title: Single-cell ATAC-seq analysis of mouse embryonic stem cells before and after Cohesin depletion

status: Public on May 01 2024

city: Shenzhen

first: PENG

last: DONG

state: Guangdong

address: 1068 Xueyuan Avenue, Shenzhen University Town

institute: Shenzhen Institute of Advanced Technology

summary: The contrast between the disruption of genome topology upon cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic co-expression tendency of cis-linked genes in single cells. Notably, through single-cell ATAC-seq analysis of mouse embryonic stem cells, we found that cohesin loss induces chromatin co-opening tens of million bases apart in cis. Our results support that cohesin arranges nuclear topology to control gene co-expression in single cells.

overall_design: JM8.N4 mouse embryonic stem cells (mESCs) from the C57BL/6N strain were edited by using CRISPR/Cas9 to fuse cohesin subunit RAD21 to the auxin-induced degron (AID) system. The derived cells were used in Smart-SCRB (Smart-Single Cell RNA Barcoding, a highly sensitive single-cell transcriptome assay) analysis and single-cell ATAC-seq analysis under control and acute cohesin loss (6hrs after the auxin treatment) conditions.

first: Peng

last: Dong

first: Zhe

last: Liu


title: Xenografted human iPSC-derived neurons with the familial Alzheimer’s disease APPV717I mutation reveal dysregulated transcriptome signatures linked to synaptic function and implicate LINGO2 as a disease signaling mediator

status: Public on May 03 2024

city: New York

first: Gunnar

last: Hargus

state: NY

address: 650W 168th Street

department: Pathology and Cell Biology

country: USA

institute: Columbia University

type: Expression profiling by high throughput sequencing

summary: Alzheimer’s disease (AD) is the most common cause of dementia, and disease mechanisms are still not fully understood. Here, we explored pathological changes in human induced pluripotent stem cell (iPSC)-derived neurons carrying the familial AD APPV717I mutation after cell injection into the mouse forebrain. APPV717I mutant iPSCs and isogenic controls were differentiated into neurons revealing enhanced Aβ42 production, elevated phospho-tau, and impaired neurite outgrowth in APPV717I neurons. Two months after transplantation, APPV717I and control neural cells showed robust engraftment but at 12 months post injection, APPV717I grafts were smaller and demonstrated impaired neurite outgrowth compared to controls, while plaque and tangle pathology were not seen. Single-nucleus RNA-sequencing of microdissected grafts, performed two months after cell injection, identified significantly altered transcriptome signatures in APPV717I iPSC-derived neurons pointing towards dysregulated synaptic function and axon guidance. Interestingly, APPV717I neurons showed an increased expression of genes, many of which are also upregulated in postmortem neurons of AD patients including the transmembrane protein LINGO2. Downregulation of LINGO2 in cultured APPV717I neurons rescued neurite outgrowth deficits and reversed key AD-associated transcriptional changes related but not limited to synaptic function, apoptosis and cellular senescence. These results provide important insights into transcriptional dysregulation in xenografted APPV717I neurons linked to synaptic function, and they indicate that LINGO2 may represent a potential therapeutic target in AD.

overall_design: We performed gene expression analysis on differentiated neurons in vitro and single-nucleus RNA-seq (snRNA-seq) on micro-dissected grafts.

first: Wenhui

last: Qu


title: Sequencing-Guided Design of Genetically Encoded Small RNAs Targeting CAG Repeats for Selective Inhibition of Mutant Huntingtin

city: Winston-Salem

first: Keith

middle: T.

last: Gagnon

state: North Carolina

address: 575 Patterson Ave

department: Biochemistry and Molecular Biology

institute: Wake Forest University School of Medicine

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Bulk gene expression profiles of pupal antennae in wild-type and orco mutant Harpegnathos saltator

status: Public on May 02 2024

first: Bogdan

last: Sieriebriennikov

address: 100 Washington Sq E

institute: New York University

summary: H. saltator orco mutant has a reduced number of cells in the antennae. We performed RNA-seq of the developing pupal antennae to describe the developmental context of cell death in the mutant pupae.

overall_design: RNA was extracted from the antennae of wild-type and orco mutant H. saltator at 10, 15, 20, and 25 days after pupal formation.

first: Kayli

middle: R

last: Sieber

first: Olena

last: Kolumba

first: Jakub

last: Mlejnek

first: Shadi

last: Jafari

first: Hua

last: Yan


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

Alzheimer’s disease (AD) is the most common cause of dementia, and disease mechanisms are still not fully understood. Here, we explored pathological changes in human induced pluripotent stem cell (iPSC)-derived neurons carrying the familial AD APPV717I mutation after cell injection into the mouse forebrain. APPV717I mutant iPSCs and isogenic controls were differentiated into neurons revealing enhanced Aβ42 production, elevated phospho-tau, and impaired neurite outgrowth in APPV717I neurons. Two months after transplantation, APPV717I and control neural cells showed robust engraftment but at 12 months post injection, APPV717I grafts were smaller and demonstrated impaired neurite outgrowth compared to controls, while plaque and tangle pathology were not seen. Single-nucleus RNA-sequencing of microdissected grafts, performed two months after cell injection, identified significantly altered transcriptome signatures in APPV717I iPSC-derived neurons pointing towards dysregulated synaptic function and axon guidance. Interestingly, APPV717I neurons showed an increased expression of genes, many of which are also upregulated in postmortem neurons of AD patients including the transmembrane protein LINGO2. Downregulation of LINGO2 in cultured APPV717I neurons rescued neurite outgrowth deficits and reversed key AD-associated transcriptional changes related but not limited to synaptic function, apoptosis and cellular senescence. These results provide important insights into transcriptional dysregulation in xenografted APPV717I neurons linked to synaptic function, and they indicate that LINGO2 may represent a potential therapeutic target in AD.

overall_design: Refer to individual Series.


title: BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation [ATAC-Seq]

city: Boston

first: Swati

last: Garg

state: MA

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity.  BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

overall_design: To investigate the function of BRD9 in human CD34+ hematopoietic stem and progenitor cells, cord blood derived CD34 cells were lentivirally infected in triplicates with GFP tagged vector containing either control (shNT) or hairpin against BRD9 (shBRD9#33 and shBRD9#81), sorted for GFP at 72 hours post infection and collected for nuclei preparation followed by Tn5 tagmentation. Differential accessibility assessment using DiffBind was performed on CD34 cells with BRD9 knockdown compared to control.

first: James

middle: D

last: Griffin


title: Mutant IDH inhibitors induce lineage differentiation in IDH-mutant Oligodendroglioma I

city: Charlestown

first: Masashi

last: Nomura

address: 149 13th street

department: Pathology

institute: Massachusetts General Hospital

summary: A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation towards the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
Fresh or frozen IDH-mutant gliomas treated with IDHi were dissociated and profiled by single-cell or single-nucleus RNA sequencing (Smart-seq2 or 10x). In-vitro IDH-mutant glioma models treated with or without IDHi was profiled by single-cell RNA-seq (10x) and single-cell multi-omics protocol (DNA methylome and transcriptome). In-vitro IDH-mutant glioma models that we knock out Notch1 (Notch1-KO) and the control (Notch1-WT) were profiled by single-cell RNA-sequencing (10x).

***************************************************************
no raw for human samples due to patient privacy concerns
***************************************************************

first: Avishay

last: Spitzer

first: Simon

last: Gritsch

middle: L

last: Ligon

first: Daniel

middle: P

last: Cahill

first: Mario

last: Suva

first: Itay

last: Tirosh


title: Sequencing-Guided Design of Genetically Encoded Small RNAs Targeting CAG Repeats for Selective Inhibition of Mutant Huntingtin [II]

summary: Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal but reduction of wild-type HTT (wtHTT) is undesirable as it compromises gene function and potential therapeutic efficacy. One promising allele- selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing to monitor processing precision of a limited set of CAG repeat- targeted small RNAs expressed from multiple scaffold contexts. Small RNA sequencing identified expression constructs with high guide strand 5' processing precision that also conferred promising allele-selective inhibition of mutHTT. However, mRNA-seq revealed varying degrees of transcriptome-wide off-target effects, including certain CAG repeat-containing mRNAs. These results support the continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process.

overall_design:
HD fibroblasts (GM04281) were obtained from Coriell Institute and used as the cellular context for this mRNA-Seq experiment.
Patient-derived HD fibroblast cells were transduced with small RNA expressing lentiviral particles. RNA was harvested by trizol methods and mRNA was isolated by poly-A magnetic bead selection.
The sequencing library was prepared by cDNA-PCR methods using an Oxford Nanopore Technologies kit and sequenced on a PromethION 2 Solo (ONT).
Sequence analysis was performed using DESeq2 to identify transcriptome changes between mock, shMCHR, and shHD2.4 control samples against  shHD33-full mimic, shHD33-full nb, and shHD33-full UAC as HTT allele selective treatments.
DESeq2 result tables, expression profile plots and gene counts were generated and used for interpretation of data in R Studio running the DESeq2 analysis program.

first: Mansi

middle: A

last: Parasrampuria

first: Adam

last: White

first: Ramadevi

last: Chilamkurthy

first: Adrian

last: Pater

first: Fatima

last: El-Azzouzi

first: Katy

middle: N

last: Ovington

first: Philip

middle: J

last: Jensik

middle: T


title: BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation [RNA-Seq]

overall_design: To investigate the function of BRD9 in human CD34+ hematopoietic stem and progenitor cells, cord blood derived CD34 cells were lentivirally infected in triplicates with GFP tagged vector containing either control (shNT) or hairpin against BRD9 (shBRD9#33 and shBRD9#81), sorted for GFP at 72 hours post infection and collected for RNA sequencing. Differential gene expression analysis was performed on CD34 cells with BRD9 knockdown compared to control.


title: Peripheral blood CD8 T cells in chronic HCV infection with cirrhosis exhibit lasting alterations in gene expression changes, with insights into the role of Hedgehog signaling

