title: Divergent aging of nulliparous and parous mammary glands reveals IL33+ hybrid epithelial cells

status: Public on Sep 12 2024

city: Santa Cruz

first: Paloma

last: Medina

address: 1156 High St.

institute: University of California Santa Cruz

summary: Aging increases breast cancer risk while an early first pregnancy reduces a woman   s life-long risk. While several studies have explored the effect of either aging or pregnancy on mammary stem/progenitor cells, the combined effect of both remains unclear. Here, we interrogate the functional and transcriptomic changes at single-cell resolution in the mammary gland of aged nulliparous and parous mice to discover that pregnancy normalizes age-related imbalances in lineage composition, while also inducing a differentiated cell state. Importantly, we uncover a minority population of Il33-expressing epithelial cells that express both luminal and basal markers (i.e. hybrid), which accumulate in aged nulliparous mice but are significantly reduced in aged parous mice. Functionally, IL33 treatment of mammary epithelial cells from young mice phenocopies aged nulliparous epithelial cells, induces proliferation and promotes formation of organoids with Trp53 knockdown. Collectively, our study demonstrates that pregnancy blocks the age-associated imbalances in lineage integrity in the basal layer, including a decrease in Il33+ hybrid cells, that could potentially contribute to pregnancy-induced breast cancer protection.

overall_design: We performed scRNA-seq from mammary glands of 18m NP and 18m P mice using 10x chromium scRNA-seq (n=3 mice/group) as per manufacture’s protocol using the 5’ Gene expression workflow (1 GEM per condition, 3 mice pooled in equal proportions per condition) Libraries were sequenced on the Illumina NovoSeq 6000 Platform by Medgenome, Inc. Reads were mapped to the mouse genome (mm10). Additionally, we integrated an 18m NP data set from Tabula Muris Senis. For quality control, we used Scanpy’s ‘scanpy.pp.calculate_qc_metrics’, ‘scanpy.pp.filter_genes’, and scanpy.pp.filter_cells. We applied filters to eliminate (1) genes that are detected in less than 3 cells, (2) cells that have less than 200 genes, (3) cells with gene counts < 600 or > 8,000, (4) cells with total counts of UMIs per cell < 2,000 or > 12,000, and (5) cells with mitochondrial gene ratio > 1.5%. The mitochondrial gene ratio was defined as the percentage of UMIs mapped to mitochondrial genes compared to non-mitochondrial genes within each cell; cells with a high ratio are indicative of non-viable or apoptotic cells. Doublets were identified using Scrublet, these cells were then filtered out. After filtering, the datasets were concatenated, the UMI counts for each cell were then normalized using a target sum of 1e4, and then log transformed using ‘scanpy.pp.log1p’, which computes X=log(X+1), where log denotes the natural logarithm.

first: Andrew

last: Olander

first: Veronica

last: Haro Acosta

first: Sara

last: Kaushik

first: Matijis

last: Dijkgraaf

first: Shaheen

last: Sikandar


title: CCKBR+ cancer cells contribute to the intertumor heterogeneity of gastric cancer and confer sensitivity to FOXO inhibition

status: Public on Sep 02 2024

city: Hefei

first: Zhenya

last: Tan

address: 81th Meishan Road

country: China

institute: Anhui Medical University

type: Expression profiling by high throughput sequencing

summary: Background: The presence of heterogeneity and poor diagnosis in gastric adenocarcinoma poses a significant challenge to cancer treatment. To address the limited therapeutic options and enhance complex clinical management, we investigated the effects of targeting FOXO to inhibit CCKBR+ cancer cells in vitro and in vivo. Methods: We conducted single-cell sequencing (scRNA-seq) analysis on malignant epithelial cells from five gastric adenocarcinoma patients, revealing the notable heterogeneity within gastric adenocarcinoma (GA). We identified the oncological features of CCKBR+ tumors through immunohistochemistry and clinical statistical analysis. To validate the biological similarity of CCKBR+ tumors, we performed single-cell sequencing on datasets GSE183904 and TCGA. The mutational hallmarks of CCKBR+ tumors were detected using OncoDriveFML and GISTIC 2.0. To further investigate the potential target of CCKBR+ tumors, we utilized organoids and tumor xenograft mouse models, assessing the impact of FOXO inhibition. Finally, we employed CUT&Tag and flow cytometry to determine the genes regulated by FOXO in CCKBR+ tumors.  Results: We identified a subset of CCKBR+ stem cell-like cancer cells associated with poorly differentiated features and a worse prognosis in gastric adenocarcinoma. This finding was validated through additional single-cell RNA sequencing (scRNA-seq), TCGA analysis, and histopathology, underscoring the heterogeneity of GA. The unique propensity for gene mutations, such as TP53, APC, SMAD4, and GLI3, was observed in CCKBR+ tumors. Mechanistically, our investigation revealed that FOXOs serve as key transcription factors in maintaining the homeostasis of CCKBR+ tumors. Inhibition of FOXO was found to suppress the growth of CCKBR+ organoids and xenograft tumors. Notably, FOXOs were identified as regulators of α2,3 sialylation levels in CCKBR+ tumor cells, shedding light on a crucial aspect of their homeostasis. Conclusions: CCKBR+ tumor cells significantly contribute to the intertumor heterogeneity of gastric cancer, leading to lower differentiation potential and a poorer prognosis. FOXO plays a crucial role in maintaining the homeostasis of CCKBR+ tumors by upregulating sialylation levels mediated by ST3GAL3/4/5 and ST6GALNAC6. Targeting FOXO effectively suppresses the progression of CCKBR+ tumors. This insight opens new avenues for developing targeted therapeutic strategies aimed at treating cancers associated with CCKBR, providing a novel perspective for precision medicine in this context.

overall_design:
We conducted single-cell sequencing (scRNA-seq) analysis on malignant epithelial cells from five gastric adenocarcinoma patients.
10X Genomics Chromium Single-Cell 3’ kit (V3) was used to load 10x Chromium for capturing a maximum of 13000 single cell form single cell suspensions, and following cDNA amplification and library construction were performed according to the manufacturer’s instructions. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. Cell Ranger (vision 5.0.0) pipeline was used for sample demultiplexing, barcode processing and gene counting. For dimensional reduction, clustering and downstream analysis, Cell Ranger output was loaded to Seurat (version 3.1.1) after genome aligning. The STAR aligner was used for generating elementary data, including reads to transcriptome, normalized matrix of gene counts versus cells across samples.


title: Prototypical innate immune mechanism hijacked by leukemia-initiating mutant stem  cells for selective advantage and immune evasion in Ptpn11 mutation-associated juvenile myelomonocytic leukemia

summary: Juvenile myelomonocytic leukemia (JMML), a clonal hematologic malignancy, originates from mutated hematopoietic stem cells (HSCs) with genetic alterations in the Ras signaling pathway. The mechanism sustaining the persistence of these mutant stem cells leading to leukemia development remains poorly understood. In this study, we conducted comprehensive examination of gene expression profiles, transcriptional factor regulones, and cell compositions throughout various stages of tumor cell development in a mouse model of Ptpn11 mutation-associated JMML. Our analyses unveiled that leukemia-initiating Ptpn11E76K/+ mutant stem cells exhibited de novo activation of the myeloid transcriptional program and aberrant developmental trajectories. Intriguingly, these mutant stem cells displayed significantly elevated expression levels of anti-microbial molecules and pro-inflammatory proteins, particularly S100a9 and S100a8. These proteins conferred a selective advantage to leukemia-initiating cells through autocrine effects and facilitated immune evasion by recruiting and promoting immune suppressive myeloid-derived suppressor cells (MDSCs) to the microenvironment. Importantly, pharmacological inhibition of S100a9/S100a8 signaling effectively impeded leukemia development from Ptpn11E76K/+ mutant stem cells. These findings collectively suggest that JMML tumor-initiating cells exploit evolutionarily conserved innate immune and inflammatory mechanism to establish clonal dominance.

overall_design: Fresh BM cells were collected from Ptpn11E76K/+/Mx1-Cre+ and Ptpn11+/+/Mx1-Cre+ mice, bone marrow cells from 3 mice per group were mixed together, The Erythrocytes were lysed, followed by the execution of the recommended protocol for the scRNA-seq 10x Genomics platform using v3 chemistry.


title: Lysine Crotonylation Acts as an Epigenetic Switch for Glutamate Neurotransmission and Spatial Memory

status: Public on Nov 13 2024

city: Iowa City

first: Snehajyoti

last: Chatterjee

state: IA

address: 169 Newton Road

department: Neuroscience & Pharmacology

country: USA

institute: University of Iowa

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Using a single-nucleus multiomics approach, this study demonstrates that enhancing histone lysine crotonylation modulates cell type–specific chromatin accessibility and drives the expression of genes encoding key proteins involved in glutamatergic neurotransmission during memory consolidation.

overall_design: To study the impact of crotonate administration on memory enhancement, mice were trained in Spatial Object Recognition (SOR) task using a sub-threshold training paradigm, and oral administration of crotonate (200 mg/kg) or vehicle (Saline) was given immediately after the completion of the training trials. 1hr after crotonate administration, the dorsal hippocampus was harvested, and nuclei were isolated for single nuclei multiomics. We performed multiomic sequencing (snRNA-seq + snATAC-seq) obtained from the dorsal hippocampus of 2 groups (Crotonate1+Crotonate2 vs. Saline1+Saline2). Chromatin Immunoprecipitation followed by sequencing (ChIP-Seq) was performed using a pan-Kcr antibody on the dorsal hippocampus tissue from the mice that were either housed in homecage or trained in the SOR task.

first: Utsav

last: Mukherjee

first: Budhaditya

last: Basu

first: Stacy

middle: E

last: Beyer

first: Saaman

last: Ghodsi

first: Nathan

last: Robillard

first: Yann

last: Vanrobaeys

first: Eric

middle: B

last: Taylor

first: Ted

last: Abel


title: A Co-Culture Model of Dopaminergic and Glutamatergic Neurons Derived from Patients with Idiopathic Schizophrenia reveals a Hypodopaminergic Phenotype

status: Public on Aug 27 2024

city: Reutlingen

first: Hansjuergen

last: Volkmer

address: Markwiesenstr 55

country: Germany

institute: NMI at the University of Tuebingen

summary:
To identify morphological and functional phenotypes relevant for SCZ, we generated iPSC-derived dopaminergic neurons from three healthy controls and four patient with schizophrenia.
We then performed gene expression profiling analysis using data obtained from RNA-seq of four schizophrenia patients and three controls to determine significantly deregulated genes in schizophrenia dopaminergic neurons.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for healthy control- and schizophrenia patient-derived iPSC-based dopaminergic neurons

first: Sophia-Marie

last: Hartmann

first: Patricia

last: Pizarro Garcia

first: Johanna

last: Heider

first: Sabrina

last: Vogel

first: Lisa-Sophie

last: Wüstner

first: Richard

last: Wüst

first: Andreas

middle: J

last: Fallgatter

first: Hansjürgen


title: Long-read sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations in cancer (SF3B1 & U2AF2 iCLIP)

status: Public on Aug 19 2024

city: Würzburg

first: Julian

last: Konig

address: Am Hubland

institute: Würzburg University

summary: Alternative splicing plays a critical role in generating transcriptome diversity, and aberrant splicing is frequently observed in cancer. Mutations in the splicing factor SF3B1 are particularly common in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS), with different survival prognoses. We applied long-read sequencing for the investigation of the SF3B1 mutation effect on the transcriptome of MDS and CLL patients, as well as isogenic cell lines. Our results revealed that SF3B1 mutation effect was common across the different diseases and specifically altered the usage of 3’ alternative splice sites within short proximity to the canonical splice sites. Based on computational simulations, the most common K700E mutation led to destabilization of the SF3B1-mRNA binding. Furthermore, we combined the full isoform information with genome-wide SF3B1-RNA binding maps to predict the functional consequences of the aberrant splicing and gain mechanistic insights into the role of mutated SF3B1 in splicing.

