title: FZD5 is an essential regulator of intestinal crypt homeostasis and surrogate Wnt agonist response

status: Public on Oct 18 2024

city: Leiden

first: Vincent

last: van Unen

state: ZH

address: Albinusdreef 2

department: Immunology

country: Netherlands

institute: Leiden University

summary: The rapid regeneration of the small intestinal epithelium is sustained by crypt intestinal stem cells (ISCs).  Wnt/b-catenin signaling is essential for intestinal crypt homeostasis and maintenance of Lgr5+ ISC, and yet no single or combinatorial knockout of Frizzled (FZD) genes, representing Wnt receptors, has phenocopied the severe intestinal epithelial effects of Wnt signaling blockade.  The elusive identification of specific Frizzled (Fzd) receptor(s) underlying homeostatic proliferation and ISC function would greatly inform therapeutic mucosal repair strategies.  In prior pharmacologic studies, bioengineered antagonists that block Wnt binding to both FZD5 and FZD8 receptors induced lethal crypt and villus loss, implicating FZD5 and/or FZD8 as essential for ISCs maintenance.  Here, the potential function of Fzd5 in during intestinal homeostasis was examined by epithelial-specific Fzd5 ko, which rapidly elicited lethal pan-intestinal crypt and villus loss, and by Lgr5-specific Fzd5 cKO, which strongly reduced Lgr5+ ISC while inducing their premature differentiation. In parallel, Fzd5 cKO potently repressed Wnt target gene expression, with phenotypic rescue by constitutive activation of b-catenin in vivo and confirmation upon in vitro organoid culture.  Fzd5 cKO but not Fzd8 cKO in organoids ablated responsiveness to dual specificity bioengineered FZD5/FZD8-selective Wnt surrogate agonists, which reversed DSS-induced colitis phenotypes in both wild-type and Fzd8 cKO mice.  Overall, our results implicate the FZD5 receptor as an essential regulator of crypt homeostasis, Lgr5+ ISCs and intestinal response to bioengineered Wnt surrogate agonists.

overall_design: Lgr5+ Intestinal stem cells were isolated by flow cytometry from the jejunum of Lgr5-eGFP-IRES-CreER mice (n=3 mice per condition). RNA isolation used the PicoPure RNA Isolation Kit (Applied Biosystems, Cat: KIT0204), followed by 10x Genomics Chromium 3’ library preparation and NGS sequencing. Sequences from scRNA-seq were processed with Cell Ranger software (10x Genomics) with UMI (unique molecular identifier) collapsing and alignment to the GRCm38 mouse transcriptome. scRNA-seq data were loaded into Seurat objects following their standard pipeline

first: Qinghui

last: Mu

first: Calvin

last: Kuo


title: Predictive prioritization of enhancers associated with pancreatic disease risk

status: Public on Oct 15 2024

city: bethesda

first: Efsun

last: Arda

state: Maryland

address: 41 medlars drive

country: USA

institute: National Institutes of Health

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series

first: H

middle: E

first: Li

last: Wang

first: Songjoon

last: Baek


title: Transcriptomic Landscape of Leptospira Forming Biofilm Reveals Adaptation to Starvation and General Stress while Maintaining Virulence.

status: Public on Oct 09 2024

city: Paris

first: Marc

last: Monot

address: 28 rue du docteur roux

country: France

institute: Institut Pasteur

type: Expression profiling by high throughput sequencing

summary: The life-threatening pathogen Leptospira interrogans navigates a dual existence: surviving in environmental reservoirs and infecting mammalian hosts. Leptospira biofilm formation is thought to be an important survival strategy in environmental contexts and may also contribute to the persistence of leptospirosis in maintenance hosts. Examining the correlation between biofilm formation and the virulence of pathogenic strains might improve our comprehension of the epidemiology of leptospirosis. To further explore Leptospira’s survival strategy, our study focused on elucidating the biological state of pathogenic Leptospira within biofilms, particularly aiming to uncover the adaptations and regulatory mechanisms that are involved in such complex microenvironments. To determine the transcriptional profile of pathogenic Leptospira in biofilm, we compared the genome-wide transcriptomic profiles in late biofilms (21 days old) with those in exponential planktonic cultures (5 days old), revealing a pronounced transcriptomic shift. While genes linked to motility, energy production, and metabolism were downregulated, those governing the general stress response, defense against metal stress, and redox homeostasis showed a significant upsurge, hinting at a tailored defensive strategy against stress in late biofilms. A standout finding was the increased expression of the csoR, copZ, and copA locus, integral to copper ion stress response in other bacterial genera, suggesting a unique adaptation to metal-induced stress. Further, despite a reduced metabolic state in biofilms, their disruption swiftly restored metabolic activity. Crucially, bacteria either in late biofilms or resulting from biofilm disruption retained virulence in a hamster infection model, defying the notion that biofilm maturation abolishes pathogenicity. In summary, our study highlights Leptospira's adaptive equilibrium in biofilms: minimizing cellular energy expenditure to conserve resources, potentially aiding in withstanding stresses while maintaining its pathogenicity. These insights are important for explaining the survival strategies of Leptospira, revealing that a biofilm lifestyle may confer an advantage in maintaining virulence. This understanding is essential for managing leptospirosis across both environmental reservoirs and mammalian hosts.

first: Grégoire

last: Davignon


title: Predictive prioritization of functional enhancers in human pancreas cells [HiChIP]

summary: We mapped enhancer-promoter interactions genome-wide in purified human pancreas cell types

overall_design: We performed HiChIP using the H3K27ac antibody to identify enhancer-promoter interactions in pancreatic alpha, beta, delta, acinar and duct cells, isolated and purified from organ donors. We followed the protocol described in Mumbach et al (PMID: 27643841)


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation [SLAM-seq]

status: Public on Oct 07 2024

city: La Jolla

first: Jesse

middle: R

last: Dixon

state: CA

address: 10010 N. Torrey Pines Rd.

institute: Salk Institute for Biological Studies

summary: The organization of the genome in three-dimensional space is highly dynamic, yet how these dynamics are regulated and the role they play in genome function is poorly understood. Here, we utilized acute depletion of NIPBL to characterize the role of cohesin-mediated loop extrusion in vivo. Using this approach, we find that many chromatin loops are rapidly diminished upon loss of NIBPL, consistent with recent single locus imaging studies showing that chromatin loops are transient. However, we also identified a subset of cohesin-dependent chromatin loops that are anchored in repressive chromatin and may be “long-lived”, given that they require NIPBL for their establishment upon mitotic exit, but are persistent when NIPBL is depleted from interphase cells. In addition to the reformation of 3D genome structures, mitotic exit coincides with widespread transcriptional activation. We found that NIPBL is essential for establishing the expression of lineage-defining genes during the M-G1 transition but has diminished impact in the steady-state maintenance of their expression. At genes sensitive to its depletion, NIPBL supports a unique local genome organization defined by greater spatial proximity to nearby enhancers and weaker transcription start site insulation of genomic contacts. Overall, we show that NIPBL-mediated loop extrusion is critical to genome organization and transcriptional regulation in vivo.

overall_design: Role of NIPBL in post-mitotic transcriptional reactivation, chromatin association of cohesin, and 3D genome organization.

first: Tessa

middle: M

last: Popay


title: Implantation of engineered adipocytes that outcompete tumors for resources suppresses cancer progression

status: Public on Oct 17 2024

city: San Francisco

first: Nadav

last: Ahituv

address: 1550 4th St., Rock Hall, room RH584C

department: Bioengineering and Therapeutic Sciences & Institute for Human Genetics

institute: UCSF

summary: Tumors acquire an increased ability to obtain and metabolize nutrients. Here, we engineered adipocytes to outcompete tumors for nutrients and show that they can substantially reduce cancer progression. Growing cells from several cancers (breast, colon, pancreas, prostate) with engineered human adipocytes or xenografts alongside adipose organoids significantly suppresses cancer progression and reduces hypoxia and angiogenesis. Transplanting modulated adipocyte organoids in pancreatic or breast cancer genetic mouse models significantly suppressed their growth and decreased angiogenesis and hypoxia. To further showcase therapeutic potential, we demonstrate that co-culturing engineered patient-derived adipocytes with tumor organoids from dissected human breast cancers significantly reduces cancer growth and reduces proliferation of organoids from donors with BRCA1/2 mutations. In addition, inducing adipose organoids to outcompete tumors via tetracycline or placing them in an integrated cell-scaffold delivery platform and implanting them next to the tumor, both significantly suppress cancer growth. Finally, to display how this approach can be customized for different tumor-associated metabolic programs, we show that upregulating UPP1 in adipose organoids can outcompete a uridine-dependent pancreatic ductal adenocarcinoma for resources and suppress its growth. Combined, our results introduce a novel cancer therapeutic approach, termed adipose modulation transplantation (AMT), that can be utilized for a broad range of cancers.

overall_design: RNA samples isolated MCF7 xenograft tumors that were co-implanted with either UCP1-CRISRPa modualted-  or dCas9-VP64 (control) treated- adipose organoids in SCID micce fed with chow, high-fet diet, or 15% glucose water

first: Hai

last: Nguyen

first: Kelly

last: An

first: Yusuke

last: Ito

first: Bhushan

middle: N

last: Kharbikar

first: Rory

last: Sheng

first: Breanna

last: Paredes

first: Elizabeth

last: Murray

first: Kimberly

last: Pham

first: Michael

last: Bruck

first: Xuija

last: Zhou

first: Cassidy

last: Biellak

first: Aki

last: Ushiki

first: Daniel

middle: A

last: Bernards

first: Filipa

last: Lynce

first: Deborah

last: Dillon

first: Mark

middle: J

last: Magbanua

first: Laura

last: Huppert

first: Heinz

last: Hammerlindl

last: Esserman

first: Tejal

last: Desai

first: Sook

middle: W

last: Yee

first: Jennifer

last: Rosenbluth


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation

first: Jesse

last: Dixon


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation [Hi-C]

first: Tessa

last: Popay


title: Predictive prioritization of functional enhancers in human pancreas cells [ATAC-Seq]

first: Efsun

last: Arda

summary: We mapped accessible chromatin regions genome-wide in purified human pancreas cell types

overall_design: We performed ATAC-seq on alpha, beta, delta, acinar and duct cell populations, isolated and purified from donor pancreas. We used the protocol described in Corces et al (PMID: 28846090).

