title: ELAC2 is a prostate cancer rheostat that regulates nuclear and mitochondrial RNA metabolism (miRNA-Seq)

first: Stefan

last: Siira

summary: Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer deaths in men. GWAS studies have identified variants associated with prostate cancer susceptibility, however, mechanistic and functional validation of these mutations are lacking. Mitochondrial energy metabolism plays an important role in the onset and development of cancer. A missense variant was identified in the ELAC2 gene, which encodes a dually localized nuclear and mitochondrial RNA processing enzyme, with predicted impact on metabolism and tumorigenesis. We used CRISPR/Cas9 genome editing to introduce this variant into the mouse Elac2 gene as well as to generate a prostate-specific gene knockout of Elac2. The mutations caused enlargement and inflammation in the prostate and nodule formation. Multi-omic profiling revealed defects in RNA and energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired processing of mitochondrial and nuclear encoded non-coding RNAs and reduced protein synthesis. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer and led to metastasis. Our systems biology analyses reveal a miRNA-mediated molecular mechanism by which specific non-coding RNAs elicit metabolic changes that drive prostate tumorigenesis and metastasis. We conclude that this ELAC2 variant is a predisposing factor for prostate cancer and provide a detailed molecular mechanism and physiologically relevant models of this cancer.

overall_design: miRNA-Seq performed on ELAC2 knockout, wild type and A537T mutant mice on TRAMP -/- and TRAMP+/- backgrounds at 10 and 30 weeks age, in triplicate.

first: Maike

last: Stentenbach

first: Judith

last: Ermer

first: Kara

last: Perks

first: Sam

last: Raven

first: Richard

first: Danielle

last: Rudler

first: Timothy

last: McCubbin

first: Esteban

last: Marcellin

first: Oliver

last: Rackham

first: Aleksandra

last: Filipovska


title: ELAC2 is a prostate cancer rheostat that regulates nuclear and mitochondrial RNA metabolism (RNA-Seq II)

status: Public on Mar 21 2023

city: Perth

middle: J

state: Western Australia

address: 15 Hospital Ave, Nedlands

department: Precision Health

country: Australia

institute: The Kids Research Institute Australia

type: Expression profiling by high throughput sequencing

overall_design: RNA-Seq performed on ELAC2 knockout, wild type and A537T mutant mice on a TRAMP+/- background at 10 and 30 weeks age, in triplicate.

middle: A

middle: L

middle: G

last: Lee


title: Mitotic bookmarking by SWI/SNF subunits

status: Public on Mar 18 2023

city: Memphis

first: Zhexin

last: Zhu

address: 262 Danny Thomas Pl

country: USA

institute: St Jude Children's Research Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: For cells to initiate and sustain a differentiated state, it is necessary that a “memory” of this state is transmitted through mitosis to the daughter cells. Mammalian SWItch/ Sucrose Non- Fermentable (SWI/SNF) complexes, also called Brg1/ Brg- associated factors (BAF), control cell identity by modulating chromatin architecture to regulate gene expression, but whether they participate in cell fate memory is unclear. Here, we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but bound on promoters during mitosis and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets, and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 plays a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.

overall_design: GSE189563 was designed to study the mitotic bookmarking role of SWI/SNF subunit SMARCE1 in mouse ES cells. GSE189563 contains ChIP-seq, Cut&Run, bulk RNA-seq, nascent RNA-seq, and ATAC-seq data to uncover the new function of SWI/SNF in mitosis.


title: Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation [SELEX]

status: Public on Mar 22 2023

city: Beijing

first: Yuanchao

last: Xue

address: Datun Road #15

department: Institute of Biophysics

country: China

institute: Chinese Academy of Sciences

summary: RNA-binding proteins (RBPs) bind at different positions of pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These “positional rules” can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.

overall_design: We developed a capture RIC-seq (CRIC-seq) method for the global mapping of RBP-associated in situ proximal RNA-RNA fragments. Two biological replicates of CRIC-seq libraries were generated for PTBP1 IP (+pCp), hnRNPA1 IP (+pCp), SRSF1 IP (+pCp), IgG IP (+pCp), PTBP1 IP (-pCp), hnRNPA1 IP (-pCp), and SRSF1 IP (-pCp) in HeLa cells. In addition, we also constructed two biological replicates of RIC-seq libraries for PTBP1 and hnRNPA1 knockdown cells to test the specificity of PTBP1- or hnRNPA1-associated RNA loops identified by CRIC-seq. To identify PTBP1 Cys23-mediated RNA loops and their influence on splicing regulation, we performed RNA-seq and CRIC-seq in the PTBP1-C23S mutant replaced (C23S+Dox) or not replaced (C23S-Dox) HeLa cells.

first: Rong

last: Ye

first: Naijing

last: Hu


title: St3gal1 and bII-spectrin pathways control CAR-T cell migration to target tumor sites [1]

status: Public on Mar 17 2023

city: Rochester

first: Cameron

last: Baker

state: NY

address: 601 Elmwood Ave

institute: University of Rochester

summary: Adoptive transfer of genetically engineered chimeric antigen receptor (CAR)-T cell is emerging as a promising treatment option for hematological malignancies. However, T cell immunotherapies have largely failed in patients with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 b-galactoside a-2,3-sialyltransferase 1 (St3gal1) as a key negative regulator of the cancer-specific migration of CAR-T cells. Analysis of glycosylated proteins reveals that CD18 is a major effector of St3gal1 in activated CD8 T cells. St3gal1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR-T cells with enhanced expression of bII-spectrin, a key LFA-1 associated cytoskeleton molecule, reversed St3gal1-mediated nonspecific T cell migration and significantly reduced tumor growth in mice by improving tumor-specific homing of CAR-T cells. These findings identify a role of the St3gal1- bII-spectrin axis as the key cell-intrinsic program for cancer targeting CAR-T cell migration and as a promising strategy for effective T cell immunotherapy.

overall_design: WT mice bearing B16-OVA tumors received freshly prepared hHER2 CAR-T cells and CAR-T cells isolated from the each organ 24 hr after adoptive transfer (n=3).

first: Hong

last: Yeonsun


title: The chromatin and single-cell transcriptional landscapes of CD4 T cells in inflammatory bowel disease link risk loci with a proinflammatory Th17 cell population

status: Public on Mar 20 2023

city: Sao Paulo

first: Tiago

middle: da Silva

last: Medina

state: SP

address: 440 Tagua Street

department: International Research Center

country: Brazil

institute: A. C. Camargo Cancer Center

summary:
Objective The imbalance between Th17 cells and regulatory T cells in the gut of inflammatory bowel disease (IBD) patients promotes intestinal epithelial cell damage. In this scenario, T helper cell lineage commitment is accompanied by dynamic changes to the chromatin that facilitate, enforce or repress gene expression patterns and ultimately promote the induction and maintenance of dysregulated intestinal CD4 T cells.
Design Here, we characterized the chromatin landscape and the heterogeneity of CD4 T cells from blood samples and biopsies of IBD patients using in house generated ATAC-Seq and single cell RNA-Seq libraries as well as publicly available datasets.
Results We found that chromatin accessibility profiles specific to CD4 T cells from inflamed intestinal biopsies associate to inflammatory genes capable of promoting γδT, NK, T and B cell activation and proliferation. Our data show that the chromatin accessibility changes of CD4 T cells are associated with a higher predominance of pathogenic Th17 cells (pTh17 cells) in inflamed biopsies of IBD patients. In addition, we found that IBD risk loci in CD4 T cells are colocalized with accessible chromatin changes near pTh17-related genes. For instance, the IBD risk locus rs12942547 lies within an intronic STAT3 region enriched in areas of active intestinal inflammation from IBD patients. A similar profile was observed in a region near IL23R. Moreover, single cell RNA-Seq analysis of CD4 T cells from patient biopsies shows a population of CD4 T cells that resembles pTh17 cells with co-expression of Th1 and Th17 transcriptional programs, including a cytotoxic gene signature.
Conclusion Altogether, our data suggest that cytotoxic pTh17 cells are associated with intestinal inflammation of IBD patients and specifically associate some IBD genetic variants with this CD4 T cell subset.

overall_design:
scRNA-seq profiling of CD4 T cells isolated from matched non-inflamed and inflamed biopsy of three Crohn’s disease patients.

*** Submitter declares that the fastq files are being deposited separately at the EGA due to patient confidentiality concerns." ***

middle: S


title: E protein binding at the Tcra enhancer promotes Tcra repertoire diversity

status: Public on Mar 16 2023

city: Durham

first: Ariana

last: Mihai

state: North Carolina

address: 207 Research Dr

institute: Duke University School of Medicine

summary: The Tcra gene enhancer (Eα) is required for Tcra rearrangment. Our study assesses the role of E protein binding at Eα. Tcra repertoire sequencing was used to determine whether loss of Eα E protein binding sites alters recombination at Tcra.

overall_design: Pre-selection DP thymocytes (Live Lin- CD4+ CD8+ CD3-) were FACS-sorted from three pairs of Eα(+/+) and Eα(ΔE1ΔE3/ΔE1ΔE3) 3-4 week old mice, six samples total. Each sample was obtained from an individual mouse. Sorted cells were resuspended in TRIzol and RNA purified. RNA was processed to assess Tcra repertoire by high-throughput sequencing.


title: ELAC2 is a prostate cancer rheostat that regulates nuclear and mitochondrial RNA metabolism (RNA-Seq I)

first: Stefan

last: Siira

overall_design: RNA-Seq performed on ELAC2 knockout, wild type and A537T mutant mice at 10 and 30 weeks age, in triplicate.

first: Maike

last: Stentenbach

first: Judith

last: Ermer


title: Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations (RNA)

city: Seattle

first: Jeremy

middle: Aspin

last: Schofield

state: Washington

address: 1100 Fairview Ave N

department: Basic Sciences Division

institute: Fred Hutchinson Cancer Center

summary: Three general classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA and Mediator (MED) Tail, but little is known about whether this dependence is determined by the core promoter, Upstream activation sites (UAS), or other gene features. It is also unclear whether UASs can broadly activate transcription from the different promoter classes or whether efficient transcription requires matching UASs and promoters of similar gene class. Here we measure transcription and cofactor specificity for tens of thousands of UAS-core promoter combinations. We find that few UASs display strong core promoter specificity while most UASs can broadly activate promoters regardless of regulatory class. However, we find that matching UASs and promoters from the same gene class is generally important for optimal expression. We find that MED Tail and SAGA are dependent on the identity of both UAS and promoter while dependence on TFIID localizes to only the core promoter.

