title: Epigenetic evolution of the Ly49 gene family [NanoPore]

status: Public on Apr 20 2024

city: Saint Louis

first: Changxu

last: Fan

state: Missouri

address: 4515 McKinley Avenue, Room 5213

department: Genetics

institute: Washington University in St. Louis

summary: Comparative genomics has revealed the rapid lineage-specific expansion of multiple gene families involved in immunity, presumably due to the complexity of ensuring pathogen defense while maintaining self-tolerance. Paralogs within these gene families often evolved distinct roles in immunity. However, less is known about how the epigenome and cis-regulation of these gene families evolved. Here we focus on the epigenetic evolution of the rapidly evolving murine Ly49 gene family. Ly49 genes mainly encode either inhibitory or activating surface receptors in natural killer (NK) cells. Together, they fine-tune NK cell activation. We systemically mapped Ly49 transcription initiation, chromatin accessibility, DNA methylation and 3D chromatin interactions. Our data revealed that each Ly49 gene forms a cis-regulatory unit consisted of one or more lowly accessible promoters and a proximal enhancer. Importantly, parallel to the functional divergence of the encoded proteins, inhibitory and activating Ly49 genes have evolved two separate sets of proximal enhancers, likely as a result of lineage-specific losses of enhancer activity. Finally, we show that some Ly49 genes can be cross-regulated by the enhancers of other Ly49 genes, suggesting that the Ly49 family have evolved a concerted regulatory mechanism. Collectively, we demonstrate the different modes of epigenetic evolution for a rapidly expanding gene family essential for host immunity, and provide a rich epigenetic dataset for NK cell research.

overall_design: Assembling the Ly49 loci of the MAP8.B6.Ly49h or the MAP8.B6.Ly49m KO mice using targeted nanopore sequencing

first: Sytse

middle: J

last: Piersma

first: Xiaoyun

last: Xing

first: Wayne

last: Yokoyama

first: Ting


title: Differential expression analysis of mutated genes at different loci of H2B

status: Public on Apr 22 2024

city: 南京

first: 冯

last: 刚

address: 江苏省南京市仙林街道南京师范大学

country: China

institute: 南京师范大学

type: Expression profiling by high throughput sequencing

summary: To investigate the differential analysis of gene expression of mutations at different sites of H2B, we constructed six strains with point mutations at different positions of H2B, and then, we performed the gene expression analysis from the RNA-Seq data.

overall_design: Gene expression analysis of the six mutant strains (H2BG52D, H2BP102L, H2BE112K, H2BE75Q, H2BE34K, and H2BD67N) was compared with the wild-type.

first: Gang

last: Feng


title: Epigenetic evolution of the Ly49 gene family [nanoCAGE]

country: USA

overall_design: Transcription start site profiling of murine natural killer cells using nanoCAGE

middle: M

last: Wang


title: Epigenetic evolution of the Ly49 gene family [Bisulfite-Seq]

type: Methylation profiling by high throughput sequencing

overall_design: DNA methylation profiling of murine natural killer cells using whole genome bisulfilte sequencing and amplicon based bisulfite sequencing

first: Jennifer

last: Poursine-Laurent


title: Placental T cells retain enhanced stemness and altered cytokine response favoring durable efficacy of CD19 CAR-T

status: Public on Apr 21 2024

city: Florham Park

first: Kathy

last: Karasiewicz

state: New Jersey

address: 1700 Ave Park

institute: Celularity

summary: We explored transcriptional profile of CD19 CAR-T cells generated from different starting material source (placenta vs. adult peripheral blood) with same protocol and transduced with same CAR construct

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CD19 CAR-T cells derived from placenta vs. peripheral blood

first: William

last: van der Touw

first: Natalia

last: Ruggeri Barbaro


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection

city: Palo Alto

first: Benjamin

state: CA

address: 299 Campus Drive

institute: Stanford

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

sample_organism: Salmonella enterica subsp. enterica serovar Typhi str. Ty2

sample_organism: Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. D23580

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. ST4/74

overall_design: Refer to individual Series


title: IL-6 blockade prevents costimulation blockade-resistant allograft rejection by promoting intragraft regulation in T cell-depleted recipients

city: Vienna

first: Moritz

last: Muckenhuber

state: Vienna

address: Währinger Gürtel 18-20

department: General Surgery

country: Austria

institute: Medical university of vienna


title: Epigenetic evolution of the Ly49 gene family

type: Genome binding/occupancy profiling by high throughput sequencing

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus


title: Epigenetic evolution of the Ly49 gene family [ATAC-Seq]

overall_design: Open chromatin profiling of mouse/rat natural killer cells and mouse CD8 Tregs using ATAC-seq


title: IL-6 blockade prevents costimulation blockade-resistant allograft rejection by promoting intragraft regulation in T cell-depleted recipients [FFPE]

summary: The efficacy of costimulation blockade (CTLA4Ig/belatacept) in transplantation is reduced by an increased incidence of T cell-mediated rejection, which also persists after induction therapy with anti-thymocyte globulin (ATG). Herein, we investigate why ATG fails to prevent costimulation blockade-resistant rejection and how this barrier can be overcome. ATG did not prevent graft rejection in a murine heart transplant model of CTLA4Ig therapy and induced a pro-inflammatory cytokine environment. While ATG improved the balance between Tregs and effector T cells in the peripheral compartment, it had no such effect within cardiac allografts, which showed signs of inflammation. Neutralizing IL-6 alleviated inflammation, increased intragraft Treg frequencies long-term, and enhanced intragraft IL-10 and Th2 cytokine expression. IL-6 blockade together with ATG led to long-term, rejection-free heart graft survival under CTLA4Ig therapy. Combining ATG with IL-6 blockade prevents costimulation blockade-resistant rejection, thereby eliminating a major impediment to clinical use of costimulation blockers in transplantation.

overall_design: to investigate the effects of IL-6 blockade on intragraft inflammation, we performed gene expression profiling of data obtained via RNAseq of FFPE tissue sections of fully mismatched murine cardiac allografts explanted from recipients treated with or without IL-6 blockade isolated 14 days after transplantation.

first: Thomas

last: Wekerle

first: Sophia

last: Derdak


title: FOXA2/AP-1 drives prostate cancer lineage plasticity [ChIP-seq]

city: Boston

first: Zifeng

state: Massachusetts

address: 100 Morrissey Blvd, ISC/4610

department: CPCT

institute: UMASS Boston

summary: FOXA (Forkhead Box Protein A) family proteins function as pioneer transcription factors by loosening the compact chromatin structure and facilitating access for other transcription factors. The role of FOXA1 has been intensively studied in normal prostate epithelial cells and the adenocarcinoma subtype of prostate cancer (PCa) where it acts as a critical pioneer factor for the chromatin binding of androgen receptor (AR). Recent studies have indicated the emergence of FOXA2 as an adaptive response to AR signaling inhibition, particularly in prostate tumors that have undergone lineage reprogramming to a neuroendocrine PCa subtype. However, the molecular basis for this transition from FOXA1 to FOXA2 and its role in regulating the development of PCa lineage plasticity remains unclear. In this study, we show that FOXA2 binds to distinct chromatin regions in multiple AR-null PCa models with different molecular subtypes and that its binding is dependent on an epigenetic factor, LSD1. More importantly, we demonstrate that FOXA2 can function as a major pioneer factor of JUN and govern the chromatin binding of AP-1 complex in PCa exhibiting lineage plasticity. Mechanistically, differential reprogramming of JUN activates lineage-specific super-enhancers that may promote PCa progression by enhancing cell state transitions to multiple lineages. Overall, our study reveals a pivotal function of the LSD1-FOXA2 axis in rewiring AP-1 to induce differential transcriptional reprogramming required for PCa lineage plasticity.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: ChIP-seq analysis of H3K27Ac in PC3 cells. ChIP-seq analysis of FOXA2, JUN, and FOSL1 in PC3 cells treated with siFOXA2 versus siNTC. ChIP-seq analysis of FOXA2 treated with vehicle versus LSD1 inhibitor (ORY-1001, C12) in PC3cells. ChIP-seq analyses of H3K4me2 in NCI-H660 cells. ChIP-seq analyses of FOXA2 in NCI-H660 cells with vehicle versus LSD1 inhibitor (ORY-1001). ChIP-seq analyses of JUN in NCI-H660 cells treated with siFOXA2 versus siNTC. ATAC analysis in PC3 and NCI-H660 cells. ChIP-seq analyses of FOXA2, JUN in LNCaP cells stably expressing FOXA2. ChIP-seq analysis of FOXA2 treated with vehicle versus LSD1 inhibitor (ORY-1001) in DKO cells.

first: Changmeng

last: Cai


title: Time Series of differentiation from human iAT2s to iAT1s in L+DCI medium

status: Public on Apr 19 2024

first: Pushpinder

last: Bawa

address: 670 Albany St

department: Boston University Medical Campus

institute: Center for Regenerative Medicine

summary: The goal of this study is to understand the transcriptomic shift during differentiatiton of iAT2s to iAT1s in 3D

overall_design: BU3 NGAT iPSCs were differentiated into iPSC derived alveolar epithelial type 2 cells (iAT2s) in 3D Matrigel. Cells were passaged into iAT2 medium (CK+DCI) and the first sample was taken 3 days post passage. Media on remaining cells was changed to iAT1 differentiation medium (L+DCI) and samples were taken at 24, 48, and 72 hours post media change. Cells were collected for 10x Genomics cDNA prep on different days, but sequenced together.

first: Claire

last: Burgess

first: Darrell

last: Kotton


title: Replication-attenuated r3LCMV vectors potentiate tumor control via IFN-I

city: Chicago

first: Slim

last: Fourati

state: Illinois

address: 300 E Superior St

department: Medicine

institute: Northwestern University

summary: Viral vectors are being used for the treatment of cancer. Yet their efficacy varies among tumors and their use poses challenges in immunosuppressed patients, underscoring the need for alternatives. We report striking antitumoral effects by a nonlytic viral vector based on attenuated lymphocytic choriomeningitis virus (r3LCMV). We show in multiple tumor models that injection of tumor-bearing mice with this novel vector results in improved tumor control and survival. Importantly, r3LCMV also improved tumor control in immunodeficient Rag1-/- mice. Single cell RNA-Seq analyses, antibody blockade experiments, and KO models revealed a critical role for host IFN-I in the antitumoral efficacy of r3LCMV vectors. Collectively, these data demonstrate potent antitumoral effects by a replication-attenuated LCMV vector and unveil mechanisms underlying its antitumoral efficacy.

