: Public on Feb 15 2024

city: Amsterdam

first: Chun-Pu

last: Lin

address: Plesmanlaan 121

department: Molecular Oncology and Immunology

country: Netherlands

institute: NKI-AvL

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Multimodal stimulation screens reveal unique and shared regulators limiting T-cell fitness [Icam1]

summary: It has not been systematically studied whether T-cell fitness regulators uniquely or broadly contribute to specific traits limiting antitumor activity. We performed genome-scale CRISPR/Cas9 knockout screens in primary CD8 T-cells to uncover genes negatively impacting on their fitness upon three modes of stimulation: (1) intense stimulation, causing activation-induced cell death (AICD); (2) acute stimulation, triggering T-cell expansion; (3) chronic stimulation, resulting in dysfunction. Besides established regulators, we uncovered several genes controlling T-cell fitness either specifically or commonly under various stimulation modalities. Ablation of Dap5, ranking highly in all three screens, increased translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T-cell clustering amplified T-cell expansion and effector functions after both acute and intense stimulation. Finally, inactivation of Ctbp1 induced T-cell persistence exclusively upon chronic stimulation. Our results functionally annotate T-cell fitness regulators based on their unique or shared contribution to traits limiting T-cell antitumor activity.

overall_design: Total RNA was isolated in parallel from resting and 24-hour antiCD3-activated Ctrl or Icam1-depleted OT-I/Cas9 T cells. RNA-Seq libraries were prepared after polyA-bead enrichment of mRNAs and sequenced on a NextSeq 550 using High Output Kit v2.0 (Illumina Inc.).

first: Astrid

last: Alflen

first: Pierre

last: Levy

first: Oscar

last: Krijgsman

first: Daniel

middle: S

last: Peeper


title: Multimodal stimulation screens reveal unique and shared regulators limiting T-cell fitness [Ctbp1]

overall_design: Total RNA were isolated in parallel from pure FACS-sorted 3w antigen-matching tumor stimulated Ctrl or Ctbp1-depleted OT-I/Cas9 T cells. RNA-Seq libraries were prepared after polyA-bead enrichment of mRNAs and sequenced on a Illumina HiSeq 2500 machine.

first: Joleen

middle: J

last: Traets


title: Identification of spatially-resolved markers of malignant transformation in Intraductal Papillary Mucinous Neoplasms. [Visium]

city: Rome

first: Antonio

last: Agostini

address: Largo Francesco Vito, 1

country: Italy

institute: Università Cattolica del Sacro Cuore

summary: Spatial transcriptomics characterization of IPMN samples by Visium Gene Expression FFPE

overall_design: We performed Visium spatial transcriptomics  in  TMA cohorts of IPMN samples grouped by morphology and degree of dysplasia :   18 samples from 14 patients: 4 low-grade IPMN (3 low-grade dysplasia (LGD), 1 intermediate dysplasia/Borderline), 9 high-grade IPMN characterized by a high-grade of dysplasia (HGD), including 4 invasive IPMN with associated PDAC, and 1 PDAC-associated normal duct).

first: Carmine

last: Carbone


title: Single-cell multiome sequencing of human kidney anatomical regions with SHARE-seq

country: USA

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We developed an optimized, low-cost, split-pool barcoding-based multimodal profiling protocol based upon SHARE-seq (concurrent single-cell ATAC/RNA-seq). With SHARE-seq, we profiled human kidney samples from multiple different anatomical regions. Therefore, we develop a large-scale multimodal single-cell atlas for 3D anatomy of the human kidney.

overall_design: We profiled 48 human kidney samples from kidney cortex, medulla, papilla, ureter and renal artery. Sequencing of the SHARE-seq libraries were done on two Novaseq-S4 flowcells and one Novaseq-S2 flowcell.


title: Identification of spatially-resolved markers of malignant transformation in Intraductal Papillary Mucinous Neoplasms. [GeoMx]

summary: Spatial transcriptomics characterization of IPMN samples by GeoMx

overall_design: 80 IPMN  samples were analyzed by Nanostring GeoMx using the Whole Transcriptome Atlas Kit


title: Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells

status: Public on Feb 14 2024

city: Guangzhou

first: Yuting

last: Fang

state: Guangdong, China

address: No. 1023-1063 Shatai South Road, Baiyun District

country: China

institute: School of Public Health, Southern Medical University

summary: Attaching/effacing(A/E) pathogens induce DNA damage and colorectal cancer by injecting effector proteins into host cells via the type III secretion system (T3SS). EspF is one of the T3SS-dependent effector proteins exclusive to A/E pathogens, which include enterohemorrhagicEscherichia coli(EHEC). The role of EspF in the induction of double-strand breaks (DSBs) and the phosphorylation of the repair protein SMC1 has been demonstrated previously. However, the process of damage accumulation and DSB formation has remained enigmatic, and the damage response is not well understood. Here, we first showed a compensatory increase in the mismatch repair (MMR) proteins MutS homolog 2 (MSH2) and MSH6, as well as poly(ADP-ribose) polymerase 1 (PARP1), followed by a dramatic decrease, threatening cell survival in the presence of EspF. Flow cytometry revealed that EspF arrested the cell cycle at the G2/M phase to facilitate DNA repair. Subsequently, 8-oxoguanine (8-oxoG) lesions, a marker of oxidative damage, were assayed by ELISA and immunofluorescence, which revealed the accumulation of 8-oxoG from the cytosol to the nucleus. Furthermore, the status of single-stranded DNA (ssDNA) and DSBs was confirmed.  We observed that EspF accelerated the course of DNA lesions, including 8-oxoG and unrepaired ssDNA, which were converted into DSBs; this was accompanied by the phosphorylation of Replication protein A 32 (RPA32) in repair-defective cells. Collectively, these findings reveal that EspF triggers various types of oxidative DNA lesions with impairment of the DNA damage response and may result in genomic instability and cell death, offering novel insight into the tumorigenic potential of EspF.

overall_design: To investigate the funtion of EspF in EHEC infection, we performed gene expression profiling analysis using data obtained from RNA-seq of Caco-2 cells after exposure to EHEC and ΔespF strains and compared gene expression between two groups.

first: Muqing

last: Fu

first: Bao

last: Zhang

first: Chengsong

last: Wan


title: Multimodal stimulation screens reveal unique and shared regulators limiting T-cell fitness [Dap5]

overall_design: Total RNA and Polysomal RNA were isolated in parallel from resting and 24-hour antiCD3-activated Ctrl or Dap5-depleted OT-I/Cas9 T cells. RNA-Seq libraries were prepared after polyA-bead enrichment of mRNAs and sequenced on a Illumina HiSeq 2500 machine.

first: Georgi

last: Apriamashvili

first: Alex

last: van Vliet

first: Yuval

last: Malka

first: Ferhat

last: Alkan


title: Knockdown of NCAPD3 inhibits the tumorigenesis of non-small cell lung cancer by regulation of the PI3K/Akt pathway

city: Fuzhou

first: Xiangqi

last: Chen

address: NO.29 of Xinquan Road, Gulou District

institute: Fujian Medical University Union Hospital

summary: Background: Accumulating evidence indicates that aberrant non-SMC condensin II complex subunit D3 (NCAPD3) is associated with carcinogenesis of various cancers. Nevertheless, the biological role of NCAPD3 in the pathogenesis of non-small cell lung cancer (NSCLC) remains unclear.Methods: Immunohistochemistry and Western blot were performed to assess NCAPD3 expression in NSCLC tissues and cell lines. The ability of cell proliferation, invasion, and migration was evaluated by CCK-8 assays, EdU assays, Transwell assays, and scratch wound healing assays. Flow cytometry was performed to verify the cell cycle and apoptosis. RNA-sequence and rescue experiment were performed to reveal the underlying mechanisms.Results: The results showed that the expression of NCAPD3 was significantly elevated in NSCLC tissues. In NSCLC patients, high NCAPD3 expression was substantially associated with a worse prognosis. Functionally, knockdown of NCAPD3 resulted in cell apoptosis and cell cycle arrest in NSCLC cells as well as a significant inhibition of proliferation, invasion, and migration. Furthermore, RNA-sequencing analysis suggested that NCAPD3 contributes to NSCLC cancer carcinogenesis by regulating PI3K/Akt/FOXO4 pathway. Insulin-like growth factors-1 (IGF-1), a PI3K/Akt signaling activator, could reverse NCAPD3 silence-mediated proliferation inhibition and apoptosis in NSCLC cells.Conclusion: NCAPD3 suppresses apoptosis and promotes cell proliferation via the PI3K/Akt/FOXO4 signaling pathway, suggesting a potential use for NCAPD3 inhibitors as NSCLC therapeutics.

overall_design: To investigate the role of NCAPD3 in regulating the progression of non-small cell lung cancer, we established A549 and SPCA-1 cell lines with target genes knocked down by shRNA

first: Fan

last: Yang

first: Yunfeng

last: Zheng

first: Qiong

last: Luo

first: Suyun


title: TBXT dose sensitivity and the decoupling of mesoderm specification from EMT progression in 2D human gastruloids

city: San Francisco

first: Emily

last: Bulger

state: CA

address: 1650 Owens St.

institute: Gladstone Institutes/UCSF

summary: In the nascent mesoderm, levels of Brachyury (TBXT) expression must be precisely regulated to ensure cells exit the primitive streak and pattern the anterior-posterior axis, but how this varying dosage informs morphogenesis is not well understood.  In this study, we define the transcriptional consequences of TBXT dose during early gastrulation using human induced pluripotent stem cell (hiPSC)-based models of early gastrulation and mesoderm differentiation. Multiomic single-nucleus RNA and single-nucleus ATAC sequencing reveal that TBXT is required for the temporal progression of epithelial-to-mesenchymal transition (EMT) in a dose-dependent manner and that this transition occurs independently of the acquisition of mesodermal identity. These results demonstrate that EMT can be decoupled from mesoderm development in the early gastrula and shed light on the mechanisms underlying human embryogenesis.

overall_design: 2D gastruloids derived from WT, TBXT-Het, or TBXT-KO human iPSCs were isolated after 48hrs of BMP4 treatment for multiomic snRNA-seq and snATAC-seq. Nuclei from two bioreplicates of about 288 2D gastruloids per genotype per replicate were sequenced.

