title: Modeling Lung Adenocarcinoma Metastases Using Patient-Derived Organoids

city: New York

first: Yuan

last: Liu

address: 1275 York Avenue

institute: Memorial Sloan Kettering Cancer Center

summary: Approximately 50% of patients with surgically resected early-stage lung cancer develop distant metastasis. At present, there is no in vivo metastasis model to investigate the biology of human lung cancer metastases. Using well-characterized patient-derived organoids (PDOs) from patients with lung adenocarcinoma (LUAD), we establish an in vivo metastasis model that preserves the biologic features of human LUAD metastases. Results of whole-genome and RNA sequencing performed in this study establish that our in vivo PDO metastasis model can be used to study clonality and tumor evolution and to identify biomarkers related to organotropism. Investigation of the response of KRASG12C PDOs to Sotorasib demonstrates that the model can examine the efficacy of treatments to suppress metastasis and identify mechanisms of drug resistance. Finally, our PDO model cocultured with autologous peripheral blood mononuclear cells can potentially be used to determine the optimal immune-priming strategy for individual patients with LUAD.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of human tumors, patient-derived organoids, and metastatis-derived organoids from  in vivo metastatic models.

first: Manendra

last: Lankadasari

first: Joel

last: Rosien

first: Kofi

last: Johnson

first: Juan

last: Zhou

first: Samhita

last: Bapat

first: Lai-Fong

last: Chow-Tsang

first: Huasong

last: Tian

first: Brooke

last: Mastrogiacomo

first: Di

last: He

first: James

last: Connolly

first: Harry

last: Lengel

first: Raul

last: Caso

first: Elizabeth

last: Dunne

first: Cameron

middle: N

last: Fick

first: Gaetano

last: Rocco

first: Smita

last: Sihag

last: Isbell

first: Mathew

last: Bott

first: Bob

middle: T

last: Li

first: Piro

last: Lito

middle: W

last: Brennan

first: Mark

middle: H

last: Bilsky

first: Natasha

last: Rekhtman

first: Prasad

last: Adusumilli

first: Marty

last: Mayo

first: Marcin

last: Imielinski

first: David

last: Jones


title: TGFb-induced transcriptome profile in cancer cell lines

status: Public on Sep 12 2024

city: Umeå

first: Yabing

last: Mu

address: Sjukhusområdet

country: Sweden

institute: Umeå University

type: Expression profiling by high throughput sequencing

summary: TGFb plays many roles during tumor progression. It inhibits epithelial cell proliferation in the early stage but promotes tumor invasion by inducing epithelial-to-mesenchymal transition (EMT) in the late stage. Coupled with the strong EMT effect, TGFb exerts an overwhelming influence on modifying the extracellular matrix (ECM), which is generally thought to favor the adhesion and migration of tumor cells [4]. However, the regulation and function of ECM deposited in the tumor microenvironment by TGFb signaling have not been elucidated. Here, we performed a transcriptomic analysis using the human prostate cancer PC3U cell line and the human A549 lung carcinoma cell line. The top-upregulated genes in PC3U cells and A549 cells upon TGFb stimulation were identified to analyze the main regulatory function elicited by TGFb signaling. The GO enrichment analysis showed that extracellular matrix organization and the positive regulation of cell migration were the top regulatory functions.

overall_design: PC3U and A549 cells were treated with or without TGFb , and TGFb receptor I kinase inhibitor LY2157299 was used as a negative control.

first: Marene

last: Landström


title: Analysis of cellular and exosomal circRNAs in MCF7 cells regulated by LSD1 (RNA-Seq)

city: Chang Chun

first: Xin

last: Hu

address: 2699 Qianjin road,china

country: China

institute: Jilin university

summary: Lysine-specific demethylase 1 (LSD1), a histone demethylating enzyme, plays a crucial role in cancer metastasis. While studies have demonstrated that the knockout of LSD1 significantly enhances lung metastasis of breast cancer cells, it remains unclear whether LSD1-deficient breast cancer cells can remodel the lung microenvironment to allow metastasis. In this study, we isolated exosomes from LSD1-knockdown breast cancer cells (LSD1 KD) and investigated their effects on the formation of pre-metastatic niches. The intravenous injection of exosomes from LSD1 KD cells in mice resulted in a substantial increase in lung colonization by breast cancer cells, while treatment with exosomes derived from LSD1 KD cells decreased the expression of the tight junction proteins ZO-1 and occludin in vascular endothelial cells, leading to increased pulmonary vascular permeability. In addition, the knockdown of LSD1 reduced the expression of the circular RNA circDOCK1, which augmented the levels of miR-1270 in exosomes. Further investigation showed that miR-1270 inhibited ZO-1 expression in human umbilical vein endothelial cells, which enhanced their permeability. In conclusion, our study uncovered a novel mechanism in which the epigenetic modifier LSD1 promotes the formation of pre-metastatic niches via the regulation of exosomal miRNA.

overall_design: In order to find the miRNAs regulated by LSD1 in exosomes, and also to find the mechanism of miRNA regulation, we analysed circRNA (RNA-seq) expression in cells and miRNA expression in exosomes. The sequencing data from MCF7 cells were divided into 3 groups: Con, kd and Re. After analysing the differentially expressed miRNAs in the exosomes of the 3 groups of cells, the circRNAs were also differentially analysed to explore the corresponding mechanisms.


title: Spatial transcriptomics of primary human hepatoblastomas

city: Stanford

first: Peng

middle: Vijaykumar

last: Wu

state: CA

address: 265 Campus Dr., Rm G2145

country: USA

institute: Stanford University School of Medicine

summary: Spatial transcriptomics was performed on human hepatoblastomas to determine features associated with histologic heterogeneity.

overall_design: Spatial transcriptomics was performed on FFPE sections from two human hepatoblastomas and one adjacent normal liver using the 10x Visium platform.

middle: V

first: Roel

last: Nusse

first: Dhananjay

last: Wagh

first: John

last: Coller


title: Evasion of antiviral bacterial immunity by phage tRNAs

city: Tokyo

first: Kotaro

last: Chihara

address: 1-23-1 Toyama

department: Research Center for Drug and Vaccine Development

country: Japan

institute: National institute of infectious diseases

type: Non-coding RNA profiling by high throughput sequencing

summary: Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNA-Tyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNA-Tyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNA-Lys, to counteract a tRNA anticodon defense system (PrrC170).

overall_design: PtuAB was cloned under an arabinose inducible promoter pBAD. 0.2% Arabinose and 0.2% Glucose were used to induce and repress the PtuAB expression, respectively.

first: Azam

last: Aa Haeruman

first: Kohei

last: Kondo

first: Tomohiro

last: Nakamura

first: Shinjiro

last: Ojima

first: Wenhan

last: Nie

first: Azumi

last: Tamura

first: Wakana

last: Yamashita

first: Yo

last: Sugawara

first: Motoyuki

last: Sugai

first: Longzhu

last: Cui

first: Yoshimasa

last: Takahashi

first: Koichi

last: Wabash

first: Kiga

last: Kotaro


title: Analysis of cellular and exosomal miRNAs in MCF7 cells regulated by LSD1 (miRNA-Seq)

first: Xin

last: Hu

overall_design: In order to find the miRNAs regulated by LSD1 in exosomes, and also to find the mechanism of miRNA regulation, we analysed circRNA expression in cells and miRNA expression in exosomes (miRNA-seq). The sequencing data from MCF7 cells were divided into 3 groups: Con, kd and Re. After analysing the differentially expressed miRNAs in the exosomes of the 3 groups of cells, the circRNAs were also differentially analysed to explore the corresponding mechanisms.


title: A three-dimensional vessel-on-chip model to study Puumala orthohantavirus pathogenesis

city: Rotterdam

first: Barry

last: Rockx

address: Wytemaweg 80

department: Viroscience

country: Netherlands

institute: Erasmus MC

summary: Puumala orthohantavirus (PUUV) infection in humans can result in hemorrhagic fever with renal syndrome. Endothelial cells (ECs) are primarily infected with increased vascular permeability as a central aspect of pathogenesis. Historically, most studies included ECs cultured under static two-dimensional (2D) conditions, thereby not recapitulating the physiological environment due to their lack of flow and inherent pro-inflammatory state. Here, we present a high-throughput method for culturing primary human umbilical vein ECs in 3D vessels-on-chip in which we compared host responses of these ECs to those of static 2D-cultured ECs on a transcriptional level. The phenotype of ECs in vessels-on-chip more closely resembled the in vivo situation due to higher similarity in expression of genes encoding described markers for disease severity and coagulopathy, including IDO1, LGALS3BP, IL6 and PLAT, and more diverse endothelial-leukocyte interactions in the context of PUUV infection. In these vessels-on-chip, PUUV infection did not directly increase vascular permeability, but increased monocyte adhesion. This platform can be used for studying pathogenesis and assessment of possible therapeutics for other endotheliotropic viruses even in high biocontainment facilities.

overall_design: To investigate endothelial cell responses in the context of Puumala orthohantavirus in a physiologically relevant model, we infected primary human umbilical vein endothelial cells, cultured either in three-dimensional (3D) endothelial vessels-on-chip or static 2D in 96-wells plates, in triplicate and performed bulk RNA-Sequencing. Additionally, to demonstrate the added value of the vessel-on-chip system, we included parallel conditions where ECs were treated with tumor necrosis factor alpha (TNFα) in both culture systems. We then compared differential gene expressions and performed pathway enrichment analyses between virus-, mock-infected and TNFα-treated samples.

first: Danny

last: Noack

first: Anouk

last: van Haperen

first: Mirjam

middle: C

last: van den Hout

first: Eleanor

middle: M

last: Marshall

first: Rosanne

last: Koutstaal

first: Vincent

last: van Duinen

first: Lisa

last: Bauer

first: Anton

middle: J

last: van Zonneveld

first: Wilfred

middle: F

last: van IJcken

first: Marion

middle: P

last: Koopmans


title: Base-excision repair pathway shapes 5-methylcytosine deamination signatures in pan-cancer genomes (CUT&RUN)

city: Paris

first: Andre

last: Bortolini Silveira

address: 26 rue d'Ulm

country: France

institute: Institut Curie

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Transition of cytosine to thymine in CpG dinucleotides is the most frequent type of mutation in cancer. This increased mutability is commonly attributed to the spontaneous deamination of 5-methylcytosine (5mC), which is normally repaired by the base-excision repair (BER) pathway. However, the contribution of 5mC deamination in the increasing diversity of cancer mutational signatures remains poorly explored. Here, we integrate mutational signatures analysis in a large series of tumor whole genomes with lineage-specific epigenomic data to draw a detailed view on 5mC deamination in cancer. We uncover tumor type-specific patterns of 5mC deamination signatures in CpG and non-CpG contexts. We demonstrate that the BER glycosylase MBD4 preferentially binds to active chromatin and early replicating DNA, which correlates with lower mutational burden in these domains. We validate our findings by modeling BER deficiencies in isogenic cell models. Overall, we establish MBD4 as the main actor responsible for 5mC deamination repair in humans.

