title: Mouse Models of Pediatric Acute Megakaryoblastic Leukemia Reveal Novel Therapeutic Vulnerabilities [DSP750]

city: Montreal

first: Heather

last: Melichar

state: Quebec

address: 5415 Boulevard de l'Assomption

department: Immunology-Oncology Unit

institute: Maisonneuve-Rosemont Hospital Research Center

summary: Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. We developed CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. Oncogenic RAS cooperates with GLIS2 to trigger AMKL, enhancing the penetrance and decreasing the latency of CBFA2T3-GLIS2-dependent AMKL. We find that the FDA-approved drug pyrvinium pamoate, a GLI protein inhibitor, can be repurposed to impair CBFA2T3-GLIS2 levels and promote AMKL cell death. In addition, consistent with our findings that both CBFA2T3-GLIS2 and GLIS2 alter the expression of numerous BH3-only proteins, murine and patient-derived AMKL cells are sensitive to the BCL-2 inhibitor navitoclax. We observe a striking synergistic response to combined treatment with pyrvinium pamoate and navitoclax, suggesting a novel therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

overall_design: Comparative gene expression analysis of RNA-seq data from leukemic bone marrow (BM) cells expressing CBFA2T3-GLIS2-mCherry+luciferase, CBFA2T3-GLIS2-mCherry+Nras(G12D)-luciferase, or mCherry+luciferase, and fetal liver (FL) cells expressing CBFA2T3-GLIS2-mCherry+GFP, CBFA2T3-GLIS2-mCherry+Nras(G12D)-GFP, mCherry+Nras(G12D)-GFP or mCherry+GFP. BM cells were sorted based on the expression of mCherry, and FL cells were sorted based on the expression of mCherry (MIC), c-Kit, and depleted for Gr1, CD3, B220 and Ter119.

first: Mathieu

last: Neault

first: Marilaine

last: Fournier

first: Charles-Étienne

last: Lebert-Ghali

first: Frédérick

last: Mallette


title: DARESOME enables concurrent profiling of multiple DNA modifications in single cells and  multi-omics detection of cancer signatures in cell-free DNA

status: Public on Aug 04 2023

city: Singapore

first: Lih Feng

last: Cheow

address: 4 Engineering Drive 3

department: Biomedical Engineering

country: Singapore

institute: National University of Singapore

type: Methylation profiling by high throughput sequencing

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Bulk samples undergo DARESOME protocol and single cells undergo scDARESOME. Cells were lysed and protease treated prior to HpaII digestion. Digested unmethylated sites were ligated with unmethylation-tag (U-tag) adapters. Excess adapters were then removed using NEB Thermolabile USER II enzyme. This was followed by the second round of digestion with MspI and NlaIII enzymes. Hydroxymethylation-tag (H-tag) and Nla-tag (N-tag) adapters were ligated with the digested fragments. Excess adapters and oligonucleotides were removed with Thermolabile USER. 5hmC CCGG sites were glucosylated by glusyl transferese. The samples then undergo MspI digestion, methylated CCGG sites are digested and liagated to Methylated-tag (M-tag). The Read1 and Read2 Illumina sequencing adapters were added through PCR. The amplified library was size selected using BluePippin and quantified using Kapa Library Quantification kit.

first: Ramya

last: Viswanathan

first: Elsie

last: Cheruba

first: Pui

middle: M

last: Wong

first: Dawn

middle: Q

last: Chong

first: Yao

last: Yi

first: Jonathan

middle: Y

last: Loh

first: Iain

middle: B

last: Tan

first: Lih

middle: F


title: Exercise Training Influences Inflammatory and Immune Systems in Patients at High Risk for Colorectal Cancer: The CYCLE-P Study

status: Public on Aug 07 2023

city: Houston

first: Eduardo

last: Vilar-Sanchez

state: TX

address: 1515 Holcombe Blvd

country: USA

type: Expression profiling by high throughput sequencing

summary: This CYCling Lynch patients for Exercise and Prevention (CYCLE-P) trial was a prospective, non-randomized controlled trial conducted at the Familial High-Risk Gastrointestinal and Cancer Prevention Clinic of The University of Texas MD Anderson Cancer Center. Twenty-one LS participants with a confirmed pathogenic mutation in one of four DNA mismatch repair (MMR) genes were enrolled between April 2018 and January 2019. Follow-up was conducted 1-year from the start of the intervention. Statistical analysis by both per-protocol and intention-to-treat was performed from March to May 2021.

overall_design: CYCLE-P was a prospective, non-randomized controlled trial that enrolled 21 consecutive participants diagnosed with LS in a 12-month aerobic exercise intervention involving cycling classes in a local community (n=11) followed by enrollment of patients to usual care (n=10) between April 2018 to January 2019. All eligible participants provided written informed consent before enrollment, and the conduction of the trial complied with the Declaration of Helsinki. The trial protocol and all amendments were approved by the MD Anderson Cancer Center institutional review board. We followed the Transparent Reporting of Evaluations with Nonrandomized Designs (TREND) reporting guidelines.

last: Deng


title: Mouse Models of Pediatric Acute Megakaryoblastic Leukemia Reveal Novel Therapeutic Vulnerabilities [DSP601-649-749]

country: Canada

overall_design: Comparative gene expression analysis of RNA-seq data from fetal liver cells expressing MIC, CBFA2T3-GLIS2, CBFA2T3, GLIS2 or GLIS2(C265G). Cells were sorted based on the expression of mCherry (MIC), c-kit, and depleted for Gr1, CD3, B220 and Ter119.

last: J Melichar

middle: A


title: Mouse Models of Pediatric Acute Megakaryoblastic Leukemia Reveal Novel Therapeutic Vulnerabilities [DSP798]

overall_design: Comparative gene expression analysis of RNA-seq data from leukemic BM cells expressing CBFA2T3-GLIS2 or BM cells from healthy mice. BM cells were sorted based on the expression of mCherry (MIC), CD41, c-Kit, and depleted for Gr1, CD3, B220 and Ter119.

middle: J


title: Whole transcriptome analysis of endometroid cell lines after ERK5 inhibition

status: Public on Aug 03 2023

city: Sabadell

first: IDOIA

middle: BOLINAGA

last: AYALA

state: Barcelona

address: CARRER DE JESUS, 8, 2

department: Bioquímica, biología molecular facultad de medicina

country: Spain

institute: UNIVERSIDAD AUTONOMA DE BARCELONA

summary: The transcriptomic network modulated after the inhibition of the nuclear tyrosin kinase ERK5 was analyzed Ishikawa endometroid cell line, in order to characterize and reveal any relevant role of this kinase at the transcriptional level.

overall_design: Ishikawa cells were treated with ERK5 inhibitor JWG-071 3µM during 24h. Later RNA was extracted in biological triplicates for RNA-Seq analysis.

first: Sergio

last: Espinosa-Gil

first: Saska

last: Ivanova

first: Elisenda

last: Alari-Pahissa

first: Melek

last: Denizli

first: Beatriz

last: Villafranca-Magdalena

first: Maria

last: Viñas-Casas

first: Idoia

last: Bolinaga-Ayala

first: Andrés

last: Gámez-García

first: Eva

last: Colas

first: Miguel

last: Lopez-Botet

first: Antonio

last: Zorzano

first: José

last: Lizcano


title: Integrated BATF transcriptional network regulates suppressive intratumoral regulatory T cells

city: Pittsburgh

first: Feng

last: Shan

state: Pennsylvania

address: Ford Building, 5051 Centre Ave

institute: University of Pittsburgh

summary: Human regulatory T cells (Tregs) are crucial regulators of tissue repair, autoimmune diseases, and cancer. However, it is challenging to inhibit the suppressive function of Tregs for cancer therapy, without impacting immune homeostasis. Identifying pathways that may distinguish tumor-restricted Tregs is important, yet the transcriptional programs that control intratumoral Treg gene expression, and that are distinct from Tregs in healthy tissues, remain largely unknown. We profiled single-cell transcriptomes of CD4+ T cells in tumors and peripheral blood from patients with head and neck squamous cell carcinomas (HNSCC), in non-tumor tonsil tissues and in peripheral blood from healthy donors. We identified a subpopulation of activated Tregs expressing multiple tumor necrosis factor receptor (TNFR) genes (TNFR+ Tregs) that is highly enriched in the tumor microenvironment (TME) compared with non-tumor tissue and the periphery. TNFR+ Tregs are associated with worse prognosis in HNSCC and across multiple solid tumor types. Mechanistically, the transcription factor BATF is a central component of a gene regulatory network that governs key aspects of TNFR+ Tregs. CRISPR/Cas9-mediated BATF knockout in human activated Tregs in conjunction with bulk RNA sequencing, immunophenotyping and in vitro functional assays, corroborated the central role of BATF in limiting excessive activation and promoting the survival of human activated Tregs. Finally, we identified a suite of surface molecules reflective of the BATF-driven transcriptional network on intratumoral Tregs in patients with HNSCC. These findings uncover a primary transcriptional regulator of highly suppressive intratumoral Tregs, highlighting potential opportunities for therapeutic intervention in cancer without impacting immune homeostasis.

overall_design: We leveraged single-cell RNA sequencing (scRNA-seq) technology to provide a detailed view on the heterogeneity of Tregs in the TME by profiling CD4+ T cells from tumors and peripheral blood mononuclear cells (PBMC) taken from patients with HNSCC, and comparing them to CD4+ T cells isolated from inflamed tonsil tissues from patients with tonsilitis, and from non-inflamed tonsil tissues from patients with sleep apnea and to healthy donor (HD) PBMC. To dissect the phenotypic and functional effects of BATF in human activated Tregs, we performed RNA-seq of BATF KO activated Tregs and scrambled controls from six cord blood samples.

first: Anthony

middle: R

last: Cillo

first: Carly

last: Cardello

first: Arjun

last: Pennathur

first: James

middle: D

last: Luketich

first: John

last: Kirkwood

first: Tullia

middle: C

last: Bruno

first: Dario

last: Vignali


title: Reciprocal regulation of phosphatidylcholine synthesis and H3K36 methylation programs metabolic adaptation

city: Hangzhou

first: Zhu

last: Yibing

state: Zhe Jiang

address: Xihu

department: Life Of Science

country: China

institute: Zhejiang University

summary: Our findings demonstrated how phospholipid metabolism enabled metabolic transformation through an epigenetic modification, illuminating an underappreciated metabolic intersection for environmental adaptation in eukaryotic cells.