overall_design: Tens of thousands of UAS-core promoter combinations were cloned into a barcoded transcriptional reporter. Transcription was measured using short 4-TU labeling and MTS-biotin enrichment +/- acute auxin-mediated depletion of Taf13 (TFIID), Spt7 (SAGA) or Med15 (Mediator) subunits. Reporter transcripts were matched to UAS-core combinations by sequencing reporter plasmid library, which was also used to normalize transcription values.

first: Steven

last: Hahn


title: Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations [III]


title: Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations

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

overall_design: Refer to individual Series


title: St3gal1 and bII-spectrin pathways control CAR-T cell migration to target tumor sites [3]

overall_design: Mouse CD8 T cell were isolated and prepared with/without in vitro activation by CD3/CD28 Abs (n=3).

first: Brandon

last: Walling


title: Decoding pancreatic endocrine cell differentiation and beta-cell regeneration in zebrafish

status: Public on Mar 14 2023

city: Stockholm

first: Olov

last: Andersson

state: Other (Non U.S.)

address: Biomedicum Quarter 5D, Tomtebodavagen 16

country: Sweden

institute: Karolinska Institute

summary: The aim of the study is to investigate the endocrinogenesis process in zebrafish pancreas during normal development and regenerative condition.

overall_design: Using lineage tracing methodology (TgKI(krt4-p2a-mNeonGreen-t2a-iCre); ubb:loxp-CFP-stop-loxp-H2BmCherry), we used FACS to sort out mCherry positive cells in adult zebrafish pancreas, indicating cells in the krt4+ cell lineage

first: Jiarui

last: Mi


title: Defining the fine structure of promoter activity on a genome-wide scale with CISSECTOR

city: Amsterdam

first: Bas

last: van Steensel

address: Plesmanlaan 121

department: division of Molecular Biology

country: Netherlands

institute: Netherlands Cancer Institute

summary: Classic promoter mutagenesis strategies can be used to study how proximal promoter regions regulate the expression of particular genes of interest. This is a laborious process, in which the smallest sub-region of the promoter still capable of recapitulating expression in an ectopic setting is first identified, followed by targeted mutation of putative transcription factor binding sites. Massively parallel reporter assays such as Survey of Regulatory Elements (SuRE) provide an alternative way to study millions of promoter fragments in parallel. Here we show how a generalized linear model (GLM) can be used to transform genome-scale SuRE data into a high-resolution genomic track that quantifies the contribution of local sequence to promoter activity. This coefficient track helps identify regulatory elements and can be used to predict promoter activity of any sub-region in the genome. It thus allows in silico dissection of any promoter in the human genome to be performed. We developed a web application, available at cissector.nki.nl, that lets researchers easily perform this analysis as a starting point for their research into any promoter of interest.

overall_design: For generating the model, previously published SuRE-seq analysis was used (GSE128325). For validation of our GLM models, a second SuRE dataset was generated in K562 cells from a focused SuRE library consisting of fragments from 4 bacterial artificial chromosomes (BACs; CTD-3252A18, CTD-3156P24, CTD-2153L18, CTD-3075C4) containing 4 genomic regions covering several housekeeping genes (chr5:139,961,667-140,166,117; chr1:109,571,381-109,687,521; chr6:26,115,655-26,242,415; chr1:155,087,497-155,235,687, respectively).  Mapping errors cause a small number of BAC fragments to map to positions outside the original BAC regions; these elements were excluded from our analysis.

first: Vincent

middle: D

last: FitzPatrick

first: Christ

last: Leemans

first: Joris

last: van Arensbergen

first: Harmen

last: Bussemaker


title: SIRT6 is required for the integrated stress response and resistance to transcriptional inhibition [ChIP-Seq]

status: Public on Mar 15 2023

first: Matt

last: Fitzgibbon

institute: Fred Hutchinson Cancer Research Center

summary: We report a detailed analysis of enhancers marked with H3K27Ac in both basal and classical PDA

overall_design: Profiling of H3K27Ac binding in three pancreatic cancer cell lines.

first: Sita

last: Kugel


title: SIRT6 is required for the integrated stress response and resistance to transcriptional inhibition [RNA-Seq]

summary: We report a detailed analysis of the transcriptomes of both basal and classical PDA treated with THZ1

overall_design: Effect of THZ1 treatment on gene expression in three pancreatic cancer cell lines


title: St3gal1 and bII-spectrin pathways control CAR-T cell migration to target tumor sites

first: Cameron

last: Baker


title: Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization (Microarray)

status: Public on Mar 08 2023

city: New York

first: Anna

last: Gaponova

address: 345 Park Avenue South

institute: Insilico Medicine

summary: Differentiation is critical for epithelial cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, lysosomal cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of Rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in a zebrafish model of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 signaling and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data on kidney cortex from CTNS KO Mouse and Rat model vs WT control

first: Alessandro

last: Luciani


title: Robustness of Single-cell RNA-Seq for Identifying Differentially Expressed Genes

status: Public on Feb 27 2023

city: Milwaukee

first: Jing

last: Huang

state: WI

address: 8701 W Watertown Plank Rd

institute: Medical College of Wisconsin

summary: We found that single-cell RNA-seq (scRNA-seq)dataneeded to have2,000or more cellsin a cluster to identify the majority of differentially expressed genes (DEGs) with modest differences, while as few as 50-100cells weresufficient for identifying the majority of DEGs withextremely small p values or high abundance.The findings provide a quantitativereference for designing and interpreting studies that usescRNA-seq datato identify DEGs.

overall_design: We performed scRNA-seq and poly(A)-dependent bulk RNA-seq in comparable aliquots of human induced pluripotent stem cells (iPSC)-derived, purifiedvascular endothelial and smooth muscle cells.

first: Yong

last: Liu

first: Rajan

last: Pandey

first: Pengyuan

first: Qiongzi

last: Qiu

first: Mingyu

last: Liang


title: Single-cell transcriptomics of resected human traumatic brain injury tissues reveals acute activation of endogenous retroviruses in oligodendroglia

status: Public on Mar 03 2023

city: Lund

first: Johan

last: Jakobsson

address: Sölvegatan 17

department: Wallenberg Neuroscience Center

institute: Lund University

summary: Traumatic brain injury (TBI) is a leading cause of chronic brain impairment and results in a robust, but poorly understood, neuroinflammatory response that contributes to the long-term pathology. We used snRNA-seq to study transcriptomic changes in different cell populations in human brain tissue obtained acutely after severe, life-threatening TBI. This revealed a unique transcriptional response in oligodendrocyte precursors and mature oligodendrocytes, including the activation of a robust innate immune response, indicating an important role for oligodendroglia in the initiation of neuroinflammation. The activation of an innate immune response correlated with transcriptional upregulation of endogenous retroviruses in oligodendroglia. This observation was causally linked in vitro using human glial progenitors, implicating these ancient viral sequences in human neuroinflammation. In summary, this work provides a unique insight into the initiating events of the neuroinflammatory response in TBI, which has new therapeutic implications.

overall_design:
To investigate changes in cell-type composition and cell-type specific transcriptional responses after severe, acute TBI in humans, we performed single-nucleus RNA sequencing (snRNA-seq) from fresh frozen human brain tissue. We recruited 12 severe TBI patients, defined as post resuscitation Glasgow Coma Scale (GCS) score ≤ 8. The mean age of TBI patients (10 males, 2 females) was 49.5 ± 18.2 years. In these patients decompressive surgery was a life-saving measure to remove injured and swollen space-occupying brain tissue causing marked mass effect or increased intracranial pressure (ICP) refractory to conservative, medical neurointensive care treatment. The injured and contused brain regions (typically the injured part of a temporal or frontal lobe) were surgically removed between 4 hours and 8 days after injury 20. The raw data of these patients is not included in the GEO repository as these are alive individuals protected by GDPR, processed files are available. As control tissue, we used five fresh-frozen post-mortem samples from the frontal and temporal lobe obtained from three non-neurological deaths aged 69, 75 and 87 years. We isolated nuclei from frozen human brain tissue using ultracentrifugation and FACS (see methods for details) and performed snRNA-seq using the 10X Genomics pipeline.

To investigate a mechanistic link between interferon activation and ERV expression we decided to perform in vitro experiments in human glial progenitor cells (hGPCs). We differentiated human embryonic stem cells (hESCs) into hGPCs using a 135-day differentiation protocol. The hGPCs were treated with interferon gamma (IFN?, 5ng/ml) for 48 hours before harvested for 2×150 bp paired-end, strand-specific bulk RNA-seq analysis.

***Raw data not provided for GSM6376811-GSM6376826 due to patient privacy concerns***

The records have been updated with the following files on Sep 5, 2023:
TBI_gene_count_matrix_2.csv
hGPC_gene_count_matrix_2.csv

first: Raquel

last: Garza

first: Yogita

last: Sharma

first: Diahann

last: Atacho

first: Arun

last: Thiruvalluvan

first: Sami

last: Hamdeh

first: Marie

last: Jönsson

first: Vivien

last: Horvath

first: Anita

last: Adami

first: Martin

last: Ingelsson

first: Patric

last: Jern

first: Molly

last: Hammell

first: Elisabeth

last: Englund

first: Agnete

last: Kirkeby

first: Niklas

last: Marklund


title: The gelatinases, matrix metalloproteinases 2 and 9, play individual roles in skeleton development

status: Public on Mar 07 2023

city: Rehovot

first: Jerome

last: Janssen

address: Herzl 229

country: Israel

institute: Hebrew University of Jerusalem

summary: The gelatinases, a subgroup of the matrix metalloproteinases (MMPs) superfamily are composed of two members; MMP2 and MMP9. They are known to degrade gelatin among other components of the extracellular matrix. Recently, the two gelatinases were found to be necessary for neural crest cell migration and to compensate for each other loss in these cells. To characterize their involvement in the skeletal system, and to better reveal their individual or common roles, we have generated double knockout (dKO) mice, lacking both MMP2 and MMP9. The transcriptome of calvaria, growth plate and femoral cortical bone of P21 C57BL/6J WT, MMP2-/-, MMP9-/- and dKO mice was analyzed by bulk RNA-Seq.