overall_design: CUT&RUN on FLAG-tagged MBD4 exogenously expressed in HAP-1 cells.Negative (IgG) and positive (H3K4me3) CUT&RUN control reactions are included.


title: MrgprA3 neurons selectively control myeloid-derived IL-33 for IL-17 dependent cutaneous immunity [bulk RNA-Seq]

city: Philadelphia

first: Juan

middle: M.

last: Inclan-Rico

state: PA

address: 3800 Spruce St

department: Pathobiology

institute: University of Pennsylvania, School of Veterinary Medicine

summary: Skin contains poorly understood interdependent cellular networks that facilitate barrier integrity and host immunity, including neurons, and myeloid cells; the latter of which intrinsically express the pleiotropic cytokine IL-33. This work shows selective suppression of IL-33 expression in myeloid cells by activation of itch-sensing neurons bearing the Mas-related G protein receptor A3 (A3).  Optogenetic activation of A3 neurons also increased IL-17-expressing γδ T cells, epidermal thickening, and resistance to the human pathogen Schistosoma mansoni, partially through the neuropeptide CGRP. Cell-intrinsic loss of IL-33 in myeloid cells alters chromatin conformation, basally elevates expression and release of IL-17-inducing cytokines (e.g., IL-1β, IL-6), and expands macrophage and cDC2 differentiation, driving both tissue pathology (e.g., epidermal thickening, keratinocyte hyperplasia) and resistance to helminth infection. Our findings suggest a mechanism of cellular cross-talk allowing “itch” neuron activation to alter myeloid composition and cytokine expression patterns for reshaping skin architecture and driving immunity

overall_design:
3 abdominal skin biopsies from naïve CD11c control or CD11c-specific IL-33KO mice

Raw data not provided for GSM6757194 and GSM6757197

first: Camila

last: Napuri

first: Li-Yin

last: Hung

first: Christopher

last: Pastore

first: Cailu

last: Lin

first: Annabel

last: Ferguson

first: Heather

middle: L

last: Rossi

first: Taku

last: Kambayashi

first: Danielle

last: Reed

first: De’Broski

middle: R

last: Herbert


title: Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded non-coding RNAs [L6 mito]

status: Public on Sep 11 2024

city: Parkville

first: Adam

middle: James

last: Trewin

state: Victoria

address: Royal Pde

department: Anatomy and Physiology

country: Australia

institute: University of Melbourne

summary: MiRNAs are non-coding RNAs that regulate gene expression. MiRNAs mostly localise within the cytosol but are also found in the mitochondria where they can regulate the expression of mitochondrial-encoded transcripts. Small RNA library preparation protocols are well described when using total cellular RNA as the template, however, these methods are not directly applicable to total RNA extracted from fractionated cells, such as isolated mitochondria. The aim of this study was to optimise the small RNA library preparation protocol for use with small (<60ng) amounts of total mitochondrial RNA.

overall_design: L6 cells were proliferated to 80% confluence, harvested, and immediately processed for the isolation of mitochondria and extraction of mitochondrial RNA which then underwent a series of dilutions (1.8-120ng). Small RNA libraries were then prepared from a range of mitochondrial RNA inputs with different ratios of adapters (0.1x, 0.3x, 0.5x) and qPCR cycles.

first: Jessica

last: Silver

first: Stella

last: Loke

first: Larry

last: Croft

first: Mark

last: Ziemann

first: Megan

last: Soria

first: Hayley

last: Dillon

first: Søren

last: Nielsen

first: Séverine

last: Lamon

first: Glenn

middle: D

last: Wadley


title: MrgprA3 neurons selectively control myeloid-derived IL-33 for IL-17 dependent cutaneous immunity [ATAC-Seq]

first: Juan

last: Inclan-Rico

overall_design: Bone marrow-derived macrophages were differentiated from CD11c control or CD11c-specific IL-33KO mice for 5-7 days following treatment with MCS-F

first: Camila

last: Napuri

first: Li-Yin

last: Hung

first: Christopher

last: Pastore


title: Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded non-coding RNAs [SMvsMT]

summary: Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimised to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a high degree of purity. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.

overall_design: Samples are ribo-depleted libraries construced from the total RNA of isolated mitochondria (MT) and the corresponding gastrocnemius skeletal muscle (SM) whole tissue from wistar kyoto rats.


title: Post-transcriptional regulation by the gut microbiota decoded by nanopore direct RNA sequencing [RNA methylation]

city: Guangzhou

first: Xiaoyun

last: Wang

state: Guangdong

address: 190 Kaiyuan Avenue, Huangpu

department: Infection and Immunity

institute: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

summary: It has been widely recognized that the microbiota has the capacity to shape host gene expression and physiological functions. However, there remains a paucity of comprehensive study revealing host transcriptional landscape regulated by the microbiota. Here, we comprehensively examined mRNA landscapes in mouse tissues (brain and cecum) from specific pathogen free (SPF) and germ-free mouse (GF) using Nanopore direct RNA sequencing. Our results show that the microbiome has global influence on host’s RNA modifications (m6A, m5C, Ψ), isoform generation, poly(A) tail length (PAL), and transcript abundance in both brain and cecum tissues. Moreover, the microbiome exerts tissue-specific effects on various post-transcriptional regulatory processes. In addition, the microbiome impacts the coordination of multiple RNA modifications in host brain and cecum tissues. In conclusion, we establish the relationship between microbial regulation and gene expression, our results help the understanding of the mechanisms by which the microbiome reprograms host gene expression.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Decoding the landscape of RNA modifications and abundance in the brain and cecum of SPF and GF mice using RNA bisulfite-seq.

first: Zihe

last: Xu

first: Xiaoqi

last: Zheng

first: Jiajun

last: Fan

first: Yuting

last: Jiao


title: Post-transcriptional regulation by the gut microbiota decoded by nanopore direct RNA sequencing

overall_design: Decoding the landscape of RNA modifications and abundance in the brain and cecum of SPF and GF mice using Nanopore direct RNA sequencing


title: Effects of Simulated Microgravity on Colorectal Cancer Organoids Growth and Drug Response

city: Seoul

first: JA-LOK

last: KU

address: 101 Daehak-ro, Jongno-gu

department: Cancer Research Institute

country: South Korea

institute: Seoul National University College of Medicine

summary:
Cellular and molecular dynamics of human cells are constantly affected by gravity. Alteration of the gravitational force disturbs the cellular equilibrium, which might modify physiological and molecular characteristics. Nevertheless, biological responses of cancer cells to reduced gravitational force remains obscure.
Here, we aimed to comprehend not only transcriptomic patterns but drug responses of colorectal cancer (CRC) under simulated microgravity. We established four organoids directly from CRC patients, and organoids cultured in 3D clinostat were subjected to genome wide expression profiling and drug library screening.
Our observations revealed changes in cell morphology and an increase in cell viability under simulated microgravity compared to their static controls. Transcriptomic analysis highlighted a significant dysregulation in the TBC1D3 family of genes. The upregulation of cell proliferation observed under simulated microgravity conditions was further supported by enriched cell cycle processes, as evidenced by the functional clustering of mRNA expressions using cancer hallmark and gene ontology terms. Our drug screening results indicated an enhanced response rate to 5-FU under conditions of simulated microgravity, suggesting potential implications for cancer treatment strategies in simulated microgravity.

overall_design: Four colorectal cancer (CRC) organoids were cultured under microgravity using clinostat and compared the organoids cultured in normal gravity. In the operational context of the 3D clinostat, certain forces such as Coriolis, radial, and frictional forces were deemed negligible. This assessment is based on the relatively minimal angular velocities achieved by the two axes of the clinostat, specifically, the inner frame rotating at 0.683 revolutions per minute (rpm) and the outer frame at 0.913 rpm. The transcriptomic landscape of four CRC organoids cultured under microgravity was analyzed using RNA-seq.

first: Soon-Chan

last: Kim

first: Min

first: Ji

last: Park

first: Young-Kyoung

last: Shin

first: Seung-Yong

last: Jeong

first: Sungwan

first: Ja-Lok

last: Ku


title: Unraveling the Phenotypic States of Human innate-like T Cells in thymus and blood: Insights from Single-Cell Transcriptomics

city: Aurora

first: Tonya

last: Brunetti

state: CO

address: 12800 East 19th Avenue

department: Immunology and Microbiology

institute: University of Colorado Anschutz Medical Campus

summary: The "innate-like" T cell compartment, known as Tinn, encompasses a diverse group of T cells that straddle the boundary between innate and adaptive immunity. We explored the transcriptional landscape of Tinn compared to conventional T cells (Tconv) in the human thymus and blood using scRNA sequencing

overall_design: iNKT cells, MAIT, gd T cells, CD4 SP and CD8 SP T cells were isolated from pediatric human thymus of adult blood by fluorescence-activated cell sorting (FACS) using appropriate tetramers, anti-TCRalpha chains and cell surface antibodies.

first: Gapin

last: Laurent

first: Meyer

last: Hannah


title: Manual therapy improves fibromyalgia symptoms by downregulating SIK1

first: Elisa

middle: Elisa

last: Oltra

state: VALÈNCIA

address: C/ Quevedo 2

department: Pathology

country: Spain

institute: Universidad Catolica de Valencia San Vicente Martir

summary: Fibromyalgia (FM), classified by ICD-11 with code MG30.0, is a chronic debilitating disease characterized by wide-spread pain, fatigue, cognitive impairment, sleep and intestinal alterations, among other. FM affects a large proportion of the world-wide population, with increased prevalence among women. The lack of understanding of its etiology and pathophysiology hampers the development of effective treatments. Our group had developed a manual therapy (MT) pressure-controlled custom manual protocol on FM showing hyperalgesia/allodynia, fatigue and patient’s quality of life benefits in a cohort of 38 FM cases (NCT04174300). With the aim of understanding the therapeutic molecular mechanisms triggered by MT, this study interrogated PBMC transcriptomes from FM participants of this clinical trial using RNAseq and RT-qPCR technologies. The results showed that the salt-induced kinase SIK1 gene was consistently downregulated by MT in FM, correlating with improvement of patient symptoms. In addition, the study compared the findings in a non-FM control cohort subjected to the same MT protocol evidencing that changes in SIK1 expression with MT only occurred in individuals with FM. This positions SIK1 as a potential biomarker to monitor response to MT, and as a therapeutic target of FM, to be further explored by continuation studies. Keywords: fibromyalgia; myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS); pressure point threshold (PPT); physiotherapy; manual therapy (MT); NCT04174300; SIK1

