title: Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of NFIX As a Novel Fetal Hemoglobin Repressor

city: Boxborough

first: Nisha

last: Rajagopal

address: 1069 Liberty Square Road

institute: Syros Pharmaceuticals

summary: Human genetics has validated de-repression of fetal gamma globin (HBG) in adult erythroblasts as a powerful therapeutic paradigm in diseases involving defective adult beta globin (HBB)1. To identify factors involved in the switch from HBG to HBB expression, we performed Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq)2 on sorted erythroid lineage cells derived from bone marrow (BM) or cord blood (CB), representing adult and fetal states, respectively. BM to CB cell ATAC-seq profile comparisons revealed genome-wide enrichment of NFI DNA binding motifs and increased NFIX promoter chromatin accessibility, suggesting that NFIX may repress HBG. NFIX knockdown in BM cells increased HBG mRNA and fetal hemoglobin (HbF) protein levels, coincident with increased chromatin accessibility and decreased DNA methylation at the HBG promoter. Conversely, overexpression of NFIX in CB cells reduced HbF levels. Identification and validation of NFIX as a new target for HbF activation has implications in the development of therapeutics for hemoglobinopathies.

overall_design: Global transcriptional analysis of 1) Primary bone marrow derived erythroblasts and cord blood derived eyrthroblasts across erythrocyte differention; and 2) Primary bone marrow derived erythroblasts transduced with NFIX shRNA or a non-targeting control.

first: Mudit

last: Chaand

first: Chris

last: Fiore

first: Brian

last: Johnston

first: Anthony

last: D'Ippolito

first: Diane

middle: H

last: Moon

first: John

last: Carulli

first: Jeffrey

middle: R

last: Shearstone


title: YTHDF2-m6A-NF-kB axis controls anti-tumor immunity through regulating intratumoral Treg survival and function

status: Public on Jun 07 2023

city: Shanghai

first: Xiaoyang

last: Dou

address: 320 Yue Yang Road

country: China

institute: CAS Center for Excellence in Molecular Cell Science, CAS

summary: N6-methyladenosine (m6A) in messenger RNA (mRNA) regulates immune cells in homeostasis and in response to infection and inflammation. The function of YTHDF2 in the tumor microenvironment (TME) in these contexts has not been explored. We discovered that loss of Ythdf2 in regulatory T (Treg) cells can reduce tumor growth in mice while maintaining peripheral homeostasis. In the tumor microenvironment, Ythdf2-deficient Treg cells have impaired function and poor survival. The elevated tumor necrosis factor (TNF) signaling in the TME grants the location-specific function of YTHDF2, which participates in the feedback regulation of NF-kB signaling by accelerating the degradation of m6A-modified, NF-kB negative regulators, Nlrc3, Nfkbie, and Traf3. TME-specific regulation of Treg by YTHDF2 points to YTHDF2 as a target for anti-cancer immunotherapy, where intratumoral Treg cells can be targeted to enhance antitumor immune response, while avoiding Treg cells in the periphery to minimize undesired inflammations.

overall_design: We performed m6A RIP-seq to examine m6A marked transcripts

first: Linda

last: Zhang

first: Chuan

last: He


title: Human SAMD9 is a Poxvirus-Activatable Anticodon Nuclease Inhibiting Codon-Specific Protein Synthesis

city: San Antonio

first: Yan

last: Xiang

state: TX

address: 7703 FLOYD CURL DR

department: Microbiology, Immunology and Molecular Genetics

country: USA

institute: UT Health San Antonio

type: Expression profiling by high throughput sequencing

summary: As a defense strategy against viruses or competitors, some microbes employ anticodon nucleases (ACNases) to deplete essential tRNAs, effectively halting global protein synthesis. However, this mechanism has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is an ACNase that specifically cleaves phenylalanine tRNA (tRNAPhe), resulting in codon-specific ribosomal pausing and stress signaling. While SAMD9 ACNase activity is normally latent in cells, it can be activated by poxvirus infection or rendered constitutively active by SAMD9 mutations associated with various human disorders, revealing tRNAPhe depletion as an antiviral mechanism and a pathogenic condition in SAMD9 disorders. We identified the N-terminal effector domain of SAMD9 as the ACNase, with substrate specificity primarily determined by a eukaryotic tRNAPhe-specific 2’-O-methylation at the wobble position, making virtually all eukaryotic tRNAPhe susceptible to SAMD9 cleavage. Notably, the structure and substrate specificity of SAMD9 ACNase differ from known microbial ACNases, suggesting convergent evolution of a common immune defense strategy targeting tRNAs.

overall_design: Ribo-seq and RNA-seq of uninfected HeLa cells as well as HeLa cells infected with vK1-C7- and WT VACV. HeLa cells in 2x150 mM dishes were left uninfected or infected with vK1-C7- or WT VACV WR in the absence or presence of AraC (50 ug/ml final) for 8 hours. The cells were treated with cycloheximide (0.1 mg/ml) for 1 min at 37oC. The cells were lysed with 800 ul mammalian lysis buffer. 100 ul of lysate was used for extracting total RNA, while 200 ul of lysate was used for ribosome foot printing.

first: Yuanhui

last: Mao


title: Genome-wide host loci regulate M. tuberculosis fitness in immunodivergent mice.

city: Durham

first: Clare

last: Smith

state: North Carolina

address: 207 Research Drive

department: MGM

institute: Duke University

summary: The factors that determine the outcome of clinical tuberculosis lie within both the host and the pathogen, Mycobacterium tuberculosis (Mtb). The advent of recombinant inbred mouse panels and next-generation transposon mutagenesis and sequencing approaches has enabled dissection of the host-pathogen interface for mammalian and pathogen genetic determinants of disease outcome. To identify host and pathogen genetic drivers of Mtb infection, we infected 19 genotypes from the BXD panel, bred from Mtb-resistant C57BL/6J (B6) and Mtb-susceptible DBA/2J (D2), with a comprehensive library of transposon mutants (TnSeq). The survival of each of the ~4000 bacterial mutants within each distinct host was quantified and leveraged as refined “endophenotypes”, directly reporting on the infection microenvironment. We leveraged QTL mapping to associate each varying bacterial fitness endophenotype to the host genome and identified 140 significant host-pathogen quantitative trait loci (hpQTL). This host-pathogen interaction screen reinforces the utility of bacterial mutant libraries as precise reporters of host immunological microenvironment during infection and highlights host gene candidates for further investigation.

overall_design: Subject mice (19 BXD strains, C57BL/6J, and DBA/2J) were infected via tail vein injection with a saturated M. tuberculosis Himar1 transposon library. Animals were sacrificed after 4 weeks, and TnSeq was performed after library recovery from harvested spleens. In the majority of cases, two replicates were obtained per mouse strain, 5 replicates in the case of in vitro-cultured libraries.

first: Rachel

middle: K

last: Meade

first: Jarukit

middle: E

last: Long

first: Adrian

last: Jinich

first: Kyu

middle: Y

last: Rhee

first: David

middle: G

last: Ashbrook

first: Robert

middle: W

last: Williams

first: Christopher

middle: M

last: Sassetti


title: A super-enhancer regulated RNA-binding protein cascade drives pancreatic cancer

city: La Jolla

first: Ruth

middle: T

last: Yu

state: CA

address: 10010 N Torrey Pines Rd

department: Gene Expression Lab

institute: Salk Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We uncovered a super-enhancer (SE)-mediated pathway, integrated by Myc, that regulates protein translation to drive pancreatic tumor growth. A SE associated with heterogeneous nuclear ribonucleoprotein F (HNRNPF) controls its expression to regulate protein arginine methyltransferase 1 (PRMT1) mRNA stability. In turn, PRMT1 asymmetrically dimethylates ubiquitin-associated protein 2-like (UBAP2L) to mediate protein translation and thus tumor growth. Deletion of any of these 3 genes, or of the super-enhancer itself, all lead to a drastic reduction in tumor burden in orthotopic PDAC models. Inhibition of PRMT1 induces apoptosis and significantly reduces tumor growth. This unexpected role of HNRNPF, PRMT1 and UBAP2L in regulating tumorigenesis by coordinating protein translation underpins this pathway, downstream of Myc, as a potential therapeutic target for PDAC.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: ChIP-Seq analysis of PDAC and PDAC cell lines.

first: Corina

last: Antal

first: Ronald

last: Evans


title: Symbiont-host interactome mapping reveals effector-targeted modulation of hormone networks and activation of host benefits

city: Birmingham

first: Rory

last: Osborne

address: Edgbaston

country: United Kingdom

institute: University of Birmingham

summary:
To identify candidate secreted proteins by the plant symbiont Serendipita indica, we infected the roots of the model plant Arabidopsis thaliana with fungal spores.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 2 conditions (mock and treated) and 2 timepoints (3 and 10 days after infection).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Serendipita colonised Arabidopsis thaliana roots at 3 and 10 days after infection.

first: Patrick

last: Schafer


title: Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation [QuantSeq-Pool]

city: Philadelphia

first: Bin

last: Tian

state: Pennsylvania

address: 3601 Spruce Street

institute: The Wistar Institute

summary: Cleavage and polyadenylation (CPA) defines the 3’ end of almost all eukaryotic mRNAs. CPA inhibition, or CPAi, leads to transcriptional readthrough. Here, we show that the CPSF-73 inhibitor JTE-607 globally perturbs gene expression, especially for those with a high GC content and located in high gene density regions. Based on regulated alternative polyadenylation (APA) events, we found that more frequently used CPA sites are inhibited by JTE-607 to a greater extent. Consistently, cells with elevated CPA activities, as indicated by preferential usage of proximal APA sites, display greater transcriptional readthrough and gene expression disturbance upon JTE-607 treatment. Remarkably, overexpression of the core CPA factor FIP1 enhances global CPA activity in the cell and leads to greater JTE-607 sensitivity. Taken together, our data indicate that CPAi selectively impacts genes based on their genomic features and the CPA activity of a cell is a key determinant of sensitivity to CPAi.

overall_design: For QuantSeq analysis, HeLa and HepG2 cells were treated with DMSO or JTE-607 (1μM or 10μM) for 8 hrs before harvest for total RNA extraction. U937 and U937MP cells were treated with DMSO or 1 μM JTE-607 for 7 hrs before total RNA extraction.

first: Yange

last: Cui

first: Luyang

last: Wang

first: Qingbao

last: Ding

first: Jihae

last: Shin

first: Joel

last: Cassel

first: Qin

last: Liu

first: Joseph

last: Salvino


title: Gene expression profile at single cell level of CD45+ cells isolated from twitcher (twi) mouse brain (postnatal day 21)

city: Farmington

first: Stephen

last: Crocker

state: Connecticut

address: 263 Farmington Ave.