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

country: Canada

institute: Ottawa Hospital Research Institute

summary: The impact of chronic hepatic infection on antigen non-specific immune cells, especially in association with liver fibrosis severity, remains poorly described. We previously reported lasting hyperfunction of bulk peripheral blood CD8 T cells in HCV-infected individuals with cirrhosis. RNA sequencing of blood CD8 T cells from HCV-infected individuals with minimal (Metavir F0-1, Fibroscan liver stiffness ≤ 7.0 kPa) or advanced fibrosis or cirrhosis (F4 ≥ 12.5 kPa), before and after direct-acting antiviral therapy, was performed. Principal component analyses determined robust differences in 362 genes expressed by CD8 T cells from HCV-infected individuals with minimal or advanced fibrosis and data suggests this remains relatively stable after viral clearance. Gene ontology analyses identified differential gene expression related to cellular metabolism, including upregulated phospholipase, phosphatidyl-choline/inositol activity and second-messenger-mediated signaling, while genes in pathways associated with nuclear processes, RNA transport and cytoskeletal dynamics were reduced. Gene Set Enrichment Analysis identified decreased expression of genes regulated by the cMyc and E2F transcription factors in cirrhotics, compared to the minimal fibrosis group, as well as reduced expression of genes linked to oxidative phosphorylation, mTOR signaling, and more. Upregulated gene sets in cirrhotics included IFN-α, -γ, TGF-β response genes, apoptosis, and apical surface pathways, among others. The Hedgehog (Hh) signaling pathway was the top featured gene set upregulated in cirrhotics, wherein hallmark genes GLI1 and PTCH1 ranked highly. This analysis of bulk CD8 T cell gene expression in chronic HCV infection suggests considerable reprogramming of the CD8 T cell pool in liver cirrhosis. Increased Hh signaling in cirrhosis may contribute to generalized CD8 T cell hyperfunction observed in chronic HCV infection. Understanding the lasting nature of immune cell dysfunction may help mitigate remaining clinical challenges after HCV clearance and more generally, improve long term outcomes for individuals with severe liver disease.

overall_design: Study participants were chronically infected HCV patients (>6 months HCV RNA+), before direct-acting antiviral (DAA) treatment (pre-DAA) or 24 weeks after sustained virologic response (24W post-SVR). PBMCs of participants were isolated by density gradient centrifugation. CD8 T cells were isolated by magnetic bead positive selection, and stimulated with 0.5 µg/ml phytohemaglutanin-L for 18h. Total RNA was isolated using TRIzol Reagent prior to RNA-sequencing.

first: Jiafeng

last: Li

first: Agatha

last: Vranjkovic

last: Read

first: Sean

last: Delaney

first: Christopher

last: Porter

first: William

last: Stanford

first: Curtis

last: Cooper

first: Angela

middle: M

last: Crawley


title: CDC7 inhibition impairs neuroendocrine transformation in lung and prostate tumors through MYC degradation II

first: Yingqian

middle: Ada

last: Zhan

state: New York

address: 430 East 67th Street

department: EPIGENETICS RESEARCH CENTER

institute: MSKCC

summary: Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Upregulation of the cell cycle kinase CDC7 occurred in tumors undergoing NE transformation, after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of MYC, implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a strategy to constrain lineage plasticity and to effectively treat NE tumors. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk.

overall_design: 4-10 female (22PC, PDXs) or male (LnCap/AR) 6-week-old NOD.Cg-Prkdc<scid> Il2rg<tm1Wjl>/SzJ (NSG) mice (PDXs) or female 6-week-old athymic nude mice (cell line xenografts) were subcutaneously engrafted per treatment arm and until tumors reached 100-150 mm3. At that point, mice were randomized into groups and treated with either vehicle, cisplatin (2 mg/kg i.p. once/week), etoposide (3 mg/kg i.p. QDx3), simurosertib (40 mg/kg p.o. QDx3), irinotecan (5 mg/kg i.p. once/week), enzalutamide (10 mg/kg p.o. QDx5), osimertinib (25 mg/kg p.o. QDx5) or the combinations of cisplatin + etoposide, cisplatin + simurosertib, irinotecan + simurosertib, enzalutamide + simurosertib or osimertinib + simurosertib at the previously mentioned doses. Mice weights and tumor volumes were measured twice a week and mice were sacrificed when tumors reached humane endpoint (volume = 1000 mm3). The number of mice per treatment arm were selected according to previous experience with the models and response to treatments. Blinding was not performed. All animal experiments were approved by the Memorial Sloan Kettering Cancer Center (MSKCC) Animal Care and Use Committee (#13-07-007). Frozen tissues or cell pellets were weighed and homogenized in RLT and nucleic acids were extracted using the AllPrep DNA/RNA Mini Kit (QIAGEN, #80204) according to the manufacturer’s instructions. RNA was eluted in nuclease-free water.

first: Alvaro

last: Quintanal-Villalonga

first: Charles

last: Rudin

first: Richard

last: Koche


title: CDC7 inhibition impairs neuroendocrine transformation in lung and prostate tumors through MYC degradation  III

overall_design:
For H146, cells were treated with the IC20 for each drug, during 7 days before cell collection. For Lx33 and Lx761, 4-10 female 6-week-old NOD.Cg-Prkdc<scid> Il2rg<tm1Wjl>/SzJ (NSG) mice were subcutaneously engrafted per treatment arm and until tumors reached 100-150 mm3. At that point, mice were randomized into groups and treated with either vehicle, cisplatin (2 mg/kg i.p. once/week), etoposide (3 mg/kg i.p. QDx3), simurosertib (40 mg/kg p.o. QDx3), irinotecan (5 mg/kg i.p. once/week), enzalutamide (10 mg/kg p.o. QDx5), osimertinib (25 mg/kg p.o. QDx5) or the combinations of cisplatin + etoposide, cisplatin + simurosertib, irinotecan + simurosertib, enzalutamide + simurosertib or osimertinib + simurosertib at the previously mentioned doses. Mice weights and tumor volumes were measured twice a week and mice were sacrificed when tumors reached humane endpoint (volume = 1000 mm3). The number of mice per treatment arm were selected according to previous experience with the models and response to treatments. Blinding was not performed. All animal experiments were approved by the Memorial Sloan Kettering Cancer Center (MSKCC) Animal Care and Use Committee (#13-07-007). Frozen tissues or cell pellets were weighed and homogenized in RLT and nucleic acids were extracted using the AllPrep DNA/RNA Mini Kit (QIAGEN, #80204) according to the manufacturer’s instructions. RNA was eluted in nuclease-free water.
-----------------------------
Authors state "the raw data was lost".


title: CDC7 inhibition impairs neuroendocrine transformation in lung and prostate tumors through MYC degradation I


title: m6A RNA Methylation Regulates Mitochondrial Function

city: Chicago

first: Michael

last: Kahl

state: IL

address: 303 E Superior St

institute: Northwestern University

summary: Methylation of RNA on N6-adenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.

overall_design: To understand the role of m6A RNA methylation in the nervous system, we utilized a Nestin-Cre driven Mettl14 KO mouse model. We then performed m6A-sequencing on both WT and Mettl14 KO P5 mouse cortex/hippocampus to determine the landscape of m6A RNA methylation in these tissues. m6A-IP as well as Input samples were sequenced.

first: Yongchao

middle: C

last: Ma

first: Zhaofa

last: Xu


title: Patient-derived mini-colons enable long-term modeling of tumor-microenvironment complexity

city: Salamanca

first: L. Francisco

last: Lorenzo-Martín

state: Castilla y León

address: Campus Miguel de Unamuno

country: Spain

institute: University of Salamanca (USAL) – Centro de Investigación del Cáncer (CIC)

summary: The compromise to reduce, replace, and refine (3Rs) animal usage in cancer research demands the development of biologically capable ex vivo cancer models. The establishment of three-dimensional organoid-based systems has paved the way in this direction by bringing some realistic in vivo features to the in vitro context. However, faithful modeling of cancer requires a degree of tissue-level organization, multi-cellular diversity, biological durability, and experimental flexibility that far exceeds the capabilities of any existing in vitro systems. Here we implemented tissue engineering and microfabrication technologies to develop topobiologically complex patient-specific avatars of colorectal cancer able to overcome those limitations. These miniature tissues consist of long-lived gut-like-shaped healthy colon epithelia (“mini-colons”) that allow the stable integration of cancer cells and their native tumor microenvironment on a platform designed for the real-time high-resolution evaluation of cellular dynamics. This provides an unprecedented repertoire of ex vivo experimental possibilities, which we illustrate through different applications, including the discovery of a cancer associated fibroblast-triggered mechanism driving colorectal cancer invasion. As a whole, our mini-colon system pushes the boundaries of ex vivo cancer research in both basic and pre-clinical settings.

overall_design: Gene expression profiling analysis of bulk RNA-seq data from CRC mini-colons cultured: in control conditions (control), with autologous cancer-associated fibroblasts (CAF), and with conditioned-medium from autologous cancer-associated fibroblasts (CM).

first: L

middle: F

first: Nicolas

last: Broguiere

first: Lucie

last: Tillard

first: Jakob

last: Langer

first: Mikhail

last: Nikolaev

first: Krisztian

last: Homicsko

first: George

last: Coukos

first: Matthias

last: Lutolf


title: Adenine effect on Enterohemorraghic E.coli

city: Madison

first: Thibaut

last: Rosay

state: Wisconsin

department: MMI

institute: UW Madison

summary: Previous experiments have shown that E. feacalis increases EHEC virulence by secreting adenine, this RNAseq aims to understand the molecular mechanism underlaying adenine role on EHEC

overall_design: 6h culture of EHEC in DMEM low glucose in anaerobic condition in presence or not of Adenine 50uM

first: Martins

last: Fernando H

first: Rosay

last: Thibaut

first: Sperandio

last: Vanessa


title: Adiponectin inhibits acquisition of the Lyme disease agent by ticks [Tick]

city: Hangzhou

first: Xiaotian

last: Tang

state: Zhejiang

address: Agro-Bio-Environ Building, Zhejiang University

department: Entomology

country: China

institute: Zhejiang University

summary: Tick gut and salivary gland global gene expression change after feeding on adiponectin wildtype and knockout mice

overall_design: Examination of tick gut and salivary gland response after feeding on adiponectin wildtype and knockout mice for 48 hours, using RNA-seq

first: Yongguo

last: Cao

first: Erol

last: Fikrig


title: ABCG2 is an itaconate exporter that limits TFEB-mediated lysosomal biogenesis to impair antibacterial innate immunity

city: Beijing

first: Chao

last: Chen

address: Datun

department: CAS Key Laboratory of Infection and Immunity

institute: Institute of Biophysics, Chinese Academy of Sciences

summary: Itaconate is a metabolite that synthesized from cis-aconitate in mitochondria and transported into cytosol to exert multiple regulatory effects in macrophages. However, the mechanism by which itaconate exits from the cytosol of activated macrophages remains unknown. Using a genetic screen, we reveal that cytosolic itaconate is exported by ABCG2, an ATP-binding cassette (ABC) transporter, in an ATPase-dependent manner in human and mouse macrophages.

