title: Effect of depletion of atp-2 by RNA interference on gene expression during the development of Caenorhabditis elegans

city: Taipei

first: chun-liang

last: Pan

address: Taipei City , Taiwan (R.O.C.)

country: Taiwan

institute: National Taiwan University

summary: Disruption of physiological homeostasis elicits cellular stress response and behavioral changes of the animal. We recently showed that mitochondrial stress induces aversive associative memory for food bacteria that C. elegans inherently prefers without stress. Depletion of atp-2 by RNA interference in the non-neural tissues causes mitochondrial disruption and induces bacterial avoidance in C. elegans. Our transcriptome provide several metabolic pathways that may are critical for aversive memory.  Among these pathwaies, we found that peroxisomal β-oxidation mediates serotonergic neuron,NSM, to promote stress-induced aversive memory.

overall_design:
To dissect the potentail pathways that drive aversive memory, We harvested the control worms and worms under atp-2 depletion by RNAi
We then performed gene expression profilling using the data from RNA sequencing of control and atp-2 RNAi group
We then performed Gene ontology analysis of the RNA sequencing data and found the pathways which may be critical for stress-induced aversive memory

first: Yu-Chun

last: Wu

first: Shang-Heng

last: Tsai


title: BRG1 programs PRC2-complex repression and controls oligodendrocyte differentiation and remyelination

status: Public on Mar 25 2024

city: Cincinnati

first: Richard

last: Lu

state: OH

address: 3333 Burnet Ave

department: CBDI

country: USA

institute: Cincinnati Children's Hospital Medical Center

type: Expression profiling by high throughput sequencing

summary: Mutations in Brg1 can cause Coffin-Siris syndrome, where a patient had white matter defects and partial agenesis of the corpus callosum; however, Brg1 functions in CNS myelination and remyelination is unsure. We show that PDGFRa expressed prior than NG2, depletion of Brg1 at PDGFRα+ OPC leads to OPC differentiation restriction and myelin defects, also, Brg1 is critical for oligodendrocyte remyelination. Genomic occupancy and transcriptome analyses indicate that Brg1 promotes H3K27me3 and neuronal genes. Thus our findings reveal that Brg1 is a critical epigenetic programmer of CNS myelination and repair through recruiting H3K27me3 and neuronal genes, suggesting potential strategies of therapeutic intervention for Brg1-associated white matter defects.

overall_design: 2 Control Samples and 2 Mutant

first: Jiajia

last: Wang

first: Lijun

last: Yang

first: Qing

middle: R


title: Temporal dynamics of signaling and transcription underlying early adaptation to oncogenic BRAF inhibition in melanoma

city: SEATTLE

first: Wei

last: Wei

state: Washington

address: 401 Terry Ave N

institute: Institute For Systems Biology

summary: Despite the recent remarkable progress of targeted therapies for the clinical management of many cancers, response rates remain lower than desired and long-term response durability is often poor due to drug resistance. A major contributor to treatment failure is drug-induced cellular adaptation to therapy, whereby systems-level reprogramming permits a drug-tolerant phenotype that may gradually lead to cell cycle re-entry and portend relapse. Drug adaptation is known to rapidly implicate complex signaling and transcriptional regulatory networks, but the scale and temporal dynamics of these remodeling events have yet to be fully resolved. Here, we used mass spectrometry-based phosphoproteomics and RNA sequencing to capture molecular snapshots within the first minutes, hours, and days of BRAF kinase inhibitor exposure in a drug-tolerant human BRAF-mutant melanoma model. By enriching specific phospho-motifs associated with mitogenic kinases, we were able to detect and monitor thousands of protein phosphorylation sites over a three-day period of drug treatment, followed by a six-day drug holiday to characterize the plasticity of the adaptive response. We observed early and sustained inhibition of the BRAF-ERK axis, gradual downregulation of canonical cell cycle-dependent phosphorylation events, and a clear delineation between three distinct phase transitions toward drug tolerance, which was almost completely reversible following drug removal. Through measuring the time evolution of large-scale networks, we were able to exploit phosphoproteome and transcriptome dynamics to infer kinetically-defined regulatory modules, revealing a concerted response to oncogenic BRAF inhibition involving cellular metabolism, RNA processing, and mitogenic signaling that demonstrated strong agreement with prior knowledge. The adaptive response to BRAF inhibition was dominated by a compensatory induction of SRC-family kinase (SFK) signaling, which we found to be at least partially driven by impaired redox homeostasis and accumulation of reactive oxygen species – providing a fine degree of temporal, mechanistic, and phosphorylation site-specific context to an important axis in tumor cell survival. This induction sensitized cells to co-treatment with an SFK inhibitor, and combination therapy significantly outperformed single-agent BRAF inhibition across a panel of patient-derived melanoma cell lines and in an orthotopic mouse xenograft model, underscoring the high translational potential for measuring the temporal dynamics of signaling and transcription networks under therapeutic challenge.

overall_design: To characterize concomitant transcriptomic alterations following signaling network rewiring, bulk mRNA sequencing (RNA-seq) was performed on VEM-treated M397 cells at select timepoints

first: Cameron

middle: T

last: Flower

first: Chunmei

last: Liu


title: Interferon signaling in the nasal epithelium distinguishes among lethal and common cold respiratory viruses and is critical for viral clearance

city: Philadelphia

first: David

middle: Michael

last: Renner

state: Pennsylvania

address: 3610 Hamilton Walk

department: Microbiology

institute: University of Pennsylvania

summary: All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at air-liquid interface (ALI). HCoV-229E, HCoV-NL63 and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33ºC) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally-directed IFNs as potential therapeutics.

overall_design:
To investigate and comapre the differences in interferon signaling in nasal cell cultures infected with multiple respiratory viruses, we pooled primary nasal epithelial cells from four to six donors and grew them in air-liquid interface cultures. These cultures were then mock infected or infected with the indicated viruses at MOI = 1 for 96 hours (MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-NL63) or 48 hours (IAV and HRV-16).
We examined differences in gene expression using data obtained from bulk RNA sequencing from each infection in triplicate (duplicate for IAV).
Differential gene expression analysis was done by comparing gene expression levels in each infection condition compared to the respective mock infection for that experiment (Mock vs. MERS-CoV, Mock vs. IAV, etc.).

middle: M

first: Clayton

middle: J

last: Otter

first: Susan

last: Weiss

first: Noam

middle: A

last: Cohen


title: Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells [CRISPRi]

status: Public on Mar 24 2024

city: San Francisco

first: Alexander

last: Marson

state: CA

address: 1650 Owens Street

institute: Gladstone-UCSF Institute of Genomic Immunology

summary: Cis-regulatory elements (CRE) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of adjacent T cell costimulatory genes CD28, CTLA4, and ICOS encoding regulators of cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells – both Conventional and Regulatory subsets – uncovered gene-, cell subset- and stimulation-specific CREs. Integrating these data with CRISPR knockout (KO) screens and ATAC-seq characterization identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. Lastly, we discovered and extensively validated a critical CTCF boundary that governs this locus, serving to reinforce CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.

overall_design: Primary human Conventional and Regulatory T cells were isolated, stimulated as indicated, and sorted for low and high target protein expression. Short guide RNA abundances were quantified to map CRISPRi Responsive Elements.

first: Cody

last: Mowery


title: Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells [ATAC-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Primary human Treg cells were isolated, subjected to AAVS1 control or FOXP3 knockout, and harvested for ATAC-seq.

first: Bence

last: Daniel

first: Katalin

last: Sandor

first: Ansuman

last: Satpathy


title: Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells

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

overall_design: Refer to individual Series


title: Gene expression profiles of CAR-T cells engineered with chimeric cytokine receptors to induce JAK-STAT signaling

city: Tokyo

first: Yuki

last: Kagoya

address: 35 Shinanomachi, Shinjuku

department: School of Medicine

country: Japan

institute: Keio University

summary: The efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy is insufficient in most cancers. However, enhancing the antitumor T cell response inevitably increases the risk of cytokine release syndrome associated with monocyte-derived IL-6 secretion. In this study, we developed a chimeric cytokine receptor consisting of the extracellular domains of GP130 and IL6RA linked to the transmembrane and cytoplasmic domain of IL-7R mutant (insertion of PPCL) (G6/7R). We also generated a modified receptor with the M452L mutation in the IL7R cytoplasmic domain (G6/7R-M452L). CAR-T cells with G6/7R or G6/7R-M452L efficiently absorbed monocyte-derived IL-6 and induced a superior therapeutic response compared to conventional CAR-T cells. Our strategy can be broadly applied to CAR-T cell therapy to enhance its efficacy and safety, irrespective of the target antigen.

overall_design: Human CD8+ T cells derived from three different healthy donors were retrovirally transduced with an anti-CD19 CAR gene (FMC63-28z) or the CAR gene + G6/7R with or without the M452L mutation. CAR-T cells were then stimulated with the CD19+ B-cell acute lymphoblastic leukemia cell line NALM-6. RNA was collected, and gene expression profiles were analyzed by RNA sequencing.

first: Toshiaki

last: Yoshikawa


title: Differential genome-wide profile of histone modifications upon treatment with gamma-secretase inibithor (GSI) DAPT [ChIP-seq]

first: Tobias

last: Friedrich

address: 421 Curie Boulevard

summary: The Notch signaling pathway regulates several processes including development and differentiation and its aberrant regulation leads to several diseases. Here, we investigated the differential chromatin accessibility, genome-wide distribution of the transcription factor RBPJ and the differential regulation of H3K4me1 and H3K4me3 upon pharmacological inhibition of the Notch pathway with gamma-secretase inibibitor (GSI) DAPT in mouse pre-T (Beko) cells.

overall_design: Mouse pre-T cells (Beko) were treated with gamma-secretase inhibitor (GSI) DAPT) and subsequently analyzed by ChIP-Seq using antibody directed against different histone modifications.

first: Benedetto

middle: D

last: Giaimo

first: Francesca

last: Ferrante

first: Marek

last: Bartkuhn

first: Tilman

last: Borggrefe


title: Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells [4C-seq]

overall_design: Primary human T cells were isolated, transduced with dCas9-ZIM3 and either control or CTCF-2 sgRNAs, and 4C-seq libraries prepared.