overall_design: To understand the RNA binding behavior of SF3B1, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to map SF3B1 binding sites throughout the transcriptome. For this, we immunoprecipitated SF3B1 from K562-SF3B1wt/wt and K562-SF3B1K700E/wt cells,

first: Heike

last: Hänel

last: König

first: Anke

last: Busch

first: Kathi

last: Zarnack

first: Christina

last: Grimm

first: Michal

middle: R

last: Schweiger

first: Mirko

last: Brüggemann


title: Xist RNA targets selective autosomal genes to modulate expression in a Repeat B-dependent manner [CHART-seq]

status: Public on Aug 23 2024

city: Boston

first: Shengze

last: Yao

state: MA

address: 185 Cambridge St

department: Molbio

institute: Massachusetts General Hospital

summary: Xist, a pivotal player in X chromosome inactivation (XCI), has long been perceived as a cis-acting long noncoding RNA that binds exclusively to the inactive X chromosome (Xi). However, Xist’s ability to diffuse under select circumstances has long been documented, leading us to suspect that Xist RNA may have targets and functions beyond the Xi. Here, using female mouse embryonic stem cells (ES) and mouse embryonic fibroblasts (MEF) as a model, we demonstrate that Xist RNA indeed can spread beyond the Xi. However, its binding is limited to ~100 genes in differentiating ES and MEF cells. The target genes are diverse in function but are unified by their active chromatin status. Although active, Xist binding is associated with a downregulation of gene expression. Unlike on the Xi, Xist binding does not lead to full silencing and also does not spread beyond the target gene. Deleting Xist’s Repeat B (RepB) domain reduces Xist’s diffusion away from the Xi and inhibits Xist’s localization to autosomal targets. Nonetheless, Xist transcript lacking RepB fails to down-modulate autosomal gene expression. Altogether, our findings reveal Xist targets beyond the Xi and identify Repeat B as a crucial domain for its in-trans function.

overall_design:
Xist localization patterns in various cell lines were examined at nucleotide resolution by CHART-seq. And RNA-seq was used to detect the gene expression level in the cells.

UPDATE: [Dec-18-2025] The genotypes of Samples GSM8368731-GSM8368736 were changed to wild-type.

first: Yesu

last: Jeon

first: Barry

last: Kesner

first: Jeannie

middle: T

last: Lee


title: m6A sites in the coding region trigger translation-dependent mRNA decay.

status: Public on Nov 20 2024

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

overall_design: Refer to individual Series


title: m6A sites in the coding region trigger translation-dependent mRNA decay (ribosome profiling)

summary: N6-Methyladenosine (m6A) is the predominant internal RNA modification in eukaryotic messenger RNAs (mRNAs) and plays a crucial role in mRNA stability. In this study, we reveal that m6A sites in the coding sequence (CDS) trigger CDS–m6A decay (CMD), a novel mRNA decay pathway that is distinct from previously reported m6A-dependent degradation mechanisms. Importantly, CDS m6A sites act considerably faster and more efficiently than those in the 3' untranslated region, which to date have been considered the main effectors of m6A-mediated RNA decay. Mechanistically, CMD depends on translation whereby m6A deposition in the CDS induces ribosome pausing and transcript destabilization. We found that the target transcripts of CMD are recognized by the m6A reader protein YTHDF2, selectively enriched in processing bodies (P-bodies) and degraded via the decapping factor DCP2. Our findings highlight CMD as a previously unknown pathway for m6A-mediated decay, which is particularly important for controlling the expression of developmental regulators and retrogenes.

overall_design: To investigate changes in translation in response to m6A depletion, we performed ribosome profiling (Ribo-seq) and matched RNA-seq in HEK293T cells subjected to m6A depletion for 6 h using METTL3 inhibitor STM2457. The Ribo-seq experiment performed in duplicate and the matched RNA-seq performed with 4 replicates.

first: Miona

last: Corovic

first: Mikhail

last: Mesitov

first: You

last: Zhou


title: Spatially-resolved gene expression patterns of fibrosing ILDs [RNA-Seq]

status: Public on Sep 30 2024

city: London

first: Marco

last: Mura

state: Ontario

address: 800 Commissioners Road E

department: Medicine, Respirology

country: Canada

institute: Western University

summary: Fibrosing interstitial lung diseases (ILDs) encompass a diverse range of scarring disorders that lead to progressive lung failure. Previous gene expression profiling studies focused on idiopathic pulmonary fibrosis (IPF) and bulk tissue samples.  We employed digital spatial profiling to gain new insights into the spatial resolution of gene expression across distinct lung microenvironments (LMEs) in IPF, chronic hypersensitivity pneumonitis (CHP) and non-specific interstitial pneumonia (NSIP). We identified differentially expressed genes between LMEs within each condition, and across histologically similar regions between conditions. Uninvolved areas in IPF and CHP were remarkably distinct from normal controls, and displayed potential therapeutic targets. Hallmarks LMEs of each condition retained a distinct gene signature, but these could not be reproduced in matched lung tissue samples. Based on these gene expression signatures and unsupervised clustering, we grouped previously unclassified ILD cases into NSIP or CHP. Lastly, we characterized a gene expression pattern associated with poor outcome .  Overall, our work uniquely dissects gene expression profiles between LMEs within and across different types of fibrosing ILDs. This new spatial transcriptomics approach has the potential to reclassify unclassifiable cases, to qualify the transcriptional relevance of smaller biopsies for clinical use, and to predict outcome at the time of diagnosis.

overall_design:
Of the 12 cases of fibrosing ILD analyzed with Nanostring DSP, seven fresh-frozen, whole lung tissue samples (2 IPF, 2 CHP and 3 NSIP) were obtained at the time of intial SLB.
Differential gene expression analysis was performed on bulk RNA-seq of the seven samples to compare the sensitivty between bulk and Nanostring DSP in identifying differentially expressed genes.
Comparative gene expression analysis of RNA-seq from three different types of fibrosing ILDS (IPF, CHP and NSIP)


title: mRNA expression profiles in hearts from sepsis-induced myocardial dysfunction mouse modles and controls

status: Public on Aug 09 2024

city: Changsha

first: Weizhi

last: Zhang

state: Hunan

address: Department of Cardiovascular Surgery, The Second Xiangya Hospital of Central South University, No. 139 Middle Renmin Road, Changsha, Hunan, 410011, China.

department: Department of Cardiovascular Surgery

summary: In order to explore the genes and molecular mechanisms involved in the development and progression of sepsis-induced myocardial dysfunction, RNA sequencing was used to examine the total RNA in heart tissues of disease model mice and control individuals, and to generate this transcriptome dataset to compare gene expression differences between the two groups of tissues at the transcriptional level.

overall_design: 10-week-old healthy, specific pathogen-free, and wild-type C57BL/6 male mice were randomly assigned to the PBS group and the LPS group (n=5/group). In order to construct mouse models of sepsis-induced myocardial dysfunction, the LPS group were given a single intraperitoneal injection of 10 mg/kg LPS, while the PBS group were given an equal volume of PBS as controls.  After 12 hours of treatment, heart tissue from the mice were collected for the RNA extraction, purification, quality assessment, reverse transcription, cDNA library construction, and RNA sequencing.

first: Hanyi

first: Zixi

last: Xiao

first: Dongping

last: Li


title: Multi-omics analysis of early reperfused ischemic heart reveals ERRβ/γ activation protects against acute myocardial infarction injury

status: Public on Aug 01 2024

city: Hangzhou

first: Li

last: Shen

state: Zhejiang Province

address: 866 Yuhangtang Road

institute: Life Sciences Institute, Zhejiang University


title: Multi-omics analysis of early reperfused ischemic heart reveals ERRβ/γ activation protects against acute myocardial infarction injury [bulk RNA-seq]

summary: The rats were subjected to left anterior descending (LAD) coronary artery ligation for 1 hour, 6 hours (MI1h and MI6h) and 24 hours (MI24h) along with Sham controls (Sham), and 1-hour, 6-hour ischemia followed by reperfusion (MI1h/R and MI6h/R, mimicking early time reperfusion (ETR) and late time reperfusion (LTR), respectively) followed by heart tissue collection 24 hours post MI to mimic the MI/R injury in AMI patients and pretreatment with FDA-approved PPARα agonist fenofibrate for each group.

overall_design: Bulk RNA sequencing performed with rats subjected to left anterior descending (LAD) coronary artery ligation for 1 hour, 6 hours (MI1h and MI6h) and 24 hours (MI24h) along with Sham controls (Sham), and 1-hour, 6-hour ischemia followed by reperfusion (MI1h/R and MI6h/R, mimicking early time reperfusion (ETR) and late time reperfusion (LTR), respectively) followed by heart tissue collection 24 hours post MI to mimic the MI/R injury in AMI patients and pretreatment with FDA-approved PPARα agonist fenofibrate for each group.

first: Yun

last: Gao

first: Fei

last: Huang

first: Fanwei

last: Ruan


title: Therapeutic silencing Spp1 in cardiac macrophages suppresses atrial fibrillation

first: I-Hsiu

state: Massachusetts

address: 185 Cambridge Street

department: Center for Systems Biology

institute: Mass General Hospital/Harvard University

summary: Atrial fibrillation (AFib) and the risk of its lethal complications are worsened by atrial fibrosis. A recent study implicates osteopontin (encoded by Spp1) secreted by atrial TREM2+ macrophages in this fibrosis. Here, we show that silencing Spp1 in TREM2+ cardiac macrophages using an antibody-siRNA conjugate (ARC) reduces atrial fibrosis and suppresses AFib, thus offering a new immunotherapy for this common arrhythmia.

overall_design:
Single-nucleus RNA sequencing was used to investigate the transcriptomic differences between nuclei isolated from left atrial tissues collected from 3 vehicle- and 3 anti-Trem2-siSpp1-treated HOMER mice.
Bulk RNA sequencing was used to investigate the transcriptomic differences between mouse bone marrow-derived macrophages treated with vehicle (lipofectamine) or siSpp1 for 24 hours.