overall_design: Transcriptome evaluation of PBMCs from fibromyalgia patients after (post-) manual therapy  with respect to basal values (pre-)

first: J

last: Bonastre-Férez

first: K

last: Giménez-Orenga

first: F

last: Falaguera-Vera

first: M

last: Garcia-Escudero

first: E


title: Laser capture microdissection of the ganglion cell layer for analyzing transcriptional responses induced by demyelination

city: Portland

first: Gregory

last: Duncan

state: Oregon

address: 3181 SW Sam Jackson Park Road, BRB 453

institute: OHSU

summary: Demyelination and loss of oligodendrocytes deprives neurons of crucial metabolic and trophic support and contributes to neurodegeneration. However, the precise mechanisms that trigger neurodegeneration during demyelination remain unclear. Here, we examine transcriptional differences in neurons that have been demyelinated relative to myelinated neurons. Demyelination is induced by the knockout of Myrf, a transcription factor essential for myelin gene expression and oligodendrocyte identity, from oligodendrocyte lineage cells using Myrffl/fl Sox10 CreERT mice. We performed laser capture microdissection of the ganglion cell layer, which contains the majority of the retinal ganglion cells (RGC), a nearly ubiquitously myelinated population of neurons. We find that dissection of the ganglion cell layer greatly enriches for RGC transcripts relative to controls.  Demyelinated Myrffl/fl Sox10 CreERT mice have upregulation of Hrk and Ecel1 in the ganglion cell layer, which are known to be upregulated during axonal injury and driven by dual leucine zipper kinase (DLK) signaling. These data suggest that demyelination may activate a DLK-mediated signaling cascade that culminates in transcriptional changes that are often associated with apoptosis of neurons in the central nervous system.

overall_design: To understand how demyelination affects neuronal transcription, Myrffl/fl Sox10 CreERT mice were injected with tamoxifen at 8 weeks of age to induce CNS-wide demyelination, including of the visual system. Myrffl/fl littermates were used as controls. Transcriptional analysis was performed on laser-microdissected ganglion cell layer or whole retina at 18 weeks of age. The ganglion cell layer of n=6 Myrffl/fl Sox10 CreERT mice and n=6 Myrffl/fl controls, as well as n=4 whole retinae were sequenced. Comparative gene expression was performed between whole retina and the ganglion cell layer, and between the ganglion cell layer of Myrffl/fl Sox10 CreERT and Myrffl/fl mice.

first: Greg

first: Ben

last: Emery


title: Single-molecule chromatin configurations link transcription factor binding to expression in human cells [RNA-seq]

status: Public on Sep 10 2024

first: Benjamin

middle: Rosenstock

last: Doughty

address: 279 Campus Drive W

department: Genetics

institute: Stanford University

summary: The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TF’s capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.

overall_design: K562 cells were cultured in 12-well plates and treated with 10 ng/mL interferon beta (PeproTech, #300-02BC) for 24 hours, after which 1 x 106 cells were harvested by centrifugation at 300 x g for 5 minutes. Cells were lysed and homogenized using QIAshredder columns (Qiagen #79654) and RNA was extracted with the RNeasy Mini Kit (Qiagen #74104). The RNA Integrity Number (RIN) for all samples was 10 as assessed by an RNA Nano Kit (Agilent #5067-1511) on an Agilent Bioanalyzer, provided by the Stanford Protein and Nucleic ACID (PAN) Biotechnology Facility. 500 ng of purified RNA for each sample was enriched for polyadenylated mRNA with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and then used as input for the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770S). Sample preparation was performed as per manufacturer’s specifications, with nine PCR cycles used for final library amplifications. Library size distributions were confirmed using the High Sensitivity DNA Kit (Agilent #5067-4626) on an Agilent Bioanalyzer. Sample concentrations were measured using the Qubit dsDNA HS Assay Kit on a Qubit 4 Fluorometer, and all samples were pooled at equimolar ratios and sequenced on a NextSeq 550 with 2 x 37 cycles.  Sequencing reads were demultiplexed with bcl2fastq. Hisat2-build was used to build a reference transcriptome with a FASTA of the GRCh38 human reference genome and the accompanying GTF genome annotation file, and hisat2 was used to align the paired reads to the reference. Output SAM files were converted to BAM files using samtools and differential expression analysis was performed in R with the Bioconductor DESeq2 package72 using a set of custom R scripts (IFN_RNA_processing.R) that were largely based on the workflow and commands described in the following tutorial: http://bioconductor.org/help/course-materials/2016/CSAMA/lab-3-rnaseq/rnaseq_gene_CSAMA2016.pdf.


title: Single-molecule chromatin configurations link transcription factor binding to expression in human cells [ChIP-seq]

overall_design: ChIP-seq was performed as previously described73 with modifications. For each ChIP reaction, 2x10^7 cells were used as input together with a spike-in of mouse chromatin used for orthogonal normalization. Cells were crosslinked with 1% formaldehyde for 15 min at room temperature, followed by quenching in glycine (final concentration 0.125 M). Cells were then centrifuged, resuspended in 1xPBS, centrifuged again, and stored at -80C. On the first day of the ChIP procedure, 10 uL Protein A Dynabeads (Thermofisher 10002D) were added to DNA Lo-Bind tubes and washed three times on a magnetic rack with 1 mL mg/mL BSA. Beads were resuspended in 1 mL BSA, 5 ug anti-H3K27ac (Abcam ab4729) or anti-Pol2-S5P (Abcam ab5408) antibody were added, and the antibodies were coupled to the beads overnight on a rotor at 4 °C. On the second day, crosslinked chromatin was resuspended in 1 mL Farnham Lysis Buffer (FLB; 5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NP-40/IGEPAL, Roche Protease Inhibitor Cocktail), centrifuged, then resuspended in 1 mL FLB and centrifuged again, then resuspended in 880 uL RIPA buffer (1x PBS, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SGS, Roche protease inhibitor cocktail), and sheared using Covaris E220 Focused-Ultrasonicator. Beads were washed again three times with 1 mL BSA on a magnetic rack, resuspended in 100 uL BSA, and the chromatin in RIPA buffer was added to them, then incubated overnight at 4 °C on a rotor. On the third day, beads were washed five times with LiCl buffer (10mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% Sodium deoxycholate) for 10 minutes on a rotator at 4 °C followed by removal of the buffer using a magnetic rack, then washed once with 1 mL 1x TE buffer, and resuspended in 200 uL IP elution buffer (1% SDS, 0.1 M NaHCO3). Chromatin was eluted off the beads by incubation at 65 °C followed by centrifugation at max speed for 3 minutes, and transfer to fresh DNA Lo-Bind tubes. Crosslinks were reversed by addition of 2 uL Proteinase K (Promega) and incubation at 65 °C for 12-16 hours in a Thermomixer. DNA was purified by adding an equal volume (200 uL) 25:24:1 Phenol:Chloroform:Isoamyl alcohol, vortexing, and centrifugation for 3 minutes at max speed. The top phase was then purified using the MinElute kit (Qiagen), eluting in 50 uL 55°C EB buffer. Sequencing libraries were prepared using the NEBNext Ultra II kit (NEB E7645) following the manufacturer’s instructions. Sequencing was performed on a NextSeq in 2x38 bp format.


title: RNA-seq analysis of mouse bone marrow hematopoietic progenitors after bone marrow transplant

city: Barcelona

first: Cristina

last: Lopez-Rodriguez

address: Carrer Dr. Aiguader, 88

department: Medicine and Life Sciences

institute: Universitat Pompeu Fabra

summary: RNA-seq analysis of differentially expressed genes in wild-type, NFAT5-deficient, IFNAR1-deficient and double deficient (NFAT5 and IFNAR1) hematopoietic stem cells (HSC) and multipotent progenitors (MPP)

overall_design: 8 samples of bone marrow (BM) hematopoietic progenitors (2 cell types and 4 mouse genotypes) after bone marrow transplantation

last: López-Rodríguez

first: Jose

last: Aramburu

first: Laia

last: Traveset

first: Victor

last: Cerdán Porqueras


title: MrgprA3 neurons selectively control myeloid-derived IL-33 for IL-17 dependent cutaneous immunity [scRNA-Seq]

first: Juan

last: Inclan-Rico

overall_design: Sort-purified CD45+ CD11c+ MHC-II+ cells from naïve abdominal skin preparations from CD11c control mice or CD11c-specific IL-33KO mice

first: Camila

last: Napuri

first: Li-Yin

last: Hung

first: Christopher

last: Pastore

first: Cailu

last: Lin

first: Annabel

last: Ferguson

first: Heather

last: Rossi

first: Taku

last: Kambayashi

first: Danielle

last: Reed

first: De’Broski

last: Herbert


title: RAS-mutant AML LSCs originate from GMPs and drive clinical resistance to BH3 mimetics [scRNA-Seq]

first: Maria

last: Sirenko

state: NY

address: 550 1st Avenue

institute: NYU Langone

summary: Cancer driver mutations often show distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in the course of acute myeloid leukemia (AML), upon progression or relapsed/refractory disease1-6. Here, by employing synthetic leukemogenesis in human cells, we first show that RAS mutations are obligatory late events that need to succeed earlier cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed progenitors of the myelomonocytic lineage (granulocyte-monocyte progenitors, GMPs) harboring previously acquired driver mutations, revealing that advanced leukemic clones originate from a different cell type than more ancestral clones. Furthermore, we demonstrate that RAS-mutant leukemia stem cells (LSCs) give rise to monocytic disease, as frequently observed in patients with poor responses to treatment with the BCL2 inhibitor drug Venetoclax (VEN). We show that this is because RAS-mutant LSCs, in contrast to RAS-WT LSCs, have altered BCL2 family gene expression profiles and are resistant to VEN, driving clinical resistance and relapse with monocytic features. Our findings demonstrate that a specific genetic driver by imposing a specific LSC target cell restriction shapes the non-genetic cellular hierarchy of AML and critically impacts therapeutic outcomes in patients.

overall_design: Single cell RNA-sequencing of AML patient bone marrow mononuclear cells using Chromium 10X Genomics 5' v1 protocol.

first: Sirenko

last: Maria

first: Papapetrou

last: Eirini


title: RAPIDASH: Tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues, cancer cells, and macrophages [RNA-Seq and Ribo-Seq]

city: Singapore

first: Teodorus Theo

last: Susanto

address: 60 Biopolis St

country: Singapore

institute: Genome Institute of Singapore

summary: Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts.