overall_design: Saccharomyces cerevisiae (CEN.PK) mRNA profiles of WT/rph1Δ/set2Δ/H3K36A in YPL (rich culture condition) at 0h and SL (minimal culture condition) at 1h and 3h

first: Yibing

last: Zhu

first: Wen

last: Fang


title: Decreased myelin-related gene expression in the nucleus accumbens during spontaneous neonatal opioid withdrawal in the absence of long-term behavioral effects in adult outbred CFW mice

city: Boston

first: Camron

last: Bryant

state: MA

address: 140 The Fenway

department: Department of Pharmaceutical Sciences - Center for Drug Discovery

institute: Northeastern University

summary: Prenatal opioid exposure is a major health concern in the United States, with the incidence of neonatal opioid withdrawal syndrome (NOWS) escalating in recent years. NOWS is revealed upon cessation of in utero opioid exposure and is characterized by increased irritability, disrupted sleep patterns, high-pitched crying, and erratic feeding. The main pharmacological treatment strategy for alleviating symptoms is opioid maintenance therapy. The neural mechanisms mediating NOWS and the long-term neurobehavioral effects are poorly understood. We used a third trimester-approximate model in which neonatal outbred pups (Cartworth Farms White; CFW) were administered once-daily morphine (15 mg/kg, s.c.) from postnatal day (P) day 1 through P14 and were then assessed for behavioral and transcriptomic adaptations within the nucleus accumbens (NAc) on P15. We also investigated the long-term effects of perinatal morphine exposure on adult learning and reward sensitivity. We observed significant weight deficits, spontaneous thermal hyperalgesia, and altered ultrasonic vocalization (USV) profiles following repeated morphine and during spontaneous withdrawal. Transcriptome analysis of NAc from opioid-withdrawn P15 neonates via bulk mRNA sequencing identified an enrichment profile consistent with downregulation of myelin-associated transcripts. Despite the neonatal behavioral and molecular effects, there were no significant long-term effects of perinatal morphine exposure on adult spatial memory function in the Barnes Maze, emotional learning in fear conditioning, or in baseline or methamphetamine-potentiated reward sensitivity as measured via intracranial self-stimulation. Thus, the once daily third trimester-approximate exposure regimen, while inducing NOWS model traits and significant transcriptomic effects in neonates, had no significant long-term effects on adult behaviors.

overall_design: To determine the effects of once-daily morphine exposure on neonatal transcriptome, we collected nucleus accumbens tissue during spontaneous withdrawal on P15.

first: Kristyn

middle: N

last: Borrelli

first: Kelly

middle: K

last: Wingfield

first: Emily

last: Yao

first: Catalina

last: Zamorano

first: Katherine

last: Sena

first: Jacob

last: Beierle

first: Michelle

last: Roos

first: Huiping

last: Zhang

first: Elisha

last: Wachman


title: Manipulating the 3D organization of the largest synthetic yeast chromosome [WGS]

city: New York

first: Weimin

state: NY

address: 435 E 30th street

department: Institute for Systems Genetics

institute: NYU Langone Health

type: Genome variation profiling by high throughput sequencing

summary: Whether synthetic genomescan power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we reportde novosynthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bpSaccharomyces cerevisiaechromosome resulting from extensive genome streamlining and modification. During the construction ofsynIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.

overall_design: This submission includes the whole genome sequencing data of synIV intermediate strains and draft and final strains.

first: Jef

last: Boeke


title: ChIPseq analysis of ParB binding to parSs and half-parSs in Pseudomonas aeruginosa under different environmental conditions and RNAseq analysis of selected half-parS removal on transcriptome

city: Warszawa

first: Adam

last: Kawałek

address: Pawinskiego 5A

department: Department of Microbial Biochemistry

country: Poland

institute: Institute of Biochemistry and Biophysics, PAS

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Control of nutrient uptake by IRF4 orchestrates innate immune memory - scRNA-seq

first: Endi

last: Santosa

state: New York

address: 417 E 68th St

department: Immunology

institute: Memorial Sloan Kettering Cancer Center

summary: Natural Killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen-specificity, clonal expansion, and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8+ T cell siblings. Various signals ranging from antigen, co-stimulation, and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified the transcription factor IRF4 as a signal integrator to coordinate the NK cell response during viral infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection.

overall_design:
Spleens were dissociated with glass slides and filtered through 100-μm cell strainer. Flow cytometry and cell sorting were performed on the Cytek Aurora (Cytek Biosciences) and Aria II cytometers (BD Biosciences), respectively. Before cell sorting, NK cells were enriched by incubating whole splenocytes with the following antibodies at 20 μg/ml against CD3ε (17A2), CD4 (GK1.5), CD8 (2.43), Ter119 (TER-119), CD19 (1D3), Ly6G (1A8) (BioXCell) followed by magnetic depletion using goat anti-rat beads (QIAGEN). For scRNA-seq enriched splenocytes were then stained with surface markers to identify Ly49H+ NK cells (CD3/TCRb/CD19- NK1.1+ CD49b+ Ly49H+) and sorted based on congenic markers (CD45.1 and CD45.2) to identify WT and Irf4−/− cells with > 95% purity.
Ly49H+ NK cells (CD3/TCRb/CD19− NK1.1+ CD49b+ Ly49H+) from either WT or Irf4−/− NK cells were sorted from spleens of MCMV-infected WT:Irf4−/− mBMC mice as described from day 0, 2, 4 and 7 PI, and stained with barcoded antibodies (Total-Seq B, Biolegend). After hash-staining, WT and Irf4−/− Ly49H+ NK cells were mixed at 1:1 ratio for each time point. The single-cell RNA-seq from these pooled FACS-sorted cell suspensions was then performed on Chromium instrument (10X genomics) following the user guide manual for 3′ v3.1. In brief, FACS-sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) trypan blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification per manual instruction. Samples were multiplexed together on one lane of 10X Chromium (using Hash Tag Oligonucleotides - HTO) following a previously published protocol. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles). The cell-gene count matrix was constructed using the Sequence Quality Control (SEQC) package.

first: Joseph

last: Sun


title: Transcriptome analysis of Pseudomonas aeruginosa strain, with 25 mutated half-parSs (ParB binding sites)

summary: Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4), which is positioned within the cell by ParA. Remarkably, ParB of P. aeruginosa binds to multiple heptanucleotides (half-parSs) scattered in the genome. In this work we analysed the transcriptome of P. aeruginosa with mutated 25 half-parSs forming the strongest ParB ChIP-seq peaks. Inactivation of ParB binding to even a small fraction of these sites modulated the gene expression, however this effect is most likely indirect. Overall this work suggests complex relation between ParB binding to genome and P. aeruginosa transcriptome.

overall_design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR)  as well as its derivative half-parS25 were grown in LB medium at 37oC, and cells were harvested at the logarithmic phase of culture growth (OD600 of 0.5).  Three independent biological replicates for each strain were used and the whole transcriptome comparison was repeated twice (Samples are labeled as Exp1 or Exp2).

last: Kawalek

first: Aneta

last: Bartosik

first: Grazyna

last: Jagura-Burdzy


title: Trans-Golgi protein TVP23B regulates host-microbe interactions via Paneth cell homeostasis and Goblet cell glycosylation

city: Dallas

first: Jin Huk

last: Choi

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center

summary: A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides.  The mechanisms underlying the maintenance and function of this intestinal barrier are not completely understood.  Using a mouse forward genetic screen for defects of intestinal homeostasis, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. In the intestine, the protein is localized to the epithelium and its deficiency in the hematopoietic extrinsic compartment was essential to the colitis phenotype. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells in vivo, leading to a decrease in antimicrobial peptides as well as a more penetrable mucus layer. As a result, Tvp23b-/- mice displayed decreased barrier function and a loss of host-microbe separation.  TVP23B-deficient colonocytes have a loss of core-3 O-glycosylation of colonic proteins, which is the major O-glycosylation present on gel forming mucins. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficent colonocytes have a common deficiency of several critical glycosylation enzymes, including those necessary for core-3 glycosylation of mucins. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.

overall_design: Transcriptome analysis of the colonic epithelial cells from control and Tvp23b deficient mice

first: Ran

last: Song

first: Emre

middle: E

last: Turer


title: Cell fate determinant Llgl1 is required for propagation of acute myeloid leukemia

city: Hannover

first: Tina

middle: M.

last: Schnoeder

address: Carl-Neuberg-Str. 1

country: Germany

institute: Hannover Medical School

summary: Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members appear to be conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Loss of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in leukemic cells. Finally, re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for FLT3-ITD/ITD Llgl1-WT/WT Mx-Cre+ LSK (Lineage-, Sca-1-high, cKit-high) cells, FLT3-ITD/ITD Llgl1-WT/WT Mx-Cre- LSK cells and FLT3-ITD/ITD Llgl1-/- Mx-Cre+ LSK cells.

first: Theresa

last: Eifert

first: Qirui

first: Anna

last: Dolnik

first: Florian

middle: H

last: Heidel


title: The ligation between ERMAP, galectin-9 and dectin-2 promotes Kupffer cell phagocytosis and antitumor immunity

city: Shanghai

first: Ji-hang

last: Yuan

address: 800 Xiangyin Road

department: Department of Medical  Genetics

institute: Naval medical university

summary: The innate immune system functions as the first line of defense against cancer cells, particularly the detection and clearance of cancer cells by phagocytosis. The liver resident macrophages, known as Kupffer cells, have the largest population of body’s tissue resident macrophages and constitute the first line of defense against cancer cells invading the liver. However, the molecule mechanisms underlying the detection and phagocytosis of cancer cells by Kupffer cells are still unclear. Here, using in vivo genome-wide CRISPR-Cas9 knockout screening, we found that Ermap expressed on cancer cells delivered the pro-phagocytosis ‘eat me’ signal to promote Kupffer cells phagocytosis and control over liver metastasis of various cancer cells. Ermap ligated Galectin-9 expressed on Kupffer cells surface in a glycosylation dependent manner. Galectin-9 further formed a bridging complex with Ermap and transmembrane receptor Dectin-2 expressed on Kupffer cells to promote the detection and phagocytosis of cancer cells by Kupffer cells. Patients with tumors expressing low ERMAP had more liver metastases. Thus, our study identifies the Ermap–Galectin-9–Dectin-2 axis as an ‘eat me’ signal for Kupffer cells to engulf cancer cells and control liver metastasis.

overall_design: CRISPR screen identifies novel targets regulating Hepa1-6 cells liver metastasis

first: Jie

last: Li


title: Impact of culture conditions on ParB distribution in Pseudomonas aeruginosa and analysis of ParB distribution in spreading deficient strains as well as half-parS25 strain

summary: Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4), which is positioned within the cell by ParA. Remarkably, ParB of P. aeruginosa binds to multiple heptanucleotides (half-parSs) scattered in the genome. In this work we analysed the influence of culturing conditions on ParB binding to DNA. Using chromatin immunoprecipitation-sequencing (ChIP-seq), we analysed patterns of genome occupancy by ParB in cells, with either coupling or uncoupling between replication and cell division. Our data indicated no altered preference of ParB to bind to individual half-parS sites under varying growth conditions, however a shift from parSs to half-parSs was evident in response to extended cell division time. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 27 half-parSs forming the strongest ParB ChIP-seq peaks was constructed and analysed showing changes in the ParB coverage of oriC region. Overall this work suggests the role of half-parSs in retaining ParB on the nucleoid within P. aeruginosa cells.