first: Bulger

last: Emily

first: Vasic

last: Ivana

first: Libby

last: Ashley

first: McDevitt

last: Todd

first: Bruneau

last: Benoit


title: CDX2 dose-dependently influences the GRN underlying vascularization in human extraembryonic mesoderm-like cells

summary: Proper regulation of gene dosage is critical for the development of the early embryo and the extraembryonic tissues that support it. Specifically, loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure that is critical for nutrient delivery and waste removal in the early embryo. In this study, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By generating an allelic series for CDX2 in human induced pluripotent stem cells consisting of WT, heterozygous, and null CDX2 genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to multiomic single nucleus RNA and ATAC sequencing, we identify several genes regulating cytoskeletal integrity, adhesiveness, and polarity of the extraembryonic mesoderm and in a dose-dependent manner, including CDH1 and WNT5B. Despite these dose-dependent gene expression patterns, snATAC-seq reveals that heterozygous CDX2 expression is capable of inducing a WT-like chromatin accessibility profile, suggesting accessibility is not sufficient to drive gene expression when the CDX2 dose is reduced. Finally, because the loss of CDX2 or TBXT phenocopy one another in vivo, we compare differentially expressed genes in our CDX2 knock-out model to those from TBXT knock-out hiPSCs differentiated in an analogous experiment. This comparison identifies several genes critical for cytoskeletal integrity and vasculogenesis, including ANK3 and ANGPT1. Taken together, these results inform how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and suggest these genes may underlie defects in vascular development and allantoic elongation seen in the absence or reduction of CDX2 in vivo.

overall_design: 2D gastruloids derived from WT, CDX2-Het, or CDX2-KO human iPSCs were isolated after 48hrs of BMP4 treatment for multiomic snRNA-seq and snATAC-seq. Nuclei from two bioreplicates of about 288 2D gastruloids per genotype per replicate were sequenced.

first: Todd

last: McDevitt

first: Benoit

last: Bruneau


title: Planococcus citri mealybug virgin and mated female transcriptome sequencing

city: Ljubljana

first: Marko

last: Petek

address: Večna pot 111

department: Department of Biotechnology and Systems Biology

country: Slovenia

institute: National Institute of Biology

summary: P. citri mealybugs cause severe damage in citrus production. We aimed at indentifying genes involved in mealybug sex pheromone biosynthesis. As these should be expressed in virgin females and are presumably downregulated after mating, we looked at genes differentially expressed between mated and virgin females.

overall_design: Design: 4 biological replicate samples of virgin females and 4 biological replicate samples of mated females.   Each replicate included a pooled sample of approx. 150 females (ca. 100 mg), either virgin or mated.

first: Mojca

last: Juteršek

first: Sandra

last: Vacas Gonzalez


title: Tyrosine 1 phosphorylated RNA polymerase II transcribes PROMPTs to promote proximal promoter pausing and induce global transcriptional repression in response to DNA damage

status: Public on Feb 13 2024

city: Oxford

first: Monika

last: Gullerova

address: S Parks Rd

department: Sir William Dunn School of Pathology

country: United Kingdom

institute: University of Oxford

summary: DNA faces continuous threats from various endogenous and exogenous stresses. When damaged, DNA requires repair to enable proper cell functioning. Cells have evolved multiple repair pathways to maintain genome stability. Transcription mediated by RNA polymerase II (RNA Pol II), which is conventionally linked with gene expression, plays an integral role in DNA repair. Transcription occurring at sites of DNA damage facilitates repair by generating functional transcripts, recruiting DNA repair factors, or modulating chromatin architecture. Conversely, the shutdown of transcription in nearby genes is crucial to ensure effective DNA repair. UV-induced DNA lesions have been shown to lead to a genome-wide suppression of transcription through RNA Pol II ubiquitylation and subsequent degradation. However, the global transcriptional response to double-strand breaks remains unclear.In this study, we employed mNET-Seq analysis to examine nascent transcripts bound to the CTD Y1P, S2P and S5P of RNA Pol II. Our data reveal a global transcriptional shutdown, as evidenced by a greater number of downregulated protein coding genes in response to irradiation. Interestingly, we observed a significant increase in Y1P antisense transcripts near promoters, known as PROMPTs, which are associated with the downregulated protein coding genes upon irradiation. Furthermore, an increased promoter pausing index correlated with elevated expression of PROMPTs in response to double strand breaks induction. Motif enrichment analysis uncovered binding sites for the Polycomb repressive complex 2 (PRC2) in upregulated PROMPTs.  Using proximity ligation assays, we demonstrated an increased proximity between Y1P RNA Pol II and PRC2 upon irradiation. Finally, we also report that inhibition of Y1P or depletion of PRC2 results in the loss of transcriptional repression of protein coding genes, associated with PROMPTs.

overall_design: mNet-Seq sample preparation using Tyrosine 1 phosphorylated (Y1P) RNA Pol II, Serine 2 phosphorylated(S2P) RNA Pol II,Serine 5 phosphorylated(S5P) RNA Pol II antibodies. Samples were either irradiated or non-irradiated for each these antibody types. The extracted RNA Pol II was then processed using mNET-Seq protocol (Nojima 2016) to study the nascent RNA transcripts bound to them

first: Kamal

last: Ajit

first: Adele

last: Alagia

first: Kaspar

last: Burger


title: CLIP-seq of Salmonella ProQ in intracellular-like conditions

city: Uppsala

first: Erik

last: Holmqvist

state: Uppland

address: Husargatan 3, Box 596, Uppsala University, Dept. of Cell and Molecular Biology

country: Sweden

institute: Uppsala University

summary: The aim of the project is to identify transcriptome-wide binding sites for the global RNA-binding protein ProQ in Salmonella during intracellular-like conditions.

overall_design: Salmonella enterica strain SL1344 carrying a chromosomal proQ-3xFLAG allele was grown in intracellular-like growth medium until and OD600 of 0.3 and thereafter subjected to crosslinking immunoprecipitation sequencing (CLIP-seq). Specifically, the bacteria were subjected to UV-light-induced crosslinking, anti-FLAG-mediated immunoprecipitation, RNase treatment, gel purification, and RNA purification, followed by Illumina library preparation using the NEBNext Small RNA Prep kit and sequencing on an Illumina NextSeq500 instrument at vertis Biotechnologie AG (Freising, Germany).

first: Liis

last: Andresen

first: Jonas

last: Kjellin


title: Resting CD4+ T cells exposed to oleic acid upregulate genes involved in immunometabolism resulting in increased IL-9+ frequencies post-activation

city: Leiden

first: Thomas

middle: B

last: Kuipers

address: Einthovenweg 20

institute: LUMC

type: Methylation profiling by genome tiling array


title: The immunopathological landscape of human pre-TCRα deficiency: from rare to common variants

city: Bethesda

first: Chaim

middle: A

last: Schramm

state: MD

address: 40 Convent Dr

department: Vaccine Research Center

institute: NIAID, NIH

summary: We report humans with biallelic loss-of-function PTCRA rare variants impairing pre-TCR-⍺ expression. Low naive ⍺β T cell blood counts at birth persist over time with normal memory ⍺β and high γδ T cell counts. Early productive TCR-δ or TCR-⍺ rearrangements can rescue ⍺β T cell differentiation by stabilizing low levels of cell surface CD3. Only a minority of them are sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that ~1/4,000 individuals from the Middle East and South Asia are homozygous for a hypomorphic PTCRA common variant. They have normal naive ⍺β T cell but high γδ T cell blood counts. Residual expression of their pre-TCR-⍺ enables differentiation of more ⍺β T cells. However, these patients have autoimmune conditions more commonly than the general population.

overall_design:
Cryopreserved peripheral blood mononuclear cells (PBMC) in R10 medium (RPMI 1640, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) were thawed and immediately centrifuged to obtain a cell pellet.  CD3+ T cells were sorted by flow cytometry from the PBMCs.  Cells were then labeled with oligonucleotide-linked hashing antibody and stained by incubation with antibody cocktail diluted in Brilliant Stain Buffer.  12,000 T cells (CD3+CD14−CD19−CD56−Live/Dead−) were sorted from each sample with a BD FACSymphony S6 Cell Sorter running BD FACSDiva Software version 9.5.1. Sorted cells were pooled four by four, and each pool was loaded in a different lane of the 10x Genomics Chromium Chip for sequencing.  For the sequencing of single-cell V(D)J repertoires for sorted T cells, the cell suspension was loaded on the 10x Genomics Chromium Instrument according to the manufacturer’s protocol for the Next GEM Single-Cell 5′ Kit v1.1 to generate gel bead-in-emulsions, and to perform GEM-RT and the amplification of total cDNA. Following purification with SPRIselect beads, specific TCR targets were amplified from the cDNA with the PTCR1 primer (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3′) and constant region primers: TRB (5′-TGCTTCTGATGGCTCAAACACAGCGACCT-3′), TRA (5′-TCTCAGCTGGTACACGGCAGGGTCAGGGT-3′), TRG (5′-GAAGGAAGAAAAATAGTGGGCTTGGGGGAAAC-3′), or TRD (5′-CACCAGACAAGCGACATTTGTTCC-3′) with a barcode and the P7 sequence added to the constant region primers. The Illumina-ready libraries were sequenced by paired-end MiSeq with 2×300 base-pair reads to obtain VDJ sequences.  10X single-cell transcriptome libraries were processed with Cellranger (v6.1.1) and Seurat (v4.0.4).  The TCR-⍺β and TCR-γδ libraries were demultiplexed and cell barcodes were assigned with Minnn (v10.1).  TCR libraries were annotated with MiXCR (v3.0.13) and then separated by subject. The numbers of αβ or γδ TCRs for the patients and controls were calculated by counting the numbers of cells expressing both TRA and TRB V-J genes and both TRG and TRD V-J genes. The final counts corresponded to the intersection of cells expressing combinations of TRA, TRB, TRG or TRD genes, accounting for 10365, 21755, 2233 and 1440 cells, respectively, for the controls (n=11) and 5963, 11416, 2095 and 529 cells, respectively, for the patients (n=5). The diversity of α, β, γ and δ TCRs was estimated by calculating Shannon’s entropy (H) index. Entropy was calculated by summing the frequencies of each clone (CDR3 amino-acid sequence) and multiplying by the base 2 logarithm of the same frequency over all cells expressing TRA, TRB, TRG or TRD V-J genes. Higher H index values indicate a more diverse distribution of CDR3 clones.