overall_design: RAPIDASH characterization by short read sequencing of polyA-selected RNAs differentially enriched by the additional sulfhydryl-charged resin chromatography step compared to sucrose cushion alone. Refer to Figure S2

first: Teodorus

first: Victoria

first: Andrew

middle: G

last: Levine

first: Yuxiang

last: Chen

first: Craig

last: Kerr

first: Yongjin

last: Yoo

middle: A

last: Oses-Prieto

first: Lisa

last: Fromm

first: Zijian

last: Zhang

first: Travis

last: Lantz

first: Kotaro

last: Fujii

first: Marius

last: Wernig

first: Alma

last: Burlingame

first: Davide

last: Ruggero

last: Barna


title: Increased EZH2 function in regulatory T cells promotes their capacity to suppress autoimmunity by driving effector differentiation prior to activation [CUT&RUN]

city: Berkeley

first: Janneke

last: Peeters

address: University Avenue and Oxford St

institute: University of California Berkeley

summary: The immunosuppressive function of regulatory T (Treg) cells is essential for maintaining immune homeostasis. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27) methyltransferase, plays a key role in maintaining Treg cell function upon CD28 co-stimulation, and Ezh2 deletion in Treg cells causes autoimmunity. Here we assessed whether increased EZH2 activity in Treg cells would improve Treg cell function. Using an Ezh2 gain-of-function mutation, Ezh2Y641F, we found that Treg cells expressing Ezh2Y641F displayed an increased effector Treg phenotype and were poised for improved homing to organ tissues. Expression of Ezh2Y641F in Treg cells led to more rapid remission from autoimmunity. H3K27me3 profiling and transcriptomic analysis revealed a redistribution of H3K27me3, which prompted a gene expression profile in naïve Ezh2Y641F Treg cells that recapitulated aspects of CD28-activated Ezh2WT Treg cells. Altogether, increased EZH2 activity promotes the differentiation of effector Treg cells that can better suppress autoimmunity.

overall_design:
H3K27me3 CUT&RUN was performed on Treg cells (sorted based on CD4+Foxp3GFP+RFP+CD62L+) or CD4+ T effector cells (sorted as CD4+Foxp3GFP-RFP-CD62L+) from Foxp3-GFP-hCre;Ezh2Y641F/+;R26RFP (Ezh2-Y641F) or Foxp3-GFP-hCre;Ezh2+/+;R26RFP (WT) mice either collected after sorting (naive samples) or after 4 days of activation with anti-CD3 and anti-CD28 coated beads at a ratio of 1:3 (cell:bead) in the presence of 2000 IU/mL recombinant human IL-2 (activated samples).
Please note that processed data file generated from replicates is linked to the corresponding *rep1 sample records.

first: Michel

last: DuPage

first: Srinivas

last: Ramachandran


title: Increased EZH2 function in regulatory T cells promotes their capacity to suppress autoimmunity by driving effector differentiation prior to activation.

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

overall_design: Refer to individual Series


title: Arp2/3 Complex Activity Enables Nuclear YAP for Naïve Pluripotency of Human Embryonic Stem Cells

city: San Francisco

first: Nathaniel

middle: Paul

last: Meyer

state: California

address: 513 Parnassus Ave

department: Cell and Tissue Biology

institute: University of California, San Francisco

summary: Our understanding of transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency that includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, uniform cell mechanics within naïve colonies, nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restored in actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

overall_design: HUES8 human embryonic stem cells were dedifferentiated to a naïve state of pluripotency over the course of 6 days with or without 80 μM CK666. CK666 is a known Arp2/3 complex activity inhibitor. Samples were submitted for bulk RNAseq in quadruplicate with the following conditions: control primed cells, control D6 naive cells, and cells which were dedifferentiated in naive media with 80 μM CK666 for 6 days. RNA was isolated using RNeasy Plus kits and assessed for quality using Nanodrop and Bioanalyzers prior to submission to ensure quality.

first: Tania

last: Singh

first: Matthew

last: Kutys

first: Todd

last: Nystul

first: Diane

last: Barber


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [ChIP-seq_primed,RSeT,RSeT+DT_hESCs]

status: Public on Sep 09 2024

city: Toronto

first: Juan

state: Ontario

address: 25 Orde Street, Room 6-1001 MRS

country: Canada

institute: LTRI

summary: In this study, we performed ChIP-seq to compare histone modifications in primed and naive hES cells.

overall_design:
H3K27me3, H3K27ac and H3K9me3 ChIP-seq in wild-type RSeT+DT, RSeT and primed hESCs was carried out in partnership with CEEHRC, following their sample preparation guidelines:
https://thisisepigenetics.ca/for-scientists/community-access-program

first: Kirti

last: Mittal

first: Marie-Michelle

last: Simon

first: Tony

last: Kwan

first: Miguel

last: Ramalho-Santos


title: Detection of 5mC and 5hmC in DNA templates generated by PCR using modified dCTPs by linear PCR using KTq DNA polymerase variant RIV A8

city: Konstanz

first: Melanie

last: Henkel

state: Baden-Württemberg

address: Universitätsstraße 10

department: Department of Chemistry

country: Germany

institute: University of Konstanz

summary: We have engineered the thermostable KlenTaq DNA polymerase variant called RIV A8 that produces error signatures specific for 5-methylcytosine (5mC) and 5-hydroxycytosine (5hmC) without prior chemical treatment of the DNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish C from 5mC and C from 5hmC in DNA templates generated by PCR using modified dCTPs (unmodified by using dCTP, methylated by using d5mCTP, hydroxymethylated by using d5hmCTP).

overall_design: DNA templates generated by PCR using modified dCTPs (unmodified by using dCTP, methylated by using d5mCTP, hydroxymethylated by using d5hmCTP) were linear amplified by RIV A8. Respective ssDNA product was used to prepare DNA amplicon libraries which were analyzed by NGS to detect 5mC and 5hmC by reading an increased error rate.

first: Andreas

last: Marx

first: Yuri

last: Motorin

first: Virginie

last: Marchand


title: Complement and microglia activation mediates stress-induced synapse loss in layer 2/3 of the medial prefrontal cortex

city: San Antonio

first: Gek Ming

last: Sia

state: Texas

address: 7703 Floyd Curl Drive, MED 237B

institute: University of Texas Health at San Antonio

summary: Spatially heterogeneous synapse loss is a characteristic of many psychiatric and neurological disorders, but the underlying mechanisms are unclear. Here, we show that spatially-restricted complement activation mediates stress-induced heterogeneous microglia activation and synapse loss localized to the upper layers of the mouse medial prefrontal cortex (mPFC). Single cell RNA sequencing also reveals a stress-associated microglia state marked by high expression of the apolipoprotein E gene (Apoehigh) localized to the upper layers of the mPFC. Mice lacking complement component C3 are protected from stress-induced layer-specific synapse loss, and the Apoehigh microglia population is markedly reduced in the mPFC of these mice. Furthermore, C3 knockout mice are also resilient to stress-induced anhedonia and working memory behavioral deficits. Our findings suggest that region-specific complement and microglia activation can contribute to the disease-specific spatially restricted patterns of synapse loss and clinical symptoms found in many brain diseases.

overall_design: Microglia were isolated from cerebral cortices of control/corticosterone treated mice, 4 replicates per treatment, 3-5 mice per replicate


title: Transcriptional coactivator MED15 is required for beta cell maturation

city: Vancouver

first: Francis

last: Lynn

state: BC

address: 950 28th Ave W

department: Surgery

institute: University of British Columbia

summary: Mediator, a co-regulator required for RNA pol II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We sought to understand whether Mediator subunit MED15 acts as a node that controls tissue-specific gene expression, using pancreatic insulin-producing β-cell maturation as a model. We found Med15 to be expressed during mouse pancreatic organogenesis and β-cell maturation; moreover, islets from human T2D donors feature reduced MED15 expression. Loss of Med15 in mouse β-cells caused defects in maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses determined that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. Human embryonic stem cell derived β-like cells, genetically engineered to express high levels of MED15, had increased maturation markers and improved insulin secretion. We provide the first evidence of the importance of Mediator in β-cell maturation and demonstrate an additional layer of control that tunes transcription factor function.

overall_design:
1)ChIP-seq on mouse MIN6 cells and islets
2)RNAseq on control and beta cell-specific Med15 knockout islets

first: A

middle: Z

last: Kadhim

first: E

middle: E

last: Xu

first: S

last: Sasaki

first: X

last: Cheng

first: H

last: Winata

last: Sproul

first: T

last: Speckmann

first: C

last: Nian

first: R

last: Cullen

last: Shi

first: D

middle: S

last: Luciani

first: B

last: Hoffman

last: Taubert

first: FC


title: Immunogenomics and spatial proteomic mapping highlight distinct neuro-immune architectures in melanoma vs. non-melanoma-derived brain metastasis

city: Los Angeles

first: Daria

last: Kessler

address: 9139 Hargis St.

institute: Saint John's Health Center

summary: Brain metastases (BrMs) are a devastating complication of solid tumors with challenging clinical management. In this study, immunogenomic and digital spatial analyses were applied to interrogate the peripheral blood and tumor specimens derived from 53 unique patients with metastatic brain tumors originating from different solid tumors including melanoma, breast cancer, lung cancer and renal cell carcinoma. In the peripheral blood, lower levels of neutrophil -lymphocytes ratio (NLR) were detected at time of craniotomy in patients with melanoma-derived brain metastasis (MBM) vs. non-melanoma- derived brain metastasis (non-MBM). Independently from the primary tumor of derivation, patients with BrMs and increased NLR levels were characterized by shorter overall survival (OS) following craniotomy. In the tumor microenvironment (TME), molecular evaluations performed on FFPEs revealed higher expression of genes and mRNA signatures identifying NK cells, CD8 cells and B cells in MBM (n=13) vs. non-MBM brain metastasis (n=41). Focusing on CD8 cells, higher infiltration of CD8+ cells were observed in patients with MBM with longer OS following craniotomy. Spatial proteomic analysis further highlighted the infiltration of CD8+ cells, antigen presenting cells- (HLA-DR+, CD11c+, B2M+), agonists of T cell activity (CD137+, CD40+) and B cells (CD20+) enriched in MBM vs non-MBM. On the contrary, an increased expression of genes associated with neuro-development, cell- cell adhesion, neutrophil enrichment together with the increased infiltrations of cells promoting neuro-differentiation (Neun+, S100+), immune regulatory functions (CD25+, CD127+), and granulocytes aggregation (CD66b+) were observed in non-MBM vs. MBM. These findings highlight that the TME of BrMs plays a pivotal role in the pathogenesis and therapeutic resistance of BrMs derived from different solid tumors. Our results also suggest that distinct neuro-immune interplay may contribute to treatment resistance in BrMs.