first: Noor

last: Momin

first: Steffen

last: Pabel

first: Kamila

last: Naxerova

first: Matthias

last: Nahrendorf

first: Maarten

last: Hulsmans


title: The impact of vaping on adolescent lung function and nasal epithelial gene expression [Genome wide DNA methylation]

status: Public on Jul 05 2024

city: Aurora

first: Sunita

last: Sharma

state: CO

address: 13001 East 17th Place

department: Anschutz Campus - Sch of Med - Pulmonary Sciences

institute: University of Colorado - Anschutz Medical Campus

type: Methylation profiling by genome tiling array

summary: Genome wide DNA methylation profiling of nasal-epithelial swabs collected from vaping and non-vaping (within 6 months) adolescents from Denver, Aurora, and Pueblo, CO . The Illumina Infinium Human Methylation 850K beadchip profiling microarray  was used to obtain DNA methylation profiles across approximately 868,000 CpGs. Samples included 11 swabs from adolescents who had vaped in the last 6 months, and 37 who had not.

overall_design: Identifying differentially methylated positions and regions (DMPs and DMRs) along the genome.

first: Sarah

last: Commodore

first: Jonathan

last: Hawkins

first: Cheyret

last: Wood

first: Cuining

last: Liu

first: Nancy

last: Perez

first: Margaret

last: Cruse

first: Vong

last: Smith

first: Eszter

last: Vladar

first: WeiChu

last: Hong

first: Russell

middle: P

last: Bowler

first: Laura

last: Alexander Crotty

first: Peter

last: Castaldi

first: Patricia

last: Valverde

first: Fernando

last: Holguin

first: Katerina

last: Kechris


title: The impact of vaping on adolescent lung function and nasal epithelial gene expression


title: Effect of RNAi-mediated knockdown of unc-13 and Rbp on gene expression in motor neurons of Drosophila melanogaster third instar larva

status: Public on Aug 28 2024

city: Berkeley

first: Caroline

last: Cypranowska

state: CA

address: 271 Weill Hall #3200

department: Molecular & Cell Biology

institute: University of California, Berkeley

summary: Reliable neurotransmission is dependent on coordinating entry of calcium into the terminal boutons of a presynaptic neuron with the exocytosis of vesicles into the synaptic cleft. This function fulfilled by an assembly of proteins in a specialized region of the presynaptic plasma membrane called the active zone (AZ). Here we use the fruit fly neuromuscular junction as a model synapse to test the consequences of specific depletion of two key AZ proteins, unc-13 and RIM-binding protein (Rbp), by RNAi knockdown on the transcriptome of these motor neurons. We find that decreased expression of Rbp results in concomittant decrease of expression in unc-13 and that the sets of differentially expressed genes between the two knockdowns are highly similar. We show that these knockdowns result in increased expression of genes involved in neuropeptide signalling, while reducing expression of other genes encoding the components of the AZ and voltage-gated K+ channels. Taken together, our findings suggest that knockdown of AZ components results in gene expression changes that bias the presynaptic neuron towards increased firing to compensate for the loss of AZs.

overall_design: To investigate the consequences of impaired neurotransmission on the transcriptome of motor neurons, we made use of two fruit fly lines, one with Gal4-mediated control of nuclear mCherry driven by the motor neuron-specific OK6 promoter and another with dsRNA constructs against unc-13 or Rbp in the attP2 insertion site. Rbp and unc-13 knockdowns were compared to controls with an empty attP2 site. Motor neurons were isolated from dissected and dissociated brain tissue from each genotype by FACS and each population profiled by RNA-seq.

first: Ehud

last: Isacoff


title: The impact of vaping on adolescent lung function and nasal epithelial gene expression [RNA-seq]

first: Sunita

last: Sharma

summary: Electronic cigarette use (vaping) among adolescents is a pressing public health issue. We aimed to investigate the impact of vape use on lung function and nasal epithelial gene expression in adolescents. We hypothesized that vaping results in abnormal lung function and differential expression of inflammatory genes in the nasal epithelium of adolescent vape users in comparison to non-vape users. We assessed airflow obstruction with impulse oscillometry (IOS) and collected nasal epithelial brush samples for genome-wide gene expression and targeted DNA methylation analyses in middle and high school students in Colorado. Written informed consent was obtained from participants and Institutional Review Board approval was obtained. Vape users [mean age (standard deviation): 14.8 (1.4) years] reported vaping within the past 6 months while non-vape users [14.8 (1.4)] years did not report any vape use in the past 6 months. Mean airway resistance identified by the IOS R5 measure revealed that vape users (n=13) had higher values than non-vape users (n=37) [p=0.026]. Of 16,860 nasal epithelial genes tested, 7,136 were significantly differentially expressed between vape and non-vape users (false discovery rate (FDR) <0.05), after covariate adjustment. Enrichment analyses identified overexpression of inflammatory response genes and underexpression of ciliogenesis genes in vape users compared to non-vape users. DNA methylation analysis revealed that REXO1 (FDR=0.01) and CERK (FDR=0.06) were differentially methylated among vape users compared to non-vape users. Vaping during adolescence may increase airway resistance and dysresgulation of nasal epithelial genes, including genes involved in airway inflammation and ciliary function.

overall_design: Gene expression profiling analysis for nasal-epithelial tissue swabs collected from 11 adolescents who self-reported vape use in the past 6 months and 36 adolescents who did not self-report vape use.

first: Sarah

last: Commodore

first: Wei

middle: C

first: Bowler

last: Russell


title: RNA splicing and alternative polyadenylation profile during sheep zygotic genome activation

status: Public on Jun 20 2024

city: Nanjing

first: Mingtian

last: Deng

address: 1st weigang

institute: Nanjing Agricultural University

summary: Zygotic genome activation (ZGA) is essential for early embryo development. Here, we reported that ZGA is initiated at 16-cell stage in sheep, as extensive alterations in gene expression and DNA methylation patterns, along with elevated levels of RNA polymerase II and developmental arrest was found at the 16-cell embryos. To uncover the sophisticated RNA metabolism during ZGA, we conducted weighted gene co-expression network analysis and identified 1957 critical maternal genes. Using dapars, we found 1058 and 933 lengthened alternative polyadenylation (APA) events, and genes exhibiting shorten APA were highly expressed in both sheep and mice ZGA-stage embryos. Using rMATs, we reported 2675 and 1963 genes showed exon skipping during ZGA in sheep and mice. These genes are related to RNA binding, translation, gamete generation, and reproduction.

overall_design: Briefly, 20 mature oocytes were transferred to a 100-μL micro-drop of BO-IVF medium (#71004, IVF Bioscience, Falmouth, UK) with mineral oil covering the surface. Then, capacitated sperm were added to the micro-drops and cultured for 16 h at 38.5℃, 5% CO2, and saturated humidity. The zygotes were transferred to BO-IVC embryo culture medium (#71005, IVF bioscience) for further culture. The 2-, 4-, 8-, and 16-cell stage embryos, as well as the mature oocytes, morula, early blastocysts, and the expanded blastocysts were collected at defined points for RNA sequencing (RNA-seq).


title: Translation dysregulation in cancer as a source for targetable antigens.

city: Palo Alto

first: Christopher

last: McGinnis

address: Biomedical Innovations Building 4600, 240 Pasteur Drive

department: Pathology

institute: Stanford University

summary: We used MULTI-seq (McGinnis, Patterson, et al., Nature Methods, 2019) to perform multiplexed single-cell RNA-sequencing analysis of cryopreserved primary melanoma tumor samples isolated from mice inoculated with TYW2 WT or KO tumors. Presented data represent CD45+ immune cells isolated from both tumor backgrounds at two timepoints (D18 and D21).

overall_design: MULTI-seq barcoded CD45+ mouse immune cells were isolated using FACS and analyzed using 3' scRNA-seq (10x Genomics)

first: Chen

last: Weller

first: Osnat

last: Bartok

first: Ansuman

last: Satpathy

first: Yardena

last: Samuels


title: Effects of the receptor-like protein tyrosine phosphatase PTPRH overexpression on gene expression in non-small cell lung cancer cell lines

status: Public on Jun 18 2024

city: East Lansing

first: Mylena

last: Ortiz

state: Michigan

address: 567 Wilson Road

department: Genetics and Genomics Sciences

institute: Michigan State University

summary: The delicate balance of protein phosphorylation is often disrupted in cancers, with hyperactivity of kinases and inactivation of phosphatases driving cell proliferation and survival pathways. PTPRH, a receptor-like protein tyrosine, is deregulated or mutated in certain cancers, including non-small cell lung cancer (NSCLC). Nonetheless, the biological processes in which PTPRH is involved and how it may contribute to tumorigenesis are unknown. We aimed to understand how overexpression of PTPRH affects tyrosine kinase signaling and in which biological processes this tyrosine phosphatase is involved, shedding light on how its inactivation may support cancer progression.

overall_design: To understand how PTPRH affects cell signaling pathways, we generated a NSCLC cell line PTPRH knockout (NCI-H23 PTPRH knockout) and stably overexpressed human PTPRH wild-type in two NSCLC cell lines (NCI-H23 PTPRH knockout and NCI-H2023). The parental cells expressing endogenous PTPRH were used as controls. Cells were serum starved for 24 hours, followed by FBS stimulation for 16 hours prior to collection of total RNA. Sequencing of the mRNA was performed in the Illumina platform (PE150) with a minimum coverage of 30M reads. We used n=3 technical replicates/group.

first: Mylena M.

middle: O

first: Matthew

last: Swiatnicki

first: Eran

last: Andrechek

first: Deeya

middle: M

last: Patel

first: Jesus

middle: A

last: Garcia-Lerena

first: Andrew

last: Nelson


title: Hypoxia drives HIF2-dependent reversible macrophagecellcycle entry

status: Public on Jun 13 2024

city: Cambridge

first: Niek

last: Wit

state: Not US or Canada

address: Puddicombe Way

department: Department of Medicine

country: United Kingdom

institute: University of Cambridge

summary: Macrophages play critical roles across health and disease. Low oxygen conditions (hypoxia) have been associated primarily with cell cycle arrest in dividing cells. Macrophages are typically quiescent in G0, though yolk sac and bone marrow derived macrophages frequently proliferate and monocyte-derived tissue macrophages are able to proliferate in response to tissue signals. Here we show that hypoxia (1% oxygen tension) results in reversible entry into the cell cycle in human monocyte derived macrophages (MDM) and mouse peritoneal macrophages. Cell cycle progression is largely limited to G1/S phase with little progression to G2/M. Mechanistically, this cell cycle transitioning is triggered by a HIF2A-directed transcriptional program. The response is accompanied by increased expression of cell cycle-associated proteins, including CDK1, and reversible activation of the canonical mitogen-activated MEK-ERK proliferation pathway. CDK1 associated SAMHD1 phosphorylation at T592 in hypoxic macrophages renders them hyper-susceptible to lentiviral transduction. Furthermore, PHD inhibitors, which activate HIFs, are able to recapitulate HIF2A-dependent cell cycle entry in macrophages, as well as susceptibility to lentiviral transduction. Finally, we demonstrate that tumour associated macrophages (TAM) in lung cancers exhibit transcriptomic profiles representing responses to low oxygen and cell cycle progression at single cell level. This work uncovers HIF2A driven macrophage cell cycle progression in low oxygen conditions that culminates in SAMHD1 phosphorylation and high susceptibility to lentiviral transduction. These findings have additional implications for inflammation and tumour progression/metastasis where low oxygen environments are common.