overall_design: As the human membrane transporter CRISPRi library (customized by Corus Biotechnology Inc., Nanjing in China) contains 7441 sgRNAs targeting 1241 membrane transporter genes, Cytosolic BioITA (cBioITA)-expressing THP-1 cells (5×107) were transduced with lentivirus from the library at a transduction effificiency of 30% and an average of 400-fold coverage of the library. The cells were stimulated with LPS (100 ng ml–1) for 8 hours. The cells were sorted into two populations by GFP expression: the top 25% of cBioITA’s intensity cells and bottom 25% of cBioITA’s intensity cells. Cells were washed in PBS and stored at -80°C for isolation of genomic DNA (gDNA). PCR were performed for identification of sgRNA inserts. The resulting amplicons were pooled and extracted from 2% agarose gels using the Gel Extraction Kit (Omega), and sequenced by Novogene Bioinformatics Institute (Beijing, China).

first: Xinjian

last: Li


title: miR394 modulates brassinosteroid signaling to regulate hypocotyl elongation in Arabidopsis

city: Jinan

first: Fengning

last: Xiang

state: Shandong

address: 27, Shandanan Road

department: School of life science

institute: Shandong University

summary: miR394 and its target gene LEAF CURLING RESPONSIVENESS are involved in BR signaling by affecting BRASSINOSTEROID INSENSITIVE2 accumulation.

overall_design: Col-0, MIR394A OE and 2 μM Brz treated Col-0, 5-day-old light-grown seedlings, 3 replicates each

first: Shuo


title: EGFR pathway regulates the cellular interactome within the liver fibrotic niche

status: Public on Apr 30 2024

city: L'Hospitalet de Llobregat

first: Isabel

last: Fabregat

state: Barcelona

address: Avinguda de la Granvia de l’Hospitalet, 199

department: Oncobell Program

institute: Bellvitge Biomedical Research Institute - IDIBELL

summary: RNA-seq analysis was performed in samples from WT and ΔEGFR mice (which expresses an inactive truncated form of the EGFR in hepatocytes). Samples were either treated with Mineral oil or CCl4.

overall_design: mRNA sequencing of hepatocytes from WT and transgenic ΔEGFR mice, treated with either mineral oil or CCl4


title: Mitochondria regulate proliferation in adult cardiac myocytes

first: Paul

last: Schumacker

address: 303 E. Superior SQBRC 4-526

department: Pediatrics

institute: Northwestern University Feinberg School of Medicine

summary: Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske Iron-Sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined and glucose utilization increased.  Simultaneously, they underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy.  Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident.  In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA-seq revealed upregulation of genes associated with cardiac development and proliferation.  Metabolomic analysis revealed a decrease in alpha-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity).  Analysis revealed an increase in methylated CpGs near gene transcriptional start sites.  Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.

overall_design: We deleted Uqcrfs1, which encodes the Rieske Iron-sulfur proteint (RISP) in adult mouse hearts in the C57BL/6 background using a tamoxifen-inducible a-mysin heavy chain promoter-driven Cre.  Hearts were harvested at 60 days after completing 5 tamoxifen doses, and RNA was isolated from bulk heart homogenates for RNAseq analysis.  All mice received tamoxifen; the mice designated as wild type were Cre-negative.

first: Gregory

middle: B

last: Waypa

first: Kimberly

last: Smith

first: Vincent

last: Dudley

last: Mungai

first: Kathryn

last: Helmin

first: Benjamin

last: Singer

first: Clara

last: Bien  Peek

first: Joseph

last: Bass

first: Lauren

last: Nelson

first: Sanjiv

last: Shah

first: Gaston

last: Ofman

first: J

last: Wasserstrom

first: William

last: Mulleer

first: Alexander

middle: V

last: Misharin

first: G

last: Budinger

first: Hiam

last: Abdala-Valencia

first: Navdeep

middle: S

last: Chandel

first: Danijela

last: Dokic

first: Elizabeth

last: Bartom

first: Shuang

last: Zhang

first: Yuki

last: Tatekoshi

first: Amir

last: Mahmoodzadeh

first: Hossein

last: Ardehali

first: Edward

last: Thorp


title: Lin-CD117+CD34+FceRI+ progenitor cells are increased in chronic spontaneous urticaria and predict clinical responsiveness to anti-IgE therapy

city: Dublin

first: Conor

last: Finlay

address: Trinity Translational Medicine Institute, Trinity College Dublin, St. James' Hospital Campus, Trinity College Dublin, Dublin 8

department: School of Medicine

country: Ireland

institute: Trinity College Dublin

summary: Background: Chronic spontaneous urticaria (CSU) is a common, debilitating skin disorder characterised by recurring episodes of raised, itchy and sometimes painful wheals lasting longer than 6 weeks. CSU is mediated by mast cells which are absent from peripheral blood. However, lineage-CD34hiCD117int/hiFcεRI+ cells in blood have previously been shown to represent a mast cell precursor.  Methods: We enumerated FcεRI-, FcεRI+ and FcεRIhi lineage-CD34+CD117+ cells using flow cytometry in blood of patients with CSU (n=55), including 12 patients receiving omalizumab and 43 not receiving omalizumab (n=43). Twenty-two control samples were studied. Disease control and patient response to omalizumab was evaluated using the Urticaria Control Test. We performed single cell RNA sequencing (scRNA-seq) on lineage-CD34hiCD117hi blood cells from a subset of patients with CSU (n=8) and healthy controls (n=4).  Results: CSU patients had more Lineage-CD34+CD117+FcεRI+ blood cells than controls. Lineage-CD34+CD117+FcεRI+ cells were significantly higher in patients with CSU who had an objective clinical response to omalizumab when compared to patients who had poor disease control 90 days after initiation of omalizumab. scRNA-seq revealed that lineage-CD34+CD117+FcεRI+ cells contained both lymphoid and myeloid progenitor lineages, with omalizumab responsive patients having proportionally more myeloid progenitors. The myeloid progenitor lineage contained small numbers of true mast cell precursors along with more immature FcεRI- and FcεRI+ myeloid progenitors.  Conclusion: Increased blood CD34+CD117+FcεRI+ cells may reflect enhanced bone marrow egress in the setting of CSU. High expression of these cells strongly predicts better clinical responses to the anti-IgE therapy, omalizumab.

overall_design:
PBMC were isolated from controls and paitents with CSU prior to treatment with omalizumab. PBMC were labelled with fluorescent antibodies and propidium iodide (PI) and purified using the FACSAria Fusion cell sorter (BD Biosciences). The lineage (Lin) channel included antibodies targeting the markers CD4, CD8, CD14 and CD19, all conjugated to V500. Cells were sorted at low pressure (100 µM nozzle at 20 psi) into PBS. For each sample, two populations of single, live (PI-) cells were sorted; (i) CD34+CD117+Lin- and (ii) Lin+, we recombined these populations at a ratio of 1:1.
Data is from a single BD Rhapsody run, containing 12  samples multiplexed using the human multiplexing kit. Fastq files contain all three library types, WTA (RNA), AbSeq (CITE-Seq) and SMK (sample tag). Fastq files should be processed via the BD Rhapsody pipeline. Supplementary data contains outputs of this pipeline (revision 11), including UMI gene counts table (DBEC_MolsPerCell), sample tag reads and calls (specifying sample information for each barcode). We also include minimal sample information (Sample_info) to identify samples and their original clinical groupings.


title: Targeting a Lineage-Specific PI3Kɣ/AKT Signaling Module in Acute Myeloid Leukemia Using a Heterobifunctional Degrader Molecule

city: Paris

first: Camille

last: Lobry

address: 16 rue de la Grange aux Belles

department: IRSL Inserm U944 / CNRS UMR7212

country: France

institute: Inserm

summary: Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in non-malignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue- and/or tumor-restricted expression. Here, we show that in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG/p110𝛾-PIK3R5/p101 axis blocks AKT signaling, compromises cell fitness, and sensitizes to established AML therapies. Importantly, we find that existing small molecule inhibitors against PIK3CG are insufficient to achieve a sustained long-term anti-leukemic effect. To address this concern, we developed a PROteolysis-TArgeting Chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary AML patient samples, and syngeneic mouse models.

overall_design: MOLM14 and OCI-AML2 AML cell lines were treated for 24h with ARM-165, AZ-2 or DMSO. Cells were subsequently lysed, total RNA extracted and RNA-sequencing libraries were prepared using TruSeq Stranded mRNA LT Sample Prep Kit. Sequencing librarie were sequenced on Illumina NovaSeq 600 sequencer.