department: Neuroscience

institute: University of Connecticut School of Medicine

summary: Gene expression profile at single cell level of CD45+ cells isolated from wildtype (twi littermate) mouse brain (postnatal day 21) to evaluate and compare differences in immune cells at the onset of spontaneous clinical disease in a mouse model of globoid cell leukodystrophy.

overall_design: CD45+ cells were isolated from single cell suspension of hemi-brain tissues by positive selection using flow cytometery

first: Pearl

middle: A

last: Sutter

first: Alexadra

last: Nicaise

middle: J


title: Distinct Chromatin Scanning Modes Lead to Targeting of Compacted Chromatin by Pioneer Factors FOXA1 and SOX2 [ChIP-seq]

first: Kenneth

middle: S.

last: Zaret

state: PA

address: 9-132, SCTR, 3400 Civic Center Boulevard

department: Cell and Developmental Biology

institute: University of Pennsylvania School of Medicine

summary:
Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity.  However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes.
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for HNF4A, FOXA1, FOXA1-DBD, FOXA1-NHAA, FOXA1-RRAA, as well as H2B-HALO after ectopic expression in human BJ fibroblast cells.

overall_design: Jonathan,Lerner

first: Andrew

last: Katznelson


title: Distinct Chromatin Scanning Modes Lead to Targeting of Compacted Chromatin by Pioneer Factors FOXA1 and SOX2 [Cut&Run]

summary:
Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity.  However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes.
Cleavage under Targets and Release Using Nuclease (CUT&RUN) of SOX2 and SOX2-DBD after ectopic expression in BJ fibroblasts.

overall_design: Andrew,Katznelson

first: Jonathan

last: Lerner


title: Visium Spatial Transcriptomics expression profiles from IPMN samples of the pancreas

city: New York

first: Anirban

last: Maitra

state: NY

address: Smilow 910, 522 1st Ave

department: Perlmutter Cancer Center

institute: New York University Grossman School of Medicine

summary: Intraductal Papillary Mucinous Neoplasms (IPMN)  are pancreatic mucinous cysts that can progress to cancer. We used spatial transcriptomics to characterize the epithelium and microenvironment of IPMN samples of different grades and histological subtypes.

overall_design: Spatial transcriptomic experiments were conducted on fresh frozen IPMN specimens with the Visium Spatial Gene Expression platform from 10x genomics.

first: Marta

last: Sans

first: Kimal

middle: I

last: Rajapakshe

first: Paola

last: Guerrero


title: Germ-cell specific eIF4E1b regulates maternal mRNA translation to ensure zygotic genome activation [RNA-seq]

status: Public on Jun 08 2023

city: Bethesda

first: Guanghui

last: Yang

state: Maryland

address: 50 South Drive

department: National Institute of Diabetes and Digestive and Kidney Diseases

institute: National Institutes of Health

summary: Translation of maternal mRNAs is detected before transcription of zygotic genes and is essential for mammalian embryo development. How certain maternal mRNAs are selected for translation instead of degradation and how this burst of translation affects zygotic genome activation remains unknown. Using gene-edited mice, we document that the oocyte-specific eukaryotic translation initiation factor 4E family member 1b (eIF4E1b) is the regulator of maternal mRNA expression that ensures subsequent reprogramming of the zygotic genome. In oocytes, eIF4E1b binds to transcripts encoding translation machinery proteins, chromatin remodelers and reprogramming factors to promote their translation in zygotes and protect them from degradation. The protein products are thought to establish an open chromatin landscape in one-cell zygotes to enable transcription of genes required for cleavage stage development. Our results define a program for rapid resetting of the zygotic epigenome that is regulated by maternal mRNA expression and provides new insights into the mammalian maternal-to-zygotic transition.

overall_design: RNA profiles in single Eif4e1b+/- or Eif4e1b-/- eggs/embryos collected at different developmental stages.

first: Qiliang

last: Xin

first: Jurrien

last: Dean


title: Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation [QuantSeq]

first: Bin

last: Tian

first: Yange

last: Cui

first: Luyang

last: Wang

first: Qingbao

last: Ding

first: Jihae

last: Shin


title: Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation [ChrRNA-seq]

overall_design: For chromatin RNA sequencing, HeLa, HepG2, U937 and U937MP cells were treated with DMSO or 1 μM JTE-607 for 2 hrs before cells were collected for chromatin RNA extraction.

first: Joel

last: Cassel

first: Qin

last: Liu

first: Joseph

last: Salvino


title: Contribution of 6mer seed toxicity to HIV-1 induced cytotoxicity (DISE-69)

city: Chicago

first: Marcus

last: Peter

state: IL

address: 303 East Superior Street, Lurie 6-123

institute: Northwestern University Feinberg School of Medicine

summary: Analysis of total RNA by RNA seq

overall_design: Samples of HCT116 cells transfected with NC1 and miRNA HIV-miR-TAR-3p

first: Elizabeth

last: Bartom

first: Aparajitha

last: Vaidyanathan


title: Transcriptomic profiles after Nkx6-2 overexpression in IPMN-associated cancer cells derived from murine pancreas [bulk RNA-Seq]

first: Anirban

last: Maitra

summary: To investigate transcriptomic changes associated with Nkx6-2, we overexpressed Nkx6-2 in murine IPMN-associated pancreatic cancer cells by lentivirus transduction and performed bulk RNA-sequencing

overall_design: We performed differential gene expression analysis and geneset enrichment analysis using data obtained from RNA-seq of 3 different cell lines with two groups, control and Nkx6-2 overexpression

first: Marta

last: Sans

first: Makino

last: Yuki

first: Jimin

last: Min


title: Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of The Pancreas Identifies NKX6-2 Expression as a Driver of Gastric Differentiation and Indolent Biological Potential

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

overall_design: Refer to individual Series


title: Contribution of 6mer seed toxicity to HIV-1 induced cytotoxicity

type: Non-coding RNA profiling by high throughput sequencing


title: Human SAMD9 is a Virus-Activatable Anticodon Nuclease Inhibiting Codon-Specific Protein Synthesis

city: Ithaca

first: Saori

last: Uematsu

state: New York

address: 526 Campus Road, Cornell University

institute: Cornell University

summary: Transfer RNAs (tRNAs) are one of the most conserved components of protein synthesis machinery. An effective strategy for microbes to defend against viruses or competitors is to employ anticodon nucleases (ACNases) to deplete essential tRNAs and thereby shut off global protein synthesis, but this strategy has not been observed in multicellular eukaryotes. Here, we report that human SAMD9 is a virus-activatable ACNase that specifically depletes phenylalanine tRNA (tRNAPhe), causing codon-specific ribosomal pausing and inducing stress signaling. SAMD9 ACNase can be activated by poxvirus infection or by SAMD9 mutations associated with a spectrum of developmental or immunological disorders, implicating tRNAPhe deficiency as a major toxic effect in these human diseases and suggesting a new therapeutic strategy. The specificity of the ACNase, located to an N-terminal effector domain of SAMD9, is largely determined by a eukaryotic tRNAPhe specific 2’-O-methylation at the wobble position, making eukaryotic tRNAPhe a universal but specific target for SAMD9. The SAMD9 ACNase differs from the microbial ACNases in structure and substrate specificity, suggesting convergent evolution resulted in a universal immune defense strategy targeting tRNAs.

overall_design: tRNA-seq, Ribo-seq and RNA-seq. For tRNA-seq, HeLa cells were uninfected or infected with either WT or vK1-C7- VACV for 8 hours. tRNAs were isolated from total RNAs resolved on a denaturing gel, untreated or treated with a tRNA demethylase and subjected to tRNA-seq. For Ribo-seq and RNA-seq,  BT20 cells transduced with SAMD9-R1293W were uninduced or induced for SAMD9-R1293W expression.

first: Yan

last: Xiang

first: Yuanhui

last: Mao


title: Orphan quality control shapes network dynamics and gene expression

city: Berkeley

first: Michael

last: Rapé

address: 330F Li Ka Shing Center

department: Molecular and Cell Biology

institute: University of California Berkeley

summary: All eukaryotes require intricate protein networks to translate developmental signals into accurate  cell fate decisions. Mutations that disturb interactions between network components often result  in disease, but how composition and dynamics of complex networks are established remains  poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade  unpaired subunits of multiple transcriptional regulators that act within a network centered on the  c-MYC oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only  become available upon complex dissociation. By rapidly turning over orphan transcription factor  subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow  cells to effectively execute gene expression, while remaining receptive to environmental signals.  We conclude that orphan quality control plays an essential role in establishing dynamic protein  networks, which may explain the conserved need for protein degradation during transcription and  offers unique opportunities to modulate gene expression in disease.

overall_design: To investigate the global effect of UBR5 loss on gene expression, we generated a UBR5 knockout HEK 293T cell line using CRISPR. We then performed comparative gene expression profiling analysis via RNA-seq from both unedited (WT) and ∆UBR5 HEK 293T cells in biological triplicate. We also performed a similar anaylsis in H1 hESCs treated with either non-targeting control siRNAs or siRNAs against UBR5.

first: Kevin

last: Mark

first: SriDurgaDevi

last: Kolla

first: Jacob

middle: D

last: Aguirre

first: Danielle

last: Garshott

first: Stefan

last: Schmitt

first: Diane

middle: L

last: Haakonsen

first: Christina

last: Xu

first: Lukas

last: Kater

first: Georg

last: Kempf

first: Brenda

last: Martínez-González

first: David

last: Akopian

first: Stephanie

last: See

first: Nicolas

last: Thomä


title: Contribution of 6mer seed toxicity to HIV-1 induced cytotoxicity [DISE-36]

first: Marcus

last: Peter

summary: Analysis of RISC bound short (s)RNAs in cells infected with HIV-1 reveals a contribution of 6mer seed toxicity to HIV-1 induced cytopathicity

overall_design: Ago protein associated short RNAs were pulled down using a GW182 peptide and subjected to smRNA Seq

first: Elizabeth

last: Bartom

first: Aparajitha

last: Vaidyanathan


title: Coding and noncoding transcriptomes of NODULIN HOMEOBOX (NDX) deficient Arabidopsis inflorescence [ncRNA-Seq]

city: Debrecen

first: Lorant

last: Szekvolgyi

address: Egyetem ter 1.

department: Department of Pharmacy

country: Hungary

institute: University of Debrecen

summary: Arabidopsis NODULIN HOMEOBOX (NDX) is a plant-specific transcriptional regulator whose role in small RNA biogenesis and heterochromatin homeostasis has recently been described. Here we extend our previous transcriptomic analysis to the flowering stage of development. We performed mRNA-seq and small RNA-seq measurements on inflorescence samples of wild-type and ndx1-4 mutant (WiscDsLox344A04) Arabidopsis plants. We identified specific groups of differentially expressed genes and noncoding heterochromatic siRNAs (hetsiRNAs) whose transcriptional activity were significantly changed in the absence of NDX. Overall, we provide a comprehensive data source on the coding and noncoding transcriptomes of NDX-deficient Arabidopsis flowers to serve as a basis for further studies and analyses on NDX function.