overall_design: To investigate the transcriptional responses of macrophages in response to hypoxia, we generated RNA-Seq data sets from normoxic and hypoxic conditions.

first: Bo

last: Meng

first: James

last: Nathan

first: Ravindra

middle: K

last: Gupta


title: Small RNA-mediated genetic switches coordinate ALG-3/4 small RNA pathway function

status: Public on Jun 27 2024

city: Arlington

first: Alicia

middle: Kathryn

last: Rogers

state: Texas

address: 1225 W Mitchell Street

department: Department of Biology

institute: University of Texas at Arlington

type: Non-coding RNA profiling by high throughput sequencing


title: Investigating the factors that regulate dengue virus infection in Aedes aegypti female mosquitoes with Aedes aegypti transcriptomic analysis

status: Public on Jun 03 2024

city: Taipei

first: Yu-Ning

last: Huang

address: No.1 Jen Ai road section 1

department: Biochemistry and molecular biology

country: Taiwan

institute: National Taiwan University

summary:
Understanding the molecular determinants of the mosquitoes’ competence to host the viruses holds promise to prevent their transmission.
We performed transcriptomic analysis with data obtained from RNA-seq of dengue- or naïve- infected mosquitoes on day 1, 2, and 7 post infections

overall_design: Based on the gene expression profile of RNA-seq analysis, we compared different days of the gene expression ratio between dengue and naïve infection. The different groups of transcripts were clustered and were analyzed with KEGG pathway analysis.

first: Kuan-Ying

last: Lee

first: Shin-Hong

last: Shiao

first: Chun-Hong

last: Chen

first: Guann-Yi

last: Yu

first: Ming-Jiun


title: ILC2s navigate tissue redistribution during infection using stage-specific S1P receptors [RNA-seq]

status: Public on Jun 05 2024

city: New York

first: Yuefeng

state: NY

address: 701W 168th Street

department: Microbiology & Immunology

institute: Columbia University

summary: Lymphocytes can circulate as well as take residence within tissues. The mechanisms by which circulating populations are recruited to infected tissue sites have been characterized, but the extent and molecular basis of tissue-resident cell recirculation is enigmatic. Here, we show that helminth infection- or IL-25-induced redistribution of intestinal tissue-localized group 2 innate lymphoid cells (ILC2s) requires access to the lymphatic vessel network even though secondary lymphoid structures, which are essential signal hubs for adaptive lymphocyte differentiation and dispatch, are dispensable for tissue ILC2 mobilization. IL-25 signals induce a dramatic change in epigenetic landscape of intestinal ILC2s, enabling the expression of sphingosine-1-phosphate (S1P) receptors. S1PR5 is critical for ILC2 exit from intestinal tissue to lymph, whereas S1PR1 plays a dominant role in ILC2 egress from mesenteric lymph nodes to blood circulation and then to lung, where redistributed ILC2s contribute to tissue repair. The requirement of two S1PRs is largely due to the dynamic expression of the tissue-retention marker CD69, which mediates S1PR1 internalization. This stage-specific requirement of different S1P receptors for ILC2 redistribution during infection illustrates that innate and adaptive lymphocytes share a circulatory network frame but employ specialized navigation cues for their distinct migratory requirements.

overall_design: iILC2s were sort-purified from the gut, mesenteric lymph node, and the lung of C57BL/6 mice (iILC2: Lin- CD45+ KLRG1+ ST2-) on day 4 after IL-25 injection. ILC2 were sort-purified from the gut of C57BL/6 mice ((ILC2: Lin- CD45+ KLRG1+ ST2+).

first: Yoshihiro

last: Ishida

first: Takamasa

last: Ito


title: Development of a novel culture medium for embryo transfer using a dye library.

status: Public on May 28 2024

city: Kagoshima

first: Sumito

last: Matsuya

address: 1-21-24 Korimoto

department: Joint Faculty of Veterinary Medicine

country: Japan

institute: Kagoshima university

summary: The effects of dyes that enhance visibility during embryo transfer on embryonic development were analyzed.

overall_design: Each dye was added to M16 medium and cultured, and the variation of expressed genes in blastocysts was analyzed.

first: Hiroyuki

last: Imai

first: Tatsumi

last: Uemura

first: Kaoru

last: Fujino

first: Toshiaki

last: Kawabe

first: Kiyoshi

last: Kano

first: Ken

last: Kusakabe


title: Bulk RNA sequencing of kidney in newborn non-CF and CF pigs

status: Public on Jun 07 2024

city: Iowa city

first: Wenjie

address: 200 Hawkins Dr, 6330 PBDB

institute: The University of Iowa

summary: We did bulk RNA sequencing in newborn cystic fibrosis (CF) and non-CF pig kidney. We compared kidney gene expression profiling between non-CF and CF pigs. RNA sequencing results showed that there is not significant difference between non-CF and CF in terms of gene expression, suggesting that CFTR knockout does not affect kidney development in newborn pigs.

overall_design: Kidney cortex tissue were collected from 6 wild-type (WT), 6 CFTR+/- (HET), and 11 CFTR-/- (CF) newborn pigs. We tried the best to collect the tissue from a similar position of the kidneys. RNA was extracted using a RNeasy Lipid Tissue Kit (Qiagen). cDNA libraries were prepared using the Illumina TruSeq Stranded Total RNA with RiboZero, followed by sequencing on NovaSeq 6000 (Illumina).

first: Bo

last: Kim

first: Michael

last: Welsh


title: Microglia protect against age-associated brain pathologies

status: Public on May 22 2024

city: Edinburgh

first: Nadine

last: Bestard-Cuche

address: 5 Little France Drive

institute: University of Edinburgh

summary: Microglia are brain-resident macrophages that contribute to central nervous system development, maturation, and preservation. Here, we examine the consequences of lifelong absence of microglia on ageing using the Csf1rΔFIRE/ΔFIRE mouse model. In juvenile Csf1rΔFIRE/ΔFIRE mice, we show that microglia are largely dispensable for the transcriptomic maturation of other brain cell types. In contrast, with advancing age, multiple pathologies accumulate in Csf1rΔFIRE/ΔFIRE brains, astrocytes and oligodendrocyte-lineage cells become increasingly dysregulated, and white matter integrity declines, mimicking many of the pathological features of human CSF1R-related leukoencephalopathy. The thalamus is particularly sensitive to neuropathological changes in the absence of microglia, with atrophy, neuron loss, vascular disturbances, macroglial dysregulation, and severe calcifications all detected in this region. Thalamic calcification formation, which often occurs with normal ageing, is dramatically accelerated in Csf1rΔFIRE/ΔFIRE brains but can be prevented via transplantation of wild-type microglia. Our results indicate that lifelong absence of microglia results in an age-related neurodegenerative condition that can be prevented by the transplantation of healthy microglia.

overall_design: FIREΔ/Δ (KO), FIREΔ/+(HET) and Fire+/+(WT) mice were processed for single cell RNA sequencing with 10X. For 6-7-week-old “young” samples, hippocampi from both hemispheres and the remainder of the left hemisphere were dissected (after removal of olfactory bulbs and cerebellum).  for the 11-12-month-old “adult” samples, the left hemisphere was processed (after removal of olfactory bulbs and cerebellum); for the 16-18-month-old “old” samples, the whole brain was processed (after removal of olfactory bulbs and cerebellum); for the 17-19-month-old “thalamus” samples, thalami were dissected from both hemispheres and processed.

first: David

middle: D

last: Munro

first: Anna

last: Williams

first: Josef

last: Priller


title: Epigenetic heterogeneity hotspots in human liver disease progression [RNA-seq]

status: Public on Jul 30 2024

city: Rochester

first: Keith

middle: Darrell

last: Robertson

state: MN

address: 200 First Street SW, Stabile 12-70

department: Pharmacology

institute: Mayo Clinic

summary: Disruption of the epigenome is a hallmark of human disease including liver cirrhosis and hepatocellular carcinoma (HCC). While genetic heterogeneity is an established effector of pathologic phenotypes, epigenetic heterogeneity is less well understood. Environmental exposures including hepatitis C viral (HCV) infection and metabolic syndrome alter the liver-specific DNA methylation landscape and influence the onset of liver cancer. Given that currently available treatments are unable to target the most frequently mutated genes in HCC, there is an unmet need for novel therapeutic strategies to prevent or reverse hepatic tumorigenesis, which the epigenome may provide.  We performed genome-wide profiling of DNA methylation, copy number, and gene expression landscapes from multiple liver regions from 31 patients with liver disease to examine their crosstalk and define the individual and combinatorial contribution of these processes to liver disease progression. We identify epigenetic heterogeneity hotspots that are conserved across patients. Elevated epigenetic heterogeneity associates with increases in gene expression heterogeneity. Cirrhotic regions comprise two distinct cohorts, one exclusively epigenetic, and a second where epigenetic and copy number variations collaborate. Epigenetic heterogeneity hotspots are enriched for genes central to liver function (e.g., HNF1A) and known tumor suppressors (e.g., RASSF1A). These hotspots encompass genes including ACSL1, ACSL5, MAT1A, and ELFN1, which have phenotypic effects in cell line functional screens, supporting their relevance to hepatocarcinogenesis. Substantial epigenetic heterogeneity arises early in liver disease development, targeting key pathways in the progression and initiation of both cirrhosis and HCC. Integration of epigenetic and transcriptional heterogeneity unveils putative epigenetic regulators of hepatocarcinogenesis.

overall_design: Gene expression from multi-regional sampling of normal and disease livers

first: Ryan

last: Hlady


title: Epigenetic heterogeneity hotspots in human liver disease progression [MethylationEPIC Beadchip]

summary: Disruption of the epigenome is a hallmark of human disease including liver cirrhosis and hepatocellular carcinoma (HCC). While genetic heterogeneity is an established effector of pathologic phenotypes, epigenetic heterogeneity is less well understood. Environmental exposures including hepatitis C viral (HCV) infection and metabolic syndrome alter the liver-specific DNA methylation landscape and influence the onset of liver cancer. Given that currently available treatments are unable to target the most frequently mutated genes in HCC, there is an unmet need for novel therapeutic strategies to prevent or reverse hepatic tumorigenesis, which the epigenome may provide.  We performed genome-wide profiling of DNA methylation, copy number, and gene expression landscapes from multiple liver regions from 31 patients with liver disease to examine their crosstalk and define the individual and combinatorial contribution of these processes to liver disease progression.  We identify epigenetic heterogeneity hotspots that are conserved across patients. Elevated epigenetic heterogeneity associates with increases in gene expression heterogeneity. Cirrhotic regions comprise two distinct cohorts, one exclusively epigenetic, and a second where epigenetic and copy number variations collaborate. Epigenetic heterogeneity hotspots are enriched for genes central to liver function (e.g., HNF1A) and known tumor suppressors (e.g., RASSF1A). These hotspots encompass genes including ACSL1, ACSL5, MAT1A, and ELFN1, which have phenotypic effects in cell line functional screens, supporting their relevance to hepatocarcinogenesis.   Substantial epigenetic heterogeneity arises early in liver disease development, targeting key pathways in the progression and initiation of both cirrhosis and HCC. Integration of epigenetic and transcriptional heterogeneity unveils putative epigenetic regulators of hepatocarcinogenesis.