overall_design: Single-cell RNA-Sequencing of a tumor sample pooled from 5 mice challenged with tumor cells and then treated with r3LCMV and a tumor sample pooled from 5 different mice challenged with tumor cells and then treated with PBS. Each of the two tumor samples was enriched for CD45+ cells and then sequenced.

first: Pablo

last: Penaloza-MacMaster

first: Ching

last: Wai


title: SAC-seq for transcriptome-wide quantitative sequencing of mRNA m6A at base resolution in Arabidopsis

first: CHUAN

last: HE

address: 929 East 57th Street, GCIS E313

institute: University of Chicago

summary: Here, we reported transcriptome-wide m6A modification maps within single-base resolution using m6A-SAC-seq in rice and Arabidopsis. Our analysis uncovered a total of 205,691 m6A sites distributed across 22,574 genes in rice, and 188,282 m6A sites across 19,984 genes in Arabidopsis.

overall_design: To establish a comprehensive atlas to investigate the tissue specificity and evolutionary conservation of poly(A) RNA m6A regulation in plants, we first extracted the total RNA of nine Arabidopsis tissues (seedling, shoot, root, rosetta leaf, cauline leaf, stem, flower, silique and seed) as well as eight rice tissues (plumule dark, plumule light, Seedling at 8 days, Seedling at 2 weeks, panicle, flag leaf at 10 days after anthesis, endosperm at 10 days after anthesis, and embryo at 10 days after anthesis with two biological replicates for each sample (Fig. 1b, c ). poly(A) RNA of each biological replicate was captured first for LC-MS/MS to measure the m6A/A ration in the whole transcriptome.


title: SAC-seq for transcriptome-wide quantitative sequencing of mRNA m6A at base resolution


title: Epigenetic evolution of the Ly49 gene family [Hi-C]

first: Changxu

last: Fan

overall_design: 3D chromatin interaction profiling of murine natural killer cells using capture Hi-C

first: Sytse

last: Piersma

first: Xiaoyun

last: Xing

first: Wayne

last: Yokoyama

first: Ting


title: Epigenetic evolution of the Ly49 gene family [RNA-seq]

overall_design: Gene expression profiling of murine natural killer cells using RNA-seq (Ly49h/m enhancer KO vs WT)

first: Jennifer

last: Poursine-Laurent

last: Wang


title: Human Muse cells isolated from preterm- and term-umbilical cord delivered therapeutic effects in rat bleomycin-induced lung injury model without immunosuppressant

status: Public on Apr 18 2024

city: Sendai

first: Yoshihiro

last: Kushida

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

country: Japan

institute: Tohoku University

summary: Background Bleomycin (BLM)-induced lung injury is characterized by the mixed histopathologic changes with inflammation and fibrosis. These changes are also observed in human patients with bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Although no curative therapies for these lung vsdiseases exist, stem cell therapy has emerged as a potential therapeutic option. Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent-like/macrophage-like stem cells distributed in various adult and fetal tissues as stage-specific embryonic antigen-3-positive cells. They selectively home to damaged tissue by sensing sphingosine-1-phosphate and replace the damaged/apoptotic cells by in vivo differentiation. Clinical trials for some human diseases suggest the safety and therapeutic efficacy of intravenously injected human leukocyte antigen-mismatched allogenic-Muse cells from adult bone marrow (BM) without immunosuppressant. Here, we evaluated the therapeutic effects of human Muse cells from preterm- and term-umbilical cord (UC), and adult BM in a rat BLM-induced lung injury model. Methods Rats were endotracheally administered BLM to induce lung injury on day 0. On day 3, human preterm UC-Muse, term UC-Muse, or adult BM-Muse cells were administered intravenously without immunosuppressants, and rats were subjected to histopathologic analysis on day 21. Body weight, serum surfactant protein D (SP-D) levels, and oxygen saturation (SpO2) were monitored. Histopathologic lung injury scoring by the Ashcroft and modified American Thoracic Society document scales, quantitative characterization of engrafted Muse cells, RNA sequencing analysis, and in vitro migration assay of infused Muse cells were performed. Results Rats administered preterm- and term-UC-Muse cells exhibited a significantly better recovery based on weight loss, serum SP-D levels, SpO2, and histopathologic lung injury scores, and a significantly higher rate of Muse cell homing to the lung and alveolar marker expression (podoplanin and prosurfactant protein-C) than those administered BM-Muse cells. Rats receiving preterm-UC-Muse cells showed statistically superior results to those receiving term-UC-Muse cells in many of the measures. These findings are thought to be due to higher expression of genes related to cell migration, lung differentiation, and cell adhesion. Conclusion Preterm UC-Muse cells deliver more efficient therapeutic effects than term UC- and BM-Muse cells for treating the BLM-induced lung injury in a rat model.

overall_design: To evaluate the characteristics of human Muse cells from preterm- and term-UC, and adult BM, three replicates of cultured preterm-UC-Muse, term-UC-Muse, and BM-Muse cells were subjected to RNA-seq.

first: Kaung

last: NWin

first: Keiji

last: Yamana

first: Sota

last: Iwatani

first: Makiko

last: Yoshida

first: Nanako

last: Nino

first: Cho

middle: Y

last: Mon

first: Hiroyuki

last: Ohsaki

first: Shingo

last: Kamoshida

first: Kazumichi

last: Fujioka

first: Mari

last: Dezawa

first: Noriyuki

last: Nishimura


title: Transcriptome of E9.5 WT and Smyd1-KO mouse hearts

city: Chapel Hill

first: Wei

last: Shi

state: NC

address: 221 Fordham Hall

department: Biology

institute: UNC-Chapel Hill

summary: Purpose: The goal of this study is to identify the differential  cardiac transcriptome profiling between WT and Smyd1 null (Smyd1-KO) hearts at E9.5 using RNA-seq. Methods: mRNA profiles of E9.5 WT and Smyd1-KO mouse hearts were generated by deep sequencing, n=3 for each genotype, using Illumina HiSeq2500. The sequence reads were aligned to the mm10 reference genome using STAR via the bcbio-nextgen RNA-sequencing pipeline. Differential gene expression was determined by DEseq2. Results: 1756 genes were differentially expressed between WT and Smyd1-KO hearts [adjusted P value <0.05, |log2(Fold Change)| > 0.5], with 1130 upregulated and 626 downregulated in E9.5 Smyd1-KO hearts.

overall_design: WT and Smyd1-KO hearts at E9.5, n=3 for each genotype, were dissected in cold PBS and immediately stored in RNAlater (Thermo, AM7020) solution, and were kept in -20C until use. RNA from individual embryonic hearts was extracted using the RNAqueous-Micro total RNA isolation kit (Thermo, AM1931). The concentration and quality of purified RNA were assessed by Tape Station (Agilent). RNA samples with RNA integrity numbers (RIN) ranged from 8.0 to 10.0 were used for library prep and sequencing.

first: Frank

last: Conlon

first: Kerry

last: Dorr


title: ATAC-seq of E9.5 WT and Smyd1-KO mouse hearts

summary: Purpose: The goal of this study is to identify the differential  cardiac chromatin accessibility between WT and Smyd1 null (Smyd1-KO) hearts at E9.5 using ATAC-seq. Methods: Four hearts at E9.5 were pooled per genotype per replicate, and were then dissociated into single cells. 40,000 viable cells were taken for were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA. Fragmented DNA was then amplified using bar-coded PCR primers and libraries were seuqenced. Results: 25851 differential peaks (2-fold change) were identified between E9.5 WT and Smyd1-KO hearts.

overall_design: Each embryonic mouse heart was dissected in cold PBS and individually cryopreserved in 200 mL of DMEM medium with 10% FBS and 10% DMSO (vol/vol) using a Mr. Frosty isopropyl alcohol chamber. Hearts from each genotype were thawed in a 37℃-water bath and pooled (n = 4 for E9.5 hearts per genotype per replicate). Samples were pelleted at 1000g for 3 min at 4℃. The medium was aspirated, and the hearts were washed with cold PBS. Hearts were then dissociated in 200 mL of 0.05% Trypsin/EDTA at 37℃ for 10 min, with gentle agitation every 3 min. Dissociation was stopped by adding 400 mL of DMEM/10% FBS (vol/vol), and the samples were passed through a 100-micron cell strainer to remove connective tissue. Cells were quantified using Trypan blue, and 40,000 viable cells were taken for downstream procedures. Dissociated embryonic cardiac cells were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA Fragmented DNA was then amplified using bar-coded PCR primers and libraries were pooled. Pooled libraries were then sequenced (Illumina Next-seq) to a depth of 100 million reads per sample. Reads were aligned to the mm10 reference genomes using BWA-MEM and peaks were called using HOMERv4.10.3. Annotation and analysis were performed using ChIP-Seeker v1.20.0.


title: Caspase-mediated processing of TRBP regulates apoptosis during viral infection

city: Bunkyo-ku, Tokyo

first: Tomoko

last: Takahashi

institute: The University of Tokyo

summary: RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

overall_design: Total RNAs were extracted from TRBP knockout cells expressing TRBP-WT, -N, or -C and performed microarray analysis.

first: Kumiko

last: Ui-Tei


title: Activation of TLR9 signaling suppresses the immuno modulating functions of CD55lo fibroblastic reticular cells during bacterial peritonitis

city: Manhasset

first: Hai

last: Huang

state: NY

address: 350 community drive

department: Institute of Molecular Medicine

institute: Feinstein Institutes for Medical Research

summary: Fibroblastic reticular cells (FRCs) are heterogeneous. We use single cell RNA sequencing to identify subsets of FRCs in fat-associated lymphoid clusters (FALC) in the mesenteric adipose tissue

overall_design: Cells of FALC were isolated from the mesenteric adipose tissues of Naïve C57BL/6 mice and subjected to single cell RNA sequencing.