first: Li

last: Wang

first: Songjoon

last: Baek

first: H


title: Triptolide sensitizes cancer cells to nucleoside DNA methyltransferase inhibitors through inhibition of DCTPP1 mediated cell-intrinsic resistance

status: Public on Oct 01 2024

city: Baltimore

first: Jianyong

last: Liu

address: 1550 Orleans St

department: Oncology

institute: Johns Hopkins University

type: Methylation profiling by genome tiling array

first: Ajay

last: Vaghasia


title: Genome-wide in vivo dynamics of cohesin-mediated loop extrusion and its role in transcription activation [ChIP-seq]


title: Epigenomic, transcriptomic and proteomic characterizations of reference samples

status: Public on Oct 10 2024

city: Loma Linda

first: Charles

address: 11021 Campus Street

department: Department of Basic Sciences

institute: Loma Linda University

type: Methylation profiling by high throughput sequencing

summary: A variety of newly developed next-generation sequencing technologies are making their way rapidly into the research and clinical applications, for which accuracy and cross-lab reproducibility are critical, and reference standards are much needed. Our previous multicenter studies under the SEQC-2 umbrella using a breast cancer cell line with paired B-cell line have produced a large amount of different genomic data including whole genome sequencing (Illumina, PacBio, Nanopore), HiC, and scRNA-seq with detailed analyses on somatic mutations, single-nucleotide variations (SNVs), and structural variations (SVs). However, there is still a lack of well-characterized reference materials which include epigenomic and proteomic data. Here we further performed ATAC-seq, Methyl-seq, RNA-seq, and proteomic analyses and provided a comprehensive catalog of the epigenomic landscape, which overlapped with the transcriptomes and proteomes for the two cell lines. We identified >7,700 peptide isoforms, where the majority (95%) of the genes had a single peptide isoform. Protein expression of the transcripts overlapping CGIs were much higher than the protein expression of the non-CGI transcripts in both cell lines. We further demonstrated the evidence that certain SNVs were incorporated into mutated peptides. We observed that open chromatin regions had low methylation which were largely regulated by CG density, where CG-rich regions had more accessible chromatin, low methylation, and higher gene and protein expression. The CG-poor regions had higher repressive epigenetic regulations (higher DNA methylation) and less open chromatin, resulting in a cell line specific methylation and gene expression patterns. Our studies provide well-defined reference materials consisting of two cell lines with genomic, epigenomic, transcriptomic, scRNA-seq and proteomic characterizations which can serve as standards for validating and benchmarking not only on various omics assays, but also on bioinformatics methods. It will be a valuable resource for both research and clinical communities.

overall_design: HCC1395 and HCC1395BL cell lines were sequenced by RRBS and ATAC-seq


title: RNA Sequencing FVB.B6 vs. CRYAB-R120G mutant mouse heart apex at 1,3, and 6 months of age

city: Vermillion

last: Bouska

state: SD

address: 414 E Clark St.

institute: Mark Bouska

summary: To investigate the transcriptional response to proteotoxic stress from CRYAB-R120G mutation in the heart with age, RNA sequencing of the heart apex was performed.

overall_design: RNA sequencing of the heart apex for FVB.B6 background and CRYAB-R120G mutant mice from 3 replicates of each genotype at 1,3, and 6 months of age followed by gene expression profiling analysis was performed.

first: Xuejung


title: Triptolide sensitizes cancer cells to nucleoside DNA methyltransferase inhibitors through inhibition of DCTPP1 mediated cell-intrinsic resistance [RNA-Seq]

summary: We show that high DCTPP1 expression was associated with cell-intrinsic resistance to nucleoside DNMT inhibition, and that triptolide and its analogs could overcome this resistance.

overall_design: mRNA profiles of DU145 treated with the DNMT inhibitor decitabine alone or in combination with triptolide

first: Liu

last: Jianyong

first: Chikarmane

last: Roshan

first: Steinberg

last: Ruchama

first: Nelson

middle: G

last: William

first: Yegnasubramanian

last: Srinivasan


title: Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity

status: Public on Oct 05 2024

city: Shanghai

first: Haochen

last: Yang

state: Shanghai

address: 320 Yue-yang Road

country: China

institute: Shanghai Institute of Biochemistry and Cell Biology

summary: Major challenges of CAR-T cell therapy include poor antigen sensitivity and cell persistence. Here we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of an engineered CD3e motif, EB6I, to the conventional 28Z or BBZ CAR induced self-phase separation through cation-π interactions. EB6I CAR can form a mature immunological synapse with CD2 corolla to transduce efficient antigen and costimulatory signaling, while its tonic signaling remains at low levels. Functionally, EB6I CAR-T cells exhibited improved signaling and cytoxicity against low-antigen tumor and persistent tumor killing function. In multiple primary and relapsed murine tumor models, EB6I CAR-T cells exerted better antitumor functions than conventional CAR-T cells against blood and solid cancers. This study thus unveils a new CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.

overall_design: To assess the long-term antritumor functions of the CD19.28Z/E28Z/EB6I28Z CAR-T cells, we continuously challenged CAR-T cells with multiple rounds of tumor cells. CAR-T cells with/without tumor cell challenge were sorted for RNA-seq.

first: Xinyi

last: Xu

first: Zhengxu

last: Ren

first: Chenqi


title: Transcriptomic analysis of effects of cancer cell detachment after treatment with 2-deoxy-D-glucose alone or in co-treatment with metformin

status: Public on Oct 20 2024

city: Ljubljana

first: Mojca

last: Pavlin

address: Vrazov Trg 2

country: Slovenia

institute: University of Ljubljana,

summary: Detachment of cancer cells from tumor, anchorage-independent survival, and maintenance of proliferative capacity upon reattachment are crucial steps in metastases formation. Better understanding of the underlying molecular mechanisms could help to improve targeted cancer therapies. We show reversible detachment of cancer cells using N-glycosylation inhibitors 2-deoxy-D-glucose (2DG) and tunicamycin, with re-attached cells retaining their proliferating capacity. This phenomenon was enhanced by combining with inhibitors of the electron transport chain.  Analysis of the detached MDA-MB-231 cells treated with 2DG or 2DG combined with metformin showed ER stress, upregulation of pentose phosphate pathway, stemness, and alterations in genes involved in EMT.

overall_design: MDA-MB-231 cells were plated on 6-well plates in RPMI-1640 medium supplemented with 25 mM glucose, 2 mM glutamine and 10 % FBS. Cells were washedand the medium was changed to complete RPMI medium with 5.6 mM glucose. Cells were treated for 72 hours with 4.8 mM 2DG or co-treated with 5 mM metformin and 0.6 mM 2DG. As additional sample, cells were grown on poly-HEMA (Poly(2-hydroxyethyl methacrylate)) coated plates were used. Medium was renewed daily, and the detached cells were collected and returned to the attached cell counterpart. After 72 hours, attached and detached cells were collected and counted separately. Both, attached and detached cells were harvested and washed twice with ice cold PBS, snap freezed in liquid nitrogen, and stored at – 80 °C. Total cell RNA was isolated from cell pellets using PureLink™ RNA Mini Kit (Invitrogen, ThermoFisher Scientific, Waltham, USA). Transcriptome sequencing of MDA-MB-231 cells was performed by Novogene (Cambridge, UK) and the sequencing was performed in triplicates in each group.

first: Tadeja

last: Snedec


title: Genetic changes through spheroid formation in ascites - interaction with ovarian cancer cell line, OV90, and human primary mesothelial cells (HPMC)-

city: Lund

first: Kaname

last: Uno

address: Medicon Village Building 404:B3

department: Clinical Sciences

country: Sweden

institute: Lund University

summary: More than 70% of patients with epithelial ovarian cancer (EOC) are diagnosed with peritoneal metastasis and ascites, which is the accumulation of intraperitoneal fluid containing non-malignant cells. However, the interactions between EOC and non-malignant cells before the development of peritoneal metastasis remain unclear. This study investigated the mechanism by which EOC cells survive through interactions with the surrounding cells in ascites. Whole EOC spheroids were observed, and their invasion into collagen layers and mesothelial monolayers was examined. RNA sequencing revealed the molecular mechanisms associated with aggressiveness of EOC cells. In malignant ascites, almost 100% of EOC cells were spheroids, 60% of which contained mesothelial cells. EOC cells quickly generated aggregated spheroids with mesothelial cells, and these aggregated cancer-mesothelial spheroids (ACMS) rapidly invaded collagen or mesothelial layers. Mesothelial cells forming ACMS initiated the invasion, with EOC cells following the route created by the mesothelial cells. RNA sequence revealed dramatically RNA expression changes in mesothelial cells, whereas the changes in EOC cells were relatively small. TGF-β1-stimulated mesothelial cells showed increased invadopodia formation along with fascin-1 upregulation. These findings suggest that EOC cells alter gene expression in mesothelial cells through ACMS formation, which explains the rapid spread of EOC in the abdominal cavity.

overall_design: We used OV90-GFP cells and three different kinds of primary human mesothelial cell (HPMC). To investigate the genetic changes through spheroid formation, three types of cell components were  incubated for 72 hours to form spheroids in ultra-low attachement 96-well round-bottome plates; (i) OV90-GFP only (2,000 cells/well), (ii) HPMC cells only (4,000 cells/well), (iii) a co-culture of OV90-GFP and HPMC cells (2,000 cells/well each). Before analyzing, these spheroids were disaggregated with trypsin and collected by FACS. Comparative gene expression profile analysis of RNA-seq data for (i) OV90-GFP cells with or without interaction with HPMC, and (ii) HPMC cells with or without interaction with OV90-GFP.

first: Yoshihiro

last: Koya

first: Mai

last: Sugiyama


title: Molecular Profiling Reveals Immunosuppressive Tumor Immune Microenvironment in Primary versus Paired Asynchronous Metastatic Clear Cell Renal Cell Carcinoma

status: Public on Sep 27 2024

city: Ann Arbor

first: Simpa

middle: S

last: Salami

state: MI

address: 1500 E Medical Center Dr

department: Urology

institute: University of Michigan

summary: Clear cell renal cell carcinoma (ccRCC) is a highly immune infiltrated cancer, but how the tumor immune microenvironment (TIME) evolves during disease progression is unknown. Insights into heterogeneity within primary ccRCC raise questions about potential heterogeneity between primary and paired metastatic tumors. This information may improve understanding of immune evasion leading to metastatic disease and response to immunotherapy. In this study, we characterize and compare the TIME of primary ccRCC with paired asynchronous metastases. We demonstrate higher infiltration of immunosuppressive cells in the TIME of primary ccRCC compared with metastatic sites. Further studies are necessary to determine the prognostic and therapeutic significance of these findings. Understanding the heterogeneity of the tumor immune microenvironment in RCC, both intratumorally and between metastatic sites, has important clinical implications for selecting appropriate therapies and developing predictive biomarkers. Future research should focus on elucidating the impact of these findings on immunotherapeutic responses and patient outcomes in ccRCC.