Contributors: Marie Materna, Ottavia M. Delmonte, Marita Bosticardo, Mana Momenilandi, Peyton E. Conrey, Bénédicte Charmeteau-De Muylder, Clotilde Bravetti, Rebecca Bellworthy, Axel Cederholm, Frederik Staels, Christian A. Ganoza, Samuel Darko, Samir Sayed, Corentin Le Floc’h, Masato Ogishi, Darawan Rinchai, Andrea Guenoun, Alexandre Bolze, Taushif Khan, Adrian Gervais, Renate Krüger, Mirjam Völler, Boaz Palterer, Mahnaz Sadeghi-Shabestari, Anne Langlois de Septenville, Chaim A. Schramm, Sanjana Shah, John J. Tello-Cajiao, Francesca Pala, Kayla Amini, Jose S. Campos, Noemia Santana Lima, Daniel Eriksson, Romain Lévy, Yoann Seeleuthner, Soma Jyonouchi, Manar Ata, Fatima Al Ali, Caroline Deswarte, Anaïs Pereira, Jérôme Mégret, Tom Le Voyer, Paul Bastard, Laureline Berteloot, Michaël Dussiot, Natasha Vladikine, Paula P. Cardenas, Emmanuelle Jouanguy, Mashael Alqahtani, Amal Hasan, Thangavel Alphonse Thanaraj, Jérémie Rosain, Fahd Al Qureshah, Vito Sabato, Marie Alexandra Alyanakian, Marianne Leruez-Ville, Flore Rosenberg, Elie Haddad, Jose R. Regueiro, Maria L. Toribio, Judith R. Kelsen, Mansoor Salehi, Shahram Nasiri, Mehdi Torabizadeh, Hassan Rokni-Zadeh, Majid Changi-Ashtiani, Nasimeh Vatandoost, Hossein Moravej, Seyed Mohammad Akrami, Mohsen Mazloomrezaei, Aurélie Cobat, Isabelle Meyts, Toyofuku Etsushi, Madoka Nishimura, Kunihiko Moriya, Tomoyuki Mizukami, Kohsuke Imai, Laurent Abel, Bernard Malissen, Fahd Al-Mulla, Fowzan Sami Alkuraya, Nima Parvaneh, Horst von Bernuth, Christian Beetz, Frédéric Davi, Daniel C. Douek, Rémi Cheynier, David Langlais, Nils Landegren, Nico Marr, Tomohiro Morio, Mohammad Shahrooei, Rik Schrijvers, Sarah E. Henrickson, Hervé Luche, Luigi D. Notarangelo, Jean-Laurent Casanova, Vivien Béziat


title: Resting CD4+ T cells exposed to oleic acid upregulate genes involved in immunometabolism resulting in increased IL-9+ frequencies post-activation [RNA-seq]

summary: T cells are the most common immune cell in atherosclerotic plaques. Furthermore, T cell function is influenced by fatty acids that are abundant in the circulation and atherosclerotic plaques. Here, we define the effect of oleic acid, one of the most abundant fatty acids in the human circulation, on the transcriptome of resting CD4+ T cells and subsequently the polarization of the cells into T cell subsets post-activation. We first performed RNA sequencing of resting CD4+ T cells from 9 donors after 0.5, 3, 24, 48, and 72 hours exposure to 30µg/mL oleic acid. This revealed the upregulation of genes involved in cholesterol biosynthesis (HMGCR, SQLE, MVD and MVK) and de novo fatty acid biosynthesis (ACACA and FASN). Both pathways point to a metabolic reprogramming of the cells towards a pro-inflammatory state. Subsequently, a 26-marker spectral flow cytometry panel showed increased frequencies of IL-9+ and IL-17A+ cells among activated CD4+ T cells pre-exposed to oleic acid. IL-9 and IL-17A are primarily produced by the respective T helper (TH) subsets TH9 and TH17, where, especially TH9, has been implicated in the aggravation of atherosclerosis. Our data shows that oleic acid may induce a shift in immunometabolism in resting CD4+ T cells towards an activated profile that leads to more pro-inflammatory and pro-atherogenic polarization of these cells post-activation. Taken together, our study signals a potential role for the interaction between circulating fatty acids and CD4+ T cells in the development and pathogenesis of atherosclerosis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data and comparative DNA methylation analysis of 850k DNA methylation array data for resting human buffy coat derived CD4+ T cells exposed to control or 30μg/mL for either 0.5, 3, 24, 48, or 72 hours. The RNA and DNA was simultaneously extracted from the cell samples and thus the RNA seq and DNA methylation data is representative of the same bulk cell sample.

first: Nathalie

last: Reilly

first: Koen

middle: F

last: Dekkers

first: Friederike

last: Sonnet

first: Joanneke

middle: C

last: Kwekkeboom

first: Lucy

last: Sinke

first: Stan

last: Hilt

first: Hayat

last: Suleiman

first: Marten

last: Hoeksema

first: Hailiang

last: Mei

middle: W

last: van Zwet

first: Bart

last: Everts

first: Andreea

last: Ioan-Facsinay

first: J

last: Jukema

first: Bastiaan

middle: T

last: Heijmans


title: High-specificity CRISPR-mediated genome engineering in CB-011, an anti-BCMA allogeneic CAR-T cell, suppresses allograft rejection in preclinical models

status: Public on Feb 12 2024

city: EMERYVILLE

first: Ryan

middle: Tevia

last: Davis

state: California

address: 2929 Seventh St

institute: Caribou Biosciences

summary: Allogeneic chimeric antigen receptor (CAR)-T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR-T cells. Key considerations in the development of allogeneic CAR-T cell therapies include prevention of GvHD and suppression of allograft rejection. Here we describe preclinical data supporting the ongoing first-in-human clinical trial (CaMMouflage) in relapsed/refractory multiple myeloma patients evaluating CB-011, a hypoimmunogenic, allogeneic anti–B cell maturation antigen (BCMA) CAR-T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR-T cells were engineered to prevent endogenous human leukocyte antigen (HLA) class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. Additionally, T cell receptor expression was disrupted at the T cell receptor alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell co-cultures derived from patients with multiple myeloma. Additionally, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer (NK) cell–mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M–HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma.

overall_design: For profiling of NK and T cells following infusion in vivo, 6 different cohorts were established in NOG-Tg (Hu-IL15) mice according to the protocol described above: (i) 2 cohorts of CB-011 cells without pNK cells (CB-011 only, n = 2 mice/cohort), (ii) 2 cohorts of CB-011 cells with NK cells (CB-011+NK, n = 3 mice/cohort), (iii) 1 cohort of B2M KO CAR-T cells without NK cells (B2M KO only, n = 2 mice), and (iv) 1 cohort of B2M KO CAR-T cells with NK cells (B2M KO+NK, n = 3 mice). Additionally, NK and CB-011 cells were isolated for single-cell RNA sequencing (scRNA-Seq; NK IP and CB-011 IP, n = 1/group) prior to infusion. NK and CAR-T cells from either the CB-011 or the CAR KI B2M KO cohorts were isolated from the spleens of NOG-Tg (Hu-IL15 mice) mice 4 days post CAR-T cell infusion by FACS using a human CD45-specific antibody. Cells were resuspended in PBS + 0.4% BSA at a concentration of 1x106 cells/mL and single-cell RNA sequencing libraries were generated (Supplemental methods).

first: Davis

last: Ryan T

first: Kanner

last: Steven B


title: Sall4 regulates posterior trunk mesoderm development by promoting mesodermal gene expression and repressing neural genes in the mesoderm

city: Minneapolis

first: Matthew

last: Pappas

address: 321 Church St. SE

institute: University of Minnesota

summary:
The trunk axial skeleton develops from paraxial mesoderm cells. Our
recent study demonstrated that conditional knockout of the stem cell
factor Sall4 in mice by TCre caused tail truncation and a disorganized
axial skeleton posterior to the lumbar level. Based on this phenotype,
we hypothesized that, in addition to the previously reported role of
Sall4 in neuromesodermal progenitors, Sall4 is involved in the
development of the paraxial mesoderm tissue. ATAC-seq in
TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4
knockout reduces chromatin accessibility. We found that Sall4-
dependent open chromatin status drives activation and repression of
WNT signaling activators and repressors, respectively, to promote
WNT signaling. Moreover, footprinting analysis of ATAC-seq data
suggests that Sall4-dependent chromatin accessibility facilitates
CTCF binding, which contributes to the repression of neural genes
within the mesoderm. This study unveils multiple mechanisms by
which Sall4 regulates paraxial mesoderm development by directing
activation of mesodermal genes and repression of neural genes.

overall_design: ATAC-seq in E9.5-E9.75 posterior mesoderm of TCre driven Sall4 conditional knockout mice.

middle: P

first: Hiroko

last: Kawakami

first: Dylan

last: Corcoran

first: Katherine

middle: Q

last: Chen

first: Earl

last: Scott

first: Julia

last: Wong

first: Micah

last: Gearhart

first: Ryuichi

last: Nishinakamura

first: Yasushi

last: Nakagawa

first: Yasuhiko


title: Reciprocal activity of AgRP and POMC neurons governs coordinated control of feeding and metabolism

city: Köln

first: Jens

last: Brüning

address: Gleueler Str. 50

department: Neuronal Control of Metabolism

country: Germany

institute: Max Planck Institute for Metabolism Research

summary: Agouti-related peptide (AgRP)- and proopiomelanocortin (POMC)-expressing neurons reciprocally regulate food intake. Here, we combined non-interacting recombinases to simultaneously express functionally opposing chemogenetic receptors in AgRP and POMC neurons allowing to compare metabolic responses in mice with simultaneous activation of AgRP and inhibition of POMC neurons with isolated activation of AgRP neurons or isolated inhibition of POMC neurons. These experiments revealed that food intake is regulated by the additive effect of AgRP-neuron activation and POMC-neuron inhibition, while systemic insulin sensitivity and gluconeogenesis are differentially modulated by isolated versus simultaneous regulation of AgRP and POMC neurons. We identified a neurocircuit engaging Npy1R-expressing neurons in the paraventricular nucleus of the hypothalamus (PVH), where activated AgRP- and inhibited POMC neurons synergize to promote food consumption and activate neurons in the nucleus tractus solitarii (NTS). We then performed single-nuclei RNA sequencing to define the molecular nature of Fos+ cells in the posterior NTS/AP area that respond to simultaneous chemogenetic intervention over AgRP and POMC neurons and identified TH+ neurons as candidates for receiving neuronal inputs initiated by the simultaneous and coordinated interplay between AgRP and POMC neurocircuits and relayed to the NTS area by the silenced glutamatergic Npy1R neurons.

overall_design: All experiments were performed in adult mice, age 12-20 weeks, and both sexes were assayed, always considering balanced sex and body weight distribution in both experimental groups. For this experiment wildtype mice and AgRP-Gq;POMC-Gi (AgRP-IRES-Cre+/-;hM3DGqfl/- ;POMC-Dre+/-;hM4DGifl/-) mice were included. Both groups were injected with CNO (clozapine-N-oxide, 3mg/kg; Abcam ab141704) intraperitoneally (ip) at the start of each experiment. Mice were sacrificed by cervical dislocation 1h after CNO injection in the absence of food, and a portion of the hindbrain containing the AP and NTS was dissected. Samples from the same experimental condition were pooled together (n = 2 mice/group).