overall_design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR) derivative of PAO1, was used in the experiment (Kawalek et al., 2020; BMC Genomics, 21:14). ParB ChIP-seq experiments were performed using cells of PAO1161 (WT) strain grown on rich medium (LB) as well as minimal M9 medium with 0.5% glucose at two different temperatures (37°C and 30°C), harvested at exponential and stationary phases of culture growth.  ΔparB strain was used as a background control. Additionally strains expressing spreading defficient variants (ParB R94C and ParB A97T) as well as C-terminally truncated ParB1-283 variant impaired in dimerization was analyzed (these strains were grown only in LB at 37°C , and harvested at log phase of the culture growth). The immunoprecipitation was performed with polyclonal anti ParB antibodies, raised in rabbit and affinity purified (as in Reznekov et al., 1996, Genes Cells 1:529-542) and two replicates were performed for each strain/conditions, with the exception of ΔparB .


title: Lethal phenotype-based database screening identifies ceramide as a negative regulator of primitive streak formation

city: Bunkyo-ku

first: Satoshi

last: Kofuji

state: Tokyo

address: 1-5-45 Yushima

country: Japan

institute: Institute of Science Tokyo

summary: In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation had been challenging. We had identified ceramide metabolism to regulate PrS formation. We investigated how C2 ceramide, a cell-permeable form of ceramide, affects gene expression for PrS formation.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for embryoid bodies treated with C2 ceramide.

first: Jing

last: Pu

first: Hiroshi

last: Nishina


title: Androgen Receptor is a Determinant of Melanoma BRAFi/MEKi Resistance [Clariom_1]

city: Biella

first: Paola

last: Ostano

address: via Malta 3

country: Italy

institute: Fondazione Edo ed Elvo Tempia

summary: The majority of BRAFV600 mutant melanomas regress in response to BRAF/MEK inhibitors (BRAFi/MEKi). Yet nearly all relapse within the first two years. Most BRAFi/MEKi-resistant tumors are cross-resistant to immunotherapies, highlighting the need to prevent and circumvent resistance.  We recently showed that androgen receptor (AR) activity is required for sustained melanoma cells proliferation and tumorigenesis. Here we find that AR expression is markedly increased in BRAFi resistant melanoma cells as well as in sensitive cells already at very early times of BRAFi exposure. Proliferation and tumorigenicity of BRAFi resistant melanoma cells are blunted by genetic or pharmacologic suppression of AR activity, while AR overexpression is by itself sufficient to rendersmelanoma cells BRAFi/MEKi-resistant. Increased AR elicits transcriptional changes linked with AXL-positive BRAFi resistant subpopulations and induces expression of PAI-1 and EGFR, two determinants of melanoma progression that associate with elevated AR expression in clinical cohorts. Our results point to increased AR signaling as a determinant of melanoma BRAFi resistance, which can be counteracted by AR as well as PAI-1 and EGFR inhibitors.

overall_design: Three different melanoma lines (A375, WM9 and M14) were transduced with an AR overexpressing lentivirus (ARoe) versus LacZ-expressing control (LacZ), followed by treatment with Dabrafenib versus vehicle control (DMSO).

first: Samarkina

last: Anastasia

first: Markus

last: Youssef

first: Gian

last: Dotto


title: DOCK8 is a Novel Regulator of Lysosome-Mediated Pancreatic Cancer Invasion

city: Rochester

first: Cheng

last: Zhang

state: MN

address: 200 1st street SW

institute: Mayo Clinic

summary: We found that oncogenic KRAS significantly enriches levels of the guanine nucleotide exchange factor (GEF) DOCK8 on lysosomes. DOCK8 is aberrantly expressed in a subset of Pancreatic Ductal Adenocarcinoma where it promotes cell invasion in vitro and in vivo.

overall_design: mRNA was extracted from 15 untreated pancreatic cancer patient-derived organoids and RNA profiles were created using Illumina HiSeq 4000. Transcripts were aligned using STAR version 2.6.0a to GRCh38 using the basic 2-pass mode.

first: Omar

middle: L

last: Gutierrez-Ruiz

first: Tara

last: Hogenson

first: Martin

last: Fernandez-Zapico

first: Mark

last: McNiven

first: Gina

last: Razidlo


title: Genotoxic effects of base and prime editing in human hematopoietic stem cells [NHEJ_BaseE_MultiLocus]

status: Public on Aug 02 2023

city: Milan

first: Stefano

last: Beretta

address: Via Olgettina, 60

institute: San Raffaele Telethon Institute for Gene Therapy

summary: Emerging base and prime editing may provide safer and more precise genetic engineering than nuclease-based approaches bypassing the dependence on DNA double strand breaks (DSBs). However, little is known about cellular responses and genotoxicity. Here, we comparatively assessed state-of-the-art base and prime editors (B/PE) versus Cas9 in human hematopoietic stem/progenitor cells (HSPCs). BE and PE induced detrimental transcriptional responses constraining editing efficiency and/or HSPC repopulation in xenotransplants, albeit to a lesser extent than Cas9. DNA DSBs and their genotoxic byproducts, including deletions and translocations, were less frequent but not abrogated by BE and PE, particularly for cytidine BE due to suboptimal inhibition of base excision repair. Tailoring timing and B/PE expression enabled highly efficient and precise editing of long-term repopulating HSPCs. However, we uncovered a genome-wide effect of BEs on the mutational landscape of HSPCs, raising concerns for a potential genotoxic impact and calling for further investigations and improvements in view of clinical application.

overall_design: Targeted deep sequencing on mPB HSPC donors, treated with different editing conditions, namely, mock electroporation (mock electro) or Base Editing after 7 days of in vitro culture in different target regions

first: Martina

last: Fiumara

first: Samuele

last: Ferrari

first: Ivan

last: Merelli

first: Luigi

last: Naldini


title: Mouse Models of Pediatric Acute Megakaryoblastic Leukemia Reveal Novel Therapeutic Vulnerabilities

first: Heather

last: Melichar

address: 5415 Boulevard de l'Assomption


title: Basonuclin-2 regulates extracellular matrix production and degradation

city: Adelaide

first: Katherine

middle: Anne

last: Pillman

state: SA

address: North Terrace

department: Centre for Cancer Biology

country: Australia

institute: Adelaide University

summary: Epithelial-Mesenchymal Transition (EMT) is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the extracellular matrix (ECM); a complex environment of proteoglycans and fibrous proteins that promotes tissue homeostasis, regulates signaling in response to chemical and biomechanical stimuli and is often dysregulated in diseases such as cancer and fibrosis. Here, we demonstrate that Basonuclin-2 (BNC2), a mesenchymal-expressed gene that has been widely associated with cancer and developmental defects by genome-wide association study (GWAS), is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the deposition and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for BT549 cells, transfected (in triplicate) with control or BNC2 siRNAs. Gene expression was measured 6 days post-transfection.

first: Ayla

last: Orang

first: Cameron

last: Bracken


title: The psychosis risk gene RBM12 encodes a novel repressor of GPCR/cAMP signal transduction

city: Durham

first: Nikoleta

middle: G

last: Tsvetanova

state: NC

address: 308 Research Drive

department: Pharmacology & Cancer Biology

institute: Duke University

summary: A deeper understanding of the regulation of G protein-coupled receptors (GPCRs) and their associated downstream cascades will provide critical insights into how these pathways shape human physiology and disease, and could yield novel therapeutic targets. Here, we validate and characterize RNA-binding motif 12 (Rbm12) as a repressor of GPCR/cAMP signaling. We established Rbm12 CRISPR KO HEK293 cells using two independent gRNAs and human iPSC-derived neurons (iNeuron) depleted of Rbm12 via CRISPR interference. Then, we performed basal gene expression profiling (HEK293) and basal + beta-2-adrenergic receptor induced (1 hour of 1 uM Isoproterenol) conditions (neurons).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HEK293 cells (WT, RBM12 KO 1, RBM12 KO 2) and neurons (WT, RBM12 knockdown), treated with isoproterenol or untreated.

first: Khairunnisa

middle: M

last: Semesta

first: Angelica

last: Garces


title: DPPA3-HIF1a axis controls colorectal cancer chemoresistance  by imposing a slow cell-cycle phenotype

city: Barcelona

first: Isabel

last: Puig

state: Catalonia

address: C/Natzaret, 115-117

department: Preclinical and Translational Research

institute: Vall d'Hebron Institute of Oncology (VHIO)

summary: Comparative transcriptome of SW1222 colorectal cancer (CRC) cells that overexpress or are depleted of DPPA3 to identify genes involved in regulating of the slow-cycling cancer cell properties to uncover the molecular mechanism of the non-mutational chemoresistant phenotype.

overall_design: Microarray gene expression analysis was performed on DPPA3 gain-of-function (overexpression (OE)) and loss-of-function (knockdown (shDPPA3) and 2 CRISPR clones (DPPA3 KO C66 and C75)) SW1222 CRC cell models and respective controls. All cell models were grown as Matrigel-embedded megacolonies (3D cultures). In the case of DPPA3-OE cell model and its respective control, cells were treated with 5 µg/ml of doxycycline (DOX) during the last 5 days of culture to induce DPPA3 expression. In order to study the short-term effects on gene expression of SW1222 cells by DPPA3 induction, DPPA3-OE and control cells cultured in 2D and treated with 5 µg/ml of DOX during 4 hours were also included in the microarray analysis.

first: Estefania

last: Cuesta-Borràs

first: Cándida

last: Salvans

first: Héctor

last: Palmer


title: Transcription Across the H19/Igf2 Imprinting Control Region Initiates Paternally Methylated Imprinting in Mouse Prospermatogonia (RNA-Seq)

city: Grand Rapids

first: Piroska

last: Szabó

state: MI

address: 333 Bostwick Ave NE

department: Department of Epigenetics

institute: Van Andel Insitute

summary: Silver-Russell syndrome(SRS, OMIM 180860) is an imprinting disordermanifesting in fetal and postnatal growth retardation. Loss of methylation (LOM) is detected in the imprinting control region 1 (ICR1) that controls the H19/IGF2 domain, in about 65% of SRS cases. The cause of this epigenetic deficiency is unknown. It may affect the process of imprint establishment --de novo methylation of ICR1 in the male germ line. In the paralogous mouse locus this process involves DNA methyltransferase DNMT3A and H3K36me2 histone methyltransferase NSD1, but the mechanism that targets these enzymes to this ICR is unknown. Based on our genome-wide deep-sequencing data of transcription and DNA methylation, we hypothesized that low-level transcription is required in prospermatogonia for targeting H3K36me2 and de novo methylation to ICR. We truncated this transcript at the entry point to ICR by gene-targeting. In response, the establishment of DNA methylation imprint was incomplete. The sporadic LOM persisted from the male germ line into the paternal allele of the soma, resulting in reduced Igf2, and increased, biallelic H19 RNA in the fetal organs, consistent with the insulator model. Fetus weight was also variably reduced upon paternal transmission of this mutation, consistent with the role of IGF2 as a fetal growth factor. We identify low-level transcription-through as the mechanism that initiates paternal imprint establishment at the H19/Igf2 ICR in the male germ line. The findings predict that RNA-dependent imprint establishment may be affected in human fetuses resulting in sporadic hypomethylation of the ICR1 in SRS.