overall_design: sRNA expression analysis of RNA-seq data for A. thaliana Col-0 wild type and ndx1-4 mutant (5-week-old plant, flower, 3 replicas).

first: Orsolya

last: Feró

first: Zsolt

last: Karányi

first: Éva

last: Nagy

first: Ágnes

last: Mosolygó-L

first: Henrik

last: Szaker

first: Tibor

last: Csorba

first: Lóránt

last: Székvölgyi


title: The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells

city: Bellinzona

first: Cristina

last: Leoni

state: Ticino

address: Via F. Chiesa 5

department: Molecular Immunology

country: Switzerland

institute: Institute for Research in Biomedicine

type: Methylation profiling by high throughput sequencing


title: Distinct Chromatin Scanning Modes Lead to Targeting of Compacted Chromatin by Pioneer Factors FOXA1 and SOX2 [Mnase-seq]

first: Kenneth

last: Zaret

summary:
Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity.  However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes.
Micrococcol nuclease digestion and sequencing (MNase-seq) of human BJ fibroblasts

first: Jonathan

last: Lerner


title: Contribution of 6mer seed toxicity to HIV-1 induced cytotoxicity [DISE-56]


title: Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation

first: Bin

last: Tian


title: Contribution of 6mer seed toxicity to HIV-1 induced cytotoxicity [DISE-42]


title: Contribution of 6mer seed toxicity to HIV-1 induced cytotoxicity (DISE-45)


title: IBD single cell data from colonic mucosa

city: Barcelona

first: Azucena

last: Salas

address: Rosselló, 149-153

department: Gastroenterology

country: Spain

institute: IDIBAPS

summary: Inflammatory bowel diseases (IBDs) including  ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory diseases with increasing worldwide prevalence that show a perplexing heterogeneity in manifestations and response to treatment. We applied single cell RNA sequencing (scRNAseq) a to colonic tissue from a combined healthy, UC and CD cohort.

overall_design: Colonic cells from Healthy controls (6), Ulcerative Colitis (6) and Crohn's disease (6) patients were isolated and analyzed using scRNAseq.

first: Alba

last: Garrido-Trigo


title: YTHDF2-m6A-NF-kB axis controls anti-tumor immunity through regulating intratumoral Treg survival and function [RNA-seq]

first: Xiaoyang

last: Dou

summary: N6-methyladenosine (m6A) in messenger RNA (mRNA) regulates immune cells in homeostasis and in response to infection and inflammation. The function of YTHDF2 in the tumor microenvironment (TME) in these contexts has not been explored. We discovered that loss of Ythdf2 in regulatory T (Treg) cells can reduce tumor growth in mice while maintaining peripheral homeostasis. In the tumor microenvironment, Ythdf2-deficient Treg cells have impaired function and poor survival. The elevated tumor necrosis factor (TNF) signaling in the TME grants the location-specific function of YTHDF2, which participates in the feedback regulation of NF-kB signaling by accelerating the degradation of m6A-modified, NF-kB negative regulators, Nlrc3, Nfkbie, and Traf3. TME-specific regulation of Treg by YTHDF2 points to YTHDF2 as a target for anti-cancer immunotherapy, where intratumoral Treg cells can be targeted to enhance antitumor immune response, while avoiding Treg cells in the periphery to minimize undesired inflammations.

overall_design: We performed total RNA-seq to examine gene expression changes upon Ythdf2 knockout in Treg cells

first: Linda

last: Zhang

first: Chuan

last: He


title: Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion [scRNAseq]

city: Houston

first: Jae-Il

last: Park

state: Texas

address: 6565 MD Anderson BLVD

department: Dept. of Experimental Radiation Oncology

institute: M.D. Anderson Cancer Center

summary: Accumulated genetic mutations or copy number alterations are frequently observed in esophageal squamous cell carcinoma (ESCC) patients. However, it is still elusive which gene is the driver to initiate ESCC. We identified key genetic determinants for ESCC development using CRISPR/Cas9-based multiple genes KO mouse esophageal organoid model.  Trp53, Cdkn2a, and Notch1 triple KO (PCN) organoid showed the phenotypes of ESCC. These triple genes KO served for cell proliferation through building multiple root cell clusters and remodeling the immune landscape beneficial for tumorigenesis by Ccl2-Ccr2 mediated intercellular interactions. Ccl2-releasing PCN tumors were surrounded by exhausted T cells and M2 macrophages leading to immune evasion. The PCN-type tumor was observed in more than 30% of ESCC patients who express high levels of B2M, CCL2, and NF-kB. Our study unveiled genetic determinants for ESCC development crucial for cell-autonomous growth as well as non-cell autonomous interactions with immune cells.

overall_design: organoids from WT, PC, PN, and PCN were collected 7 days after seeding and digested with 0.05% trypsin-EDTA at 37 ℃ for 30 min. After inactivating trypsin with 10% FBS DMEM, single-cell suspension was collected by passing through the 35 m cell strainer. Each group was tagged with 2 CMO tags from CellPlex kit (10x Genomics). The tagged cells of each group were pooled together with same number of cells after counting cells. cDNA library was prepared with the 10x Genomics 3’ v2 kit, cDNA library was sequenced on an Illumina NovaSeq (Novogene) and mapped to GRCm38/mm10 genome and demultiplexed using Cellranger.

first: Kyung-Pil

last: Ko

first: Yuanjian

last: Huang

first: Shengzhe

first: Gengyi

last: Zou

first: Bong

last: Kim

first: Jie

first: Sohee

last: Jun


title: YTHDF2-m6A-NF-kB axis controls anti-tumor immunity through regulating intratumoral Treg survival and function [RIP-seq]

address: 320 Yue Yang Road

institute: CAS Center for Excellence in Molecular Cell Science, CAS

overall_design: We performed YTHDF2 RIP-seq to examine YTHDF2 targets


title: Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion


title: Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging (Cxxc1-deficient mice)

city: hangzhou

first: lie

last: wang

state: zhejiang

address: Zijingang Campus, Zhejiang University，Yuhangtang Road 866

department: immunology

institute: zhejiang universiry

summary: To investigate the role of Cxxc1 in the regulation of ILC3 development and function, we used Cxxc1flox/floxRorccre mice and carried out single-cell RNA sequencing  (BD Rhapsody) on small intestinal ILC3s.

overall_design: ILC3s from 6-8 weeks-old Cxxc1flox/floxRorccre mice are isolated from the small intestine and analyzed by single-cell RNA sequencing technology (BD Rhapsody).

first: Xin

last: Shen

first: Xianzhi

last: Gao

first: Lie

last: Wang


title: Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging [H3K4me3]

summary: To investigate the role of aging in the regulation of ILC3 development and function, we carry out cleavage under targets and tagmentation (CUT&Tag) to characterize the genome-wide profiles of the H3K4me3 modification  on aged and young gut ILC3s.

overall_design: ILC3s from 18-months-old and 6-8 weeks-old male C57BL/6 mice were isolated from the small intestine and analyzed by cleavage under targets and tagmentation (CUT&Tag).


title: Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance

first: Irfan

last: Asangani

address: 421 Curie Blvd

department: Cancer Biology

institute: University of Pennsylvania

summary: The first line of therapy for advanced prostate cancer (PCa) is androgen-deprivation therapy (ADT) through surgical or chemical castration; however, in the majority of cases, tumors relapse in a hormone refractory or castration resistant prostate cancer (CRPC) form. Once the PCa has recurred in CRPC form, it progresses to a highly aggressive disease with frequent metastasis and poses an increased risk of morbidity and death This study shows that the loss of PP2AcÎ± methylation in enzalutamide (Enza)-resistant CRPC cells plays a central role in imparting the resistant to the cancer cells by stabilizing the interaction of MED1, BRD4, and AR associated transcriptional complex, thereby amplifying the oncogenic AR transcriptional output through chromatin re-modulatory mechanism. Further, this study demonstrates that targeting the PP2ACÎ± regulatory mechanisms or its downstream epigenetic effectors/mechanisms abolish the enzalutamide-resistance phenotype, thus paving the way for the development of more effective therapeutics to curtail mCRPC. The below given experiments  validate the above findings.

overall_design: LNCaP and VCaP enzalutamide-resistant (LNCaP-EnzaR and VCaP-EnzaR) cell line was generated by culturing their parental lines in presence of enzalutamide for 4-6 months and a comparative gene expression profiling analysis of RNA-seq data for DMSO verses ARD69 or DT061 treated LNCaP-EnzaR and VCaP-EnzaR was performed.  Similar kind of comparative transcription profiling was performed for PTEN-WT verses PTEN-null mouse prostate organoids, LNCaP cells stably over-expressing LCMT1 verses LNCaP cells over-expressing LacZ,  and VCaP cells verses VCaP-EnzaR cells.

first: Reyaz

last: Ur Rasool

first: Mohammed

last: Alhusayan


title: Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging [Cxxc1 KO CUT&TAG]

summary: To investigate the role of Cxxc1 in the regulation of ILC3 development and function, we carry out cleavage under targets and tagmentation (CUT&Tag) to characterize the genome-wide profiles of the H3K4me3 modification  on Cxxc1flox/floxRorccre and Cxxc1flox/flox ILC3s.

overall_design: ILC3s from Cxxc1flox/floxRorccre and Cxxc1flox/flox  mice were isolated from the small intestine and analyzed by cleavage under targets and tagmentation (CUT&Tag).


title: Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging


title: Sex blind: bridging the gap between drug exposure and sex-related gene expression in Danio rerio using next-generation sequencing (NGS) data and a literature review to find the missing links in pharmaceutical and environmental toxicology studies

status: Public on Jun 06 2023

city: Muttenz

first: Armin

middle: K.

last: Zenker

state: Baselland

address: Hofackerstrasse 30

department: School of Life Sciences - Institute for Ecopreneurship

institute: University of Applied Sciences and Arts Northwestern Switzerland

summary: Abstract: The sex of both humans and Danio rerio has previously been shown to affect the way individuals respond to drug exposure. Genes which allow identification of sex in juvenile zebrafish show potential to reveal these confounding variables between sex in toxicological and preclinical trials but the link between these is so far missing. These sex-specific, early expressed genes where expression is not altered by drug exposure must be carefully selected for this purpose. We aimed to discover genes which can be used in pharmaceutical trials and environmental toxicology studies to uncover sex-related variations in gene expression with drug application using the model organism Danio rerio. Previously published early sex-determining genes from King et al. were evaluated as well as additional genes selected from our zebrafish Next-generation sequencing (NGS) data which are known from previously published works not to be susceptible to changes in expression with drug exposure. NGS revealed a further ten female-specific genes (vtg1, cyp17a1, cyp19a1a, igf3, ftz-f1, gdf9, foxl2a, Nr0b1, ipo4, lhcgr) and five male-related candidate genes (FKBP5, apobb1, hbaa1, dmrt1, spata6) which are also expressed in juvenile zebrafish, 28 days post fertilisation (dpf). Following this, a literature review was performed to classify which of these early-expressed sex-specific genes are already known to be affected by drug exposure in order to determine candidate genes to be used in pharmaceutical trials or environmental toxicology testing studies. Discovery of these early sex-determining genes in Danio rerio will allow identification of sex-related responses to drug testing to improve sex-specific healthcare and the medical treatment of human patients.