first: Alain

last: De Solis

first: Almudena

last: Del Rio-Martin

first: Lukas

last: Steuernagel


title: Cancer-associated fibroblasts metabolic targeting overcomes T-cell exclusion and chemoresistance [Mouse]

status: Public on Feb 10 2024

city: Houston

first: Jlenia

last: Guarnerio

state: TX

address: 6767 Bertner Ave

department: Genetics

institute: MD Anderson Cancer Center

summary: T cell-based immunotherapies have exhibited promising outcomes in tumor control; however, their efficacy is limited in immune-excluded tumors. Cancer-associated fibroblasts (CAFs) play a pivotal role in shaping the tumor microenvironment and modulating T cell infiltration. Despite the identification of distinct CAF subtypes using single-cell RNA-sequencing, their specific impact on hindering T cell infiltration remains unclear, particularly in soft-tissue sarcomas (STS) characterized by low response rates to T cell-based therapies. In this study, we comprehensively characterized the STS microenvironment using murine models with distinct immune composition and scRNA-seq, leading to the identification of a unique subset of CAFs termed glycolytic cancer-associated fibroblasts (glyCAFs). GlyCAFs rely on glycolysis to impede cytotoxic T cell infiltration into the tumor parenchyma. Targeting glycolysis in glyCAFs enhances T-cell infiltration and augments the efficacy of chemotherapy. These findings highlight new avenues for combinatorial therapeutic interventions in sarcomas and other solid tumors. Further investigations and clinical trials are needed to validate these potential strategies and translate them into clinical practice.

overall_design: Mouse Undifferentiated Pleiomorphic Sarcoma (UPS) tumors bearing one of two tumor genetics (p53-/-Ccne1+ or p53-/-Vgll3+) were collected for single-cell RNA-sequencing. Digested tumor cell suspensions were enriched for specific cell types of interest. In experiments where the abundance of the cell type of interest was low (cancer-associated fibroblasts), multiple mouse tumors were first pooled together into a single sample, and processed only in aggregate. Where the abudance of the targeted cell type was sufficiently high, individual mouse tumors were processed as separate biological replicates, and tagged with an oligonucleotide hashtag to preserve sample identity. Hashtagged samples were finally pooled together prior to capture. Where indicated, technical replicates represent capture of additional cells and in a separate channel of the 10X Chromium chip, but drawn from the same overall pool of cells.

first: Marina

last: Broz

first: Emily

middle: Y

last: Ko

first: Marco

last: DeSimone


title: Cancer-associated fibroblasts metabolic targeting overcomes T-cell exclusion and chemoresistance [Human]

overall_design: Human sarcoma samples (n=1 tumor each Sample) from surgical resections were singly collected and processed for single-cell RNA sequencing.


title: Persistent backtracking by RNA polymerase II

city: NYC

first: Kevin

last: Yang

state: New York

institute: New York University Medical Center

summary: RNA polymerase II (pol II) can backtrack during transcription elongation, exposing the 3’ end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome wide distribution of more persistent backtracking is unknown. Consequently, we developed a novel method to directly sequence backtracked RNA. Our data shows that pol II slides backwards more than 20 nucleotides in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where pol II pauses near promoters and intron-exon junctions, and is enriched in genes involved in translation, replication, and development, where gene expression is inhibited if these events are unresolved. Histone genes are highly prone to persistent backtracking, where the accumulation and resolution of such events is required for timely expression during cell division. These results demonstrate that persistent backtracking has the potential to affect diverse gene expression programs.

overall_design: LORAXseq, crosslinked footprinting, and mNETseq was performed in cells lacking TFIIS to measure the genomic distribution of persistent backtracking and nascent transcription.

first: Evgeny

last: Nudler


title: Select EZH2 inhibitors enhance the viral mimicry effects of DNMT inhibition through a mechanism involving Calcium-Calcineurin-NFAT signaling [siQ-ChIP]

status: Public on Feb 26 2024

city: Grand Rapids

first: Scott

last: Rothbart

state: MI

address: 333 Bostwick Avenue NE

institute: Van Andel Research Institute

summary: DNA methyltransferase inhibitors (DNMTi) are FDA-approved for various hematological malignancies but have limited efficacy in solid tumors. DNA hypomethylation with these drugs is associated with elevated lysine 27 tri-methylation on histone H3 (H3K27me3). We hypothesized that this EZH2-dependent repressive mark limits the full potential of DNMTi. Here, we show in cell line and tumoroid models of colorectal cancer, that low-dose DNMTi sensitizes cells to selective EZH2 inhibitors (EZH2i) that have limited single agent toxicity, and that EZH2i enhances DNMTi-driven molecular and therapeutic effects. Through integrative epigenomic analyses, we reveal that DNMTi induces H3K27me3 accumulation at genomic regions poised with EZH2. Unexpectedly, combined treatment alters the epigenome landscape to promote transcriptional upregulation of the Calcium-Calcineurin-NFAT signaling pathway. Blocking this pathway limits the transcriptional activating effects of the drug combination, including expression of transposable elements and innate immune response genes within a viral defense pathway. Consistently, we demonstrate positive correlations between DNMTi- and viral infection-associated transcription profiles and Calcium signal activation in colon cancer patient samples. Collectively, our study demonstrates that compensatory EZH2 activity following DNA hypomethylation presents a barrier to the therapeutic action of DNMTi in colon cancer, reveals a new application of EZH2i beyond cancers associated with PRC2 hyperactivity, and links Calcium-Calcineurin-NFAT signaling to epigenetic therapy-induced viral mimicry.

overall_design: Chromatin Immunoprecipitation for H3K27me3 was performed on HCT116 cells treated with: Vehicle (DMSO), 5'aza-2'deoxycytidine (DAC) 30 nM, DAC 300 nM, Tazemetostat (TAZ) (1 uM), or combination DAC + TAZ for 72 hours.

first: Alison

last: Chomiak

first: Rochelle

middle: L

last: Tiedemann

first: Yanqing

last: Liu

first: Xiangqian

last: Kong

first: Ying

last: Cui

first: Kate

last: Thurlow

first: Evan

middle: M

last: Cornett

first: Michael

last: Topper

first: Stephen

last: Baylin


title: LMO3 is a suppressor of the basal-like/squamous PDAC subtype and reduces disease aggressiveness of pancreatic cancer through glycerol 3-phosphate metabolism

status: Public on Feb 08 2024

first: Stefan

last: Ambs

address: Building 37, Room 3050B

department: CCR

institute: NCI

summary: Pancreatic cancer is a heterogenous disease and consists of distinct subtypes. Here, we searched for candidate suppressor genes of the basal-like/squamous subtype, a more aggressive molecular subtype of pancreatic ductal adenocarcinoma (PDAC). Through an integrated transcriptome analysis, we identified the LMO3, as being downregulated in the basal-like/squamous PDAC using a discovery and validation approach. LMO3 downregulation associated with decreased patient survival. The goal of this study was aimed to identify LMO3 induced molecular signatures in PDAC.

overall_design: RNA was isolated from PDAC cell lines (n = 8 Panc 10.05_Ctrl vs LMO3) using TRIzol method. The mRNA profiles of the above human PDAC cell lines were generated by deep sequencing using Illumina NextSeq.

first: Ohara

last: Yuuki

first: Craig

last: Amanda J.

first: Liu

last: Huaitian

first: Yang

last: Shouhui

first: Moreno

last: Paloma

first: Dorsey

last: Tiffany H.

first: Cawley

last: Helen

first: Azizian

last: Azadeh

first: Gaedcke

last: Jochen

first: Ghadimi

last: Michael

first: Hanna

last: Nader

first: Ambs

last: Stefan

first: Hussain

last: S Perwez


title: Molecular patterns of resistance to immune checkpoint blockade in melanoma

status: Public on Feb 06 2024

city: Lund

first: Martin

last: Lauss

address: Barngatan 2B

department: Dept of Oncology

institute: Lund University

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

overall_design: Refer to individual Series


title: Molecular patterns of resistance to immune checkpoint blockade in melanoma [scRNA-Seq]

summary: Immune checkpoint blockade (ICB) has improved outcome for patients with metastatic melanoma but not all benefit from treatment. Several immune- and tumor intrinsic features are associated with clinical response at baseline. However, we need to further understand the molecular changes occurring during development of ICB resistance. Here, we collected biopsies from a cohort of 44 melanoma patients after progression to anti-CTLA4 or anti-PD1 monotherapy. Genetic alterations of antigen presentation and interferon gamma signaling pathways were observed in approximately 25% of ICB resistant cases. Anti-CTLA4 resistant lesions had a sustained immune response, including immune-regulatory features, as suggested by multiplex spatial and TCR clonality analyses. One anti-PD1 resistant lesion harbored a distinct immune cell niche, however, anti-PD1 resistant tumors were generally immune poor with non-expanded TCR clones. Such immune poor microenvironments were associated with melanoma cells having a de-differentiated phenotype lacking expression of MHC-I molecules. In addition, anti-PD1 resistant tumors had reduced fractions of PD1+ CD8+ T cells as compared to ICB naïve metastases. Collectively, these data show the complexity of ICB resistance and highlight differences between anti-CTLA4 and anti-PD1 resistance that may underlie differential clinical outcomes of therapy sequence and combination.

overall_design:
Single cell transcriptomics from four samples that contain B cell rich regions, two of which having progressed on Immune Checkpoint Blockade, generated by single cell RNAseq.

***************************************************************
Authors state that due to Swedish and Danish laws, the patient consent, and the risk that the sequencing data contains personally-identifiable information and hereditary mutations, we cannot deposit the short sequencing read data in a public access repository
***************************************************************

first: Göran

last: Jönsson

first: Katja

last: Harbst


title: Molecular patterns of resistance to immune checkpoint blockade in melanoma [bulk RNA-Seq]

overall_design:
Gene expression profiles of 41 samples after progression on Immune Checkpoint Blockade treatment, generated by RNAseq.

***************************************************************
Authors state that due to Swedish and Danish laws, the patient consent, and the risk that the sequencing data contains personally-identifiable information and hereditary mutations, we cannot deposit the short sequencing read data in a public access repository
***************************************************************


title: Heterozygous missense variant in GLI2 impairs human endocrine pancreas development

city: London

first: Francesca

middle: M.

last: Spagnoli

address: Great Maze Pond

department: CGTRM

institute: King's College London

summary: To study the impact of an heterozygous missense variant in GLI2 in pancreatic cell differentiation and function, we established a patient-like pluripotent stem cell model. Interestingly, engineered iPSC lines carrying the identified missense variant in the gene GLI2 showed impaired differentiation into endocrine progenitors.  To further characterize putative downstream mechanisms underlying the impaired endocrine differentiation of iPSCs, we performed bulk RNA-seq at three stages of differentiation (iPSC, gut tube and endocrine progenitor stages). At early stages (iPSC and gut tube stage), the transcriptome of GLI2-CTRL and GLI2-MUT-derived cells was highly comparable, with only 48 genes significantly dysregulated at gut tube stage. By contrast, differences at the transcriptome level became evident at endocrine progenitor stage [928 genes upregulated and 1070 downregulated in mutant versus control cells].