overall_design: Cortical bone, growth plate and calvarial bone samples were collected from 5-7 P21 females of all four genotypes: WT, MMP2-/-, MMP9-/- and dKO. Cortical bone samples were taken from femur bone and were cleaned from all adjacent tissues (muscle, tendon and ligaments) and the diaphyseal bone marrow was removed by washing it out with ultra-pure water, using a small needle and syringe. Growth plate samples were collected from cleaned tibiae and for the calvarial samples the right/left frontal bone was taken. The samples were individually flash frozen in liquid nitrogen and were subjected to manually pulverization. Total RNA was extracted and purified using TRI reagent (Sigma, USA) according to the manufacturer’s protocol.

first: Rotem

last: Kalev-Altman

middle: N

first: Nir

last: Ben-Haim

first: Tamar

last: Levy

first: Astar

last: Shitrit-Tovli

first: Joshua

last: Milgram

first: Ron

last: Shahar

first: Dalit

last: Sela-Donenfeld

first: Efrat

last: Monsonego-Ornan


title: "Alk1 acts in non-endothelial VE-cadherin+ perineurial cells to maintain nerve branching during hair homeostasis" and  "Blood endothelial ALK1-BMP4 signaling axis regulates adult hair follicle stem cell activation"

status: Public on Mar 01 2023

city: Ithaca

first: Tudorita

last: Tumbar

address: 526 Campus Rd

department: Molecular biology and genetics

institute: Cornell University

summary:
Purpose: To comprehensively characterize VE-cadherin expressing lineages during adult skin homeostasis, to dissect their population-specific transcriptomic heterogeneity, and to investigate role of Alk1 in these linegease in vivo.
Methods: We performed 10x genomics single-cell RNA sequencing (scRNA-seq) of FACS sorted tdTomato+ cells from tdTomato;Cdh5-CreERT2;Krt14-H2BGFP mice (for WT) or Alk1 flox flox;tdTomato;Cdh5-CreERT2 (For Alk1KO) back skin at indicated stage/genotype. FACS purified tdTomato+ single-cell suspension was processed for the barcoded single-cell 3′ cDNA libraries generation using Chromium Single Cell 3′ gel bead and library Kit v3. The final libraries were quantified using Agilent Bioanalyzer high sensitivity DNA chip and sequenced using an Illumina NextSeq-500. The raw data files were demultiplexed to generate the sample-specific FASTQ files, which were aligned to the mouse reference genome (mm10-2020-A) using the 10x Genomics Cell Ranger pipeline (v6.0.1).  The raw scRNA-seq data was processed using Cell Ranger from the 10x platform to generate expression matrix for each sample that was further analyzed in R using the Seurat package version 4.0. Only high-quality cells that had between 200 and 5000 genes expressed and had under 10% of the UMIs mapped to mitochondrial genes were retained for further analysis.
Results: We obtained more than 2000 high quality cells at each stage/genotype for further analysis by Seurat pipeline
Conclusions: Obtained high quality single cell transcriptomic data to dissect cellular and molecular heterogeneity of VE-cadherin expressing endothelial and non-endothelial lineages in adult mouse back skin.

overall_design: Single cell RNA-seq profiles of sorted VE-cadherin+ lineages at telogen and anagen stages from wild type (WT) mice and telogen stage of Alk1 knockout (KO) mice

first: Tumbar

last: Tudorita

first: Chovatiya

last: Gopal

first: Li

last: Kefei Nina


title: Striated muscle-specific base editing enables correction of mutations causing dilated cardiomyopathy

status: Public on Feb 26 2023

city: Heidelberg

first: Shengdi

last: Li

address: Meyerhofstraße 1

country: Germany

institute: EMBL

summary: Dilated cardiomyopathy (DCM) is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of DCM patients harbor heritable mutations which are amenable to CRISPR-based gene therapy1. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart2. We employed a combination of the viral gene transfer vector AAVMYO with superior targeting specificity of heart muscle tissue3 and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive and arrhythmogenic DCM4. Using optimized conditions, we could improve splice defects in human iPSC-derived cardiomyocytes (iPSC-CMs) and repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restored the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reached wildtype levels. Single-nuclei RNA sequencing (snRNA-seq) uncovered restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing (WGS) revealed no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.

overall_design: RNA sequencing was performed to compare the transcriptomes of mouse hearts treated with base editors for correcting RBM20 gene mutations that cause DCM.

first: Markus

last: Grosch

first: Lars

last: Steinmetz


title: Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations (DNA)

first: Jeremy

last: Schofield

first: Steven

last: Hahn


title: St3gal1 and bII-spectrin pathways control CAR-T cell migration to target tumor sites [2]

first: Cameron

last: Baker

overall_design: hHER2 Tg mice bearing B16-hHER2 tumors received freshly prepared hHER2 CAR-T cells and CAR-T cells isolated from the lung 72 hr after adoptive transfer. CAR-T cells were isolated from the mouse lung (T72 vs. T0 cells, n=3).

first: Hong

last: Yeonsun


title: Common activities and predictive gene signature identified for genetic hypomorphs of TP53

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Mutational inactivation of TP53 is a common event in cancer.  Germline mutations in TP53 that inactivate this protein also occur in Li Fraumeni syndrome, which predisposes to early-onset cancer.  In addition, there are dozens of other germline variants in TP53 that do not completely inactivate the function of this protein.  In many cases studies have shown strong support for an impact of these lesser-functioning hypomorphs with increased cancer risk in humans and mouse models; however, the majority of these hypomorphs have yet to be categorized as pathogenic in clinical genetics databases.  There is thus need for a functional assay to distinguish lesser-functioning hypomorphic p53 variants from wild type p53, or benign, fully-functional, variants.  We report the surprising finding that two different African-centric genetic hypomorphs of p53, which occur in distinct functional domains of the protein, share common activities.  We show that the Pro47Ser variant in the transactivation domain and the Tyr107His variant in the DNA binding domain both share increased propensity to misfold into a conformation specific for mutant p53.  Moreover, cells and tissues with these variants show increased NF-B activity.  We have identified a common gene signature from unstressed lymphocyte cell lines that is shared between these two, and other, genetic missense hypomorphs of TP53.  We show that this gene signature successfully distinguishes wild type p53 and a benign p53 variant from lesser-functioning hypomorphic variants.  These findings should allow us to better understand how hypomorphic variants contribute to cancer risk, and to better inform cancer risk in hypomorph carriers.

overall_design: RNA-seq of samples with wild type or hypomorphic p53

first: Jessica

last: Leung

first: Maureen

last: Murphy


title: DNA methylome of Alpine Dingo

status: Public on Feb 24 2023

city: Sydney

first: Ksenia

last: Skvortsova

state: NSW

address: 384 Victoria St, Darlinghurst

department: Genomics and Epigenetics

institute: Garvan Institute

type: Methylation profiling by high throughput sequencing

summary: Using WGBS we investigated blood DNA methylation profiles of Cooinda the Alpine dingo and determined putative regulatory elements (unmethylated regions, UMRs, and lowly methylated regions, LMRs).

overall_design: Whole genome bisulphite sequencing of alpine dingo's blood DNA

first: Ozren

last: Bogdanovic


title: SIRT6 is required for the integrated stress response and resistance to transcriptional inhibition

first: Matt

last: Fitzgibbon


title: RNA sequencing/Gene expression studies of prednisolone in aggressive B-cell lymphomas [JC1]

city: Bethesda

first: David

last: Huang

state: Maryland

address: 9000 Rockville Pike

institute: National Cancer Institute

summary: We determined that glucocorticoid (prednisolone) treatment affects oncogenic signaling in aggressive B-cell lymphomas. We sought to measure gene expression change in aggressive B-cell lymphoma cells by RNA-sequencing following prednisolone treatment.

overall_design: HBL1, BL60, MD8, FL318, SUDHL5 and RAMOS cells were treated in duplicates with DMSO or 10 µM prednisolone for all cells except SUDHL5 with 25nm prednisolone for 6h and 24h.

first: Jaewoo

last: Choi

first: Louis

middle: M

last: Staudt


title: Gene Expression Signatures for the Accurate Diagnosis of Peripheral T-Cell Lymphoma Entities in the Routine Clinical Practice

status: Public on Feb 23 2023

city: Omaha

first: Javeed

last: Iqbal

state: NE

address: 601 S 42nd St

institute: UNMC


title: Transcriptome profiling of the livers from Mettl3 flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice

status: Public on Mar 12 2023

city: Harbin

first: Zheng

last: Chen

state: Heilongjiang

address: 92 Xidazhijie  Harbin Institute of Technology

institute: Harbin Institute of Technology

summary: The goal of this study is to compare transcriptome profilings of liver from Mettl3 flox/flox and hepatocyte-specific Mettl3 knockout (HKO) mice. Gene ontology analysis showed that the dowregulated genes were associated with monocarboxylic acid metabolic process, small molecule catabolic process, fatty acid metabolic process, cofactor metabolic process, organic acid catabolic process, and lipid biosynthetic process, whereas the upregulated genes were associated with leukocyte migration, cell chemotaxis, leukocyte cell-cell adhesion, positive regulation of cytokine production and defense response to other organism. KEGG pathway analysis showed that the downregulated genes were associated with Steroid hormone biosynthesis, Peroxisome, Chemical carcinogenesis, proteasome, Oxidative phosphorylation and Non-alcoholic fatty liver disease (NAFLD), whereas the upregulated genes were associated with Cell adhesion molecules (CAMs), Staphylococcus aureus infection, Viral protein interaction with cytokine and cytokine receptor, and Chemokine signaling pathway.

overall_design: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of Mettl3 flox/flox and HKO mice at 8 weeks old (n=3 for each group). mRNA profiles were generated by deep sequencing using an Illumina NovaSeq 6000 platform. Paired-end clean reads were aligned to the mouse reference genome(Ensemble_grcm38_p6) with Hisat2 v2.0.5, and the aligned reads were used to quantify mRNA expression by using featureCounts v1.5.0-p3.

first: Xinzhi

last: Li

first: Chuanhong

last: Liu


title: Single cell RNAseq Adult Mouse Lumbar Dorsal Root Ganglia (DRG)

city: Chicago

first: Rachel

middle: E

last: Miller

address: 1735 W Harrison St

institute: Rush University Medical Center

summary: Single cell RNAseq was performed on naïve adult mouse lumbar dorsal root ganglia (DRG) cells. Neuronal and non-neuronal cell populations were identified.

overall_design: L3-L5 right side DRGs were pooled from 10 male C57BL/6 mice at 18 weeks of age. A single cell solution was created by standard digestion. Single cell RNAseq was performed using the 10x Chromium system.