overall_design: NGS RNA sequences were produced from two 28-day-old zebrafish (which we classified as juvenile in this paper), two adult male and two adult female zebrafish using whole-body tissue samples. Juveniles were tested to indicate whether sex was genetically visible at this stage of development. RNA quality testing and NGS were performed at the Biocentre under the supervision of Philippe Demougin (Life Sciences Training Facility (LSTF), Basel), according to Illumina’s standard protocol for RNA sequencing (Illumina Inc., San Diego, USA, Cat. # RS-100-0801). The NGS transcript data of the sampled zebrafish were compared to identify possible genes that contribute to sex determination as well as those which are expressed early in juvenile development, 28dpf (King et al., 2020). All data analyses steps were carried out using the Galaxy server (usegalaxy.org) (Jalili et al., 2020). Data from transcript sequencing was mapped to the GRCz11 reference genome (GCA_000002035.4) using HISAT2 (Galaxy Version 2.1.0+galaxy4) (Kim et al., 2019). Transcripts were assembled and merged from the mapped reads with StringTie (Galaxy Version 1.3.4) (Pertea et al., 2015). After determining gene expression with featureCount (Galaxy Version 1.6.3+galaxy2), the NGS data was normalised and differential gene expression analysis of juvenile, male and female zebrafish was carried out using DESeq2 (Galaxy Version 2.11.40.2) (Love et al., 2014; King et al., 2020). Normalisation was based on the ‘median of ratios’ - the counts divided by sample-specific size factors determined by median ratio of gene counts relative to geometric mean per gene, to account for sequencing depth and RNA composition. This allowed us to produce a table of gene expression which highlighted genes with the highest transcript count difference between males and females. From this data, genes expressed at juvenile-stage (28dpf) which were also expressed highly in male or female samples were selected as the best gene candidates for indicating early sex identity of zebrafish. Four male (Sox9a, Gapdhs, atp1b1a and cyp26b1) and nine female genes (gyg1a, rdh10b, pdia, KPNA2, ccnb1, ctsla, Chr4, bmp15 and zbp3) which were previously identified as an early sex-determining marker in zebrafish (King et al., 2020) were used within a literature search to access previous information as to whether their level of expression was known to be influenced by certain drugs. In this study a further ten female and five male genes, which could be used in early sex-determination were selected from NGS data. A literature search was also carried out on these genes to discover if they were known to be affected by drug usage. Based on published literature from ScienceDirect in early 2021, we obtained information previous studies from the past twenty years by searching for the key terms; including the gene name, drug or pharmaceutical exposure, sex, or gender influence. Then, we eliminated irrelevant literature by reading the titles and abstracts and supplemented our literature database by reading the references of the selected studies. We used the selected literature to inform whether genes highlighted from our NGS data as early expressed within juveniles could be candidates in drug trials. Finally, we grouped early expressed genes from NGS data based on the literature search into three categories. If the study was shown to affect the expression of the selected genes, then they were deemed not good to use in pharmaceutical trials. Conversely, if it was not known from previous research that drugs affect the expression of a certain gene, it was included as a potential candidate for revealing how sex response differs with drug exposure and further investigation of its use in this area is necessary in future research.

first: Alex

middle: C

last: King


summary: To investigate the role of aging in the regulation of ILC3 development and function, we used aged mice and carried out single-cell RNA sequencing  (BD Rhapsody) on aged gut ILC3s.

overall_design: ILC3s from 18-months-old male C57BL/6 mice were isolated from the small intestine and analyzed by single-cell RNA sequencing technology (BD Rhapsody).


title: A multi-omics dataset of human transcriptome and proteome stable reference

city: Guangzhou

first: Hong

last: Lu

address: No. 601, West Huangpu Avenue, Tianhe District, Guangzhou

institute: jinan university

summary:
To find the most stable cell line by comparing the gene expression quantification of the transcriptome of the 9th to 12th generations of the 5 cell lines.
We demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome.

overall_design: Quantitative analysis of gene expression of RNA-seq data for HCCLM3 cell,HCCLM6 cell,A549 cell,Hela cell and MHCC97H cell.Multi center, multi batch and multi platform comprehensive evaluation of MHCC97H cell transcriptome stability.

first: Gong

last: Zhang

first: Shaohua


title: HDAC6 deacetylates IDH1 to regulate the homeostasis of hematopoietic stem/progenitor cells [WGBS-seq]

city: Tianjin

first: hanxiao

last: Yin

address: Weijin road 94

institute: nankai university

summary: In order to further explore the mechanism by which increased 5hmC inhibits expansion of LSK cells from Hdac6-/- murine primary BM cells, we performed WGBS-seq and oxWGBS-seq analysis using LSK cells isolated from Hdac6-/- and WT mice.

overall_design: Comparative differentially methylated regions and differentially hydroxymethylated regions analysis in bone marrow hematopoietic stem/progenitor cells (LSK) of wild-type and HDAC6-deficient mice

first: Jia

last: Yang

first: Yang

last: Liu

first: Hanxiao


title: Gene expression comparison of the bulb region between different time points during bend formation of mouse zigzag hair follicle

city: Kobe

first: Makoto

last: Takeo

state: Hyogo

address: 2-2-3 Minatojima-minamimachi, Chuo-ku

department: BDR

country: Japan

institute: RIKEN

summary: To identify genes that upregulated only at bend formation, we compared gene expression profiles of the entire bulb region isolated from adult mouse zigzag hair follicle by microdissection at 11.0, 11.5, and 12.0 days after induction of hair regeneration.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for the entire bulb region at 11.0, 11.5, and 12.0 days after hair regeneration (n = 2 for each time point).

first: Takashi

last: Tsuji


title: Contribution of 6mer seed toxicity to HIV-1 induced cytotoxicity [DISE-19]

first: Marcus

last: Peter

first: Elizabeth

last: Bartom

first: Aparajitha

last: Vaidyanathan


title: Identification of cardiac developmental toxicants in differentiated human induced pluripotent stem cells [Toxicants]

city: Cologne

first: Agapios

last: Sachinidis

state: NRW

address: Robert-Kochstr. 39

department: Institute of Neurophysiology

country: Germany

institute: University of Cologne

summary:
Animal studies for embryotoxicity evaluation of potential therapeutics and environmental factors are complex, costly, and time-consuming. Often, studies are not of human relevance because of species differences. In the present study, we recapitulated the process of cardiomyogenesis in human induced pluripotent stem cells (hiPSCs) by modulation of the Wnt signaling pathway to identify a key cardiomyogenesis gene signature that can be applied to identify compounds and/or stress factors compromising the cardiomyogenesis process.
Among the 23 tested teratogens and 16 non-teratogens, we identified three retinoids including 13-cis-retinoic acid that completely block the process of cardiomyogenesis in hiPSCs. Moreover, we have identified an early gene signature consisting of 31 genes and associated biological processes that are severely affected by the retinoids. To predict the inhibitory potential of teratogens and non-teratogens in the process of cardiomyogenesis we established the “Developmental Cardiotoxicity Index” (DCI_31g) that accurately differentiates teratogens and non-teratogens to do or do not affect the differentiation of hiPSCs to functional cardiomyocytes.

overall_design: Human iPSCs wered differentiated for 14 days  according to a cardiomyogenic protocol and incubated for 24 h at day 1 with compounds at the same time. RNA was isolated from these cells, at spesific timepoints and a whole transcritpome analysis was performed. Compound-induced gene expression changes were determined with statistical and biological evalutation.

first: Anna

last: Cherianidou

first: Franziska

last: Kappenberg

first: Florian

last: Seidel

first: Panagiota

last: Papazoglou

first: Sureshkumar

middle: P

last: Srinivasan

first: Marcel

last: Leist

first: Jan

last: Hengstler

first: Jörg

last: Rahnenführer


title: Identification of cardiac developmental toxicants in differentiated human induced pluripotent stem cells

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

overall_design: Refer to individual Series


title: Identification of cardiac developmental toxicants in differentiated human induced pluripotent stem cells [CHIR]

first: Aviseka

last: Acharya


title: Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging [control scRNA-seq]

first: lie

last: wang

summary: To investigate the role of aging in the regulation of ILC3 development and function, we used the young mice as control and carried out single-cell RNA sequencing  (BD Rhapsody) on young gut ILC3s.

overall_design: ILC3s from young C57BL/6 mice(6-8 weeks-old ) are isolated from the small intestine and analyzed by single-cell RNA sequencing technology (BD Rhapsody).

first: Xin

last: Shen

first: Xianzhi

last: Gao

first: Lie

last: Wang


title: Effects of morphine exposure on the transcriptome of the dorsalmedial prefrontal cortex (dmPFC) in rats

city: Minneapolis

first: Shirelle

middle: Xinyue

last: Liu

state: Minnesota

address: 75 E River Rd

institute: University of Minnesota

summary: We investigated changes in gene transcription induced by subcutanuous morphine injection in adult male and female rats' dmPFC.

overall_design: Comparative gene expression profiling analysis of dmPFC RNA-seq data for rats receiving morphine vs. saline injection.