overall_design: Analysis of ccRCC patients who developed recurrence post radical nephrectomy and had both primary and metastatic tissue available. Whole-transcriptome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) specimens using the illumina platform. Differential expression gene (DEG) analysis was performed using R package edgeR. Gene set enrichment analyses (GSEA) to identify hallmark pathway enrichment was performed using R package fgsea. TIME deconvolution was quantified using CIBEROSRT, an in silico flow cytometry tool.

first: Brittney

middle: H

last: Cotta

first: Srinivas

last: Nallandhighal

first: Zayne

last: Knuth

first: Nathan

last: Graham

first: Yuping

last: Zhang

first: Rui

last: Wang

first: Amy

last: Kasputis

first: Xuhong

last: Cao

first: Aaron

last: Udager

first: Saravana

last: Dhanasekaran

first: Ganesh

last: Palapattu

first: Rohit

last: Mehra

first: Marcin

middle: P

last: Cieslik

first: Todd

last: Morgan


title: ATAC-seq for C42B treated with SR8278 or OE NR1D1; Lucap35CR treated with SR8278

status: Public on Sep 25 2024

city: Sacramento

first: yatian

last: yang

institute: UC Davis

summary: Genome-wide measurements of chromatin accessibility in prostate cancer cells to study the tumorigenic programs.

first: Yatian

last: Yang

first: Xiong

first: Demin

last: Cai

first: Hong-wu

last: Chen


title: Triptolide sensitizes cancer cells to nucleoside DNA methyltransferase inhibitors through inhibition of DCTPP1 mediated cell-intrinsic resistance [Array]

first: Jianyong

last: Liu

first: Roshan

last: Chikarmane

first: Ruchama

last: Steinberg

first: William

last: Nelson

first: Srinivasan

last: Yegnasubramanian


title: The H3K9me3 heterochromatin integrity and function are sustained by H3K9me3 methyltransferases-HP1 dependencies [H4K20me3 ChIP-seq]

status: Public on Sep 19 2024

city: Philadelphia

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

summary: H3K9me3-heterochromatin, compacted by HP1 proteins, restricts transcription factor binding and functions as a barrier to cell fate changes in development and reprogramming1,2. To investigate principles underlying H3K9me3-heterochromatin maintenance in mammalian cells, we applied degron engineering3,4 on mouse ESCs to conditionally deplete all three H3K9me3-methyltransferases within one hour and found an unexpectedly dynamic nature of heterochromatin maintenance. Following H3K9me3 methyltransferases degradation, HP1β dissociates from heterochromatin within three hours, and transcription factors quickly access the de-compacted heterochromatin, activating transcription of their targets, which then promotes H3K9me3 decay. Both passive dilution and active removal contribute to the H3K9me3 decay. Mathematical modeling reveals distinct H3K9me3 heterochromatin states with diverse chromatin features which influence initial H3K9me3 levels and the stability of H3K9me3 domains, thereby expanding our understanding of distinct heterochromatin domains. Rampant transposable element de-repression interferes with transcription of poised early developmental and signaling genes outside H3K9me3-domains, highlighting the intricate roles of dynamic H3K9me3-heterochromatin maintenance in safeguarding developmental programs. Our findings deconvolve the hierarchical relationships between H3K9me3 marks, methyltransferases, HP1 and transcription factors to reveal principles of H3K9me3-heterochromatin maintenance and how its remodeling enables transcription and cell fate change.

overall_design: A time-course experiment in which histone lysine 9 methyltransferases are ablated in mESCs and the decay of heterochromatin protein H4K20me3 is examined genome-wide by chIP-seq. The time-course runs 48hrs with collection points at 0hrs, 12hrs, 24hrs, and 48hrs. Three biological repeats were sequenced in three separate sequencing runs (of the same libraries) for additional genome coverage at each time point. The 0hrs time point is taken to be the baseline against which decay is measured. Matched inputs for all samples (sequenced and uploaded in GSE231845) were used to control for sonication efficiency. A drosophila chromatin spike-in is employed to properly adjust sample signal between different time points.

first: Jingchao

last: Zhang

first: Greg

first: Tomer

last: Lapidot

first: Dario

last: Nicetto

first: Kenneth

last: Zaret


title: Individual RNA-seq for different prostate cancer cells and Lucap35CR PDX treated by SR8278,JQ1,AZD5153,ENZ or by FOXA1 and NR1D1 gene knockdown or NR1D1 overexpression.

summary: Gene expression profile of different prostate cancer PDX tumor, and cell models to study tumorigenic programs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA-seq for C42B, 22RV1, Lucap35CR with different treatment.


title: Rhesus immune reference atlas (RIRA): a comprehensive single-cell atlas of rhesus macaque immune cells

status: Public on Sep 24 2024

city: Beaverton

first: Benjamin

last: Bimber

state: OR

address: 505 NW 185th Ave

summary: This is a single-cell RNA-seq atlas comprised of 10x genomics data from 47 macaques and 7 tissues

overall_design:
This dataset contains single-cell RNA-seq from bulk immune cells from rhesus macaque tissues
Please note that the series supplementary .rds data files have been updated on Feb 7, 2025.


title: Hepatokine ORM2 suppresses pathological ferritinophagy to prevent acute tissue injury

status: Public on Sep 22 2024

first: Hongyi

last: Tang

address: Chongqing Nan Road 280

department: Department of Biochemistry and Molecular Cell Biology

institute: Shanghai Jiao Tong University School of Medicine

summary: Acute tissue injuries trigger rapid cellular damage, but cell-intrinsic protective programs attenuate pathology through regulatory axes. Here, we demonstrate that Orosomucoid 2 (ORM2), a hepatokine, is significantly upregulated during drug-induced acute liver injury. Knock out Orm2 exacerbates liver damage, while its overexpression provides protection, identifying ORM2 as an endogenous hepatoprotective factor during acute liver injury. Mechanistically, ORM2 disrupts the FTH-NCOA4-TAX1BP1 interaction, thereby blocking ferritinophagy and preventing iron overload-induced cytotoxicity. Furthermore, acute ischemia-reperfusion injuries in other organs also trigger hepatic ORM2 upregulation and secretion, demonstrating liver-organ crosstalk via the circulatory system. Exogenous administration of ORM2 effectively ameliorates ferroptosis and tissue damage in multiple ischemia-reperfusion injury models. Collectively, our results identify the hepatokine ORM2 as a key suppressor of pathological ferritinophagy. This function positions ORM2 protein as a potential therapeutic candidate for acute tissue injuries driven by ferritinophagy-mediated iron overload.

overall_design: WT and Orm2 KO mice were injected with APAP or PBS, and liver tissues were collected 24 hours later for RNA-seq.

first: Xuxu

last: Sun

first: Bing

last: Zhou

first: Jun

last: Wu

first: Jie


title: The H3K9me3 heterochromatin integrity and function are sustained by H3K9me3 methyltransferases-HP1 dependencies

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


title: The H3K9me3 heterochromatin integrity and function are sustained by H3K9me3 methyltransferases-HP1 dependencies [ATAC-Seq]

overall_design: A time-course experiment in which histone lysine 9 methyltransferases are ablated in mESCs and newly open chromatin is examined genome-wide by ATAC-seq. The time-course runs 48hrs with collection points at 0hrs, 3hrs, 6hrs, 12hrs, 24hrs, and 48hrs. Three biological repeats were sequenced per time point. The 0hrs time point is taken to be the baseline against which decay is measured.


title: Single cell RNA sequencing data of SNCA triplication cortical organoids

status: Public on Sep 18 2024

city: Jacksonville

last: Zhao

state: FL

address: 4500 San Pablo Rd S

department: Neuroscience

institute: Mayo Clinic

summary: Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we generated human cortical LBD models by differentiating induced pluripotent stem cells (iPSCs) from SNCA triplication LBD patients into cerebral organoids and observed increased levels of pathological α-SYN in these organoids. Single-cell RNA sequencing revealed prominent expression of the SNCA gene in excitatory neurons, which exhibited synaptic and mitochondrial dysfunction, consistent with findings in the cortex of LBD human brains. Furthermore, screening 1280 FDA-approved drugs identified four candidates, which inhibited α-SYN seeding in RT-QuIC assay, reduced α-SYN aggregation and alleviated mitochondrial dysfunction in SNCA triplication iPSC models. Our findings provide valuable insights into the development of cortical LBD models and the discovery of potential drugs targeting α-SYN aggregation.

overall_design: Single cell RNA sequencing of isolated cells from SNCA triplication and control cortical organoids. Three replicates per subclone, two subclones per cell line, and two cell lines per triplication or control group

first: Yunjung

last: Jin

first: Tadafumi

middle: C

last: Ikezu


title: The H3K9me3 heterochromatin integrity and function are sustained by H3K9me3 methyltransferases-HP1 dependencies [ChIP-Seq]

overall_design: A time-course experiment in which histone lysine 9 methyltransferases are ablated in mESCs and the decay of heterochromatin proteins H3K9me3 and HP1b is examined genome-wide by chIP-seq. The time-course runs 48hrs with collection points at 0hrs, 1hr, 3hrs, 12hrs, 24hrs, and 48hrs. Three biological repeats were sequenced in three separate sequencing runs (of the same libraries) for additional genome coverage at each time point. The 0hrs time point is taken to be the baseline against which decay is measured. Matched inputs for all samples were used to control for sonication efficiency. A drosophila chromatin spike-in is employed to properly adjust sample signal between different time points.


title: Single nucleus RNA sequencing data of Lewy Body Dementia (LBD) brains

overall_design:
Single nucleus RNA sequencing of isolated nuclei from superior cortext region of LBD patients including SNCA duplication and SNCA triplication carriers and healthy individuals. Three brains from control individuals and five brains from LBD patients including one SNCA duplication and one SNCA triplication carrier were used.