first: Anne-Marie

last: Malfait


title: Dynamic expression of smRNA from fecal exosome in disease progression of an inflammatory bowel disorder mouse model

status: Public on Feb 19 2023

city: Hershey

first: Yuka

last: Imamura Kawasawa

address: 500 University Dr.

department: College of Medicine, Pharmacology

institute: Penn State University

type: Non-coding RNA profiling by high throughput sequencing

summary: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract affecting over 3 million adults in the United States. Despite being widespread, reliable early diagnostic tests are not available. In order to remedy this, we examined exosomal small RNA (smRNA), specifically targeting microRNA (miRNA) and piRNA from the stool samples of IBD model mice, interleukin 10 knockout mice (IL-10 KO), as a potential diagnostic marker. Stool samples were specifically chosen because they are readily available and collection is noninvasive.

overall_design: Total 17 samples were analyzed; 7 and 10 replicates each for end-point and start-point samples, respectively.

first: Sean

last: Manning

first: Shisui

last: Torii

first: Hannah

last: Atkins

middle: I

last: Kawasawa


title: Changes in social behaviour with alterations of MAPK3 and KCTD13/CUL3 pathways in two new outbred rat models for the 16p11.2 syndromes with autism spectrum disorders [Long Evans]

status: Public on Feb 18 2023

city: ILLKIRCH

first: Yann

last: HERAULT

address: 1 rue Laurent Fries

country: France

institute: IGBMC/PHENOMIN-ICS

summary: Copy number variations (CNVs) of the human 16p11.2 locus are associated with several developmental/neurocognitive syndromes. Particularly, deletion and duplication of this genetic interval are found in patients with autism spectrum disorders, intellectual disability and other psychiatric traits. The high gene density associated with the region and the strong phenotypic variability of incomplete penetrance, make the study of the 16p11.2 syndromes extremely complex. To systematically study the effect of 16p11.2 CNVs and identify candidate genes and molecular mechanisms involved in the pathophysiology, mouse models were generated previously and showed learning and memory, and to some extent social deficits. To go further in understanding the social deficits caused by 16p11.2 syndromes, we engineered deletion and duplication of the homologous region to the human 16p11.2 genetic interval in two rat outbred strains, Sprague Dawley (SD) and Long Evans (LE). The 16p11.2 rat models displayed  convergent defects in social behaviour and only a few cognitive defects. Interestingly major pathways affecting MAPK3 and CUL3 were found altered in the rat 16p11.2 models with additional changes in males compared to females. Altogether, the consequences of the 16p11.2 genetic region dosage on social behaviour are now found in three different species: humans, mice and rats. In addition, the rat models pointed to sexual dimorphism, with lower severity of phenotypes in rat females compared to male mutants. This phenomenon is also observed in humans. We are convinced that the two rat models will be key to further investigating social behaviour and understanding the brain mechanisms and specific brain regions that are key to controlling social behaviour.

overall_design:
Comparison of hippocampal RNA profile in the rat Long Evans models of 16p11.2 between Del/+ and control in males and females (n=5 per genotype).
Hippocampus from 5 Del/+  and 5 wt young adult male and female littermates, were isolated at 24weeks of age and flash frozen in liquid nitrogen. Total RNA was prepared using an RNA extraction kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Samples quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA).

first: Sandra

last: Martin Lorenzo

last: Herault


title: Vessel Forming Mesenchymal Stromal Cell Populations

city: Palo Alto

first: Liming

last: Zhao

state: CA

address: G3105, Lokey Stem Cell Research Building, 265 Campus Drive, 450 Serra Mall, Stanford

institute: Stanford University

summary: We have identified two vessel forming mesenchymal stromal cell populations. Population 1 (P1) is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low and gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and Population 2 (P2) which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. To test the homogeneity and signal interactions of these populations we performed 10X single cell RNA-sequencing on them.

overall_design: Two vessel forming mesenchymal stromal cell populations were FACS sorted according to their surface markers, P1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low and P2 is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high,10x single cell RNA-sequencing were performed after sorting.

first: Koki

last: Sasagawa

first: Charles

middle: K

last: Chan


title: In situ tumor arrays reveal early environmental control of cancer immunity [Whole Exome Sequencing]

status: Public on Feb 17 2023

city: South San Francisco

first: Joaquin

last: Pechuan jorge

address: 1 dna way

department: Cancer Immunology

institute: Genentech

summary: We use a laser to implant tumor cells in a regular pattern on the ears of the animal. When GFP expressing tumor cells are implanted in a CD4-Cre Tdtomato mouse we can perform live imaging of red T Cells trafficking in and out of each micro-tumor of the array. Thanks to live imaging we were able to classified each of the implanted tumors as: 1-Rejected tumors (no GFP+ tumor cells anymore but lots of immune cells), 2- Inflamed tumors (GFP+ tumors infiltrated with lots of immune cells), 3-Excluded Tumors (GFP+ tumors with immune cells segregated around the tumor area) or Desert tumors (GFP+ tumor cells without any immune cells). As these tumors are implanted just under the epidermis, we were able to biopsy them out and pool them by categories (Rejected, Inflamed, Excluded and Desert). We froze down biopsies of pooled tumors for each category (Rejected, Inflamed, Excluded and Desert) and extracted DNA. We wish to characterize these 4 kinds of tumors (DNA seq) to evaluate how much tumors grown in the same animals from a clonal KPP tumor cell line differ from each other in terms of mutation load.

overall_design: Whole Exome Sequencing

first: Markus

last: Brown

first: Catherine

middle: B

last: Carbone

first: Ximo

last: Pechuan-Jorge

first: Vincent

last: Rouilly

first: Henrik

last: Lindberg

first: Alex

middle: T

last: Ritter

first: Gautham

last: Ragupathi

first: Qianbo

last: Sun

first: Tess

last: Nicotra

first: Shreya

middle: R

last: Mantri

first: Angela

last: Yang

first: Deepti

last: Nagarkar

first: Sanjeev

last: Mariathasan

first: Yulei

last: Wang

first: Carlos

last: Gomez-Roca

first: Calors

last: Eduardo de Andrea

first: Davie

last: Spigel

first: Thomas

last: Wu

first: Lelia

last: Delamarre

first: Johannes

last: Schöneberg

first: Zora

last: Modrusan

first: Richard

last: Price

first: Shannon

last: Turley

first: Ira

last: Mellman

first: Christine

last: Moussion


title: In situ tumor arrays reveal early environmental control of cancer immunity [STAMP D8]

first: Joaquin

last: Pechuan jorge

summary: KPP-GFP (C57Bl/6) cells were implanted into the laser-induced micropores ears of B6.Cg-Foxn1nu/J (C57Bl/6J Jackson background) that were adoptively transferred with 4x10^6 naive CD4tdTomato-positive Tcells (B6 N/J background) the same day. The STAMP microtumors were imaged daily starting at day 3 after tumor cell implantation. On day 8 after tumor cell implantation tumors were classified as either (1) Inflamed, (2) Excluded, (3) Ignored/Desert or (4) Resolved.

overall_design: Bulk RNA Sequencing

first: Markus

last: Brown

first: Catherine

last: Carbone

first: Ximo

last: Pechuan-Jorge

first: Vincent

last: Rouilly

first: Henrik

last: Lindberg

first: Alex

last: Ritter

first: Gautham

last: Ragupathi

first: Qianbo

last: Sun

first: Tess

last: Nicotra

last: de Andrea


title: In situ tumor arrays reveal early environmental control of cancer immunity [NGS3077]

summary: The immune phenotype of a tumor is a key predictor of its response to immunotherapy. Patients who respond to immune checkpoint blockade generally present with tumors infiltrated by T cells, a phenotype referred to as ‘inflamed’. However, not all inflamed tumors respond to therapy, and even lower response rates occur among patients with tumors that lack T cells (‘desert’) or that spatially exclude T cells to the periphery of the tumor lesion (‘excluded’). Despite the importance of these tumor immune phenotypes in patients, little is known about their development, heterogeneity or dynamics due to the technical difficulty of modeling and tracking these features in situ. Here, we introduce STAMP (skin tumor array by microporation), a preclinical approach that combines in vivo high-throughput time-lapse imaging with next generation sequencing of tumor arrays.  Using this approach, we follow the early formation of thousands of tumors to show that development of a given immune phenotype is not under strict control of tumor genetics or transcriptional state, but is also influenced by local features of the tumor microenvironment. Only the spatial organization of T cells, specifically early infiltration of T cells recruited by fibroblasts and monocytes into the core of a tumor, was predictive of functional T cell attack and tumor rejection. Evaluating the dynamic immune-history of tumors revealed that early conversion to the immune inflamed phenotype was predictive of therapy-induced or spontaneous tumor regression. Thus, STAMP captures the dynamic and complex relationships of spatial, cellular and molecular components of tumor progression and has the potential to translate therapeutic concepts into successful clinical strategies.

overall_design: Deciphering the molecular regulators of tumor T cell infiltration in the pre-clinical STAMP (Skin Tumor Array by Microporation) model. The classification of immune infiltrated, immune excluded or immune desert tumors is a powerful predictor of clinical response to immunotherapy. Patients who respond to immune checkpoint blockade generally present with tumors that are infiltrated by activated T cells. However, the majority of patients present with tumors that are poorly (immune desert) or improperly (immune excluded) infiltrated by CD8 T cells. In order to mimic the clinical observations and perform live Imaging of the Immune cell infiltration in tumors, we developed a new pre-clinical mouse model called STAMP (Skin Tumor Array by Microporation). We use a laser-assisted technique to deposit tumor cells in a regular pattern into the superficial dermis of the ear of the animal. When GFP expressing KPP tumor-cells are implanted into a CD4-Cre Tdtomato mouse, we can perform live imaging of TdTomato+ T Cells trafficking in and out of each micro-tumor of the array. Using live imaging we were able to classify each of the implanted tumors as: 1 Hot/Inflamed tumors (GFP+ tumors infiltrated with T cells in the tumor core), 2-Excluded Tumors (GFP+ tumors with T cells segregated around the tumor area) 3-Resolved tumors (no GFP+ tumor cells anymore but only a cloud of T cells) or 4-Desert tumors (GFP+ tumor cells without any T cells infiltration). Even though the KPP mother tumor cell line used for the implantation is derived from a single cell clone, the skin microenvironment is able to dictate the infiltration pattern of individual tumors implanted into the same recipient. Even 2mm apart, those tumors behave as independent compartments. As the tumors are implanted in the superficial dermis, we were able to biopsy them out and separate them by categories (Resolved, Inflamed, Excluded and Desert). We harvested RNA directly from biopsies of individual tumors of each category. We performed this experiment 7 times independently, and have a total of 33 samples (cf below). We wish to characterize the differences in Transcriptome profile to determine regulators, drivers and potential targets of these 4 tumor categories. KPP GFP+ tumor cells were implanted into the ear of a B6.Cg-Foxn1nu/J (JAX: 000819) animal with the STAMP technic. The same day naÃÂ¯ve TdTomato T cells (colony 7976; CD4.cre.tg_Rosa26.LSL.tdTomato.cki_OT-I.TCR.tg) were adoptively transferred i.v into the same mouse. Tumor infiltration is imaged from day 5-14 of each individual tumor. Day 13 after implantation, we performed live imaging of the whole Tumor array to be able to annotate tumors as Rejected, Inflamed, Excluded and Desert. 1mm Skin tumor biopsies were harvested and pooled by category (Resolved, Hot/Inflamed, Excluded and Desert) or treated individually.