first: Zeyu

last: Chen

first: Salvador

last: Casaní-Galdón

first: Bradley

middle: E

last: Bernstein


title: ID3 is a novel target gene of p53 and modulates lung cancer cell metastasis

status: Public on Mar 26 2024

city: Nagoya

first: Mai

last: Nagasaka

address: 3-1 Tanabe-dori, Mizuhoku

institute: Nagoya City University

summary: The tumor suppressor p53 prevents cancer development through regulation of dozens of target genes with diverse biological functions. Numerous p53 target genes have been identified to date, whereas the dynamics and function of the regulatory network centered on p53 have not yet been fully elucidated. To identify novel p53 target genes, we performed RNA sequencing and differential expression analysis on MCF7 cells treated with DMSO or Nutlin-3, a nongenotoxic small molecule that activates p53 through disruption of p53-MDM2 interaction. Among the top 15 differentially expressed genes, we found a potent transcriptional target of p53.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for human breast MCF7 cells treated with DMSO and those of treated with Nutlin-3.

first: Chiharu

last: Miyajima

first: Yasumichi

last: Inoue

first: Sakura

last: Hashiguchi

first: Yuya

last: Suzuki

first: Daisuke

last: Morishita

first: Hiromasa

last: Aoki

first: Kohki

last: Toriuchi

first: Ryohei

last: Katayama

first: Mineyoshi

last: Aoyama

first: Hidetoshi

last: Hayashi


title: Circulating Hematopoietic Stem/Progenitor Cell subsets contribute to human hematopoietic homeostasis

city: Milano

first: Guido

last: Pacini

address: Via Olgettina 60

department: Pathogenesis and therapy of primary immunodeficiencies

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

summary: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPC) are present in peripheral blood (PB) and their role remain unsolved. By integrating advanced immunophenotyping, high-throughput transcriptome profiling and functional assays we characterized the biological properties of human cHSPC. We found distinct composition but comparable bone marrow (BM) homing and differentiation potential after xenotransplantation of cHSPC with respect to their BM counterpart. Trafficking HSPC are in a low-cycling pre-activated state with primitive and committed progenitors being transcriptionally and functionally skewed towards erythroid differentiation. Moreover, a subset of circulating lymphoid progenitors are primed for seeding lymphoid organs and can actively contribute to T-cell production in patients treated with gene therapy. In case of hematopoietic impairment, cHSPC content and composition correlate with BM-HSPC state. Altogether, our data unveiled the cHSPC contribution to steady-state hematopoiesis, with a key function in sustaining extramedullary lympho- and erythro-poiesis, and supported their clinical exploitation as biomarker for BM-HSPC.

overall_design: To compare the intrinsic molecular properties of HSPC subsets derived from BM and PB, we analyzed their transcriptional profiles by CITE-seq, which allows unbiased single-cell transcriptome and immunophenotypic analysis. To this aim, we exploited antibody derived tag (ADT)-labeled antibodies directed toward HSPC phenotypic markers. Human BM CD34+ (n=5) or PB CD34+ cells (n=4) or total PBMC (n=1) samples were thawed and stained with a mix of FACS antibodies and ADT-barcoded antibodies directed toward surface antigens used for the phenotypic classification of human HSPC. Then, we FACS sorted primitive (Lin- CD34+ CD38-) and/or bulk (Lin- CD34+) or committed (Lin- CD34+ CD38+) HSPC fractions, depending on the sample. Sorted fractions were processed according to Chromium 10x protocol, generating for each sample separated sequencing libraries from cDNA or ADT enriched fractions. Libraries were subsequently sequenced and resulting data were analyzed and integrated by bioinformatic approaches.

first: Pamela

last: Quaranta

first: Luca

last: Basso-Ricci

first: Raisa

last: Jofra Hernandez

first: Matteo

last: Naldini

last: Barcella

first: Giulia

last: Pais

first: Giulio

last: Spinozzi

first: Fabrizio

last: Benedicenti

last: Seffin

first: Carlo

last: Pietrasanta

first: Jin-Gyu

last: Cheong

first: Andrea

last: Ronchi

first: Lorenza

last: Pugni

first: Francesca

last: Dionisio

first: Ilaria

last: Monti

first: Stefania

last: Giannelli

first: Silvia

last: Darin

first: Federico

last: Fraschetta

first: Graziano

last: Barera

last: Ferrua

first: Valeria

last: Calbi

first: Marco

last: Ometti

first: Raffaella

last: Di Micco

first: Fabio

last: Mosca

first: Steven

last: Zvi Josefowicz

first: Eugenio

last: Montini

last: Calabria

first: Maria

last: Ester Bernardo

last: Pia Cicalese

first: Bernhard

last: Gentner

first: Ivan

last: Merelli

first: Alessandro

last: Aiuti

first: Serena

last: Scala


title: Gene expression profiles of chimeric cytokine receptor-engineered CAR-T cells

first: Yuki

last: Kagoya

summary: The efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy is suboptimal in most of the cancer types , necessitating further improvement in their therapeutic actions. However, enhancing antitumor T cell response inevitably confers an increased risk of cytokine release syndrome associated with monocyte-derived IL-6 secretion. Thus, a novel approach to simultaneously enhance therapeutic efficacy and safety is warranted. In this study, we developed a chimeric cytokine receptor composed of the extracellular domains of GP130 and IL6RA linked to the transmembrane and cytoplasmic domain of IL-7R mutant that constitutively activate JAK-STAT pathway (G6/7R). CAR-T cells with G6/7R efficiently absorbed and degraded monocyte-derived IL-6 both in vitro and in vivo. The G6/7R-expressing CAR-T cells showed superior expansion and persistence in vivo, which resulted in durable antitumor response in multple tumor models. Our strategy can be widely applicable to CAR-T cell therapy to enhance its efficacy and safety, irrespective of the target antigen.

overall_design: Human CD8+ T cells derived from three different healthy donors were retrovirally transduced with an anti-CD19 CAR gene (FMC63-28z) and stimulated with the CD19+ B-cell acute lymphoblastic leukemia cell line NALM-6 for three times. RNA was collected after the third stimulation, and gene expression profiles were analyzed by RNA sequencing.

first: Toshiaki

last: Yoshikawa


title: Differential genome-wide profile of RBPJ, H3K4me1 and H3K4me3 as well as differential chromatin accessibility upon treatment with gamma-secretase inibithor (GSI) DAPT (ATAC-Seq).

first: Tobias

last: Friedrich

overall_design: Mouse pre-T cells (Beko) were treated with gamma-secretase inhibitor (GSI) DAPT) and subsequently analyzed by ChIP-Seq using an RBPJ-specific antibody or antibody directed against H3K4me1 or H3K4me3 or ATAC-Seq to analyze chromatin accessibility.

first: Benedetto

last: Giaimo

first: Francesca

last: Ferrante

first: Marek

last: Bartkuhn

first: Tilman

last: Borggrefe


title: The microbiota and T cells non-genetically modulate inherited phenotypes transgenerationally

first: Natalie

middle: Alyce

last: Trigg

address: 421 Curie Blvd

institute: Children's Hospital of Philadelphia

type: Non-coding RNA profiling by high throughput sequencing

summary: The host-microbiota relationship has evolved to shape mammalian physiology, including immunity, metabolism, and development. Germ-free models are widely used to study microbial effects on host processes such as immunity. Here, we find that both germ-free and T cell-deficient mice exhibit a robust sebum secretion defect persisting across multiple generations despite microbial colonization and T cell repletion. These phenotypes are inherited by progeny conceived during in vitro fertilization using germ-free sperm and eggs, demonstrating that non-genetic information in the gametes is required for microbial-dependent phenotypic transmission. Accordingly, gene expression in early embryos derived from gametes from germ-free or T-cell deficient mice are strikingly and similarly altered. Our findings demonstrate that microbial and immune-dependent regulation of non-genetic information in the gametes can transmit inherited phenotypes transgenerationally in mice. This mechanism could rapidly generate phenotypic diversity to enhance host adaptation to environmental perturbations.

overall_design: To investigate transcriptomic, transgenerational differences in the presence of an altered microbiome, we performed several sequencing experiments comparing tissues from mice derived from conventionally raised (CR) parents, germ-free (GF) parents, and/or a GF male parent crossed to a CR female parent. We performed mRNA sequencing on F0 and F1 sebaceous glands, F1 and F2 back skin, F1 small intestine, F1 liver, and 4 cell and morula stage embryos. To further investigate the influence of the immune system on RNA expression in gametes and embryos, we performed mRNA sequencing on embryos derived from WT eggs fertilized with RAG knockout sperm.

first: Jordan

middle: C

last: Harris

first: Bruktawit

last: Goshu

first: Yuichi

last: Yokoyama

first: Lenka

last: Dohnalová

first: Ellen

middle: K

last: White

first: Adele

last: Harman

first: Sofia

last: Murga-Garrido

first: Jamie

last: Ting-Chun Pan

first: Preeti

last: Bhanap

first: Christoph

last: Thaiss

first: Elizabeth

last: Grice

first: Colin

last: Conine

first: Taku

last: Kambayashi


title: Notch

first: Tobias

last: Friedrich


title: The intellectual disability risk gene Kdm5b regulates long term memory consolidation in the hippocampus

city: London

first: Leticia

last: Perez Sisques

address: Great Maze Pond

department: Centre for Craniofacial and Regenerative Biology

country: United Kingdom

institute: King's College London

summary: The histone lysine demethylase KDM5B has been implicated in recessive intellectual disability disorders and heterozygous, protein truncating variants in KDM5B are associated with reduced cognitive function in the normal adult population. To help distinguish between developmental and demethylase-dependent functions of KDM5B in hippocampus-dependent learning and memory, we studied mice homozygous for a Kdm5bDARID allele that lacks demethylase activity. Demethylase-deficient Kdm5bDARID/DARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in mice in the home cage (baseline) and hyperactivated upon learning stimulus compared to wildtype mice. The expression of other learning-associated genes was also significantly altered in the Kdm5bDARID/DARID hippocampus. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on learning and memory mechanisms.