overall_design: Comparative gene expression profiling analysis of RNA-seq data for human iPSC cells [control and mutant (MUT) lines] at two stages of pancreatic differentiation (PD)

first: Laura

last: Müller

first: Abigail

last: Isaacson

first: Klemens

last: Raile


title: Gene expression changes in injured myocardium of Ciona intestinalis

city: Charleston

first: Jeremy

last: Barth

state: SC

address: 173 Ashley Ave, BSB 627

institute: MUSC

summary: Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages for the study of cardiovascular biology.  The regulatory mechanisms of cardiac myocyte proliferation in Ciona are intriguing since Ciona are capable of regeneration throughout their lifespan. To identify important regeneration factors in Ciona, microarray analysis was conducted on RNA from adult Ciona hearts with normal or damaged myocardium using custom Affymetrix GeneChips. After a 24- or 48-hour recovery period, total RNA was isolated from damaged and control hearts.  Initial results indicate significant changes in gene expression in hearts damaged by ligation in comparison to cryoinjured or control hearts.  Ligation injury shows differential expression of 223 genes as compared to control (fold change >2, p<0.01, Student’s t-test) with limited false discovery (5.8%).  Among these 223 genes, 117 have known human orthologs of which 68 were up-regulated and 49 were down-regulated.   Notably, FGF 9/16/20 and Ras were significantly upregulated in injured hearts. Histological analyses of injured myocardium were conducted in parallel to the microarray study.  Taken together, these studies will coordinate differences in gene expression to cellular changes in the regenerative myocardium of Ciona, which will help to elucidate the regulatory mechanisms of cardiac myocyte proliferation across chordates.

overall_design: Samples were prepared from Ciona intestinalis adult (7-12 cm) control hearts (exposed but not ligated) or injured hearts (ligated or cryo injury) at 24h or 48h following treatment with two biological replicates per sample type. A minimum of 4 hearts per group was done for each experiment. Microarray profiling was performed using the Affymetrix Ciona intestinalis custom-designed array (CINT06a520380F; GEO platform GPL15657).

first: Heather

last: Evans-Anderson


title: Cancer-associated fibroblasts metabolic targeting overcomes T-cell exclusion and chemoresistance

first: Jlenia

last: Guarnerio

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Systematic mapping of TF-mediated cell fate changes by a pooled inductioncoupled with scRNA-seq and multi-omics approaches

status: Public on Feb 05 2024

city: AUSTIN

first: Qingqing

last: Guo

address: 2506 Speedway

institute: The University of Texas at Austin

summary: Transcriptional regulation controls various cellular functions via the interactionsbetween cell-type-specific transcription factors (TFs) and their chromosomaltargets. While multiple TFs capable of converting cell fates from one to anotherhave been reported, only a few systematic studies aiming to understand thefate conversion potential of numerous individual TFs have been reported. Here,we propose an iTF-seq method (pooled induction of individual TFs followed bysingle-cell RNA sequencing) to identify potent TFs capable of converting cellfates and elucidate their lineage specification potential at a single-cell level.Enabling metabolic biotinylation of TFs during the process, the method allowssubsequent multi-omics approaches to understand underlying actionmechanisms of TFs during cell fate changes. Our pilot iTF-seq of 80 TFs inmouse embryonic stem (ES) cells has identified multiple previously known andunknown TFs triggering transcriptome changes within one day of TF induction.Subsequent tests revealed that Gcm1 and Otx2 function as activators andpioneer factors, while they resulted in distinct cell fates and gene expressionprograms upon induction.

overall_design: BioChIP-seq was performed on Gcm1 and Otx2 SBFB cells after 24h of Dox induction with uninduced control cells as well as Nanog FB mESCs. ATAC-seq was performed on Gcm1 and Otx2 SBFB cells after 24h of Dox induction with uninduced control cells.

first: Jonghwan

last: Kim


title: Massively parallel screen uncovers many rare 3’ UTR variants regulating mRNA abundance of cancer driver genes [rare_cosmic]

city: Los Angeles

first: Xinshu

middle: Grace

last: Xiao

address: 610 Charles E. Young Drive South, Room 2000E

department: Integrative Biology and Physiology

institute: UCLA

summary: Understanding the function of rare non-coding genetic variants represents a significant challenge. Here, we developed MapUTR, a screen to identify rare 3’ UTR variants affecting mRNA abundance post-transcriptionally. Among 17,301 rare variants, an average of 24.5% were functional, with 70% in cancer-related genes, many in critical cancer pathways. This observation motivated a further interrogation of 11,929 cancer somatic mutations, uncovering 3,928 (33%) functional mutations in well-established cancer driver genes, such as CDKN2A. Functional MapUTR variants were enriched in miRNA targets and protein-RNA interaction sites. Based on MapUTR, we define a new metric, untranslated tumor mutation burden (uTMB), reflecting the amount of somatic functional MapUTR variants of a tumor. We showed the potential of uTMB in predicting patient survival. Through prime editing, we characterized three variants in cancer-relevant genes (MFN2, FOSL2, and IRAK1), illustrating their cancer-driving potential. Our study elucidates the function of thousands of non-coding variants, nominates non-coding cancer driver mutations, and demonstrates their potential contributions to cancer.

overall_design:
To test a variant of interest, both the reference and the alternative allele were designed into 200nt oligos synthesized by Twist Biosciences. Each oligo contains a 164nt flanking sequence centered around the variant and adaptor sequences for cloning. The oligos were then clonedinto the 3’ UTR region of the eGFP gene, whose expression is driven by the CAG promoter, in the plasmid reporters. We introduced 3ug plasmid libraries into 15M HeLa cells via cell electroporation for each biological replicate, with a total number of three replicates. Total mRNA was isolated from the cells 24h after electroporation, reverse-transcribed into cDNA, and made into sequencing libraries through a stepwise PCR. Specifically, a unique molecular identifier (UMI) was added to each cDNA transcript in the first round of PCR. The PCR products were further amplified to add Illumina sequencing adaptors in the second round of PCR. A similar stepwise PCR protocol was also applied to the plasmid DNA to generate DNA sequencing libraries. Both DNA and RNA sequencing libraries were pooled together to be sequenced on  Novaseq S1 PE150 with 15% PhiX spike-in. For data analysis, we extracted UMIs from each read to remove PCR duplicates.

Please note that the C#Sp#.fasta contains Chip# Subpool# library sequences (e.g. C4Sp1.fasta = Chip4 Subpool1 library sequences etc.).

first: Ting

last: Fu

first: Kofi

last: Amoah

first: Tracey

last: Chan

first: Jae-Hoon

last: Bahn

first: Jae-Hyung

last: Lee

first: Sari

last: Terrazas

first: Rockie

last: Chong

first: Sriram

last: Kosuri


title: Real-time quantitative PCR analysis of Tomato stress response

status: Public on Feb 04 2024

city: Iksan

first: Sujeevan

last: Rajendran

address: Muwang-ro

country: Sri Lanka

institute: Wonkwang University

summary: To extract RNA from shoots and roots, 3 weeks old control and treated plants were harvested at 2pm in the greenhouse. Total RNA was extracted using the AccuPrep® Universal RNA extraction kit (Bioneer, Daejeon, Korea) and treated with RNase-free DNase to remove DNA fragments (Qiagen, Hilden, Germany). One microgram of total RNA was used to synthesize cDNA with AccuPower® RT PreMix (Bioneer, Daejeon, Korea). Quantitative real-time RT-PCR was conducted using a T100TM Thermocycler system (Bio-Rad, Hercules, CA, USA). Primer information is given in Table S7. Reactions (10 µL final volume) were prepared using 5 µL of LaboPass™ SYBR Green Q Master Kit (Cosmogenetech, Dajeon, Korea). 0.5 pmol of a primer pair, and 0.5 µL of cDNA template. 4 biological samples and two technical replicates were used for the quantifications. Ubiquitin was used as the reference. gene expression analysis was performed with the 2^−ΔΔCt method using Bio-Rad CFX Maestro software v.4.0 (Bio-Rad). Baseline and threshold levels were set according to the manufacturer’s instructions.

overall_design: qPCR gene expression profiling.  Soot and root samples were acquired and cDNA synthezised using 1microgram RNA. qPCR was conducted using CYBR green with Biorad C-1000.

first: Chul


title: Massively parallel screen uncovers many rare 3’ UTR variants regulating mRNA abundance of cancer driver genes


title: Massively parallel screen uncovers many rare 3’ UTR variants regulating mRNA abundance of cancer driver genes [rare_3utr_gnomad.motif_activity]

overall_design:
To test a variant of interest, both the reference and the alternative allele were designed into 200nt oligos synthesized by Twist Biosciences. Each oligo contains a 164nt flanking sequence centered around the variant and adaptor sequences for cloning. The oligos were then cloned into the 3’ UTR region of the eGFP gene, whose expression is driven by the CAG promoter, in the plasmid reporters. We introduced 3ug plasmid libraries into 15M HEK293/HeLa cells via cell electroporation for each biological replicate, with a total number of three replicates. Total mRNA was isolated from the cells 24h after electroporation, reverse-transcribed into cDNA, and made into sequencing libraries through a stepwise PCR. Specifically, a unique molecular identifier (UMI) was added to each cDNA transcript in the first round of PCR. The PCR products were further amplified to add Illumina sequencing adaptors in the second round of PCR. A similar stepwise PCR protocol was also applied to the plasmid DNA to generate DNA sequencing libraries. Both DNA and RNA sequencing libraries were pooled together to be sequenced on Hiseq3000 PE150 or Novaseq SP PE150 with 15% PhiX spike-in. For data analysis, we extracted UMIs from each read to remove PCR duplicates.

Please note that the C#Sp#.fasta contains Chip# Subpool# library sequences (e.g. C1Sp1.fasta = Chip1 Subpool1 library sequences etc.).


title: Massively parallel screen uncovers many rare 3’ UTR variants regulating mRNA abundance of cancer driver genes [prime_editing_3utr]

overall_design: Whole transcriptome sequencing (RNA-Seq) for genome edited HEK293T single cell clones of vatiants of interest (MFN2, FOSL2, and IRAK1).


title: Yeast zinc cluster proteins paralogs involved in the switch from fermentation to respiration show interdependency for DNA binding revealing a novel type of DNA recognition

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

country: Canada

institute: IRCM

type: Genome binding/occupancy profiling by array

summary: In budding yeast, fermentation is the most important pathway for energy production. Under low glucose, ethanol is used for synthesis of this sugar requiring a shift to respiration. This process is controlled by the transcriptional regulators Cat8, Sip4, Rds2, and Ert1. We characterized Gsm1, a paralog of Rds2 and Ert1. Genome-wide analysis showed that Gsm1 has a DNA binding profile highly similar to Rds2, suggesting that these factors bind to DNA as heterodimers. Indeed, binding of Gsm1 and Rds2 is interdependent at the gluconeogenic gene FBP1. However, Rds2 is required for Gsm1 to bind at other promoters but not the reverse. Gsm1 and Rds2 also bind to DNA independently of each other. We showed that the DNA binding domains of Gsm1 and Rds2 bind cooperatively in vitro to the FBP1 promoter. In contrast, at the HAP4 gene, Ert1 cooperates with Rds2 for DNA binding. Mutational analysis suggests that Gsm/Rds2 and Ert1/Rds2 bind to short common DNA stretches revealing a novel mode of binding for this class of factors. Two-point mutations in a HAP4 site convert it to a Gsm1 binding site. In summary, our observations add another layer of transcriptional regulation with the formation of different heterodimers at specific promoters.