first: Lois

last: Kelly

first: Alexandre

last: Puissant


title: Combined inhibition of IL-1, IL-33 and IL-36 signaling by targeting IL1RAP ameliorates skin and lung fibrosis in preclinical models of Systemic Sclerosis

city: Düsseldorf

first: Jörg

last: Distler

address: Merowingerplatz 1A

department: Department of Rheumatology

country: Germany

institute: University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University

summary: Background: The IL-1 receptor accessory protein (IL1RAP) is an essential co-receptor required for signaling through the IL-1, IL-33 and IL-36 receptors. Here, we investigate the antifibrotic potential of the combined inhibition of these cytokines by an anti-IL1RAP antibody to provide a scientific background for clinical development in systemic sclerosis (SSc). Methods: The expression of IL1RAP-associated signaling molecules was determined by data mining of publicly available RNAseq data as well as by imaging mass cytometry (IMC). Efficacy of therapeutic dosing of anti-IL1RAP antibodies was determined in three complimentary mouse models: chronic sclerodermatous graft-versus-host-disease (cGvHD), bleomycin-induced dermal fibrosis model and topoisomerase-I (topo)-induced fibrosis.  Results: SSc skin showed upregulation of IL1RAP and IL1RAP-related signaling molecules on mRNA and protein level compared to normal skin. IL-1, IL-33, and IL-36 all regulate distinct genes sets related to different pathophysiological processes in SSc. The responses of human fibroblasts and endothelial cells to IL-1, IL-33, and IL-36 were completely blocked by treatment with an anti-IL1RAP-antibody in vitro. Moreover, anti-IL1RAP antibody treatment reduced dermal and pulmonary fibrosis in cGvHD-, bleomycin- and topoisomerase-induced fibrosis. Importantly, RNAseq analyses revealed effects of IL1RAP inhibition on multiple processes related to inflammation and fibrosis that are also deregulated in human SSc skin.  Conclusion: This study provides first evidence for the therapeutic benefits of targeting of IL1RAP in SSc. Our findings have high translational potential as the anti-IL1RAP antibody CAN10 has recently entered a phase 1 clinical trial.

overall_design: The allogeneically transplanted mice (cGVHD mice) were given i.p twice a week for 4 weeks of ddH2O (Allo_H), anti-mouse IL1RAP antibody (mCAN10), Nintedanib (Nin), or isotype mIgG2a LALA-PG antibody (Allo). The syngeneically transplanted mice treated with isotype mIgG2a LALA-PG antibody were used as control (Syn).

first: Thuong

last: Trinh-Minh

first: Sara

last: Rattik

first: Caitríona

last: Grönberg


title: Structure and Dynamics of Enterovirus Genotype Networks

city: Bethesda

first: Philip

last: Murphy

state: MD

address: 10 CENTER DR RM 11N111

department: National Institute of Allergy and Infectious Diseases

institute: National Institutes of Health

summary: Like all biological populations, viral populations exist as networks of genotypes connected through mutation. Mapping the topology of these networks and quantifying population dynamics across them is crucial to understanding how populations adapt to changes in their selective environment. The influence of mutational networks is especially profound in viral populations which rapidly explore their mutational neighborhoods via high mutation rates. Using a novel single-cell sequencing method, scRNAseq-Enabled Acquisition of mRNA and Consensus Haplotypes Linking Individual Genotypes and Host Transcriptomes (SEARCHLIGHT), we captured and assembled viral haplotypes from hundreds of individual infected cells to reveal the complexity of viral populations. We obtained these genotypes in parallel with host cell transcriptome information, enabling us to link host cell transcriptional phenotypes to the genetic structures underlying virus adaptation.

overall_design: The template-switch oligonucleotide (TSO) conjugated to the Gel Beads in the 10X Genomics 5′ scRNA sequencing protocol (Fig. 1B) captures and barcodes transcripts generated during cDNA synthesis, addressing each read to an individual source cell and transcript, by a ‘cell barcode’ and ‘unique molecular identifier’ (UMI), respectively. We add virus-specific primers tiled at regular 2kb intervals across the viral RNA genome alongside poly-dT primers during in-droplet reverse transcription. Taking advantage of the TSO, reverse transcription generates barcoded cDNA transcripts (of ~2kb) that cover the entire viral genome and can be amplified for long-read sequencing. Short-read and long-read library preparation from the same barcoded scRNAseq cDNA pools allows us to reconstruct the consensus genotype from each cell and map them directly onto the transcriptome and viral RNA count information from matched infected cells.

first: Nathania

last: Dabilla

first: Patrick

last: Dolan


title: Transcriptome changes in Olig2-3 over-expression retina ganglion cells (RGCs) by AAV-Olig1/2.

city: Shanghai

first: Xiuqing

last: Fu

state: Shanghai

address: 393 Middle Huaxia Road, Pudong

department: School of Life Science and Technology (SLST)

institute: ShanghaiTech University

summary: To gain mechanistic insights into the effects of Olig family, we performed gene expression profiling analysis in RGCs. Transcriptome changes in Olig1-3 over-expression RGCs showed up-regulating genes related to axon projection development and regeneration-associated genes (RAGs), such as Stmn2, Gal, Gap43, and Sprr1a.

overall_design: Adult male mice were anesthetized with avertin (300-500mg/kg) and positioned in a stereotaxic apparatus. Under microscopic illumination, injections were made through the sclera and retina with a 5μL Hamilton syringe with a 32-gauge needle, avoiding injuring lens for excluding the false-positive possibility of regeneration. In this work, all pacaged AAV2 virus (10^13 gc/mL, 1.5μL) were injected into the vitreous chamber to infect RGCs. 14 days after ONC, we separated RGCs, which were transfected with AAV-Olig2-3 or AAV-GFP, by magnetic beads coupled with CD90.2 antibody and picked 15~20 cells by Unipick in each sample.

first: Wenrong

last: Zhan

first: Weiya

last: Tian

first: Pengming

last: Zeng

first: Zhenge

last: Luo


title: Comparison of transcriptomic profiles between HFPO-DA and positive controls for PPARa, PPARg or cytotoxicity in mouse, rat, and pooled human hepatocytes

city: Asheville

first: Melissa

middle: Marie

last: Heintz

state: NC

address: 31 College Place, Suite B118

institute: ToxStrategies

summary: Like many per- or polyfluorinated alkyl substances (PFAS), toxicity studies with HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate; CASRN 62037-80-3), a short-chain PFAS used in the manufacture of some types of fluorinated polymers, indicate that the liver is the primary target of toxicity in rodents following oral exposure. Although the current weight of evidence supports the PPARa mode of action (MOA) for liver effects in HFPO-DA-exposed mice, alternate MOAs have also been hypothesized including PPARg or cytotoxicity. To further evaluate the MOA for HFPO-DA in rodent liver, transcriptomic analyses were conducted on samples from primary mouse, rat and pooled human hepatocytes treated for 12, 24 or 72 hours with various concentrations of HFPO-DA, or established agonists of PPARa (GW7647), PPARg (rosiglitazone), or cytotoxic agents (i.e., acetaminophen or d-galactosamine). Concordance analyses of enriched pathways across chemicals within each species demonstrated greatest concordance between HFPO-DA and PPARa agonist GW7647-treated hepatocytes compared to the other chemicals evaluated. These findings were supported by benchmark concentration modeling and predicted upstream regulator results. In addition, transcriptomic analyses across species demonstrated a greater transcriptomic response in rodent hepatocytes treated with HFPO-DA or agonists of PPARa or PPARg, indicating rodent hepatocytes are more sensitive to HFPO-DA or PPARa/g agonist treatment. These results are consistent with previously published transcriptomic analyses and further support that liver effects in HFPO-DA-exposed rodents are mediated through rodent-specific PPARa signaling mechanisms as part of the MOA for PPARa activator-induced rodent hepatocarcinogenesis.

sample_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Comparative gene expression profiling analysis of RNA-seq data from lysed mouse, rat and human primary hepatocytes treated for 12, 24 or 72 h with supplemented MCM media containing solvent control in the presence or absence of HFPO-DA (0.1, 5, 50 or 500 μM) or one of the following positive controls: GW7647 (0.01, 0.1, 1 or 10 μM), rosiglitazone (0.01, 0.1, 1 or 10 μM), acetaminophen (0.3, 1, 3 or 10 mM) or d-galactosamine (0.3, 1, 3 or 10 mM).

first: William

last: Klaren

first: Alexander

middle: W

last: East

first: Chad

last: Thompson

first: Laurie

last: Haws


title: The soluble factor milieu in IPF and in COPD exacerbations dysregulates epithelial differentiation

city: Groningen

first: Reinoud

last: Gosens

address: Antonius Deusinglaan 1

department: Molecular Pharmacology

country: Netherlands

institute: University of Groningen

summary: In idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), epithelial abnormalities are present including bronchiolization and alveolar cell death and dysfunction. As epithelial progenitor cells are directed by their microenvironment, we hypothesized that changes in the microenvironment disrupt normal epithelial growth and differentiation. We therefore mimicked the soluble factor microenvironment in IPF using an IPF cocktail (IPFc), composed of 9 factors which are increased in IPF lungs (CCL2, IL-1β, IL-4, IL-8, IL-13, IL-33, TGF-β, TNFα and TSLP) and in COPD exacerbations using an exacerbation cocktail (EC) composed of 4 factors that are increased during an exacerbation of COPD (TNFα, IL-1β, IL-6, IL-8). We asked whether the soluble factor milieu in IPF and COPD exacerbations affects epithelial growth and differentiation. Mouse lung organoids (primary EpCAM+ cells co-cultured with CCL206 fibroblasts) were used to study epithelial growth and differentiation. Organoids exposed to IPFc, EC or TGF-β (as a comparator) were resorted into EpCAM+ and CCL206 fractions, and subjected to RNA-sequencing.

overall_design: Mouse lung organoids (primary EpCAM+ cells co-cultured with CCL206 fibroblasts) were used to study epithelial growth and differentiation. Organoids exposed to IPFc, EC and TGF-β for 72 hours were resorted into EpCAM+ and CCL206 fractions, and subjected to RNA-sequencing.