first: Ji

last: Liao

first: Sangmin

last: Song

first: Zhen

last: Fu

last: VanderKolk

first: Kin

last: Lau


title: Novel method of isolating nuclei of human oligodendrocyte precursor cells reveals substantial developmental changes in gene expression and H3K27ac histone modification. [RNA-Seq]

status: Public on Aug 01 2023

city: Bronx

first: Stella

last: Dracheva

address: James J. Peters VA Medical Center,130 W Kingsbridge Rd

department: Dept. of Psychiatry

institute: Icahn School of Medicine at Mount Sinai

summary: Oligodendrocyte precursor cells (OPCs) generate differentiated mature oligodendrocytes (MOs) during development. In adult brain, OPCs replenish MOs in adaptive plasticity, neurodegenerative disorders, and after trauma. The ability of OPCs to differentiate to MOs decreases with age and is compromised in disease. Here we explored the cell specific and age-dependent differences in gene expression and H3K27ac histone mark in these two cell types. H3K27ac is indicative of active promoters and enhancers. We developed a novel flow-cytometry-based approach to isolate OPC and MO nuclei from human postmortem brain and profiled gene expression and H3K27ac in adult and infant OPCs and MOs genome-wide.In adult brain, we detected extensive H3K27ac differences between the two cell types with high concordance between gene expression and epigenetic changes. Notably, the expression of genes that distinguish MOs from OPCs appears to be under a strong regulatory control by the H3K27ac modification in MOs but not in OPCs. Comparison of gene expression and H3K27ac between infants and adults uncovered numerous developmental changes in each cell type, which were linked to several biological processes, including cell proliferation and glutamate signaling. A striking example was a subset of histone genes that were highly active in infant samples but fully lost activity in adult brain. Our findings demonstrate a considerable rearrangement of the H3K27ac landscape that occurs during the differentiation of OPCs to MOs and during postnatal development of these cell types, which aligned with changes in gene expression. The uncovered regulatory changes justify further in-depth epigenetic studies of OPCs and MOs in development and disease.

overall_design: RNA sequencing (RNA-seq) of 2 brain cell populations (oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (MOs)) from the gray matter of dorsolateral prefrontal cortex of human adult and infant (0-2 years old) control donors. The populations were isolated using fluorescence-activated nuclei sorting.

first: Alexey

last: Kozlenkov


title: Transcription Across the H19/Igf2 Imprinting Control Region Initiates Paternally Methylated Imprinting in Mouse Prospermatogonia (ChIP-Seq)

summary: The insulator model explains the workings of the H19 and Igf2 domain in the soma, where insulation of the Igf2 promoter from its enhancers occurs by CTCF in the maternally inherited unmethylated chromosome but not the paternally inherited methylated allele. The molecular mechanism that targets paternal methylation imprint establishment to the imprinting control region (ICR) in the male germline is unknown. We tested the function of a prospermatogonia-specific broad low-level transcription in this process using mouse genetics. Paternal imprint establishment was abnormal when transcription was stopped at the entry point to the ICR. The germline epimutation persisted into the paternal allele of the soma, resulting in reduced Igf2 in fetal organs, and reduced fetal growth, consistent with the insulator model and IGF2’s role as fetal growth factor. In summary, broad low-level transcription through the ICR initiates paternal imprint establishment at the H19/Igf2 ICR in the male germ line, with implications for Silver-Russell syndrome. The superseries contains RNAseq and ChIPseq data from 15.5 dpc fetal male germ cells that carry the RNA terminator cassette and also from control wild type mice.

overall_design: Four different antiboies and input ChIP for wild type and H19 ter/H19 ter homozygous genotypes.

first: Samuel

last: Gusscott

first: Julie

last: Brind’Amour


title: Novel method of isolating nuclei of human oligodendrocyte precursor cells reveals substantial developmental changes in gene expression and H3K27ac histone modification. [ChIP-Seq]

overall_design: ChIP-seq sequencing (H3K27ac histone modification mark) of 2 brain cell populations (oligodendrocyte precursor cells and mature oligodendrocytes) from the gray matter of dorsolateral prefrontal cortex of human adult and infant (0-2 years old) control donors. The populations were isolated using fluorescence-activated nuclei sorting.


title: CONSTANS alters the circadian clock in Arabidopsis thaliana

city: Sevilla

first: Federico

last: Valverde

state: Sevilla

address: 49th Americo vespucio Avenue

department: Institute for Plant Biochemistry and Photosynthesis (IBVF)

institute: Spanish national research Council (CSIC)

summary: Plants are sessile organisms that have acquired highly plastic developmental strategies to adapt to the environment. Among these processes, the floral transition is essential to ensure reproductive success and is finely regulated by several internal and external genetic networks. The photoperiodic pathway, which controls the plant response to day length, is one of the most important pathways controlling flowering. In Arabidopsis photoperiodic flowering, CONSTANS (CO) is the central gene activating the expression of the florigen FLOWERING LOCUS T (FT) in the leaves at the end of a long day. CO expression is strongly regulated by the circadian clock. However, to date, no evidence has been reported regarding a feedback loop from the photoperiod pathway back to the circadian clock. Using transcriptional networks, we have identified relevant network motifs regulating the interplay between the circadian clock and the photoperiod pathway. Gene expression, chromatin immunoprecipitation experiments and phenotypic analysis allowed us to elucidate the role of CO over the circadian clock. Plants with altered CO expression showed a different internal clock period, measured by daily rhythmic movements in the leaves. We show that CO is able to activate key genes related to the circadian clock, such as CCA1, LHY, PRR5 and GI, at the end of a long day by binding to specific sites on their promoters. Moreover, a significant number of PRR5 repressed target genes are upregulated by CO, and this could explain the phase transition promoted by CO. The CO-PRR5 complex interacts with the bZIP transcription factor HY5 and helps to localize the complex in the promoters of clock genes. Our results indicate that there may be a feedback loop in which CO communicates back to the circadian clock, feeding seasonal information to the circadian system.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for seedlings of Col-0 (WT), 35S:CO, and SUC2:HA:CO

first: Pedro

last: de los Reyes

first: Francisco

last: Romero-Campero

first: He

last: Gao

first: Gloria

last: Serrano-Bueno

first: Jose

last: Romero


title: Novel XBP1s-independent function of IRE1 RNase in HIF-1a-mediated glycolysis upregulation in human macrophages

city: LIEGE

first: Sylvie

last: Legrand-Poels

address: Avenue de l'Hôpital

department: GIGA

country: Belgium

institute: Uliege

summary:
We investigated the immunometabolic reprogramming in SFA-treated human macrophages.
As expected, RNA sequencing highlighted a pro-inflammatory profile but also metabolic
signatures including glycolysis and hypoxia as well as a strong unfolded protein response.

overall_design:
We then performed comparative gene expression profiling analysis of RNA-seq data for human
monocyte-derived macrophages(MDMs) treated with BSA (Bovin serum albumin, vehicle) and MDMs
treated with a saturated fatty acid (C18:0, 100 µM) for 16h. Three replicates are included.

first: Margaud

last: Iovino

first: Megan

last: Colonval

first: Chloé

last: Wilkin

first: Laurent

last: L’homme

first: Jacques

last: Piette


title: The psychosis risk gene RBM12 encodes a novel repressor of GPCR/cAMP signal transduction II

first: Nikoleta

last: Tsvetanova

summary: To investigate beta-2-adrenergic targets in RBM12-depleted neurons derived from the parental WTc11 iPSC line

overall_design: Comparative gene expression profiling analysis of RNA-seq data of RBM12-depleted glutamatergic excitatory neurons derived from the parental WTc11 iPSC line, untreated or treated with isoproterenol (beta-2-adrenergic receptor agonist)

first: Khairunnisa

last: Semesta

first: Angelica

last: Garces


title: Semaglutide has beneficial effects on non-alcoholic steatohepatitis in Ldlr-/-.Leiden mice

city: Leiden

first: Anita

last: van den Hoek

state: Zuid-Holland

address: Zernikedreef 9

department: MHR

country: Netherlands

institute: TNO

summary: Abstract.   Background and aims: Semaglutide, a glucagon-like peptide-1 receptor agonist, is an antidiabetic medication that has recently been approved for treatment of obesity as well. Semaglutide is also postulated to be a promising candidate for treatment of non-alcoholic steatohepatitis (NASH). Here, we evaluated the effects of semaglutide in a translational diet-induced model with advanced NASH and fibrosis.  Methods: Ldlr-/-.Leiden mice received a fast food diet (FFD) for 25 weeks, followed by another 12 weeks on FFD with daily subcutaneously injections of semaglutide or vehicle (control). Plasma parameters were evaluated, livers and hearts were examined and hepatic transcriptome analysis was performed.  Results: In the liver, semaglutide significantly reduced macrovesicular steatosis (-74%, p<0.001), inflammation (-73%, p<0.001) and completely abolished microvesicular steatosis (-100%, p<0.001). Histological and biochemical assessment of hepatic fibrosis showed no significant effects of semaglutide. However, digital pathology revealed significant improvements in the degree of collagen fiber reticulation (-12%, p<0.001). Semaglutide did not affect atherosclerosis relative to controls. Additionally, we compared the transcriptome profile of FFD-fed Ldlr-/-.Leiden mice with a human gene set that differentiates human NASH patients with severe fibrosis from those with mild fibrosis. In FFD-fed Ldlr-/-.Leiden control mice, this gene set was upregulated as well, while semaglutide predominantly reversed this gene expression.  Conclusions: Using a translational model with advanced NASH, we demonstrated that semaglutide is a promising candidate with particular potential for treatment of hepatic steatosis and inflammation, while for reversal of advanced fibrosis, combinations with other NASH agents may be necessary.