first: Shreya

last: Mantri

first: Angela

last: Yang

first: Deepti

last: Nagarkar

first: Sanjeev

last: Mariathasan

first: Yulei

last: Wang

first: Carlos

last: Gomez-Roca

first: Calors

last: Eduardo de Andrea

first: Davie

last: Spigel

first: Thomas

last: Wu

first: Lelia

last: Delamarre

last: Schöneberg


title: Gene Expression Signatures for the Accurate Diagnosis of Peripheral T-Cell Lymphoma Entities in the Routine Clinical Practice [validation]

first: Javeed

last: Iqbal

summary:
Peripheral T-cell lymphoma (PTCL) includes heterogeneous clinicopathologic entities with numerous diagnostic and treatment challenges. We previously defined robust transcriptomic signatures that distinguish common PTCL entities and identified two novel biologic and prognostic PTCL-not otherwise specified subtypes (PTCL-TBX21 and PTCL-GATA3). We aimed to consolidate a gene expression–based subclassification using formalin-fixed, paraffin-embedded (FFPE) tissues to improve the accuracy and precision in PTCL diagnosis.
We assembled a well-characterized PTCL training cohort (n = 105) with gene expression profiling data to derive a diagnostic signature using fresh-frozen tissue on the HG-U133plus2.0 platform (Affymetrix, Inc, Santa Clara, CA) subsequently validated using matched FFPE tissues in a digital gene expression profiling platform (nCounter, NanoString Technologies, Inc, Seattle, WA). Statistical filtering approaches were applied to refine the transcriptomic signatures and then validated in another PTCL cohort (n = 140) with rigorous pathology review and ancillary assays.

overall_design: We developed a novel transcriptomic approach for PTCL subclassification that facilitates translation into clinical practice with higher precision and uniformity than conventional pathology diagnosis.

first: Wing

middle: C

last: Chan


title: In situ tumor arrays reveal early environmental control of cancer immunity [NGS2751]

first: Joaquin

last: Pechuan jorge

overall_design: KPP-GFP (C57Bl/6) cells were implanted into the ears of B6.129S6-Rag2tm1Fwa N12 mice (C57Bl/6NTac background) that were adoptively transferred with 4x10^6 naive CD4tdTomato-positive Tcells (B6 N/J background) the same day. The STAMP microtumors were imaged daily starting at day 3 after tumor cell implantation. On day 8 after tumor cell implantation tumors were classified as either (1) Inflamed, (2) Excluded, (3) Ignored/Desert or (4) Resolved. Thereafter a pool of 3-4 tumors (ranging from from a size of 0.7 - 1.2 um diameter) of each phenotype (including small amounts of surrounding healthy stroma) were biopsied from the two ears of one single mouse and enzymatically digested (in the presence of Actinomycin D) to generate single cell suspensions. The cells were then incubated in a staining solution containing Calcein Blue and 7AAD and enriched for live and non-dead cells by flow cytometry. The viability of single cells suspensions as determined by Vicell were (1) 59.3%, (2) 71.4%, (3) 90.6% and (4) 80%. The cell number ranged from 5-6x10^4 for (3) and (4) to about 10-11x10^4 for (1) and (2) as determined by flow cytometry and Vicell. Comparing 5' single cell RNAseq data between the four STAMP microtumor immune phenotypes should provide insight into differences of the cellular compositions of their tumor-immune and -stromal microenviornments, reveal differences of the associated TCR repertoire, help in the generation of transcriptome-based classifiers of the different immune phenotypes and lead to the discovery of important and or novel pathways associated with the different immune phenotypes.

first: Guadalupe

last: Ortiz-Muñoz

first: Markus

last: Brown

first: Catherine

last: Carbone

first: Ximo

last: Pechuan-Jorge

first: Vincent

last: Rouilly

first: Henrik

last: Lindberg

last: Ritter

last: Ragupathi

first: Johannes

first: Zora

last: Modrusan

first: Richard

last: Price

first: Shannon

middle: J

last: Turley

first: Ira

last: Mellman

first: Christine

last: Moussion


title: Gene Expression Signatures for the Accurate Diagnosis of Peripheral T-Cell Lymphoma Entities in the Routine Clinical Practice [training]

overall_design:
We developed a novel transcriptomic approach for PTCL subclassification that facilitates translation into clinical practice with higher precision and uniformity than conventional pathology diagnosis.

Matched transcriptomic samples on the HG-U133plus2.0 platform (Affymetrix) are available in GSE19069 and GSE58445. GSMs include links to matched GSM analyzed on HG-U133plus2.0.


title: FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity

city: Cambridge

first: Matthew

state: MA

address: 75 Ames St

institute: Broad Institute

summary: Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies), an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.

overall_design: Examination of gene expression in mouse beta cells (min6) exposed to 61 structurally diverse free fatty acids.

first: Nicolas

last: Wieder

first: Juiliana

last: Coraor Fried

first: Choah

last: Kim

first: Eriene-Heidi

last: Sidhom

first: Jamie

last: Marshall

first: Carlos

last: Arevalo

first: Moran

last: Dvela-Levitt

first: Maria

last: Kost-Alimova

first: Jonas

last: Sieber

first: Katlyn

last: Gabriel

first: Julian

last: Pacheco

first: Clary

last: Clish

first: Hamdah

last: Abbasi

first: Shantanu

last: Singh

first: Justine

last: Rutter

first: Martine

last: Therrien

first: Haejin

last: Yoon

first: Zon

middle: W

last: Lai

first: Aaron

last: Baublis

first: Renuka

last: Subramanian

first: Ranjan

last: Devkota

first: Jonnell

last: Small

first: Vedagopuram

last: Sreekanth

first: Myeonghoon

last: Han

first: Donghyun

last: Lim

first: Anne

last: Carpenter

first: Jason

last: Flannick

first: Hilary

last: Finucane

first: Marcia

last: Haigis

first: Melina

last: Claussnitzer

first: Eric

last: Sheu

first: Beth

last: Stevens

first: Bridget

last: Wagner

first: Amit

last: Choudhary

first: Jillian

last: Shaw

first: Juan

last: Pablo

first: Anna

last: Greka


title: A phase Ib trial evaluating the safety, efficacy, and immunologic effects of pembrolizumab plus paclitaxel or flat-dose capecitabine in 1st/2nd line metastatic triple-negative breast cancer

city: Portland

first: venkatesh

last: rajamanickam

state: OR

address: 4805 NE Glisan St, Suite 2N35

institute: Earle A. Chiles Research Institute

summary:
Chemoimmunotherapy with anti-programmed cell death 1/ligand 1 and cytotoxic chemotherapy is a promising therapeutic modality for women with triple-negative breast cancer, but questions remain regarding optimal chemotherapy backbone and biomarkers for patient selection. We report final outcomes from a phase Ib trial evaluating pembrolizumab (200mg IV every 3 weeks) with either weekly paclitaxel (80mg/m2 weekly) or flat-dose capecitabine (2000mg orally twice daily for 7 days of every 14-day cycle) in the 1st/2nd line setting. The primary endpoint is safety (receipt of 2 cycles without grade III/IV toxicities requiring discontinuation or ≥21-day delays). The secondary endpoint is efficacy (week 12 objective response). Exploratory aims are to characterize immunologic effects of treatment over time, and to evaluate novel biomarkers.
The trial demonstrates that both regimens meet the pre-specified safety endpoint (paclitaxel: 87%; capecitabine: 100%). Objective response rate is 29% for pembrolizumab/paclitaxel (n=4/13, 95% CI: 10-61%) and 43% for pembrolizumab/capecitabine (n=6/14, 95% CI: 18-71%). Partial responses are observed in two subjects with chemo-refractory metaplastic carcinoma (both in capecitabine arm). Both regimens are associated with significant peripheral leukocyte contraction over time. Response is associated with clinical PD-L1 score, non-receipt of prior chemotherapy, and the H&E stromal tumor infiltrating lymphocyte score, but also by a novel 27 gene IO score and spatial biomarkers (lymphocyte spatial skewness). In conclusion, pembrolizumab with paclitaxel or capecitabine is safe and clinically active. Both regimens are lymphodepleting, highlighting the competing immunostimulatory versus lymphotoxic effects of cytotoxic chemotherapy. Further exploration of the IO score and spatial TIL biomarkers is warranted.

overall_design:
RNA sequencing of tumor from patients treated with pembrolizumab plus paclitaxel or capecitabine in 1st / 2nd line metastatis triple-negative breast cancer
*** Raw data are not available due to patient privacy concerns. ***

first: Venkatesh

last: Rajamanickam

first: David

last: Page


title: In situ tumor arrays reveal early environmental control of cancer immunity [STAMP Pre and Post Implantation Tumor Cells]

first: Joaquin

last: Pechuan jorge

summary: The immune phenotype of a tumor is a key predictor of its response to immunotherapy1–4. Patients who respond to immune checkpoint blockade generally present with tumors infiltrated by T cells, a phenotype referred to as ‘inflamed’5–7. However, not all inflamed tumors respond to therapy, and even lower response rates occur among patients with tumors that lack T cells (‘desert’) or that spatially exclude T cells to the periphery of the tumor lesion (‘excluded’)8. Despite the importance of these tumor immune phenotypes in patients, little is known about their development, heterogeneity or dynamics due to the technical difficulty of modeling and tracking these features in situ. Here, we introduce STAMP (skin tumor array by microporation), a preclinical approach that combines in vivo high-throughput time-lapse imaging with next generation sequencing of tumor arrays.  Using this approach, we follow the early formation of thousands of tumors to show that development of a given immune phenotype is not under strict control of tumor genetics or transcriptional state, but is also influenced by local features of the tumor microenvironment. Only the spatial organization of T cells, specifically early infiltration of T cells recruited by fibroblasts and monocytes into the core of a tumor, was predictive of functional T cell attack and tumor rejection. Evaluating the dynamic immune-history of tumors revealed that early conversion to the immune inflamed phenotype was predictive of therapy-induced or spontaneous tumor regression. Thus, STAMP captures the dynamic and complex relationships of spatial, cellular and molecular components of tumor progression and has the potential to translate therapeutic concepts into successful clinical strategies.