first: Rosa

middle: K

last: Kortekaas

first: Kerstin

middle: E

last: Geillinger-Kästle

first: Janette

last: Burgess


title: Genome-Wide CRISPR Screen Identifies an NF2-adheren junction mechanistic dependency for cardiac lineage

city: Singapore

first: Roger

last: Foo

address: 14 Medical Drive, MD6

country: Singapore

institute: National University of Singapore

summary: Cardiac differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear.  Here, we performed a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells (hESCs) and identify NF2, a Moesin-Ezrin-Radixin Like (MERLIN) Tumor Suppressor, as an upstream driver of cardiomyocyte specification. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of non-mesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anti-cardiac regulators CDX2, MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks.  Since YAP is a known repressor of mesendoderm genes, we found that NF2 and AMOT cooperatively bind to YAP during mesendoderm formation, thereby preventing YAP activation independent on canonical MST-LATS1 kinase activity. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 Serine-518 phospho-mutants, but not NF2 FERM-domain blue-box mutants, showing that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 for cardiomyocyte lineage specification by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

overall_design: CRISPR-mediated NF2 KO hESC cell lines were derived from H1 MYH6-mCerulean3 reporter cell line.  Briefly, two independent monoclonal NF2 Knockout lines were generated using Plasmid pMIA3 (Addgene #109399), and sgRNAs were designed to target exons 1 and 4 of the NF2 gene locus respectively to create single indels. Independent NF2 WT and KO cell lines were subjected to cardiomyocyte differentiation using the 7-day GiWi protocol, and total RNA were isolated at Day 0, 1, 5 and 14 using Trizol Reagent and Direct-Zol RNA Miniprep kit according to manufacturer's protocol. RNA quality and yield were assessed using Agilent RNA 6000 Pico kit for quality control. Total RNA library preparations were prepared using TruSeq Stranded Total RNA Library Prep HMR Kit (20020596, Illumina) to deplete ribosomal RNA, followed by appending illumina TruSeq index adaptors to respective cDNA libraries according to manufacturer's instructions. cDNA libraries were quantified using Agilent DNA1000 kit (5067-1505, Agilent) prior to sequencing on HiSeq 4000 illumina Sequencing platform by Macrogen Asia

first: Chang Jie Mick

last: Lee


title: ASXL1/2 links MLL4 and BAP1 on enhancers

first: Kai

last: Ge

address: 50 South Dr Rm 4154

department: NIDDK

institute: NIH

summary: Histone H3K4 monomethyltransferases MLL3 and MLL4 contain a set of uncharacterized PHD fingers. By structural and biochemical assays, we found a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL3 and MLL4, revealing their direct binding to the conserved MBH (MLL binding helix) region of ASXL1/2, components of the Polycomb repressive PR-DUB complex. In mouse embryonic stem cells, we observed that BAP1, the catalytic subunit of the PR-DUB complex, physically interacts with MLL4 in an ASXL1/2 MBH-dependent manner. Genomic studies demonstrate that the ASXL1/2 MBH is required for BAP1 binding on active enhancers and suggest that MLL4 facilitates BAP1 binding on active enhancers through ASXL1/2 MBH.

overall_design: ChIP-Seq profiling of T7 tag and MLL4 as well as active enhancer marks H3K4me1 and H3K27ac in ESCs overexpressing BAP1-T7

first: Xie

last: Guojia

first: Lee

last: Ji-Eun

first: Ge

last: Kai


title: Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response [ribosome profiling]

first: Nicholas

middle: R

last: Guydosh

address: 8 Center Dr, Bldg 8, 220

summary: During viral infection, several dsRNA sensors are activated in the cell and trigger changes in gene expression. One of these sensors activates a generic endonuclease, RNase L, that cleaves many types of RNA in the cell. However, how the resultant widespread RNA decay affects gene expression is not fully understood. Here we found that gene expression changes caused by activating dsRNA sensing pathways are tuned by RNase L activation, pointing to an intricate antiviral response where multiple inputs are integrated to create an optimized output. We show that RNA fragmentation induces the activation of the Ribotoxic Stress Response, potentially through ribosome collisions. The p38 and JNK pathways that are actuated as part of this response promote outcomes that likely inhibit the virus, such as programmed cell death. We also show that RNase L appears to limit the translation of genes that are regulated by another dsRNA-induced pathway, the Integrated Stress Response. Intriguingly, we found the activity of another generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage events can directly evoke an antiviral program.

overall_design: To test if the activity of any generic endonuclease leads to increased translation initiation at alternative ORFs within the 3' UTR of genes, we electroporated RNase A into RNASEL KO A549 cells and performed ribosome profiling on those same samples after 4.5 hours. In addition, to assess changes in translation due to activation of the dsRNA sensor pathway involving RNase L,  RNASEL WT and KO A549 cells were transfected with either 2-5A or poly(I:C) followed by ribosomal profiling after 4.5 hours.

first: Agnes

last: Karasik

first: Hernan

last: Lorenzi


title: Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response

overall_design:
To measure changes in the transcriptome under activation of RNase L, but not other components of the innate immune response, we performed RNA-seq on wild type (WT) and RNASEL KO cells that were transfected with 2-5A. Then, we analyzed differential expression (DE) patterns in 2-5A treated vs untreated cells by DEseq2.
To investigate whether activation of RNase L modulates the effects of other pathways that are typically activated during infection by other dsRNA sensors, we simultaneously activated multiple pathways and tested RNase L’s role by transfecting WT and RNASEL KO A549 cells with poly I:C, a dsRNA mimic, followed by RNA-seq analysis. Then, we analyzed differential expression analysis of poly I:C treated vs untreated cells with DEseq2.
To  investigate how global RNA cleavage changes genes expression, we electroporated a generic RNase, RNase A, in RNASEL KO A549 cells and performed RNA-seq after 4.5 hours. Differential gene expression analysis between KO cells transfected wth either RNAse A or BSA (conotrols) was performed with DESeq2.


title: sequential DNA affinity purification sequencing (seq-DAP-seq) data of MADS SEP3-AG tetramerization mutants.

city: Grenoble

first: Romain

last: BLANC-MATHIEU

address: 17 Avenue des martyrs

institute: CEA

summary: The MADS genes encode transcription factors (TF) that act as master regulators of plant reproduction and flower development. The SEPALLATA (SEP) MADS subfamily is not only absolutely required for the development of floral organs, but also plays roles in inflorescence architecture and determinacy of the floral meristem. The SEPs act as organizers of MADS complexes and are able to form both heterodimers and heterotetramers in vitro. To date, the MADS TF complexes characterized in angiosperm floral organ development contain at least one SEP TF. Whether DNA-binding by SEP-containing dimeric MADS complexes are sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization impaired SEP mutants. Here we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3-AGAMOUS tetramerisation domain, structure-based mutagenesis and complementation experiments in sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers are able to bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild type binding sites genome-wide, tetramerisation is not only required for floral meristem determinacy, but also absolutely required for floral organ identity in the second, third and fourth whorls.

overall_design: In this study we applied sequential DNA affinity purification sequencing (seq-DAP-seq) to two new tetramerization mutants of SEP3AG heterotetramer: SEP3Δtet3M-AG and SEP3Δtet-AGΔtet. We compared the DNA binding intensity of these two new mutant complexes and another previsouly analyzed mutant (SEP3ΔtAG) at regions bound by the SEP3AG wildtype complex (also previsoulsy analyzed in PMID 32890394) .

first: Véronique

last: Hugouvieux

first: Xuelei

last: Lai

last: Blanc-Mathieu

first: Jérémy

last: Lucas

first: François

last: Parcy

first: Chloe

last: Zubieta


title: Olfactory neuroblastoma mimics molecular subtypes and lineage trajectories of small cell lung cancer [Single cell RNA seq on RPM-GFP and RPMA ONB tumors]

status: Public on May 15 2024

city: Durham

first: Trudy

last: Oliver

email: tgo@duke.edu, trudy.oliver@duke.edu

address: Duke University, Box 3813, LSRC Room C138B, 308 Research Drive

department: Pharmacology & Cancer Biology

institute: Duke University

summary: The olfactory epithelium relies on active neuron regeneration from basal stem cells and is susceptible to olfactory neuroblastoma (ONB), a rare, aggressive tumor of unclear origins. Here, we establish a new, highly-penetrant, genetically-engineered mouse model of ONB with alterations in Rb1/Trp53/Myc that exhibit a NEUROD1+ immature neuronal state. ASCL1 loss leads to emergence of non-neuronal histopathologies, including a POU2F3+ microvillar-like state. We find ONB tumor heterogeneity to recapitulate developmental states of multipotent globose basal cells (GBCs), which our data demonstrate is a cell of origin for ONB. Similar to small cell lung cancer (SCLC), mouse and human ONB exhibit: mutually exclusive ASCL1, NEUROD1, and POU2F3- like states, an immune-cold tumor microenvironment, intratumoral subtype heterogeneity comprising neuronal and non-neuronal lineages, and subtype plasticity—as evidenced by barcode-based lineage tracing and single-cell transcriptomics. Collectively, our findings highlight conserved developmental trajectories between ONB and SCLC subtypes with significant implications for ONB classification and treatment.

overall_design: We harvested olfactory neuroblastoma tumor cells from n=1 homozygous Rb1 fl/fl; Trp53 fl/fl; H11b-LSL-MycT58A-Ires-Luc (RPM)-Rosa26-LSL-Cas9-Ires-Gfp  (RPM-GFP) mouse in which tumor cells are GFP+, and n=3  homozygous Rb1 fl/fl; Trp53 fl/fl; H11b-LSL-MycT58A-Ires-Luc; Ascl1 fl/fl (RPMA) to perform single cell RNA-sequencing (scRNA-seq). Tumors were micro-dissected from the olfactory cavity of each mouse, processed into single cell suspensions, and subject to  10X Genomics single-cell gene expression library construction (Dual-Index 3' GEX) and downstream Illumina-based sequencing.