first: José

last: Inia

first: Geurt

last: Stokman

first: Martine

last: Morrison

first: Nicole

last: Worms

first: Lars

last: Verschuren

first: Martien

last: Caspers

first: Aswin

last: Menke

first: Mathieu

last: Petitjean

first: Louis

first: Li

last: Chen

first: J

middle: W

last: Jukema

first: Hans

last: rincen

last: an den Hoek


title: Transcriptomic and epigenomic changes following acute serine starvation in breast cancer cells

city: palo alto

first: Jiangbin

last: ye

state: California

address: 269 Campus Drive CCSR-South 1245

institute: Stanford university

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

overall_design: Refer to individual Series


title: Control of nutrient uptake by IRF4 orchestrates innate immune memory - ChIP-seq

city: New York

first: Endi

last: Santosa

address: 417 E 68th St

institute: Memorial Sloan Kettering Cancer Center

summary: Natural Killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen-specificity, clonal expansion, and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8+ T cell siblings. Various signals ranging from antigen, co-stimulation, and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified the transcription factor IRF4 as a signal integrator to coordinate the NK cell response during viral infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection.

overall_design: Splenic WT NK cells (NK1.1+ CD49b+) were expanded ex vivo for two weeks in complete IMDM media (10% FBS, 1% Pen/Strep, 1X BME, 1X MEM-NAA, 1X HEPES) in the presence of 50ng/ml of human IL-15 (Miltenyi Cat#130-095-766). 10 million IL-15 expanded NK cells were then stimulated with anti-NK1.1 + cytokines (IFNa+IL-12/18+IL-2/15) or left unstimulated in 5ng/ml of hIL-15 overnight in a 6-well plate. Upon stimulation, cells were then washed with 1X PBS and crosslinked by slowly adding 16% formaldehyde to a final concentration of 1% formaldehyde, agitated, and incubated for 10 mins at RT. Glycine was then added to quench the sample, and incubated at RT 5 min, and on ice for 15 minutes. Cells were then centrifuged, and 600 ul of cell lysis buffer (50mM HEPES-KOH, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) was added into cell pellets, and incubated for 4 minutes on ice, spun at 600 xg for 10 minutes at 4C, and supernatant was discarded. 600 ul of nuclei wash buffer (10mM Tris-Cl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5 mM EGTA) was then added to each sample and resuspended gently, spun at 600xg for 10 minutes at 4C, and supernatant was discarded. Cell pellets were then resuspended with nuclei lysis buffer and incubated for 5 minutes on ice, and sonicated for 8 cycles, 30s on and 30s off high at 4C. Upon sonication, Triton-X 100 was added to a final concentration of 1%. Nuclei were centrifuged for 20 minutes at 14000 RPM at 4C, and supernatant was transferred into a new tube where 10% of samples were then aliquoted into a new tube as ‘input’.  25 ul of Protein A (ThermoScientific), was added into each sample and agitated for 30mins at 4C on a rotator. Moreover, protein A beads were blocked with ice cold RIPA buffer by adding BSA to a final concentration of 0.1 % and agitated for 3 mins at 4C on a rotator. Upon incubation, immunoprecipitated samples were centrifuged for 30s for 4C at 2400 xg and supernatant was transferred into new tubes and combined with blocked protein A beads. Anti-IRF4 (Clone D9P5H, Cell Signaling Cat# 15106S) was added gently to prevent bead shearing and incubated on a rotator overnight at 4C. Samples were then centrifuged at 2400 xg for 1 min at 4C, and supernatant was removed gently. Beads were then washed twice with RIPA buffer, four times with ChIP wash buffer (100mM Tris-Cl pH 8.5, 500mM LiCl, 1% NP-40, 1% NaDeoxycholate, twice again with RIPA buffer, and twice with ChIP TE buffer (10mM Tris pH 7.5, 1mM EDTA, 50mM NaCl). Beads were centrifuged at 5000 RPM for 1 min at 4C between all washes and supernatant was removed after the last wash. 100ul of decrosslinking buffer (TE buffer, 0.5% SDS buffer, and 1X Proteinase K) was added into every sample and incubated for 2 hours at 55C. After incubation, samples were vortexed, and then transferred to 65C and incubated overnight. Samples were then centrifuged at 2400 xg for 1min at RT, transferred into new tubes, and purified using Qiagen PCR purification kit (Qiagen). Immunoprecipitated DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. Illumina sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (Kapa Biosystems KK8234) according to the manufacturer’s instructions with up to 5 ng input DNA and 8-12 cycles of PCR. Barcoded libraries were run on the NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina). An average of 29 million paired reads were generated per sample.

first: Joseph

last: Sun


title: Control of nutrient uptake by IRF4 orchestrates innate immune memory


title: Transcriptome changes following acute serine starvation in breast cancer cells

summary: We cultured the ER negative breast cancer cell line MDA-MB-231 and the ER positive breast cancer cell line MCF7 in serine-free media for 24h. RNA was extracted from the cells and submitted for RNA sequencing.

overall_design: Proper number of cells were plated in 100mm dishes before treatment in +Ser or -Ser media for 24h. Each group contains 3 biological replicates.

first: Albert

last: Li

last: Ye


title: Clariom S Microarray Expression Data from 4T1 parental and transduced tumors collected from WT and Nlrx1-/- mice

city: Blacksburg

first: Irving

middle: Coy

last: Allen

state: VA

address: 295 Duck Pond Dr

institute: Virginia Tech

summary:
The immune signaling protein NLRX1 can be either tumor promoting or tumor suppressing in different models of cancer. We demonstrate that in a mammary tumor model of triple-negative breast cancer, NLRX1 impacts tumor volume and lung metastasis. We used this microarray to understand what genes and pathways are impacted by NLRX1 in murine triple-negative mammary tumors to produce the in vivo phenotypes we observed.
Abstract from associated publication: Prior studies have defined multiple, but inconsistent, roles for NLRX1 in regulating cancer-associated biological functions. Here, we explore the role of NLRX1 in the highly-metastatic murine 4T1 mammary tumor model. Using Nlrx1-/- mice engrafted with 4T1 tumors, we demonstrate that NLRX1 functions as a tumor suppressor when expressed in healthy host cells. Specifically, NLRX1 attenuates tumor growth and metastasis through suppressing epithelial-mesenchymal transition, tumor-associated eosinophil recruitment, and the lung metastatic niche. Conversely, we demonstrate that NLRX1 functions as a tumor promoter when expressed in 4T1 tumor cells using gain- and loss-of-function studies. Mechanistically, NLRX1 augments 4T1 aggressiveness through regulating epithelial-mesenchymal transition, cell death, proliferation, migration, ROS levels, and mitochondrial respiration. Together, we provide critical insight into NLRX1 function in breast cancer and establish cellular context as the director of the dichotomous role of NLRX1 in mammary tumor metastasis.

overall_design: 12 pooled RNA samples extracted from murine 4T1 (murine breast cancer) tumors. Comparing gene expression between 6 groups, consisting of 2 mouse genotypes (WT, KO) and 3 4T1 cell lines (4T1, 4T1OE, 4T1OECTL). 6 groups x 2 replicates each = 12 total samples. 1 MRSL, provided by the manufacturer, was included. Based on PCA analysis, ThermoFisher Clariom S service recommended excluding sample A3 from analysis.

first: Margaret

last: Nagai-Singer


title: tRFs/tiRNAs expression signature of low-glucose-treated HPAF-II cells

city: Guangzhou

first: Shineng

last: Zhang

state: Guangdong

address: No.107 Yanjiang West Road

institute: Sun Yat-sen Memorial Hospital, Sun Yat-sen University

type: Non-coding RNA profiling by high throughput sequencing

summary: Glucose metabolism makes contributions to the development of pancreatic ductal adenocarcinoma (PDAC). Meanwhile, tsRNAs, including  tRNA-derived fragment (tRF) and tRNA-derived stress-induced RNA (tiRNA), a subset of noncoding RNAs, exerts a vital role in glucose metabolism and cancer development. Analysis of high-glucose-treated and none glucose-treated HPAF-II cells from 6 samples (3 samples each group) was conducted. Results indicate insight into molecular signature of the pathogenesis of PDAC in abberant glucose metabolism.

overall_design: High throughput sequencing analysis was carried out to identify the differentially expressed tRFs/tiRNAs between high-glucose-treated and none glucose-treated HPAF-II cells  (3 samples each group).

first: Fengting

last: Huang

first: Yanyan

last: Zhuang

first: Sihua

last: Lan


title: Epigenomic changes following acute serine starvation in breast cancer cells

summary: Genome wide H3K27ac CHIP sequencing was performed in the ER positive breast cancer cell line MCF7 cultured in serine-free media for 24h, with or without supplementation with 2 mM glycerol triacetate.

overall_design: Proper number of cells were plated in 100mm dishes before treatment in +Ser, -Ser, or -Ser+GTA media for 24h.


title: The circadian clock protein Cryptochrome 1 is a direct target and feedback regulator of the Hippo pathway

city: Charlestown

first: Xu

last: Wu

address: 149, 13th Street

department: Dermatology, Harvard Medical School,

institute: Cutaneous Biology Research Center, Massachusetts General Hospital

summary: To examine the impact of Cry1 downregulation on gene expression, empty vector (Sh Ctrl) and Sh-RNA targeting Cry1 (Sh Cry1)  were used to produce lentiviral vector. Cell were then infected with control and sh-RNA lentivirus and then selected with puromycin. Upon validation of the knockdown with real time PCR. RNA were prepared from cells stably expressing empty vector or sh-RNA against Cry1 and used gene expression profiling by RNA seq. RNA of two biological replicate were prepared from each genotype.

overall_design: To examine the impact of Cry1 downregulation on gene expression  empty vector (Sh Ctrl) and Sh-RNA targeting Cry1 (Sh Cry1)  were used to produce lentiviral vector.

first: Abdelhalim

last: Azzi

first: Tao

last: Zhipeng

first: Carla

last: Guarino


title: Fission yeast poly(A) polymerase active site mutation Y86D suppresses the rad24∆ aps1-H397A synthetic growth defect and upregulates mRNAs targeted by Mmi1/MTREC

first: Angad

last: Garg

department: Molecular Biology

summary: This RNA-Seq analysis compares gene expression of the pla1-Y86D mutant contrasting to the isogenic wild-type fission yeast (Schizosaccharomyces pombe).

overall_design: 1 test samples and 1 control sample was processed (each sample has 3 replicate experiments)

first: Stewart

last: Shuman

first: Beate

last: Schwer


title: RNA-seq of plasmacytoid dendritic cells stimulated by TLR3, TLR4, TLR7 and TLR8

status: Public on Jul 31 2023

city: Hong Kong

first: Lishi

last: Wang

state: Hong Kong

address: 30-32 Ngan Shing Street, Shatin, NT

department: Surgery

institute: The Chinese University of Hong Kong

summary: Plasmacytoid dendritic cells (pDCs) are a rare type of dendritic cells that exist antiviral functions in response to toll-like receptors (TLRs). We here report TLR4 activated pDCs similar to TLR7/8 stimulation. Despite the high resemblance, we found the unique genes that were activated by TLR4 activation.