first: Meihong

last: Deng

first: Ting

last: Jiang

first: Yiming

last: Li


overall_design: Total RNAs were extracted from mock-treated or SeV infected TRBP knockout cells expressing TRBP-WT or -D234A and performed microarray analysis.


title: The effects of JAK inhibition on SARS-CoV-2-induced kidney injury

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

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

summary: Patients often present with kidney injury in COVID-19. Although severe COVID-19 cases are treated with baricitinib, a JAK inhibitor, the effects of baricitinib on the kidneys in COVID-19 are unclear. The authors examined the pharmacological effects of baricitinib on kidney injury using an in vivo murine COVID-19 model.

overall_design: Male BALB/c mice (6–10 weeks old) were intranasally administered 2 × 10⁵ PFU/mouse mouse-adopted SARS-CoV-2 (MA10). MA10-infected mice were administered the JAK inhibitor baricitinib on days 1–3. RNA-sequencing was performed using the kidneys of non-infected, and MA10-infected mice treated with vehicle (0.5% CMC) or 10 mg/kg baricitinib.

first: Masanori

last: Obana

first: Hibiki

last: Sakai


title: FLI1 promotes IFN-γ-induced kynurenine production to impair anti-tumor immunity (ATAC-Seq)

city: Guangzhou

first: wuguo

last: deng

state: Guangdong

address: 651 Dongfengdongroad, Guangzhou, Guangdog

institute: Sun yat-sen university

summary: Nasopharyngeal carcinoma (NPC)-mediated immunosuppression within the tumor microenvironment (TME) frequently culminates in the failure of otherwise promising immunotherapies, underscoring the imperative to develop strategies to counteract the tumor's ability to foster an immunologically "cold" TME. In this study, we identify tumor-intrinsic FLI1 as a critical mediator in impairing T cell anti-tumor immunity through bioinformatic analysis. A mechanistic inquiry reveals that FLI1 orchestrates the expression of CBP and STAT1, facilitating chromatin accessibility and transcriptional activation of IDO1 in response to T cell-released IFN-γ. This regulatory cascade ultimately leads to augmented IDO1 expression, resulting in heightened synthesis of kynurenine (Kyn) in tumor cells. This, in turn, fosters CD8+ T cell exhaustion and regulatory T cell (Treg) differentiation, a process that redounds to the advantage of tumor cell survival.

overall_design: To elucidate the mechanism driving FLI1-mediated CD8+ T cell exhaustion, we conducted RNA sequencing (RNA-seq) on FLI1-KO and WT HK1 cells after co-incubation with activated CD8+ T cells.

first: Enni

last: Chen

first: Jiawei

last: Wu

first: Wuguo


title: FLI1 promotes IFN-γ-induced kynurenine production to impair anti-tumor immunity (ChIP-Seq)


title: Single-cell transcriptional profile of CD34+ hematopoietic progenitor cells from del(5q) Myelodysplastic Syndromes and impact of lenalidomide

city: Pamplona

first: Guillermo

last: Serrano

state: Navarra

address: Av. de Pío XII, 55

department: Bioinformatics

country: Spain

institute: CIMA - Universidad de Navarra

summary: While del(5q) MDS patients comprise a well-defined hematological subgroup, the molecular basis underlying its origin and the reason behind the relapse to lenalidomide remains unknown. Using scRNAseq on CD34+ progenitor cells from patients with del(5q) MDS we were able to identify cells harboring the deletion, enabling us to deeply characterize the transcriptional impact of this genetic insult on disease pathogenesis and treatment response. We found, across all patients, an enrichment of del(5q) cells in GMP and megakaryocyte-erythroid progenitors not described to date. Interestingly, both del(5q) and non-del(5q) cells presented similar transcriptional lesions when compared to progenitors from healthy individuals, indicating that all cells, and not only those harboring the deletion, are altered in these patients and may contribute to aberrant hematopoietic differentiation. However, GRN analysis revealed a group of regulons with aberrant activity in del(5q) cells that could be responsible for triggering altered hematopoiesis, pointing to a more prominent role of these cells in the phenotype of these patients. An analysis of del(5q) MDS patients achieving hematological response upon lenalidomide treatment showed that the drug reverted several transcriptional alterations in both del(5q) and non-del(5q) cells, but other lesions remained, which may be responsible for potential future relapses. Moreover, lack of hematological response was associated with the inability of lenalidomide to reverse transcriptional alterations. Collectively, this study provides a deep characterization of del(5q) and non-del(5q) cells at single-cell resolution, revealing previously unknown transcriptional alterations that could contribute to disease pathogenesis, or lack of responsiveness to lenalidomide.

overall_design: Cells from bone marrow aspirates were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of CD34+ signal and analyzed using scRNAseq.

first: Nerea

last: Berastegui

first: Aintzane

last: Diaz

first: Teresa

last: Ezponda

first: Mikel

last: Hernaez


title: RNA sequencing of Rbpm1 knockout in Plasmodium yoelii

city: Xiamen

first: jiepeng

last: guan

address: Xiamen University

institute: Xiamen University

summary: RBPm1, an RNA-binding protein, plays a crucial role in regulating axoneme assembly during male gametogenesis of Plasmodium yoelii. To elucidate the molecular mechanisms that underlie this process, we conducted RNA-seq analysis to investigate changes in the male transcriptome resulting from the loss of RBPm1.

sample_organism:
Plasmodium yoelii
Plasmodium yoelii yoelii 17XNL

overall_design: To obtain highly pure male gametocytes, we employed a DFsc7 reporter strain that selectively exhibited GFP expression within these cells. We created a mutant strain by deleting RBPm1 from the DFsc7 strain. Male gametocytes from both the DFsc7 and mutant strains were sorted using fluorescence-activated cell sorting (FACS), relying on GFP fluorescence intensity as a criterion, and subsequently subjected to RNA-seq analysis.  Addition: Through Principal component analysis (PCA), it was observed that the RNA-seq data from the initial sample, WT biol rep1, exhibited poor reproducibility. Subsequently, we re-sorted and obtained three additional WT samples (WT_biol_rep4, WT_biol_rep5, and WT_biol_rep6) for RNA-seq, resulting in improved data reproducibility.

first: Xiaoli

last: Mo

first: Jiepeng

last: Guan


title: FLI1 promotes IFN-γ-induced kynurenine production to impair anti-tumor immunity (RNA-Seq)

last: Deng


title: Plasmodium yoelii gender-specific gametocyte transcriptome

summary: In order to identify RNA binding proteins that are essential for male gametogenesis in the rodent malaria parasite P. yoelii, we conducted RNA-seq analysis to identify RBPs that are specifically expressed in male gametocytes.

overall_design: A double fluorescence reporter strain DFsc7, which allowed for specific expression of GFP in male gametocytes and mCherry in female gametocytes, was utilized to induce gametocytes in mice. Subsequently, the male and female gametocytes were sorted using fluorescence-activated cell sorting (FACS), and RNA-seq analysis was conducted to generate gender-specific transcriptomes. Addition: Through Principal Component Analysis (PCA) analysis, the initial sample, Male biol rep1, showed poor reproducibility in RNA-seq data. Subsequently, we re-sorted and obtained three samples of male gametes (Py17XNL_MG_Rep1, Py17XNL_MG_Rep2, and Py17XNL_MG_Rep3) for RNA-seq, which yielded more reliable data. The corresponding Supplementary files for the improved data are edgeR_male_vs_female.csv.gz and male_vs_female_TPM.csv.gz.

first: Zhenkui


title: Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy [RNA-seq]

status: Public on Apr 16 2024

city: AURORA

first: Etienne

last: Danis

state: CO

department: Cancer Center

institute: University of Colorado, Denver - Anschutz Medical Campus

summary: Using a combination of ChIP-, ATAC-, RNA-seq, and CUT&RUN in model systems of diffuse intrinsic pontine glioma and other pediatric high-grade gliomas, we observe a rapid genome-wide reorganization of active chromatin within hours of exposure to therapeutic ionizing radiation. This redistribution facilitates PTEFb-mediated pause-release of Pol II to drive nascent transcriptional induction of canonical DNA damage response programs. Concurrent inhibition of PTEFb imparts a transcription elongation defect, disrupting this chromatin reorganization and blunting transcriptional induction. Functionally, concurrent PTEFb inhibition abrogates key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival.

overall_design: RNAseq was conducted in the SU-DIPG4, HSJD-DIPG007 and SF8628 cell lines after 6 Gy radiation in the presence or absence of AZD4573.

first: Lays

last: Sobral

first: Faye

last: Walker

first: Nathan

last: Dahl


title: Multi-center Korean cohort study based on RNA-sequencing data targeting COPD patients

status: Public on Apr 17 2024

city: Sejong

first: Sinwoo

last: Park

address: Sejongro

country: South Korea

institute: Korea Universiy

summary: To determine gene expression changes that contribute to the development of COPD among smokers using high-throughput RNA sequencing in the peripheral blood of COPD patients. Total RNA sequencing was investigated by prospectively collecting lung function test results and whole blood samples from smokers who visited the outpatient department of respiratory medicine at three hospitals since 2020. This study confirmed gene expression differences in non-coding RNA in COPD patients according to the degree of airflow limitation through RNA-Seq transcriptome analysis. These results may provide valuable predictive resources for future COPD research.

overall_design: A transcriptomic epidemiologic study was conducted on a total of 294 patients with mild to severe COPD at three hospitals located in South Korea.

first: Jinkyeong

first: Minseok

last: Seo


title: FLI1 promotes IFN-γ-induced kynurenine production to impair anti-tumor immunity

first: wuguo

last: deng

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

overall_design: Refer to individual Series


title: shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq PC-3]

city: Moscow

first: Anton

last: Zhiyanov

address: 20 Myasnitskaya ulitsa

department: Faculty of Biology and Biotechnology

country: Russia

institute: HSE University

type: Non-coding RNA profiling by high throughput sequencing

summary: miRNA overexpression strategy based on RNA Polymerase (Pol) III mediated expression of short hairpin RNA (shRNA) coding particular miRNA is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that extra \texttt{U} residues (up to five) added Pol III at the 3$'$-end of the transcribed shRNA associated with a shift in the Dicer cleavage position of the shRNA. This results in the undesirable formation of 5$'$-end miRNA isoforms (5$'$-isomiRs), which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. The predominant expression of 5$'$-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5$'$-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved.