overall_design: Genome-wide DNA methylation from multi-region sampling in normal and liver tissues


title: Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response

status: Public on May 17 2024

first: Niek

last: Wit

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

overall_design: Refer to individual Series


title: Esophageal adenocarcinoma

status: Public on May 25 2024

city: Amsterdam

first: Richard

last: Volckmann

state: Noord-Holland

address: van der Boechorststraat 6a

department: Laboratory of Experimental Oncology and Radiobiology

country: Netherlands

institute: msterdam UMC and University of Amsterdam


title: Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [RNA-seq]

summary: The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1 isoform, and while USP43 does not alter HIF-1 stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes.  Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia-dependent manner to increase the nuclear pool of HIF-1, independently of DUB activity. Together, our results unveil the DUB landscape in HIF signalling, and highlight the multifunctionality of DUBs, illustrating that they can provide important signalling roles outside of their catalytic activity.

overall_design: To investigate the transcriptional responses of DUB genes in response to hypoxia, we generated RNA-Seq data sets of control and HIF1B KO cells from normoxic and hypoxic conditions. We also included USP43 KO samples for these conditions as this is a hypoxia-inducible DUB and a hit in our genetic screens.

first: Tekle

last: Pauzaite

first: Rachel

last: Seear

first: James

last: Nathan


title: Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (DMS-Seq)

city: Boston

first: yongwoo

last: lee

address: 185 Cambridge St

department: Mol bio

institute: MASSACHUSETTS GENERAL HOSPITAL

summary: Whether RNA G-quadruplexes (rG4) form extensively in vivo and what roles they play remain actively debated. Among their proposed functions is recognition of Polycomb repressive complex 2 (PRC2), but how the interaction results in epigenetic regulation is not understood. Here we demonstrate that rG4s form dynamically during ES cell differentiation and require ATRX’s helicase function to unwind competing secondary structures. Mutating ATRX causes rG4 depletion on a transcriptome-wide basis and dramatically increases gene expression. We identify and mutate rG4s within Xist RNA and mechanistically separate PRC2’s recruitment versus catalysis. Surprisingly, although rG4s recruit PRC2, unfolding the rG4 structure causes PRC2 hyperactivation, entrapment of PRC2 in the S1 chromosomal compartment, and loss of gene silencing. Thus, we link dynamic rG4 folding and unfolding to PRC2 recruitment, trans-compartmental Xist migration, regulated activation of PRC2, and whole-chromosome gene silencing.

overall_design: Modified existing rG4-seq and RT-Stop protocols were performed and generated an “in vitro” and “in vivo” pipeline, based on differential sensitivity of various RNA structures to dimethyl sulfate (DMS), reverse transcriptase (RT), and the monovalent cations Li+ versus K+.

first: Yong

middle: W

last: Lee

first: Uri

last: Weissbein

first: Roy

last: Blum

first: Jeannie


title: Cell type-specific loops linked to RNA polymerase II elongation in human neural differentiation

status: Public on Jul 10 2024

city: St. Louis

first: Jennifer

middle: E.

last: Phillips-Cremins

state: MO

address: 4515 McKinley Ave

department: Bioengineering

institute: Washington University in St. Louis

summary: DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.

overall_design: We performed Hi-C, ChIP-seq (H3K27ac, RNA Pol II, and CTCF), and RNA-seq to characterize epigenetic changes, chromatin rewiring, and gene expression changes along human iPSC to neural progenitor and terminal cortical neuron differentiation.

first: Katelyn

last: Titus

first: Zoltan

last: Simandi

first: Harshini

last: Chandrashekar

first: Dominik

last: Paquet


title: G-quadruplex folding in Xist RNA antagonizes PRC2 activity for step-wise regulation of X-chromosome inactivation


title: Multi-omics analysis of early reperfused ischemic heart reveals ERRβ/γ activation protects against acute myocardial infarction injury [snRNA-seq]

city: Hangzhou

first: Li

last: Shen

address: 866 Yuhangtang Road

institute: Life Sciences Institute, Zhejiang University

summary: The rats were subjected to left anterior descending (LAD) coronary artery ligation for 1 hour, 6 hours (MI1h and MI6h) and 24 hours (MI24h) along with Sham controls (Sham), and 1-hour, 6-hour ischemia followed by reperfusion (MI1h/R and MI6h/R, mimicking early time reperfusion (ETR) and late time reperfusion (LTR), respectively) followed by heart tissue collection 24 hours post MI to mimic the MI/R injury in AMI patients.The heart infarct zones (Sham, MI1h/R, MI6h/R and MI24h) were isolated to single-nucleus transcriptome analysis. Collected nuclei for each sample were loaded onto 10X Chrominum platform. We obtained 22,234 cells and 17,003 genes. Five major cell types were identified, which included cardiomyocytes (CMs), endothelial cells (ECs), fibroblasts (FBs), macrophages (MACs) and smooth muscle cells (SMCs).

overall_design: Single-nucleus RNA sequencing performed with rats subjected to left anterior descending (LAD) coronary artery ligation for 1 hour, 6 hours (MI1h and MI6h) and 24 hours (MI24h) along with Sham controls (Sham), and 1-hour, 6-hour ischemia followed by reperfusion (MI1h/R and MI6h/R, mimicking early time reperfusion (ETR) and late time reperfusion (LTR), respectively) followed by heart tissue collection 24 hours post MI to mimic the MI/R injury in AMI patients.

first: Yun

last: Gao

first: Fei

last: Huang

first: Fanwei

last: Ruan


title: Immunotyping of microglia and macrophages in the CNS during acute SIV infection: a single-cell study of rhesus macaque brains

status: Public on May 01 2024

city: Omaha

first: Howard

middle: S

last: Fox

state: Nebraska

address: 42nd and Emile

institute: University of Nebraska Medical Center

summary: Human immunodeficiency virus (HIV) is widely recognized for its striking impact on the immune system. Even though HIV is primarily known for the impairment of the peripheral CD4 T cells, its influence on the central nervous system (CNS) also cannot be neglected. The main immune constituents in the brain, microglia and macrophages, are the target for HIV in the brain, as well as for the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological effects as well as the establishment of a virus reservoir. Given the gaps in our knowledge on how these cells respond in vivo to CNS infection, we performed single-cell RNA sequencing (scRNA-seq) on myeloid cells from the brains of 3 rhesus macaque with 12-day SIV infection as well as 3 uninfected controls. We identified six microglial clusters and two macrophage clusters by transcriptomic profiling. Intriguingly, there were two microglial clusters populated with the cells from infected animals. These two infection-specific clusters had significantly higher expression (p < 0.05) of MHC class I molecules, interferon-induced proteins, chemokines, and cytokines than did the other clusters. Although only a low proportion of SIV-infected cells were detected in microglia and macrophages (~0.14%), almost all the microglia and macrophages in infected animals were activated to certain extents. In addition, the clusters with higher expression of cytotoxic molecules increased their populations during the infection, suggesting their potential to cause HIV-associated neurological disorders.

overall_design: 92T, 104T, 111T were non-infected control samples and other three (93T, 94T, 95T) were in acute-infected group. The rhesus macaques in the acut-infected group were infected with Simian Immunodeficiency Virus (SIV) for 12 days

first: Xiaoke

last: Xu

first: Meng

last: Niu

first: Benjamin

middle: G

last: Lamberty

first: Katy

last: Emanuel


title: ILC2s navigate tissue redistribution during infection using stage-specific S1P receptors [ATAC-seq]

first: Yuefeng

first: Vincent

last: Guichard

first: Takamasa

last: Ito


title: Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [CRISPR]

first: Niek

last: Wit

overall_design: explore the contribution of DUBs to HIF signalling using CRISPR/Cas9 mutagenesis screens

first: Tekle

last: Pauzaite

first: Rachel

last: Seear

first: James

last: Nathan


title: Exploring kidney allograft rejection: A proof-of-concept study using spatial transcriptomics

status: Public on May 02 2024

city: Derio

first: Ana

middle: Maria

last: Aransay

state: BIZKAIA

address: Parque tecnologico de Bizkaia, Building 801-A

department: Genome Analysis Platform

country: Spain

institute: CIC bioGUNE

summary:
In this proof-of-concept study, spatial transcriptomics combined with public single-cell RNA sequencing
data were used to explore the potential of this technology to study kidney allograft
rejection. We aimed to map gene expression patterns within diverse pathological states by
examining biopsies classified across non-rejection, T cell-mediated acute rejection, and
interstitial fibrosis and tubular atrophy (IFTA). Our results revealed distinct immune cell
signatures, including those of T and B lymphocytes, monocytes, mast cells, and plasma cells, and
their spatial organization within the renal interstitium. We also mapped chemokine receptors
and ligands to study immune-cell migration and recruitment. Finally, our analysis demonstrated
differential spatial enrichment of transcription signatures associated with kidney allograft
rejection across various biopsy regions. Interstitium regions displayed higher enrichment scores
for rejection-associated gene expression patterns than did tubular areas, which had negative
scores. This implies that these signatures are primarily driven by processes unfolding in the renal
interstitium. Overall, this study highlights the value of spatial transcriptomics for revealing
cellular heterogeneity and immune signatures in renal transplant biopsies, and demonstrates its
potential for studying the molecular and cellular mechanisms associated with rejection.
However, certain limitations must be borne in mind regarding the development and future
applications of this technology.

overall_design: Using spatial transcriptomics, this study examined three kidney post-implantation biopsies with different histological phenotypes. The Banff criteria were used to evaluate the samples. Each sample exhibited different rejection levels with varying parameter scores, including i, t, v, and g. The diagnostic categories for biopsy samples included acute cellular rejection, interstitial fibrosis and tubular atrophy (IFTA), and unaltered (non-rejection).