UPDATE: [Mar-04-2026] The characteristics were updated for Samples GSM7511852-GSM7511857. The GSM7511853 title was changed from "LBD brain (SNCA Dup)" to "LBD brain (SNCA Tri)", and the GSM7511857 title was changed from "LBD brain (SNCA Tri)" to "LBD brain (SNCA Dup)".


title: Individual ChIP-seq for REV-ERBα, FOXA1,BRD4 and H3K27ac

first: yatian

last: yang

summary: Genome-wide measurements of histone modifications and transcripton factors binding in Lucao35CR PDX tumor and cell models to study FOXA and REV-ERBα regulate the tumorigenic programs

overall_design: ChIP-seq for different histone marks and transcriptional factors in C42B cell and Lucap35CR.

first: Yatian

last: Yang

first: Xiong

first: Demin

last: Cai

first: Hong-wu

last: Chen


title: Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma [CUT&Tag]

city: Chengdu, China

first: Ailing

last: Zhong

state: Sichuan

address: No.17 Renmin road section3

department: State Key Laboratory of Biotherapy, West China Hospital, Sichuan University.

institute: Sichuan University

summary: Signet ring cell carcinoma (SRCC) is a lethal malignancy with distinctive histologic features, characterized by mucin-enriched vacuoles in the cytoplasm and compressed nuclei. Gastric SRCC is the most common form of SRCC with increasing incidence in recent years. However, the driver genes of SRCC and the molecular mechanisms underlying its unique morphology remain poorly understood. Here, we report that AT-rich interactive domain-containing protein 1A (ARID1A), one of the most frequently mutated genes in SRCC is a bona fide tumor suppressor gene in gastric SRCC. Its loss, together with Trp53 and Pten loss, drove a SRCC in mice. Mechanistically, Arid1a loss upregulates the expressions of mucins mediated by the BRD9-containing ncBAF complex, which would compete with the ARID1A-containing cBAF complex. And Arid1a can directly regulates the expression of Scin, which is responsible for mucins. And inhibition of Brd9 can ameliorate the malignancy of SRCC. Thus, our study revealed dual roles of ARID1A in restraining SRCC by regulating both mucin production and secretion, providing new mechanistic insights and potential therapeutic vulnerabilities for ARID1A-deficient SRCC.

overall_design: We analyzed gastric organoids with two genotypes: Trp53−/−; Pten−/− (TP) and Trp53−/−; Pten−/−; Arid1a−/− (TPA). sgRNAs targeting Pten and Arid1a were introduced into Trp53−/−; Cas9-EGFP gastric organoids to generate the corresponding genotypes. TP organoids served as the control group, and an IgG control was included to account for nonspecific background binding. Organoids from each group were collected for downstream CUT&Tag-seq library preparation and comparative differential binding analyses.

first: Hongyu

last: Liu


title: Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma

summary: Signet ring cell carcinoma (SRCC) is a lethal malignancy with distinctive histologic features, characterized by mucin-enriched vacuoles in the cytoplasm and compressed nuclei. Gastric SRCC is the most common form of SRCC with increasing incidence in recent years. However, the driver genes of SRCC and the molecular mechanisms underlying its unique morphology remain poorly understood. Here, we report that AT-rich interactive domain-containing protein 1A (ARID1A), one of the most frequently mutated genes in SRCC, is a bona fide tumor suppressor gene in gastric SRCC. Its loss, together with Trp53 and Pten loss, drove SRCC in mice. Mechanistically, Arid1a loss upregulates the expression of mucins mediated by the BRD9-containing ncBAF complex, which competes with the ARID1A-containing cBAF complex. In addition, Arid1a directly regulates the expression of Scin, which is responsible for mucin secretion. Inhibition of Brd9 can ameliorate the malignancy of SRCC. Thus, our study reveals dual roles of ARID1A in restraining SRCC by regulating both mucin production and secretion, providing new mechanistic insights and potential therapeutic vulnerabilities for ARID1A-deficient SRCC.

overall_design: We analyzed RNA-seq data generated from both bulk and monoclonal Trp53⁻/⁻; Pten⁻/⁻ (TP) and Trp53⁻/⁻; Pten⁻/⁻; Arid1a⁻/⁻ (TPA) gastric organoids. Briefly, sgRNAs targeting Pten and Arid1a were introduced into Trp53⁻/⁻; Cas9-EGFP gastric organoids. Following genome editing, a subset of organoids was subjected to clonal isolation to establish monoclonal organoid lines, and individual clones were expanded for downstream analyses. RNA-seq libraries were prepared either from bulk organoid populations (without clonal isolation) or from individually expanded monoclonal organoid lines.


title: Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma [ATAC-Seq]

overall_design:
We analyzed gastric organoids derived from two engineered genotypes: Trp53-/-; Pten-/- (TP) and Trp53-/-; Pten-/-; Arid1a-/-(TPA). TP organoids served as the reference group, while TPA organoids represented additional Arid1a loss. sgRNAs targeting Pten and Arid1a were introduced into Trp53-/-; Cas9-EGFP gastric organoids to generate the respective genotypes. Organoids from each group were collected for downstream ATAC-seq library preparation and comparative chromatin accessibility analyses.
On March 3, 2026 all samples were replaced.


title: Dual roles of ARID1A in both mucin production and secretion to restrain gastric signet ring cell carcinoma

overall_design: Refer to individual Series


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: Single Cell RNA sequencing of CD11b+Gr1+ cells from the bone marrow of young and aged mice

status: Public on Sep 01 2024

city: Guangzhou

first: Siyuan

last: Zhu

state: Guangdong

address: 1838 North Guangzhou Avenu

institute: Nanfang Hospital, Southern Medical University

summary: Myeloid-derived suppressor cells (MDSCs) are recognized as one of the key mediators of immunosuppression in aging process, which induce immunosenescence and increase elderly people’s susceptibility to to infections, cancers, autoimmune diseases, and degenerative diseases. It still remains elusive whether MDSCs represent a unique subset of myeloid cells that differ from their young, healthy counterparts, which will hamper the deeper understanding of the pathophysiological functions of MDSCs. In the present study we conducted scRNAseq analysis to compare MDSCs from three aged mice and three young mice. We established the MDSC-specific gene signature of aged mouse, which revealed the general characteristics of MDSCs, and thus faciliating distinguishing them from normal myeloid cells in aging process. In addition, our study provides a single-cell transcriptional map defining the development of MDSCs in aging process, which will ultimately enable us to specifically target these cells in old individuals.

overall_design: In the present study we conducted scRNAseq analysis of mice myeloid cells collected from three old mice and three young mice using the 10X Genomics Chromium Platform and sequenced on the Illumina HighSeq 3000.


title: Frizzled5 Maintains Homeostasis of Adult Intestinal Epithelium Through Organization of Chromatin Accessibility

status: Public on Aug 31 2024

city: Kansas City

first: Shiyuan

middle: (Cynthia)

state: MO

department: Computational Biology

institute: Stowers Institute for Medical Research

summary: Analyze the gene profiles and chromatin accessibility change upon Fzd5 knockout in mouse intestinal epithelium

overall_design: ScRNA-seq of Intestinal epithelial cells, ScRNA-seq of Intestinal Lgr5 positive cells, ScRNA-seq of Ctrl and Fzd5KO Intestinal epithelial cells, and ScATAC-seq of Ctrl and Fzd5KO Intestinal nuclei

first: Lu

last: Deng

first: Linheng

last: Li


title: Single-cell RNA-sequencing of human intestinal organoid cells

status: Public on Aug 30 2024

city: Utrecht

first: Daisong

last: Wang

address: Uppsalalaan 8

institute: Hubrecht Institute

summary: We focused on a rare cell population of human intestine, the M cells. Using human intestinal organoid as a model, the M cells could be differentiated, FACS-sorted from organoids and analyzed by scRNA-seq.

overall_design: Human intestinal organoids were cultured in M cell differentiation medium. Cells derived from the differentiated M cell orgnaoids were FACS sorted and individally collected in the 384 well plates for SORT-seq based scRNA-seq analysis.

first: Sangho

last: Lim


title: The H3K9me3 heterochromatin integrity and function are sustained by H3K9me3 methyltransferases-HP1 dependencies [TT-Seq]

first: Gregory

last: Donahue

overall_design: A time-course experiment in which histone lysine 9 methyltransferases are ablated in mESCs and the expression of genes, repeats, and other transcriptional units is examined genome-wide by nascent RNA TT-seq. The time-course runs 48hrs with collection points at 0hrs, 3hrs, 12hrs, 24hrs, and 48hrs. Three biological repeats were sequenced in two separate sequencing runs (of the same libraries) for additional genome coverage at each time point. The 0hrs time point is taken to be the baseline against which decay is measured. A S. cerevisiae total RNA spike-in is employed to properly adjust sample signal between different time points.

first: Jingchao

last: Zhang

first: Greg

first: Tomer

last: Lapidot

first: Dario

last: Nicetto

first: Kenneth

last: Zaret


title: Bulk RNA-sequencing of human intestinal organoid cells

summary: We focused on a rare cell population of human intestine, the M cells. Using human intestinal organoid as a model, the M cells could be differentiated, FACS-enriched and analyzed by RNA-seq.

overall_design: Human intestinal organoids were cultured in M cell differentiation medium. Cell subpopulations derived from the differentiated M cell orgnaoids were FACS sorted and collected in TRIzol for bulk RNA-seq analysis.


title: PANoptosis, a combination of inflammatory cell death mechanisms, induced by Ophiobolin A in breast cancer cell lines

status: Public on Sep 17 2024

city: Waco

first: Joe

last: Taube

state: Texas

address: One Bear Place #97388

institute: Baylor University

summary: An unmet challenge in managing breast cancer is treatment failure due to resistance to apoptosis-inducing chemotherapies. Thus, it is important to identify novel non-apoptotic therapeutic agents. Several non-apoptotic programmed cell death pathways utilize specific cellular signaling events to trigger lytic and pro-inflammatory cell death. PANoptosis, which encompasses pyroptosis, apoptosis and necroptosis, is of paramount importance in the regulation of cell death and immune responses. Our study illustrates that ophiobolin A (OpA) is an anti-cancer agent that triggers lytic cell death in breast cancer cells, including triple-negative breast cancer (TNBC), via a mechanism dependent on RIPK1. This study reveals that OpA induces typical pyroptosis-like characteristics, including cellular swelling, plasma membrane rupture, GSDMD cleavage and release of cytokines in breast cancer cells. The involvement of caspase 3, RIPK1, and GSDMD suggests that PANoptosis is activated upon OpA treatment in breast cancer. The induction of pro-inflammatory cell death suggests potential applications for OpA in cancer treatment.