overall_design: To comprehensively examine the immune landscape of the tumor microenvironment of metastases derived from different solid tumors, we analyzed a set of surgically resected BrMs originated from different solid tumors including melanoma, lung cancer, breast cancer and renal cell carcinoma (RCC), using whole gene expression profile, digital spatial proteomics (DSP), immunohistochemistry (IHC), multiplex immunofluorescence (mIF), and spectral flow cytometry. Formalin-fixed, paraffin embedded (FFPE) tumor biposies were derived from  13 MBM and 41 non-MBM [18 lung cancer-derived brain metastasis (LBM), 18 breast cancer-derived brain metastasis (BBM) and 5 renal cancer-derived brain metastasis (RCM) biopsies. Three BBM specimens (BREBRM45-B, BREBRM46-B, BREBRM47-B) derived from the same patient (patient 26) and four LBM specimens derived from two different pts (2 specimens/each: LUNBRM14-B and LUNBRM44-B specimens from patient 34, and LUNBRM18-B and LUNBRM36-B from patient 37). RNA was isolated from the tumor regions of  the 54 FFPEs specimens derived from 50 unique patients with brain metastasis and subjected to whole gene expression profiling by RNA-seq.

first: Alberto

last: Mendoza-Valderrey

first: Ethan

last: Dettmann

first: Douglas

last: Hanes

middle: M

first: Ludmila

last: Danilova

first: Kai

last: Rau

first: Yueqin

last: Quan

first: Stacey

last: Stern

first: Mark

last: Faries

first: Daniel

last: Kelly

first: Garni

last: Barkhoudarian

first: Carlo

last: Bifulco

first: Kim

last: Margolin

first: Steven

last: Kolker

first: Maria

last: Ascierto


title: Plasma Cell-free RNA Signatures of Inflammatory Syndromes in Children

city: Ithaca

first: Conor

last: Loy

address: 237 Tower Rd, Room 314

institute: Cornell University

summary: Inflammatory syndromes, including those caused by infection, are a major cause of hospital admissions among children and are often misdiagnosed because of a lack of advanced molecular diagnostic tools. In this study, we explored the utility of circulating cell-free RNA (cfRNA) in plasma as an analyte for the differential diagnosis and characterization of pediatric inflammatory syndromes. We profiled cfRNA in 370 plasma samples from pediatric patients with a range of inflammatory conditions, including Kawasaki disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), viral infections and bacterial infections. We developed machine learning models based on these cfRNA profiles, which effectively differentiated KD from MIS-C — two conditions presenting with overlapping symptoms — with high performance (Test Area Under the Curve (AUC) = 0.97). We further extended this methodology into a multiclass machine learning framework that achieved 81% accuracy in distinguishing among KD, MIS-C, viral, and bacterial infections. We further demonstrated that cfRNA profiles can be used to quantify injury to specific tissues and organs, including the liver, heart, endothelium, nervous system, and the upper respiratory tract. Overall, this study identified cfRNA as a versatile analyte for the differential diagnosis and characterization of a wide range of pediatric inflammatory syndromes.

overall_design:
We performed plasma cfRNA-seq on samples from 370 pediatric patients with a range of inflammatory conditions

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

first: Venice

last: Servellita

first: Alicia

last: Sotomayor-Gonzalez

first: Andrew

last: Bliss

first: Joan

last: Lenz

first: Emma

last: Belcher

first: Will

last: Suslovic

first: Jenny

last: Nguyen

first: Meagan

last: Williams

first: Miriam

last: Oseguera

first: Michael

last: Gardiner

first: Jong-Ha

last: Choi

first: Hui-Mien

last: Hsiao

first: Hao

last: Wang

first: Jihoon

last: Kim

first: Chisato

last: Shimizu

first: Adriana

last: Tremoulet

first: Meghan

last: Delaney

first: Roberta

last: DeBiasi

first: Christina

last: Rostad

first: Jane

middle: C

last: Burns

first: Charles

middle: Y

last: Chiu

first: Iwijn

last: De Vlaminck


title: Detection of 5mC in DNA templates generated by PCR and HeLa human genomic DNA by linear PCR using KTq DNA polymerase variants

first: Melanie

last: Henkel

summary: We have engineered thermostable KlenTaq DNA polymerase variants called RIII H20, RIV A8 and RIV D15 that produce error signatures specific for 5-methylcytosine (5mC) without prior chemical treatment of the DNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish C from 5mC in DNA templates generated by PCR (unmodified and methylated using the CpG Methyltransferase (M.SssI)). Finally, RIV A8 was used to detect 5mC in HeLa human genomic DNA (native methylation and supplementary methylated using the CpG Methyltransferase (M.SssI))

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: DNA templates generated by PCR (unmodified and methylated using the CpG Methyltransferase (M.SssI)) and HeLa human genomic DNA (native methylation and supplementary methylated using the CpG Methyltransferase (M.SssI)) were linear amplified by RIII H20, RIV A8 and RIV D15 (genomic DNA only by RIV A8). Respective ssDNA product was used to prepare DNA amplicon libraries which were analyzed by NGS to detect 5mC by reading an increased error rate.

first: Andreas

last: Marx

first: Yuri

last: Motorin

first: Virginie

last: Marchand


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [H3K27me3 ChIP-seq,RSeT+DT_L1KD]

first: Juan

last: Zhang

summary: In this study, we performed H3K27me3 ChIP-seq to compare LINE1 knockdown naive hES cells with the control.

overall_design: RSeT media maintained naive H9 hES cells were transfected with non-specific targeting control anti-sense oligos (ASO-Ctr) and anti-sense oligos (ASO-L1), respectively. Transfected cells were maintained in the RSeT media added with 10 nM DZNep and 5nM TSA (RSeT+DT). 48 hours later, ASO transfected cells were collected and subjected to H3K27me3 ChIP-seq. ChIP steps were performed using the ChIP-lysates True MicroChIP-Seq kit (Diagenode # C01010132) following the instruction manuals. The rabbit antibody against H3K27me3 (Cell Signaling Technologies # 9733) was used for the pulldown. 5% of input from replicate sample 3 was saved before the pulldown, and used for input DNA extraction, as the instruction manuals indicated.

first: Lauren

last: Caldwell

first: Miguel

last: Ramalho-Santos


title: Targeting EGLN2/PHD1 protects motor neurons and normalizes the astrocytic interferon response

city: Leuven

first: Kristofer

last: Davie

address: Herestraat 49

department: Center for Brain and Disease Research

country: Belgium

institute: Vlaams Instituut voor Biotechnologie

summary: The egl-9 family hypoxia-inducible factor (EGLN) enzymes, also known as prolyl hydroxylase domain (PHD) enzymes, are metabolic sensors regulating inflammatory responses and cellular metabolism. Both neuroinflammation and dysregulated energy metabolism are linked to motor neuron degeneration leading to amyotrophic lateral sclerosis (ALS) and correlate with disease progression. Therefore, the aim of this study was to explore the role of EGLN2 in ALS. Using a genetic and an oligonucleotide-based approach, we showed that downregulation of EGLN2 protects motor neurons and mitigates the ALS-like phenotypes in a mouse and a zebrafish model of ALS. Single-nuclei RNA sequencing of murine spinal cord revealed that loss of EGLN2 induced an astrocyte-specific downregulation of pro-inflammatory interferon-stimulated genes. In addition, we found that genetic deletion of EGLN2 restored the disturbed interferon response in iPSC-derived astrocytes from an ALS patient, confirming the novel link between EGLN2 and astrocytic interferon signaling. In conclusion, we identified EGLN2 as a potential novel ALS therapeutic target normalizing the astrocytic interferon-dependent inflammatory axis in vivo as well as in patient-derived cells

overall_design: Nuclei were extracted from frozen mouse lumbar spinal cords (3xSOD1G93A/PHD1+/+, 3x SOD1G93A/PHD1-/-) using a protocol slightly modified from the Sweitch et al protocol. Purified nuclei were processed using the 10X genomics 3' single cell gene expression kit.

first: Christine

last: Germeys

first: Tijs

last: Vandoorne

first: Suresh

last: Poovathingal

first: Kara

last: Heeren

first: Wendy

last: Vermeire

first: Fatemeh

last: Nami

first: Matthieu

last: Moisse

first: Annelies

last: Quaegebeur

first: Annerieke

last: Sierksma

first: Laura

last: Rué

first: Adrià

last: Sicart

first: Caroline

last: Eykens

first: Bart

last: De Strooper

first: Peter

last: Carmeliet

first: Philip

last: Van Damme

first: Katrien

last: De Bock

first: Ludo

last: Van Den Bosch


title: Increased EZH2 function in regulatory T cells promotes their capacity to suppress autoimmunity by driving effector differentiation prior to activation [RNA-seq]

first: Janneke

last: Peeters

overall_design: RNA-seq was performed on Treg cells (sorted based on CD4+Foxp3GFP+RFP+CD62L+) from Foxp3-GFP-hCre;Ezh2Y641F/+;R26RFP (Ezh2-Y641F) or Foxp3-GFP-hCre;Ezh2+/+;R26RFP (WT) mice either collected after sorting (naive samples) or after 4 days of activation with anti-CD3 and anti-CD28 coated beads at a ratio of 1:3 (cell:bead) in the presence of 2000 IU/mL recombinant human IL-2 (activated samples).

first: Michel

last: DuPage

first: Srinivas

last: Ramachandran


title: Unraveling MLL1-fusion Leukemia: Epigenetic Revelations from an iPS Cell Point Mutation

city: Syracuse

first: Michael

middle: Scott

last: Cosgrove

address: 750 East Adams Street

department: Biochemistry and Molecular Biology

institute: SUNY Upstate Medical University

summary: Human iPS cells (WT ASE9203 cells) were obtained from Applied Stem Cell (ASC), and CRISPR-Cas9-mediated gene editing was completed by ASC using their proprietary CRISPR-Cas9 protocol to introduce the MLL1 Win motif R3765A. Two clones (MT1 and MT2) homozygous for R3765A were identified and sequenced. Both WT and MT iPS cells were maintained in mTeSRTM1 medium with 1x Supplement media (Stem Cell Technologies, Catalog #85850) and supplemented with 10 μM Y27632 (Stemgent) only upon thawing and passaging. RNA sequencing was performed.

overall_design: Compare gene expression in CRISPR-edited mutant and wild type human IPSC cells

first: Laila

last: Kobrossy

first: Weiyi

last: Xu

first: Chunling

last: Zhang

first: Christopher

last: Turner

first: Wenyi

last: Feng


title: Inhibition of JNK Signaling Overcomes Cancer-Associated Fibroblast-Mediated Immunosuppression and Enhances the Efficacy of Immunotherapy in Bladder Cancer [RNA-Seq]

status: Public on Sep 08 2024

city: Hefei

first: Yang

last: Li

state: Anhui

address: 81 Meishan Road

institute: Anhui Medical University

summary: Currently, only 20-40% of cancer patients benefit from immune checkpoint inhibitors. Understanding the mechanisms underlying the immunosuppressive tumor microenvironment (TME) and characterizing dynamic changes in the immunological landscape during treatment are critical for improving responsiveness to immunotherapy. Here, we identified JNK signaling in cancer-associated fibroblasts (CAFs) as a regulator of the immunosuppressive tumor microenvironment. Single-cell RNA sequencing of bladder cancer treated with a JNK inhibitor revealed enhanced cytotoxicity and effector functions of CD8+ T cells. In untreated tumors, CAFs interacted frequently with CD8+ T cells and mediated their exhaustion. JNK inhibition abrogated the immunosuppression function of CAFs by downregulating the expression of TSLP, thereby restoring CD8+ T cell cytotoxicity. In addition, blockade of CAF-derived TSLP in combination with anti-PD1 treatment promoted tumor elimination by CD8+ T cells in vivo. Collectively, these results indicate that JNK signaling plays an important immunosuppressive role in the tumor microenvironment by promoting expression of TSLP in CAFs and suggest that inhibiting JNK signaling could be a promising immunotherapeutic strategy for cancer treatment.