middle: X

first: Mari

middle: S

last: Gades

first: Yayi

last: Swain

first: Aarthi

last: Ramakrishnan

first: Andrew

last: Harris

first: Phu

middle: V

last: Tran

first: Jonathan

last: Gewirtz


title: Clarification of human blood ILC subtype interrelatedness and discovery of amphiregulin production by human NK cells shed light on HIV-1 pathogenesis

city: Worcester

first: Ozkan

last: Aydemir

state: Massachusetts

address: 366 Plantation Street, Paul J. DiMare Center  N4-1085

institute: UMass Medical School

summary: Human blood innate lymphoid cells (ILCs), which include ILCs and natural killer (NK) cells, derive from a common CD117+ILC precursor (ILCP). Yet, the relationship among the ILC subsets remains unclear. Bulk and single cell RNA-Seq and ATAC-Seq showed that blood ILC subsets cluster into ILC2s, ILCPs, a mixed cluster of CD56dim and CD56– NK cells, and a separate cluster of CD56hiNK cells that shares features with both ILCs and CD56dim/–NK cells. In surprising contrast to mice, tissue repair protein amphiregulin (AREG) was produced by human NK cells, with even higher levels in CD56hiNK cells than in ILCs. AREG expression in NK cells was driven by TCF7/WNT signaling and inhibited by TGFB1, a cytokine elevated in HIV-1+ people. Knockout of RUNX3, a WNT antagonist acting downstream of TGFB1, increased AREG production. Consistent with these findings, AREG+NK cells were decreased in people living with HIV-1. Additionally, functionally defective CD56–NK cells expanded in HIV-1+ people, in inverse correlation with CD56dimNK cells, ILCs, and CD4+T cells. Experiments in tissue culture and in humanized mice showed that CD56–NK cells derive from the epigenetically similar CD56dimNK cells, and that stimulation of MTOR by CD4+T cells or exogenous IL-2 prevents their expansion. These findings clarify how ILC subsets are related to each other and provide insight into how HIV-1 infection disrupts ILC homeostasis and contributes to pathology

overall_design: Different subsets of NK cells and ILCs from HIV-1 negatve and HIV-1+ peoples were sorted, and were subjected to bulk and single cell RNA-Seq and ATAC-Seq. PBMCs or sorted cells were used for in vitro culture, Cas12-crRNA mediated knockout and flow cytometry analysis.

first: Yetao


title: Potential roles for lncRNA Mirg/Foxp1 in an ARHL model created using C57BL/6J mice hearing research

city: Chongqing

first: xiaoqin

last: luo

address: Medical College Road, Yuzhong District

institute: chongqing medical university

summary: Age-related hearing loss (ARHL) is involved in the hair cell apoptosis, but the underlying  mechanism of hair cell apoptosis remains unclear. Here, we explored expression  profiles of lncRNAs and mRNAs in an ARHL mouse model by using RNA sequencing  and found that the cell apoptosis signaling pathway was activated in the cochlea  tissues of old mice compared with those of young mice. We further found that lncRNA  Mirg was most correlated with the cell apoptosis signaling pathway by the coexpression  analysis. LncRNA Mirg was increased in the cochlea tissues of old mice  compared with those of young mice, and its overexpression promoted hair cell  apoptosis. H2O2-induced oxidative stress increased hair cell apoptosis by upregulating  lncRNA Mirg. Furthermore, the expression of lncRNA Mirg and Foxp1 had the maximal  correlation coefficient in the cochlea tissues of old mice, and lncRNA Mirg promoted  hair cell apoptosis by increasing Foxp1 expression. In conclusion, we identified that  oxidative stress-induced lncRNA Mirg promoted hair cell apoptosis via increasing  Foxp1 expression, and these suggest that the lncRNA Mirg/Foxp1 signal may be a  potential therapeutic target for ARHL.

overall_design: Expression profiles of lncRNAs and mRNAs in the basal parts of cochlear cochleae of age-related hearing loss

first: Xiaoqin

last: Luo

first: Yaqin

last: Hu

last: Zhou

first: chanyuan

last: Zhang

first: Menglong

last: Feng

first: Ting

last: Yang

first: Wei

last: Yuan


title: An allelic series of spontaneous Rorb mutant mice exhibit a gait phenotype, changes in retina morphology and behavior, and gene expression signatures associated with the unfolded protein response

city: Bar Harbor

first: George

last: Murray

state: ME

address: 600 Main St.

department: Research

institute: The Jackson Laboratory

summary:
We have identified and characterized an allelic series of spontaneous Rorb mutations in mice
We perform RNASeq to identify gene expression changes associated with Rorb mutations in brain and spinal cord from all five mutant strains.  We also perform CNS region-specific RNASeq in the Rorbh5/h5 mutant.

overall_design: Comparative gene expression analysis of RNA-seq data for five strains of mice with spontaneous Rorb mutations and C57BL/6J and DBA/1J control mice.

first: Abigail

last: Tadenev

first: Robert

last: Burgess


title: Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion [bulkRNAseq]

first: Jae-Il

last: Park

summary: Accumulated genetic mutations or copy number alterations are frequently observed in esophageal squamous cell carcinoma (ESCC) patients. However, it is still elusive which gene is the driver to initiate ESCC. We identified key genetic determinants for ESCC development using CRISPR/Cas9-based multiple genes KO mouse esophageal organoid model.  Trp53, Cdkn2a, and Notch1 triple KO (PCN) organoid showed the phenotypes of ESCC. These triple genes KO served for cell proliferation through building multiple root cell clusters and remodeling the immune landscape beneficial for tumorigenesis by Ccl2-Ccr2 mediated intercellular interactions. Ccl2-releasing PCN tumors were surrounded by exhausted T cells and M2 macrophages leading to immune evasion. The PCN-type tumor was observed in more than 30% of ESCC patients who express high levels of B2M, CCL2, and NF-kB. Our study unveiled genetic determinants for ESCC development crucial for cell-autonomous growth as well as non-cell autonomous interactions with immune cells.

overall_design: Triplicated organoids from WT, PN, and PCN were collected 7 days after seeding.

first: Kyung-Pil

last: Ko

first: Yuanjian

last: Huang

first: Shengzhe

first: Gengyi

last: Zou

first: Bong

last: Kim

first: Jie

first: Sohee

last: Jun


title: scRNA-seq and CITE-seq of T-BET/EOMES CRISPR deleted human NK cells

city: St Louis

first: Todd

last: Fehniger

state: Missouri

address: 660 South Euclid Avenue

institute: Washington University School of Medicine

summary: Peripheral blood NK cells that were CRISPR-edited to knock-out expression of T-BET or EOMES were prepared for scRNA-sequencing(10X Genomics).   The following samples were analyzed to elucidate the role of T-BET and EOMES in regulating mature human NK cell transcriptional profiles.

overall_design: 12 samples scRNA-seq(Donors 1-3 in vitro and in vivo): TRAC and double TBX21+EOMES CRISPR edited NK cell generated from the same normal donors. 2 samples (Donors 89528 and 3227 in vitro only) CITEseq: TRAC, TBX21, EOMES, and double TBX21+EOMES CRISPR edited NK cell generated from the same normal donors are hashtagged into the same files


title: Transcriptomic and Co-expression Network Analyses on Diverse Wheat Landraces Identifies Candidate Master Regulators of the Response to Early Drought

city: Heslington, York

first: Sara

last: Franco Ortega

address: Wentworth Way

department: Department of Biology

institute: Centre for Novel Agricultural Products, University of York

summary: More than four billion people rely on bread wheat (Triticum aestivum L.) as a major constituent of their diet. However, the changing climate threatens wheat production, with periods of intense drought stress already causing widespread wheat yield losses. Much of the research into the wheat drought response has centred on the response to drought events later in development, during anthesis or grain filling. But as the timing of periods of drought stress become increasingly unpredictable, a more complete understanding of the response to drought during early development is also needed. Here, we utilized the YoGI landrace panel to identify the key genes regulating processes such as, stomatal opening, stomatal closing, stomatal morphogenesis and stress hormone signalling related to drought stress.

overall_design: Fourteen landraces where used to asses the wheat transcriptomic response to drought. The experiment was performed by growing the plant until GS13 (Zadok’s growth scale) and applying the drought stress during 10 days (withhold of water) and three days of recovery (normal watering) after that. Four replicates, per treatment, per landrace accession were used in this experiment. mRNA-Seq analysis was performed on RNA material collected at GS13, and after 10 days of drought stress.

first: Liam

last: Barratt

first: Isaac

last: Reynolds

first: Andrea

last: Harper


title: Virological characterization of the 2022 outbreak-causing monkeypox virus using human keratinocytes and colon organoids

city: Kyoto

first: Kazuo

last: Takayama

state: Kyoto

address: Shogoin Kawaharacho 53, Sakyo-ku

department: Center for iPS Cell Research and Application

institute: Kyoto University

summary: The outbreak-causing monkeypox virus of 2022 (2022 MPXV) is classified as a clade IIb strain and phylogenetically distinct from prior endemic MPXV strains (clades I or IIa), suggesting that its virological properties may also differ. Here, we used human keratinocytes and induced pluripotent stem cell-derived colon organoids to examine the efficiency of viral growth in these cells and the MPXV infection-mediated host responses. MPXV replication was much more productive in keratinocytes than in colon organoids. We observed that MPXV infections, regardless of strain, caused cellular dysfunction and mitochondrial damage in keratinocytes. Notably, a significant increase in the expression of hypoxia-related genes was observed specifically in 2022 MPXV-infected keratinocytes. Our comparison of virological features between 2022 MPXV and prior endemic MPXV strains revealed signaling pathways potentially involved with the cellular damages caused by MPXV infections and highlights host vulnerabilities that could be utilized as protective therapeutic strategies against human mpox in the future.

overall_design: The MPXV-infected colon organoids and keratinocytes were collected, and then total RNA was collected and RNA-seq was performed.


title: YTHDF2/m6A/NF-κB axis controls anti-tumor immunity by regulating intratumoral Tregs

city: Shanghai

first: Xiaoyang

last: Dou

summary: N6-methyladenosine (m6A) in messenger RNA (mRNA) regulates immune cells in homeostasis and in response to infection and inflammation. The function of the m6A reader YTHDF2 in the tumor microenvironment (TME) in these contexts has not been explored. We discovered that the loss of YTHDF2 in regulatory T (Treg) cells reduces tumor growth in mice. Deletion of Ythdf2 in Tregs does not affect peripheral immune homeostasis but leads to increased apoptosis and impaired suppressive function of Treg cells in the TME. Elevated tumor necrosis factor (TNF) signaling in the TME promotes YTHDF2 expression, which in turn regulates NF-κB signaling by accelerating the degradation of m6 A-modified transcripts that encode NF-κB-negative regulators. This TME-specific regulation of Treg by YTHDF2 points to YTHDF2 as a potential target for anti-cancer immunotherapy, where intratumoral Treg cells can be targeted to enhance anti-tumor immune response while avoiding Treg cells in the periphery to minimize undesired inflammations.