overall_design:
We profiled Rds2 and Gsm1 proteins, both tagged with HA, in yeast Saccharomyces cerevisiae cells by ChIP-chip on tiling arrays. The Rds2 protein was profiled in WT and gsm1Δ cells, whereas the Gsm1 protein was profiled  in WT and rds1Δ cells. All HA-tagged ChIP samples were labeled with Cy5 and hybridized against no tag ChIP samples (labeled with Cy3). The microarrays used were described before (Jeronimo and Robert, 2014). The data were normalized using the Limma Loess method, and replicates were combined as described previously (Ren et al. 2000). The data were subjected to one round of smoothing using a Gaussian sliding window with a standard deviation of 100 bp to generate data points in 10-bp intervals (Guillemette et al. 2005). Each experiment was done in duplicate.
Please note that each bed file generated from both replicates is linked to the corresponding *rep1 sample records.

first: Karla

last: Martinez

first: Najla

last: Gasmi

first: Célia

last: Jeronimo

first: Natalia

last: Klimova

first: François

first: Bernard

last: Turcotte


title: Single-cell gene expression of the subventricular zone (SVZ) neurogenic niche after partial reprogramming

city: Stanford

first: Lucy

last: Xu

address: 290 Jane Stanford Way

institute: Stanford University

summary: Partial reprogramming has the potential to reverse age-related decline in some tissues. Its impact on the cell types within the aging brain, particularly the subventricular zone neurogenic niche, is unknown.

overall_design: Partial reprogramming was induced in the whole body of old mice, or induced specifically in the SVZ of old mice. SVZ cells were isolated from old mice after partial reprogramming, as well as young and old controls, and analyzed by single-cell RNA-sequencing.

first: Anne

last: Brunet


title: A mouse model of Zhu-Tokita-Takenouchi-Kim syndrome reveals indispensable SON functions in organ development and hematopoiesis

city: Birmingham

first: Mostafa

middle: Gamal Saadeldin

last: Mohamed

state: Alabama

address: 6th Avenue South

department: Pathology

institute: University of Alabama at Birmingham

summary: Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/−) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/− mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. To obtain a more comprehensive understanding of the changes within HSPCs at the molecular level, we employed single-cell RNA-sequencing (scRNAseq) using both WT and Son+/−  bone marrow HSPCs isolated and sorted based on their Lin− cKit+ (LK) cell markers (24,355 total LK cells from 2 WT mice; 31,232 total LK cells from 2 Son+/− mice. The linage-marker-negative (Lin-) c-kit+ population containing hematopoietic stem and progenitor cells was sorted from dissociated mouse bone marrow cells by flow cytometry. Sorted single-cell suspension, 10x barcoded gel beads and oil (10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1 PN 1000268) were loaded into 10x Chromium™ Single Cell Chip to capture single cells into nanoliter-scale oil droplets by 10xChromium X Controller and generate Gel Bead-In-EMulsions (GEMs). cDNA from single cells were synthesized and barcoded by incubation of the GEMs in a thermocycler machine and then purified from GEMs by DynaBeads (10xGenomics PN 2000048). cDNA was pre-amplified by PCR to generate sufficient mass for sequencing library construction. The single-cell cDNA libraries were constructed by following 10xGenomics 3’ NextGem 3.1 version kit instructions. The final constructed 3’-biased single-cell libraries were sequenced by the Illumina Novaseq6000 machine, targeting 50,000 read pairs/cell. We identified hematopoietic abnormalities in Son+/− mice, including leukocytopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing genes required for lymphoid and B-cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

overall_design: The linage-marker-negative (Lin-) c-kit+ population containing hematopoietic stem and progenitor cells was sorted from dissociated mouse bone marrow cells by flow cytometry. Sorted single cell suspension, 10x barcoded gel beads, and oil (all were included in 10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1 PN 1000268) were loaded into 10x Chromium™ Single Cell Chip to capture single cells into nanoliter-scale oil droplets by 10xChromium X Controller and generate Gel Bead-In-EMulsions (GEMs). cDNA from single cells were synthesized and barcoded by incubation of the GEMs in a thermocycler machine and then purified from GEMs by DynaBeads (10xGenomics PN 2000048). cDNA was pre-amplified by PCR to generate sufficient mass for sequencing library construction. The single cell cDNA libraries were constructed by following 10xGenomics 3’ NextGem 3.1 version kit instruction, briefly by following steps of cDNA fragmentation, end repair & A-tailing, and size selection by SPRIselect beads (Beckman Coulter, PN B23318), adaptor ligation, and sample index PCR amplification, and then SPRIselect beads size selection again. The final constructed 3’-biased single cell libraries were sequenced by Illumina Novaseq6000 machine, targeting 50,000 read pairs/cell. The sequencing cycles consisted of 28bp for read 1, 90bp for read 2, and 10bp for i7 and i5.

first: Lana

last: Vukadin

first: Bohye

last: Park

first: Huashi

last: Li

first: Amr

last: Elkholy

first: Alex

last: Torrelli-Diljohn

first: Jung-Hyun

last: Kim

first: Kyuho

last: Jeong

first: James

last: Murphy

first: Caitlin

last: Harvey

first: Sophia

last: Dunlap

first: Leah

last: Gehrs

first: Hanna

first: Hyung-Gyoon

first: Jay

last: Sah

first: Seth

first: Denise

last: Stanford

first: Robert

last: Barrington

first: Jeremy

last: Foote

first: Anna

last: Sorace

last: Welner

first: Blake

last: Hildreth

first: Ssang-Taek

last: Lim

first: Eun-Young

middle: E

last: Ahn


title: Low-intensity exercise training improves systolic function of heart during metastatic melanoma-induced cachexia in mice

status: Public on Feb 03 2024

city: Xiamen

first: Binbin

last: Liu

state: Fujian

address: Jinshan Road

department: Xiamen Cardiovascular Hospital

institute: Xiamen University

summary: Cardiac dysfunction often arises during the early stages of cancer cachexia, posing a significant complication of the disease. Physical fitness is commonly recommended in these early stages of cancer cachexia due to its beneficial impacts on various aspects of the condition, including cardiac dysfunction. However, the direct functional impacts of exercise on the heart during cancer cachexia largely remain unexplored. In this study, we induced cancer cachexia in mice using a metastatic B16F10 melanoma model. Concurrently, these mice underwent a physical exercise regimen to investigate its potential role in cardiac function during cachexia. Our findings indicate that exercise training can help prevent metastatic melanoma-induced muscle loss without significant alterations to body and fat weight. Moreover, exercise improved the melanoma-induced decline in ejection fraction and fractional shortening, while also mitigating the increase in high-sensitive cardiac troponin T levels caused by metastatic melanoma in mice. Transcriptome analysis revealed that exercise significantly reversed the transcriptional alterations in the heart induced by melanoma, which were primarily enriched in pathways related to heart contraction. These results suggest that exercise can improve systolic heart function and directly influence the transcriptome of the heart during metastatic melanoma-induced cachexia.

overall_design: C57BL/6J male mice (8 weeks old) were divided into the exercise group and the control group. The mice in the exercise group underwent a treadmill running exercise protocol for 5 days per week over a period of 4 weeks. In the first week, the mice were subjected to adaptive training with the following protocol: 6 m/min for 5 min, 9 m/min for 5 min, 12 m/min for 5 min, and 15 m/min for 5 min. Subsequently, for the next 3 weeks, the mice were forced to run at 6 m/min for 5 min and 12 m/min for 35 min. Meanwhile, the mice in the control group were housed in cages without exercise. From the 2nd week of the study, 1×10^5 B16F10 cells were intravenously injected into the mice through the tail vein to induce cancer cachexia

first: Lin

last: Wang

first: Wei

last: Shao


title: Systematic mapping of TF-mediated cell fate changes by a pooled induction coupled with scRNA-seq and multi-omics approaches

city: Austin

first: Muyoung

last: Lee

department: Molecular Biosciences

institute: University of Texas at Austin

summary: Transcriptional regulation controls various cellular functions via the interactions between cell-type-specific transcription factors (TFs) and their chromosomal targets. While multiple TFs capable of converting cell fates from one to another have been reported, only a few systematic studies aiming to understand the fate conversion potential of numerous individual TFs have been reported. Here, we propose an iTF-seq method (pooled induction of individual TFs followed by single-cell RNA sequencing) to identify potent TFs capable of converting cell fates and elucidate their lineage specification potential at a single-cell level. Enabling metabolic biotinylation of TFs during the process, the method allows subsequent multi-omics approaches to understand underlying action mechanisms of TFs during cell fate changes. Our pilot iTF-seq of 80 TFs in mouse embryonic stem (ES) cells has identified multiple previously known and unknown TFs triggering transcriptome changes within one day of TF induction. Subsequent tests revealed that Gcm1 and Otx2 function as activators and pioneer factors, while they resulted in distinct cell fates and gene expression programs upon induction.

overall_design:
We generated 80 cell lines to induce individual TFs in J1 ES cells. cDNAs for the TFs were individually cloned, and 80 individual stable lines were generated using a Sleeping Beauty (SB) transposon combined with a metabolic biotinylation (SBFB) system. We performed scRNA-seq of pooled cell lines upon doxycycline treatment for 1, 3, and 5 days with wildtype ES cells.