overall_design: To investigate the impact of OpA on gene expression in MDA-MB-231 cells, we compared triplicate samples of vehicle-treated and OpA-treated cells.

first: Joseph

first: Santha

last: Ranganathan


title: The human pathome shows sex specific aging patterns post-development

status: Public on Aug 24 2024

city: Copenhagen N

first: Morten

last: Scheibye-Knudsen

state: Copenhagen N

address: Blegdamsvej 3B

country: Denmark

institute: University of Copenhagen

summary: Little is known about tissue specific changes that occur with aging in humans. Using the description of 33 million histological samples we extract thousands of age- and mortality-associated features from text narratives that we call The Human Pathome (pathoage.com). Notably, we can broadly determine when pathological aging starts, indicating a sexual dimorphism with females aging earlier but slower and males aging later but faster. Using machine learning, we employ unsupervised topic-modelling to identify terms and themes that predict age and mortality. As a proof of principle, we cross reference these terms in PubMed to identify nintedanib as a potential aging intervention and show that nintedanib reduces markers of cellular senescence, reduces pro-fibrotic gene pathways in senescent cells and extends the lifespan of fruit flies. Our findings pave the way for expanded exploitation of population datasets towards discovery of novel aging interventions.

overall_design: Examination of transcriptome in ionizing radiation (IR) induced senesecent human dermal fibroblasts treated with varying doses of Nintedanib and Axitinib versus control

first: Michael

last: Ben Ezra

first: Jonas

last: Bach Garbrecht

first: Nasya

last: Rasmussen


title: Frizzled5

status: Public on Aug 28 2024

first: Shiyuan

last: Chen


title: Bulk RNA-seq of intestinal epithelial cells from Ctrl and Fzd5KO

summary: General gene profiles change after Fzd5 knockout

overall_design: 3 replicates for each group

first: Lu

first: Linheng


title: Cancer-cell-secreted miR-9-5p regulates cholesterol homeostasis to promote tumor metastasis [TF TB Rab27AKD miR-9KO]

city: Wuhan

first: Li

last: Mei-Xin

address: College of Life Science, Wuhan University, Luojiashan Road, Wuchang District, Wuhan City, Hubei Province, China

institute: Wuhan University

summary:
To investigate the effects of breast cancer derived EVs on liver metabolism,we inoculated MDA-MB-231,231 /Rab27A KD and 231 /miR-9 KO cells into subcutaneous tumor in NSG mice.
We then performed gene expression profiling analysis using data obtained from RNA-seq of liver from mice xenografted MDA-MB-231 cells (tumor bearing) or MDA-MB-231/Rab27A KD cells (231/Rab27A KD) or MDA-MB-231 /miR-9 KO (231/miR-9 KO) and tumor free mice.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for liver (tumor free, MDA-MB-231 cells (tumor bearing), MDA-MB-231/Rab27A KD cells (231/Rab27A KD) and MDA-MB-231/miR-9 KO cells (231 /miR-9 KO)).

first: Mei-Xin

first: Wei

last: Yan


title: Myokine secretion enhancement by the addition of lavender essential oil in cultured skeletal muscle cells

city: Tsukuba

first: RANDEEP

last: RAKWAL

state: Ibaraki

address: 1-1-1 Tennodai

department: Institute of Health and Sport Sciences

country: Japan

institute: University of Tsukuba

summary: In recent years, aromatherapy for sportspersons termed as sports aromatherapy has been attracting attention in the field of sports, and aromatic baths and aromatherapy treatments are being used as a new conditioning method, mainly by athletes. Among these, aroma treatment is used to improve performance and recovery from fatigue. On the other hand, myokines (muscle-derived cytokines and chemokines) secreted from the skeletal muscles after exercise, have recently been attracting attention. Many types of myokines are known to act on various organelles and through diverse beneficial metabolic actions promote health. One of them is IL-6, which has been reported to act on muscles and promote myogenesis. Previous studies on essential oil treatment effects are thought to be due to aroma or massage stimulation, and there are no reports of direct effects on muscle at the molecular level comparing essential oils with carrier oils alone. Therefore, we aimed to elucidate the molecular mechanism of myokine secretion by the addition of an essential oil (Lavender oil, LO) using an in vivo exercise model of a mouse muscle cell line (C2C12 cells), which are known as exercise cells.

overall_design: Whole genomic DNA microarray was performed in C2C12 cells (total RNA was extracted using RNeasy Micro Kit), suggesting myokines produced by lavender essential oil and EPS(Electric Pulse Stimulation treatment).

first: Fumiko

last: Takenoya

first: Junko

last: Shibato

first: Michio

last: Yamashita

first: Makoto

last: Kanzaki

first: Seiji

last: Shioda

first: Randeep

last: Rakwal


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

last: Fallgatter

first: Hansjürgen


title: Chromatin assessibility detection in the absence of Fzd5 gene in intestinal epithelium [Cut & Tag]

first: Shiyuan

last: Chen

summary: Using CUT&TAG to assess Fzd5 knockdown effect on epithelial chromatin assessiblity change, using anitbodies targeting PoII2, H3K4trime3, and Beta-catenin.

overall_design: Two groups: Ctrl and Fzd5KO. Replicates for each antibody of each group.

first: Lu

last: Deng

first: Linheng

last: Li


title: METTL14 is a chromatin regulator independent of RNA N6-methyladenosine (nascent RNA)

status: Public on Aug 16 2024

city: Shanghai

first: Xiaoyang

last: Dou

address: 320 Yue Yang Road

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

summary: METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC, also known as m6A writer) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on mouse embryonic stem cell (mESC) self-renewal. While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-indenepent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification and PRC2 complex. Mechanically, METTL14, but not METTL3, recognizes H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression  . The regulation of H3K27me3 by METTL14 is essential to the transition of mESCs from self-renewal to differentiation. This work reveals a regulation mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance and differentiation of mESCs.

overall_design: Examination of transcription rate changes upon Mettl14 knockout in mESCs

first: Chuan

last: He

first: Jun

last: Liu


title: Tumor-derived miR-9-5p-loaded EVs regulate cholesterol homeostasis to promote breast cancer liver metastasis in mice.

first: Li

last: Mei-Xin

summary: Cancer cells secrete extracellular vesicles (EV) encapsulating bioactive cargoes to facilitate inter-organ communication in vivo and are emerging as critical mediators of tumor progression and metastasis, a condition which is often accompanied by a dysregulated cholesterol metabolism. Whether EVs are involved in the control of cholesterol homeostasis during tumor metastasis is still undefined and warrant further investigation. Here, we find that breast cancer-derived exosomal miR-9-5p induces the expression of HMGCR and CH25H, two enzymes involved in cholesterol synthesis and the conversion of 25-hydroxycholesterol from cholesterol by targeting INSIG1, INSIG2 and ATF3 genes in the liver. Notably, in vivo miR-9-5p antagomir treatment and genetic CH25H ablation prevents tumor metastasis in a mouse model of breast cancer. Thus, our findings reveal the regulatory mechanism of tumor-derived miR-9-5p in liver metastasis by linking oxysterol metabolism and Kupffer cell polarization, shedding light on future applications for cancer diagnosis and treatment.


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [RNA-seq-blastocyst]

status: Public on Aug 07 2024

city: Bethesda

first: Pedro

last: Rocha

address: 6 Center Drive Building 6B 2B220

department: NICHD

institute: National Institutes of Health

summary: Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes¬—Fgf3, Fgf4, and Fgf15—that regulate several developmental processes¬. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain¬ resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes¬—for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, our work starts to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries.  Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

overall_design: RNA sequencing was done in single blastocysts, by isolating RNA using Dynabeads mRNA DIRECT Purification Kit as previously described (Huffman et al., 2012). Blastocysts at E4.5 were flushed out of the uterine horns from super-ovulated females using M2 media and transferred into 50 µl of pre-warmed lysis buffer (100mM Tris-HCl pH-7.5, 500mM LiCl, 10mM EDTA pH-8, 1%LiDS, 5mM DTT) in DNA low binding PCR tubes. Lysed embryos were stored in -20C and processed within a few weeks. Dynabeads Oligo(dT)25 mRNA isolation beads were warmed to room temperature for 30 mins and rinsed with 100 µl of lysis buffer by vortexing continuously for 5 mins. 10 µl of bead suspension was used per embryo lysate. The poly A tail of mRNA was allowed to anneal to beads by mixing the embryo lysates and bead suspension in a vortexer for 5 mins at low speed, followed by 5 mins incubation without shaking. Tubes were briefly spun and placed in a magnetic stand to collect the clear supernatant, which was used for DNA isolation using 2X SPRI beads to determine the genotype of each embryo. mRNA bead complexes were washed twice using Wash Buffer A and twice with Wash Buffer B by vortexing for 5 mins each. cDNA and library prep were then processed using an adaptation of the smartseq2 protocol (Picelli et al., 2014; Satija et al., 2015; Trombetta et al., 2014). Buffers were removed and beads annealed with mRNA was resuspended in 12.5µl of RNA suspension mix containing 20U/µl SUPERase-In RNase inhibitor (Thermo Fisher, #AM2694), 10mM each dNTP Mix (New England Biolabs, #N0447L) and 100µM polydT oligonucleotide primer (custom sequence ‘AGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN’ synthesized by Integrated DNA Technologies). Bead suspension is heated at 75°C for 5mins to denature the RNA and supernatant is transferred into a new RNase free 0.2ml PCR tube.  To each sample tube 7.5µl of first strand reverse transcription mix with 100mM DTT, 5X SuperScript IV Buffer, SuperScriptTM IV Reverse Transcriptase (Thermo Fisher, #18090050), 20U/µl SUPERase-In RNase inhibitor (Thermo Fisher, #AM2694), 100µM of template switching oligo with 3G’s and an adaptor sequence same as polydT oligonucleotide (custom sequence ‘AGACGTGTGCTCTTCCGATCTNNNNNrGrGrG’, Integrated DNA Technologies) was added and incubated at 50°C for 60 mins to prepare the first strand of cDNA, followed by 85°C for 5mins to inactivate the enzyme. The first strand of cDNA is bound by the template switching oligo and a complementary sequence to the template switching oligo was synthesized, where a PCR oligo is hybridized (custom sequence ‘AGACGTGTGCTCTTCCGATCT’, Integrated DNA Technologies) and amplified. PCR cDNA amplification was done for 16 cycles (98°C for 15 secs, 67°C for 20 secs and 72°C for 10 mins) in a thermal cycler with KAPA Hifi HotStart ReadyMix (Roche, #9420398001). Pre-amplication cDNA mix was purified with 0.8X SPRI beads and quality of cDNA was assessed with a D5000 Screen Tape assay (Agilent, #5067-5588) using Agilent 4150 Tapestation. cDNA mix was diluted to 0.2ng/µl and used for library preparation as described earlier (Trombetta et al., 2014) using Nextera XT DNA Library Prep Kit (Illumina, #FC-131-1024). Briefly, in a 384 well plate, 1 µl of cDNA, 2µl of Tagment DNA Buffer and 1µl of Amplicon Tagment Mix was mixed in ice and tagmentation was done in a thermocycler at 55°C for 10 mins. Reaction was neutralized with 1 µl of Neutralize Tagment Buffer, incubated at room temperature for 5 mins and transferred to ice. Amplification PCR was set up with 3µl of Nextera PCR Mastermix and 2µl of each adaptor from Illumina DNA/RNA UD Indexes Set A (Illumina, #20027213) per sample. Tagmented DNA was incubated at 72°C for 3 mins, 95°C for 30 secs and 12 cycles of 95°C for 10 secs, 55°C for 30 secs, 72°C for 1 min and a final extension at 72°C for 5 mins. Library DNA was purified with 0.9X SPRI and quality was assessed using D1000 Screen Tape assay (Agilent, #5067-5585) before sequencing.