first: Abbie

last: Ireland


title: DOT1L controls NK cell maturation and lineage integrity

city: Esch-sur-Alzette

first: Sebastian

last: Scheer

address: 29, rue Henri Koch

department: Infection and Immunity

country: Luxembourg

institute: Luxembourg Institute of Health

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

overall_design: Refer to individual Series


title: DOT1L controls NK cell maturation and lineage integrity [CUT&TAG]

summary: Epigenetic modifiers are promising targets to improve therapies in patients with cancer. Targeting the methyltransferase DOT1L with small molecule inhibitors has shown promising effects on the control of cancerous cells. However, these small molecules are given systemically and may have a profound impact on non-cancerous cells, such as the cells of the immune system. In the innate immune system, natural killer (NK) cells are a critical subset of cells with important roles in controlling transformed cells and tumour inflammation. Previous studies have shown that NK cells can convert into ILC1-like cells in a TGFβ-rich tumour microenvironment (TME). Additionally, cancer patients with acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL) have increased frequencies of ILC1 cells in peripheral blood mononuclear cells (PBMCs), which show reduced production of proinflammatory cytokines and decreased granzyme B production. In this study, we identify DOT1L as a critical regulator of NK cell activation and lineage integrity. We generated NKp46-conditional DOT1L knockout mice (DOT1L.Ncr1) and observed increased frequencies of ILC1-like cells (CD49b+ CD49a+, CD62L-) and reduced frequencies of NK cells (CD49b+ CD49a-, CD62L+) in these mice. While the absence of DOT1L increases the sensitivity to TGFβ, the increased expression of CD49a is highly intrinsic and only partially dependent on TGFβ signalling. Functionally, the increased presence of ILC1-like cells in the tumour microenvironment of DOT1L.Ncr1 mice leads to decreased tumour control. Our assessment of the transcriptional program reveals alternative uses of transcription factors, such as the AP-1 family or SMAD2/3, to maintain NK cell activation and lineage integrity. Our findings provide evidence for a previously unknown role of DOT1L in NK cell biology, and demonstrate the importance of maintaining NK cell lineage integrity for effective tumour control. These findings have significant implications for the development of improved therapies for cancer or other NK cell-lineage dependent malignancies.

overall_design: Splenic NK cells of cultured for 5 days in the presence of 50 ng/ml IL-15 prior to performing Cut&Tag.


title: Transcriptional effects of TDP1 deficiency in non-replicating U2OS cells

city: Bologna

first: Giovanni

last: Capranico

state: BO

address: Via Selmi 3

department: FABIT

country: Italy

institute: University of Bologna

summary: Spinocerebellar ataxia with axonal neuropathy (SCAN1) is a rare recessive neurodegenerative syndrome associated with cerebellar atrophy and peripheral neuropathy. It is caused by a homozygous missense mutation in the tyrosyl-DNA phosphodiesterase-1 (TDP1) gene (A1478G). resulting in a substitution of histidine for arginine-493 (H493R) in the TDP1 catalytic site, leading to reduced TDP1 activity. How TDP1 H493R mutation promotes the SCAN1 phenotype, which is associated with the death of post-mitotic neurons, is unclear. We have generated models of osteosarcoma U2OS cells homozygous for TDP1 H493R employing the CRISPR-Cas9 technique (2 clones, named “1P” and “3.3”). Here, we have generated transcriptional genome wide profile in order to characterize differences in gene expression that are specific of TDP1-mutated clones.

overall_design: RNA-seq profiles of wild type (WT) and  TDP1 H493R mutated (2 clones, 1P and 3.3) U-2 OS cells

first: Marco

last: Russo

first: Simona

last: Salimbeni


title: DOT1L controls NK cell maturation and lineage integrity [RNA-seq D0 & D2]

overall_design: Splenic NK cells of either WT or DOT1L.Ncr1 mice at steady-state (d0) or 2 days after infection with MCMV (d2) were sorted for NK1.1+ CD3/TCRβ- Ly49H+ cells prior to isolation of RNA


title: The tissue-resident regulatory T cell pool is shaped by transient multi-tissue migration and a conserved residency program. Bulk RNA-Seq profiling of mouse tissue Tregs.

status: Public on Apr 29 2024

city: Cambridge

first: Adrian

last: Liston

address: Tennis Court Road

department: Department of Pathology

country: United Kingdom

institute: University of Cambridge

summary: The tissues are the site of many important immunological reactions, yet how the immune system is controlled at these sites remains opaque. Recent studies have identified Foxp3+ regulatory T cells (Tregs) in non-lymphoid tissues, with unique characteristics compared to lymphoid Tregs. However, tissue Tregs have not been considered holistically across tissues. Here we performed a systematic analysis of the Treg population residing in non-lymphoid organs throughout the body, revealing shared phenotypes, transient residency and common molecular dependencies. Tissue Tregs from different non-lymphoid organs shared T cell receptor (TCR) sequences, with functional capacity to drive multi-tissue Treg entry, and were tissue-agnostic on tissue homing. Together these results demonstrate that the tissue-resident Treg pool in most non-lymphoid organs, other than the gut, is largely constituted by broadly self-reactive Tregs, characterised by transient multi-tissue migration. This work suggests common regulatory mechanisms may allow pan-tissue Tregs to safeguard homeostasis across the body.

overall_design: For bulk RNA sequencing, 2000 CD4+ Foxp3Thy1.1+ Tregs and Foxp3Thy1.1- Tconv were sorted on a BD FACSAria from each source from perfused mice. RNA was isolated using RNeasy Mini kit (Qiagen).RNA concentration and purity were determined using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity with a Bioanalyser 2100 (Agilent). 3′mRNA-seq library preparation and transcriptome analysis was performed by Lexogen (Austria) using the QuantSeq 3′mRNA-Seq Library Prep Kit for Illumina and QuantSeq data analysis workflow.

first: Liston

last: Adrian

first: Oliver

last: Burton

first: Vaclav

last: Gergelits

first: Samar

last: Tareen


title: Sequencing of the influenza PR8 virus hemagglutinin Sb site mutant library

first: Nicholas

last: Heaton

address: 213 Research Drive, 426 CARL Building

department: Molecular Genetics and Microbiology

institute: Duke University School of Medicine

summary: Current flu vaccine induces strong response to plastic and higly variable hemagglutinin (HA) head. However, response to conserved epitopes in HA elicited by current vaccine or infection is very limited. We hypothesized that change the relative "molarity" of immunodominant epitopes and conserved domain  might increase response to conserved domains. Therfore, we made a library with the most ommunodominant domian Sb site randomized and tested its efficacy in vivo. Here we show the diversity of the library.

sample_organism: Influenza A virus (A/Puerto Rico/8/1934(H1N1))

overall_design: Four codons in Sb site of PR8 hemagglutinin were randomized by mutagenesis. Viable mutants were sorted with hemaglutinin stalk trimer monoclonal 6F12 by megnetic-activated cells  sort. Library before purification and after purification were sequenced.

first: Zhaochen

last: Luo

first: Madison

last: Berry

first: Kevin

last: Wiehe


title: Fetal brain response to maternal inflammation requires microglia

status: Public on May 07 2024

first: Paola

last: Arlotta

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology

institute: Harvard University


title: Expression data from mice cortical microglia and astrocytes.

city: Seville

first: Juan Jose

last: Perez-Moreno

address: Instituto de Biomedicina de Sevilla (IBiS), Campus Hospital Universitario Virgen del Rocío, Avda. Manuel Siurot

department: Cell Biology

institute: University of Seville

summary:
Functional recruitment of microglia depends on OXPHOS in vivo. OXPHOS upregualtion correlates with phagocytic activity of microglia in physiologycal conditions and with proinflammatory microglia in pathological conditions.
We used microarrays to detail the global programme of gene expression underlying loss of mitochondrial Complex I in microglia cells and its impact in astrocytes gene expression.

overall_design: Mice brains were dissected to use the whole cortex for isolation of micrgolia and astrocytes by fluorescence-activated cell sorting (FACS) (microglia: CD11b + CD45 postive cells; astrocytes: ACSA2 positive cells).

first: Juan

last: Pérez-Moreno

first: Bella

last: Mora-Romero

first: María

middle: I

last: Álvarez-Vergara

first: Alberto

last: Pascual


title: LC3-associated phagocytosis of neutrophils trigger tumor ferroptotic cell death in glioblastoma

city: Hershey

first: Wei

last: Li

state: PA

address: 500 University Drive

institute: Penn State College of Medicine

summary: Necrosis is commonly found in various solid tumors and predicts worse outcome. Chronic ischemia can initiate tumor necrosis, but how the damaged tissue further expands is unclear. Previous studies found that neutrophils associate with necrosis and could contribute to necrosis development in glioblastoma (GBM) by transferring myeloperoxidase (MPO)-containing granules into tumor cells and inducing tumor cell ferroptosis. How the neutrophilic granule transfer occurs is unknown. Here, through an unbiased small molecule screen, we found that statins can inhibit neutrophil-induced tumor cell death by blocking the neutrophilic content transfer into tumor cells. Surprisingly, we found that neutrophils are engulfed by tumor cells before they are fragmented and release the MPO-containing contents in tumor cells. This process involves LC3-associated phagocytosis (LAP) and can be blocked by inhibiting the Vps34-UVRAG-containing PI3K complex. Inhibition of MPO or depletion of Vps34 in an orthotopic xenograft GBM mouse model reduced necrosis formation and allowed tumor-bearing mice to survive longer. Therefore, this study revealed that the neutrophilic granule transfer involves LAP-mediated neutrophil internalization, which then triggers tumor ferroptotic cell death in glioblastoma. Blocking this process may improve prognosis of GBM.

overall_design: To examine gene expression changes associating with differentiation of HL-60 cells, we performed RNA-seq analysis of gene expression in parental HL-60 cells and HL-60 cells which were induced for differentiation for 7 days under the differentiation protocol as indicated in the manuscript. The gene expression changes were identified by comparing the gene expression in these two types of cells.

first: Tong

last: Lu

first: Patricia

last: P Yee

first: Stephen

middle: Y

last: Chih

first: Dawit

middle: G

last: Aregawi

first: Michael

last: Glantz

first: Brad

last: Zacharia

first: Hong-Gang

last: Wang


title: Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation

city: Pittsburgh

first: Benjamin

middle: Alexander

last: Nacev

address: 5117 Centre Avenue, Suite 2.6b

department: Medicine

institute: University of Pittsburgh


title: Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation [scRNA-Seq]

summary: Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants was exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupted broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss led to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrated impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.