first: Cristina

last: Martin-Martin

first: Beatriz

last: Suarez-Alvarez

first: Monika

last: González

first: Irina

last: Torres

first: Oriol

last: Bestard

first: José

last: Ezequiel Martín

first: Gwendolyn

last: Barceló-Coblijn

first: Francesc

last: Moreso

first: Carlos

last: Lopez-Larrea

first: Ramon

last: Rodriguez


title: Integrated miRNA-mRNA analysis reveals critical miRNAs and targets in the diet-induced obesity-related glomerulopathy

status: Public on Jun 12 2024

city: Sant Joan Despí

first: Maruja

last: Navarro-Díaz

state: Barcelona

address: Oriol Martorell, 12

department: Nephrology

institute: Hospital Sant Joan Despí Moisès Broggi

summary: Background:The objective of this study was to investigate obesity-related glomerulopathy (ORG) at a cellular, structural and transcriptomic level. Methods:Thirty Wistar rats were randomized into two groups: control rats(n=15), which were fed a standard diet(SD) and study rats(n=15), which were fed a high-fat diet (HFD). After 10 weeks, weight, parameters of kidney function, renal histological features, transcriptomic changes, miRNA and mRNA isolation were compared. Results:HFD gained more weight(55.8%) than SD(29.2%), p<0.001. Albuminuria was also significantly higher in HFD(10,384.04 ng/ml) compared with SD(5,845.45 ng/ml), p<0.001. HFD showed typical lesions of early stages of ORG, with a predominance of mesangial matrix increase (MMI) and podocyte hypertrophy (PH). All histologic lesions correlated with genes differentially expressed (DE) in kidneys of HFD group and PH, also correlated with specific miRNAs DE in the urine of HFD. The functional analysis showed 4 miRNAs DE in the kidneys of HFD group that negatively regulates PTEN gene, which promotes podocyte endocytosis of lipids in ORG. The electronic microscope confirmed the spaces of lipid vacuoles in the podocytes of HFD. Between those 4 miRNAs DE, miR-205 was also found to be upregulated in the urine of HFD group.  Conclusions: Wistar rats fed a HFD developed early-stages of ORG, with a specific targetome of miRNAs and gene expression. The upregulation of miR-205 in kidney and its isolation in urine is associated with lipid endocytosis of podocytes, which could become a plausible biomarker of early-stages of ORG and open new avenues for future therapeutics research. Translational Statement: The findings of the basic research in this study can be replicated in human models of obesity. The results of the study on miRNA in humans may contribute to improving the understanding of the pathophysiology of obesity-related glomerulopathy, aiding in the search for early biomarkers, and exploring new therapeutic targets. Keywords: MicroRNA, Biomarker, targetome, obesity-related glomerulopathy, mesangial matrix increase, podocyte hypertrophy, Wistar rats

overall_design: To investigate the interactome of miRNAs, their targetome, and the presence of obesity-related renal disease, we established a non-genetically modified animal model of obesity. Thirty male Wistar rats were included in the study and randomized into two groups: 15 rats fed a standard diet (control group) and 15 rats fed a high-fat diet (experimental group) for 10 weeks. Total RNA and small RNA were extracted from rat kidneys, while small RNA was also extracted from urine. Comparative gene expression profiling analysis of RNA-seq and small RNA data was performed.

last: Navarro

first: Maria

last: Armengol

first: Mireia

last: Ferrer


title: An integrated single-nucleus and spatial transcriptomics atlas reveals the molecular landscape of the human hippocampus [spatial-visium]

status: Public on Apr 27 2024

city: Baltimore

first: Keri

last: Martinowich

state: Maryland

address: 855 N Wolfe St Suite 300

institute: Lieber Institute for Brain Development

summary: The hippocampus contains many unique cell types, which serve the structure’s specialized functions, including learning, memory and cognition. These cells have distinct spatial topography, morphology, physiology, and connectivity, highlighting the need for transcriptome-wide profiling strategies that retain cytoarchitectural organization. Here, we generated spatially-resolved transcriptomics (SRT) and single-nucleus RNA-sequencing (snRNA-seq) data from adjacent tissue sections of the anterior human hippocampus across ten adult neurotypical donors. We defined molecular profiles for hippocampal cell types and spatial domains. Using non-negative matrix factorization and transfer learning, we integrated these data to define gene expression patterns within the snRNA-seq data and probe their expression in the SRT data. Using this approach, we leveraged existing rodent datasets that feature information on circuit connectivity and neural activity induction to make predictions about axonal projection targets and likelihood of ensemble recruitment in spatially-defined cellular populations of the human hippocampus. Finally, we integrated genome-wide association studies with transcriptomic data to identify enrichment of genetic components for neurodevelopmental, neuropsychiatric, and neurodegenerative disorders across cell types, spatial domains, and gene expression patterns of the human hippocampus. To make this comprehensive molecular atlas accessible to the scientific community, both raw and processed data are freely available, including through interactive web applications.

overall_design: We obtained tissue blocks of postmortem human brain containing anterior hippocampus from 10 neurotypical adult brain donors. We used the 10x Genomics Visium and 3’ Single Cell Gene Expression platforms to generate paired spatial transcriptomics and single-nucleus RNA-seq data of the human hippocampus. We performed a second spatial transcriptomics experiment using the Visium Spatial Proteogenomics (Visium-SPG) platform for two of the 10 donors.

first: Erik

last: Nelson

first: Madhavi

last: Tippani

first: Anthony

last: Ramnauth

first: Heena

last: Divecha

first: Ryan

last: Miller

first: Nicholas

middle: J

last: Eagles

first: Elizabeth

last: Pattie

first: Sang

middle: H

last: Kwon

first: Svitlana

middle: V

last: Bach

first: Uma

last: Kaipa

first: Jianing

last: Yao

first: Joel

middle: E

last: Kleinman

first: Leonardo

last: Collado-Torres

first: Shizhong

last: Han

first: Kristen

last: Maynard

first: Thomas

last: Hyde

first: Stephanie

last: Page

last: Hicks


title: Mutant IDH1 inhibition induces reverse transcriptase and dsDNA sensing to activate tumor immunity

status: Public on Apr 24 2024

city: Dallas

first: Samuel

middle: Kent

last: McBrayer

address: 6000 Harry Hines Boulevard, NL11.110K

department: Children's Research Institute

institute: UT Southwestern Medical Center

summary: Isocitrate Dehydrogenase 1 (IDH1) is the most commonly mutated metabolic gene across human cancers. Mutant IDH1 (mIDH1) generates the oncometabolite (R)-2-hydroxyglutarate, disrupting enzymes involved in epigenetics and other processes. A hallmark of IDH1-mutant solid tumors is T cell exclusion, whereas mIDH1 inhibition in preclinical models restores anti-tumor immunity. Here, we define a cell-autonomous mechanism of mIDH1-driven immune evasion. IDH1-mutant solid tumors show striking, selective hypermethylation and silencing of the cytoplasmic dsDNA sensor, CGAS, compromising innate immune signaling. mIDH1 inhibition restores DNA demethylation, derepressing CGAS and transposable element (TE) subclasses. dsDNA produced by TE-reverse transcriptase activates cGAS, triggering viral mimicry and stimulating anti-tumor immunity. Thus, we demonstrate that mIDH1 epigenetically suppresses innate immunity and link endogenous reverse transcriptase activity to the mechanism of action of an FDA-approved oncology drug.

overall_design: We performed RNA sequencing on a panel of IDH mutant and IDH WT glioma stem-like cells.

first: Milan

middle: R

last: Savani

first: Bailey

middle: C

last: Smith


title: The TNFSF12/TWEAK modulates colonic inflammatory fibroblast differentiation and promotes fibroblast-monocyte interactions

status: Public on Apr 23 2024

city: Dublin

first: Mario

last: Manresa

address: UCD Belfield

country: Ireland

institute: School of Biomolecular and Biomedical Sciences

summary: Fibroblasts acquire a pro-inflammatory phenotype in inflammatory bowel disease (IBD), but the factors driving this process and how fibroblasts contribute to the immune response is incompletely understood. The TNF superfamily factor 12 (TWEAK) has gained interest as a mediator of chronic inflammation. Here, we explore its role as a driver of inflammatory responses in fibroblasts and its contribution to fibroblast-monocyte interaction using human primary colonic fibroblasts, THP1 and peripheral blood mononuclear cells (PBMC). TWEAK induced the expression of cytokines, chemokines and immune receptors in fibroblasts. The TWEAK up-regulated transcriptome shared 29% homology with the previously published transcriptional profile of inflammatory fibroblasts from ulcerative colitis. TWEAK elevated surface expression of activated fibroblasts markers and adhesion molecules (PDPN, ICAM-1 and VCAM-1) and secretion of IL-6, CCL2 and CXCL10. In co-culture, fibroblasts induced monocyte adhesion and promoted a CD14high/ICAM-1 high phenotype in THP1 cells, both of which were enhanced when fibroblasts were pre-stimulated with TWEAK. Medium from TWEAK-treated fibroblast-THP1 co-cultures had elevated levels of CXCL1 and IL-8. PBMCs in co-culture with TWEAK-treated fibroblasts had altered surface expression of CCR2 and CD16, and increased CXCL1 and CXCL10. Our results indicate that TWEAK promotes and inflammatory fibroblast-monocyte crosstalk that may be amenable for therapeutic intervention.

overall_design: To test the role of TWEAK in colonic fibroblast differentiation, human colonic fibroblasts were treated with TWEAK for 24 hours and the transcriptional profile compared to vehicle was assessed using bulk RNA sequencing.

first: Carlos

last: Matellan

first: Ciarán

last: Kennedy


title: Gene expression profile at single cell level of syngeneic ovarian tumors from WT and Tsk mice [scRNA-seq]

status: Public on Apr 08 2024

city: los angeles

first: Sandra

last: Orsulic

state: california

address: 10833 Le conte ave

department: OBGYN

institute: UCLA

summary: Patients with systemic sclerosis have a significantly increased incidence of developing various solid malignancies within a few years of systemic sclerosis onset but the mechanism of tumor promotion is not well understood. The tight skin (TSK) mouse has been a valuable model for investigating systemic sclerosis-related pathologies due to increased extracellular matrix deposition, fibrosis in connective tissues, and altered immune cell activation. Despite the role of extracellular matrix and fibrosis in cancer progression, the potential of the TSK mouse as a model for cancer studies is unexplored. Our RNA sequencing analysis of adult dorsal skin samples from TSK and wild-type (WT) mice revealed a notable enrichment for genes typically associated with cancer aggressiveness and desmoplasia. To investigate the impact of the altered microenvironment in TSK mice on cancer progression, we compared the tumor-forming capabilities in TSK mice and WT mice using a syngeneic mouse model of ovarian cancer. TSK mice exhibited larger and more invasive subcutaneous and intraperitoneal tumors in comparison to WT controls, suggesting the role of the microenvironment in promoting cancer progression. Single-cell RNA sequencing analysis of tumors in TSK and WT mice revealed a higher neutrophil-to-lymphocyte ratio, and an enrichment in profibrotic subpopulations of myofibroblasts and macrophages in TSK mice.