overall_design:
PC-3 (prostate cancer) cell line was transduced by lentiviral particles coding one of the two types of shRNA to achieve stable overexpression of hsa-miR-93-5p.
All shRNA constructs contained the sequence of the canonical hsa-miR-93-5p and differed in their stem lengths. The first one contained the full sequence of hsa-miR-93-5p: 5'-CUACCUGCACGAACAGCACUUAG-3' -- upper (5p) stem, 5'-CUUCAAGAGAG-3' -- stem loop, 3'-...UUGAUGGACGUGCUUGUCGUGAAAC-5' -- lower (3p) stem, while the second one contained 4 nucloetides 3'-end shortened version: 5'-CUGCACGAACAGCACUUAG-3' -- upper (5p) stem, 5'-CUUCAAGAGAG-3' -- stem loop, 3'-...UUGACGUGCUUGUCGUGAAAC-5' -- lower (3p) stem.
Both shRNA-overexpressed cell lines were sequenced to understand the impact of shRNA length on isomiR production.

first: Diana

last: Maltseva

first: Ivan

last: Kirillov

first: Roman

last: Suvorov

first: Daria

last: Gubani

first: Anna

last: Kudriaeva

first: Alexey

last: Belogurov

first: Alexander

last: Tonevitsky


title: Pancreatic Beta-Cell IL-22 Receptor Deficiency Induces Age-Dependent Dysregulation of Insulin Biosynthesis and Systemic Glucose Homeostasis

city: Brisbane

first: haressh

last: sajiir

state: QLD

address: 37 Kent Street

country: Australia

institute: Mater Research Institute - The University of Queensland

summary: The IL-22RA1 receptor is highly expressed in the pancreas, and exogenous IL-22 has been shown to reduce endoplasmic reticulum and oxidative stress in human pancreatic islets and promote secretion of high-quality insulin from beta-cells. However, the endogenous role of IL-22RA1 signaling on these cells remains unclear. Here, we show that antibody neutralisation of IL-22RA1 in cultured human islets leads to impaired insulin quality and increased cellular stress. Through the generation of mice lacking IL-22ra1 specifically on pancreatic alpha- or beta-cells, we demonstrate that ablation of murine beta-cell IL-22ra1 leads to similar decreases in insulin secretion, quality and islet regeneration, whilst increasing islet cellular stress, inflammation and MHC II expression. These changes in insulin secretion led to impaired glucose tolerance, a finding more pronounced in female animals compared to males. Our findings attribute a regulatory role for endogenous pancreatic beta-cell IL-22ra1 in insulin secretion, islet regeneration, inflammation/cellular stress and appropriate systemic metabolic regulation.

overall_design: Murine Pancreatic beta cells (MIN6N8) were treated with 100ng/mL mIL-22 or vehicle (PBS) for 4 hours and confirmation of IL-22 activated pathways was performed via westerm blotting. Subsequently, total RNA was extracted and RNA quality was confirmed by Nanodrop and the Agilent bioanalyzer.

first: Haressh

last: Sajiir

first: Kuan Yau

last: Wong

first: Alexandra

last: Müller

first: Sahar

last: Keshvari

first: Lucy

last: Burr

first: Elena

last: Aiello

first: Teresa

last: Mezza

first: Andrea

last: Giaccari

first: Guido

last: Sebastiani

first: Francesco

last: Dotta

first: Grant

middle: A

last: Ramm

first: Graeme

last: Macdonald

first: Michael

last: McGuckin

first: Johannes

middle: B

last: Prins

first: Sumaira

middle: Z

last: Hasnain


title: Ciliogenesis defects after neurulation impact brain development and neuronal activity in larval zebrafish

city: Trondheim

first: Nathalie

last: Jurisch-Yaksi

address: Erling Skjalgsons gate 1

country: Norway

institute: NTNU

summary: Cilia are slender, hair-like structures extending from cell surfaces and playing essential roles in diverse physiological processes. Within the nervous system, primary cilia contribute to signaling and sensory perception, while motile cilia facilitate cerebrospinal fluid flow. Here, we investigated the impact of ciliary loss on neural circuit development using a zebrafish line displaying ciliogenesis defects. We found that cilia defects after neurulation affects neurogenesis and brain morphology, especially in the cerebellum, and lead to altered gene expression profiles. Using whole brain calcium imaging, we measured reduced light-evoked and spontaneous neuronal activity in all brain regions. By shedding light on the intricate role of cilia in neural circuit formation and function in the zebrafish, our work highlights their evolutionary conserved role in the brain and set the stage for future analysis of ciliopathy models.

overall_design: To analyse the impact of cilia loss on gene expression, we performed RNA sequencing analysis of 4 days old elipsa mutant and sibling control zebrafish larvae. We extracted total RNA from mutant (selected based on their curly tail down phenotype) and sibling controls (mixture of heterozygous and wild-type). RNAsequencing was performed on 4 different clutches.

first: N

first: P

middle: P

last: D'Gama


title: DNA Microarray and Bioinformatic Analysis Reveals the Potential of Whale Oil in Enhancing Hair Growth in C57BL/6 Mice

city: Tsukuba

first: RANDEEP

last: RAKWAL

state: Ibaraki

address: 1-1-1 Tennodai

department: Institute of Health and Sport Sciences

institute: University of Tsukuba

summary: Much research has been conducted to determine how hair regeneration is regulated, as this could provide therapeutic, cosmetic, and even psychological interventions for hair loss. The current study focused on the hair growth effect and effective utilization of fatty oil obtained from Bryde's whales through high-throughput DNA microarray approach in conjunction with immunohistochemical observations. The research also examined the mechanisms and factors involved in hair growth. In an experiment using female C57BL/6J mice, the vehicle control group (VC: propylene glycol: ethanol: water), the positive control group (MXD: 3% minoxidil), and the experimental group (WO: 20% Whale Oil) were topically applied to the back of the mouse. The results showed that 3% MXD and 20% WO were more effective than VC in promoting hair growth, especially 20% WO. Furthermore, in hematoxylin and eosin-stained skin tissue, an increase in the number of hair follicles and subcutaneous tissue thickness was observed with 20% WO. Whole-genome transcriptomic analysis also showed that 20% WO may have lower stress- and inflammation-related responses than 3% MXD. Therefore, whale oil can be expected to be used as a safe hair growth agent.

overall_design: Whole genome DNA microarray was carried out on the skin samples (the back whole skin tissue of C57BL/6J mice was ground into a fine powder in liquid nitrogen and stored at -80°C until RNA isolation, and total RNA was extracted from powdered mouse skin tissue using the RNeasy Mini Kit ) to suggest molecular factors underlying the hair-promoting effects of WO.  Total RNA extracted from mouse skin tissue for each control and treatment (n=6) was pooled in each group, prior to DNA microarray analysis.

first: Junko

last: Shibato

first: Randeep

last: Rakwal

first: Fumiko

last: Takenoya

first: Michio

last: Yamashita

first: Ai

last: Kimura

first: Satoshi

last: Hirako

first: Seiji

last: Shioda


title: XRN1 deletion induces PKR-dependent cell lethality in interferon-activated cancer cells

first: Tao

last: Zou

address: 450 Brookline Avenue

institute: Dana-Farber Cancer Institute

summary: Emerging data suggest that induction of viral mimicry responses through activation of double-stranded RNA (dsRNA) sensors in cancer cells is a promising therapeutic strategy. One approach to induce viral mimicry is to target molecular regulators of dsRNA sensing pathways. Here, we show that the exoribonuclease XRN1 is a negative regulator of the dsRNA sensor protein kinase R (PKR) in cancer cells with high interferon-stimulated gene (ISG) expression. XRN1 deletion causes PKR pathway activation and consequent cancer cell lethality. Disruption of interferon signaling with the JAK1/2 inhibitor ruxolitinib can decrease cellular PKR levels and rescue sensitivity to XRN1 deletion. Conversely, interferon-b stimulation can increase PKR levels and induce sensitivity to XRN1 inactivation. Lastly, XRN1 deletion causes accumulation of endogenous complementary sense/anti-sense RNAs, which may represent candidate PKR ligands. Our data demonstrate how XRN1 regulates PKR, and how this interaction creates a vulnerability in cancer cells with an activated interferon cell state.

overall_design:
To determine the RNA transcripts that are regulated by XRN1 in human cancer cell lines, we performed strand-specific RNA sequencing on rRNA-depleted samples from control, XRN1 knockout, PKR knockout, or XRN1/PKR double knockout HCC366 cells and control or XRN1 knockout NCI-H1650 cells.
To understand the gene expression changes induced with each condition, we performed standard differential gene expression analysis between samples within each cancer cell line. To identify candidate double-stranded RNA transcripts that may stimulate PKR activation after XRN1 deletion, we developed a novel computational pipeline with which we found that XRN1 deletion causes increased accumulation of complementary sense and anti-sense RNA transcript pairs.

first: Meng

last: Zhou

first: Akansha

last: Gupta

first: Patrick

last: Zhuang

first: Alyssa

middle: R

last: Fishbein

first: Hope

last: Wei

first: Zhouwei

last: Zhang

first: Diego

last: Capcha-Rodriguez

first: Andrew

middle: D

last: Cherniack

first: Matthew

last: Meyerson


title: IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T-cell engager immunotherapy in solid tumors

city: Chuncheon

first: Hawon

last: Song

address: Gangwon Daehak-gil 1

institute: Kangwon National University

summary: Bispecific T-cell engagers (TCEs) show promising clinical efficacy in blood tumors, but their application to solid tumors with immunosuppressive environments remains challenging. Here, we show that Fc-fused IL-7 (rhIL-7-hyFc) changes the intratumoral CD8 T cell landscape, allowing TCE-immunotherapy to function effectively in solid tumors. In order to explore the transcriptomic changes induced by rhIL-7-hyFc or a combination of rhIL-7-hyFc and TCE treatment at the clonal level, we performed scRNA/TCR-seq.