overall_design: To investigate the mechanism of the JNK signaling-mediated immunosuppressive role of CAFs. Primary CAF1 cells treated with or without SP600125 were collected and analyzed by RNA sequencing.

first: Chengying

last: Cui


title: Inhibition of JNK Signaling Overcomes Cancer-Associated Fibroblast-Mediated Immunosuppression and Enhances the Efficacy of Immunotherapy in Bladder Cancer [scRNA-Seq]

overall_design: To systematically investigate the cellular diversity and molecular variation in the bladder tumor microenvironment following JNK inhibition, we collected whole bladders from N-Butyl-N(4-hydroxybutyl) nitrosamine-induced mice (2 samples per group) and performed single-cell RNA sequencing using 10x genomics platform.


title: Unveiling the association between HMG-CoA reductase inhibitors and bladder cancer

city: Wuhan

first: Yu

last: Xiao

address: Donghu Road 169

department: Department of Urology

institute: Zhongnan Hospital of Wuhan University

summary: This study utilized Mendelian randomization (MR) analysis and genome-wide association study (GWAS) data to investigate the association between commonly prescribed drugs and bladder cancer (BLCA) risk. Our results revealed that HMGCR inhibitors, specifically simvastatin, are significantly associated with a reduced BLCA risk. We further showed that simvastatin could significantly inhibit BLCA proliferation and epithelial-mesenchymal transition in animal models, with transcriptomic data identifying several associated pathways. Higher levels of HMGCR were linked with BLCA development and progression, and certain blood lipids, such as lipoprotein particles and VLDL cholesterol, might influence BLCA risk. These findings suggested that HMGCR inhibitors, particularly simvastatin, could be potential treatment options or adjuvant therapies for BLCA. Meanwhile, RhoB is a key protein involved in the regulation of bladder cancer cell metastasis by simvastatin. Therefore, we tested the changes of key genes in bladder cancer cells after simvastatin (HMGCR inhibitor) treatment and overexpression of RhoB, respectively, using RNA sequencing.

overall_design: To investigate the role of HMGCR and RhoB in BLCA, we treated the T24 BLCA cell line with 2 μM simvastatin for 48 hours. In addition, T24 cells were transfected with empty plasmid and RhoB overexpression plasmid for 48 hours. Then, in order to ensure the reliability of the results, we performed independent experiments three times for each sample. Finally, the 12 samples were analyzed by RNA sequencing. (O = DMSO, S = simvastatin, TN = empty vector, TR = RHOB overexpression).


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

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in distinct MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of catalytic SET domain of MLL1 (known for H3K4 mono- and dimethylation), remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human induced pluripotent stem cells. Remarkably, this mutation mimics the histone methylation, gene expression, and EMT phenotypes of MLL1F-LSCs- without the need for a translocation or functional wild-type MLL1. This observation underscores the essential role of MLL1's enzymatic activity in restraining the cascade of epigenetic changes associated with the gene activating H3K4 trimethylation mark, which we show is catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1’s enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1's histone methyltransferase activity. We advocate for a therapeutic paradigm shift, targeting SETd1a for precision medicine. This work opens new avenues for addressing the complexities of MLL1-associated leukemias and improving targeted therapies.


title: DNAJB1-PRKACA fusion drives fibrolamellar liver cancer through impaired SIK signaling and CRTC2/p300-mediated transcriptional reprogramming

city: Boston

first: Nabeel

last: Bardeesy

state: MA

address: 185 Cambridge St.

institute: Massachusetts General Hospital/Harvard Medical School

summary: Fibrolamellar carcinoma (FLC) is a liver cancer of adolescents and young adults defined by fusion of the DNAJB1 heat shock protein and protein kinase A (PKA) catalytic subunit (DNAJB1-PRKACA). The resulting chimeric protein has increased kinase activity and is essential for FLC xenograft growth. However, the critical oncogenic pathways controlled by DNAJB1-PRKACA have not been defined. Here, we explored this question by studying patient-derived FLC models and engineered systems and analyzing patient samples. We show that the core function of DNAJB1-PRKACA is the direct phosphorylation and inactivation of the Salt-inducible kinases. This leads to deregulation of the CRTC2 co-activator and p300 acetyltransferase, resulting in transcriptional reprogramming and global increases in histone acetylation necessary for malignant growth. Our studies establish a central oncogenic mechanism of DNAJB1-PRKACA and suggest opportunities for therapeutic targeting of CRTC2/p300 in FLC. Notably, these findings link this signature fusion oncoprotein of a rare cancer type to more common cancer gene alterations involving the STK11 tumor suppressor and GNAS oncogene, which also function via SIK suppression.

overall_design: To investigate the effects of PKA and SIK inhibitors on FLX1 cell lines, we treated these cells under four different conditions: DMSO (control), PKA inhibitor, SIK inhibitor, and a combination of both inhibitors. Subsequently, we harvested triplicate biological replicates for RNA-seq and analyzed the data.

first: Ilaria

last: Gritti


title: Comparison of human pluripotent stem cell differentiation protocols to generate neuroblastoma tumors.

first: Miller

last: Huang

address: 4640 West Sunset Blvd Off Campus

department: Pediatrics

institute: Keck School of Medicine of Usc

summary: Neuroblastoma is the most common extra cranial solid tumor derived from sympathoadrenal (SA) cells and characterized as either adrenergic or mesenchymal. Here, we compared four different protocols to differentiate induced pluripotent stem cells (iPSC) toward SA cells and intermediate cell states (neuromesodermal progenitors [NMP], trunk neural crest cells [tNCC]) as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two iPSC lines of different genders. This protocol also generated tumors with the highest level of the neuroblastoma-specific marker, PHOX2B.  Transcriptomally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma.

overall_design:
We first compared four different tNCC/SA differentiation protocols in vitro by RT-qPCR for tNCC/SA markers. WT PSCs were then transduced with MYCN, differentiated, and implanted orthotopically into immunocompromised mice.
We also analyzed the tumors by RNA-seq and compared the transcriptomes with neural crest derived tumors.

first: Bo

last: Cheng


title: Convergent evolution of MDP-monocyte differentiation in adult skin permits repair of the Langerhans cell network [scRNA-seq]

city: London

first: Stephen

last: Henderson

address: Paul O'Gorman Building

department: Cancer Institute

country: United Kingdom

institute: University College London

summary: Langerhans cells (LCs) are a unique population of phagocytes programmed within embryonic skin to maintain tissue and immunological homeostasis at the epidermal barrier site.  Unique amongst tissue-resident macrophages, LCs play roles in skin innervation and repair, while also functioning as migrating professional antigen presenting cells, a capability classically assigned to dendritic cells (DC).  We current lack insight into the molecular pathways that determine this dichotomy, when they are established, and whether precursors recruited to the adult skin niche access the same instructive signals laid down in utero.   Tracking differentiation of monocyte-derived (m)LCs  after destruction of embryo-derived cells in murine adult skin, revealed intrinsic intra-epidermal heterogeneity and selection of MDP-monocytes to by-pass gene programmes specifying a macrophage fate.  Adopting a process unique to adult skin, subsequent extrinsic specification of EpCAM+ precursors via follicular Notch ligands, and aryl hydrocarbon receptor (Ahr) signalling, combined to determine commitment to resident mLCs.  Using a unique human LC dataset, we demonstrated that embryo-derived (e)LCs in newborn human skin retained transcriptional evidence of their macrophage origin, which was superseded by a distinct DC-like immune modules following proliferation in infant skin.  Thus, convergent differentiation of monocytes within adult skin exploits novel molecular pathways to replicate conditioning of eLC towards more immunogenic DC-like cells within post-natal skin.

overall_design: Lethally irradiated male CD45.2 C57BL/6 mice received female CD45.1 C57BL/6 bone marrow cells, CD4 T cells, with CD8 Matahari (Mh) T cells administered by intravenous injection through the tail vein. Single-cell RNA-sequencing (scRNA-seq) was carried out on donor and host CD11b+MHCII+ myeloid cells, as well as CD45- keratinocytes FACS purified from the epidermis 3 weeks post-BMT using the 10X Genomics platform.

first: Anna

last: Appios

first: James

last: Davies

first: Marta

last: Polak

first: Clare

last: Bennett


title: Convergent evolution of MDP-monocyte differentiation in adult skin permits repair of the Langerhans cell network

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

overall_design: Refer to individual Series


title: Transcriptomic analysis reveals a critical role for activating Gsα mutations in spontaneous feline hyperthyroidism

status: Public on Sep 07 2024

city: Hatfield

first: Lucy

last: Davison

address: Hawkshead Lane

department: CSS

institute: Royal Veterinary College

summary: Feline hyperthyroidism (FHT) is a debilitating disease affecting >10% of elderly cats. It is generally characterised by chronic elevation of thyroid hormone in the absence of circulating TSH. Understanding of the molecular pathogenesis of FHT is currently limited. However, FHT shares clinical and histopathological similarities with human toxic multinodular goitre, which has been associated with activating mutations in TSH receptor (TSHR) and Gsα encoding genes. Using RNA-seq transcriptomic analysis of thyroid tissue from hyperthyroid and euthyroid cats, we identified differentially expressed genes and dysregulated pathways in FHT, many of which are downstream of TSHR. In addition, we detected missense variants in thyroid RNA-seq reads that alter the structure of both TSHR and Gsα. All FHT-associated mutations were absent in germline sequence from paired blood samples. Only a small number of hyperthyroid cats demonstrated TSHR variation, however all thyroids from advanced cases of FHT carried at least one missense variant affecting Gsα.  The activating nature of the acquired Gsα mutations was demonstrated by increased cAMP production in vitro. These data indicate that constitutive activation of signalling downstream of TSHR is central to the TSH-independent production of thyroid hormone in FHT, offering a novel therapeutic target pathway in this common disease.