overall_design: To better understand the differences in cell populations in TSK and WT mice (i.e. subtypes of fibroblasts and specific functional subsets of immune cells), we conducted single-cell RNA sequencing analysis of tumors isolated from TSK and WT mice after injection of 1x10^6 SO cells (syngeneic ovarian cancer cell line)

first: Maria

last: Recouvreux


title: Targeting hepatic IRE1a for Treating GDF15-driven Anorexia in Chemotherapy

status: Public on Apr 22 2024

city: china

first: Yao

last: Tao

state: zhejiang

address: zhejiang province

institute: First Affiliated Hospital, Zhejiang University

summary: Anorexia and other side effects of chemotherapy significantly limit the clinical applications of these chemotherapeutic agents, with underlying mechanisms still unclear. A growing body of research reveals that elevated levels of circulating GDF15 after chemotherapy are essential in initiating anorexia and other side effects by activating GFRAL+ neurons in the brainstem. However, the full array of context-dependent transcriptional regulators controlling Gdf15 expression remains to be defined. This study reports that chemotherapy drugs acutely stimulate liver GDF15 production via selective activation of the hepatic stress sensor IRE1α, thereby controlling circulating GDF15 levels. Genetic ablation of hepatic IRE1α reduces circulating GDF15 and alleviates anorexia and body weight loss following chemotherapy drug treatments in tumor-bearing mice. Mechanistically, chemotherapy drugs activate hepatic IRE1α RNase activity to produce the active form of the transcription factor XBP1 to promotes the expression of Gdf15 gene in hepatocytes. Moreover, treatment with pharmacological IRE1α RNase inhibitor effectively suppresses liver Gdf15 expression and circulating GDF15 levels, resulting in improvements in chemotherapy-induced anorexia and body weight loss. Our results reveal a stress-responsive mechanism that mediates communication between the liver and brain. This mechanism can be targeted pharmacologically to alleviate anorexic side effects that accompany chemotherapy-induced body weight loss.

overall_design:
For the evaluation of chemotherapy effects in healthy mice, the liver samples were collected from the mice 1 day after doxorubicin and chemotherapy. Bulk RNA-seq experiment was performed to analyze gene expression profile in these liver samples.
A-Saline B-Cisplatin C-Doxorubicin
*Samples do not include information about treatment with doxorubicin.

first: Ying

last: Wu

first: Bo

last: Shan

first: Yuexiao

last: Tang

first: Tao


title: Gene expression profile at single cell level of phenotypic LT-HSCs from single-mutant JAK2 V617F and double mutant JAK2 V617F;Dnmt3a-/- mice

status: Public on Mar 29 2024

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

country: Switzerland

institute: University of Basel

summary: We investigated if Dnmt3a loss leads to alterations in JAK2 V617F LT-HSCs heterogenity and expression profile using single-cell RNA sequencing.

overall_design: LT-HSCs (Lineage-, c-Kit+, Sca-1+, CD150+, CD48-) were FACS-sorted from bone marrow of mice treated with pegylated IFN alpha (25µg/kg, s.c. in 100µl) for 24 hours or vehicle (saline) and used for downstream single-cell RNAseq.

first: Jan

last: Stetka

first: Damien

last: Luque Paz

first: Anastasiya

last: Börsch

first: Radek

last: Skoda


title: Gene expression profile at single cell level from the dorsal skin of WT and Tsk mice [bulk RNA-seq]

overall_design: To better understand the differences in cell populations in TSK and WT mice (i.e. subtypes of fibroblasts and specific functional subsets of immune cells), we conducted Bulk RNA sequencing analysis of dorsal skin from 3 TSK and 3 WT mice


title: A Brucella ovis HWE histidine kinase is important for detergent resistance.

status: Public on Mar 26 2024

city: East Lansing

first: Sean

last: Crosson

address: 567 Wilson Rd

department: Dept. Microbiology and Molecular Genetics

institute: Michigan State University

summary: Through a system-level analysis of non-essential two-component signal transduction systems (TCS) genes in Brucella ovis, we have discovered that deletion of an HWE histidine kinase, BOV_1602, leads to significant increase in resistance to SDS detergent and other anionic or zwitterionic detergents. To better understand this detergent resistance phenotype, we performed RNA-seq experiment using both WT and ∆BOV_1602 cultured on no stress (TASB) or TSAB supplemented with 0.004% SDS.

overall_design:
This RNA-seq experiment was designed to investigate the transcriptome of WT and ∆BOV_1602 mutant under no stress condition and under SDS treatment. Cells were cultured on both TSAB (no stress) and TSAB + 0.004% SDS and collected after incubation.
We compared the differential gene expression between ∆BOV_1602 and WT grown on TSAB and TSAB supplemented with 0.004% SDS. Each strain cultured on each condition had three replicates

first: Xingru

last: Chen

first: Aretha

last: Fiebig


title: Under pressure: altered endothelial flow response.

status: Public on Mar 12 2024

city: Seattle

first: Brandon

last: Hadland

state: WA

address: 1100 Fairview Avenue N., D2-115, P.O. Box 19024

institute: Fred Hutchinson Cancer Center

summary: Blood flow within the vasculature is a critical determinant of endothelial cell (EC) identity and functionality, yet the intricate interplay of various hemodynamic forces and their collective impact on endothelial and vascular responses are not fully understood. Specifically, the role of hydrostatic pressure in the context of flow response is understudied, despite its known significance in vascular development and disease. To address this gap, we developed in vitro models to investigate how pressure influences EC responses to flow. Our study demonstrates that elevated pressure conditions significantly modify shear-induced flow alignment and increase endothelial cell density, a phenomenon often observed in vascular diseases. Utilizing both bulk and single-cell RNA sequencing, we found that while flow is the primary driver of transcriptional changes from static conditions, pressure distinctly modulates this flow response by upregulating gene sets linked to arterial cell phenotypes. Conserved pressure-responsive transcriptional signatures identified in human ECs were upregulated during the onset of circulation in early mouse embryonic vascular development, where pressure was notably associated with transcriptional programs essential to arterial and hemogenic EC fates. Our findings emphasize the necessity of an integrative approach to endothelial cell mechanotransduction, one that encompasses the effects induced by pressure alongside other hemodynamic forces.

overall_design: single cell RNA-sequencing (10X Genomics platform) of HUVECs cultured in microfluidic vessels and subjected to various levels of hydrostatic pressure (0, 20 and 60 mmHg), while maintained under constant shear stress (flow). A static sample was also included as a negative control.

first: Hadland

last: Brandon

first: Zheng

last: Ying


title: RNA Sequencing-based Transcriptome Profiling of TNBC Tumors from Mice Treated with EGFR-targetd CAR-T Therapy or Radiotherapy Alone and in Combination

status: Public on Mar 10 2024

city: Shanghai

first: Min

last: Zhou

state: Shanghai

address: No.25/Ln 2200 XieTu  Road

institute: Shanghai Cancer Institute

summary: We performed RNA-seq experiments on TNBC tumor tissues from mice treated with EGFR targeted CAR-T therapy or radiotherapy alone and in combination. In brief, we established E0771-EGFR TNBC cell line and inoculated the E0771-EGFR cells orthotopically into the 4th inguinal mammary fat pads of C57BL/6 mice. When the average tumor volume reached 100 mm3 on day 14, tumor bearing mice were divided to 4 groups (3 mice per group). Tumor bearing mice were given tail vein injection of 5×106 UT cells (group 1) or CAR-T cells (group 2), or exposed to 10 Gy local irradiation first then followed by tail vein injection of UT cells (group 3) or CAR-T cells (group 4). Tumor tissues were collected on day 21. Tumor tissues collected from group 1 to group 4 were named as T1, T2, T3 and T4. Total RNA from tumor was prepared using the RNeasy Plus Universal Mini Kit (QIAGEN). RNA-Seq libraries were constructed after oligo dT-based mRNA enrichment using a NEBNext mRNA Magnetic Isolation Module (New England Biolabs). The RNA-Seq Library Prep Kit (Illumina) was then used according to the manufacturer’s instructions and the cDNAs were sequenced with the Illumina Hiseq 4000.

overall_design: mRNA profiles of TNBC tumors from mice treated with EGFR-targetd CAR-T therapy or radiotherapy alone and in combination

first: Muhua

first: Hua

last: Jiang

first: Zonghai

last: Li


title: RoCK and ROI: a single-cell RNA-sequencing method with enhanced transcriptome information via targeted enrichment of transcripts and preselected, region-specific sequencing

status: Public on May 07 2024

city: Zürich

first: Giulia

middle: Arianna

last: Moro

address: Winterthurerstrasse 190

department: DMLS

institute: University of Zurich

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: Pol II pausing is a milestone on the road to complex animals

status: Public on Feb 05 2024

city: Freeville

first: Brent

last: Basso

state: NEW YORK

address: 1631 Dryden Rd.

department: Computational Biology

institute: Cornell University

summary: PRO-seq experiments in multiple organisms. Also, PRO-seq experiment of Nelfb and Nelfe degron mESC in serum/LIF with or without dTAG-13.

sample_organism:
Escherichia coli
Haloferax mediterranei
Daphnia pulex
Strongylocentrotus purpuratus
Mus musculus
Perkinsus marinus
Dryas iulia
Dictyostelium discoideum
Nematostella vectensis
Sphaeroforma arctica
Capsaspora owczarzaki
Creolimax fragrantissima

overall_design: We used Precision Run-On and Sequencing (PRO-seq) to study transcription in 20 extant organisms that represent very diverse branches on the tree of life, including multiple species near the base of the Metazoa phylogeny and the transition between plants and animals. We also used PRO-seq to study mESCs after degrading NELF-B or NELF-E.

first: Alexandra

last: Chivu

first: James

last: Lewis

first: John

middle: T

last: Lis

first: Charles

last: Danko


title: Mechanoresponsive genomic enhancers potentiate the cellular response to matrix stiffness [migration]

status: Public on Jan 31 2024

city: Durham

middle: A

last: Gersbach

state: North Carolina

address: 101 science drive rm 2323

institute: Duke University

summary: Epigenetic control of gene regulation and cell phenotype is influenced by changes in the mechanical microenvironment. Yet how mechanical forces precisely influence epigenetic state to regulate transcriptional responses remains largely unmapped. Here, we combine genome-wide epigenome profiling, epigenome editing, and phenotypic and single-cell RNA-seq screening to identify a sub-class of genomic enhancers that is responsive to the mechanical microenvironment. These enhancers regulate genes that act as key drivers for a variety of functional behaviors including apoptosis, mechanotransduction, proliferation, and migration. We find distinct patterns wherein subsets of mechano-enhancers can be preferentially active on either soft or stiff extracellular matrix (ECM) contexts. Epigenetic editing of these mechano-enhancers on rigid materials precisely tunes mechanically-induced gene expression to levels observed on softer materials, and broadly enables reprogramming of cellular response to the mechanical microenvironment. These mechanically-activated enhancers act as key signal transducers and may constitute a new class of targets that can be modulated to alter mechanically-driven disease states.

overall_design: To determine the genes regulated by the pREs identified in the proliferation and migration screens, we made a sub-library of gRNAs targeting 87 of the pREs (10 gRNAs/pRE) with the largest effect sizes in either or both of the previous screens. This library also included positive controls of promoter-targeting and known enhancer-targeting gRNAs, and 100 non-targeting negative control gRNAs, for a total of 1,005 gRNAs in the sub-library. We transduced HFFs expressing dCas9KRAB cultured on TCP with this gRNA library at 0.33 MOI, and eight days post-transduction we profiled 103,440 quality single cell transcriptomes. We recovered a median of 1 gRNA per cell and identified an average of 159 cells containing each gRNA. To identify significant gene linkages to each pRE, we tested cells that had each gRNA versus all cells that did not receive that gRNA, and compared expression for all genes within +/- 1Mb from the targeted pRE.