overall_design: Tumor-infiltrating CD8 T cells from mice bearing MC38 tumors were isolated following two treatment conditions: (1) treatment with either buffer or rhIL-7-hyFc, and (2) treatment with either rhIL-7-hyFc or a combination of rhIL-7-hyFc and TCE. For the enrichment of CD8 T cells, cells were isolated by positive selection methods with IMag (condition 1) or sorted by FACS (condition 2) after single-cell preparation. The purified CD8 T cells were analyzed by scRNA-seq paired with scTCR-seq.

first: Kun-Joo

last: Lee

first: Donghoon

last: Choi

first: Nara

last: Tae

first: Ha

middle: W

first: Sujeong

last: Park

first: Yeon-Woo

last: Kang

first: Dain

last: Moon

first: Youngsik

last: Oh

first: Ji-hae

last: Kim

first: Dahee

last: Hing

first: Joohyuk

last: Sohn

first: Minji

first: Sun-Kyoung

last: Im

first: Yunji

first: Sun

middle: S

first: Dae

middle: H

first: Seung-Woo


title: Effect of systemic depletion of glutamine via PEGylated glutaminase on CT26 tumor tissue.

city: Houston

first: Navin

last: Varadarajan

state: TX

address: 4726 Calhoun Rd

department: William A Brookshire Chemical and Biomolecular Engineering Department

institute: University of Houston

summary: Proliferating cancer cells are addicted to glutamine for biomass synthesis and energy generation. We here enzymatically deplete circulating glutamine in mice and report how cancer cells adapt to low circulating glutamine and document immunological changes in the tumor microenvironment (TME) upon glutamine depletion in CT26 tumor model. We show that cancer cells adapt to low glutamine by diverting intracellular glutamine flux to nucleotide synthesis and downregulating global translation. Through immune cell deconvolution and GSEA, we show that systemic glutamine depletion is immunsosuppressive and marked by accumulaton of PMN-MDSC in the TME.

overall_design: We implanted CT26 tumor in six BALB/c mice subcutaneously. When, the tumors were palpable, we treated three mice with 50 IU/kg PEGylated glutaminase and three with vehichle (PBS, 10% glycerol) every three days. After two treatmetns, we euthanized the mice, froze the tumor tissue and confirmed glutamine depletion in mouse serum.

first: Monish

last: Kumar


title: Germline-like TCR alpha chains shared between autoreactive T cells in blood and pancreas [dataset1]

city: Seattle,

first: Stephanie

last: Osmond

state: WA

address: 1201 9th Ave

institute: Benaroya Research Institute

summary: How human islet antigen reactive CD4+ memory T cells (IAR T cells) from peripheral blood affect Type 1 Diabetes (T1D) progression in the pancreas is poorly understood. We identified paired alpha/beta (TRA/TRB) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new onset, and established T1D donors, and measured sequence overlap with TCRs in pancreata from organ donors. We detected extensive TRA junction sharing between IAR T cells and pancreatic infiltrating T cells (PIT), with perfect or single mismatched TRA junction amino acid sequences comprising ~34% of unique IAR TRA junctions. PIT-matched TRA junctions were largely public, and showed significant nucleotide sequence convergence, increased use of germline-encoded residues in epitope engagement, and a potential for cross-reactivity. The link with T cells in the pancreas implicates autoreactive IAR T cells with shared TRA junctions in the prediabetic and new onset phases of T1D progression.

overall_design: Islet antigen reactive CD4 T cells were isolated using a CD154 activation assay from cryopreserved PBMC of a cross-sectional cohort of islet autoantibody (Aab) negative (n=6), 1 Aab+ >18y (n=6), 1 Aab+ <14y (n=3), and >1 Aab+ (n=6) donors with increasing risk for progression to T1D. Activated IAR T cells were single-cell sorted and analyzed by single-cell RNA-sequencing to identify T cell receptor α/β pairs in parallel with full transcript profiles.

first: Peter

last: Linsley

first: Karen

last: Cerosaletti


title: Unbiased profiling of clinical kinase inhibitors’ effects in activated macrophages using chromatin modifications as high-content readouts [RNA-Seq]

city: Milan

first: Francesco

last: Gualdrini

address: Via Adamello, 16

department: Department of Experimental Oncology

country: Italy

institute: European Institute of Oncology

summary: More than 500 kinases are implicated in the control of virtually every cellular process in mammals and deregulation of their activity is causally linked to diseases ranging from cancer to inflammatory and degenerative disorders. In the last three decades, massive worldwide efforts led to the approval of 62 clinical kinase inhibitors (CKI), with hundreds of additional molecules in various stages of pre-clinical or clinical development. In addition to the intended target(s), small molecule CKI commonly inhibit multiple additional kinases, resulting in both enhanced clinical effects and undesired side effects that are largely unpredictable based on in vitro selectivity profiling. To complement current CKI profiling methods, we set out a novel experimental and analytical approach grounded on the use of chromatin modifications as unbiased and information-rich readouts of the functional effects of CKI on macrophage activation, a complex biological response of biomedical relevance. This approach allowed us to characterize and dissect the distinctive behaviors of CKI with identical intended targets as well as to discover undescribed activities of CKI on macrophage activation programs.

overall_design: PolyA RNAseq of mouse bone marrow-derived macrophages (BMDM) were pre-treated with DMSO or the individual Kinase Inhibitors for 1h and then stimulated with LPS for 2h.

first: Stefano

last: Rizzieri

last: Pileri

first: Sara

last: Polletti

first: Gioacchino

last: Natoli


title: Unbiased profiling of clinical kinase inhibitors’ effects in activated macrophages using chromatin modifications as high-content readouts [ChIP-Seq]

overall_design: H3K27ac ChIPseq of mouse bone marrow-derived macrophages (BMDM) were pre-treated with DMSO or the individual Kinase Inhibitors for 1h and then stimulated with either LPS or IL4. Samples were retrieved at 30 minutes, 1h, 2h and 4h post-stimulation.


title: Unbiased profiling of clinical kinase inhibitors’ effects in activated macrophages using chromatin modifications as high-content readouts [ATAC-Seq]

overall_design: ATACseq of mouse bone marrow-derived macrophages (BMDM) were pre-treated with DMSO for 1h and then stimulated with either LPS or IL4. Samples were retrieved at 30 minutes, 1h, 2h and 4h post-stimulation.


title: Unbiased profiling of clinical kinase inhibitors’ effects in activated macrophages using chromatin modifications as high-content readouts


title: m5C methylated lncRncr3–MeCP2 interaction restricts miR124a-initiated neurogenesis

city: Boulder

first: Jing

last: Zhang

address: 1945 Colorado ave.

department: MCDB

institute: CU Boulder

summary: Coordination of neuronal differentiation with expansion of the neuroepithelial/neural progenitor cell (NEPC/NPC) pool is essential in early brain development. Our in vitro and in vivo studies identify independent and opposing roles for two neural-specific and differentially expressed non-coding RNAs derived from the same locus: the evolutionarily conserved lncRNA Rncr3 and the embedded microRNA miR124a-1. Rncr3 regulates NEPC/NPC proliferation and controls the biogenesis of miR124a, which determines neuronal differentiation. Rncr3 conserved exons 2/3 are cytosine methylated and bound by methyl-CpG binding protein MeCP2, which restricts expression of miR124a embedded in exon 4 to prevent premature neuronal differentiation, and to orchestrate proper brain growth. MeCP2 directly binds cytosine-methylated Rncr3 through previously unrecognized lysine residues and suppresses miR124a processing by recruiting PTBP1 to block access of DROSHA-DGCR8. Thus, miRNA processing is controlled by lncRNA m5C methylation along with the defined m5C epitranscriptomic RNA reader protein MeCP2 to coordinate brain development.

overall_design: Three individual clones of CRISPR-Cas9 DNA edited Rncr3 exons 2/3 deletion or Rncr3 full-length deletion in NE-4C cells were used. Three biological replicates of non-manipulated NE-4C cells were used as wild type control. All cell samples were cultured under proliferation conditions for three days and then collected and subjected to total RNA extraction.

first: Huili

last: Li

first: Lee

last: Niswander


title: Germline-like TCR alpha chains shared between autoreactive T cells in blood and pancreas


title: Profiling of exosomal microRNAs expression in culture medium of human esophageal cancer cell lines under hypoxic conditions

status: Public on Apr 15 2024

city: Chiba-Shi

first: Yasunori

last: Matsumoto

state: Chiba

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

department: Frontier Surgery

institute: Chiba University

summary:
The purpose of this analysis is to analyze the changes that occur in microRNAs contained in exosomes released from cancer cells in the tumor hypoxic environment of esophageal squamous cell carcinoma(ESCC).
We used microarrays to perform a comprehensive analysis of microRNAs in exosomes released from human ESCC cell lines under normoxic or hypoxic conditions.

overall_design: Two human ESCC cell lines (KYSE-960, T.Tn) were cultured in 20% oxygen or 1% oxygen for 48 hours, and microRNA array analysis of RNA contained in exosomes extracted from the culture supernatant by ultracentrifugation was performed using the GeneChip miRNA 4.0 Array to identify common variable microRNA groups.

first: Maiyulan

last: Abula


title: Tumor mitochondrial oxidative phosphorylation stimulated by the nuclear receptor RORγ represents an effective therapeutic opportunity in osteosarcoma

first: zheng

last: jianwei

address: School of Pharmaceutical Sciences, Sun Yat-sen University, No.132 East Huanhuan Road, Guangzhou

institute: SYSU

summary: Osteosarcoma (OS) is the most common malignant bone tumor with a poor prognosis. The treatment strategy has remained virtually unchanged over the past 40 years. Here, we show that the nuclear receptor RORγ may serve as a potential therapeutic target in osteosarcoma. OS exhibits a hyperactivated OXPHOS program, which fuels the carbon source to promote tumor progression. We found that RORγ is overexpressed in OS tumors and is linked to hyperactivated OXPHOS. RORγ induces the expression of PGC-1β and physically interacts with it to activate the OXPHOS program by upregulating the expression of respiratory chain component genes. Knockdown or pharmacological inhibition of RORγ strongly inhibits OXPHOS activation, downregulates mitochondrial functions and increases ROS production, which results in OS cell apoptosis and ferroptosis. RORγ inverse agonists strongly suppressed OS tumor growth and progression in multiple cell-based xenograft models and in chemotherapy-resistant, patient-derived xenograft (PDX) models and sensitized OS tumors to chemotherapy without obvious toxicity in mice. Taken together, our results indicate that RORγ is a critical regulator of the OXPHOS program in OS and provide a potential therapeutic strategy for this deadly disease.