overall_design: The KPP-EGFP tumor cell line is derived from a single cell clone and gives rise to diverse tumor immune phenotypes (Inflamed, Desert, Excluded) in the STAMP tumor model 8 days after implantation. Within this project we would like to determine the degree of non-genetic heterogeneity before (D0) and after tumor cell implantation (Day8) and evaluate the possibility of non-genetic heterogeneity driving the emergence of diverse tumor immune phenotypes.

first: Guadalupe

last: Ortiz-Muñoz

first: Markus

last: Brown

first: Catherine

last: Carbone

first: Ximo

last: Pechuan-Jorge

first: Vincent

last: Rouilly

first: Henrik

last: Lindberg

first: Alex

last: Ritter

first: Gautham

last: Ragupathi

first: Qianbo

last: Sun

first: Tess

last: Nicotra

first: Shreya

last: Mantri

first: Angela

last: Yang

first: Deepti

last: Nagarkar

first: Sanjeev

last: Mariathasan

first: Yulei

last: Wang

first: Carlos

last: Gomez-Roca

first: Calors

last: Eduardo de Andrea

first: Davie

last: Spigel

first: Thomas

last: Wu

first: Lelia

last: Delamarre

first: Johannes

last: Schöneberg

first: Zora

last: Modrusan

first: Richard

last: Price

first: Shannon

last: Turley

first: Ira

last: Mellman

first: Christine

last: Moussion


title: Pol II Preferentially Regulates Ribosomal Protein Expression by Trapping Disassociated Subunits

status: Public on Feb 16 2023

city: Beijing

first: Xiong

last: Ji

address: Haidian District, Beijing Summer Palace Road No. 5

department: School of Life Sciences

institute: Peking University

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: In situ tumor arrays reveal early environmental control of cancer immunity [STAMP time course (Day1,3,8) bulk RNAseq ]

first: Joaquin

last: Pechuan jorge

summary: The STAMP tumor model utilizes laser-guided microporation to deposit tumor cells into the dermis of the mouse ear. This project is aimed to characterize the early molecular and cellular changes associated with microporation and tumor cell seeding along the time course of 8 days in WT C57Bl6 animals. The data generated in this project will allow us to decipher which changes are associated with microporation and how it compares to the tumor progression.

first: Markus

last: Brown

first: Catherine

last: Carbone

first: Ximo

last: Pechuan-Jorge

first: Vincent

last: Rouilly

first: Henrik

last: Lindberg

first: Alex

last: Ritter

first: Gautham

last: Ragupathi

first: Qianbo

last: Sun

first: Tess

last: Nicotra

first: Shreya

last: Mantri

last: de Andrea


title: Impact of elraglusib (9-ING-41) treatment on gene expression in human colorectal cancer and immune cell lines.

city: Providence

first: Kelsey

middle: Elizabeth

last: Huntington

state: RI

address: 70 Ship Street, Campus Box G-E5

department: Pathology and Laboratory Medicine

institute: Brown University

summary:
A growing body of literature evaluates the potential anti-cancer and immunostimulatory effects of GSK-3 inhibition. Accordingly, we sought to evaluate GSK-3 inhibitor elraglusib (9-ING-41) and its impact on gene expression in both human colorectal cancer (CRC) cell lines and human immune cell lines.
We used microarrays to detail the differential gene expression that occurs post-treatment with GSK-3 inhibitor 9-ING-41 (elraglusib).

overall_design: We utilized microarray analysis to gain insights into gene expression changes in colorectal cell lines and immune cell lines post-GSK-3 inhibition with elraglusib (9-ING-41). Several CRC cell lines (HCT-116, HT-29, KM12C) and immune cell lines (TALL-104, NK-92) were treated with elraglusib or DMSO as a vehicle control for 24 hours and treated versus untreated samples were compared in triplicate.

first: Christoph

last: Schorl


title: RPS19 binding with and without Diamond-Blackfan anemia variants

status: Public on Feb 14 2023

city: La Jolla

first: Gene

last: Yeo

address: 2880 Torrey Pines Scenic Dr

department: Cellular & Molecular Medicine

institute: University of California San Diego

summary: We found that endogenously expressed RPS19 binds evolutionarily constrained intronic elements. By assessing overexpression of WT RPS19 and RPS19 harboring the most common Diamond-Blackfan anemia missense mutations, we confirmed that disease-relevant mutations alter RPS19 binding preferences in human cells.

overall_design:
eCLIP of WT and mutant overexpressed RPS19 in HEK
Please note that processed data generated from all replicates is linked to the corresponding *rep1 sample records.

first: Evan

middle: A

last: Boyle

first: Hsuan-Lin

last: Her

first: Jasmine

last: Mueller

first: Jack

last: Naritomi

first: Grady

middle: G

last: Nguyen


title: Pol II Preferentially Regulates Ribosomal Protein Expression by Trapping Disassociated Subunits [ChAR-seq]

first: Xiong

last: Ji

summary: Pol II has been recognized as a passively regulated holoenzyme. However, whether Pol II plays specific regulatory roles remain unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, Specific Degradation of Disassociated Subunits (SDDS), we demonstrated that dRPB3 functions as a regulatory component of Pol II to enable the preferential control of 3’ end processing of ribosomal protein genes directly through N-terminal domain of RPB3. Machine learning analysis of large-scale genomic features revealed that the little elongation complex helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3’ end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that Pol II gains specific regulatory functions by trapping disassociated subunits.

overall_design:
We performed ChAR-seq, TT-seq, RNA-seq, and ChIP-seq to investigate the roles of various specialized subunits in transcription and post-transcriptional regulation. The V6.5 mouse ES (mES) cell line was used for all the high-throughput analyses. Degron mES cells were pretreated with 1 μg/ml Doxycycline for 12h to induce TIR1 expression, and treated with or without 500 μM IAA for different hours; refer to individual Series. For TT-seq, ChAR-seq, and RNA-seq, the Drosophila S2 cells were used as the spike-in cells, and for ChIP-seq, the HEK293T cells were used as the spike-in cells.
This dataset includes the ChAR-seq data.

first: Yuanjun

last: Li

first: Jie

last: Huang

first: Lijun

last: Bao

first: Junyi

last: Zhu

first: Wenjia

last: Duan


title: In situ tumor arrays reveal early environmental control of cancer immunity

first: Joaquin

last: Pechuan jorge


title: Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons [Bulk RNA-seq]

status: Public on Feb 13 2023

city: Baltimore

first: Andrew

middle: S.

last: McCallion

address: 733 North Broadway

department: Genetic Medicine

institute: Johns Hopkins University School of Medicine

summary: To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate of disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques – karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq – to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo, mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable a proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes.

overall_design: 8 Bulk RNA-seq samples: 4 from SN4741 cells cultured at 37C, 4 from SN4741 cells cultured at 39C

first: Rachel

last: Boyd

first: Sarah

last: McClymont

first: Nelson

last: Barrientos

first: Paul

last: Hook

first: William

last: Law

first: Rebecca

last: Rose

first: Eric

middle: L

last: Waite

first: Jay

last: Rathinavelu

first: Dimitrios

last: Avramopoulos

middle: S


title: Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons [scRNA-seq]

overall_design: 8 Single Cell RNA-seq samples: 4 from SN4741 cells cultured at 37C, 4 from SN4741 cells cultured at 39C

middle: D


title: Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization (RNA-Seq)

city: New York

first: Anna

last: Gaponova

address: 345 Park Avenue South

institute: Insilico Medicine

summary: Differentiation is critical for epithelial cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, lysosomal cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of Rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in a zebrafish model of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 signaling and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: Comparative gene expression profiling analysis of RNA-seq data on kidney cortex from CTNS KO Mouse and Rat model vs WT control

first: Alessandro

last: Luciani


title: Single-cell and spatial transcriptome analysis reveals the cellular heterogeneity of liver metastatic colorectal cancer

status: Public on Feb 22 2023

city: Guangzhou

first: Jie

last: Long

address: No.106 Zhongshan 2nd Road

institute: Guangdong Provincial People's Hospital

summary: In this study, we comprehensively charted the cellular landscape of colorectal cancer (CRC) and well-matched liver metastatic CRC using single-cell and spatial transcriptome RNA sequencing. We generated 41892 CD45- non-immune cells and 196473 CD45+ immune cells from 27 samples of 6 CRC patients, and found that CD8_CXCL13 and CD4_CXCL13 subsets increased significantly in liver metastatic samples that exhibited high proliferation ability and tumor-activating characterization, contributing to better prognosis of patients. Distinct fibroblast profiles were observed in primary and liver metastatic tumors. The F3+ fibroblasts enriched in primary tumors contributed to worse overall survival by expressing pro-tumor factors. However, the MCAM+ fibroblasts enriched in liver metastatic tumors might promote generation of CD8_CXCL13 cells through Notch signaling. In summary, we extensively analyzed the transcriptional differences of cell atlas between primary and liver metastatic tumors of CRC by single-cell and spatial transcriptome RNA sequencing, providing different dimensions of the development of liver metastasis in CRC

overall_design:
For single-cell RNA sequencing, 6 CRC patients with liver metastasis were enrolled after written informed consent was obtained. Primary CC, CN (at least 2 cm from matched tumors), LM, LN (at least 2 cm from matched tumors) and PB of the enrolled patients were collected following resection. All the patients received preoperative chemotherapy and/or radiotherapy. For spatial transcriptome sequencing, formalin-fixed paraffin embedded (FFPE) tissues of 6 CRC patients were prepared.