title: Different chromatin scanning modes lead to targeting of compacted chromatin by pioneer factors FOXA1 and SOX2

city: Philadelphia

first: Kenneth

last: Zaret

address: 9-132, SCTR, 3400 Civic Center Boulevard

institute: University of Pennsylvania School of Medicine


title: Mutual antagonism between glucocorticoid and canonical Wnt signaling pathways impacts steroid resistance in acute lymphoblastic leukemia [RNA-Seq]

city: Memphis

first: Daniel

last: Savic

state: Tennessee

address: 262 Danny Thomas Pl

department: Pharmaceutical Sciences

institute: St. Jude Children's Research Hospital

summary: Glucocorticoids (GCs) are a mainstay of contemporary, multi-drug chemotherapy in the treatment of acute lymphoblastic leukemia (ALL). Although overall survival rates of childhood ALL have improved, resistance to antileukemic agents remains a major clinical concern. In particular, resistance to GCs is predictive of ALL relapse and poor clinical outcome, and it therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes and transcriptional signatures implicated in ALL GC resistance, there remains an insufficient understanding of the impact of glucocorticoid response element (GRE) alterations in GC resistance. In this work (which is a follow-up of our recent study- Bergeron et al. Leukemia 2022), we uncovered the genomic mechanism linking disruptions to the Wnt repressor gene TLE1 with glucocorticoid (GC) drug resistance in acute lymphoblastic leukemia. In this process we uncovered extensive crosstalk and mutual antagonism between GC signaling and canonical Wnt signaling in ALL cells at the DNA sequence, transcriptome and proteome levels.

overall_design: RNA-seq performed in the parental Nalm6 ALL cell line and TLE1 KO Nalm6 cell line. Nalm6 parental cells grown in standard culture conditions were treated with a Wnt agonist CHIR-99021 at 0.5uM for 24 hours and RNA was extracted. RNA from untreated control samples was also extracted. Three independent experiments were conducted. TLE1 KO Nalm6 cells grown in standard culture conditions were treated with prednisolone at 5uM for 24 hours and RNA was extracted. RNA from untreated control samples was also extracted. Three independent experiments were conducted.


title: Mutual antagonism between glucocorticoid and canonical Wnt signaling pathways impacts steroid resistance in acute lymphoblastic leukemia [CUT&RUN]

overall_design: CUT&RUN was performed in Nalm6  cells with or without prednisolone treatment. Cells grown in standard culture conditions were treated with prednisolone (5uM for Nalm6 cells) for 24 hours. Half a million cells were collected for each replicate from independent experiments. 1ul of LEF1 antibody or 0.5ug of IgG was used for each replicate.


title: TEQUILA-seq: a versatile and low-cost method for targeted long-read RNA sequencing

first: Yi

last: Xing

state: PA

address: 9054 Colket Translational Research Bldg. 3501 Civic Center Blvd.

department: Executive Director, Department of Biomedical and Health Informatics

institute: The Children’s Hospital of Philadelphia

summary: Long-read RNA sequencing (RNA-seq) is a powerful technology for transcriptome analysis, but the relatively low throughput of current long-read sequencing platforms limits transcript coverage. We present TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq. TEQUILA-seq can be broadly used for targeted sequencing of full-length transcripts in diverse biomedical research settings.

overall_design: Targeted long-read sequencing on the Oxford nanopore platform was used to profile full-length transcript isoforms of preselected genes in poly(A)-selected RNA from human brain tissue (Clontech) and SH-SY5Y human neuroblastoma cells, each with three replicates. Targeted long-read sequencing was also used to examine full-length transcript isoforms of 468 actionable cancer genes in poly(A)-selected RNA extracted from 40 breast cancer cell lines (ATCC).

first: Feng

last: Wang

first: Yang

last: Xu

first: Robert

first: Lan

last: Lin


title: FOXP1 Orchestrates Neurogenesis in Human Cortical Basal Progenitors

city: Dallas

first: Genevieve

last: Konopka

state: TX

address: 5323 Harry Hines Blvd.

department: Neuroscience

institute: UT Southwestern Medical Center

summary: We report the application of single-molecule-based sequencing technology for high-throughput profiling of Forkhead Box P1, in human iPSC-derived forebrain organoids with and without FOXP1. We generated cell type-specific gene expression profiles of FOXP1 in progenitors and excitatory neurons of dorsal telencephalic lineage.

overall_design: Examination of differentially expressed genes regulated by FOXP1 by comparing scRNA-seq results of WT organoids to FOXP1 KO organoids in different neural cell types

first: Seon

last: Park

first: Ashwinikumar

last: Kulkarni


title: The SWI/SNF subunit Dpf2 licenses Nrf2-dependent gene expression to enforce multilineage control of inflammation [CUT&RUN]

city: Miami

first: Gloria

last: Mas Martin

state: Florida

address: 1501 NW 10th Avenue

institute: University of Miami

summary:
Purpose: Multi-omics characterization of the consequences of deleting the SWI/SNF subunit Dpf2 in the transcriptome (RNAseq), accessibility of the genome (ATACseq) and binding of SWI/SNF subunits (CUT&RUN) in hematopoietic stem/progenitor cells, resting and polarized bone-marrow derived macrophages and resting and activated CD4+ T cells obtained from Dpf2f/f and Dpf2D/D mice.
Methods: Lin- cKit+ cells were sorted from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Bone-marrow derived macrophages were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and polarized with IFNg and LPS, or IL-4 for 24hours. CD4+ splenic T helper cells were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and activated by treatment with PMA and Ionomycin.
Methods: For CUT&RUN experiments, Lin- cKit+ cells were obtained from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Cells were crosslinked (1 min, 1% formaldehyde), quenched (125mM Glycine) and washed in buffers following the CUTANA ChIC/CUT&RUN kit according to the manufacturer’s CUT&RUN cross-linking protocol (EpiCypher, 14-1048).
Results: For CUT&RUN experiments, 5 million (paired-end, 75bp) reads were obtained per sample. Pair-end fastq files were processed with the ENCODE Transcription Factor and Histone ChIP-Seq processing pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2). Reads were trimmed using cutadapt v2.5. and aligned to the mm10 genome using Bowtie2 v2.3.4.3. SAMtools v1.9 were used to convert the output file to BAM format. Duplicates were removed using Picard Tools v2.20.7. Peak calling was performed with MACS2 v2.2.4.
Conclusions: The absence of Dpf2 in LK cells results in downregulation of anti-oxidative and anti-inflammatory gene expression programs in bone marrow LK cells, bone-marrow derived macrophages and CD4+ T cells.

overall_design:
Samples were obtained from primary bone marrow or spleen cells isolated or sorted from Dpf2f/f and Dpf2D/D mice of approximately 28 days of age. For RNAseq and ATACseq datasets, each file corresponds to data from 1 independent mouse/genotype. At least 3 mice (independent biological replicates) per condition and genotype were used. CUT&RUN samples were obtained from a pool of LK cells.
Antibodies used for Cut&Run are as follows: anti-Brg1 (Bethyl A300-813A, lot#4), anti-Dpf2 (Millipore SIGMA, SAB4502621, lot#3111434), anti-Nrf2 (R&D Biosystems, AF3925, lot#WID0121081), anti-IgG (Diagenode, C15410206, lot#RIG001), anti-H3K27ac (Diagenode, C15410196, lot#A1723-0041D), anti-H3K4me1 (Diagenode, C15410194, lot#A1862D).


title: Next Generation Sequencing Facilitates Quantitative Analysis of transcriptome profiling (RNA-seq) of  adult hippocampus from offspring derived from control and preeclampsia mothers

first: Wen-Long

last: Zhao

address: Guangyuan road 145#

institute: The International Peace Maternity and Child Health Hospital

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling (RNA-seq) of adult hippocampus from offspring from control and preeclampsia mother mice.
Methods: Adult hippocampus RNA profiles of offspring from control and preeclampsia mother mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks.
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the adult hippocampus of offspring from control and preeclampsia mother mice with BWA workflow and 34,115 transcripts with TopHat workflow. R
Conclusions: Our study represents the first detailed analysis of adult hippocampus transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of RNA content within tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: Adult hippocampus RNA profiles of offspring from  Control (Con) and preeclampsia (PE) mother mice were generated by deep sequencing, in quadruplication, using HiSeq X Ten

first: Xueyuan

last: Liu

first: Wenlong


title: Distinct transcriptome, translatome, and proteome reprogramming during the oocyte-to-embryo transition

city: Beijing

first: Wei

last: Xie

address: Zhongguancun street, Haidian

department: THU-PKU Center for Life Sciences

institute: Tsinghua University

summary: Oocyte-to-embryo transition plays a critical role in oocyte maturation and embryogenesis. It is a highly regulated process in part due to a transcription silenced period followed by zygotic genome activation. How transcriptome, translatome, and proteome interplay in this critical developmental window remains poorly understood. Utilizing a highly sensitive mass spectrometry, we obtained a high-quality proteome landscape spanning 10 stages, from the mouse full-grown oocyte (FGO) to blastocyst, using 100 oocytes/embryos at each stage. By integrative analysis with corresponding transcriptome and translatome, we found transcription and translation levels can not reflect protein abundance in most cases. From FGO to 4-cell embryos, proteomes are predominated by FGO-produced proteins, while the transcriptome and translatome are much more dynamic. FGO inherited proteins frequently persist after the corresponding transcripts are already downregulated or decayed. Improved concordance between protein and RNA is observed for genes starting translation only upon meiotic resumption (OET upregulated) or transcribed only in embryos, although the detected protein dynamics often lag behind transcription and translation. Concordance between protein and transcription/tranlation is associated with protein half-lives. Finally, a kinetic model well predicts protein dynamics when incorporating both the initial protein abundance in FGO and translation kinetics across developmental stages. In sum, our study reveals multilayer control of gene expression during oocyte maturation and embryogenesis.

overall_design: By employing RNA-seq, Ribo-seq, and LC-MS/MS across 10 developmental stages from FGO to blastocyst, we investigated the protein, translation and transcription dynamics during oocyte maturation and early embryo development. We also invetigated the interplay between proteome and translatome or transcriptome.

first: Hongmei

last: Zhang

first: Shuyan

last: Ji

first: Ke


title: The SWI/SNF subunit Dpf2 licenses Nrf2-dependent gene expression to enforce multilineage control of inflammation [RNA-seq]

summary:
Purpose: Multi-omics characterization of the consequences of deleting the SWI/SNF subunit Dpf2 in the transcriptome (RNAseq), accessibility of the genome (ATACseq) and binding of SWI/SNF subunits (CUT&RUN) in hematopoietic stem/progenitor cells, resting and polarized bone-marrow derived macrophages and resting and activated CD4+ T cells obtained from Dpf2f/f and Dpf2D/D mice.
Methods: Lin- cKit+ cells were sorted from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Bone-marrow derived macrophages were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and polarized with IFNg and LPS, or IL-4 for 24hours. CD4+ splenic T helper cells were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and activated by treatment with PMA and Ionomycin.
Methods: For RNAseq, total RNA was extracted using RNEasy Plus micro kit (Qiagen). Ribosomal RNA was removed using NEBNext rRNA Depletion kit and RNAseq libraries were prepared using Illumina's TruSeq total stranded RNAseq prep kit (NuGEN Technologies). RNAseq libraries were sequenced on an Illumina NextSeq500 prep kit (Paired-end, 75bp) or NovaSeq 6000.
Results: For RNAseq samples, we obtained more than 40 million paired-end reads per sample. Reads were aligned to the mm10 transcriptome and ERCC spike-in indexes using RSEM (v1.3.0) and STAR (v2.6.0c) with Gencode annotations. Differentially expressed genes were determined by DESeq2 (v1.18.1, Wald Test, p-adj<0.05) after gene counts were corrected based on ERCC variances using RUVseq (v1.12.0).
Conclusions: The absence of Dpf2 in LK cells results in downregulation of anti-oxidative and anti-inflammatory gene expression programs in bone marrow LK cells, bone-marrow derived macrophages and CD4+ T cells.