overall_design: Total RNA was extracted from 143B and MG63 cells, which were treated with vehicle or the inverse agonist GSK805 or XY018 or SR2211 for 48 h. RNA-seq libraries from 2 µg total RNA were prepared by using Illumina Tru-Seq RNA Sample, according to the manufacturer’s instructions. RNA sequencing (RNAseq) analysis was performed by BGI (Beijing Genomics Institute, Shenzhen, China). Briefly, mRNA with a polyA tail was enriched by magnetic beads with OligodT. cDNA was synthesized in a high-temperature system and purified by the kit, then using the BGISEQ platform for RNA-seq. To generate clean reads, raw reads were filtered by using SOAPnuke software. Then, we used HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) and Bowtie2 to compare clean reads and the reference genome sequence. RSEM was used to calculate the gene expression level of each sample. Transcripts were assembled, differentially expressed genes were generated, and heatmaps were drawn.

first: Jianwei

last: Zheng

first: Qianqian

last: Wang

first: Jianghe

last: Chen

first: Guodi

last: Cai


title: Determinants of gastric cancer immune escape identified from non-coding immune-landscape quantitative trait loci

city: Boston

first: Christos

last: Miliotis

state: MA

address: 330 Brookline Ave

institute: BIDMC

summary: Only a portion of cancer patients respond favorably to immunotherapy, indicating the need for new biomarkers and targets. Here, we identify common somatic and germline 3´ untranslated region (3´UTR) variants across the human transcriptome from 375 gastric cancer patients from The Cancer Genome Atlas (39.5% somatic). By performing gene expression quantitative trait loci (eQTL) and immune landscape QTL (ilQTL) analysis, we discover 3´UTR variants with cis effects on expression and immune landscape phenotypes, such as immune cell infiltration. To distinguish between causal and correlative effects of 3´UTR eQTLs in immune-related genes, we perform a massively parallel reporter assay. Our approach identifies numerous 3´UTR eQTLs and ilQTLs (e.g. in ADAR1), providing a unique resource for the identification of immunotherapeutic targets and biomarkers. Our prioritized ilQTL variant signature predicts response to immunotherapy better than standard-of-care PD-L1 expression in independent patient cohorts, showcasing the untapped potential of non-coding mutations in cancer.

overall_design: To assess the effect of 3'UTR variants on gene expression we performed a massively parallel reporter assay (MPRA) in two stomach cancer cell lines (AGS, SNU719) in triplicate.

first: Dimitra

last: Karagkouni

first: Kaia

last: Mattioli

first: Frank

last: Slack

first: Ioannis

last: Vlachos


title: Unbiased profiling of clinical kinase inhibitors’ effects in activated macrophages using chromatin modifications as high-content readouts [Chromatin associated RNA-seq]

first: Francesco

last: Gualdrini

overall_design: Chromatin associated RNAseq of mouse bone marrow-derived macrophages (BMDM) were pre-treated with DMSO for 1h and then stimulated with either LPS or IL4. Samples were retrieved at 30 minutes, 1h, 2h and 4h post-stimulation.

first: Stefano

last: Rizzieri

last: Pileri

first: Sara

last: Polletti


title: RNA-seq data of BRAF wild-type and BRAF mutant tumor cells treated with the ERK inhibitor GDC-0994

city: guangzhou

first: Yulu

state: guangdongsheng

address: No. 58 Zhongshan Second Road

department: Precision Medicine Research Institute

institute: The First Affiliated Hospital of Sun Yat-sen University

summary: BRAF or RAS mutation-induced aberrant activation of the mitogen-activated protein kinase (MAPK) pathway is frequently observed in human cancers. As the key downstream node of MAPK pathway, ERK1/2 is as an important therapeutic target. GDC-0994 (ravoxertinib), an orally bioavailable, highly selective small-molecule inhibitor of ERK1/2, showed acceptable safety and pharmacodynamic profile in a recent phase I clinical trial. In this study, we investigated dependence of the anti-tumor effect of ERK inhibitor GDC-0994 on genetic alterations in the MAPK pathway. Our date showed that the expression of a large number of genes, particularly the genes in the cell cycle pathway, were significantly changed after GDC-0994 treatment in BRAF mutant cells, while no remarkable expression change of such genes was observed in wild-type cells.

overall_design: To investigate the mechanism of the anti-tumor effect of ERK inhibitor GDC-0994, we selected the BRAF mutant cell KHM-5M and the BRAF wild-type cell TTA-1 as the research objects. These two cells were treated with vehicle (DMSO) or 1μM of GDC-0994 for 24 h in triplicate. RNA-seq was then performed and analyzed.

first: Ye

last: Sang

first: Shiyong

last: Li

first: Junyu

last: Xue

first: Mengke

first: Shubin

last: Hong

first: Weiming

last: Lv

first: Kartik

last: Sehgal

first: Haipeng

last: Xiao

first: Rengyun

last: Liu


title: Germline-like TCR alpha chains shared between autoreactive T cells in blood and pancreas [dataset2]

first: Stephanie

last: Osmond

first: Peter

last: Linsley

first: Karen

last: Cerosaletti


title: Galectin-3 Marks Protection from Lethal Bacterial Infection in Mice and Acute Respiratory Failure in Humans

city: Pittsburgh

first: Dhivyaa

last: Rajasundaram

state: PA

address: 4401 Penn Avenue

department: Pediatrics

institute: University of Pittsburgh

summary: Infection by the Gram-negative bacterium Pseudomonas aeruginosa is common in hospitalized immunocompromised as well as immunocompetent ventilated patients and is often life-threatening because of resistance of the bacteria to antibiotics. This prompts the question whether the host’s immune system can be educated to combat this bacterium. Bacterial lipopolysaccharide (LPS) is known to promote host resistance to bacterial infections although an understanding of the underlying mechanism is lacking. Here we show that prior exposure to a single low dose of LPS protects mice from a lethal infection by P. aeruginosa. These mice displayed expansion of a neutrophil and an interstitial macrophage population that were barely detectable in mice that did not receive LPS prior to infection. Both cell populations were distinguishable from other immune cell populations by being enriched in gene sets that included phagocytosis- and cell-killing-associated genes. The cell killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging of neutrophils for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Interrogating the relevance of these findings in hospitalized patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs. Taken together, our study provides impetus to harnessing the potential of galectin-3-expressing neutrophils to protect the lung from lethal infections and respiratory failure.

overall_design: CD11b+ cells from mouse lungs were sequenced, 3 biological replicates each of LPS+PA14 and PA14 infected samples

first: Prabir

last: Ray

first: Anuradha

first: Georgios

last: Kitsios

first: Sudipta

last: Das


title: Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy [ChIP-Seq]

first: Etienne

last: Danis

institute: University of Colorado, Denver - Anschutz Medical Campus

summary: Using a combination of ChIP-, ATAC-, RNA-seq, and CUT&RUN in model systems of diffuse intrinsic pontine glioma and other pediatric high-grade gliomas, we observe a rapid genome-wide reorganization of active chromatin within hours of exposure to therapeutic ionizing radiation. This redistribution facilitates PTEFb-mediated pause-release of Pol II to drive nascent transcriptional induction of canonical DNA damage response programs. Concurrent inhibition of PTEFb imparts a transcription elongation defect, disrupting this chromatin reorganization and blunting transcriptional induction. Functionally, concurrent PTEFb inhibition abrogates key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival.

overall_design: H3K27ac ChIP-seq was conducted in the SU-DIPG4 cell line after 6 Gy radiation in the presence or absence of AZD4573.

first: Lays

last: Martin Sobral

first: Faye

last: Walker

first: Nathan

last: Dahl


title: Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy [CUT&RUN]

summary: Using a combination of ChIP-, ATAC-, and RNA-seq in model systems of diffuse intrinsic pontine glioma and other pediatric high-grade gliomas, we observe a rapid genome-wide reorganization of active chromatin within hours of exposure to therapeutic ionizing radiation. This redistribution facilitates PTEFb-mediated pause-release of Pol II to drive nascent transcriptional induction of canonical DNA damage response programs. Concurrent inhibition of PTEFb imparts a transcription elongation defect, disrupting this chromatin reorganization and blunting transcriptional induction. Functionally, concurrent PTEFb inhibition abrogates key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival.

overall_design: Phosphorylated RNA polymerase II, Brd4 and ENL CUT&RUN was conducted with the DIPG4 cell line in the presence or absence of AZD or DMSO as control and subjected or not to radiation.


title: RNAseq on Human APAP-ALF

city: Kansas City

first: David

last: Umbaugh

email: dumbaug@gmail.com, hjaeschke@kumc.edu

state: KS

address: 3901 Rainbow Blvd

institute: University of Kansas Medical Center

summary: Fifty percent of all acute liver failure (ALF) cases in the United States are due to acetaminophen (APAP) overdose. Assessment of canonical features of liver injury, such as plasma alanine aminotransferase activities are poor predictors of acute liver failure (ALF), suggesting the involvement of additional mechanisms independent of hepatocyte death. Previous work demonstrated a severe overdose of APAP results in impaired regeneration, the induction of senescence by p21, and increased mortality. We hypothesized that a discrete population of p21+ hepatocytes acquired a secretory phenotype that directly impedes liver recovery after a severe APAP overdose. Leveraging in-house human APAP explant liver and publicly available singlenuclei RNAseq data, we identified a subpopulation of p21+ hepatocytes enriched in a unique secretome of factors, such as Cxcl14. Spatial transcriptomics in the mouse model of APAP overdose confirmed the presence of a p21+ hepatocyte population that directly surrounded the necrotic areas. In both male and female mice, we found a dose-dependent induction of p21 and persistent circulating levels of the p21-specific constituent, Cxcl14, in the plasma after a severe APAP overdose. In parallel experiments, we targeted either the putative senescent hepatocytes with the senolytic drugs, dasatinib and quercetin, or Cxcl14 with a neutralizing antibody. We found that targeting Cxcl14 greatly enhanced liver recovery after APAP-induced liver injury, while targeting the senescent hepatocyte had no effect. This data supports that the sustained induction of p21 in hepatocytes with persistent Cxcl14 secretion are critical mechanistic events leading to ALF in mice and human patients.