overall_design: To investigate the plasmacytoid dentritic cells (pDCs) activation by toll-like receptors (TLRs), we either unstimulated (UNS) or stimulated CAL-1, PMDC05 and THP-1 cell lines with CL097, FLA, LPS, or PLOY, and then measure gene expression with RNA-seq.

first: Kwan

middle: T

last: Chow

first: King

last: Lim


title: Hyperinsulinemia acts via acinar INSR to drive obesity-associated PDAC initiation by promoting digestive enzyme production and inflammation

city: Vancouver

first: Howard

last: Cen

state: BC

address: 2350 Health Sciences Mall

institute: University of British Columbia

summary: The rising incidence of pancreatic cancer is largely driven by the skyrocketing prevalence of obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of both conditions, and is independently associated with increased cancer incidence and mortality. Our previous studies demonstrated that genetically reducing insulin production suppressed formation of pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice with mutant Kras. However, we found that hyperinsulinemia affected many cell types in the pancreatic microenvironment. Thus, it remained unclear whether hyperinsulinemia exerted its effects directly on the cells that give rise to PanINs or indirectly on the tumor microenvironment, and molecular mechanisms involved were unknown. Here, we tested whether insulin receptors (Insr) in KrasG12D-expressing pancreatic acinar cells are necessary for the effects of hyperinsulinemia on obesity-associated pancreatic cancer development. Loss of Insr in KrasG12D-expressing acinar cells did not prevent hyperinsulinemia or weight gain associated with high fat diet (HFD) consumption in mice. However, solely reducing Insr in KrasG12D-expressing acinar cells significantly reduced formation of PanIN and tumors, in a gene dose-dependent manner. Mechanistically, proteomic analyses showed that hyperinsulinemia acts through Insr to drive the excess production of digestive enzymes in acinar cells by modulating the activity of the spliceosome, ribosome, and secretory machinery. This resulted in increased inflammation, which was abrogated by acinar-specific Insr knockout. We confirmed that insulin increased the conversion of wild-type acinar cells into acinar-to-ductal metaplasia (ADM) in a trypsin-dependent manner. Collectively, these data demonstrate that hyperinsulinemia acting via acinar cells insulin receptors promotes inflammatory conditions that cooperate with Kras signaling to increase the risk of developing pancreatic cancer, mechanistically linking obesity and pancreatic cancer.

overall_design: Total RNA of a piece of the tail of pancreata were collected from 12-week old female mice for the following 3 genotypes: Insr WT (Ptf1aCreER;KrasLSL-G12D;Insrw/w;nTnG), Insr Het (Ptf1aCreER;KrasLSL-G12D;Insrw/f;nTnG), Insr KO(Ptf1aCreER;KrasLSL-G12D;Insrf/f;nTnG).

first: Janel

last: Kopp

first: Anni


title: Molecular and phenotypic changes in FLExDUX4 mice

city: Washington

first: Yi-Wen

last: Chen

state: District of Columbia

address: 111 Michigan Ave NW

department: Center for Genetic Medicine Research

institute: Children's National Hospital

summary: We investigated differences in the quadriceps transcriptome of 13-week-old heterozygous B6(Cg)-Gt(ROSA)26Sortm1.1(DUX4*)Plj/J (FLExDUX4) mouse model of Facioscapulohumeral Muscular Dystrophy (FSHD) and their wild type (WT) littermates to determine if there were characterize the structural/functional adaptations to aerobic exercise training and their molecular foundations using RNA sequencing

overall_design: Comparative gene expression profiling analysis of RNA-seq data from the quadriceps of  13-week-oldmale FLExDUX4 and WT littermates. Samples were collected from n=3 FLExDUX4 mice and n= 3 WT mice.

first: Kelly

last: Murphy

first: Aiping

first: Adam

middle: J

last: Bittel


title: Loss of flrt2 gene leads to microphthalmia in zebrafish

city: Shenzhen

first: Siyu

last: Yang

address: No.628, Zhenyuan Rd, Guangming (New) Dist., Shenzhen

institute: The Seventh Affiliated Hospital of Sun Yat-Sen University

summary: As a member of the flrt gene family, Fibronectin Leucine-Rich Transmembrane 2 (flrt2) is strongly expressed in a subset of sclerotome cells, and the resultant protein interacts with FGFR1 in the FGF signaling pathway during development. Studies on flrt2 have focused mainly on its roles in the brain, heart and chondrogenesis. However, reports on its expression and function in the zebrafish retina are lacking. Here, we detected the high expression of flrt2 in zebrafish retina using in situ hybridization technique and developed an flrt2-knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. Quantitative real-time PCR was used to measure the expression levels of flrt2, which results in an approximately 60% mRNA reduction. The flrt2-KO zebrafish eyes' altered morphological, cellular, and molecular events were identified using BrdU labeling, TUNEL assay, immunofluorescent staining, fluorescent dye injection and RNA sequencing. Abnormal eye development, known as microphthalmia, was found in flrt2-KO larvae, and the retinal progenitor cells exhibited increased apoptosis, perhaps owing to the combined effects of crx, neurod4, atoh7, and pcdh8 downregulation and Casp3a and Caspbl upregulation. In contrast, the retinal neural development, as well as retinal progenitor cell differentiation and proliferation, were not affected by the flrt2 deletion. Thus, flrt2 appears to play important roles in retinal development and function, which may provide the basis for further investigations into the molecular mechanisms of retinal development and evolution.

overall_design: We created and described a novel zebrafish flrt2 mutant line to further characterize the effects of flrt2 on the development of, and physiological function in, zebrafish retina. The flrt2-KO zebrafish eyes' altered morphological, cellular, and molecular events were identified using BrdU labeling, TUNEL assay, immunofluorescent staining, fluorescent dye injection and RNA sequencing.


title: HDACi-dependent microenvironmental normalization overcomes tumor burden-induced T-cell exhaustion

status: Public on Jul 29 2023

city: Hamilton

first: Yonghong

last: Wan

state: Ontario

address: 1280 Main St W

department: Medicine

institute: McMaster University


title: HDACi-dependent microenvironmental normalization overcomes tumor burden-induced T-cell exhaustion [RNA-seq]

summary:
Purpose: T-cell exhaustion limits immunotherapy for the treatment of solid tumors. While immune checkpoint blockade and adoptive T cell therapy (ACT) can mediate tumor regression, their potency is often determined by tumor burden. Here, we identified tumor burden-related pathway changes that are conducive to T-cell exhaustion. We then determined if microenvironmental reprogramming via epigenetic modulation could reverse T-cell exhaustion and improve immunotherapeutic responsiveness.
Experimental Design:
We developed a murine syngeneic tumor model wherein an increased burden ablated therapeutic responsiveness to ACT, which corresponded with systemic induction of T cell exhaustion. Transcriptome analysis of these large tumors allowed us to characterize changes to immunosuppressive pathway expression during class I histone deacetylase inhibitor (HDACi) MS-275 treatment. We then measured the therapeutic impact of MS-275 during ACT and assessed T-cell exhaustion by transcriptome/phenotypic analysis.
Results:
ACT durably regressed small tumors but failed to control large tumors, which were associated with systemic T-cell exhaustion and ablation of T-cell responses. Large tumors were defined by an immunosuppressive pathway signature. MS-275 reversed this pathway signature and promoted durable regression of large tumors during ACT. Prototypical exhaustion marker Tim-3 was selectively up-regulated in transferred T cells despite displaying a reduced exhaustion signature. Instead, we observed enhanced activation-dependent signaling correlating with enrichment of the IL2-STAT5 signaling axis. Activated CD8+ T-cell responses were predominantly skewed towards terminal effector cell-like CD44+ Tim-3hi TCF1- CD127- KLRG1+ differentiation.
Conclusion:
Tumor burden-induced pathway changes can be reversed through epigenetic reprogramming, enabling the conversion from T-cell exhaustion to effector lineage differentiation.

overall_design: We sought to understand the local changes that predisposed large tumors to therapeutic resistance. We harvested RNA from 7- and 14-day old CMS-5 tumors and analyzed them using bulk-cell RNA-seq.

first: Andrew

last: Nguyen


title: Transcriptome signatures of wastewater effluent exposure in larval zebrafish vary with seasonal mixture composition in an effluent-dominated stream

city: Milwaukee

first: Rebecca

last: Klaper

state: WI

address: 600 E Greenfield

department: School of Freshwater Sciences

institute: University of Wisconsin - Milwaukee

summary: Wastewater treatment plant (WWTP) effluent-dominated streams provide critical habitat for aquatic and terrestrial organisms but also continually expose them to complex mixtures of pharmaceuticals that can potentially impair growth, behavior, and reproduction. Currently, few biomarkers are available that relate to pharmaceutical-specific mechanisms of action. In the experiment reported in this paper, zebrafish (Danio rerio) embryos at two developmental stages were exposed to water samples from three sampling sites (0.1 km upstream of the outfall, at the effluent outfall, and 0.1 km below the outfall) during base-flow conditions from two months (January and May) of a temperate-region effluent-dominated stream containing a complex mixture of pharmaceuticals and other contaminants of emerging concern. RNA-sequencing identified potential biological impacts and biomarkers of WWTP effluent exposure that extend past traditional markers of endocrine disruption. Transcriptomics revealed changes to a wide range of biological functions and pathways including cardiac, neurological, visual, metabolic, and signaling pathways. These transcriptomic changes varied by developmental stage and displayed sensitivity to variable chemical composition and concentration of effluent, thus indicating a need for stage-specific biomarkers. Some transcripts are known to be associated with genes related to pharmaceuticals that were present in the collected samples. Although traditional biomarkers of endocrine disruption were not enriched in either month, a high estrogenicity signal was detected upstream in May and implicates the presence of unidentified chemical inputs not captured by the targeted chemical analysis. This work reveals associations between bioeffects of exposure, stage of development, and the composition of chemical mixtures in effluent-dominated surface water. The work underscores the importance of measuring effects beyond the endocrine system when assessing the impact of bioactive chemicals in WWTP effluent and identifies a need for non-targeted chemical analysis when bioeffects are not explained by the targeted analysis.

overall_design: We investigate associations between gene expression in zebrafish larvae (3 and 6 dpf) and evolving seasonal pharmaceutical mixtures in a wastewater effluent-dominated stream. Embryos were exposed to stream water samples at three sites: 100 meters above the effluent outfall, at the the WWTP effluent outfall, and 100 meters below the effluent outfall. There were 5 replicate exposures per site with 20 individual per replicate for separate 3- and 6-day exposures. Samples used for RNA sequencing met the following quality control parameters: OD260/OD280 ratio 1.8-2.0 and OD260/OD230 ratio 2.0-2.2 and a RIN >7.