first: Shreeta

last: Chakraborty

first: Alicia

last: Evans

first: Ryan

middle: K

last: Dale


title: Early Decline of Conventional NK Cells During Acute Pseudomonas aeruginosa Lung Infection

status: Public on Aug 13 2024

city: Beijing

first: Duo

last: Su

address: Street Dongdajie

institute: Beijing Institute of Microbiology and Epidemiology

summary: To assess gene expression changes, RNA-sequencing technology was employed to map the temporal shifts in the transcriptional profile of the host lung post-infection. Our findings provide novel insights into the pathogenesis of PA pulmonary infections and offer valuable suggestions for inhalational infection protection and immunotherapy.

overall_design: Lung mRNA profiles of 8 week-old female wild type (WT) mice infected with PAO1 by aerosolized intratracheal inoculation and control mice were genreated by deep sequencing on the Illumina sequencing platform NovaSeq 6000. RT–PCR validation was performed using  SYBR Green assays.

first: Fuliang

last: Zong

last: Xiao

first: Yifeng

last: Wang

first: Huiying

last: Yang


title: A convenient single-cell newly synthesized transcriptome assay reveals FLI1 regulon downregulation during T-cell activation

status: Public on Aug 23 2024

last: Lyu

address: 37 Convent Drive

department: Laboratory of Biochemistry and Molecular Biology

institute: National cancer institute

summary: We invented NOTE-seq, a method for simultaneous profiling of regular and newly synthesized transcriptomes in single cells with high cellular throughput. NOTE-seq integrates 4-thiouridine labeling of newly synthesized RNA, thiol-alkylation-based chemical conversion, and a streamlined 10X Genomics workflow, making it accessible and convenient for biologists without extensive single-cell expertise. Using NOTE-seq, we investigated the temporal dynamics of gene expression during early-stage T-cell activation, identified transcription factors and regulons in Jurkat and naïve T cells, and uncovered the down-regulation of FLI1 as a master transcription factor upon T-cell stimulation. Notably, topoisomerase inhibition led to the depletion of both topoisomerases and FLI1 in T cells through a proteasome-dependent mechanism. This degradation was driven by topoisomerase cleavage complexes rather than topoisomerase catalytic inhibition, highlighting potential complications topoisomerase-targeting cancer chemotherapies could pose to the immune system.

overall_design:
eHAP cells were cultivated and subjected to NOTE-seq and 10xGenomics 3' expression v3.1 kit. Jurkat T cells were cultivated and subjected to PMA/ION treatment versus DMSO treatment, followed by NOTE-seq profiling. Mouse naïve T cells were subjected to antiCD3 and antiCD28 stimulation, followed by NOTE-seq profiling.  For the barnyard experiment, Jurkart T cells and NIH3T3 cells were mixed at a 1:1 ratio, followed by the NOTE-seq workflow without T-to-C conversion.

newly synthesized transcriptome profiling at single cell level for eHAP1 cells fixed with formaldehyde (NOTE-seq) or methanol (MeOH-4sU-10X). NOTE-seq profiling of K562 cells with or without chemical conversion. NOTE-seq profiling of Jurkat cells with or without PMA/ION stimulation

first: Xiaoyan

last: Xu

first: Chongyi


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [RCMC]

summary: Chromatin domain boundaries delimited by CTCF motifs have been shown to restrict the range of enhancers. However, disruption of domain structure often results in mild gene expression changes, and predicting the impact of boundary rearrangements on animal development remains challenging. By targeting clusters of CTCF motifs in a domain with three FGF genes—Fgf3, Fgf4, and Fgf15—we tested whether gene regulation and mouse development may be particularly sensitive to disruption of chromatin domains with multiple developmental regulators. Deletion of a cluster of four CTCF motifs that defines the centromeric boundary of this domain, resulted in ectopic interactions of the FGF genes with brain enhancers located across the deleted boundary, and a strong induction of FGF expression that led to perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary loss was enough to cause these fully penetrant phenotypes, and strikingly, loss of the single CTCF motif within the cluster that is oriented towards the brain enhancers, was sufficient to induce ectopic FGF expression and cause encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF-mediated boundaries of this domain nor to all developmental processes controlled by these three FGF genes. Although precise control of FGF4 levels is essential to regulate blastocyst development, none of our structural perturbations affected implantation. In fact, deletion of a CTCF boundary between Fgf3 and Fgf4, showed that the ability of these neighboring genes to have fully divergent expression patterns in blastocysts is achieved through CTCF-independent mechanisms and relies on remarkable enhancer-promoter specificity in vivo. Our work highlights how small sequence variants at certain domain boundaries can have a surprisingly outsized phenotypic effect and provides important insight into how different developmental contexts affect phenotypic sensitivity to perturbation of chromatin structure.

overall_design: RCMC was performed in the following tissues: blastocyst-derived mouse embryonic stem cells and midbrain of E11.5 and E12.5 embryos. For ES cells, 2 individual homozygous embryos were processed and analyzed seperately.  WT controls are littermates from isolated embryos. Midbrain samples were pooled from several WT and homozygous mutant embryo.


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [Micro-C]

overall_design: Promoter capture was performed in the following tissues: Midbrain of E11.5 and E12.5 embryos. Each  sample was pooled from several WT and homozygous mutant embryos.


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [CHiC]

overall_design: Capture HiC (CHiC) was performed in the following tissues: blastocyst-derived mouse embryonic stem cells, differentiated XEN cells and midbrain of E11.5 embryos. For ES and XEN cells, 2 individual homozygous embryos were processed and analyzed seperately.  WT controls are littermates from isolated embryos. Midbrain samples were pooled from several WT and homozygous mutant embryo.


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [RNA-seq-brain]

overall_design: RNA sequencing was performed in the following tissues: forebrain and midbrain of E11.5 embryos. For each line homozygous embryos and WT littermates were processed using trizol reagent.After confirming that the RNA integrity number for each sample was above 8, libraries were prepared using TruSeq Stranded mRNA prep kit with PolyA purification and sequenced on Novaseq6000.


title: QTL Mapping and Bulk Segregant Analysis identifies CO2 tolerance genes associated with virulence in the global pathogen Cryptococcus neoformans

status: Public on Aug 09 2024

city: Iowa City

first: Damian

last: Krysan

state: IA

address: 25 South Grand, 2040 ML

department: Division of Pediatrics & Infectious Disease

institute: University of Iowa Hospitals and Clinics

summary: Cryptococcus neoformans is a ubiquitous free-living soil yeast and opportunistic pathogen that causes ~223,100 cases of cryptococcal meningitis per year, killing over 180,000 people. The pathogenicity of C. neoformans relies on its adaptation to the host conditions. An important difference between its natural environment and the mammalian host is the concentration of CO2. CO2 levels in the host fluctuate around 5%, which is ~125-fold higher than in ambient air. We recently found that while clinical isolates are tolerant to host levels ofCO2, many environmental isolates are CO2-sensitive and virulence-attenuated in animal models. The genetic basis responsible for cryptococcal adaptation to high levels of CO2 is unknown. Here, we utilized quantitative trait loci (QTL) mapping with 374 progeny from a cross between a CO2-tolerant clinical isolate and a CO2-sensitive environmental isolate to identify genetic regions regulating CO2 tolerance. To identify specific quantitative trait genes (QTGs), we applied fine mapping through backcrossing and bulk segregant analysis coupled with pooled genome sequencing of near-isogenic progeny but with distinct tolerance levels to CO2. The roles of the identified QTGs in CO2 tolerance were verified by targeted gene deletion. We further demonstrated that virulence levels among near-isogenic strains in a murine model of cryptococcosis correlate with their levels of CO2 tolerance. Moreover, we discovered that sensitive strains may adapt in vivo to become more tolerant to increased CO2 levels and more virulent. These findings highlight the underappreciated role of the host CO2 tolerance and its importance in the ability of an opportunistic environmental pathogen to cause disease.

overall_design: To investigate the effects of host levels of CO2 compared to enviornmental levels of CO2 on Cryptococcus neoformans gene expression in environmental isolates A1-84-14 and A7-35-23 compared to a clinical isolate and reference strain, H99 at 24 hours, in biological triplicate.

first: Benjamin

last: Chadwick

first: Xiaofeng

last: Xie

first: Laura

last: Ristow

first: Xiaorong

last: Lin


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [Cut&Run]

overall_design: CUT&RUN sequencing was performed in blastocyst derived mouse embryonic stem cells.