overall_design: Histone mutations of interest were introduced into mesenchymal progenitor cells (C3H 10T1/2) and the effects gene expresion in differentiation assay were determined.

middle: A

first: Nicholas

middle: D

last: Socci


title: Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation [CUT&RUN]

overall_design: Histone mutations of interest were introduced into mesenchymal progenitor cells (C3H 10T1/2) and the effects on chromatin were determined.


title: Spatial transcriptomics analysis of neoadjuvant cabozantinib and nivolumab in advanced hepatocellular carcinoma identifies independent mechanisms of resistance and recurrence

city: Baltimore

first: Luciane

middle: Tsukamoto

last: Kagohara

address: 1650 Orleans St - Rm 485

department: Oncology

institute: Johns Hopkins University

summary: Novel immunotherapy combination therapies have improved outcomes for patients with hepatocellular carcinoma (HCC), but responses are limited to a subset of patients. Little is known about the inter- and intra-tumor heterogeneity in cellular signaling networks within the HCC tumor microenvironment (TME) that underlie responses to modern systemic therapy. We applied spatial transcriptomics (ST) profiling to characterize the tumor microenvironment in HCC resection specimens from a prospective clinical trial of neoadjuvant cabozantinib, a multi-tyrosine kinase inhibitor that primarily blocks VEGF, and nivolumab, a PD-1 inhibitor in which 5 out of 15 patients were found to have a pathologic response at the time of resection. ST profiling demonstrated that the TME of responding tumors was enriched for immune cells and cancer associated fibroblasts (CAF) with pro-inflammatory signaling relative to the non-responders. The enriched cancer-immune interactions in responding tumors are characterized by activation of the PAX5 module, a known regulator of B cell maturation, which colocalized with spots with increased B cell markers expression suggesting strong activity of these cells. HCC-CAF interactions were also enriched in the responding tumors and were associated with extracellular matrix (ECM) remodeling as there was high activation of FOS and JUN in CAFs adjacent to tumor. The ECM remodeling is consistent with proliferative fibrosis in association with immune-mediated tumor regression. Among the patients with major pathologic response, a single patient experienced early HCC recurrence. ST analysis of this clinical outlier demonstrated marked tumor heterogeneity, with a distinctive immune-poor tumor region that resembles the non-responding TME across patients and was characterized by HCC-CAF interactions and expression of cancer stem cell markers, potentially mediating early tumor immune escape and recurrence in this patient. These data show that responses to modern systemic therapy in HCC are associated with distinctive molecular and cellular landscapes and provide new targets to enhance and prolong responses to systemic therapy in HCC.

overall_design:
Spatial transcriptomics with Visium (10x Genomics) was performed on HCC samples with neoadjuvant therapy. Spatial gene expression analyses was performed between patients that responded versus patients that did not respond to the neoadjuvant therapy.

Submitter states that missing raw data are being made available for controlled access in dbGaP.

Please note that processed data files have been updated on July 19, 2024.


title: Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation [RNA-Seq]

overall_design: Histone mutations of interest were introduced into mesenchymal progenitor cells (C3H 10T1/2) and the effects on genes expression were determined.


title: Senescent stroma mediates immunosuppression and drives breast cancer progression

city: Saint Louis

first: Sheila

last: Stewart

address: 660 South Euclid Avenue

institute: Washington University School of Medicine

first: Jiayu

last: Ye

first: Qihao

last: Ren


title: DOT1L controls NK cell maturation and lineage integrity [RNA-seq D5]

first: Sebastian

last: Scheer

summary: In the innate immune system, natural killer (NK) cells represent a highly important subset of cells with utmost importance in the control of transformed cell and tumour inflammation. Previous studies showed that NK cells can convert into ILC1-like cells in a TGF-β-rich tumour microenvironment (TME). In addition, cancer patients with acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL) have increased frequencies of ILC1 cells in peripheral blood mononuclear cells (PBMCs), which show reduced production of the proinflammatory cytokines IFN-γ and TNF in addition to decreased granzyme B production. Here, we identify the histone methyltransferase DOT1L as a critical regulator of NK cell lineage integrity in vitro and in vivo. NK cells from NKp46-conditional DOT1L knockout mice (DOT1L.Ncr1) show increased frequencies of ILC1-like cells (CD49b+ CD49a+), and also express more of the ILC1 markers CD200R, TRAIL, while showing reduced expression of Ly49H. We further identify that the increased ILC1-like phenotype is largely independent of TGFβ. Assessment of transcription factor (TF) availability in the absence of DOT1L shows that the Myocyte-specific enhancer factor (MEF)2C is the highest downregulated TF in the absence of DOT1L. CRISPRCas9-mediated deletion of MEF2C ultimately provides evidence that this TF is limiting NK cell plasticity in vivo.   Our findings provide evidence for a previously unknown role of DOT1L and MEF2C in NK cell biology for maintaining NK cell lineage integrity and may ultimately result in improved therapies for patients with leukemia or other NK cell-lineage dependent malignancies.

overall_design: RNA-seq of enriched splenic NK cells after culturing for 5 days in the presence of 50 ng/ml IL-15; Three replicates each for WT (Ncr1-Cre- DOT1L fl/fl) and DOT1L knockout (DOT1L.Ncr1; Ncr1-Cre+ DOT1L fl/fl).


title: Olfactory neuroblastoma mimics molecular subtypes and lineage trajectories of small cell lung cancer [Single nucleus RNA-seq on human ONB tumor]

first: Trudy

last: Oliver

overall_design: We harvested one olfactory neuroblastoma tumor specimen from the operating room and flash froze the sample. The flash frozen pellet was dissociated at a later time, and nuclei were isolated using the standard 10X Genomics Nuclear Isolation Kit. Dissociated nuclei were then loaded onto a 10X Chromium controller and subject to 10X Genomics single-cell gene expression library construction (Dual-Index 3' GEX) and downstream Illumina-based sequencing.

first: John

last: Finlay

first: Bradley

last: Goldstein


title: Senescent stroma mediates immunosuppression and drives breast cancer progression [MMF]

summary: To investigate the gene expression profile of normal versus senescent mouse mammary fibroblasts (MMF), we induced senescence in vitro and harvested cells for RNA-sequencing.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for normal versus doxorubicin- or hydrogen peroxide-induced senescencent fibroblasts.


title: H3K27ac ChIP-seq for lung ILC2 and CD4 T cells in HDM induced asthma

status: Public on May 14 2024

city: Chiba-shi

first: Arifumi

last: Iwata

state: Chiba

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

department: Department of Allergy and Clinical Immunology

country: Japan

institute: Chiba University

summary: Lung ILC2 and Th2 cells exhibited similar chromatin activation. However, small fraction of super-enhancers showed ILC2-specific, or T cell specific activation.

overall_design: H3K27ac ChIP-seq were performed in ILC2 and CD4 T cells obtained from HDM-induced murine asthma models and control mice.

first: Hiroki

last: Furuya

first: Yosuke

last: Toda


title: Senescent stroma mediates immunosuppression and drives breast cancer progression [scRNA-seq]

summary: Tumor-bearing mammary fat pads from MMTV-PyMT mice were digested with 2mg/mL Collagenase A and 2U/mL Dnase.

overall_design:
FVB background MMTV-PyMT+/INK-ATTAC- (WT) and MMTV-PyMT+/INK-ATTAC+ (INK+)  mice were weaned and recruited to studies by 28-30 days of age. Administration of AP20187 (10 mg/kg, 3 doses/week) was delivered through intra peritoneal inections . By 7 weeks of age, tumor-bearing mammary fat pads were digested to single cell suspension, staied, and sorted into CD45+ and CD45- populations. Samples from 2 mice were pooled for each sample.
FVB background MMTV-PyMT+ mice were weaned and recruited to studies by 28-30 days of age. Administration of ABT737 (25 mg/kg/week) or vehicle (Veh) was delivered through intra peritoneal inections . By 7 weeks of age, tumor-bearing mammary fat pads were digested to single cell suspension, staied, and sorted into CD45+ and CD45- populations. Samples from 2 mice were pooled for each sample.


title: Senescent stroma mediates immunosuppression and drives breast cancer progression [CAF]

summary: To investigate the gene expression profile of different cancer associated fibroblasts (CAF) subpopulations, we harvested tumors and sorted 4 CAF populations from 3 FVB background 10-week-old MMTV-PyMT mice.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 4 different CAF populations in spontanous MMTV-PyMT tumors.


title: Single-cell RNA-seq analysis of mouse embryonic stem cells before and after Cohesin depletion

city: Shenzhen

first: PENG

last: DONG

state: Guangdong

address: 1068 Xueyuan Avenue, Shenzhen University Town

institute: Shenzhen Institute of Advanced Technology

summary: The contrast between the disruption of genome topology upon cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic co-expression tendency of cis-linked genes in single cells. Notably, through single-cell RNA-seq profiling of genes in mouse embryonic stem cells, we found that cohesin loss induces widespread gene co-activation in cis. Our results support that cohesin arranges nuclear topology to control gene co-expression in single cells.

overall_design: JM8.N4 mouse embryonic stem cells (mESCs) from the C57BL/6N strain were edited by using CRISPR/Cas9 to fuse cohesin subunit RAD21 to the auxin-induced degron (AID) system. The derived cells were used in Smart-SCRB (Smart-Single Cell RNA Barcoding, a highly sensitive single-cell transcriptome assay) analysis and single-cell ATAC-seq analysis under control and acute cohesin loss (6hrs after the auxin treatment) conditions.

