title: Unveiling novel conserved HIV-1 open reading frames encoding T-cell antigens using ribosome profiling.

city: Orsay

first: Olivier

last: Namy

address: Rue Gregor Mendel, bat 400

institute: CNRS, Université Paris-Sud

summary: Alternative reading frames (ARFs) were described in several viral genomes using ribosome profiling such as HCMV, KSV, and SARS-CoV-2 but no demonstration has been made on HIV genome. Using ribosome profiling, we uncovered 98 conserved HIV ARFs distributed across the genome, with high conservation among HIV clade B and C isolates. Our analysis revealed that at least 42 ARFs encode viral polypeptides, as demonstrated by T-cell responses targeting 45 ARF-derived peptides in patients under treatment or naturally controlling the infection. At the same time, we identified a ligand of HLA-A*0201 derived from an identified ARF on primary infected cells. These responses were mediated by polyfunctional CD4+ T-cells secreting at least 3 cytokines simultaneously. Our discovery expands the list of conserved viral polypeptides that might be potential targets for vaccination strategies.

overall_design: RiboSeq of supT1 cells infected by the virus HIV NL4-3 XCS. Cells are harvested 20h post-infection. Two independant infections have been done, which were sequenced in triplicate

first: Lisa

last: Bertrand

first: Annika

last: Nelde

first: Bertha

middle: C

last: Ramirez

first: Isabelle

last: Hatin

first: Hugo

last: Arbes

first: Pauline

last: François

first: Emiliano

last: Ricci

first: Emmanuel

last: Labaronne

first: Sylvie

last: Grégoire

first: Anne

last: Bet

first: Guillaume

last: Beauclair

first: Antoine

last: Gross

first: Maja

last: Ziegler

first: Mathias

last: Pereira

first: Yann

last: Verdier

first: Joelle

last: Vinh

first: Stéphanie

last: Graff-Dubois

first: Audrey

last: Esclatine

first: Cécile

last: Gouttefangeas

first: Marcus

last: Altfeld

first: Laurent

last: Hocqueloux

first: Assia

last: Samri

first: Brigitte

last: Autran

last: Lambotte

first: Hans-Georg

last: Rammensee

first: Juliane

last: Walz

first: Arnaud

last: Moris


title: DNA methylation profiles in FFPE brain tumor samples

status: Public on Jan 15 2025

city: Alicante

first: Luis

middle: M

last: Valor

state: Alicante

address: Av. Pintor Baeza, 12

country: Spain

institute: ISABIAL

type: Methylation profiling by genome tiling array

summary: Primary brain cancers are highly heterogeneous and include the most prevalent and aggressive glioblastomas. To understand the key factors underlying brain tumourigenesis, we have built our own database based on hybridization of DNA methylation beadchips from FFPE-derived biomaterial.

overall_design: We performed DNA methylation profiling using Infinium Methylation EPIC v2.0 beadchips to obtain the gene expression profiles of a variety of primary brain tumour subtypes.


title: Transcriptomic profiling of brain-infiltrating and peripheral CD4+Foxp3+ Tregs following traumatic brain injury and nasal anti-CD3 treatment

city: Boston

first: Omar

last: Albastaki

state: MA

address: 60 Fenwood Rd

country: USA

institute: Brigham and Women's Hospital

type: Expression profiling by high throughput sequencing

summary: We performed bulk RNA-sequencing analysis of brain and blood CD4+Foxp3+ Treg cells from sham and injured mice to characterize the gene expression profile of Tregs following traumatic brain injury and nasal anti-CD3 treatment

overall_design: CD4+Foxp3(GFP)+ Treg cells were FACS-isolated from the pericontusional brains tissue and from blood of the sham and injured mice at 7 days post-TBI. For brain samples, each RNA sequencing sample is a pool of 5 ipsilateral hemispheres and 20 sham brain hemispheres. For blood samples, each RNA sequencing sample is a pool of 5 mice. Preparation for RNA libraries for sequencing was performed using standard Illumina protocols.


title: From Local to Systemic: The Journey of Tick Bite Biomarkers in Australian patients.