We performed RNA-seq on SBFB cells. First, we conducted time-course experiments involving the removal of Dox for Dlx5, Gata3, Gcm1, Otx2, and Pdx1 cases. In addition to maintaining continuous Dox treatment for 7 days as a control, we generated samples by discontinuing Dox after 1 day, 3 days, and 5 days of culture. Second, we conducted global gene expression profiling upon the induction of Otx2, Gcm1, and Dlx5. The samples were collected from Dox-treated cells at various time points: 0h, 3h, 6h, 12h, day 1, day 3, and day 5.

first: Jonghwan

last: Kim


title: Impact of partial reprogramming, aging, and differentiation on primary neural stem cells

first: Lucy

last: Xu

summary: Partial reprogramming is of interest as a potential strategy to improve or erase aging hallmarks. The mechanisms underlying its effect on specific cell types remain unknown. Here, we investigate early transcriptional changes that could mediate the effects of reprogramming on old neural stem cells (NSCs) and their differentiation.

overall_design: To investigate early transcriptional changes that could underlie the impact of partial reprogramming on the function of old neural stem cells, we isolated primary neural stem cells from young (3-4 months; n=5) and old (25 months; n=4) transgenic mice. We performed bulk RNA-seq for a time course of partial reprogramming in neural stem cells (3 time points) and during differentiation (3 time points). Differential expression analysis reveals genes that change during differentiation to neuronal and glial lineages; genes that differ with aging; and age-associated changes that are restored by partial reprogramming at various time points.

first: Anne

last: Brunet


title: Alternative Polyadenylation Determines the Functional Landscape of Inverted Alu Repeats

status: Public on Feb 02 2024

city: Daejeon

first: Yoosik

state: Daejeon

address: Daejeon

country: South Korea

institute: KAIST

summary: Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3′ UTRs. Yet, the biological significance of IRAlus embedded in 3′ UTRs remains largely unknown. Here, we find that 3′ UTR IRAlus silence genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3′ UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3′ UTR IRAlus and its role in human pathophysiology.

overall_design: mRNA-seq following subcellular fractionation in HeLa cells or A549 cells as well as J2 fCLIP-seq in NPCs or A549 cells. siLuc or a mixture of siCSTF2&siCSTF2T was transfected.

first: Jayoung

last: Ku


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment [scATAC-seq]

city: Shinjuku-ku

first: Taeko

last: Hayakawa

state: Tokyo

address: 35 Shinanomachi

department: Division of Tumor Immunology, Institute for Advanced Medical Research

country: Japan

institute: Keio University School of Medicine

summary: Cytotoxic T cells (CTLs) play a crucial role in the elimination of cancer cells. However, within the intra-tumor microenvironment, these cells eventually undergo cell death or enter a state of dysfunction known as "exhaustion". T cell exhaustion not only impairs the efficacy of tumor immunity but also contributes to resistance against immune-checkpoint inhibitors. Our previous investigations have demonstrated that the NR4a family of nuclear receptors, induced by chronic TCR stimulation, plays a pivotal role in driving exhaustion. These receptors enhance the transcription of inhibitory molecules, such as PD1, while suppressing the expression of functional molecules, including cytokines. However, it remains unclear at which stage of T cell differentiation NR4a exerts its influence and whether the loss of NR4a can "rejuvenate" T cell fate. In the present study, we confirmed that simultaneous deletion of NR4a1 and NR4a2 in CD8+ T cells led to a less exhausted phenotype characterized by reduced expression of PD1, Tim3, and Tox, as well as increased oxidative phosphorylation (OXPHOS) and glycolysis activities, resulting in potent suppression of tumor growth. Interestingly, the transfer of activated NR4a1-/-NR4a2-/-CD8+ T cells into tumor-bearing mice gave rise to TCF1+ (Tcf7+) early memory T cells. Wild-type PD1+TIM3+CD8+ tumor-infiltrating lymphocytes (TILs) diminished in the secondary transfer to tumor-bearing mice, while NR4a-deficient CD8+ TILs expanded even during the secondary transfer, indicating a strong stemness of memory T cells due to NR4a loss. We discovered that NR4a directly repressed the expression of Tcf7 by binding to the enhancer region of the Tcf7 gene. Collectively, these findings indicate that inhibiting NR4a in tumors represents a potent strategy for immuno-oncotherapy by enhancing stemness and reducing exhaustion of memory T cells.

overall_design: Rag2-/- mice, were subcutaneously injected with 1×106 B16-OVA cells 10 days before adoptive transfer with CD8+ T cells from WT and Cd8Cre NR4a1flox/floxNR4a2flox/flox  OT-ITg/+ mice.

first: Tanakorn

last: Srirat

first: Akihiko

last: Yoshimura


title: Versatile human cardiac tissues engineered with perfusable heart extracellular microenvironment for biomedical applications

city: Seoul

first: Sungjin

last: Min

address: 50, Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea

institute: Yonsei University

summary: To explore genetic profiles in the cardiac tissues fabricated by heart extracellular matrix (HEM) hydrogel and dynamic flow in microfluidic chip (Chip flow), we analyzed and compared differences in mRNA expression levels of the each cardiac tissue (No HEM-Plate static, HEM-Plate static, No HEM-Chip flow, HEM-Chip flow).

overall_design:
Examination of mRNA expression levels in 4 different groups
1) fabricated without HEM hydrogel and cultured in static well plate
2) fabricated with HEM hydrogel and cultured in static well plate
3) fabricated without HEM hydrogel and cultured in microfluidic chip with flow
4) fabricated with HEM hydrogel and cultured in microfluidic chip with flow

first: Suran

first: Woo-Sup

last: Sim

first: YiSun

last: Choi

first: Hyebin

last: Joo

first: Jae-Hyun

last: Park

first: Su-Jin

first: Hyeok

first: MiJeong

first: Sung-Hyun

last: Jo

first: Baofang

last: Cui

first: Jin-Ju

first: SeokBeom

last: Hong

first: Yeon-Jik

first: Kiwon

last: Ban

first: Yun-Gon

first: Hyang-Ae

first: Hun-Jun

first: Seung-Woo

last: Cho


title: Post-transcriptional splicing can occur in a slow-moving zone around the gene

city: Philadelphia

first: Allison

last: Cote

state: PA

address: 415 Curie Blvd

institute: University of Pennsylvania

summary: Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single molecule RNA FISH, expansion microscopy, and live cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription site proximal zone through which RNA move more slowly than in the nucleoplasm. Full length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

overall_design: To corroborate the use of dispersed RNA FISH signal to measure post-transcriptional splicing, we used nascent RNA sequencing. We isolated nascent RNA through metabolic labelling and cellular fractionation, then calculated a "splicing index" on a per-intron basis (see Drexler et al, 2019).

last: Coté

first: Heather

last: Drexler

first: L

middle: S

last: Churchman

first: Arjun

last: Raj


title: Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory [total RNA-seq]

city: Trento

first: CIBIO

last: Zippo

state: Not Applicable

address: via sommarive 9

department: Cellular, Computation and Integrative Biology (CIBIO)

institute: University of Trento

summary: With this experiment, we aimed to decipher the changes in gene expression during tumour progression in a model of basal-like breast cancer, based on immortalized mammary epithelial cells (IMEC). The tumorigenic IMEC, xenograft-derived and metastasis-derived cells were used for the total RNA-seq analysis.

overall_design: total RNA-seq

first: Alessio

first: Sven

last: Beyes

first: Luca

last: Fagnocchi

first: Leonardo

last: Morelli


title: ANTI-CD40 DENDRITIC CELL-TARGETING VACCINE INDUCES PROTECTIVE IMMUNITY AGAINST NIPAH DISEASE

city: CRETEIL

first: Cecile

last: LEFEBVRE

address: 51 Avenue du Maréchal de Lattre de Tassigny, Bât R+1 Eq16 U955

country: France

institute: INSERM

summary: Nipah virus (NiV) is an emerging paramyxovirus which causes severe respiratory illness and deathly encephalitis in humans.Improving the ability of vaccines to induce strong, cellular, and humoral immune responses, remains the challenge to respond to Nipah and future Henipavirus infections rapidly and efficiently. A CD40.NiV vaccine has been engineered by fusing to the anti-CD40 monoclonal Ab the ectodomain of the Nipah G protein (Bangladesh strain) and immunogenic and conserved NiV F and N peptides. In mice, CD40.NiV promotes poly-antigenic T cell responses and significantly improves anti-NiV G IgG responses compared to the non-targeted NiV G immunogenic protein, in terms of avidity and neutralization potency. Immunogenicity was confirmed in the AGM (African Green Monkey) model, with induction of cross-neutralizing sera against circulating NiV strains and Hendra virus (HeV). Challenge experiment using NiV-B strain demonstrated the high protective efficacy of the vaccine with 100% survival in vaccinated group as compared to 100% lethality in controls. Surviving animals did not exhibit NiV viral replication in the blood, organs and swabs suggesting a sterilizing immunity conferred by the CD40.NiV vaccine. Taken together, results obtained with CD40.NiV vaccine are highly promising in terms of breadth and viral efficacy against NiV.

overall_design: we have performed RNA-sequencing analysis of peripheral whole blood at D0 (before prime), and one day post prime (PP, D1), before boost (D21) and one day post boost, (PB, D22) in 9 African green monkeys (AGM) vaccinated with CD40.NiV+Poly-ICLC  at D0 and D21

first: Sylvain

last: CARDINAUD

first: Cécile

last: Lefebvre

first: Hakim

last: Hocini

first: Nora

last: El-Jahrani

first: Yadira

last: PASTOR

first: Yves

last: LEVY


title: Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory

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

overall_design: Refer to individual Series


title: Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory [HiChIP]

summary: With this experiment, we aimed to decipher the changes in 3D chromatin interactions between active regulatroy elements (enhancer, promoter) during tumour progression by performing H3K27ac HiChIP in a model of basal-like breast cancer, based on immortalized mammary epithelial cells (IMEC). The tumorigenic IMEC, xenograft-derived and metastasis-derived cells were used for the analysis.

overall_design: HiChIP

first: Daniela

last: Michelatti


title: Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory [nascent RNA-seq]

summary: We aimed to investigate the transcriptional memory of metastatic basal-like breast cancer cells  by performing nascent RNA-seq by treating with all-trans Retinoic acid (pulse, priming, priming + pulse)

overall_design: RNA-seq 5-EU labeled nascent RNA of 4 replicates per timepoint and


title: Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory [Cut&Run]

summary: With this experiment, we aimed to decipher the epigenetic changes occuring during tumour progression in a model of basal-like breast cancer, based on immortalized mammary epithelial cells (IMEC). The tumorigenic IMEC, xenograft-derived and metastasis-derived cells were used for the analysis

overall_design: Cut&Run


title: APOE4/4 is linked to damaging lipid droplets in Alzheimer's microglia

status: Public on Feb 01 2024

first: Tony

last: Wyss-Coray

department: Neurology

summary: Several genetic risk factors for Alzheimer’s Disease (AD) implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells. However, the relationship between lipid metabolism in glia and AD pathology remains poorly understood. Through single-nucleus RNA-sequencing of AD brain tissue, we have identified a microglial state defined by the expression of the lipid droplet (LD) associated enzyme ACSL1 with ACSL1-positive microglia most abundant in AD patients with the APOE4/4 genotype. In human iPSC-derived microglia (iMG) fibrillar Aβ (fAβ) induces ACSL1 expression, triglyceride synthesis, and LD accumulation in an APOE-dependent manner. Additionally, conditioned media from LD-containing microglia leads to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for AD with microglial LD accumulation and neurotoxic microglial-derived factors, potentially providing novel therapeutic strategies for AD.

overall_design: (1) Single-nucleus RNA-sequencing (snRNA-seq) on fresh-frozen frontal cortex tissue from individuals diagnosed with AD with the APOE4/4 genotype, individuals with AD and an APOE3/3 genotype and age and sex-matched control individuals with the APOE3/3 genotype. (2) RNA-seq of APOE4/4 iPSC-derived microglia (un)treated with fAβ in the presence or absence of GNE-317. (3) FACS sorted lipid droplet high and low APOE4/4 and isogenic APOE3/3 iPSC-derived microglia (iMG) followed by ATAC-seq and RNA-seq.