overall_design: Total RNA was extracted using Trizol Reagent. RNA was next purified with the Monarch total RNA miniprep kit (New England Biolabs). Hippocampal mRNA profiles of WT and Kdm5bDARID/DARID mice at different timepoints (baseline and 1h and 3h post fear conditioning) were generated by sequencing on the Illumina Nextseq 6000.

last: Perez-Sisques

first: Shail

last: Bhatt

first: Rugile

last: Matuleviciute

first: Talia

last: Gileadi

first: Eniko

last: Kramar

first: Andrew

last: Graham

first: Franklin

middle: G

last: Garcia

first: Ashley

last: Keiser

first: Dina

middle: P

last: Matheos

first: James

last: Cain

first: Alan

last: Pittman

first: Cathy

last: Fernandes

first: Marcelo

last: Wood

first: K

last: Giese

first: M

last: Basson


title: Divergent local and systemic antitumor response in primary uveal melanomas

status: Public on Mar 23 2024

city: PARIS

first: Anne-laure

last: Le gac

address: 26 rue d'ulm

country: France

institute: Institut Curie

summary: Uveal melanoma (UM) is the most common cancer of the eye. The loss of chromosome-3 (M3) is associated with high risk of metastases. M3-tumors are more infiltrated by T-lymphocytes than low-risk disomic-3 (D3) tumors, contrasting with other tumor types in which T-cell infiltration correlates with better prognosis. Whether these T-cells represent an anti-tumor response and how these T-cells would be primed in the eye are both unknown. Herein, we characterized the T-cells infiltrating primary UMs. CD8+ and Treg cells were more abundant in M3- than in D3-tumors. CD39+PD-1+CD8+ T-cells were enriched in M3-tumors suggesting specific responses to tumor-antigen (Ag) as confirmed using HLA-A2:Melan-A tetramers. ScRNAseq-VDJ analysis of T-cells evidenced high numbers of proliferating CD39+PD1+CD8+ clonal expansions suggesting in-situ anti-tumor Ag responses. TCRseq and tumor-Ag tetramer staining characterized the recirculation pattern of the anti-tumor responses in M3- and D3-tumors. Thus, tumor-Ag responses occur in localized UMs raising the question of the priming mechanisms in the absence of known lymphatic drainage.

overall_design:
we studied the phenotype of the T cells found in primary UM at the time of diagnosis using multicolor flow cytometry, single cell transcriptomic (scRNAseq) associated to single cell TCR repertoire analysis with multiplex tetramer staining and bulk TCR sequencing for alpha and beta chains.
CITE_Seq Demultiplexing HLA-A2:Melan-A HTO Barcode_sequence
COH4_84 HLA-A2:Melan-A AACCTTTGCCACTGC
COH4_56 HLA-A2:Melan-A AACCTTTGCCACTGC
COH4_76 HLA-A2:Melan-A AACCTTTGCCACTGC

first: Francesca

last: Lucibello

first: Ana

middle: I

last: Lalanne

first: Anne-Laure

last: Le Gac

first: Olivier

last: Lantz


title: Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury

city: New York City

first: Alessandro

last: Vasciaveo

state: NY

address: 1130 Saint Nicholas Ave., 910

department: Department of Systems Biology

institute: Columbia University

summary: The currently accepted intestinal epithelial cell organization model equates crypt base columnar (CBC) cells, marked by high levels of Lgr5 expression, with the intestinal stem cell (ISC). However, recent intestinal regeneration studies have uncovered limitations of the ‘Lgr5-CBC’ model, leading to two major views: one favoring the presence of a quiescent reserve stem cell population, the other calling for differentiated cell plasticity. To test if an alternative model may help reconcile these perspectives, we studied the hierarchical organization of crypt epithelial cells in an unbiased fashion, by combining high-resolution, single-cell profiling and lineage tracing in multiple transgenic mouse models. These show that Lgr5 is not a specific ISC marker; rather, cells located in the crypt isthmus, which include Lgr5low cells, comprise the ISCs that sustain tissue homeostasis. Following irradiation or intestinal injury, surviving ISCs and progenitors, but not differentiated cells, participate in intestinal regeneration, suggesting that neither de-differentiation nor reserve stem cell populations are drivers of intestinal regeneration. Our results provide a novel viewpoint for the intestinal crypt epithelium, in which ISCs localize to the crypt isthmus, and ISC potential is restricted to stem and progenitor cells.

overall_design: Single-cell RNA-Seq data of highly purified mouse intestinal crypt epithelial cells

first: Ermanno

last: Malagola

first: Andrea

last: Califano

first: Timothy

last: Wang


title: Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells [Transko]

first: Alexander

last: Marson

overall_design: Primary human T cells were isolated, transduced with a transcription factor sgRNA library, nucleofected with Cas9 complexes, and sorted for low and high target protein expression. Short guide RNA abundances were quantified to identify trans regulators that significantly act on the target genes.

first: Cody

last: Mowery

first: Jacob

last: Freimer

first: Christian

last: Garrido


title: Differential Notch target genes expression profile upon GSI washout.

first: Tobias

last: Friedrich

summary: The Notch signaling pathway regulates several processes including development and differentiation and its aberrant regulation leads to several diseases. Here, we analyzed the gene expression profile upon washout of the Notch inhibitor DAPT in mouse pre-T (Beko) cells.

overall_design: Mouse pre-T cells (Beko) were treated with gamma-secretase inhibitor (GSI) DAPT for 48 hours. After washing out the inhibitor, cells were placed back in culture for 24 hours before analyzing the gene expression profile by RNA-seq.

first: Benedetto

last: Giaimo

last: Ferrante

first: Marek

last: Bartkuhn

first: Tilman

last: Borggrefe


title: A stress sensor IRE1α is required for bacterial exotoxin-induced interleukin-1β production in tissue-resident macrophages

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarified the roles of IRE1α , an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production from RPMs. In RPMs, CTB is incorporated into ER and induced ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α -deficient RPMs showed a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α  was required for CT- or CTB-induced IL-1β production from RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

overall_design: comprehensive analysis of CT or CTB-induced genes in LPS-primed RPMs, comprehensive analysis of CT or Tm-induced genes in resting RPMs

first: Izumi

last: Sasaki

first: Yuri

last: Fukuda-Ohta

first: Chihiro

last: Nakai

first: Naoko

last: Wakaki-Nishiyama

first: Chizuyo

last: Okamoto

last: Okuzaki

first: Shuhei

last: Morita

first: Shiori

last: Kaji

first: Yuki

last: Furuta

first: Hiroaki

last: Hemmi

first: Takashi

last: Kato

first: Asumi

last: Yamamoto

first: Emi

last: Tosuji

first: Shin-Ichiroh

last: Saitoh

last: Tanaka

first: Katsuaki

last: Hoshino

first: Shinji

last: Fukuda

first: Kensuke

last: Miyake

first: Etsushi

last: Kuroda

first: Ken

last: Ishii

first: Takao

last: Iwawaki

first: Koichi

last: Furukawa

first: Tsuneyasu

last: Kaisho


title: Mapping putative enhancers in mouse oocyte and early embryo reveals TCF3/12 as key folliculogenesis regulators

city: Beijing

first: Bofeng

last: Liu

address: Haidian District

department: School of Life Science

country: China

institute: Tsinghua University

summary: Dynamic epigenomic reprogramming occurs during mammalian oocyte maturation and early development. However, the underlying transcription circuitry remains poorly characterized. By mapping cis-regulatory elements using H3K27ac, we identified putative enhancers in mouse oocytes and early embryos distinct from those in adult tissues, enabling global transitions of regulatory landscapes around fertilization and implantation. Gene deserts harbor prevalent putative enhancers in fully-grown oocytes linked to oocyte-specific gene and repeat activation. Embryo-specific enhancers are primed prior to zygotic genome activation and are restricted by oocyte-inherited H3K27me3. Putative enhancers in oocytes often manifest H3K4me3, bidirectional transcription, Pol II binding, and can drive transcription in STARR-seq and a reporter assay. Finally, motif analysis of these elements identified crucial regulators of oogenesis – TCF3 and TCF12, the deficiency of which impairs activation of key oocyte genes and folliculogenesis. These data reveal distinctive regulatory landscapes and their interacting TFs that underpin the development of mammalian oocytes and early embryos.

overall_design: STAR ChIP-seq, ATAC-seq , RNA-seq, STARR-seq, and DamID were applied on mouse oocytes, pre-implantation embryos, and cell lines.

first: Wei

last: Xie


title: Differential genome-wide profile of RBPJ, H3K4me1 and H3K4me3 as well as differential chromatin accessibility upon treatment with gamma-secretase inibithor (GSI) DAPT (ChIP-Seq).

first: Tobias

last: Friedrich

first: Benedetto

last: Giaimo

first: Francesca

last: Ferrante

first: Marek

last: Bartkuhn

first: Tilman

last: Borggrefe


title: IFRD1 promotes tumor cells “low-cost” survival under glutamine starvation via inhibiting histone H1.0 nucleophagy

city: hefei

first: sun

last: xuedan

address: daoxiangcun

institute: ustc

summary: Glutamine addiction represents a metabolic vulnerability of cancer cells although effective therapeutic targeting of the pathways involved remains to be realized. Here, we disclose the critical role of IFRD1 (interferon-related developmental regulator 1) in the adaptive survival of hepatocellular carcinoma cells (HCC) during glutamine starvation. The induction and translocation of IFRD1 to the endoplasmic reticulum inhibits autophagy by promoting proteasomal degradation of the key autophagy regulator ATG14 in a TRIM21-dependent manner. On the contrary, enforced IFRD1 loss increases autophagy flux leading to cell death. This effect is largely realized through the autophagy-dependent degradation of histone H1.0 causing globally enhanced chromatin accessibility associated with unchecked increases in ribosome and protein biosynthesis . This in turn leads to metabolic exhaustion and death under glutamine starvation. Practically, IFRD1 depletion in preclinical HCC models potentiates glutamine limiting strategies including synergizing with the glutaminase-1 selective inhibitor CB-839. Thus, IFRD1 supports the adaptive survival of cancer cells under glutamine starvation, with potential as a therapeutic target in anti-cancer applications.