Submitter states: Raw data are not included due to patient privacy concerns, which are deposited in the CNGB Nucleotide Sequence Archive (CNSA: https://db.cngb.org/cnsa; accession number: CNP0002540 for single cell RNA sequencing data, and CNP0003321 for spatial transcriptome RNA sequencing data)

first: Fei

last: Wang


title: Serial whole blood transcriptomic analysis demonstrates activation of neutrophils, platelets and coagulation in severe and critical COVID-19 – submitted

city: Oslo

first: Yohan

last: Lefol

state: Oslo

address: Sognsvannsveien 20 Rikshospitalet

department: Institute of Clinical Medicine

country: Norway

institute: University of Oslo

summary:
Introduction: A maladaptive inflammatory response has been implicated in the pathogenesis of severe and critical COVID-19. This study aimed to characterize the temporal dynamics of this response and investigate whether critical disease is associated with distinct gene expression patterns.
Methods: We performed microarray analysis of serial whole blood RNA samples from 19 patients with critical COVID-19, 15 patients with severe but non-critical disease and 11 healthy controls. We assessed whole blood gene expression patterns by differential gene expression analysis, gene set enrichment, two clustering methods and estimated relative leukocyte abundance using CIBERSORT.
Results: Neutrophils, platelets, cytokine signaling, and the coagulation system were activated in COVID-19, and more pronounced in critical vs. non-critical disease. We observed two different trajectories of neutrophil-associated genes, indicating the emergence of a more immature neutrophil phenotype over time. Interferon-associated genes were strongly enriched in early COVID-19 before falling markedly, with modest severity-associated differences in trajectory.
Conclusions: Severe COVID-19 is associated with a broad inflammatory response, which is more pronounced in critical disease. Our data suggest a progressively more immature circulating neutrophil phenotype over time. Interferon signaling is enriched in COVID-19 but does not seem to drive critical disease.

overall_design: Between March and May 2020, 135 patients admitted to Akershus University Hospital with COVID-19 confirmed by SARS-CoV-2 RT-PCR were prospectively recruited to the Coronavirus Disease Mechanisms (COVID MECH) observational cohort study. Thirty-six patients (27%) were admitted to the ICU and 8 (6%) died. Inclusion predated the use of corticosteroids in severe COVID-19. This substudy included 19 patients with critical disease, defined as requiring invasive mechanical ventilation, and 15 patients with non-critical disease receiving supplemental O2. Patients were selected based on the availability of sequential whole blood RNA samples, and time from symptom onset to baseline sampling between five and 15 days. RNA samples from 11 healthy volunteers matched to patients by age and gender served as controls.

first: Torben

last: Lüders

first: Peder

last: Myhre

first: Jan-Erik

last: Berdal


title: Single-cell genomics improves the discovery of risk variants and genes of Atrial Fibrillation [scATAC-seq]

status: Public on Feb 12 2023

first: Sebastian

last: Pott

state: IL

address: 920 E 58th St

institute: The University of Chicago

summary: Genome-wide association studies (GWAS) have linked hundreds of loci to cardiac diseases. However, in most loci the causal variants and their target genes remain unknown. We developed a combined experimental and analytical approach that integrates single cell epigenomics with GWAS to prioritize risk variants and genes. We profiled accessible chromatin in single cells obtained from human hearts and leveraged the data to study genetics of Atrial Fibrillation (AF), the most common cardiac arrhythmia. Enrichment analysis of AF risk variants using cell-type-resolved open chromatin regions (OCRs) implicated cardiomyocytes as the main mediator of AF risk. We then performed statistical fine-mapping, leveraging the information in OCRs, and identified putative causal variants in 122 AF-associated loci. Taking advantage of the fine-mapping results, our novel statistical procedure for gene discovery prioritized 45 high-confidence risk genes, highlighting transcription factors and signal transduction pathways important for heart development. We further leveraged our single-cell data to study genetics of gene expression. An unexpected finding from earlier studies is that expression QTLs (eQTLs) are often shared across tissues even though most regulatory elements are cell-type specific. We found that this sharing is largely driven by the limited power of eQTL studies using bulk tissues to detect cell-type-specific regulatory variants. This finding points to an important limitation of using eQTLs to interpret GWAS of complex traits. In summary, our analysis provides a comprehensive map of AF risk variants and genes, and a general framework to integrate single-cell genomics with genetic studies of complex traits.

overall_design: scATAC-seq

first: Alan

last: Selewa

first: Kaixuan

last: Luo

first: Xin

last: He


title: Gastric single-cell RNA-sequencing of mice with Helicobacter pylori infection and/or KRAS-driven metaplasia

status: Public on Feb 11 2023

city: Seattle

first: Taran

last: Gujral

state: WA

address: 1100 Fairview Ave N

institute: Fred Hutch Cancer Center

summary: Stomachs from mice +/- H. pylori infection, +/- induction of active KRAS in gastric Mist1-expressing cells, were used for gastric single-cell RNA-sequencing. Samples were obtained six and 12 weeks after infection/KRAS induction. Six samples were frozen, then thawed before sequencing (and in some cases pooled from multiple mice) and two samples were freshly prepared and never frozen before sequencing.

first: Valerie

middle: P

last: O'Brien

first: Yuqi

last: Kang

first: Meera

last: Shenoy

first: Greg

last: Finak

first: William

last: Young

first: Julien

last: Dubrulle

first: Lisa

last: Koch

first: Armando

last: Rodriguez Martinez

first: Jazmine

last: Snow

first: Jeffery

last: Williams

first: Elizabeth

last: Donato

first: Surinder

last: Batra

first: Cecilia

last: Yeung

first: Susan

last: Bullman

first: Meghan

first: Raphael

last: Gottardo

first: Nina

last: Salama


title: RNA-seq of two maize inbred lines B73 and Mo17

status: Public on Feb 10 2023

first: Qingyu

last: Xu

address: Changjiang Road,Xiangfang district

department: College of Agronomy

institute: Northeast Agricultural University

summary: Find differentially expressed genes under low temperature (10℃) and normal conditions (25℃) during maize germination

overall_design: The low temperature resistant lines B73 and one low temperature sensitive line Mo17 were used in this study. The seeds were sampled imbibition within the germinating paper under the ‘normal’ condition (25℃) for CK_B and CK_M and low temperature condition (10℃) for LT_B and LT_M,biologically repeated three times.Comparison of differentially expressed genes of two maize lines at low temperature by RNA-seq

first: Zhou

last: Yu

first: Wang

last: Zhenhua

first: Xu

last: Qingyu


title: Single-cell genomics improves the discovery of risk variants and genes of Atrial Fibrillation

first: Sebastian

last: Pott


title: Changes in social behaviour with alterations of MAPK3 and KCTD13/CUL3 pathways in two new outbred rat models for the 16p11.2 syndromes with autism spectrum disorders [Sprague Dawley]

first: Yann

last: HERAULT

summary: Copy number variations (CNVs) of the human 16p11.2 locus are associated with several developmental/neurocognitive syndromes. Particularly, deletion and duplication of this genetic interval are found in patients with autism spectrum disorders, intellectual disability and other psychiatric traits. The high gene density associated with the region and the strong phenotypic variability of incomplete penetrance, make the study of the 16p11.2 syndromes extremely complex. To systematically study the effect of 16p11.2 CNVs and identify candidate genes and molecular mechanisms involved in the pathophysiology, mouse models were generated previously and showed learning and memory, and to some extent social deficits. To go further in understanding the social deficits caused by 16p11.2 syndromes, we engineered deletion and duplication of the homologous region to the human 16p11.2 genetic interval in two rat outbred strains, Sprague Dawley (SD) and Long Evans (LE). The 16p11.2 rat models displayed  convergent defects in social behaviour and only a few cognitive defects. Interestingly major pathways affecting MAPK3 and CUL3 were found altered in the rat 16p11.2 models with additional changes in males compared to females. Altogether, the consequences of the 16p11.2 genetic region dosage on social behaviour are now found in three different species: humans, mice and rats. In addition, the rat models pointed to sexual dimorphism, with lower severity of phenotypes in rat females compared to male mutants. This phenomenon is also observed in humans. We are convinced that the two rat models will be key to further investigating social behaviour and understanding the brain mechanisms and specific brain regions that are key to controlling social behaviour.

overall_design:
Comparison of hippocampal RNA profile in the Sprague Dawley rat models for 16p11.2 syndromes between 4 genotypes (n=4 to 6 per genotype): wt, Del/Dup, Del/+ and Dup/+
Hippocampus from 4 Del/+ , 5 Dup/+  and 4 Del/Dup  and 6 wt littermates, were isolated and flash frozen in liquid nitrogen. Total RNA was prepared using an RNA extraction kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Samples quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA).

first: Sandra

last: Martin Lorenzo

last: Herault


title: Changes in social behaviour with alterations of MAPK3 and KCTD13/CUL3 pathways in two new outbred rat models for the 16p11.2 syndromes with autism spectrum disorders


title: Evolution of antagonism among DUX family members from an ancestral toxic single  homeodomain protein II

status: Public on Feb 08 2023

city: Bad Nauheim

first: Carsten

last: Kuenne

address: Ludwigstrasse 43

institute: Max Planck Institute for Heart and Lung Research

summary: Double homeobox genes are unique to eutherian mammals, transiently expressed in early blastomere-stage embryos, and comprise 3 clades (DUXA, B, C) evolved by homeobox duplication and divergence from an ancestral single homeobox gene, sDUX, present in other vertebrates. We test platypus sDUX and find that it drives cytotoxicity and inhibition of myogenesis identical to DUX4, the human DUXC gene. sDUX binds DNA as a head-to-head dimer, with protein core and DNA nearly overlapping the DUX4-DNA crystal structure. Although DUXA lacks transcriptional activity, DUXA-VP64 transcriptome and chromatin accessibility profiles significantly overlap those of DUX4 and sDUX, including expression of ZGA genes and LTR elements. Furthermore, DUXA binds DUX4 sites at most DUX4 target genes and counteracts transcriptional activity of DUX4, including in FSHD patient cells, potentiating a feedback inhibitory loop. The DUX gene family therefore comprises cross-regulating members of opposing function, with implications for their roles in ZGA, FSHD, and cancer.

overall_design: ChIP-seq was performed in duplicates starting from HEK293 cells that were transfected with a plasmid encoding for a V5-epitope tagged DUXA.

first: Felipe

last: Luettmann

first: Stefan

last: Günther

first: Johnny

last: Kim


title: Evolution of antagonism among DUX family members from an ancestral toxic single  homeodomain protein I

overall_design: ChIP-seq was performed in duplicates starting from HEK293 cells that were transfected with a plasmid encoding for a V5-epitope tagged DUX4.