overall_design: Samples were obtained from primary bone marrow or spleen cells isolated or sorted from Dpf2f/f and Dpf2D/D mice of approximately 28 days of age. For RNAseq and ATACseq datasets, each file corresponds to data from 1 independent mouse/genotype. At least 3 mice (independent biological replicates) per condition and genotype were used. CUT&RUN samples were obtained from a pool of LK cells


title: The SWI/SNF subunit Dpf2 licenses Nrf2-dependent gene expression to enforce multilineage control of inflammation [ATAC-seq]

summary:
Purpose: Multi-omics characterization of the consequences of deleting the SWI/SNF subunit Dpf2 in the transcriptome (RNAseq), accessibility of the genome (ATACseq) and binding of SWI/SNF subunits (CUT&RUN) in hematopoietic stem/progenitor cells, resting and polarized bone-marrow derived macrophages and resting and activated CD4+ T cells obtained from Dpf2f/f and Dpf2D/D mice.
Methods: Lin- cKit+ cells were sorted from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Bone-marrow derived macrophages were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and polarized with IFNg and LPS, or IL-4 for 24hours. CD4+ splenic T helper cells were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and activated by treatment with PMA and Ionomycin.
Methods: For ATACseq, Lin- cKit+ cells were processed following the OMIM-ATAC-seq protocol (Corces MR et al., Nature Methods 2017). Libraries were sequenced on a NextSeq (75bp, paired end reads) to obtain more than 40 million reads/sample.
Results: For ATACseq, we obtained more than 40 million reads per sample. ATAC-Seq chromatin accessible regions were determined using ENCODE pipeline standards (https://github.com/ENCODE-DCC/atac-seq-pipeline; git commit 2b693ab).
Conclusions: The absence of Dpf2 in LK cells results in downregulation of anti-oxidative and anti-inflammatory gene expression programs in bone marrow LK cells, bone-marrow derived macrophages and CD4+ T cells.


title: The SWI/SNF subunit Dpf2 licenses Nrf2-dependent gene expression to enforce multilineage control of inflammation [ChIP-seq]

summary:
Purpose: Multi-omics characterization of the consequences of deleting the SWI/SNF subunit Dpf2 in the transcriptome (RNAseq), accessibility of the genome (ATACseq) and binding of SWI/SNF subunits (CUT&RUN) in hematopoietic stem/progenitor cells, resting and polarized bone-marrow derived macrophages and resting and activated CD4+ T cells obtained from Dpf2f/f and Dpf2D/D mice.
Methods: Lin- cKit+ cells were sorted from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Bone-marrow derived macrophages were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and polarized with IFNg and LPS, or IL-4 for 24hours. CD4+ splenic T helper cells were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and activated by treatment with PMA and Ionomycin.
Methods: For ChIPseq in SKNO1 cell lines (AML), 10 million SKNO1 cells carrying shLuciferase or shDpf2#2487 were grown in the presence of doxycycline for 7 days to induce Dpf2 depletion. At day 7 of culture, cells were crosslinked with 1% formaldehyde for 15 minutes, quenched with 125mM Glycine for 5 minutes, washed in PBS and flash frozen at -80C. Crosslinked cell pellets were processed for ChIPseq following a previously described protocol (Boulay G et al., Cell 2017).
Results: For ChIPseq, reads were trimmed using Cutadapt (v1.15--nextseq-trim=20 -m 18), aligned to the Human h38 genome using BWA (v0.7.13) with parameters aln -q 5 -l 32 -k 2, and deduplicated using Picard Mark Duplicates (v2.10.6). Peaks were called using narrow peaks by macs2 (v2.1.1.20160309) with IgG used as background.
Conclusions: The absence of Dpf2 in LK cells results in downregulation of anti-oxidative and anti-inflammatory gene expression programs in bone marrow LK cells, bone-marrow derived macrophages and CD4+ T cells.

overall_design:
Samples were obtained from primary bone marrow or spleen cells isolated or sorted from Dpf2f/f and Dpf2D/D mice of approximately 28 days of age. For RNAseq and ATACseq datasets, each file corresponds to data from 1 independent mouse/genotype. At least 3 mice (independent biological replicates) per condition and genotype were used. CUT&RUN samples were obtained from a pool of LK cells.
For ChIP-seq, antibodies used are as follows: Rabbit anti-ARID1A (BAF250A) (D2A8U, Cell Signaling Technology, Cat#12354, lot#1; RRID:AB_2637010) and Rabbit IgG (Diagenode, C15410206, lot#RIG001).


title: Haplodeficiency of the 9p21 Tumor Suppressor Locus Causes Myeloid Disorders Driven by Bone Marrow Microenvironment

city: New York City

first: Boris

last: Reizis

address: 550 1st Ave

department: Pathology

institute: NYU Grossman School of Medicine

summary:
The chromosome 9p21 locus comprises several tumor suppressor genes including MTAP, CDKN2A and CDKN2B, and its homo- or heterozygous deletions are associated with reduced survival in multiple cancer types. We report that mice with germline monoallelic deletion or induced biallelic deletion of the 9p21-syntenic locus (9p21s) developed fatal myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN)-like disease associated with aberrant trabecular bone formation and/or fibrosis in the bone marrow (BM). Reciprocal BM transfers and conditional targeting of 9p21s suggested that the disease originates in the BM stroma. Single-cell analysis of 9p21s-deficient BM stroma revealed the expansion of chondrocyte and osteogenic precursors, reflected in increased osteogenic differentiation in vitro. It also showed reduced expression of factors maintaining hematopoietic stem/progenitor cells, including Cxcl12. Accordingly, 9p21s-deficient mice showed reduced levels of circulating Cxcl12 and a concomitant upregulation of the pro-fibrotic chemokine Cxcl13. Our study highlights the potential of mutations in the BM microenvironment to drive MDS/MPN-like disease.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at two time points.

overall_design: For Bulk RNA-seq, comparative gene expression profiling analysis of bulk RNA-seq data for 9p21s+/-  moribund and age-matched wild type mice bone marrow-derived mesenchymal  stromal cells (BM-MSCs) at 0 and 7days after osteogenic diffrentiation. For CITE-seq, BM cells were flushed from two 9p21s+/-  moribund and  age-matched wild type mice bones including two tibias, two femurs and two humerus. The left bones were digested with Liberase TM (0.2 mg/ml) and DnaseI (200U/ml) at 37°C for 45 mins at 550 rpm shaking. BM cells from the digested bones were combined with the flushed BM cells and treated with 1X RBC lysis buffer to remove red blood cells. Cells were stained with biotinylated antibodies to lineage markers cocktail (CD3, CD11b, CD19, B220, Gr1, TCRb and Ter119) at 4°C for 15 mins and then negatively selected using selection cocktail from EasySep Mouse Mesenchymal Stem/Progenitor enrichment Kit (STEMCELL Technologies). Each sample of lineage-depleted cells was incubated with Fc blocking antibody and stained with sample-specific cell hashing reagent and a cocktail of FACS antibodies (lineage cocktail described above, CD71, CD31) at 4°C for 30 mins. All mice samples were washed and Lin-CD71-CD31- cells were sorted for CITE-seq analysis.

first: Jue

last: Feng

first: Eduardo

last: Esteva


title: ATAC-seq of T-BET and EOMES CRISPR edited Primary human NK cells

first: Todd

last: Fehniger

summary: ATAC-seq was performed on peripheral blood NK cells that were CRISPR-edited to knock-out expression of T-BET and EOMES.   The following samples were analyzed to elucidate the role of T-BET and EOMES in regulating mature human NK cell chromatin accessibility.

overall_design: Control and double TBX21+EOMES CRISPR edited NK cell generated from the same normal donors were prepared for ATAC-sequencing at 2 time points, Day 7 and Day 10 post CRISPR electroporation.


title: p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism (RNA-Seq II)

status: Public on Jun 05 2023

first: Jonuelle

last: Acosta

address: 421 Curie Boulevard

institute: University of Pennsylvania

summary: The p53 tumor suppressor regulates multiple context-dependent tumor  suppressive programs. Although p53 is mutated in ~90% of small cell lung cancer  (SCLC) tumors, how p53 mediates tumor suppression in this context is unknown. Here,  using a mouse model of SCLC in which endogenous p53 expression can be  conditionally and temporally regulated, we show that SCLC tumors maintain a  requirement for p53 inactivation. However, we identified tumor subtype heterogeneity  between SCLC tumors such that p53 reactivation induces a canonical senescence  response in a subset of tumors, while, in others, p53 induces a non-apoptotic form of  cell death that culminates in necrosis. We pinpointed the cyclophilin family of peptidyl  prolyl cis-trans isomerases as critical determinants of a p53-induced transcriptional  program that is specific to SCLC tumors and cell lines that are poised to undergo p53-  mediated necrosis. Importantly, inhibition of cyclophilin isomerase activity, or genetic  ablation of specific cyclophilin genes, suppresses p53-mediated necrosis by limiting p53  transcriptional output without impacting p53 chromatin binding. Our study demonstrates  that a previously unappreciated intertumoral heterogeneity in SCLC can influence the  biological response to p53 restoration, describes a novel mechanism of p53-regulated  cell death, and uncovers putative mechanisms for the treatment of this most-recalcitrant  tumor type.

overall_design: RNA-sequencing analysis of Bgal or Ppia CRISPR-knockout Type D cells after 72hrs of p53 restoration.

first: Qinglan

last: Li

first: Nelson

middle: F

last: Freeburg

first: Nivitha

last: Murali

first: Alexandra

last: Indeglia

first: Grant

last: Grothusen

first: Michelle

last: Cicchini

first: Hung

last: Mai

first: Amy

last: Gladstein

first: Keren

last: Adler

first: Katherine

middle: R

last: Doerig

first: Jinyang

first: Miguel

last: Ruiz-Torres

first: Kimberly

last: Manning

first: Ben

middle: Z

last: Stanger

first: Luca

last: Busino

first: Maureen

last: Murphy

first: Liling

last: Wan

first: David

last: Feldser


title: The study of nascent RNA transcripts in asynchronous cells and cells released from mitosis at the indicated timepoints between IAA or dmso treatment in the SMARCE1 - AID system

first: Xiaolong

last: Chen

state: TN

institute: St Jude Children's Research Hospital

summary: SMARCE1 binding on mitotic chromatin is required for the timely reinitiation of expression of SMARCE1-bookmarked genes and maintaining identity memory in cell division.