overall_design: 1. RNA seq for mice bearing HepG2 xenografts with the glutaminase inhibitor CB-8392. 2.  ATAC-seq for  IFRD1 knockout and wildtype PLC/PRF/5 cells under glutamine starvation

first: Yabin

last: Huang

first: Mian

last: Wu

first: Xuedan

last: Sun

first: lianxin


title: RNA-seq analysis of Sphingosine 18:1 treated mice bone marrow-derived macrophages

status: Public on Mar 21 2024

first: Chuan

last: Ye

address: 38 Xueyuan Road, Haidian District

institute: Peking University

summary:
Purpose: Study of the influence of Sphingosine 18:1 in macrophages
Method: 2μM Sphingosine 18:1 was added to BMDMs for 6 hours, solvent was added as control. mRNA profiles were generated by deep sequencing.
Results: Several signaling pathways were changed after Sphingosine administration, including hypoxia-related signal pathway. RT-quantitive PCR was performed to validate the expression of Epas1 and its downstream target genes, results showed that Sphingosine 18:1 significantly inhibits Epas1 expression.

overall_design: Expression profiling by RNA-seq, profiling of BMDMs after Sphingosine administration.

first: J

last: Xia

first: H

last: Chen

first: X

last: Wang

last: Rao

first: C

last: Jiang

first: Y

last: Pang


title: Genome organization around nuclear speckles drives mRNA splicing efficiency

status: Public on Mar 22 2024

city: Pasadena

first: Mitchell

last: Guttman

address: 1200 E. California Blvd

department: Biology and Biological Engineering

institute: California Institute of Technology

summary: The nucleus is highly organized such that factors involved in transcription and processing of distinct classes of RNA are organized within specific nuclear bodies. One example is the nuclear speckle, which is defined by high concentrations of protein and non-coding RNA regulators of pre-mRNA splicing. What functional role, if any, speckles might play in the process of mRNA splicing remains unknown. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs, and higher co-transcriptional splicing levels relative to genes that are located farther from nuclear speckles. We show that directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Finally, we show that gene organization around nuclear speckles is highly dynamic with differential localization between cell types corresponding to differences in splicing efficiency. Together, our results integrate the longstanding observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a critical role for dynamic 3D spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
10 minutes of 5EU was used to sequence nascent RNA in mouse embryonic stem cells.

RNA-DNA or DNA SPRITE was performed on human H1 embryonic stem cells or mouse skeletal myoblasts.

first: Prashant

last: Bhat


title: Gene expression profile at single cell level of FACS-sorted blood NK cells from kidney transplant recipients

city: Pittsburgh

first: Elodie

last: Bailly

state: PA

address: BST 200 lothrop street

department: Surgery

institute: Starzl Transplant Institute

summary: We explored NK cell differential gene expression profiles in patients with antibody-mediated rejection versus T-cell mediated rejection.

overall_design: From frozen PBMC, circulating NK cells from 8 patients (4 antibody-mediated rejection and 4 T-cell mediated rejection) were FACS-sorted. Samples were at the time of biopsy-proven rejection. Cells were processed for 3 prime V3 single cell RNA sequencing. Cell were hashed to process 4 samples together. Fastq files were demultiplexed by Cell Ranger v6 multi.

first: Tracy

last: Tabib

first: Diana

last: Metes

first: Robert

last: Lafyatis


title: Targeting the HSP47-Collagen Axis Inhibits Brain Metastasis by Reversing M2 Microglial Polarization and Restoring Anti-Tumor Immunity

city: Guangzhou

first: Li

address: 1023 S Shatai Rd, Life Science BLDG 15B03

department: Department of Cell Biology

institute: Southern Medical University

summary: Brain metastases (BrMs) are the leading cause of death in patients with solid cancers. BrMs exhibit a highly immunosuppressive milieu and poor response to immunotherapies; however, the underlying mechanism remains largely unclear. Here, we show that upregulation of HSP47 in tumor cells drives metastatic colonization and outgrowth in brain by creating an immunosuppressive microenvironment. HSP47-mediated collagen deposition in the metastatic niche promotes microglial polarization to M2 phenotype via the α2β1 integrin/NF-κB pathway, which upregulates the anti-inflammatory cytokines and represses CD8+ T cell anti-tumor responses. Depletion of microglia reverses HSP47-induced inactivation of CD8+ T cells and abolishes brain metastasis. Col003, an inhibitor disrupting HSP47-collagen association restores an anti-tumor immunity and enhances the efficacy of anti-PD-L1 immunotherapy in BrMs-bearing mice. Our study supports that HSP47 is a critical determinant of M2 microglial polarization and immunosuppression, and blocking the HSP47-collagen axis represents a promising therapeutic strategy against brain metastatic tumors.

overall_design: To elucidate the mechanism underlying that COL1 promotes M2 microglial polarization, we cultured BV2 cells in a 3D COL1 gel and then profiled gene expression by RNA-seq.

first: Cuiying

last: Li

first: Hongchao

last: Zhan


title: Parental conflict driven regulation of endosperm cellularization by a family of Auxin Response Factors

city: Uppsala

first: Juan

last: Santos-González

state: Uppsala

address: Almas Allé 5

department: Department of Plant Biology and Forest Genetics

country: Sweden

institute: SLU

summary: The endosperm is a reproductive tissue supporting embryo development. In most flowering plants, the initial divisions of endosperm nuclei are not succeeded by cellularization; this process occurs only after a specific number of mitotic cycles have taken place. The timing of cellularization significantly influences seed viability and size. Previous research implicated auxin as a key factor in initiating nuclear divisions and determining the timing of cellularization. Here we uncover the involvement of a family of clustered auxin response factors (cARFs) as dosage-sensitive regulators of endosperm cellularization. cARFs, maternally expressed and paternally silenced, are shown to induce cellularization, thereby restricting seed growth. Our findings align with the predictions of the parental conflict theory, suggesting that cARFs represent major molecular targets in this conflict. We further demonstrate a recurring amplification of cARFs in the Brassicaceae, suggesting an evolutionary response to parental conflict by reinforcing maternal control over endosperm cellularization. Our study highlights that antagonistic parental control on endosperm cellularization converges on auxin biosynthesis and signalling.

overall_design: Each library was made from around 500 seeds harvested from different crosses. For pPHE1:ARF22, Col-0 x Col-0 and osd1 x Col-0, two biological replicates were used for library preparation. For the CRISPR/Cas9 induced arf13 arf20 mutations, Col-0 x Col-0 and Col-0 x osd1 three biological replicates were used for library preparation.

first: Nicolas

last: Butel

first: Yichun

last: Qiu

first: Wenjia

last: Xu

first: Claudia

last: Köhler


title: Differential squamous cell fates elicited by NRF2 gain-of-function versus KEAP1 loss-of-function

city: Sendai

first: Takafumi

last: Suzuki

address: 2-1 Seiryo-machi, Aoba-ku

department: Department of Biochemistry and Molecular Biology

institute: Tohoku Medical Megabank Organization, Tohoku University

summary: Clinical evidence has revealed that high-level activation of NRF2 caused by somatic mutations in NRF2 is frequently detected in esophageal squamous cell carcinoma (ESCC), whereas that by somatic mutations in KEAP1, a negative regulator of NRF2, is not. Here, we challenged to generate a mouse model of NRF2-activated ESCC using the cancer-derived NRF2L30F mutation and cancer-driver mutant Trp53R172H. Concomitant expression of NRF2L30F and Trp53R172H induced proliferation of squamous cell epithelia and resulted in NRF2-activated ESCC-like lesions. In contrast, while squamous cell-specific deletion of KEAP1 induced similar NRF2 hyper-activation, the loss-of-KEAP1 combined with Trp53R172H did not elicit the proliferation and formation of ESCC-like lesions. Instead, KEAP1-deleted cells disappeared from the esophageal epithelium over time by cell competition. These findings provide insights into the observation that somatic mutations are more frequently observed in NRF2 than in KEAP1, and the mouse model developed here will be instrumental in elucidating the mechanistic basis leading to NRF2-activated ESCC.

overall_design:
To examine how mutations in NRF2 gene affect esophageal squamous cell carcinogenesis, we generated a knock-in mouse line with human cancer-derived NRF2L30F, in which leucine (L) at codon 30 in DLGex motif was replaced with phenylalanine (F).To iduce NRF2L30F-expression by Cre-mediated recombination in squamous cells, we exploited K5Cre mice, and K5CreERT2 mice which harbor tamoxifen-inducible squamous cell specific CreERT2 recombinase system. We generated NRF2L30/FL30F::K5Cre, NRF2L30F/L30F::K5CreERT2, and NRF2L30F/+::K5CreERT2 mice.To compare the phenotype of squamous cell specific NRF2L30F-expressing mice with that of conditionally KEAP1-deficiency mice, we also generated KEAP1flox/flox::K5Cre and KEAP1flox/flox::K5CreERT2 mice.
We performed RNA-sequence analysis using RNA samples from the esophagus. In the first experiment, we utilized RNA samples from esophagus of Wild-type, NRF2L30F/L30F::K5Cre, and KEAP1flox/flox::K5Cre mice, in age of 7-10 day-old. Second, we utilized RNA samples of esophagus derived from wild-type, NRF2L30F/+::K5CreERT2, NRF2L30F/L30F::K5CreERT2, and KEAP1flox/flox::K5CreERT2 mice at 1 week after tamoxifen treatment (100 μg/g body weight, tyree constitutive days), in age of 6-9 week-old.

first: Jun

last: Takahashi

first: Miu

last: Sato

first: Shuji

last: Nitta

first: Nahoko

last: Yaguchi

first: Tatsuki

last: Muta

first: Kouhei

last: Tsuchida

first: Hiromi

last: Suda

first: Masanobu

last: Morita

first: Shin

last: Hamada

first: Atsushi

last: Masamune

first: Satoru

first: Takashi

last: Kamei

first: Masayuki

last: Yamamoto


title: Functionally-instructed modifiers of response to ATR inhibition in experimental glioma

city: Tübingen

first: Bianca

last: Walter

state: Deutschland

address: Otfried-Müller-Str. 27

country: Germany

institute: Hertie Institut für klinische Hirnforschung

summary: We investigated the influence of ATR inhibition by AZD6738 treatment in LN229 and LNZ308 glioma cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for LN229 and LNZ308 cells treated with AZD3678 and respective vehicle control in three replicates (A, B, C) per sample of three time points (2h, 24h, 72h) and an untreated control.