first: Joyce

last: Thompson


title: Metabolic reprogramming driven by TFE3 fusions unveils novel vulnerabilities in translocation renal cell carcinoma [RNA-Seq]

status: Public on Aug 10 2024

city: Boston

first: Dipanjan

last: Chowdhury

state: MA

address: 4 Blackfan Circle

institute: Dana-Farber Cancer Institute

summary: Translocation renal cell carcinoma (tRCC) is an aggressive subtype of kidney cancer driven by TFE3 gene fusions, which act via poorly characterized downstream mechanisms. Here we report that TFE3 fusions transcriptionally rewire tRCCs toward oxidative phosphorylation (OXPHOS), contrasting with the highly glycolytic metabolism of most other RCCs. The transcriptional program driven by TFE3 fusions sustains high NRF2 signaling and glutathione production, which offsets reactive oxygen species generated by OXPHOS but renders tRCC cells sensitive to reductive stress. Genome-scale CRISPR screening identifies tRCC-selective vulnerabilities linked to maintaining this metabolic balance, including EGLN1, which hydroxylates HIF-1α and targets it for proteolysis. Inhibition of EGLN1compromises tRCC cell growth by stabilizing HIF-1a and promoting glycolytic reprogramming. Our study defines a distinctive tRCC-essential metabolic program driven by TFE3 fusions and nominates EGLN1inhibition as a therapeutic strategy to counteract fusion-induced metabolic rewiring.

overall_design: To inverstigate the regulatory role of TFE3 fusion in tRCC, we performed RNA-Seq upon TFE3 fusion knockout/knockdown in tRCC.

first: Jiao

last: Li

first: Srinivas

last: Viswanathan


title: Transcriptional Profile Tumor Evolution in Metastatic and Non Metastatic MYC driven HCC

status: Public on Aug 06 2024

city: Stanford

first: Renumathy

last: Dhanasekaran

address: 300 Pasteur Drive, Alway M211

institute: Stanford University

summary: Comparative transcriptional profiling of MYC driven HCC and MYC/Twist1 driven HCC during tumor progression, regression and recurrence.

overall_design: RNA sequencing of MYC-HCC and MYC/Twist1-HCC was performed.

first: Renu

first: Dean

last: Felsher


title: Minimal Residual Disease in Mouse Hepatocellular carcinoma

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

overall_design: Refer to individual Series


title: Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development

first: Pedro

last: Rocha


title: Highly sensitive mapping of type II topoisomerase DNA cleavage sites with SHAN-seq

first: Ian

middle: L

last: Morgan

state: MD

address: 50 Center Dr.

institute: National Heart Lung and Blood Institute

summary: Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability, and a number of clinically important anticancer and antibacterial drugs, e.g., quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50 fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e., negative vs. positive supercoiling. SHAN-seq’s exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.

overall_design: Supercoiled pBR322 DNA was cleaved with E. coli gyrase or topoisomerase IV and spiked with three DNA oligonucleotides. The DNA was subject to standard DNA-seq library preparation to label the enzyme-cleaved ends, fragmented, and subject to a second round of standard DNA-seq library preparation to label the fragmented ends. The labelled DNA was pair-end sequenced and mapped to the plasmid and spike-in oligonucleotide reference sequences. The reads from the topo-cleaved plasmid DNA ends were normalized by the reads from the spike-in ends and were used to determine the gyrase or topo IV cleavage levels at each position on the plasmid.

first: Shannon

last: Mckie

first: Rachel

last: Kim

first: Yeonee

last: Seol

first: Gabor

last: Harami

first: Anthony

last: Maxwell

first: Keir

last: Neuman


title: Minimal Residual Disease in Mouse Hepatocellular carcinoma [in vitro]

summary: Residual tumor cells which remain dormant after MYC inactivation in a primary MYC-HCC derived cell line.

overall_design: MYC-HCC clonal primary celll line treated with doxycyline for one week to inactivate the MYC oncogene. Viable cells used for scRNAseq.


title: An engineered ovarian somatic organoid model exhibits age-dependent changes in cellular organization and function

status: Public on Aug 02 2024

city: Chicago

first: Shweta

last: Dipali

state: IL

address: 303 E. Superior St., Suite 10-220

institute: Northwestern University

summary: Ovarian somatic cells are essential for tissue function, but there are no ex vivo models which maintain the cellular heterogeneity, in addition to the cell-cell and cell-matrix interactions of this compartment. We engineered a novel ovarian somatic organoid model in which a stroma-enriched fraction of primary mouse ovarian somatic cells was cultured in scaffold-free agarose micromolds. We sought to use this ovarian somatic organoid model to investigate mechanisms of ovarian aging and performed single cell RNA sequencing using primary ovarian somatic cells isolated from reproductively young and old mice, as well as organoids generated from young and old mice at days 1 and 6 of culture to assess age-dependent changes to cellular composition.

overall_design: Primary ovarian somatic cells isolated from reproductively young (6-12 weeks) or old (10-14 months) mice were lifted from 2D culture overnight or dissociated from organoids at day 1 or day 6 of culture and analyzed by scRNAseq

first: Madison

middle: Q

last: Gowett

first: Pratik

last: Kamat

first: Aubrey

last: Converse

first: Emily

last: Zaniker

first: Abigail

last: Fennell

first: Teresa

last: Chou

first: Michele

middle: T

last: Pritchard

first: Mary

last: Zelinski

first: Jude

last: Phillip

first: Francesca

last: Duncan


title: Minimal Residual Disease in Mouse Hepatocellular carcinoma [in vivo]

first: Renumathy

last: Dhanasekaran

state: CA

summary: Transgenic MYC/Twist1 HCC uncergoes tumor regression with persistence of minimal residaul disease (MRD) in the liver upon oncogene deprivation.

overall_design: Microdissected area of residual tumor in mouse HCC subjected to single nuclear RNA sequencing


title: ACSL6-activated IL18R1-NF-κB promotes IL18-mediated tumor immune evasion and tumor progression

first: Ziyang

last: Wang

state: --- Select One ---

address: 58 Zhongshan 2nd Road

institute: Ziyang Wang, Doctor, The First Affiliated Hospital, Sun Yat-sen University

summary: RNAseq analysis of MHCC97H cells infected with shNT or shACSL6 lentivirus.

overall_design: mRNA profiles of MHCC97H cells infected with shNT or shACSL6 were generated by deep sequencing, in duplicate, using Illumina Navoseq 6000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using  SYBR Green assays.

first: Yuqin

last: Di

first: Jing

last: Xiao

first: Xiang

last: Zhang

first: Lvlan

last: Ye

first: Xiangqiong

last: Wen

first: Jiale

last: Qin

first: Ligong

last: Lu

first: Xiongjun

first: Weiling

last: He


title: LAIR1 prevents excess inflammatory tissue damage in S. aureus skin infection and Cutaneous T-cell Lymphoma [abscess]

status: Public on Jul 31 2024

city: Saint Louis

first: Hannah

last: Dorando

state: Missouri

address: 660 South Euclid Avenue, Campus Box 8118-0020-07

institute: Washington University in St. Louis

summary: Patients with cutaneous T cell lymphoma (CTCL) experience high morbidity and mortality due to S. aureus skin infections and sepsis, but the causative immune defect is unclear. We propose that high levels of LAIR2 in CTCL suppress LAIR1 inhibitory signaling, promoting inflammation and tissue damage, which increases S. aureus susceptibility. Mice do not have a LAIR2 homolog, so we used Lair1 KO mice to model LAIR2 overexpression. In a model of subcutaneous S. aureus skin infection, Lair1 KO mice had  significantly larger abscesses and areas of dermonecrosis compared to WT. Lair1 KO exhibited a pattern of increased inflammatory responses in infection and sterile immune stimulation, including increased production of proinflammatory cytokines and myeloid chemokines, neutrophil ROS, and collagen/ECM remodeling pathways. Notably, compared to WT, Lair1 KO infected skin had a similar bacterial burden and neutrophils and monocytes had equivalent S. aureus phagocytosis. These findings support a model in which lack of LAIR1 signaling results in an excessive inflammatory response that does not improve infection control. CTCL tissues harbored similar patterns of increased cytokine and collagen production, suggesting that high levels of LAIR2 in CTCL recapitulates Lair1 KO, causing inflammatory tissue damage and compromising host defense against S. aureus infection.

overall_design: To investigate the impact of LAIR1 loss in S. aureus skin and soft tissue infection, we collected skin punch biopsies from 2 WT and 2 Lair1 KO mice 48 hours-post infection with S. aureus. We then performed gene expression profiling analysis from RNA-seq of these lesions.

first: Jacqueline

last: Payton


title: A small protein encoded by lncRNA PCBP1-AS1 promotes the replication of influenza virus via regulating the expression of proviral effectors (Ribo-Seq)

status: Public on Aug 03 2024

city: Fuzhou

first: Song

state: Fujian

address: Cangshan District

institute: Fujian Agriculture and Forestry University

summary: Many annotated long noncoding RNAs (lncRNAs) contain small open reading frames (sORFs), some of which have been demonstrated to encode small proteins or micropeptides with fundamental biological importance. While the small proteins or micropeptides hidden in lncRNA are gradually being revealed, their functions in influenza virus infection remain largely unexplored. Here, we identified and characterized a small 110-amino acid protein named PAESP that is encoded by the putative lncRNA PCBP1-AS1. We found that both PCBP1-AS1 and PAESP were upregulated by influenza virus infection as well as type I interferon treatment. Overexpression of PCBP1-AS1 or PAESP enhanced influenza virus replication. Conversely, knockdown of PCBP1-AS1 or knockout of PAESP inhibited viral production. In addition, loss-of-function experiment demonstrated that PAESP is essential for PCBP1-AS1 to facilitate influenza virus replication. More importantly, the overexpression of PAESP increases the mRNA level of several genes such as IFIT2 and IFIT3, which have been proved to be retasked by influenza virus from canonical antiviral factors into proviral effectors. Interestingly, when IFIT2 or IFIT3 expression was knocked down in PAESP-overexpressed cells, the virus titers significantly decreased compared to control cells. These findings reveal a novel lncRNA-derived small protein that is exploited by influenza virus and provide new insights into the virus-host regulatory network.