first: Michael

last: Haney

first: Robert

last: Palovics


title: SNORA69 is up-regulated in the lateral habenula of individuals with major depressive disorder

status: Public on Jan 31 2024

first: Gustavo

last: Turecki

state: Quebec

address: 6875 Boulevard LaSalle

department: Psychiatry

institute: McGill University

type: Non-coding RNA profiling by high throughput sequencing

summary: Major depressive disorder (MDD) is a complex and potentially debilitating illness whose etiology and pathology remains unclear. Non-coding RNAs have been implicated in MDD, where they display differential expression in the brain and the periphery. In this study, we quantified small nucleolar RNA (snoRNA) expression by small RNA sequencing in the lateral habenula (LHb) of individuals with MDD (n=15) and psychiatrically-healthy controls (n=15). We uncovered five snoRNAs that exhibited differential expression between MDD and controls (FDR<0.01). Specifically, SNORA69 showed increased expression in MDD and was technically validated via RT-qPCR. We further investigated the expression of Snora69 in the LHb and peripheral blood of an unpredicted chronic mild stress (UCMS) mouse model of depression. Snora69 was specifically up-regulated in mice that underwent the UCMS paradigm. SNORA69 is known to guide pseudouridylation onto 5.8S and 18S rRNAs. We quantified the relative abundance of pseudouridines on 5.8S and 18S rRNA in human post-mortem LHb samples and found increased abundance of pseudouridines in the MDD group. Overall, our findings indicate the importance of brain snoRNAs in the pathology of MDD, and the first study to investigate snoRNA expression in MDD. Future studies characterizing SNORA69’s role in MDD pathology is warranted.

overall_design: We utilized small RNA-seq to investigate the expression profiles of snoRNAs. Small RNA-seq was performed on human post-mortem brain tissue of 15 controls and 15 MDD cases who died by suicide.

first: Rixing

last: Lin

first: Haruka

last: Mitsuhashi

first: Laura

last: Fiori

first: Ryan

last: Denniston

first: El

last: Ibrahim

first: Catherine

last: Belzung

first: Naguib

last: Mechawar


title: Patch-Seq of synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment

city: Shanghai

first: Chun-Xiao

last: Huang

address: 65 Chifeng Road, Yangpu District

department: School of Medcine, Tongji University

summary: The goals of this study is to compare transcriptome profiles (RNA-seq) of synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment.Tg(HuC:H2B-GCaMP6f) fish of 4-5 weeks old were were fixed in a recording chamber containing extracellular fluid, and neurons with synchronized or non-synchronized calcium activity after CAS treatment were identified by two-photon imaging and collected separately with glass pipette. Single cells were transferred to the lysis buffer solution. Total RNA was extracted using TRIzol (Invitrogen) and then purified on RNeasy columns (Qiagen). Total RNA quality was assessed on a bioanalyzer (Thermofisher). RNA-Seq libraries (N=10) for each group were prepared using the Illumina TruSeq Strand mRNA Prep Kit according to the manufacturer's instructions. RNA-Seq libraries were sequenced using Illumina NextSeq 500, generating 75 bp paired-end reads for each sample. RNA-Seq reads untrimmed. Differential expression analysis was performed using the CPM (counts per million) function in the Bioconductor package edgeR (v 3.14.0). Low expression genes were excluded to make a simple correction for gene counts. Genes with P-value (instead of adjusted P-value) < 0.05 were assigned as differentially expressed. We identified differentially expressed genes (DEGs) between synchronized and non-synchronized neurons in zebrafish dorsal pallium in response to CAS treatment. Synchronized neurons expressed much higher level of glutamate transporter genes (slc17a7a, slc17a6a), while non-synchronized neurons showed significantly higher expression of gad1b, suggesting that synchronized neurons are primarily glutamatergic, while non-synchronized neurons are mainly GABAergic.

overall_design: To distinguish neurons with synchronized or non-synchronized calcium activity after CAS treatment, Tg(HuC:H2B-GCaMP6f) fish of 4-5 weeks old were fixed in a recording chamber containing extracellular fluid and subjected to two-photon imaging, and neurons were collected separately with glass pipette. RNA-Seq libraries (N=10) for each group were prepared using the Illumina TruSeq Strand mRNA Prep Kit according to the manufacturer's instructions.

first: Jianren

last: Song

last: Guan

first: Yang

last: Zhao


title: Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting a transcriptional memory [ATAC-seq]

first: CIBIO

last: Zippo

summary: With this experiment, we aimed to decipher the changes in chromatin accessibility during tumour progression in a model of basal-like breast cancer, based on immortalized mammary epithelial cells (IMEC). The tumorigenic IMEC, xenograft-derived and metastasis-derived cells were used for the analysis

overall_design: ATAC-seq

first: Alessio

first: Sven

last: Beyes

first: Luca

last: Fagnocchi

first: Naiara

last: Garcia Bediaga


title: Repositioning the early pathology of type 1 diabetes to the extra-islet vasculature.

city: La Jolla

first: Luc

last: Teyton

address: 10550 North Torrey Pines Road

department: Immunology and Microbiology

institute: Scripps Research

overall_design:
Blood was obtained from NOD mice via retro-orbital bleeding. Blood was then ficolled and PBMCs were isolated. CD4 T cells were then single cell sorted from PBMCs via MHC tetramers. Macrophages and Endothelial cells were isolated from pancreatic islets. In short, pancraetic islets were isolated form NOD pancreas via tissue harvesting and processing via enzymatic digestion and ficoll.
Cells were either sorted with pMHC tetramers or antibody surface staining.
There is no processed data file because all analysis was completed using custom R scripts and there is no need for normalization of our data.


title: Translation variation across genetic backgrounds reveals a post-transcriptional buffering signature in yeast

city: Wako

first: Shintaro

last: Iwasaki

state: Saitama

address: 2-1, Hirosawa

department: Center for Pioneering Research

institute: RIKEN

summary: Gene expression is known to vary among individuals, and this variability can impact the phenotypic diversity observed in natural populations. While the transcriptome and proteome have been extensively studied, little is known about the translation process itself. Here, we therefore performed ribosome and transcriptomic profiling on a genetically and ecologically diverse subset of natural isolates of the Saccharomyces cerevisiae yeast. Interestingly, we found that the Euclidean distances between each profile and the expression fold changes in each pairwise isolate comparison were approximately 10% higher at the transcriptomic level. This observation clearly indicates that the transcriptional variation observed in the different isolates is buffered through a phenomenon known as post-transcriptional buffering at the translation level. Furthermore, this phenomenon seemed to have a specific signature by preferentially affecting essential genes as well as genes involved in complex-forming proteins, and low transcribed genes. We also explored the translation of the S. cerevisiae pangenome and found that the accessory genes related to introgression events displayed similar transcription and translation levels as the core genome. By contrast, genes acquired through horizontal gene transfer events tended to be less efficiently translated. Together, our results highlight both the extant and signature of the post- transcriptional buffering.

overall_design: Ribosome profiling and RNA-seq of eight natural Saccharomyces cerevisiae isolates

first: Elie

last: Teyssonniere

first: Yuichi

last: Shichino

first: Anne

last: Friedrich

first: Joseph

last: Schacherer


title: NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment

first: Taeko

last: Hayakawa

relation:
SuperSeries of: GSE242050
SuperSeries of: GSE242051
SuperSeries of: GSE242052
SuperSeries of: GSE242053
SuperSeries of: GSE242054
SuperSeries of: GSE242055
SuperSeries of: GSE247640
SuperSeries of: GSE247641


title: Xrp1 governs the stress response program to spliceosome dysfunction

city: Cologne

first: Mirka

last: Uhlirova

state: NRW

address: Joseph-Stelzmann Str. 26

department: Institute for Genetics

institute: CECAD Research Center  Cologne

summary: We investigated the changes in gene expression in response to reduced levels of Ecdysoneless (Ecd), and the role of Xrp1 in induction of transcriptional changes upon loss of Ecd in the developing wing disc of Drosophila. To do this we utilized the GAL4/UAS system to express UAS-ecd-RNAi alone and in combination with UAS-xrp1-RNAi in the nubbin-domain (nub-Gal4, UAS-mRFP, UAS-ecd-RNAi and nub-Gal4, UAS-mRFP, UAS-ecd-RNAi, xrp1-RNAi) of the wing disc and used nub-Gal4, UAS-mRFP wing discs as controls.  We quantified transcriptomic expression analysis from four biological replicates for both experimental groups

overall_design: mRNA profiles were generated by single-end, total RNA Illumina sequencing (HiSeq2500) from wandering L3 stage wing discs of nub-Gal4, UAS-mRFP, UAS-ecd-RNAi; nub-Gal4, UAS-mRFP, UAS-ecd-RNAi, xrp1-RNAi and nub-Gal4, UAS-mRFP Drosophila larvae.

first: Dimitrije

last: Stankovic

first: Luke

last: Tain


title: SARS-CoV-2 causes dysfunction in human iPSC-derived brain microvascular endothelial like cells potentially by signal modulation

city: Kawasaki

first: Yasunari

last: Kanda

state: Kanagawa

address: 3-25-26 Tonomachi, Kawasaki-ku

department: Division of Pharmacology

institute: National Institute of Health Science

summary: SARS-CoV-2 is considered to affect the central nervous system (CNS) by interacting with the blood–brain barrier (BBB), which is defined by tight junctions that seal paracellular gaps between brain microvascular endothelial like cells (BMECs). Although SARS-CoV-2 infection of BMECs has been reported, the mechanism has not been fully elucidated. Here, we investigated the mechanism using iPSC derived brain microvascular endothelial like cells (iPSC-BMELCs). We observed that iPSC-BMELCs were infected with SARS-CoV-2, which resulted in inflammatory responses. RNA-seq analysis revealed that SARS-CoV-2 modulated the expression of signal molecules in iPSC-BMELCs. These findings suggest that SARS-CoV-2 infection causes BBB dysfunction in humans.

overall_design:
We investigated the infection mechanism of SARS-CoV-2 in iPSC-BMELCs.
We performed RNA-seq in SARS-CoV-2-infected iPSC-BMELCs.
Gene analysis using RNA-seq data.

first: Shigeru

last: Yamada

first: Tadahiro

last: Hashita

first: Shota

last: Yanagida

first: Hiroyuki

last: Sato

first: Yukuto

last: Yasuhiko

first: Kaori

last: Okabe

first: Takamasa

last: Noda

first: Motohiro

last: Nishida

first: Tamihide

last: Matsunaga


title: Rejuvenating Aged Immune Systems

city: Palo Alto

first: Jason

last: Ross

address: 265 Campus Drive

summary: We performed gene expression analysis of mouse HSCs isolated from aged (11 month) Control mice or Treated mice, which received antibody conditioning.

overall_design: We performed gene expression analysis of mouse HSCs (KLS FLT3-CD34-CD150+) isolated from aged (11 month) mice that are Control (n=3) or Treated (n=3). Treated mice receiving antibody conditioning and isolation was performed at day 9.

middle: B

first: Lara

last: Myers

first: Kim

middle: J

last: Hasenkrug

first: Irving

last: Weissman