first: Laurence

last: Kuhlburger

first: Daniel

last: Merk


title: A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations [Parse scRNA-seq]

city: Vienna

first: Florian

last: Halbritter

address: Zimmermannplatz 10

country: Austria

institute: St. Anna Children's Cancer Research Institute (CCRI)

summary: Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

overall_design:
1 multiplexed scRNA-seq dataset comprising 96 samples from hESCs differentiation toward trunk neural crest and sympathoadrenal derivatives at 9 key stages (day 0, 3, 6, 9, 10, 12, 14, 19, 28) and in different experimental conditions (H7 hESCs: wild-type, 17q gain, 17q+1q gain, all with/without MYCN overexpression; H9 hESCs: WT, 17q+1q, 17q+1q+MYCN overexpression); most sample groups are represented by 2 replicates (independent differentiation experiments with the same cell lines). Single-cell libraries were prepared using split-pool barcoding by Parse Bioscience (WT Mega kit).
Please note that all samples were pooled in this scRNA-seq reaction using split Parse Biosciences pool combinatorial barcoding
The data stem from a combinatorial indexing scRNA-seq experiment (Parse Bio), which means that all files contain reads from many single cells and all samples mixed up into one. Demultiplexing of both samples and cells is done in one step by Parse's pipeline as described in the sample data processing field.

first: Ingrid

last: Saldana-Guerrero

first: Luis

middle: F

last: Montano-Gutierrez

first: Katy

last: Boswell

first: Christoph

last: Hafemeister

first: Evon

last: Poon

first: Lisa

last: Shaw

first: Dylan

last: Stavish

first: Rebecca

last: Lea

first: Sara

last: Wernig-Zorc

first: Eva

last: Bozsaky

first: Irfete

middle: S

last: Fetahu

first: Peter

last: Zoescher

first: Ulrike

last: Pötschger

first: Marie

last: Bernkopf

first: Andrea

last: Wenninger-Weinzierl

first: Caterina

last: Sturtzel

first: Celine

last: Souilhol

first: Sophia

last: Tarelli

first: Mohamed

last: Shoeb

first: Polyxeni

last: Bozatzi

first: Magdalena

last: Rados

first: Maria

last: Guarini

first: Michelle

last: Buri

first: Wolfgang

last: Weninger

last: Putz

first: Miller

last: Huang

first: Ruth

last: Ladenstein

middle: W

last: Andrews

first: Ivana

last: Barbaric

first: George

last: Cresswell

first: Helen

last: Bryant

first: Martin

last: Distel

first: Louis

last: Chesler

first: Sabine

last: Taschner-Mandl

first: Matthias

last: Farlik

first: Anestis

last: Tsakiridis


title: Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development.

city: Mainz

first: Wolfram

last: Ruf

address: Langenbeckstrasse 1

institute: CTH Mainz

summary: Antiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia. Here we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the the development of persistent aPL and circulating phospholipid-reactive B1 cells in latent viral infection and lupus prone MRL-lpr mice. NGS mRNA profiling of splenic DC in lupus mice treated with NAPc2 versus saline control uncovered a suppression of dendritic cell activation in the spleen.

overall_design: NGS sequencing of splenic DCs used spleens digested with collagenase II and DNase I followed by dissociated cell enrichment on an Optiprep density gradient and FACS of live DCs identified by MHCII and CD11c in the CD3/CD19/NK1.1 negative population. DC RNA of 4 saline and 4 NAPc2 treated mice was sequenced by Novogene and mapped to the GRCm39 mouse genome with STAR on the fly.

first: Ruf

last: Wolfram

first: Posma

last: Jens

first: Nguyen

last: T Son


title: Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development

summary: Antiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) areis a major cause for acquired thrombophilia. Here we test the effect of specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) on the activation of monocytes by aPL. NGS data show that aPL-induced proinflammatory and prothrombotic activation of monocytes, but not interferon regulated gene induction is prevent by NAPc2.

overall_design: Monocytic MM1 cells were stimulated with 400 ng/ml aPL HL5B without (n=4) and with 200 nM NAPc2 (n=3) or HL7G without (n=4) or with (n=4) NAPc2, 10 ng/ml LPS (n=3), control IgG (n=4), no addition (n=4), NAPc2 (n=4). RNA isolated from MM1 cells was sequenced at the MDC Berlin and NGS data were mapped to GRCh38.p13 with STAR on the fly.  NGS sequencing of splenic DCs used spleens digested with collagenase II and DNase I followed by dissociated cell enrichment on an Optiprep density gradient and FACS of live DCs identified by MHCII and CD11c in the CD3/CD19/NK1.1 negative population. DC RNA of 4 saline and 4 NAPc2 treated mice was sequenced by Novogene and mapped to the GRCm39 mouse genome with STAR on the fly.

first: Nadine

last: Müller-Calleja

first: Kathrin

last: Saar

first: Norbert

last: Hübner

first: Jens

last: Posma

first: T

last: Nguyen


title: Neopetrotaurines A-C, Isoquinoline Alkaloids with an Unprecedented Taurine-Bridged Isoquinoline Quinone Moiety from the Sponge Neopetrosia sp.

city: Bethesda

first: Javed

last: Khan

state: MD

address: 37 Convent Dr.

department: Genetics Branch

institute: NCI, NIH

summary: Neopetrotaurines A-C, isoquinoline quinone-linked isoquinoline alkaloids that possess a unique taurine bridge connecting the two bicyclic components, were isolated from a Neopetrosia sp. marine sponge. These new compounds have proton-deficient scaffolds, so their structure elucidation was facilitated by utilizing LR-HSQMBC and HMBC NMR experiments optimized to detect 4- and 5-bond long-range 1H-13C heteronuclear correlations. Neopetrotaurines A-C showed potent inhibition of transcription driven by the oncogenic fusion protein PAX3-FOXO1 in alveolar rhabdomyosarcoma.

overall_design: RNA-seq with no replicates.  Drug treatment versus control analysis.

first: Yong

middle: Y

last: Kim

first: Dongdong

last: Wang

first: Kirk

middle: R

last: Gustafson


title: Pharmacogenomic synthetic lethal screens reveal hidden vulnerabilities and new therapeutic approaches for treatment of NF1-associated tumor

city: Minneapolis

first: David

last: Largaespada

state: MN

address: 515 Delaware St SE

department: Department of Pediatrics

institute: University of Minnesota

summary: Neurofibromatosis Type 1 (NF1) is a common cancer predisposition syndrome, caused by heterozygous loss of function mutations in the tumor suppressor gene NF1. Individuals with NF1 develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage after somatic loss of the wild type NF1 allele, some of which progress further to malignant peripheral nerve sheath tumors (MPNST). There is only one FDA approved targeted therapy for symptomatic plexiform neurofibromas and none approved for MPNST. The genetic basis of NF1 syndrome makes associated tumors ideal for using synthetic drug sensitivity approaches to uncover vulnerabilities. We developed a drug discovery pipeline to identify therapeutics for NF1-related tumors using isogeneic pairs of NF1 proficient and deficient immortalized human Schwann cells. We utilized these in a large-scale high throughput screen (HTS) for drugs that preferentially kill NF1 deficient cells, which identified 23 compounds capable of killing NF1 deficient Schwann cells with selectivity. Multiple hits from this screen clustered into classes defined by method of action. Four clinically interesting drugs from these classes were tested in vivo using both a genetically engineered mouse model of high-grade peripheral nerve sheath tumors and human MPNST xenografts. All trugs tested showed single agent efficacy in these models as well as significant synergy when used in combination with the MEK inhibitor selumetinib. This HTS platform has yielded novel therapeutically relevant compounds for the treatment of NF1-associated tumors and can serve as a tool to rapidly evaluate new compounds and combinations in the future.

overall_design: To understand the transcriptional response to a variety of drug treatments in MPNST cells, we treated S462TY cells with a variety of drug treatments and combinations. RNAseq was captured at both 2 hour and 24 hour timepoints for Digoxin, Rigosertib, and Selumetinib, as well as combinations of Digoxin with Selumetinib and Rigosertib with Selumetiinib. DMSO (vehicle) controls were captured for both time points. Untreated cell line controls were captured at 0, 2, and 24 hour timepoints.

first: Kyle

middle: B

last: Williams

first: Tyler

last: Jubenville


title: Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants

city: Edinburgh

first: Grzegorz

last: Kudla

address: Crewe Road

department: MRC HGU

institute: University of Edinburgh

summary: Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2,700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position Ile71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting to moderate evidence to suggest that 1,306 variants are likely benign, and 977, likely pathogenic. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.

sample_organism:
Saccharomyces cerevisiae
synthetic construct

overall_design: To investigate the effects on DNA-binding of amino acid substitutions in paired domain of the human transcription factor PAX6, we established a deep mutational scanning assay that employed a yeast one-hybrid functional readout and a competitive growth assay. Each yeast cell contained one of two genomically integrated PAX6-binding sequences (one assay per binding sequence; LE9 and BLX) upstream of an antibotic resistance gene, and expressed, from plasmid DNA, a single PAX6 variant fused to GAL4 activation domain. Binding of PAX6 to the binding sequence drove expression of the antibiotic resitance gene, growth rate in the presence of antibotic served as a proxy for PAX6:DNA perturbation. Each PAX6 variant was phased with a 30-nt barcode. Enrichment or depletion of each variant in the culture was quanitified using deep sequencing of barcodes before (timepoint 0) and after (timepoints 1-3) the competitive growth assay. Libraries were prepared from plasmids isolated from aliquots of each timepoint and PCR-amplified to append illumina-compatible adapters and dual indexes.

first: Alexander

last: McDonnell

first: Marcin

last: Plech

first: Benjamin

last: Livesey

first: Lukas

last: Gerasimavicius

first: Liusaidh

last: Owen

first: Hildegard

middle: N

last: Hall

last: FitzPatrick

first: Joseph

last: Marsh


title: Single nuclei gene expression profile for adult C. elegans and several longevity mutants

city: Ashburn

first: Shihong

middle: Max

last: Gao

address: 19700 Helix Dr

institute: Janelia Research Campus

summary: We presents a complete single-cell atlas of aging worms, encompassing both somatic and germ cell types. This atlas was built upon 241K nuclei across the worm lifespan at day 1, 6, 12, and 14 (100%, 99%, 61%, and 14% survival rates, respectively). The worms were aged under normal physiological conditions, without interfering with their reproductive processes.