first: Brian

last: Cosgrove

first: Lexi

last: Bounds

first: Carson

middle: K

last: Taylor

first: Alan

middle: L

last: Su

first: Anthony

last: Rizzo

first: Alejandro

last: Barrera

first: Gregory

last: Crawford

first: Brenton

last: Hoffman


title: Mechanoresponsive genomic enhancers potentiate the cellular response to matrix stiffness [MYH9 facs}

middle: D


title: Mechanoresponsive genomic enhancers potentiate the cellular response to matrix stiffness


title: A novel approach for simultaneous detection of structural and single-nucleotide variants based on a combination of chromosome conformation capture and exome sequencing

status: Public on Jan 24 2024

city: Novosibirsk

first: Nikita

last: Torgunakov

address: Prospekt Lavrentyeva 10

country: Russia

institute: Institute of Cytology and Genetics

summary: Effective molecular diagnosis of congenital diseases hinges on comprehensive genomic analysis, traditionally reliant on various methodologies specific to each variant type — whole exome or genome sequencing for single nucleotide variants (SNVs), array CGH for copy-number variants (CNVs), and microscopy for structural variants (SVs). We introduce a novel, integrative approach combining exome sequencing with chromosome conformation capture, termed Exo-C. This method enables the concurrent identification of SNVs in clinically relevant genes and SVs across the genome and allows analysis of heterozygous and mosaic carriers. Enhanced with targeted long-read sequencing, Exo-C evolves into a cost-efficient solution capable of resolving complex SVs at base-pair accuracy. Through several case studies, we demonstrate how Exo-C's multifaceted application can effectively uncover diverse causative variants and elucidate disease mechanisms in patients with rare disorders.

overall_design:
Exo-C protocol includes a 3C part followed by exome enrichment. Applied on a cohort of 66 human samples, including one K562 cell line sample and the A549 cell line sample.

***************************************************************
Submitter states that missing raw files are due to patient privacy concerns.
***************************************************************

first: Maria

last: Gridina

first: Timofey

last: Lagunov

first: Polina

last: Belokopytova

first: Miroslav

last: Nuriddinov

first: Artem

last: Nurislamov

first: Lyudmila

middle: P

last: Nazarenko

first: Anna

last: Kashevarova

last: Lopatkina

first: Elena

middle: O

last: Belyaeva

first: Olga

last: Salyukova

first: Aleksandr

last: Cheremnykh

first: Natalia

middle: N

last: Suhanova

first: Marina

last: Minzhenkova

first: Zhanna

last: Markova

first: Nina

last: Demina

first: Yana

last: Stepanchuk

last: Khabarova

last: Yan

first: Emil

last: Valeev

first: Galina

last: Koksharova

last: Grigor’eva

last: Kokh

first: Tatiana

last: Lukjanova

first: Yulia

last: Maximova

first: Elizaveta

last: Musatova

last: Shabanova

first: Andrey

last: Kechin

first: Evgeniy

last: Khrapov

first: Uliana

last: Boyarskih

first: Oxana

last: Ryzhkova

last: Suntsova

first: Alina

last: Matrosova

first: Mikhail

last: Karoli

last: Manakhov

first: Maxim

last: Filipenko

first: Evgeny

last: Rogaev

first: Nadezhda

last: Shilova

first: Igor

last: Lebedev

first: Veniamin

last: Fishman


title: Mechanoresponsive genomic enhancers potentiate the cellular response to matrix stiffness [ATAC-seq]

first: Charles

last: Gersbach

overall_design:
To determine the genes regulated by the pREs identified in the proliferation and migration screens, we made a sub-library of gRNAs targeting 87 of the pREs (10 gRNAs/pRE) with the largest effect sizes in either or both of the previous screens. This library also included positive controls of promoter-targeting and known enhancer-targeting gRNAs, and 100 non-targeting negative control gRNAs, for a total of 1,005 gRNAs in the sub-library. We transduced HFFs expressing dCas9KRAB cultured on TCP with this gRNA library at 0.33 MOI, and eight days post-transduction we profiled 103,440 quality single cell transcriptomes. We recovered a median of 1 gRNA per cell and identified an average of 159 cells containing each gRNA. To identify significant gene linkages to each pRE, we tested cells that had each gRNA versus all cells that did not receive that gRNA, and compared expression for all genes within +/- 1Mb from the targeted pRE.
Human fibroblasts and A549 lung adenocarcinoma cells grown on soft (1 kPa) or stiff (50 kPa) hydrogels showed widespread differences in gene expression and chromatin accessibility by RNA-seq and ATAC-seq. CRISPRi was used to perturb differentially accessible regions surrounding stiffness-responsive genes such as MYH9, BMF, and FZD2 and identify distal mechanoenhancers that influenced gene expression. Functional high-throughput CRISPRi screening was performed to discover ~100 putative regulatory regions that influenced cell growth or migration. CRISPRi screening with single-cell RNA-seq identified 201 functional connections of these mechanoenhancers to their gene targets, including CTGF, CYR61, NF2, RFLNB, RANGAP1, SKP2 and others. Epigenetic editing of mechanoenhancers with CRISPRi in cells cultured on stiff substrates, including activated human donor-derived fibroblasts from healthy and idiopathic pulmonary fibrosis (IPF) lung tissue, resulted in gene expression patterns similar to those observed on soft substrates. Epigenetic modulation of these mechanoenhancers also resulted in functional changes in mechanosensing, migration, growth, and apoptosis.

first: Brian

last: Cosgrove

first: Lexi

last: Bounds

first: Carson

last: Taylor

first: Alan

last: Su

first: Anthony

last: Rizzo

first: Alejandro

last: Barrera

first: Gregory

last: Crawford

first: Brenton

last: Hoffman


title: EIF6 conditions drug-tolerant persister-like transdifferentiation in small cell lung carcinoma

status: Public on Apr 30 2024

city: Chengdu

first: Haoning

last: Peng

state: Sichuan

address: No.37 Guoxue Alley, Wuhou District

department: Department of Thoracic Surgery

institute: West China Hospital, Sichuan University


title: Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome [Nanopore]

status: Public on Dec 21 2023

city: St. Louis

first: Jennifer

middle: E.

last: Phillips-Cremins

address: 4515 McKinley Ave

institute: Washington University in St. Louis

summary: Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find Megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X-chromosome in induced pluripotent stem cell (iPSC)-derived neural progenitors, B-cells, and fibroblasts with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication stress-induced double strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the full-mutation CGG to premutation-length reverses H3K9me3 domains on the X-chromosome and multiple autosomes, refolds TADs, and restores expression. H3K9me3 domains also arise in a subset of normal-length iPSCs with increased STR instability burden. Our results reveal Mb-scale heterochromatinization and trans interactions among chromosomes susceptible to repeat genetic instability.

overall_design: Hi-C, H3K9me3 ChIP-seq, CTCF ChIP-seq, RNA-seq, H3K9me3 CUT&RUN, and short and long read genome sequencing were used in multiple cell types to interrogate epigenetic changes in cell lines with normal, premutation, and disease level repeat expansions in FMR1.

first: Thomas

last: Malachowski

first: Keerthivasan

last: Chandradoss

first: Linda

last: Zhou

first: Ravi

last: Boya

first: Ashley

last: Cook


title: Shifts in isoform usage underlie transcriptional differences in regulatory T cells in type 1 diabetes

status: Public on Jan 03 2024

city: Gainesville

first: Jeremy

middle: Robert Barker

last: Newman

state: FL

address: 2033 Mowry Road

department: Genetics Institute

institute: University of Florida

summary: Genome-wide association studies have identified numerous loci with allelic associations to Type 1 Diabetes (T1D) risk. Most disease-associated variants are enriched in regulatory sequences active in lymphoid cell types, suggesting that lymphocyte gene expression is altered in T1D. We assayed gene expression between T1D cases and healthy controls in two autoimmunity-relevant lymphocyte cell types, memory CD4+/CD25+ T-regulatory cells (Treg) and memory CD4+/CD25- T-cells, using a splicing event-based approach to characterize tissue-specific transcriptomes. Limited differences in isoform usage between T1D cases and controls were observed in memory CD4+/CD25- T-cells. In Tregs, 402 genes demonstrated differences in isoform usage between cases and controls, particularly RNA recognition and splicing factor genes. Many of these genes are regulated by the variable inclusion of exons that can trigger nonsense mediated decay. Our results suggest that dysregulation of gene expression, through shifts in alternative splicing in Tregs, contributes to T1D pathophysiology.

overall_design: Peripherial blood mononuclear cells from cases with type 1 diabetes and unaffected controls were sorted into lymphocyte cell types. Gene expression profiling and alternative splicing analysis was performed using RNA sequencing data obtained from each sorted cell type

first: S

last: Long

first: Cate

last: Speake

first: Carla

middle: J

last: Greenbaum

first: Karen

last: Cerosaletti

first: Stephen

middle: S

last: Rich

first: Suna

last: Onengut-Gumuscu

first: Lauren

middle: M

last: McIntyre

first: Jane

last: Buckner

first: Patrick

last: Concannon


title: Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome [WGS]

middle: E


title: Mechanoresponsive genomic enhancers potentiate the cellular response to matrix stiffness [scRNA-seq]

first: Charles

last: Gersbach

first: Brian

last: Cosgrove

first: Lexi

middle: R

last: Bounds

first: Carson

last: Taylor

first: Alan

last: Su

first: Anthony

last: Rizzo

first: Alejandro

last: Barrera

first: Gregory

last: Crawford

first: Brenton

last: Hoffman


title: Reconstitution of Human Brain Cell Diversity in Organoids via Four Protocols [time-course bulk RNA-seq]

status: Public on Nov 20 2024

city: Vienna

first: Julia

last: Naas

address: Dr. Bohr Gasse 9

department: Max Perutz Labs

country: Austria

institute: University of Vienna

summary: Systematic analysis of the cellular and transcriptional landscape of brain organoids grown from multiple cell lines using four different protocols recapitulating dorsal and ventral forebrain, midbrain, and striatum via time-course bulk-RNA sequencing.

overall_design:
Time-course bulk-RNA sequencing of brain organoids grown in four distinct growth protocols designed to generate dorsal (D) and ventral (V) forebrain, midbrain (M) and striatum (S) tissue at days 13 (for dorsal and ventral)/16 (for midbrain and striatum), 25, 40, 80 and 120. Per per cell line-protocol-timepoint combination three organoids (biological replicates) were grown. For reference, also three samples of the pluripotent stem cells across cell lines, not yet exposed to any protocol, were sequenced (day 0, pluripotent).

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

Raw data have been deposited at the European Genome-phenome Archive (EGA), under series numbers EGAS50000000663, and are available through controlled access.

first: Meritxell

last: Balmãna

first: Laurenz

last: Holcik

first: Maria

last: Novatchkova

first: Lina

last: Dobnikar

first: Thomas

last: Krausgruber

first: Sabrina

last: Ladstätter

first: Christoph

last: Bock

first: Arndt

last: von Haeseler

first: Christopher

last: Esk

first: Jürgen

last: Knoblich