status: Public on Jan 14 2025

city: Nedlands WA

first: Wenna

last: Lee

state: WA

address: Perth Children's Hospital, 15 Hospital Ave, 15 Hospital Ave

country: Australia

institute: The Kids Research Institute Australia

summary: Tick bites and tick-related diseases are on the rise. Diagnostic tests that identify well-characterised tick-borne pathogens (TBPs) possess limited capacity to address the causation of symptoms asso-ciated with poorly characterised tick-related illnesses, such as debilitating symptom complexes attributed to ticks (DSCATT) in Australia. Identification of local signals in tick-bitten skin that can be detected systemically in blood would have both clinical (diagnostic or prognostic) and research (mechanistic insight) utility, as a blood sample is more readily obtainable than tissue biopsies. We hypothesised that blood samples may reveal signals which reflect relevant local (tissue) events, and that the time course of these signals may align with local pathophysiology. As a first step to-wards testing this hypothesis, we contrasted molecular signatures in skin biopsies taken from the tick-bite location of human participants along with peripheral blood signatures obtained at the same time. This approach captures differentially expressed molecules across multiple omics da-tasets derived from peripheral blood (including cellular and cell-free transcriptomics, proteomics, metabolomics, and DNA methylation), and skin biopsies (spatial transcriptomics). Our data re-vealed that extracellular matrix organisation and platelet degranulation pathways were upregu-lated in skin within 72 hours of a tick bite. The same signals appeared in blood, where they then remained elevated for three months, displaying longitudinally consistent alterations of biological functions. Despite the limited sample size these data represent proof-of-concept that molecular events in the skin following a tick bite can be detectable systemically. This underscores the poten-tial value of blood samples, akin to liquid biopsy, to capture biomarkers reflecting local tissue processes.

overall_design: This study investigates the transcriptome of peripheral blood obtained from tick-bitten participants on enrolment (T0) and at subsequent timepoints at 1 week (T1) and 3 months (T2) post tick-bite.

first: Amanda

middle: D

last: Barbosa

first: Amy

middle: H

first: Andrew

last: Currie

first: David

last: Martino

first: John

last: Stenos

first: Michelle

last: Long

first: Miles

last: Beaman

first: Nathan

middle: T

last: Harvey

first: Nina

last: Kresoje

first: Patrycja

last: Skut

first: Peter

middle: J

last: Irwin

first: Prasad

last: Kumarasinghe

first: Roy

middle: A

last: Hall

first: Rym

last: Ben-Othman

first: Stephen

last: Graves

first: Tobias

middle: R

last: Kollmann

first: Charlotte

middle: L

last: Oskam


title: Menin maintains lysosomal and mitochondrial homeostasis through epigenetic mechanisms in lung cancer [ChIP-seq]

city: Xiamen

first: junbo

last: yuan

state: Fujian

address: Xiang'an South Road

country: China

institute: Xiamen university

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Lysosome-mediated autophagy (including mitophagy) is crucial for cell survival and homeostasis. Although the mechanisms of lysosome activation during stress are well recognized, the epigenetic regulation of lysosomal gene expression remains largely unexplored. Menin, encoded by the MEN1 gene, is a chromatin-related protein that is widely involved in gene transcription via histone modifications. Here, we report that menin regulates the transcription of specific lysosomal genes, such as CTSB, CTSE, and TFE3, through MLL-mediated H3K4me3 reprogramming, which is necessary for maintaining lysosomal homeostasis. Menin also directly controls the expression of SQSTM1 and MAP1LC3B to maintain autophagic flux in an AMPK-mTORC1-independent manner. Furthermore, loss of menin led to mitochondrial dysfunction, resulting in elevated levels of reactive oxygen species (ROS) and ultimately leading to genome instability. In genetically engineered mouse models, Men1 deficiency resulted in severe lysosomal and mitochondrial dysfunction and an impaired self-clearance ability, which further led to metabolite accumulation and spontaneous lung cancer development. SP2509, a histone demethylase inhibitor, effectively reversed the downregulation of lysosomal and mitochondrial genes caused by loss of Men1. Our study confirms the previously unrecognized biological and mechanistic importance of menin-mediated H3K4me3 in maintaining organelle homeostasis.

overall_design: The submission includes samples from chromatin immunoprecipitation sequencing (ChIP-seq) experiments conducted on A549 cells, a LUAD cell line. The primary objective of these experiments is to investigate which genomic regions' H3K4me3 can be remodeled by MI-3, an inhibitor that can specifically disrupt the menin-MLL interaction.DMSO serves as the control.

first: Jun-Bo

last: Yuan

first: Gui-Xin

last: Gu

first: Bang-Ming

last: Jin

first: Qing

last: Han

first: Bing-Hui

last: Li

first: Li

last: Zhang

first: Guang-Hui


title: Cascaded immunotherapy with implantable dual-drug depots sequentially releasing STING agonists and apoptosis inducers