overall_design: To perfom snRNAseq for wild-type and longevity worms, we harvested and homogenized ~2,000 worms for each genotype/condition. Nuclei were isolated using fluorescence-activated cell sorting (FACS) based on the DNA content signal and then proceed with 10x Chromium sequcing.

middle: M

first: Yanyan

last: Qi

first: Hongjie

last: Li

first: Yusi

last: Fu

first: Meng


title: Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations [RNA-Seq 2]

status: Public on Mar 20 2024

city: New York

first: Jahan

last: Rahman

state: New York

address: 417 E 68th St

department: Center for Hematologic Malignancies

institute: Memorial Sloan Kettering

summary: Mutations in the RNA splicing factor SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing but therapeutic means to correct this mis-splicing do not exist. Here we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SUGP1, a G-patch protein recently implicated in SF3B1 mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1 splicing defects and was sufficient to improve hematopoiesis in SF3B1 mutant mouse and human cells. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and the therapeutic impact of correcting aberrant splicing in SF3B1 mutant cancers.

overall_design: We sought to determine the impact of GPATCH8 deletion on RNA splicing genome-wide. We therefore performed RNA-seq analyses of SF3B1-mutant or WT CD34+ cells from cord blood and following 7d of differentiation into erythroid cells, with or without GPATCH8 KO.

first: Salima

last: Benbarche

first: Jose

last: Maria Bello Pineda

first: Laura

last: Baquero Galvis

first: Bo

last: Liu

first: Jeetayu

last: Biswas

first: Eric

last: Wang

first: Kadeen

last: Lyttle

first: Ariana

last: Dahi

last: Lewis

first: Martina

last: Sarchi

first: Sanjoy

last: Mehta

first: Ralph

last: Garippa

first: Juliana

last: Ortiz-Pacheco

first: Zhuoning

first: Mara

last: Monetti

first: Robert

last: Stanley

first: Sergei

last: Doulatov

last: Bradley

first: Omar

last: Abdel-Wahab


title: The disordered extensions of the Arid5a core ARID domain modulate its DNA- and RNA-binding propensities

city: Marburg

first: Julia

middle: E.

last: Weigand

address: Marbacher Weg 6

department: Pharmacy

institute: University of Marburg

summary: ARID (AT-rich interacting domain) proteins are a heterogeneous family of DNA-binding proteins involved in transcriptional regulation. No precise DNA-binding preferences have yet been defined for the aberrant member Arid5a. In addition, the protein binds to mRNA motifs for transcript stabilisation, presumably through the DNA-binding ARID domain. Here we first provide an unbiased definition of RNA motifs and a clear breakdown of nucleic acid binding by the ARID domain. An RNA Bind-n-Seq (RBNS) experiment was performed to find a consensus motif of the Arid5a domain. It reveals a preference for an unexpected CAGGCAG consensus motif, accompanied by a general preference for AU-rich motifs.

overall_design: RNA Bind-n-Seq (RBNS) experiment to to test ARID’s capability to interact with specific RNA motifs. An RNA pool (1 µM) of 20 random nucleotides flanked by short constant adapter sequences was incubated with different concentrations of the Strep-tagged Arid49-152 domain (0.25, 1, 5 µM)

first: Lasse

last: Oberstrass

first: Julian

last: von Ehr

first: Sophie

last: Korn

first: Francois

last: McNicoll

first: Ege

last: Yazgan

first: Lara

last: Schnaubelt

first: Nicole

last: Blümel

first: Kathi

last: Zarnack

first: Michaela

last: Müller-McNicoll

first: Andreas

last: Schlundt


title: A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations

first: Florian

last: Halbritter

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

overall_design: Refer to individual Series


title: Cox7a1 mediated CIV dimerization impacts skeletal muscle physiology and cardiac injury response

city: Bern

first: Nadia

last: Mercader

address: Baltzerstrasse 2

country: Switzerland

institute: University of Bern

summary: The oxidative phosphorylation (OXPHOS) system is dynamic and the respiratory complexes (RCs) coexist with super-assembled quaternary structures called supercomplexes (SCs). How assembly occurs and the physiological role of supercomplex assembly is still under intensive investigation. The Cox7a family member Cox7a2l, also known as Scaf1, promotes CIII-CIV super assembly and energetic efficiency in zebrafish, mice, and humans. Here we studied the role of a second member of the Cox7a family, Cox7a1 in SC assembly and striated muscle physiology. We found that this protein drives CIV homodimer formation, which increases CIV activity. The substantial reduction in CIV2 formation led to a profound metabolic rearrangement with a consequent non-pathological loss of skeletal muscle performance. Ablation of Cox7a1 also rewired heart metabolism.  Already in homeostatic conditions, cox7a1-/- hearts revealed a pro-regenerative metabolic profile. While overall cardiac function was not affected, the absence of Cox7a1 altered the cardiac regenerative response. The effects of cox7a1 and cox7a2l loss of function on skeletal muscle and myocardium physiology and injury response were not identical, revealing that there is  a high specificity of Cox7a isoform in controlling OXPHOS assembly and striated muscle metabolism. While in both cases overall OXPHOS activity is modified, the loss of CIII-CIV heterodimer formation or CIV homodimer formation have very distinct metabolic and physiological consequences, highlighting the complexity of OXPHOS function and the importance of cox7a1 in striated muscle maturation.

overall_design: Adult zebrafish hearts for cox7a1-/- mutants and wildtype siblings were cryoinjured. 7 days post injury the cardiac ventrilces were extracted and RNA was extracted for RNA seq.  4 pools each comprising 5 ventricles were sequenced per condition.

first: Prateek

last: Arora

first: Maria

last: Galardi-Castilla

first: José Antonio

last: Enríquez


title: Antibiotic Exposure Reprograms Metabolism to Mobilize Bacillus subtilis in competitive interactions

city: College Station

first: Paul

last: Straight

state: TX

address: 2128 TAMU

institute: Texas A&M University

summary: Antibiotics have dose-dependent effects on exposed bacteria. The medicinal use of antibiotics relies on their growth-inhibitory activities at sufficient concentrations. At subinhibitory concentrations, exposure effects vary widely among different antibiotics and bacteria. Bacillus subtilis responds to bacteriostatic translation inhibitors by mobilizing a population of cells (MOB-Mobilized Bacillus) to spread across a surface. How B. subtilis regulates the antibiotic-induced mobilization is not known. In this study, we used chloramphenicol to identify regulatory functions that B. subtilis requires to coordinate cell mobilization following subinhibitory exposure. We measured changes in gene expression and metabolism and mapped the results to a network of regulatory proteins that direct the mobile response. Our data reveal that several transcriptional regulators coordinately control the reprogramming of metabolism to support mobilization. The network regulates changes in glycolysis, nucleotide metabolism, and amino acid metabolism that are signature features of the mobilized population. Among the hundreds of genes with changing expression, we identified two, pdhA and pucA, where the magnitudes of their changes in expression, and in the abundance of associated metabolites, reveal hallmark metabolic features of the mobilized population. Using reporters of pdhA and pucA expression, we visualized the separation of major branches of metabolism in different regions of the mobilized population. Our results reveal a regulated response to chloramphenicol exposure that enables a population of bacteria in different metabolic states to mount a coordinated mobile response.

overall_design: To investigate how subinhibitory concentrations of chloramphenicol induce sliding motility of Bacillus subtilis we measured the gene expression levels at two different time points, 6 and 24 hours.

first: Yongjin

last: Liu

first: Sandra

last: LaBonte


title: Effect of stimulation with lipopolysaccharide (LPS) at 0,30, 60, and 120 minutes on miRNA expression in immortalized mouse macrophages (IMM).

first: Matthew

middle: James

last: Marino

address: 9000 Rockville Pike

department: NIAID

institute: NIH

summary: The interactions between RNA and protein within cellular signaling pathways, known as the interactome, have modulatory effects on RNA binding proteins (RBP’s) effector functions, which contribute to post-transcriptional control of inflammatory cytokine release in the innate immune response. Post-translational modifications (PTMs) have a regulatory effect on a protein’s effector function. Thus, investigating the effect of RNA-protein interactions can answer questions central to our understanding of cellular signaling. Global RNA sequencing was used to investigate the differentially expressed (DE) genes from baseline following induction with LPS.  Shotgun proteomics along with SILAC labeling was used to quantify relative abundance and differential protein expression in response to LPS.  To assess the proteins interacting with RNA after LPS induction, total RNA associated protein purification was utilized. Using the tandem MS approach and methodology mentioned above, we were able to identify a list of candidate RNA binding proteins. Global RNA sequencing provided a baseline transcriptomic profile as well as quantification of DE genes across a stimulation time course (0, 30, 60 minutes). To investigate the discordance between the transcriptome and proteome, we performed miRNA sequencing at 0,30,60, and 120 minutes of LPS stimulation to assess expression changes in LPS induced miRNA’s which may regulate the transcriptome. We then handpicked potential RBPs of immunological interest (Il1a, MARCKS, and Acod1). Interestingly, Il1a, MARCKS, and Acod1 were all highly DE compared to baseline in the RNA Seq results and displayed a tendency to disassociate from RNA as the LPS time course pursued. Il1a, MARCKS, and Acod1 can all directly regulate pro-inflammatory gene expression in macrophages. These RBPs could offer a new regulatory mechanism to tune their effector function, which could lead to new therapeutic innovations for disease pathologies these proteins are implicated in. This research was supported by the Intramural Research Program of NIAID, NIH.

overall_design: To investigate changes in the expressed miRNA's following stimulation with LPS, we examined 3 biological replicates per time point, where 0 minute was the control and 30, 60, and 120 minutes corresponded to the treated samples.