first: Emma

middle: B

last: Meade

first: Luke

last: Iwanowicz

first: Nicklaus

last: Neureuther

first: Gregory

last: LeFevre

first: Dana

last: Kolpin

first: Hui

last: Zhi

first: Shannon

last: Meppelink

first: Rachael

middle: F

last: Lane

first: Angela

last: Schmoldt

first: Aurash

last: Mohaimani

first: Olaf

last: Mueller


title: Tannerella forsythia intraoral infection and periodontitis in C57BL/6J mice

status: Public on Jul 27 2023

city: gainesville

first: Kesavalu

middle: Lakshmyya

last: Naidu

state: Florida

address: 1600 SW Archer Road

department: Dentistry

institute: University of Florida

summary: Individual miRNA analyzed were successfully constructed through nanostring technology of a total of 577 mouse miRNAs in 20 number of SHAM mice and 20 number of Tannerella forsythia infected mice, which have been euthanized on the end of 16 weeks infection study.

overall_design: The study depicted here, is a 16 weeks of total, 10 mice/group, Sham infected and Tannerella forsythia infected groups both in 8 weeks and 16 weeks, followed 16 weeks, euthanized and extracted left mandibles for miRNA expression profiles, of a total of 577 unique Nanostring miRNA panels. This leading to successful construction of supervised.

first: Aravindraja

last: Chairmandurai

first: Lakshmyya

last: Kesavalu


title: HDACi-dependent microenvironmental normalization overcomes tumor burden-induced T-cell exhaustion [Affymetrix]

first: Yonghong

last: Wan

summary:
Purpose: T-cell exhaustion limits immunotherapy for the treatment of solid tumors. While immune checkpoint blockade and adoptive T cell therapy (ACT) can mediate tumor regression, their potency is often determined by tumor burden. Here, we identified tumor burden-related pathway changes that are conducive to T-cell exhaustion. We then determined if microenvironmental reprogramming via epigenetic modulation could reverse T-cell exhaustion and improve immunotherapeutic responsiveness. Experimental Design: We developed a murine syngeneic tumor model wherein an increased burden ablated therapeutic responsiveness to ACT, which corresponded with systemic induction of T cell exhaustion. Transcriptome analysis of these large tumors allowed us to characterize changes to immunosuppressive pathway expression during class I histone deacetylase inhibitor (HDACi) MS-275 treatment. We then measured the therapeutic impact of MS-275 during ACT and assessed T-cell exhaustion by transcriptome/phenotypic analysis. Results: ACT durably regressed small tumors but failed to control large tumors, which were associated with systemic T-cell exhaustion and ablation of T-cell responses. Large tumors were defined by an immunosuppressive pathway signature. MS-275 reversed this pathway signature and promoted durable regression of large tumors during ACT. Prototypical exhaustion marker Tim-3 was selectively up-regulated in transferred T cells despite displaying a reduced exhaustion signature. Instead, we observed enhanced activation-dependent signaling correlating with enrichment of the IL2-STAT5 signaling axis. Activated CD8+ T-cell responses were predominantly skewed towards terminal effector cell-like CD44+ Tim-3hi TCF1- CD127- KLRG1+ differentiation. Conclusion: Tumor burden-induced pathway changes can be reversed through epigenetic reprogramming, enabling the conversion from T-cell exhaustion to effector lineage differentiation.
We used whole transcriptome arrays (Clariom D) to determine the effect of MS-275 on exhausted T cells in ACT+/-MS-275-treated mice

overall_design: After ACT+/-MS-275 treatment of 14d tumors, adoptively transferred tumor-specific Thy1.1+ CD8+ T cells were flow-sorted from spleens and RNA was extracted for the whole transcriptome array (Clariom D).


title: Transcriptional analysis in multiple barley varieties identifies signatures of waterlogging response

city: Dublin

first: Ailbhe

middle: Jane

last: Brazel

address: School of Natural Sciences

department: Botany Discipline

country: Ireland

institute: Trinity College Dublin

summary: Waterlogging leads to major crop losses globally, particularly for waterlogging sensitive crops such as barley. Waterlogging reduces oxygen availability and results in additional stresses, leading to the activation of hypoxia and stress response pathways that promote plant survival. Although certain barley varieties have been shown to be more tolerant to waterlogging than others and some tolerance-related QTLs have been identified, the molecular mechanisms underlying this trait are mostly unknown. Transcriptomics approaches can provide very valuable information for our understanding of waterlogging tolerance. Here, we surveyed 21 barley varieties for the differential transcriptional activation of conserved hypoxia-response genes under waterlogging, and selected five varieties with different levels of induction of core hypoxia-response genes. We further characterized their phenotypic response to waterlogging in terms of shoot and root traits. RNA-sequencing to evaluate the genome-wide transcriptional responses to waterlogging of these selected varieties led to the identification of a set of 98 waterlogging-response genes common to the different datasets. Many of these genes are orthologs of the so-called ‘core hypoxia response genes’, thus highlighting the conservation of plant responses to waterlogging. Hierarchical clustering analysis also identified groups of genes with intrinsic differential expression between varieties prior to waterlogging stress. These genes could constitute interesting candidates to study ‘predisposition’ to waterlogging tolerance or sensitivity in barley.

overall_design: Differential gene expression analysis from RNA-seq from 10-day old roots from 5 barley varieties (Golden Promise, Infinity, Passport, Pilastro, Regina) under waterlogged and control conditions, with 3 replicates per sample.

first: Alexandra

last: Miricescu

first: Joseph

last: Beegan

first: Frank

last: Wellmer

first: Emmanuelle

last: Graciet


title: MAVS positively regulates mitochondrial integrity and metabolic fitness in antigen receptor-activated B cells

status: Public on Jul 26 2023

first: Dan

last: Sturdevant

state: MT

address: 903 S 4th street

department: NIAID

institute: NIH

summary: The role of mitochondrial homeostatic control in the metabolic remodeling associated with antigen receptor stimulation in B cells remains incompletely defined. Here we report that the mitochondrial antiviral signaling (MAVS) adaptor protein is involved in B cell receptor (BCR) initiated cellular proliferation and prolonged survival. MAVS is well known as a mitochondrial tethered signaling adaptor for sensing viral double-stranded RNA and type I interferon inducer. The role of MAVS downstream of BCR stimulation was recognized in absence of interferon, suggesting a new pathway for MAVS activation that is independent of viral infection. Mitochondria of BCR-activated MAVS-deficient B cells exhibited a damaged phenotype including disrupted mitochondrial morphology, excess mitophagy and the temporal progressive blunting of mitochondrial oxidative capacity with mitochondrial hyperpolarization and cell death. Co-stimulation with anti-CD40, in addition to BCR stimulation, corrected the proliferative gap between MAVS-deficient and -sufficient B cells; however, mitochondrial structural defects and functionality were only partially restored. Our data reveal a previously unrecognized role of MAVS in controlling mitochondrial homeostasis in response to BCR initiated B cell proliferation.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for

first: Hongsheng

last: Wang

first: Wenxiang

last: Sun

first: Javier

last: Traba

first: Juan

last: Wu

first: Chen-Feng

last: Qi

first: Laura

last: Amo

first: Hemanta

last: Kole

first: Bethany

last: Scott

first: Komudi

last: Singh

first: Michael

middle: N

last: Sack

first: Silvia

last: Bolland


title: Novel method of isolating nuclei of human oligodendrocyte precursor cells reveals substantial developmental changes in gene expression and H3K27ac histone modification.

city: Bronx

first: Stella

last: Dracheva

state: New York

address: James J. Peters VA Medical Center,130 W Kingsbridge Rd

institute: Icahn School of Medicine at Mount Sinai

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

overall_design: Refer to individual Series


title: TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions [WGBS]

status: Public on Jul 24 2023

city: CHAPEL HILL

first: Gang

middle: Greg

state: North Carolina

address: 450 West Drive

department: Lineberger Comprehensive Cancer Center

institute: UNC chapel hill

summary: Trimethylation of histone H3 lysine 9 (H3K9me3) is critical for regulation of gene repression and heterochromatin formation, cell-fate determination, and organismal development. In particular, H3K9me3 provides an essential mechanism for silencing various transposon elements (TEs). However, previous studies showed that the canonical H3K9me3 readers (e.g. HP1 and MPP8) play rather limited roles in silencing endogenous retroviruses (ERVs), one of major TE classes in the mammalian genome. Here, we report that Trinucleotide Repeat Containing 18 (TNRC18), a previously under-studied chromatin regulator, recognizes H3K9me3 and directly binds ERVs, mediating ERV silencing. Our biochemical, biophysical and structural studies identified the C-terminal Bromo Adjacent Homology (BAH) domain of TNRC18 (TNRC18BAH) as a H3K9me3-specific reader, and its N-terminal segment as a platform for direct recruitment of co-repressors such as TRIM28 and HDAC-Sin3-NCoR complexes, thereby enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18BAH-mediated H3K9me3 engagement caused neonatal lethality in mice and, in multiple mammalian cell models, led to de-repressed expression of ERVs, affecting the landscape of cis-regulatory elements and thus gene-expression programs. Collectively, this study describes a previously-unknown TNRC18BAH-directed H3K9me3-sensing pathway, which operates to epigenetically silence the evolutionarily young ERVs, thereby exerting profound impacts on host genome integrity, transcriptomic regulation, immunity, and development.

overall_design: Whole Genome-wide Bisulfite Sequencing (WGBS) was performed with HEK293 cells that carry the homozygous knock-in (KI) mutation (MUT; W2858A) at TNRC18’s H3K9me3-binding BAH domain, in comparison to wildtype (WT) controls.

first: Shuai

last: Zhao

first: Bo

last: Pan


title: TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions [RNA-Seq 2]

overall_design: RNA-seq was used to assess transcriptomic profiles of HEK293 (human embryonic kidney cancer cell line), SNU-1 (Human gastric carcinoma cell line) and HT-29 (human colon adenocarcinoma cell line), either those with TNRC18 knockout (KO) or TNRC18 L760A mutation (at a Sin3/HDAC-interaction region), relative to wildtype (WT) cell controls. The processing of transcriptomic profiles employed two different methods for analysis of either regular genes or transposable elements (TEs).


title: Single cell resolution of the adult zebrafish intestine under conventional conditions, and in response to an acute, natural infection

status: Public on Jul 25 2023

city: Edmonton

first: Edan

last: Foley

state: Alberta

address: 116 st and 85 ave

department: Medical Microbiology and Immunology

institute: University of Alberta

summary: We used single cell RNA sequencing to analyze the diversity of intestinal cells under conventional conditions and the effect of an acute, natural Vibrio cholerae infection on the intestinal transcriptome.

overall_design: Adult zebrafish were infected with Vibrio cholerae for 16h then intestines dissected and dissociated into a single cell suspension and processed for single cell RNA sequencing.