overall_design: To examine the effect of mitosis-specific degradation of SMARCE1 on transcription reactivation following exit from mitosis, we used 5- ethynyluridine (EU) to pulse-label nascent transcripts in SMARCE1-AID IAA treated or dmso treated ES cells. Pulse-labeled transcripts were analyzed at 0, 30, 60, 90, 120,180 and 240 min after nocodazole washout as well as in asynchronous cells.

first: ZHEXIN

last: Zhu

first: XIAOLONG


title: Gene expression profiling of effector CD8+ T cells during viral skin infection

city: Portland

first: Jeffrey

last: Nolz

state: OR

address: 3181 SW Sam Jackson Park Road

institute: Oregon Health & Science University

summary: T cells were sorted by FACS from the skin or spleen during a Vaccinia virus infection

overall_design: We generated gene expression profiles of mouse CD8+ T cells from the skin that were or were not producing IFN-gamma and control T cells from the spleen using Clariom S microarray.

first: Samuel

last: Hobbs

first: Mahmoud

last: Abdelbary


title: p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism (ChIP-Seq)

first: Jonuelle

last: Acosta

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for p53 in 3 Type D and 3 Type V SCLC cell lines.

first: Qinglan

last: Li

first: Nelson

last: Freeburg

first: Nivitha

last: Murali

first: Alexandra

last: Indeglia

first: Grant

last: Grothusen

first: Michelle

last: Cicchini

first: Hung

first: Amy

middle: M


title: p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism

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

overall_design: Refer to individual Series


title: Next Generation Sequencing Facilitates Quantitative Analysis of transcriptome profiling (RNA-seq) of E17.5 cortex from offspring derived from control and preeclampsia mothers

first: Wen-Long

last: Zhao

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling (RNA-seq) of E17.5 cortexes of offspring from control and preeclampsia mother mice.
Methods: E17.5 cortex mRNA profiles of offspring from control and preeclampsia mother mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks.
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the E17.5 cortexes of offspring from control and preeclampsia mother mice with BWA workflow and 34,115 transcripts with TopHat workflow. R
Conclusions: Our study represents the first detailed analysis of E17.5 cortical transcriptomes, with biologic replicates, generated by mRNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within tissue. We conclude that mRNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: E17.5 cortical mRNA profiles of  offspring from  Control (Con) and preeclampsia (PE) mother mice were generated by deep sequencing, in quadruplication, using HiSeq X Ten

first: Xueyuan

last: Liu

first: Wenlong


title: Differential effects of ontamalimab versus vedolizumab on immune cell trafficking in intestinal inflammation and IBD

city: Erlangen

first: Mark

last: Dedden

state: Bayern

address: Schwabachanlage 12

department: Department of Medicine 1

institute: Universitätsklinikum Erlangen

summary: The anti-MAdCAM-1 antibody ontamalimab demonstrated efficacy in a phase II trial in UC and results of early terminated phase III trials are pending, but its precise mechanisms of action are still unclear. Thus, we explored the mechanisms of ontamalimab and compared it to the anti-α4β7 antibody vedolizumab.

overall_design: C57/BL6J mice were treated with 1.5% DSS in their drinking water for 7 days, followed by 3 days of normal drinking water. Mice were treated with 250µg of vedolizumab surrogate MECA-367, ontamalimab surrogate DATK32 or rat IgG isotype control antibody intraperitoneally (i.p.) three times a week. Mini-endoscopy and LPMC isolation were performed as described above.

first: Sebastian

last: Zundler

first: Lisa

last: Schulze


title: A SINGLE-CELL RNA-SEQ ANALYSIS UNRAVELS THE HETEROGENEITY OF PRIMARY CULTURED HUMAN CORNEAL ENDOTHELIAL CELLS

city: Maastricht

first: Vanessa

last: LaPointe

address: Universiteitssingel 40

country: Netherlands

institute: MERLN Institute

summary: The corneas is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparent. CECs remain arrested at a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies.

overall_design:
Research-grade donor human corneas and ethical statement
This study was performed in compliance with the tenets of the Declaration of Helsinki. All research-grade human donor corneas used for primary culture were obtained from the Lions Eye Institute for Transplant & Research (Tampa, USA), with informed consent from the next of kin. The research involving human-derived corneas was performed in accordance to Maastricht University and Dutch national regulations. All corneas had an endothelial cell density of at least 2800 cells/mm2, deemed unsuitable for transplantation, and were preserved in Optisol-GS at 4°C for up to 14 days prior to their use (Table 1). No corneas were procured from prisoners.
Isolation and culture of primary human corneal endothelial cells
Six paired corneas, from two male and one female donor aged 27 to 34 years, were used for isolation and primary culture of endothelial cells. Donors had no history of ocular disease, chronic systemic disease, or pathological infection such as HIV, Hepatitis B and C, HTLV-I/II, syphilis, or SARS-CoV-2.
Prior to isolation, the endothelial–Descemet’s corneal layer was manually stripped as follows: the corneas were vacuum fixed in a punch base (e.janach) endothelial cell side up and trephined with a 10 mm Ø corneal punch at a fixed depth of 100 μm (e.janach). To delimit the endothelial trephined line, corneas were stained with a trypan blue solution (0.4%) for 30 s, and washed with balanced salt solution sterile irrigating solution (BSS; Alcon). The corneal endothelium was then gently lifted using a DMEK cleavage hook (e.janach) and fully stripped using angled McPherson tying forceps.
Human corneal endothelial cells were isolated and cultured as previously reported.5 Briefly, the stripped endothelium–Descemet’s layer was incubated with 2 mg/ml collagenase A (Roche) solution in human endothelial serum free media (SFM) (Thermo Fisher Scientific) for 2–5 h at 37°C followed by a 5 min incubation in TrypLE express (Thermo Fisher Scientific) to generate small clumps of corneal endothelial cells. Cells from each cornea were seeded equally across 2 wells of a 24-well plate coated with fibronectin collagen (FNC) coating mix (Athena Enzyme Systems) in M5 stabilization media (human endothelial SFM (Thermo Fisher Scientific) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin–streptomycin, and 0.25 μg/mL amphotericin B) supplemented with 10 μM Y-27632 (STEMCELL Technologies). Subsequently, corneal endothelial cells were cultured in M4 proliferation medium (1:1 Ham’s F12 (Thermo Fisher Scientific) and M199 (Thermo Fisher Scientific) supplemented with 5% FBS, 20 μg/mL ascorbic acid (Sigma), 1 × ITS (Thermo Fisher Scientific), 10 ng/mL human recombinant bFGF (Sigma), and 10 μM Y-27632 (STEMCELL Technologies)); media was refreshed every other day. Upon reaching 90% confluency, after approximately 8–10 days of culture, cells were cultured in M5 stabilization media for 7 days. After this, corneal endothelial cells were treated with TripLE express and passaged into wells pre-coated with FNC-coating mix at a seeding density of 10,500 cells/cm2 in M5 stabilization medium. All cell culture was performed in incubators with humidified atmosphere of 37°C and 5% CO2.
Preparation of single cell suspension and methanol fixation
Cells from five different culture time points were methanol fixed for sequencing. Namely, cells at days 2 and 5 of culture after isolation in M4 proliferation media at passage 0, and cells at confluency after 7 days of culture in M5 stabilization media, at passages 0, 1, and 2.
To generate a single cell suspension, primary cultured corneal endothelial cells were treated with TripLE express for approximately 30 min at 37°C. Then cells were centrifuged for 5 min at 800 × g and resuspended in 1 mL ice-cold Dulbecco’s phosphate-buffered saline (DPBS). Next, the cells were centrifuged for 5 min at 800 × g and resuspended in ice-cold DPBS at a ratio of 200 μL DPBS/1 × 106 cells, followed by the dropwise addition of ice-cold methanol at a ratio of 800 μL DPBS/ 1 × 106 cells. The fixed cell suspensions were stored at -80°C until sequencing.
Single-cell RNA sequencing
scRNAseq of primary cultured CECs was performed at Single Cell Discoveries (Utrecht, the Netherlands) following standard 10× Genomics 3’ V3.1 chemistry protocol. Cells were rehydrated and loaded on the 10× Chromium controller as follows. Approximately 10,000 cells were loaded per each sample specified in Table 2. The resulting sequencing libraries were prepared following a standard 10× Genomics protocol and sequenced with an Illumina NovaSeq 6000 platform; read length: 150 bp, paired-end.

first: Pere

last: Catala

first: Nathalie

last: Groen

first: Mor

last: Dickman

last: Lapointe


title: Integrating single-cell sequencing and metabolomics reveals the macrophages heterogeneity in glucocorticoids-induced immunocompromised mice during Pneumocystis infection

last: Song

state: Beijing

address: 8 Gongren Tiyuchang Nanlua, Chaoyang District

department: Medical Research Center

institute: Beijing Institute of Respiratory Medicine and Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China.

summary: With the extensive use of glucocorticoids (GCs), the occurrence of immunocompromised patients is increasingly raised. Pneumonia, caused by opportunistic pathogens including Pneumocystis (PC), is a major cause of morbidity and mortality. The loss of adaptive immune responses have been demonstrated as a key reason for the infection of Pneumocystis. Nevertheless, the alterations of innate immunity by GCs treatment and the subsequent effects remain unclear.   Here we integrated the single-cell RNA sequencing and metabolomic analysis to decipher the alterations of the innate immunity in immunocompromised mice with PC infection. Our results have shown that a group of Mmp12+ macrophages was enriched in lung tissues of the immunocompetent mice upon PC infection. Pathway enrichment analysis showed obviously up-regulated pro-inflammatory pathways, suggesting that these Mmp12+ macrophages may be essential for the protection against PC infection. In the dexamethasone (DEX)-treated mice, the monocyte differentiation towards Mmp12+ macrophages was dramatically hampered via downregulating the granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. Further, DEX treatment simultaneously impaired the metabolic and functional integrity of resident macrophages through down-regulating lysophosphatidylcholine and GM-CSF. These induced suppressed host antimicrobial capacities. The results of our study provides a rich resource for understanding the heterogeneity and changes in macrophages and also provides novel insights into immune response take place in GCs-induced immunosuppressed mice during PC infection.

overall_design: Single-cell RNA profiles of immunocompetent or Dexamethasone(DEX)-induced immunocompromised mices with or without Pneumocystis infection for 2 weeks.

first: Zhaohui

last: Tong

first: Yawen

last: Wang

first: Kang

first: Huqin

last: Yang