overall_design: N=2 healthy livers, N=1 APAP-ALF liver

first: Hartmut

last: Jaeschke


title: A phase II randomized trial with autologous polyclonal expanded regulatory T cells in children with new-onset type 1 diabetes

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

overall_design: Refer to individual Series


title: The role of MEIOC at the transition from mitosis to meiosis during mammalian spermatogenesis

city: Cincinnati

first: Maria

middle: M.

last: Mikedis

state: OH

address: 240 Albert Sabin Way

institute: Cincinnati Children's Hospital Medical Center


title: Single cell RNAseq at 48h and 72h in mice

overall_design: C57BL/6J mice were treated with 300 mg/kg APAP or saline vehicle control. Then 48h or 72h later mice underwent a 2-step collagenase perfusion to isolate either a hepatocyte or non-parenchymal cell liver fraction via percoll gradient with differential centrifugation.

first: Anup

last: Ramachandran


title: A critical requirement for IκBα in controlling dormancy in Hematopoietic stem cells via retinoic acid during embryonic development [scRNA-seq_10x]

city: Barcelona

first: Anna

last: Bigas

state: Barcelona

address: Dr. Aiguader 88

department: Cancer Research

institute: Institut Hospital del Mar d'Investigacions Mèdiques

summary: Hematopoietic Stem Cells (HSCs) originate from the E11.5 aorta-gonads-and mesonephros (AGM) region during development before they migrate to the foetal liver for proliferation and maturation, and finally seed the bone marrow around birth, their final site of residence. In the AGM, HSCs reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). Molecular pathways that determine HSC fate instead of HPCs are still unknown, although inflammatory signalling has been implicated in the development of all blood cells, including NF-κB. Here, we describe a dormant phenotype of LT-HSCs in the IκBα KO. Although IκBα is critical for retaining inactive NF-κB complexes in the cytoplasm, it can regulate stem cell related genes by interacting with the PRC2 complex in the nucleus. Accordingly, we find decreased PRC2 dependent H3K27me3 accumulation at the promoters of PI3K and retinoic acid signalling molecules by cut and tag assay in AGM derived CD31+ cells, which includes HE/IAHC derived from IκBα KO embryos. Furthermore, this regulation of the retinoic acid signalling by IκBα is further confirmed by cut and tag assay for IκBα itself in CD31+ cells of the AGM and more specifically also in sorted LT-HSCs from the E14.5 foetal liver. Over-activation of the retinoic acid/PI3K levels in LT-HSCs of the IκBα KO is evident in their dormant molecular profile. Functionally, IκBα KO LT-HSCs are less proliferative and respond with delayed activation upon transplantation. Overall, we identify nuclear IκBα as an essential player specifically for HSC specification/proliferation from the onset of HSPC emergence in the AGM.

overall_design: We performed 10x scRNAseq analysis in E14.5 LT-HSC fetal liver 1xWT and IκBα 1xKO samples. WT replicate was generated from a pool of 2 embryos and the KO replicate from a pool of 3 embryos.

first: Roshana

last: Thambyrajah

first: Yolanda

last: Guillén

first: Martin

last: Proffitt

first: Neo

last: Hao

first: Zaki

last: Fadlullah

first: Patricia

last: Herrero

first: Carla

last: Brujas

first: Jessica

last: González

first: Arnau

last: Iglesias

first: Laura

last: Marruecos

first: Cristina

last: Ruiz

first: Fernando

last: Calero

first: Berthold

last: Göttgens

first: George

last: Lacaud

first: Lluis

last: Espinosa


title: Massively Parallel Dissection of RNA in RNA-protein Interactions in vivo

city: Baltimore

first: Taeyoung

last: Hwang

state: MD

address: 855 N. Wolfe St, 3rd floor, Suite 300

institute: Lieber Institute for Brain Development

summary: Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to molecular biology. Here, we present MPRNA-immunoprecipitation (MPRNA-IP), an adaptation of the previously developed massively parallel RNA assay (MPRNA) for in vivo high-throughput dissection of RNA-protein interactions, and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, we are able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest including NORAD, MS2, and human telomerase RNA, and we use it to interrogate the RNA-binding preferences of the DNA-looping factor CTCF. By blending modern and classical approaches, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions.

overall_design: Enrichment analysis of overexpressed oligonucleotides in Immunoprecipitation relative to Input by sequencing cDNA

first: Evan

last: Hass

first: Yu

last: Lee

first: John

middle: L

last: Rinn


title: Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia [bulk RNA-seq]

status: Public on Apr 14 2024

city: Munich

first: Markus

last: Joppich

state: Bavaria

address: Amalienstr. 17

department: Department of Informatics

country: Germany

institute: LMU Munich

summary: Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodelling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodelling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodelling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

overall_design: We studied macrophages sorted from hearts in CD45.1/CD45.2 chimeras 2 days after ischemia/reperfusion injury. BM transplantation was enabled by conditional deletion of the transcription factor myb as previously described (Stremmel et al, 2018). In brief, we induced BM ablation by 4 injections of polyI:C into CD45.2; Mx1CreMybflox/flox every other day, and then transplanted BM from congenic C57BL/6 CD45.1 (Ly5.1; JAX Stock No: 006584). We confirmed the success (chimerism of above 90%) of the BM-transplantation after 4 weeks.

first: Tobias

last: Weinberger

first: Christian

last: Schulz


title: Identification of SMCHD1 as a Novel Regulator of Alternative Splicing in FSHD2

status: Public on Apr 13 2024

city: Jerusalem

first: Eden

last: Engal

address: Ein Kerem

department: Faculty of Medicine

country: Israel

institute: Hebrew University of Jerusalem

summary: Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1) is a non-canonical member of the structural maintenance of chromosomes (SMC) protein family involved in the regulation of chromatin structure, epigenetic regulation and transcription.  Mutations in SMCHD1 cause facioscapulohumeral muscular dystrophy type 2 (FSHD2), a rare genetic disorder characterized by progressive muscle weakness and wasting, believed to be caused by aberrant expression of DUX4 in muscle cells. Here we suggest a new role for SMCHD1 as a regulator of alternative splicing, and demonstrate how splicing alteration caused by SMCHD1 mutations lead to DUX4 expression and FSHD pathogenesis. Analyzing RNA-seq data from muscle biopsies of FSHD2 patients and FSHD2 mouse model found that hundreds of genes were alternatively spliced upon SMCHD1 mutation. At least 20% of alternatively spliced genes were associated with abnormalities of the musculature. Moreover, we show that alternatively spliced exons tend to be bound by SMCHD1, and SMCHD1 bound exons demonstrate slower elongation rate, suggesting SMCHD1 binding promotes exon exclusion by slowing RNAPII. Specifically, we discovered that SMCHD1 mutations promotes the splicing of the DNMT3B1 isoform of DNMT3B, which leads to hypomethylation of the D4Z4 region and DUX4 expression. These results suggest that alternative splicing regulated by SMCHD1 may play a major role in FSHD2 pathogenesis by promoting alternative splicing of different targets including DNMT3B, and highlight the potential for targeting alternative splicing as a therapeutic strategy for this disorder.

overall_design: We extracted RNA from Smchd1 null NSCs and performed next-generation sequencing, using RNA ScreenTape kit (catalog #5067-5576; Agilent Technologies), D1000 ScreenTape kit (catalog #5067-5582; Agilent Technologies), Qubit RNA HS Assay kit (catalog # Q32852; Invitrogen), and Qubit DNA HS Assay kit (catalog #32854; Invitrogen) were used for each specific step for quality control and quantity determination of RNA and library preparation. For mRNA library preparation: TruSeq RNA Library Prep Kit v2 was used (Illumina). In brief, 1 µg was used for the library construction; library was eluted in 20 µL of elution buffer. Libraries were adjusted to 10 mM, and then 10 µL (50%) from each sample was collected and pooled in one tube. Multiplex sample pool (1.5 pM including PhiX 1.5%) was loaded in NextSeq 500/550 High Output v2 kit (75 cycles cartridge and 150 cycles cartridge; Illumina). Run conditions were in paired end (43 × 43 bp and 80 × 80 bp, respectively) and loaded on NextSeq 500 System machine (Illumina).

first: Maayan

last: Salton

first: Yotam

last: Drier


title: Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia [scRNA-seq]

overall_design: We studied leukocytes from hearts in Csf1r∆FIRE mice in baseline conditions and 2 days after ischemia/reperfusion injury. At the 2 specific timepoints leukocytes were harvested from the heart using MACS as well as FACS sorting strategies for CD45+ cells.


title: A critical requirement for IκBα in controlling dormancy in Hematopoietic stem cells via retinoic acid during embryonic development [RNA-seq]

first: Anna

last: Bigas

overall_design: We performed bulk RNA seq with sorted E14.5 LT-HSCs (LSKCD48-CD150+) in order to define HSCs at a molecular level considering 3xWT, 2x IκBα KO and  3x IκBα HET independent biological replicates.

first: Roshana

last: Thambyrajah

first: María

last: Maqueda

first: Zaki

last: Fadlullah

first: Martin

last: Proffitt

first: NeoWen

last: Hao

first: Yolanda

last: Guillén

first: Marta

last: Casado-Pelaez

first: Patricia

last: Herrero-Molinero

first: Carla

last: Brujas

first: Noemi

last: Castelluccio

first: Jessica

last: González

first: Arnau

last: Iglesias

last: Ruiz-Herguido

first: Manel

last: Esteller

first: Elisabetta

last: Mereu


title: Whole transcriptome sequencing for wild type and IRAIN over-expression K562 cells

city: Dalian

first: Jinsong

last: Yan

address: 9 west section, Lvshun south road, Dalian, Liaoning Province, China

department: Institute of Cancer Stem Cell

institute: Dalian Medical University

summary: The development of tyrosine kinase inhibitors (TKIs) has revolutionarily increased the overall survival of patients with chronic myeloid leukemia (CML). However, drug resistance remains a major obstacle. Here, we demonstrated that a BCR-ABL1-independent long non-coding RNA, IRAIN, is constitutively expressed at low levels in CML, resulting in imatinib resistance. IRAIN knockdown decreased the sensitivity of CD34+ CML blasts and cell lines to imatinib, whereas IRAIN overexpression significantly increased sensitivity. Mechanistically, IRAIN downregulates CD44, a membrane receptor favorably affecting TKI resistance, by binding to the nuclear factor kappa B subunit p65 to reduce the expression of p65 and phosphorylated p65. Therefore, the demethylating drug decitabine, which upregulates IRAIN, combined with imatinib, formed a dual therapy strategy which can be applied to CML with resistance to TKIs.