overall_design: To investigate the thyroid transcriptome in feline hyperthyroidism, we used RNA-seq to profile gene expression in thyroid tissue samples collected from 4 euthyroid (ET) and 11 hyperthyroid (HT) cats. We then identified differentially expressed genes between HT and ET samples.

first: Thomas

middle: K

last: Hiron

first: Joana

last: Aguiar

first: Sara

last: Falcone

first: Harriet

last: Syme


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [ChIRP-seq,RSeT+DT H9]

first: Juan

last: Zhang

summary: In this study, we profiled for LINE1 binding loci in RSeT+DT naïve hES cells with the Chromatin Isolation by RNA Purification (ChIRP)-seq strategy. We followed a previously published ChIRP protocol described (Percharde et al. 2018; Lu et al. 2020).

overall_design: RSeT H9 hES cells were witched to RSeT media added with 10 nM DZNep and 5nM TSA (RSeT+DT) for 48 hours, then collected for preparing ChIRP sequencing sample. x107 cells per ChIRP pulldown were subjected to DNAseI digestion after crosslinking the cells. After brief sonication and spinning, chromatin supernatants were collected for hybridization with probes. After 3 hour of hybridization with LINE1 probes or the control MALAT1 probes, streptavidin M280 beads were added and incubated for another 3 hours. After hybridization, the beads were washed 5 times and then treated with RNase H to elute ChIRP pulled down DNAs, which were reverse crosslinked and purified, and then used for library preparation. More protocol details are described in the methods of this study.

first: Lauren

last: Caldwell

first: Miguel

last: Ramalho-Santos


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [pA-DamID LAD-seq]

summary: To investigate LADs associated DNAs in the naïve and primed hES cells.

overall_design:
In this study, we profiled for DNA loci within the nucleus Lamina associated domain (LADs) in naïve and primed H9 hES cells for comparison. We followed a previously published pA-DamID sequencing method using LMNB1 antibody. So, Genes in close contact with LMNB1 are DamID labelled (methylated) and thus annotated via sequencing.
Naïve and primed H9 hES cells maintained in RSeT and mTeSR, respectively, were collected for pA-DamID reaction. The experiment was performed using the rabbit antibody against LMNB1 (Abcam # ab16048) to tag pA-Dam fusion protein to LADs and conducted to DamID sequencing onwards. The details of protocol is described by van Schaik et al. (van Schaik et al. 2020) for the pA-Dam step, and by Vogel, Peric-Hupkes, and van Steensel (Vogel, Peric-Hupkes, and van Steensel 2007) for the DamID sequencing step. More protocol details are described in the methods of this study. The DamID-DNA library was subjected to 100bp single-end sequencing on HiSeq 2500 with 1%PhiX spike-in.

first: Linh

last: Huynh


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [NAD-seq]

summary: To investigate NADs associated DNAs in the naïve and primed hES cells.

overall_design:
In this study, we profiled for DNA loci within the nucleolus associated domain (NADs) in naïve and primed H9 hES cells for comparison. We followed a previously published “crosslinked method of nucleoli isolation” protocol described by Vertii et al. (Vertii et al. 2019).
Naïve and primed H9 hES cells maintained in RSeT and mTeSR, respectively, were collected for preparing NADs sequencing sample. 20-30 x 10^6 cells were subjected to ultrasonic cell disruption after crosslinking the cells. Nucleolar fraction was spin pelleted after 2-3 rounds of sonication in the high magnesium (HM) buffer and then subject to another round of sonication and nucleolar pelleting in the low magnesium (LM) buffer. The final pelleted nucleolar DNA were extracted and processed to DNA library preparation with the Illumina`s TruSeq DNA PCR-free Library Preparation kit (Illumina # 20015962), and 350 bp fragments were sonicated and size-selected by sample-purifying beads following manual instruction. The library was subjected to 150bp paired-end sequencing onNovaSeq. The details of protocol is described by by Vertii et al. (Vertii et al. 2019). More protocol details are described in the methods of this study.


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [RNA-seq_primed,RSeT,RSeT+DT_hESCs]

summary: In this study, we performed RNA-seq to compare transcriptome in primed and naive hES cells.

overall_design:
RNA-seq in wild-type RSeT+DT, RSeT and primed hESCs was carried out in partnership with CEEHRC, following their sample preparation guidelines:
https://thisisepigenetics.ca/for-scientists/community-access-program

first: Kirti

last: Mittal

first: Marie-Michelle

last: Simon

first: Tony

last: Kwan


title: Effect of embryo lysates treatment on small RNA expression

city: Beijing

first: Ying

last: Liu

address: 5 Summer Palace Road, Haidian District, Beijing

department: College of Future Technology

institute: Peking University

summary: To systematically evaluate the effect of embryo lysates feeding, we conducted small RNA sequencing on day 7 N2 worms treated with either embryo or day 6 worm lysates. Our findings revealed that embryo lysates affected the 21ur-6059 expression.

overall_design: Day 7 N2 worms were treated with embryo lysates for 24h. Then worms were collected for small RNA seq and small RNA expression profiliing analysis.

first: Liankui

last: Zhou

first: Wanqiu

last: Ding


title: Scalable production of uniform and mature organoids in a 3D geometrically-engineered permeable membrane

city: Ulsan

first: Tae-Eun

last: Park

address: 50, UNIST-gil, Eonyang-eup, Ulju-gun

department: Biomedical Engineering

institute: Ulsan National Institute of Science and Technology (UNIST)

summary: The application of organoids has been limited by the lack of methods for producing uniformly mature organoids at scale. This study introduces an organoid culture platform, called UniMat, which addresses the challenges of uniformity and maturity simultaneously. UniMat is designed to not only ensure consistent organoid growth but also facilitate an unrestricted supply of soluble factors by a 3D geometrically-engineered, permeable membrane-based platform. Using UniMat, we demonstrate the scalable generation of kidney organoids with enhanced uniformity in both structure and function compared to conventional methods. Notably, kidney organoids within UniMat matured significantly better, showing increased expression of nephron transcripts, more in vivo-like cell-type balance, and better vascularization. Moreover, UniMat's design offers a more standardized organoid model for drug testing, as demonstrated by its consistent response to a polycystic-kidney-disease drug. In essence, UniMat presents a transformative platform for organoid technology, promising applications in organ development, disease modeling, and drug screening.

overall_design: We performed single-cell RNA sequencing to compare kidney organoids differentiated in AggreWell and UniMat.

first: Dohui

last: Kim

first: Hyeonji

last: Lim

first: Jaeseung

last: Youn

first: Dong Sung


first: Michael

last: Cosgrove

summary: Human iPS cells (WT ASE9203 cells) were obtained from Applied Stem Cell (ASC), and CRISPR-Cas9-mediated gene editing was completed by ASC using their proprietary CRISPR-Cas9 protocol to introduce the MLL1 Win motif R3765A. Two clones (MT1 and MT2) homozygous for R3765A were identified and sequenced. Both WT and MT iPS cells were maintained in mTeSRTM1 medium with 1x Supplement media (Stem Cell Technologies, Catalog #85850) and supplemented with 10 μM Y27632 (Stemgent) only upon thawing and passaging. ChIP seq analysis was performed.

overall_design: Compare ChIP-seq data for H3K4me3 in CRISPR-edited mutant and wild type human IPSC cells

first: Laila

last: Kobrossy

first: Weiyi

last: Xu

first: Chunling

first: Christopher

last: Turner

first: Wenyi

last: Feng


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [RNA-Seq_RSeT_H9_L1KD]

first: Juan

last: Zhang

summary: To study L1 knockdown effect in RSeT naïve hES cells

overall_design:
In this study, we investigated LINE1 (L1), a major family of transposon elements (TEs) in the human genome, in naive hES cells for its roles in regulating 8-cell (8C) stage-specific totipotent genes.
RSeT media maintained naive H9 hES cells were transfected with 6-FAM (Fluorescein) labeled non-specific targeting ASO (ASO-Ctr), L1 targeting sense oligo (SO-L1) and anti-sense oligo (ASO-L1), respectively. Cells were maintained in the RSeT media post transfection. 48 hours later, FAM+ cells were FACS collected and immediately processed for RNA extraction and RNA-seq library prep.

first: Miguel

last: Ramalho-Santos


title: PD-L2 of tumor-derived exosomes mediates the immune escape of cancer cells via the impaired T cell function

city: Nanjing

first: Shan

last: Gao

address: No. 2 Sipailou, Xuanwu District

institute: Southeast University

summary: The function and regulation of PD-1/PD-L1 axis have been widely studied for immune escape of various cancer cells. However, the underlying function and regulation of PD-L2 remain poorly to be understood. We demonstrate that PD-L2 is majorly secreted and expressed on exosomes with surface localization by tumor cell. In order to investigate the function of TDE-PD-L2 in the regulation of lymphocytes, we explored the expression profiles of TDE-PD-L2 on lymphocytes of PBMC using RNA-seq.

overall_design: To investigate the function of tumor cell-derived exosome PD-L2 (TDE-PD-L2) in the regulation of lymphocytes of PBMC, we established 786-O cell lines in which PDCD1LG2 gene has been knockout by CRISPR-Cas9. Exosomes isolated from 786-O CTR and PD-L2 KO culture supernatants of equal numbers of cells were feeded to PMA and ionomycin stimulated lymphocytes isolated from peripheral blood lymphocytes in 10cm Petri dish for 72h.

first: Tongfeng

first: Shuwen

last: Cheng

first: Bo

last: peng

first: Haojing

last: Zang

first: Xiaofeng

last: Zhu

first: Xuetong

last: Wang


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [scRNA-seq]

address: 25 Orde Street, Room 6-1001 MRS

institute: LTRI

summary: In this study, we investigated LINE1 (L1), a major family of transposon elements (TEs) in the human genome, in naive hES cells for its roles in regulating 8-cell (8C) stage-specific totipotent genes. We performed single-cell RNA-seq (scRNA-seq) to assess if L1 knockdown in RSeT+DT naive hES cells induces 8-cell-like cells (8CLCs), and profile the transcriptome changes in the cells.

overall_design: RSeT media maintained naive H9 hES cells were transfected with 6-FAM (Fluorescein) labeled SO-L1 and ASO-L1 oligos and cultured in the RSeT+DT condition. 48 hours later, 100,000 FAM+ cells were FACS collected and immediately processed on the 10x Genomics Chromium platform using the Single Cell 3’ v3 RNA-seq kit and targeting a yield of 12,000 cells. Final libraries were sequenced on a NovaSeq6000 S1 flow cell (Illumina) to an average depth of >65,000 reads/cell.