title: TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions [T18-tissue RNA-Seq]

overall_design: To assess the function of the TNRC18-BAH domain in different organs, we isolated the primary tissues, namely, the lung, liver, heart, stomach and brain, from adult WT mice (n =2) and the paired littermates carrying Tnrc18-BAH homozygous mutation (W2745A/Y2747A; n=3), followed by preparation of total RNA samples and RNA-sequencing (n=2 duplicates per sample).  The processing of transcriptomic profiles used two different methods for analysis of either regular genes or transposable elements (TEs).


title: TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions [CAGE-seq]

overall_design: Cap analysis gene expression (CAGE) followed by sequencing (CAGE-seq) was performed with HEK293 cells that carry the knock-in (KI) homozygous mutation (MUT) at TNRC18’s H3K9me3-binding BAH domain, in comparison to wildtype (WT) controls.


title: The LARP1 homolog Slr1p controls the stability and expression of proto-5’TOP mRNAs in fission yeast [RNA-seq]

status: Public on Jul 21 2023

city: Toronto

first: Farnaz

last: Mansouri-Noori

state: ON

address: 4700 Keele Street

department: Biology

institute: York University

summary: Messenger RNAs (mRNAs) in higher eukaryotes that encode highly expressed proteins important for the assembly of the translational apparatus (e.g. ribosomal proteins) often harbour a pyrimidine-rich motif at the extreme 5’ end known as a 5’ terminal oligopyrimidine (5’TOP) sequence. Members of the La-related protein 1 (LARP1) family control 5’TOP expression through a conserved DM15 motif, but the mechanism is not well understood. 5’TOP motifs have not been described in many lower organisms, but fission yeast (and many other single-celled eukaryotes) harbour a LARP1 homolog that also lacks a DM15 motif. In this work, we show that the fission yeast LARP1 homolog, Slr1p, controls the translation and stability of mRNAs encoding proteins analogous to 5’TOP mRNAs in higher eukaryotes, which we thus refer to as proto-5’TOPs. Our data suggest that the LARP1 DM15 motif and the mRNA 5’TOP motif may be features that were scaffolded over a more fundamental mechanism of LARP1 family member-associated control of gene expression that is still utilized in lower systems.

overall_design: To study RNA abundance in presence and absence of Slr1p in S. pombe, translational profiling was performed with wildtype yAS99 cells and slr1- cells. This was followed by RNA-Seq of total RNA and polysome-associating RNAs. Sequencing was performed for two biological replicates.

first: Andreas

last: Pircher

first: Danielle

last: Bilodeau

first: Lidia

last: Siniavskaia

first: Jorg

last: Grigull

first: Olivia

middle: S

last: Rissland

first: Mark

middle: A

last: Bayfield


title: TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions [RNA-seq]

last: Wang

overall_design: We used RNA-seq to assess transcriptomic profiles of HEK293 cells, either mock treated (shCtl), with the knock-down (KD) of endogenous TNRC18 (shTNRC18), or with rescued re-expression of TNRC18 in cells with TNRC18 KD (TNRC18_KD_rescue). RNA-seq analysis was also conducted with the HEK293 cells or mouse embryonic fibroblast (MEF) cells carrying the H3K9me3-binding-defective mutation at TNRC18?s BAH domain, relative to wildtype (WT) controls. The processing of transcriptomic profiles used two different methods for analysis of either regular genes or transposable elements (TEs).


title: TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions [CUT&RUN]

overall_design: We used CUT&RUN to assess the genome-wide binding of TNRC18 (with either anti-GFP antibody for GFP-tagged TNRC18 or endogenous antibody for TNRC18), H3K9me3, H3 acetylation, HDAC2, TRIM28 and SETDB1 in HEK293 cells, either parental lines or those with the engineered knock-in (KI) mutation at TNRC18?s H3K9me3-binding BAH domain. IgG CUT&RUN served as the negative control. TNRC18 binding was also assessed in HEK293 cells with the SETDB1 knockout (KO) versus WT cells.


title: Short heat shock has a long-term effect on mesenchymal stem cells’ transcriptome

city: Rehovot

first: Sharon

last: Schlesinger

address: Herzl 229

department: Animal Science

country: Israel

institute: Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem

summary: Hear shock (HS) treatment protocols were designed to examine the short- or long-term effects of exposure to mild HS conditions on transcriptome of MSCs.

overall_design: One day after seeding MSCs from early passages (P2–P4), were moved from 37°C to 40.5°C  for six-hour (ShortHS) or three days (LongHS) or a six hours HS followed by three days recovery back at 37°C (ShortHS_Recovery). Each HS treatment had a matching 37°C normothermic (NT) control (ShortNT and LongNT). The two time points were selected in order to capture the immediate transcriptional effects of HS vs. the stable and delayed ones. Immediately after the completion of the six hours or three days treatments, the cells were harvested, viability checked and pellets stored at -80° for later matched RNA extraction and sequencing. RNA sequencing and bioinformatics analysis were performed to systematically examine the transcriptional changes following each treatment and to identify specific biological features and processes.

first: Ivana

last: Ribarski-Chorev


title: ChIP-seq on ZNF506 overexpression HEK293T to verify ZNF506 binding capacity at PBS-Pro sites.

city: shanghai

first: qian

last: wu

address: siping

department: biology

institute: tongji university

summary:
To identify ZNF506 genome-wide target sites, we performed ChIP-seq assay and found that ZNF506 binding sites enriched at PBS-Pro-containing ERV subfamilies (ERVPs) and further motif calling analysis showed that ZNF506 binds to PBS-Pro sequences, promoting formation of H3K9me3 modifications at binding regions. And ChIP-seq assay also indicated that the distinction of H3K9me3 signals closed to ZNF506 peak regions between ZNF506 overexpressing (OE) HEK293T cells and ZNF506 Knockout (KO) HEK293T cells was correlated with the different recruitment of corepressor KAP1.
The ChIP-seq experiments using GFP antibodies and H3K9me3 antibodies on ZNF506-GFP OE HEK293T cell lines, and ZNF417-GFP OE HEK293T cell lines were used as controls for H3K9me3 ChIP. Also, the ChIP-seq experiments were performed using H3K9me3 antibodies and KAP1 antibodies on ZNF506 KO HEK293T cells and ZNF506 OE HEK293T cells.

overall_design: ZNF506-GFP OE HEK293T cell lines were used to perform two replicates of ChIP reaction with GFP antibodies for KZFPs. ZNF506-GFP OE HEK293T cell lines were used for anti-H3K9me3 ChIP and ZNF417-GFP OE HEK293T cells performed as controls. And the ChIP-seq experiments using H3K9me3 antibodies and KAP1 antibodies on ZNF506 KO HEK293T cells and ZNF506 OE HEK293T cells for comparison.

first: Lu

last: Fang


title: The LARP1 homolog Slr1p controls the stability and expression of proto-5’TOP mRNAs in fission yeast [RIP-seq]

status: Public on Jul 18 2023

overall_design: To identify RNA targets of Slr1p in S. pombe, wildtype yAS99 cells were used. Immunoprecipitation of Slr1p was performed after formaldehyde crosslink followed by sequencing of associated RNAs (RIP-Seq). Non-immune rabbit IgG immunoprecipitation was used as a control. Sequencing was performed for two biological replicates.


title: Elastic Fibers Define Embryonic Tissue Stiffness to Enable Buckling Morphogenesis of the Small Intestine

city: New York

first: Nandan

last: Nerurkar

state: NEW YORK

department: Biomedical Engineering

institute: Columbia University

summary: During embryonic development, tissues must possess precise material properties to ensure that cell-generated forces give rise to the stereotyped morphologies of developing organs. However, the question of how material properties are established and regulated during development remains understudied. Here, we aim to address these broader questions through the study of intestinal looping, a process by which the initially straight intestinal tube buckles into loops, permitting ordered packing within the body cavity. Looping results from elongation of the tube against the constraint of an attached tissue, the dorsal mesentery, which is elastically stretched by the elongating tube to nearly triple its length. This elastic energy storage allows the mesentery to provide stable compressive forces that ultimately buckle the tube into loops. Beginning with a transcriptomic analysis of the mesentery, we identified widespread upregulation of extracellular matrix related genes during looping, including genes related to elastic fiber deposition. Combining molecular and mechanical analyses, we conclude that elastin confers tensile stiffness to the mesentery, enabling its mechanical role in organizing the developing small intestine. These results shed light on the role of elastin as a driver of morphogenesis that extends beyond its more established role in resisting cyclic deformation in adult tissues.

overall_design: Looping morphogenesis of the small intestine is driven by geometric and mechanical properties of the mesentery.In the absence of a strong contextual understanding of mesentery biology, we opted to perform a transcriptomic analysis of the tissue throughout the stages of looping, on the grounds that genes related to regulation of mesentery growth and stiffness are likely differentially regulated before, during, and at the end of looping. We performed bulk RNA-Seq analysis on mesentery tissue collected from chick embryos at embryonic day 8 (E8)  when looping begins, E12 when looping is underway, and E16 when looping is complete.

first: Elise

last: Loffet

first: John

last: Durel

first: Hyunjee

last: Lim


title: TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions

status: Public on Jul 23 2023

first: Gang


title: Identifying genetic determinants of Streptococcus pyogenes-host interactions in a murine intact skin infection model

status: Public on Jul 17 2023

city: Aurora

first: Reid

middle: V

last: Wilkening

state: CO

department: Department of Pediatrics, Section of Critical Care Medicine

institute: University of Colorado

summary: Streptococcus pyogenes is an obligate human pathobiont associated with many disease states. Here, we present a novel model of S. pyogenes infection using intact murine epithelium. From this model, we were able to perform RNA sequencing to evaluate the genetic changes undertaken by both the bacterium and host at 5 and 24 hours post infection. Analysis of these genomic data demonstrate that S. pyogenes undergoes significant genetic adaptation to successfully infect the murine epithelium, including changes to metabolism and activation of the Rgg2/Rgg3 quorum sensing (QS) system. Subsequent experiments demonstrate that an intact Rgg2/Rgg3 QS cascade is necessary to establish a stable superficial skin infection. Furthermore, activation of this pathway results in increased murine morbidity and increased bacterial burden on the skin. This phenotype is associated with gross changes to the murine skin, as well as histopathological evidence of inflammation. Taken together, these experiments offer a novel method to investigate S. pyogenes-epithelial interactions and demonstrate that a well-studied QS pathway is critical to a persistent infection.

sample_organism:
Mus musculus
Streptococcus pyogenes NZ131

overall_design: To investigate gene expression assoicated with infection of intact murine skin by Streptococcus pyogenes, we obtained RNA from both mice and bacterium at 5 and 24 hours after infection. Dual RNAsequencing allowed for comparison of bacterial samples to their input control, as well as murine samples versus their uninfected control.

first: Christophe

last: Langouët-Astrié

first: Morgan

last: Severn

first: Michael

last: Federle

first: Alexander

middle: R

last: Horswill