overall_design: Investigation of drug resistance for over-expression IRAIN1 in CML cell line K562


title: m6a methylation drives IMP1 regulation during human neuronal differentiation.

city: London

first: Pierre

last: Klein

address: Gower Street

department: Institute of Structural and Molecular Biology

country: United Kingdom

institute: University College London


title: A critical requirement for IκBα in controlling dormancy in Hematopoietic stem cells via retinoic acid during embryonic development

first: Anna

last: Bigas


title: m6a methylation drives IMP1 regulation during human neuronal differentiation [RNA-seq]

summary: Human neurodevelopment requires differentiating neurons to establish large networks of connections in a highly stereotyped manner. Neuronal differentiation in particular requires RNA-binding proteins to spatiotemporally regulate thousands of different mRNAs. Yet how these proteins precisely relate to neuronal development and coordinate the expression of functionally coherent genes in a cell type specific manner is only partially understood. To address this, we sought to clarify how the paradigmatic RNA-binding protein IMP1/IGF2BP1, an essential developmental factor, selects and regulates its RNA targets transcriptome-wide during the differentiation of human pluripotent stem cell-derived neural precursor cells to their neuronal counterparts. We used a combination of systemic and molecular analyses to show that IMP1 directly binds to and regulates the expression of a large sets of mRNAs.

overall_design: We performed gene expression profiling analysis using data obtained from RNA-seq in hIPSC derived NPC and their neuronal contrepart either treated siRNA against IMP1 or siRNA CTRL

first: Marija

last: Petrić Howe

first: Jasmine

last: Harley

first: Harry

last: Crook

first: Sofia

middle: E

last: Serra

first: Anob

last: Chakrabati

first: Raphaëlle

last: Luisier

first: Rickie

last: Patani

first: Andres

last: Ramos


title: A critical requirement for IκBα in controlling dormancy in Hematopoietic stem cells via retinoic acid during embryonic development [Cut&Tag]

overall_design: We performed cut&tag assay for H3K27me3 (IkBa Ko and WT) and Ikba with sorted embryonic fetal liver (E14.5 LT-HSCs) or AGM (E11.5 CD31+). Specifically, we generated 2 replicates from fetal liver and 2 replicates from AGM for IkBa Cut&Tag experiments. Regarding H3K27me3 experiments, replicates were only generated for E14.5 FL-HSCs and considered 3xIkBa WT and 3xIkBa KO.

first: Roshana

last: Thambyrajah

first: María

last: Maqueda

first: Zaki

last: Fadlullah

first: Martin

last: Proffitt

first: NeoWen

last: Hao

first: Yolanda

last: Guillén

first: Marta

last: Casado-Pelaez

first: Patricia

last: Herrero-Molinero

first: Carla

last: Brujas

last: Castelluccio

first: Laura

last: Marruecos

first: Cristina

first: George

last: Lacaud

first: Lluis

last: Espinosa


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq9 (LB, gentamycin, cotrimoxazole, rifampicin, polymyxin B)

status: Public on Apr 12 2024

city: Palo Alto

first: Benjamin

last: Wang

state: CA

address: 299 Campus Drive

institute: Stanford

summary: Salmonella enterica is comprised of genetically distinct “serovars”, that together provide an intriguing model for exploring the genetic basis of pathogen evolution. While the genomes of numerous Salmonella isolates with broad variations in host range and human disease manifestations have been sequenced, the functional links between genetic and phenotypic differences among these serovars remain poorly understood. Here, we conduct high-throughput functional genomics on both generalist (Typhimurium) and human-restricted (Typhi & Paratyphi A) Salmonella at unprecedented scale in the study of this enteric pathogen. Using a comprehensive systems biology approach, we identify gene networks with serovar-specific fitness effects across 25 host-associated stresses encountered at key stages of human infection. By experimentally perturbing these networks, we characterize previously undescribed pseudogenes in human-adapted Salmonella. Overall, this work highlights specific vulnerabilities encoded within human-restricted Salmonella that are linked to the degradation of their genomes, shedding light into the evolution of this enteric pathogen.

platform_organism: Salmonella enterica subsp. enterica serovar Typhi str. Ty2

platform_organism: Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. D23580

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. ST4/74

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 2, including gentamycin, cotrimoxazole, rifampicin, polymyxin B.

middle: X

first: Denise

last: Monack


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq13 (Typhi and Paratyphi A infection of THP-1 human macrophages)

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 13, which is Typhi/Paratyphi A infection of THP-1 human macrophages.


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq12 (Typhimurium THP-1 infections)

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 12, including Typhimurium THP-1 infections.


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq3 (LB, ceftriaxone, ciprofloxacin, azithromycin)

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 2, including LB, ceftriaxone, ciprofloxacin, azithromycin.


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq2 (Bile, iron restriction, osmotic pressure, heat)

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 2, including bile stress, iron limitation, osmotic pressure, heat shock.


title: m6a methylation drives IMP1 regulation during human neuronal differentiation [iCLiP]

first: Pierre

last: Klein

overall_design: iCLIP on IPSC-derived NPC and neurons for IMP1

first: Marija

last: Petrić Howe

first: Jasmine

last: Harley

first: Harry

last: Crook

first: Sofia

last: Serra

first: Anob

last: Chakrabati

first: Raphaëlle

last: Luisier

first: Rickie

last: Patani


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq4 (InSPI2, hydrogen peroxide (H2O2), nitric oxide (NO), sodium hypochlorite (bleach))

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 2, including H2O2, NO, bleach.


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq1 (NonSPI2, InSPI2, InSPI2Mg, M9glucose)

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Ilumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 1, including NonSPI2, InSPI2, inSPI2Mg, and M9 glucose conditions.


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq5 (anaerobic LB, GMM, zinc, Nmedia, protamine)

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 5, including anaerobic LB, GMM, zinc overload, NMedia, protamine.


title: m6a methylation drives IMP1 regulation during human neuronal differentiation [miCLIP]

overall_design: miCLIP on IPSC-derived NPC and neurons

first: Andres

last: Ramos


title: High-throughput Fitness Experiments Reveal Specific Vulnerabilities of Human-Adapted Salmonella During Stress and Infection- Barseq6 (anaerobic LB, GMM, zinc, Nmedia, protamine- second set)

overall_design: This is a Rb-Tn-seq project using barcoded transposons to rapidly identify changes in gene fitness. We tested 4 Salmonella isolates against 25 stress conditions each. Tn libraries were grown to an OD~1, then back-diluted 1:50 into either a media control or stressor. The concentration of each stressor was optimized such that growth of the library decreased by ~30-50% on average. The Tn libraries were allowed to grow overnight, then DNA was extracted using a Qiagen gDNA extraction kit. A barcoding PCR was performed to amplify all Tn-associated barcodes and sent for Illumina sequencing on a Hiseq4000. Fitness changes were calculated according to published scripts. This submission includes experiments from Barseq round 6, including anaerobic LB, GMM, zinc overload, Nmedia, and protamine (second set of samples).


title: AgRP neuron epigenomes across hunger states reveal that IRF3 mediates leptin's effects

first: NORC

last: Boston

address: 3 Blackfan Circle, CLS0733

institute: Beth Israel Deaconess Medical Center

summary: AgRP neurons in the arcuate nucleus of the hypothalamus (ARC) coordinate homeostatic changes in appetite associated with fluctuations in food availability and leptin signaling. Identifying the relevant transcriptional regulatory pathways in these neurons has been a priority, yet such attempts have been stymied due to their low abundance and the rich cellular diversity of the ARC. Here we generated male mouse AgRP neuron-specific transcriptomic and chromatin accessibility profiles during three distinct hunger states of satiety, fasting-induced hunger, and leptin-induced hunger suppression. Cis-regulatory analysis of these integrated datasets enabled the identification of 28 putative hunger-promoting and 29 putative hunger-suppressing transcriptional regulators in AgRP neurons, 16 of which were predicted to be transcriptional effectors of leptin. Within our dataset, Interferon regulatory factor 3 (IRF3) emerged as a leading candidate mediator of leptin-induced hunger-suppression. Gain- and loss-of-function experiments in vivo confirm the role of IRF3 in mediating the acute satiety-evoking effects of leptin in AgRP neurons, while live-cell imaging in vitro indicate that leptin can activate neuronal IRF3 in a cell autonomous manner. Finally, we employ CUT&RUN to uncover direct transcriptional targets of IRF3 in AgRP neurons in vivo. Thus, our findings identify AgRP neuron-expressed IRF3 as a key transcriptional effector of the hunger-suppressing effects of leptin.

overall_design:
For RNA-seq and ATAC-seq data we generated a NuTRAP^AgRP mouse line, from which we could isolate AgRP neuron-specific mRNA and nuclei
We profiled RNA expression and chromatin accessibility in AgRP neurons harvested from the above animals under fed (ad-libitum), fasting (overnight), and “leptin” (fasting plus leptin administration) conditions. RNA-seq samples had an group size of n = 4 (3 in fed), while ATAC was performed in groups of size n = 2.
Comparative gene expression and chromatin-accessibility analyses were performed separately for these the above groups.
Integrated analyses demonstrating relationships between concordant changes in expressions and chromatin profiles were performed by linking ATAC peaks with gene TSSes at a range of +/- 200kb.
Profiling of IRF3 binding sites in AgRP neurons was performed via CUT&RUN in mouse models expressing a constitutively active variant of IRF3 (IRF3-2D).

first: Frankie

last: Heyward

first: Evan

last: Rosen