title: Reduced phosphorylated ATM expression is associated with poor prognosis and gemcitabine resistance in pancreatic cancer

city: Singapore

first: Daisuke

last: Douchi

address: 14 Medical Drive

department: Centre for Translational Medicine

country: Singapore

institute: Cancer Science Institute of Singapore

summary:
Background & Aims: Loss of ataxia-telangiectasia mutated, occurring in patients with multiple primary malignancies, including pancreatic cancer, is associated with poor prognosis. This study investigated the detailed molecular mechanism through which ataxia-telangiectasia mutated expression affects the prognosis of pancreatic-cancer patients
Methods: Ataxia-telangiectasia mutated and phosphorylated ataxia-telangiectasia mutated levels in pancreatic-cancer patients who underwent surgical resection were analyzed using immunohistochemistry staining. RNA sequencing was performed on ataxia-telangiectasia mutated-knockdown pancreatic-cancer cells to elucidate the mechanism underlying the involvement of ataxia-telangiectasia mutated in pancreatic cancer.
Results: Immunohistochemical analysis showed that 15.3% and 27.8% of clinical samples had low levels of ataxia-telangiectasia mutated and phosphorylated ataxia-telangiectasia mutated, respectively. Low phosphorylated ataxia-telangiectasia mutated expression substantially reduced overall and disease-free survival in pancreatic-cancer patients. Loss of ataxia-telangiectasia mutated promoted MET and NTN1 over-expression via hypoxia-inducible factor-1α, thereby enhancing pancreatic-cancer cell proliferation and migration.
Conclusions: These results demonstrate that the loss of ataxia-telangiectasia mutated activates downstream proto-oncogenes, inhibits apoptosis, and promotes tumor growth; moreover, loss of phosphorylated ataxia-telangiectasia mutated leads to poor prognosis in pancreatic-cancer patients. Thus, ataxia-telangiectasia mutated may serve as a potential molecular marker to monitor patient prognosis and as a potential target for pancreatic cancer therapy

overall_design: RNA expression profile of ATM-knockdown MIA-PaCa2 and SUIT-2 cells. Total RNA was extracted from the ATM-knockdown MIA-PaCa2 and SUIT-2 cells.

first: Hideo

last: Ohtsuka

first: Jingyu

last: Xun

first: Katsuya

last: Hirose

first: Shun

last: Nakayama

first: Masaharu

last: Ishida

first: Takayuki

last: Miura

first: Kyohei

last: Ariake

first: Masamichi

last: Mizuma

first: Kei

last: Nakagawa

first: Takanori

last: Morikawa

first: Toru

last: Furukawa

first: Michiaki

last: Unno


title: Durable Alterations of Hematopoiesis and Chromatin post-COVID-19 [Bulk ATAC-seq]

status: Public on Feb 15 2023

first: Jin-Gyu

last: Cheong

address: 1300 York Avenue

institute: Weill Cornell Medicine

summary: Acute SARS-CoV-2 infection is often highly inflammatory and protracted. Recent advances have established that inflammation can trigger innate immune memory and a persistent influence on hematopoietic development, through epigenetic mechanisms. However, these phenotypes and their molecular and cellular features are poorly described in humans. Here we reveal epigenomic alterations in innate immune and hematopoietic stem and progenitor cells (HSPC) post-COVID-19, with distinct molecular programs across disease severities. Enabled by novel approaches to study hematopoiesis from peripheral blood, we find persisting HSPC epigenetic programs conveyed, for months to a year, to short-lived progeny monocytes. These epigenetic changes are associated with increased myeloid cell differentiation and inflammatory and antiviral programs. We provide insights into post-infectious HSPC and innate immune cell epigenetic alterations that are broadly relevant.

overall_design:
To discover changes in chromatin post-SARS-CoV-2 infection, ATAC-seq was performed on CD34+ HSPCs and CD14+ monocytes sorted from PBMC of healthy, non-COVID-19 post-ICU patients, mild convalescent, and severe convalescent COVID-19 patients.

Please note that the processed data *csv files contain the bulk data alongside pseudobulked atac counts data.

first: Arjun

last: Ravishankar

first: Siddhartha

last: Sharma

first: Christopher

middle: N

last: Parkhurst

first: Steven

middle: Z

last: Josefowicz

first: Duygu

last: Ucar


title: DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration

status: Public on Feb 07 2023

city: Los Angeles

first: John

middle: Duc

last: Nguyen

address: 3711 Baldwin Street, Unit 602

department: Stem Cell

institute: USC

summary: Purpose - To profile epigenetic changes in cochlear progenitor cells, hair cells, and supporting cells across postnatal development.  Methods-   Cochlear cell types were obtained from fluorescently labeled transgenic mice. E13.5 cochlear prosensory progenitor cells (E13.5_PG, p27Kip1-GFP+), P1 hair cells (P1_HC, Atoh1-GFP+), P1 supporting cells (P1_SC, Lfng-GFP+), and P6, P8, P21 supporting cells (P6_SC, P8_SC, P21_SC, Lfng-creER, NuTRAP lineage-traced) were FACS purified for epigenetic profiling using WGBS (DNA methylation), CUT&Tag (H3K4me1, H3K4me3, H3K27me3, CUTAC H3K4me2). DNA demethylation of hair cell-specific promoters was interrogated by comparing % mCpG profiles of transdifferentiating supporting cells (P1_SC_tdt_gfp, Lfng-creER/TdTomato+, Atoh1-GFP+) against non-transdifferentiating supporting cells (P1_SC_tdt, P6_SC_tdt, Lfng-creER/TdTomato+) purified via FACS. P1 and P8 wildtype cochleas were enzymatically dissociated and used directly for scMultiome simultaneous profiling of RNA and ATAC at the single cell level (P1_scMultiome, P8_scMultiome, 10x Genomics). For scMultiome of P70 wildtype (P70_wt_scMultiome, Lfng-CreERT2, NuTRAP) and P70 deafened (P70_deaf_scMultiome, Lfng-CreERT2, NuTRAP, Pou4f3DTR) supporting cells, mice received 100 mg/kg tamoxifen (Sigma-Aldrich T5648) at P21, 0.01 mg/kg Diptheria toxin (Sigma-Aldrich D0564) at P28, and allowed to mature to P70, at which point cochlear tissues were harvested, FACS purified for NuTRAP+ supporting cells, and inputted into a scMultiome reaction.  Results-   WGBS highlighted differentially methylated regions (DMR) across E13.5_PG, P1_HC, P1_SC, P6_SC, and P21_SC. Pre-established DMRs become hypermethylated in supporting cells between P1 and P21. This corresponds with increasing heterochromatin characteristics, such as loss of accessibility (CUTAC), loss of H3K4me1, and switch between H3K27me3-mediated repression to DNA methylation-mediated silencing. WGBS of P1_SC_tdt_gfp compared to P1_SC_tdt and P6_SC_tdt showed DNA demethylation of hair cell-specific DMRs, suggesting that DNA demethylation is required for transdifferentiation of supporting cells into hair cells. P1_scMultiome, P8_scMultiome, P70_wt_scMultiome, and P70_deaf_scMultiome revealed changes in chromatin accessibility associated with age, as well as from long-term deafening at the single cell level. scMultiome datasets also highlight cell type-specific enhancers active during different stages of postnatal development.

overall_design: Examination of epigenetic modifications of FACS purified mouse cochlear cells at P1, P6, P8, and P21. scMultiome ATAC + Gene Expression (10x genomics) experiments on P1 and P8 wildtype, as well as P70 wildtype and long-deafened cochlear sensory epithelia.

first: Andrew

middle: K

last: Groves

first: Neil

last: Segil

first: Juan

last: Llamas


title: Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability II

city: Bethesda

first: Alan

last: Hinnebusch

address: 6 Center Drive

department: Eunice Kennedy Shriver National Institute of Child Health and Human Development

institute: National Institutes of Health

summary: Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, and as a translational repressor, but their functions in cells are incompletely understood. We have analyzed these questions by a combination of ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs.

overall_design: It includes total 14 samples. pat1, pat1dhh1 (triplicates) and edc3scd6 (duplicates) were subjected to RNA-Seq. Separately, WT and dcp2 in triplicates (6 samples) were subjected to Rpb1 ChIP-seq. All strains were grown in YPD at 30ºC till the OD600 ~0.6

first: Anil

last: Vijjamarri

first: Xiao

last: Niu

first: Matthew

last: Vandermeulen

first: Chisom

last: Onu

first: Fan

last: Zhang

first: Hongfang

last: Qui

first: Neha

last: Gupta

first: Swati

last: Gaikwad

first: Miriam

last: Greenberg

first: Paul

last: Cullen

first: Zhenguo

last: Lin


title: Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons

last: McCallion

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

overall_design: Refer to individual Series


title: Rapid evolution of the embryonically-expressed homeobox gene LEUTX within primates

status: Public on Feb 06 2023

city: Oxford

first: Thomas

middle: Lewin

last: Lewin

state: Oxfordshire

address: 11a Mansfield Road

department: Department of Biology

country: United Kingdom

institute: Thomas Lewin

summary: LEUTX is a homeodomain transcription factor expressed in the very early embryo with a function around embryonic genome activation. The LEUTX gene is found only in eutherian mammals, including humans, but unlike the majority of homeobox genes, the encoded amino acid sequence is very different between divergent mammalian species. However, whether dynamic evolution has also occurred between closely related mammalian species remains unclear. In this work, we perform a comparative genomics study of LEUTX within the primates, revealing dramatic evolutionary sequence change between closely related species. Positive selection has acted on sites in the LEUTX protein, including six sites within the homeodomain; this suggests that selection may have driven changes in the set of downstream targets. Transfection into cell culture followed by transcriptomic analysis reveals small functional differences between human and marmoset LEUTX, suggesting rapid sequence evolution has fine-tuned the role of this homeodomain protein within the primates.

overall_design: Human dermal fibroblasts were transfected with either human LEUTX or the LEUTX gene of Callithrix jacchus.

first: Josephine

last: Blagrove

first: Peter

last: Holland


title: Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons [Hi-C]

first: Andrew

overall_design: 4 Promoter-capture Hi-C samples from SN4741 cells cultured at 39C

first: Rachel

last: Boyd

first: Sarah

last: McClymont

first: Nelson

middle: B

last: Barrientos

last: Hook

first: William

last: Law

first: Rebecca

last: Rose

first: Eric

last: Waite

first: Jay

last: Rathinavelu

first: Dimitrios

last: Avramopoulos


title: Histone bivalency regulates the timing of cerebellar granule cell development in vivo.

first: Kärt

last: Mätlik

state: NY

address: 1230 York Ave

institute: Rockefeller University