first: Peng

last: Dong

first: Zhe

middle: J

last: Liu


title: Neutrophil-mediated hypoxia drives pathogenic CD8 T cell responses in cutaneous leishmaniasis

city: Philadelphia

first: Camila

last: Farias Amorim

state: United States

address: 380 S University Ave

department: Pathobiology

institute: University of Pennsylvania

summary: Cutaneous leishmaniasis caused by Leishmania parasites exhibits a wide range of clinical manifestations. Although parasites influence disease severity, cytolytic CD8 T cell responses mediate disease. While these responses originate in the lymph node, we found that expression of the cytolytic effector molecule granzyme B was restricted to lesional CD8 T cells in Leishmania-infected mice, suggesting that local cues within inflamed skin induced cytolytic function. Expression of Blimp-1 (Prdm1), a transcription factor necessary for cytolytic CD8 T cell differentiation, was driven by hypoxia within the inflamed skin. Hypoxia was further enhanced by the recruitment of neutrophils that consumed oxygen to produce reactive oxygen species and ultimately increased the hypoxic state and granzyme B expression in CD8 T cells. Importantly, lesions from cutaneous leishmaniasis patients exhibited hypoxia transcription signatures that correlated with the presence of neutrophils. Thus, targeting hypoxia-driven signals that support local differentiation of cytolytic CD8 T cells may improve the prognosis for patients with cutaneous leishmaniasis, as well as other inflammatory skin diseases where cytolytic CD8 T cells contribute to pathogenesis.

overall_design: CD8 T cells isolated from the ear and draning lymph node of C57BL/6 mice infected with L. major

first: Fernanda

last: Novais

first: Phillip

last: Scott


title: Proteome-scale tagging and functional screening in mammalian cells by ORFtag

status: Public on May 10 2024

city: Vienna

first: Alexander

last: Stark

address: Campus-Vienna-Biocenter 1

country: Austria

institute: The Research Institute of Molecular Pathology (IMP)

overall_design: Refer to individual Series

first: Filip

last: Nemčko

first: Moritz

last: Himmelsbach

first: Vincent

last: Loubiere

first: Ramesh

last: Yelagandula

first: Michaela

last: Pagani

first: Nina

last: Fasching

first: Julius

last: Brennecke

first: Ulrich

last: Elling

first: Stefan

middle: L

last: Ameres


title: Myocardin-related transcription factor (MRTF) mediates epithelial fibrogenesis in polycystic kidney disease

city: Toronto

first: Andras

last: Kapus

state: Ontario

address: 209 Victoria Street

department: Keenan Research Centre for Biomedical Science

institute: St. Michael's Hospital

summary: Background: Autosomal dominant polycystic kidney disease (PKD) is characterized by extensive cyst formation and progressive fibrosis. However, the molecular mechanisms whereby the loss/loss-of-function of polycystin 1 or 2 (PC1/2) provokes fibrosis are largely unknown. The small GTPase RhoA has been implicated in cystogenesis, while we have shown that the RhoA/cytoskeleton/myocardin-related transcription factor (MRTF) pathway is a key inducer of epithelium-induced fibrogenesis. Therefore, we hypothesized that MRTF 1) is activated by PC1/2 loss, and 2) plays a critical role in fibrogenic repogramming of the epithelium and the subsequent induction of fibrosis. Methods: PC1/2 loss was achieved by gene silencing in tubular cells (in vitro) and in PKD1 (RC/RC) and PKD2 (ws25/-) mice (in vivo). RhoA activation was measured by pull-down assays and immunofluorescence. MRTF localization (nuclear/cytosolic ratio, intensity) was quantified in large cell populations and in renal tissue using automated image analysis. PC1/PC2 loss-promoted, MRTF-dependent gene expression was followed by qPCR, RNAseq and RNAscope. MRTF-dependent paracrine effects were assessed by a bioassay. Results:  Loss of PC1 or PC2 activated RhoA in vitro and in vivo, resulting in cytoskeletal remodelling and robust nuclear MRTF translocation, accompanied by increased MRTF expression. Nuclear translocation occurred predominantly in dilated tubules, while overexpression in the cyst-lining epithelium. A large cohort of PC1/PC2 downregulation-induced genes was MRTF-dependent, including cytoskeletal, integrin-related, and matricellular/fibrogenic proteins. Epithelial MRTF was necessary for paracrine priming of fibroblast-myofibroblast transition.   Conclusion: The Rho/MRTF pathway is a critical novel mediator of PC1/2 loss-induced acquisition of the profibrotic epithelial phenotype and the ensuing fibrosis.

overall_design: The porcine proximal tubule cell line LLC-PK1 was subjected to treatment with siRNAs (siPC1, siPC2, and in combination with siMRTFA) along with a non-related control siRNA (siNR). This was carried out to assess the impact of MRTFA in the context of polysystic kidney disease, particularly when the Polycystin (PC1 or PC2) gene is downregulated.

first: Zsuzsanna

last: Lichner

first: Mei

last: Ding

first: Tarang

last: Khare

first: Qinghong

last: Dan

first: Raquel

last: Benitez

first: Mercédesz

last: Parszner

first: Xuewen

last: Song

first: Boris

last: Hinz

first: York

last: Pei

first: Katalin

last: Szászi

first: András


title: Increased susceptibility to ischemia causes exacerbated response to laser microinjuries in the cirrhotic liver

status: Public on Apr 28 2024

first: Ben

last: Leaker

state: MA

address: 77 Massachusetts Avenue

department: HST

institute: MIT

summary:
Background & Aims: Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested in other tissues.
As a first step towards determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response.
Methods: Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0hr up to 14d after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model.
Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion.
Results: Healthy rats showed little damage beyond the initial microinjury and healed completely by 7d without scarring.  In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6hr after ablation, resulting in enlarged and heterogeneous zones of cell death.
Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4hr. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule
microvasculature, which matched the zones of cell death.
Conclusions: The cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development.
Modifications would need to be made to account for this to continue developing a microinjury treatment for cirrhosis. Regardless, the remarkable results of microinjury treatment in skin warrant further investigation for fibrotic conditions throughout the body.

overall_design: Differential gene expression anaysis with a difference-in-differences model comparing laser-treated healthy and laser-treated cirrhotic liver samples. Paired control samples from an untreated section of the liver is used to control for differences between the untreated healthy and cirrhotic livers

first: Mozhdeh

last: Sojoodi

first: Kenneth

last: Tanabe

first: Yury

last: Popov

first: Joshua

last: Tam

first: R

last: Anderson


title: EHF is an essential driver of mammary alveolar lineage differentiation and putative suppressor of breast tumorigenesis

city: Clayton

first: Wei

last: Shi

state: VIC

address: 15 Innovation Walk

department: Department of Biochemistry and Molecular Biology

country: Australia

institute: Monash University

summary: The Ets homologous factor (Ehf) is highly expressed in the mammary gland in pregnancy and lactation. Mothers with Ehf deletion fail to lactate due to impaired lobuloalveolar development. scRNA-seq profiling of mammary epithelial cells (MECs) at late pregnancy (18.5dP) demonstrated that EhfKO mammary gland comprised fewer differentiated alveolar cells, and an accumulation of the preceding cell populations in the secretory differentiation hierarchy. EhfKO mammary alveolar cells also displayed significant downregulation of milk proteins and lipids, and increased expression of proliferation markers indicating incomplete differentiation

overall_design: Single cell RNA sequencing of mammary epithelial cells (Ter119–CD31−CD45–) from mice with and without deletion of Ehf at late pregnancy (18.5 days pregnant)

first: Yang

last: Liao

first: Rebecca

last: Nightingale

first: Camilla

last: Reehorst

first: François

last: Vaillant

first: Jane

last: Visvader

first: Bhupinder

last: Pal

first: John

last: Mariadason


title: Lrp10 suppresses IL7R limiting CD8 T cell homeostatic expansion and anti-tumor immunity

status: Public on Apr 26 2024

city: Dallas

first: Evan

last: Nair-Gill

state: Texas

address: 5323 Harry Hines Blvd

department: Center for the Genetics of Host Defense

institute: UT Southwestern Medical Center

summary: Through forward genetic screening, we found a gene called LDL receptor Related Protein 10 (Lrp10) that plays an important role in the homeostasis and differentiation of peripheral CD8 T cells through its effects on the interleukin 7 receptor (IL7R). Lrp10 knockout mice exhibit enhanced resistance to MC38 tumor growth, and we sought to better define the tumor infiltrating Lrp10+/+ and Lrp10-/- CD8T cells using single cell RNA seq and paired single cell TCR sequencing.

overall_design: Lrp10+/+ and Lrp10-/- CD8 T cells were sorted from D12 MC38 tumors and subjected to paired scRNAseq and single cell TCR sequencing.

first: Luming

last: Chen


title: Sequencing-Guided Design of Genetically Encoded Small RNAs Targeting CAG Repeats for Selective Inhibition of Mutant Huntingtin [I]

city: Winston-Salem

first: Keith

middle: T.

last: Gagnon

state: North Carolina

address: 575 Patterson Ave

department: Biochemistry and Molecular Biology

institute: Wake Forest University School of Medicine

summary: Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal but reduction of wild-type HTT (wtHTT) is undesirable as it compromises gene function and potential therapeutic efficacy. One promising allele- selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing to monitor processing precision of a limited set of CAG repeat- targeted small RNAs expressed from multiple scaffold contexts. Small RNA sequencing identified expression constructs with high guide strand 5' processing precision that also conferred promising allele-selective inhibition of mutHTT. However, mRNA-seq revealed varying degrees of transcriptome-wide off-target effects, including certain CAG repeat-containing mRNAs. These results support the continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process.

overall_design:
To identify processing of designed repeat shRNA/miRNAs in mammalian cell tissues, HEK 293T mammalian cells were transfected with repeat shRNA/miRNA encoding lentiviral plasmids.
Sequence analysis was performed using pre-existing RNA seq analysis methods as well as in house excel spreadsheets for reconstructing read constructs.
ImageJ software was used to evaluate transfection efficiency between transfected samples in order to normalize read abundance levels.
Expression profiling using sRNAtoolbox and further analysis was used to evaluate transfected small RNAs and endogenous miRNA expression levels.

first: Mansi

last: Parasrampuria

first: Adam

last: White

first: Ramadevi

last: Chilamkurthy

first: Adrian

last: Pater

first: Fatima

last: El-Azzouzi

first: Katy

middle: N

last: Ovington

first: Philip

last: Jensik

middle: T