overall_design: To identify putative lncRNAs that can encode functional proteins/peptides during the pathogenesis of  influenza virus infection, we infected A549 cells with or without PR8 influenza virus for 12h, and performed Ribo-seq to find lncRNAs bound by the ribosome.

first: Xiaojuan

last: Chi

first: Ji-Long

last: Chen


title: LAIR1 prevents excess inflammatory tissue damage in S. aureus skin infection and Cutaneous T-cell Lymphoma [BMDM]

overall_design: To investigate the impact of LAIR1 loss on the macrophage immune response to S. aureus, we treated bone marrow-derived macrophages from 3 WT and 3 Lair1 KO mice with PBS or PAM3CSK4 for 6 hours. We then performed gene expression profiling analysis from RNA-seq of these cells.


title: RNA-seq and Ribo-Seq of human iPSC-ECs treated with DMSO or sunitinib for 24 hours

status: Public on Aug 01 2024

first: Soroush

last: Tahmasebi

address: 909 S Wolcott, Chicago, IL 60607

institute: University of Illinois

summary: Sunitinib is a receptor tyrosine kinase (RTK) inhibitor that is widely used for treatment of various types of cancer. Vascular toxicity represents a debilitating side-effect of sunitinib which impends its application in some patients. Study of sunitinib has been primarily focused on transcriptional control, and there is an important gap in our understanding of its impact on translation. To interrogate the role of mRNA translation in mediating the effect of sunitinib on endothelial cells (ECs) we performed ribosome footprinting.

overall_design: Ribosome footprinting was performed on two healthy human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) that were treated with DMSO or sunitinib (2ÂµM) for 24 hours."

first: Mehdi

last: Amiri

first: Stephen

last: Kiniry

first: Negar

last: Tabatabaei

first: Sang-Ging

last: Ong


title: Thr4 phosphorylation on RNA Pol II occurs at early transcription regulating 3’-end processing

city: Austin

first: Qian

state: TX

address: 2515 Speedway

institute: The Unibersity of Texas at Austin

summary: RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on Ser2/Ser5 of the C-terminal heptad repeats is well-established, Thr4’s role remains enigmatic. Paradoxically, Thr4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and mRNA processing. Our investigation revealed that Thr4 phosphorylation detection was obstructed by flanking Ser5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via Thr4 phosphorylation, which previously were attributed to Ser2. Importantly, loss of Thr4 phosphorylation greatly reduces Ser2 phosphorylation, revealing a crosstalk between the two marks. Finally, the function analysis of the Thr4 phosphorylation highlighted its role in alternative 3’-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the CTD.

overall_design: HEK293T cells were transiently transfected with RPB1 constructs containing the wild-type CTD sequence or a T4A mutation in every repeat. These constructs also contain resistance to alpha amanitin. Alpha amanitin was added to cells for 48 hours to kill off endogenous RPB1 and total RNA was collected. The library prep strategy  isolates polyA RNA for further analysis.

first: Rosamaria

middle: Y

last: Moreno

first: Svetlana

middle: B

last: Panina

first: Yan


title: Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming

status: Public on Jul 07 2024

city: Aurora

first: Kevin

last: Ess

state: Co

address: 12700 East 19th Avenue

department: Pediatrics

institute: University Colorado Denver

summary: Primary fibroblasts are a commonly used cell type used in a variety of experimental and therapeutic studies. Patient-derived skin biopsies are an accessible way to generate fibroblasts for use in various assays as well as reprogramming to iPSCs. To understand the potential of long-term skin biopsy culture, we cultured biopsy samples for 6 to 16 months and analyzed subsequent generation of fibroblasts. We found maintenance of morphology and physiology over time. Proliferation assays showed that older generations remained proliferative but at a decreased rate. mRNA analyses revealed transcriptional changes with long-term skin culture. Deep DNA sequencing did not reveal any large deletions or amplifications. Spontaneous DNA mutations seemed to be random and not enriched for any specific signaling pathways. Older fibroblasts generated after 16 months in culture retained competency for reprogramming into iPSCs. Our results support long term culture of skin to generate very large numbers of primary fibroblasts. These cells maintain their identity and integrity enabling the study of human disorders, particularly rare ones.

overall_design:
To determine the effect of long-term skin biopsy culture and the stability of produced fibroblasts, we cultured 2 skin biopsies for 6 to 16 generations
We extracted RNA from fibroblasts from Gen1, Gen6, and Gen16 and compared expression changes over time to determine culture stability

first: Sudiksha

last: Rathan-Kumar

first: Michael

last: Ripperger

first: Grant

last: Westlake


title: Systematic evaluation of GAPs and GEFs identifies ARHGAP45 as a targetable leukemia-specific RhoGAP dependency

status: Public on Jul 26 2024

city: New York

first: Jahan

last: Rahman

state: New York

address: 417 E 68th St

department: Center for Hematologic Malignancies

institute: Memorial Sloan Kettering

summary: GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) function as negative and positive regulators of small GTPases, respectively, and play key roles in cell fate determination. The aberrant function of GAPs and GEFs has long been implicated in cancer development but their vast number and potential redundancy have hindered comprehensive understanding of their roles. Here we performed functional genetic screens of GAPs and GEFs in primary AML specimens to uncover an unexpected and selective role for ARHGAP45 in AML. Depletion of ARHGAP45 impedes AML growth without affecting normal human CD34+ cells in vivo. Epistasis screens revealed RhoA as the major substrate of ARHGAP45 and that depletion of the Rho GTPase CDC42 synergized with ARHGAP45 deletion. Consistent with this, pharmacologic CDC42 inhibition enhanced the effect of ARHGAP45 deletion. ARHGAP45 gives rise to a minor histocompatibility antigen (termed HA-1), which can be recognized by antigen-specific T cells. CDC42 inhibition not only sensitized AML cells to ARHGAP45 inhibition but also promoted the activity of HA-1 antigen-specific T cells by upregulating ARHGAP45-derived epitope in AML cells.

overall_design: To gain insights into ARHGAP45-regulated transcriptomic changes at single-cell resolution, we introduced Cas9-sgRNA RNP into cells from three human AML PDX samples with diverse genotypes and two primary patient samples. These primary samples were directly isolated from patients without propagation in mice. Both samples harbored DNMT3A, FLT3-ITD, and NPM1c mutations, which are associated with poor clinical outcomes.. Each sample was subjected to Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), for multiplex sample indexing and simultaneous measurement of single-cell transcriptomics and cell surface protein expression.

first: Pu

last: Zhang

first: Zhendong

last: Cao

first: Xiangyu

last: Pan

first: Junwei

last: Shi

first: Omar

last: Abdel-Wahab


title: Role of the STAR proteins in early mammalian development

city: Barcelona

first: Elias

last: Bechara

address: Dr Aiguader 88

country: Spain

institute: CRG


title: A multi-cancer CRISPR/Cas9 reveals distinct gene dysregulation by noncoding MSTRG transcripts

status: Public on Jun 17 2024

city: Guangzhou

first: wei

last: xiong

address: No.1023,South Shatai Road

department: Bioinformatics Section

institute: Southern Medical University

summary: Long noncoding RNAs (lncRNAs) regulate gene expression with their functional domains without being translated into proteins. Whether wrong noncoding transcripts have functions is of special interest and lacks systematic investigation.  Our study identified several MSTRG transcripts that may have regulatory functions through specific DNA-binding domains (DBDs). We selected three MSTRG noncoding transcripts and validated their roles in cancer through CRISPR/Cas9-mediated knockout experiments, using A549 and HCT116 cell lines as experimental models. Then, we conducted a comparative gene expression analysis using RNA-seq data to compare the knockout of three aberrant human non-coding transcripts to their respective wild-type controls.

overall_design: We selected three MSTRG transcripts for knockout of their DNA-binding domains (DBDs), including MSTRG.4401.1, MSTRG.2513.6, and MSTRG.34636.7. Specifically, MSTRG.4401.1 and MSTRG.2513.6 were knocked out in both A549 and HCT116 cell lines, while MSTRG.34636.7 was knocked out only in HCT116 cells. Both before and after the knockout, RNA sequencing (RNA-seq) was performed on the samples.

first: Hao

last: Zhu


title: Human Accelerated Regions regulate gene networks implicated in apical-to-basal neural progenitor fate transitions

status: Public on Jul 05 2024

city: New Haven

first: Mark

middle: Alan

last: Noble

state: CT

address: 22 Gold Street, APT 102

department: Genetics

institute: Yale University

summary: The evolution of the human cerebral cortex involved modifications in the composition and proliferative potential of the neural stem cell (NSC) niche during brain development. Genetic changes that altered the activity of transcriptional enhancers have been linked to phenotypic differences between humans and other primates. Human Accelerated Regions (HARs) consist of >1600 highly constrained noncoding regions of the genome that show significant evolutionary acceleration on the human lineage, suggesting they encode human-specific functions. Multiple studies support that HARs include neurodevelopmental enhancers with novel activities in humans, but the regulatory roles of HARs in NSC biology has not been empirically assessed at scale. Here we conducted a direct-capture Perturb-seq screen repressing 180 neurodevelopmentally active HARs in human iPSC-derived NSCs with single-cell transcriptional readout. After profiling over 188,000 NSCs, we identified a set of HAR perturbations with convergent transcriptional effects on gene networks related to the specification of basal radial glia (bRG), a progenitor population that is expanded in humans. Processes associated with these networks included the regulation of apicobasal polarity and migration. We found convergent dysregulation of specific apicobasal polarity and adherens junction regulators, including PARD3, ABI2, SETD2, and PCM1, which are critical to apical-basal RG fate decisions. Autism- and intellectual-disability-associated genes were also enriched among genes showing changes in expression due to HAR perturbations. Our findings reveal interconnected roles for HARs in NSC biology and cortical development and link specific HARs to processes implicated in human cortical expansion.

overall_design: Polyclonal human iPSC cells (line H23555, a gift from Y. Gilad) expressing KRAB:dCas9 were inducted into neural stem cells via dual SMAD inhibition for 3 days, then transduced with a direct-capture sgRNA library targeting 180 Human Accelerated Regions, 5 positive control loci, and 6 non-targeting control sequences. Cells were recovered then sorted for lentiviral BFP, and neural induction continued to 9 days. Experiments were performed in 3 batches across 8 GEM lanes each.

first: Noble

last: Mark Alan

first: Noonan

last: James