first: Rathore

last: Deepali

first: Marino

middle: J

last: Matthew

first: Aleksandra

last: Nita-Lazar


title: Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model [iPS-VEC RNA-seq]

city: Chiba

first: Naoya

last: Takayama

state: Chiba

address: 1-8-1 Inohana, Chuo-ku

department: Graduate School of Medicine

institute: Chiba University

summary: The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, is not fully understood. Here, we established an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induced endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. RNA-seq and ATAC-seq revealed accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the reduced H3K9me3 levels showed inverse correlation with retrotransposable elements, and retrotransposable element-derived dsRNA activated the DHX58-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescued cell proliferation and suppressed cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.

overall_design: RNA-sequencing (RNA-Seq)

first: Sudip

last: Paul

first: Masaki

last: Fukuyo

first: Bahityar

last: Rahmutulla

first: Atsushi

last: Kaneda

first: Motohiko

last: Oshima


title: Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model [H3K9me3 ChIP-seq]

overall_design: Chromatin immunopricipitation DNA-sequencing (ChIP-seq) for histone modifications H3K9me3.


title: Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model [VSMC RNA-seq]


title: Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model

summary: The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.


title: A complex pattern of gene expression in tissue affected by viperid snake envenoming: the emerging role of autophagy-related genes

city: Charlottesville

first: Ana Karina

last: de Olliiveirra

state: VA

address: 1340 Jefferson Park Avenue

department: Department of Pathology

institute: UVA

summary: Cellular and inflammatory events were evaluated in mouse muscle after snake venoms Daboia russelii and Bothrops asper injection over time. A murine model of muscle necrosis based on venom injection was used to investigate up to 800 genes involved in fibrosis diseases and tissue regeneration using the multiplex RNA panel Fibrosis V2 from NanoString technology.

overall_design: In this study we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 hr, 6 hr and 24 hr after injection of the venoms of Bothrops asper and Daboia russelii, two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy.

last: de Oliveira

first: Alexandra

last: Rucavado

first: Teresa

last: Escalante

first: José

last: Gutiérrez

first: Jay

last: Fox


title: Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model [iPS macrophage RNA-seq]


title: Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model [RTE RNA-seq]

overall_design: RNA sequencing (RNA-Seq)

first: Ashwini

last: Patil


title: ARID1B regulates BCL11B-mediated non-canonical Activin signaling to maintain mesenchymal stem cell homeostasis in the mouse incisor [scRNA-seq]

city: Los Angeles

first: Mingyi

last: Zhang

address: 2250 Alcazar Street, CSA103, HSC

department: Center for Craniofacial Molecular Biology

institute: University of Southern California

summary: ARID1B is a epigenetic regulator. We used the scRNA sequencing to analysis the role of ARID1B in regulating mouse incisor tissue homeostasis.

overall_design: Single-cell transcriptomes were obtained from the digested proximal region of mouse incisor of at 4 days post-tamoxifen induction for both control and Arid1b mutant mice at the age of 1 month.


title: ARID1B regulates BCL11B-mediated non-canonical Activin signaling to maintain mesenchymal stem cell homeostasis in the mouse incisor [bullk RNA-seq]

summary: ARID1B is a epigenetic regulator. We used the RNA sequencing to analysis the role of ARID1B in regulating mouse incisor tissue homeostasis.

overall_design: Transcriptomes were obtained from the proximal region of mouse incisor of at 4 days post-tamoxifen induction for both control and Arid1b mutant mice at the age of 1 month.


title: Single-cell transcriptome and TCR analysis of human Cytomegalovirus (hCMV)-specific T cell memory reveals effector and pre-effectors of CD8+ and CD4+ cytotoxic T cells

city: New Delhi

first: Veena

middle: S.

state: Delhi

address: Aruna Asaf Ali Marg

country: India

institute: National Institute of Immunology

summary: The latent human Cytomegalovirus (hCMV) infection can pose a serious threat of reactivation and disease occurrence in immune-compromised individuals, as well as burdens the immune system in immune-competent individuals. Though, T cells are at the core of the protective immune response to hCMV infection, a detailed characterization of different T cell subsets involved in protection against the hCMV infection is lacking. Here we analyzed the single-cell transcriptomes and the single-cell T cell antigen receptor (TCR) repertoires of over 8000 hCMV-reactive peripheral T cells isolated from different memory compartments. The hCMV-reactive T cells were highly heterogeneous and consisted of different developmental memory and functional T cell subsets such as, the long-term memory precursors and effectors, T helper-17, T regulatory cells (TREGs) and cytotoxic T lymphocytes (CTLs). The hCMV-antigen specific TREGs were enriched for molecules linked to their suppressive function and interferon response genes. The CTLs were of two types, the pre-effector and effector like. Of particular interest was the mixture of both CD4-CTLs and CD8-CTLs in both the pre-effector and effector cytotoxic clusters, suggesting that both CD4-CTLs and CD8-CTLs share transcriptomic signatures. The huge TCR clonal expansion of both the cytotoxic clusters imply their predominant role in protective immune response to CMV. Further the clonotype sharing between the CTL clusters and the long-term memory clusters, indicate potential progenitors of CD4-CTLs. Together our study has identified many subsets of hCMV-specific memory T cells that may have implication in better understanding the hCMV-specific T cell immunity to design vaccination strategies and therapeutics.

overall_design: PBMCs were isolated from healthy human blood bank donors and CMV-peptide reactive cells were sorted using FACS based on suface marker expression of CD137 and CD154, after 6 and 24 hours (combined). scRNA-Seq was performed on these sorted cells after pooling cells from 10 donors and equal representation from all memory compartments - CD4-Naive, CD4-TSCM, CD4-TCM, CD4-TEM, CD4-TEMRA and CD8-TEMRA.

first: Raunak

last: Kar

first: Somdeb

last: Chattopadhyay

first: Anjali

last: Sharma

first: Kirti

first: Aneeshkumar

last: Arimbasseri


title: Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model [iPS ATAC-seq]

first: Naoya

last: Takayama

overall_design: Assay for transposase-accessible chromatin with sequencing (ATAC-Seq)

first: Sudip

middle: K

last: Paul

first: Masaki


title: Apelin Stimulation of the Perivascular MuSC Niche Enhances Endogenous Repair in Muscular Dystrophy

city: Sherbrooke

first: C Florian

last: Bentzinger

state: Quebec

address: Campus de la santé, 3001 12e Avenue N

department: Pharmacologie-Physiologie

country: Canada

institute: Université de Sherbrooke

summary: Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here we describe that defects in the endothelial cell (EC) compartment of the perivascular stem cell niche in three different types of MD are associated with inefficient mobilization of MuSCs following tissue damage. Using chemoinformatic analysis, we identified the 13 amino acid form of the peptidic hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. In dystrophic mice, administration of AP-13 generates a pro-myogenic EC-rich niche that supports MuSC function and markedly improves tissue regeneration, muscle strength, and physical performance. Moreover, we demonstrate that EC specific knockout of the AP-13 receptor leads to regenerative defects that phenocopy major pathological features of MD. Altogether, we provide in vivo proof-of-concept that enhancing endogenous repair by targeting the perivascular niche is a viable therapeutic avenue for MD and characterize AP-13 as a novel drug candidate for systemic treatment of stem cell dysfunction.

overall_design: We performed RNA-sequencing of HUVEC cultures exposed to AP-13 or MM54 for 3 days.

first: Emmeran

last: Le Moal

first: Simon

last: Dumontier

first: Joris

last: Michaud

first: Jérôme

last: Feige

first: C


title: Subolesin knockdown in tick cells provides insights into vaccine protective mechanisms

city: Ciudad Real

first: Sara

last: Artigas Jerónimo

state: Ciudad Real

address: Ronda de Toledo 12

department: Sanidad y Biotecnología (SaBio)

country: Spain

institute: Instituto de Investigación en Recursos Cinegéticos (IREC)

summary: Ticks, as obligate blood-feeding arthropod vectors of pathogenic viruses, bacteria, protozoa and helminths, are responsible for prevalent tick-borne diseases (TBDs) worldwide. This arthropod constitutes the second most common that transmit pathogens among humans, after mosquitoes, and the first vector in domestic animals. Vaccines constitute the safest and more effective approach to control tick and TBDs, but this is in constant research to identify new antigens and improve vaccines formulations. The tick antigen Subolesin is a well-known vaccine protective antigen with a highly conserved sequence at both gene and protein levels in the Ixodidae and among arthropods and vertebrates. In this study, RNAseq and proteomic analyses were carried out in wild type and Subolesin knockdown tick ISE6 cells in order to identify and characterize the functional implications of Subolesin in tick cells, demonstrating once again the importance of this antigen in vaccine development against tick and TBDs.

overall_design: To study the importance and the functional role of tick subolesin which had been widely demostrated as a great vaccine antigen, we performed iRNA in ISE6 tick cells. This study comprises a comparison between wild type and subolesin knockdown ISE6 cells from a multi-omic point of view.

last: Artigas-Jerónimo

first: Margarita

last: Villar

first: Agustín

last: Estrada-Peña

first: Pilar

last: Alberdi

first: José

last: De la Fuente


title: Polysome bound mRNA Seq analysis of tolerant (APO) and susceptible (IR64) variety of Oryza sativa cv. Indica under 50% drought

city: Faridabad

first: Ramu

last: Vemanna

address: NCR Biotech Science Cluster, Faridabad

institute: Regional Centre for Biotechnology

summary: Plants evolved several acquired tolerance traits for drought stress adaptation to maintain the cellular homeostasis. The combination of constitutive and acquired traits governs drought tolerance, which is crucial for maintaining crop productivity under drought. Drought affects protein synthesis, to uncover the translational landscape with response to drought stress in rice, polysome bound mRNA sequencing at anthesis stage in resistant APO and sensitive IR64 genotypes were performed. Our results demonstrate that drought tolerant genotype maintains higher transcripts bound to poly-ribosomes which facilitate higher protien synthesis which impacted on  photosynthesis, spikelet fertility, seed filing and yield under drought stress. We identified many novel LncRNAs and relevant genes associated with translation which can play important role in manitaing grain protein content with drought tolerance.

overall_design: Samples from two categories: control, drought were analysed from both APO and IR64 variety. Each sample was run in triplicate. Total number of sample analysed is 12.