first: David

middle: P

last: Cook

first: Liang

last: Wu


title: Mevalonate pathway inhibition reduces bladder cancer metastasis by modulating RhoB protein stability and integrin β1 localization

first: Yu

last: Xiao

summary: Metastasis is an important factor affecting the prognosis and survival of bladder cancer (BLCA) patients. Our previous study found that the mevalonate pathway is associated with the migratory ability of BLCA cells, but the exact mechanism is unclear. Here, we found that BLCA patients with mevalonate pathway activation had a poorer prognosis. Inhibition of the mevalonate pathway (FDPS knockdown, simvastatin or zoledronic acid) significantly reduced the migratory ability of BLCA cells. Therefore, we tested the changes of key genes after knocking down the key enzymes of the mevalonate pathway, FDPS and SQLE1, and the transcription factor YY1 in bladder cancer cells using RNA sequencing.

overall_design: To investigate the roles of FDPS, SQLE and YY1 in BLCA, we first knocked down the target genes FDPS, SQLE or YY1 in the T24 BLCA cell line using the corresponding siRNAs, respectively, and all of these siRNAs were proved to have good knockdown efficiency. Then, in order to ensure the reliability of the results, we performed independent experiments three times for each sample. Finally, the 12 samples were analyzed by RNA sequencing. (N = negative control, S = siSQLE, F = siFDPS, Y = siYY1)


title: Convergent evolution of MDP-monocyte differentiation in adult skin permits repair of the Langerhans cell network [RNA-seq]

first: Stephen

last: Henderson

overall_design: To determine how Notch signaling combined with other environmental signals in the skin we cultured monocytes with GM-CSF/TGFβ/IL-34 with or without Jagged-1, the Ahr antagonist FICZ, or both, and compared the transcriptional changes within sorted CD11blowEPCAM+ cells

first: Anna

last: Appios

first: James

last: Davies

first: Marta

last: Polak

first: Clare

last: Bennett


title: Crinophagic granules in pancreatic β cells contribute to mouse autoimmune diabetes by diversifying pathogenic epitope repertoire

status: Public on Sep 06 2024

city: St Louis

first: Tiandao

last: Li

state: MO

address: 4444 Forest Park Ave

institute: Washington University

summary: We examined the transcriotional changes in antigen specific CD4 T cells to antigens derived from thymus or to the antigens derived from islets in the autoimmune diabetes mouse model.

overall_design: Single cells were prepared from spleen and lymph nodes of 8-12 weeks old NOD mice or HEL specfic TCR transgenic mice. Tetramer enriched cells were sorted from the NOD mice and naïve CD4 T cells were sorted from HEL TCR trangenic mice. Before surface staining, samples were incubated with 100 μl of TotalSeq™ anti-mouse Hashtag reagent (1:100 [vol/vol], BioLegend) to tag the samples: enriched cells from the islet epitope set were tagged with the TotalSeq-C0301 anti-mouse Hashtag 1 Antibody (Barcode sequence: ACCCACCAGTAAGAC); enriched cells from the thymic epitope set were tagged with the TotalSeq-C0302 anti-mouse Hashtag 2 Antibody (Barcode sequence: GGTCGAGAGCATTCA); 10E11 naïve CD4+ T cells were tagged with the TotalSeq-C0303 anti-mouse Hashtag 3 Antibody (Barcode sequence: CTTGCCGCATGTCAT).

first: Hao

last: Hu

first: Xiaoxiao

last: Wan


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [RNA-Seq_RSeT+DT_H9_L1KD]

city: Toronto

first: Juan

last: Zhang

summary: To study L1 knockdown effect in RSeT+DT naïve hES cells

overall_design:
In this study, we investigated LINE1 (L1), a major family of transposon elements (TEs) in the human genome, in naive hES cells for its roles in regulating 8-cell (8C) stage-specific totipotent genes.
RSeT media maintained naive H9 hES cells were transfected with non-specific targeting control anti-sense oligos (ASO-Ctr), L1 targeting sense oligos (SO-L1) and anti-sense oligos (ASO-L1), respectively. Transfected cells were maintained in the RSeT media added with 10 nM DZNep and 5nM TSA (RSeT+DT). 48 hours later, cells were directly processed for RNA extraction and RNA-seq library prep.

first: Miguel

last: Ramalho-Santos


title: Spns1 controls megalin dependent endocytosis via iron misregulation

city: New York

first: Katherine

last: Xu

address: 1150 St. Nicholas Ave., Room 408A/B

department: Medicine

institute: Columbia University Medical Center

summary: Proximal tubule endocytosis is essential for protecting the kidney from potentially damaging proteinuria and for mediating metabolic pathways, such as the activation of Vitamin D.  We have identified the lysosomal membrane protein, Spns1, as a novel iron conductance that is indispensable for the normal endocytic function of the proximal tubule. Conditional knockout of Spns1 with a Cre-lox system in megalin-expressing cells in mice leads to arrest of both pinocytosis and receptor-mediated endocytosis in the proximal tubule. This endocytic defect is associated with iron overload in proximal tubules as measured by immunofluorescence of ferritin abundance and by a reduction in nascent TfRc mRNA transcripts. We have recapitulated previous findings in drosophila and zebrafish that Spns1 knockout causes endolysosomal dysfunction including abnormally enlarged lysosomes. The endocytic defect resulting from conditional Spns1 knockout can be rescued nutritionally with an iron deficient diet or genetically through double knockout of both Spns1 and the Fe2+ transporter, DMT1. Surprisingly, the endocytic defect in mice with Spns1 conditional knockout occurs despite normal megalin expression at the proximal tubule apical membrane. This data raises the possibilty that post-translational regulation of megalin activation may be nutrient-responsive.

overall_design: Examining transcriptional profiles of two models of iron overload, genetic Spns1-KO and dietary Iron Overload, compared to Wildtype Controls, in the kidney proximal tubule using Megalin-Cre mediated 4-thio-Uracil tagging of RNA.

first: Andrew

last: Beenken

first: Tian

last: Shen

first: Atlas

last: Khan

first: Jonathan

last: Barasch


title: RNA Sequencing of dendritic cells treated with glycan-costumed virus-like particles

city: Cambridge

first: Valerie

last: Lensch

state: Massachusetts

address: 77 Massachusetts Avenue

institute: MIT

summary: We engineered a glycan-costumed virus-like particle (VLP) vaccine that delivers programmable peptide antigens to induce tumor-specific cellular immunity in vivo. VLPs encapsulating TLR7 agonists were decorated with a synthetic mannose-derived ligand (VLP-Man-OvaI/II) that selectively engages the lectin DC-SIGN. Lectin-TLR7 dual engagement induced robust DC activation and type 1 cellular immunity, whereas VLPs lacking this key DC-SIGN ligand (VLP-PE-OvaI/II) failed to promote DC-mediated cellular responses. We performed bulk RNA-seq experiments on moDCs treated with VLP-Man-OvaI/II and VLP-PE-OvaI/II or unstimulated control moDCs. A total of 486 genes were differentially expressed after treatment with VLP-Man-OvaI/II vs. VLP-PE-OvaI/II (log2FC >1.5 and p<0.05). The significantly upregulated genes included CXCL1, CXCL5, ISG15, IL6, CCL4, ISG20, and SOCS3, all of which are implicated in the TLR7 downstream signaling pathway, suggesting a higher extent of TLR7 activation with the VLP-Man-OvaI/II. Gene set enrichment analysis (GSEA) between VLP-Man-OvaI/II- and VLP-PE-OvaI/II-treated moDCs revealed that hallmark DC activation pathways were also upregulated in VLP-Man-OvaI/II-treated DCs, including pathways related to IFN-a, IFN-g, and TNF-a signaling via nuclear factor-κB (NF-kB). These data give insight into how lectin binding with glycan-costumed VLPs can be employed to reprogram immunity.

overall_design: 5 replicates per condition (DC alone, DC+VLP-PE-OvaI/II, DC + VLP-Man-OvaI/II)

first: Lensch

last: Valerie

first: Gabba

last: Adele

first: Hincape

last: Robert

first: Basak

last: Ankit

first: Shalek

last: Alex K.

first: Finn

first: Kiessling

last: Laura L.


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [RNA-Seq_RSeT+DT H9_EZH2i]

summary: In this study, we investigated PRC2 (polycomb repressive complex 2), the H3K27me3 modifier, in naive hES cells for its roles in regulating 8-cell (8C) stage genes expression. EZH2 inhibitor, UNC1999 and its inactive analog UNC2400 were used for the treatment.

overall_design: Naive H9 hES cells were maintained RSeT. 24 hours after cell splitting, cells were maintained in the RSeT media added with 10 nM DZNep and 5nM TSA (RSeT+DT), in combination with UNC1999 and UNC2400 added at 2.5 μM final concentration for the treatment and controls. 48 hours later, cells were directly processed for RNA extraction and RNA-seq library prep.


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [RNA-seq_PXGL H9_L1KD]

summary: In this study, we investigated LINE1 (L1), a major family of transposon elements (TEs) in the human genome, in naive hES cells for its roles in regulating 8-cell (8C) stage genes expression.

overall_design: PXGL media maintained naive H9 hES cells were transfected with L1 targeting sense oligos (SO-L1) and anti-sense oligos (ASO-L1), respectively. Transfected cells were maintained in the PXGL media. 48 hours later, cells were directly processed for RNA extraction and RNA-seq library prep.


title: LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs

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

overall_design: Refer to individual Series


title: Gene expression profile at single cell level of 4T1 WT and Mga KO tumors derived on BALB/c mice

city: Houston

first: Chang

last: Yang

address: 6565 MD Anderson BLVD

department: Experimental Radiation Oncology

institute: The University of Texas MD Anderson Cancer Center

summary: Immune evasion is critical for tumor initiation and progression, as well as determining the efficacy of immunotherapies. Through iterative in vivo CRISPR screens within seven syngeneic tumor models, we not only identified the core immune evasion pathways and context-dependent ones across cancer types but also provided a high-confidence valuable dataset for understanding tumor intrinsic immunomodulators and discovering novel anti-cancer therapeutic targets. With a focus on triple-negative breast cancer (TNBC), we found that Mga knock-out significantly enhances anti-tumor immunity and inhibits the tumor growth of TNBC model. Transcriptomics and single cell-RNA (sc-RNA) seq analyses revealed Mga function through, at least partially, repression of MHC-II genes and related immune responses. Consistently, we observed that low MGA expression in breast cancer patients correlates with a favorable prognosis, particularly in those with active interferon-g signaling. Our findings provide new insights into tumor immune escape and pave the way for further exploration of MGA inhibition for clinical benefits in triple-negative breast cancer.

overall_design: WT or Mga KO 4T1 cells were implanted into BALB/c mice and incubated for 3 weeks. Tumors were harvested and digested, and single-cell suspensions with high viability were obtained via FACS sorting. Libraries were prepared according to Chromium Single Cell Gene Expression 3v3.1 kit (10x Genomics) and sequenced on the Illumina NovaSeq platform.

first: Xu

last: Feng

first: Yuanjian

last: Huang

first: Junjie

last: Chen


title: Mga