first: Akashata

last: Dawane

first: Sanjay

last: Deshpande

first: Preethi

last: Vijayaraghavareddy


title: Regulation of mRNA translation by RNH1 in hematopoietic cells

first: Mayuresh

last: Sarangdhar

address: Murtenstrasse 40

department: DBMR

institute: University Bern

summary: Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) containing protein. It binds to pancreatic-type ribonucleases and inhibit their function. However, the entire biological role of Rnh1 is unknown. We generated RNH1 knock out K562 cells by CRISPR/Cas9 method. We isolated polysomal RNA from control and RNH1-deficient K562 cells to quantify actively translated mRNAs by RNA-seq.

overall_design: Total RNA was isolated from polysome fractions of wild type and RNH1 deficient K562 cells.

first: Ramanjaneyulu

last: Allam

middle: A


title: ARID1B regulates BCL11B-mediated non-canonical Activin signaling to maintain mesenchymal stem cell homeostasis in the mouse incisor [scATAC-seq]

first: Mingyi

last: Zhang

summary: ARID1B is a epigenetic regulator. We used the scATAC sequencing to analysis the role of ARID1B in regulating mouse incisor tissue homeostasis.

overall_design: Single-cell transposase-accessible chromatin were obtained from the digested proximal region of mouse incisor of at 4 days post-tamoxifen induction for both control and Arid1b mutant mice at the age of 1 month.


title: Attenuation of A(H7N9) influenza virus infection in mice exposed to cigarette smoke

status: Public on Mar 19 2024

city: Minato

first: Yoshihiro

last: Kawaoka

state: Tokyo

address: Shirokanedai 4-6-1

department: ERATO Influenza-induced Host Responses Project

institute: Japanese Science and Technology Agency

summary: This study aims to determine how gene expression in the lung of mice is affected by cigarette smoking during infection with an H7N9 influenza virus.

overall_design: Seven-week-old female C57BL/6j mice were exposed to cigarette smoke or air for 30, 90, or 180 days. At the end of the exposure period, the animals were either infected with A/Anhui/1/2013 (H7N9) virus or mock infected with PBS. At days 2 and 5 post infection (p.i.), the animals were humanely sacrificed and their lungs harvested for microarray analysis.

first: Satoshi

last: Fukuyama

first: Jason

middle: E

last: Shoemaker

first: Dongming

last: Zhaho

first: Noriko

last: Nagajima

first: Yuriko

last: Tomita

first: Tadashi

last: Maemura

first: Tiago

last: da Silva Lopes

first: Tokiko

last: Watanabe

first: Seiya

last: Yamayoshi

first: Hideki

last: Hasegawa


title: Gene expression in liver from WT/ACOX1 liver specific knockout mice

city: St. Louis

first: Irfan

last: Lodhi

state: MISSOURI

address: 660 S. Euclid Ave.

department: Endocrinology, Metabolism and Lipid Research

institute: Washington University School of Medicine

summary:
The target of this project is to investigate the effect of ACOX1 knockout in liver
Gene expression profiling analysis were performed using data obtained from RNA-seq of two genotypes mice liver

overall_design: Comparative liver gene expression between WT and ACOX1-LKO mice

first: Lu

last: Dongliang

first: Lodhi

last: Irfan


title: Von Hippel Lindau tumor suppressor controls m6A-dependent gene expression in renal tumorigenesis

city: Chapel Hill

first: Austin

last: Hepperla

state: NC

address: 7018B Mary Ellen Jones Building

department: Genetics

institute: University of North Carolina at Chapel Hill

summary: N6‐Methyladenosine (m6A) is the most abundant internal post‐translational modification, and its contribution to cancer evolution has recently been appreciated. Renal cancer is the most common adult genitourinary cancer, of which ~85% is accounted for by the clear cell subtype (ccRCC) characterized by VHL loss. In this study, we demonstrate for the first time that VHL binds with m6A enzymatic complex proteins and promotes METTL3-METTL14 complex formation, and VHL depletion suppresses mRNA m6A modification. m6A RIP-seq coupled with RNA-seq in renal cells with or without VHL allowed us to identify a selection of genes whose expression may be regulated by m6A. Specifically, PIK3R3 is identified to be one critical gene whose mRNA stability is regulated by VHL in a m6A-dependent but HIF-independent manner. Functionally, PIK3R3 depletion promotes renal cancer cell growth and orthotopic tumor growth while vice versa. Mechanistically, PIK3R3 suppresses tumor growth by restraining PI3K/AKT activity. Taken together, we propose a new mechanism by which VHL regulates m6A through modulation of METTL3-METTL14 protein complex formation, thereby promoting PIK3R3 mRNA stability and protein level that is critical for regulating ccRCC tumorigenesis.

overall_design: m6A RIP-seq and RNA-seq between normal (sgCtrl) and VHL KO (sgVHL_sg1 or sgVHL_sg2) HKC cells.

first: Cheng

first: Miaomiao

last: Yu

first: Zhao

first: Rishi

last: Raj

first: Hua

last: Zhong

first: Jin

last: Zhou

first: Lianxin

last: Hu

first: Jun

last: Fang

first: Hongyi

last: Liu

first: Qian

last: Liang

first: Liwei

last: Jia

first: Chengheng

last: Liao

first: Sichuan

last: Xi

first: Jeremy

last: Simon

first: Kexin

last: Xu

first: Zhijie

first: Yunsun

last: Nam

first: Payal

last: Kapur

first: Qing


title: Single-cell analysis reveals an antiviral network that controls Zika virus infection in human dendritic cells

city: Atlanta

first: Gregory

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

summary: Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10x Genomics Chromium single cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human moDCs infected with ZIKV at the single cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN dependent and independent genes (antiviral module). We modeled the ZIKV specific antiviral state at the protein level leveraging experimentally derived protein-interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per cell basis with experimental protein interaction data. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool to gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.

sample_organism:
Chlorocebus aethiops
Homo sapiens

platform_organism:
Chlorocebus aethiops
Homo sapiens

overall_design:
Experiment 1 consisted of 4 pools of 1:1 mixed mock  infected and ZIKV infected VeroE6 cells with varying (0, 0.1, 1, 10 µM) concentrations of ZIKV-specific primer
Experiment 2 consisted of 4 pools of 1:2 mixed mock infected and (mock infected/IFN-β treated/primer; ZIKV infected/no treatment/primer; ZIKV infected/IFN-β treated/primer; ZIKV infected/IFN-β treated/no primer )

first: Kathryn

last: Moore

first: Adam

last: Pelletier

first: Stacey

last: Lapp

first: Amanda

last: Metz

first: Michelle

last: Lee

first: Swati

last: Bhasin

first: Manoj

first: Rafick

last: Sékaly

first: Steven

last: Bosinger

first: Mehul

last: Suthar


title: RFX6 Regulates Human Intestinal Patterning and Function Upstream of PDX1

city: Cincinnati

first: Jim

last: Wells

state: Ohio

address: 3333 Burnet Ave

department: Developmental Biology

institute: Cincinnati Childrens Hospital

summary: The gastrointestinal (GI) tract consists of highly specialized organs from the proximal esophagus to the distal colon, each with unique functions. Rare congenital malformations of the GI tract, including organ atresia, agenesis or mispatterning are linked to gene mutations although the molecular basis of these malformations has been poorly studied due to lack of model systems to study human development. We identified a patient with compound heterozygous mutations in the transcription factor RFX6 with pancreatic agenesis as previously described. In addition, the patient had duodenal mal-rotation and atresia suggesting that establishment of the proximal small intestine was impaired in these patients. To identify the molecular basis of the intestinal malformation we generated induced pluripotent stem cell lines from this patient, and derived human intestinal organoid (HIOs) to identify how mutations in RFX6 impact intestinal patterning and function. We identified that the duodenal identity of HIOs and patient tissues had adopted a more distal small intestinal signature, including expression of SATB2, normally expressed in the ileum and colon. CRISPR-mediated correction of RFX6 restored duodenal identity, including expression of PDX1, which is required for duodenal development. Using transcriptomic approaches in HIOs and Xenopus embryos we identified that PDX1 is a downstream transcriptional target of RFX6 and that PDX1 expression in a RFX6 mutant background was sufficient to rescue duodenal identity. However, RFX6 had additional transcriptional activities that were separate from PDX1, in particular to regulate multiple components of signaling pathways that are critical for establishing early regional identity in the GI tract including WNT, HH, and BMP pathways. In summary, we have identified that RFX6 is one of the most upstream regulators early intestinal patterning in vertebrates and that it acts by regulating key transcriptional and signaling pathways.

overall_design: Human pluripotent stem cell derived intestinal and colonic organoids were grown in vitro for 7 and 35 days  and in vivo for 10wks, total RNA was extracted from pooled in vitro organoids or individuall in vivo organoids and subjected to bulk 3' RNA-sequencing. All conditions were done in biological triplicate.

first: J

middle: G

last: Sanchez

first: James


title: Absence of chromosome axis proteins recruitment prevents meiotic recombination chromosome-wide in the budding yeast Lachancea kluyveri (ChIP-Seq)

city: Marseille

first: Sylvain

middle: Quentin

last: Legrand

address: 27 Bd Lei Roure

institute: CNRS

summary: Here we studied the budding yeast Lachancea kluyveri, a cousin of the model Saccharomyces cerevisiae, in order to try to understand the mechanism responsible for the absence of meiotic recombination on its almost entire sex chromosome (Brion et al. 2017). We performed protein-DNA interaction mapping of Rec8, Red1, Hop1 and Zip3 proteins by ChIP-seq. Briefly, we observed an enrichment of Zip3, a protein known as a marker of type I crossing-overs, where peaks of DSBs were detected by CC-seq and a virtual absence of Zip3 within Lakl0C-left. For Rec8, a meiotic specific protein part of the cohesin complex, we detected enriched peaks at centromeres and inside intergenic regions flanked by convergent ORFs everywhere in the genome Lakl0C-left included. Remarkably, Red1 and Hop1, known to form the lateral element of the synaptonemal complex and important for DSB formation, were enriched mainly at Rec8 peaks except at centromeres and particurlarly on Lakl0C-left where they are completely absent.

overall_design: This data set contains maps of Rec8, Red1, Hop1, Zip3 (FLAG tagged at their carboxy-termini) and untagged DNA contacts in the L. kluyveri genome generated by ChIP-seq of meiotic CBS 10367 cells. Cells were treated 1, 4 and 6 hours after sporulation induction.

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