city: Guangzhou

first: Yanteng

last: Xu

state: Guangdong

address: 600# Tianhe Road

department: Centre for Nanomedicine

institute: The Third Affiliated Hospital of Sun Yat-sen University

summary: Non-nucleotide stimulators of interferon gene (STING) agonists hold promise as immunotherapeutic agents for postsurgical adjuvant treatment of tumors. However, their limited effect duration hampers therapeutic effectiveness, necessitating prolonged administration of multiple doses that heightens infection risk and impacts patient compliance. Here, we develop an implantable dual-drug depot in a sandwich-like configuration, with a non-nucleotide STING agonist (MSA-2) in the outer layers of 3D-printed scaffolds and an immunogenic apoptosis inducer (doxorubicin, DOX) in the inner layer of electrospun fibers. We discover that MSA-2 can elicit endoplasmic reticulum stress-mediated and general immunogenic apoptosis of cancer cells. The stimulations with tumor-associated antigens and damage-associated molecular patterns from cancer cells, along with proinflammatory factors secreted by matured dendritic cells and M1-polarized macrophages, can depolymerize intracellular microtubules guiding activated STING trafficking towards lysosomes for degradation. Collectively, the dual-drug depots can initiate a long-lasting cascaded immunotherapy and chemotherapy, suppressing postsurgical tumor recurrence and metastasis.

overall_design: To elucidate the intricate mechanisms underlying intratumor activation and potentiation of the STING-IFNβ pathway, we conducted RNA sequencing and transcriptomic analysis on tumor samples obtained from mice in two distinct cohorts: an untreated control group and a treatment group receiving a 12-h MS-DF-MS therapy.

first: Kai

first: Xuan

last: Yu

first: Weiling

last: He

first: Yu

last: Tao

first: Mingqiang


title: FGFR2 Directs Inhibition of WNT Signaling to Regulate Anterior Fontanelle Closure during Skull Development

city: Los Angeles

first: Audrey

last: Nickle

state: CA

address: 2250 Alcazar St

department: Center for Craniofacial Molecular Biology

institute: University of Southern California

summary: The calvarial bones of the infant skull are linked by transient fibrous joints known as sutures and fontanelles, which are essential for skull compression during birth and expansion during postnatal brain growth. Genetic conditions caused by pathogenic variants in FGFR2, such as Apert, Pfeiffer, Crouzon syndromes, result in calvarial deformities due to premature suture fusion and a persistently open anterior fontanelle (AF). In this study we investigated how Fgfr2 regulates AF closure by leveraging mouse genetics and single-cell transcriptomics. We find that AF cells, marked by the tendon/ligament factor SCX, are spatially organized into ecto- and endocranial domains that selectively differentiate into ligament, bone, and cartilage to form the posterior frontal suture. We show that AF cell differentiation is non-autonomously regulated by FGFR2 signaling in osteogenic front cells of the frontal bones, which regulate WNT signaling in neighboring AF cells by expressing the secreted WNT inhibitor Wif1. Upon loss of Fgfr2, Wif1 expression is downregulated, and AF cells fail to form the posterior frontal suture. This study identifies an FGF-WNT signaling circuit that that directs suture formation within the AF during postnatal development.

overall_design: RNA-seq of the E18.5 mouse anterior fontanelle to compare gene expression between WT and Wnt1-Cre; Fgfr2fl/fl (neural crest KO of Fgfr2) animals. n=5 for each group

first: Lauren

last: Bobzin

first: Sebastian

last: Ko

first: Michaela

last: Ince

first: Aaron

last: Huang

first: Arshia

last: Bhojwani

first: Ryan

last: Roberts

first: Amy

middle: E

last: Merrill


title: Menin maintains lysosomal and mitochondrial homeostasis through epigenetic mechanisms in lung cancer

first: junbo

last: yuan

summary: Lysosome-mediated autophagy (including mitophagy) is crucial for cell survival and homeostasis. Although the mechanisms of lysosome activation during stress are well recognized, the epigenetic regulation of lysosomal gene expression remains largely unexplored. Menin, encoded by the MEN1 gene, is a chromatin-related protein that is widely involved in gene transcription via histone modifications. Here, we report that menin regulates the transcription of important lysosomal genes through MLL-mediated H3K4me3 reprogramming, which is necessary for maintaining lysosomal homeostasis. Menin also directly controls the expression of SQSTM1 and MAP1LC3B to maintain autophagic flux in an AMPK/mTORC1-independent manner. Moreover, menin maintains mitochondrial genome integrity and homeostasis by tightly regulating TFAM expression. In genetically engineered mouse models, Men1 deficiency resulted in severe lysosomal/mitochondrial dysfunction and an impaired self-clearance ability, which further led to metabolite accumulation and spontaneous lung cancer development. SP2509, a histone demethylase inhibitor, effectively reversed the downregulation of lysosomal/mitochondrial genes caused by loss of Men1. Our study confirms the previously unrecognized biological and mechanistic importance of menin-mediated H3K4me3 in maintaining organelle homeostasis.

overall_design: In order to study the transcriptional regulation of lysosomal/autophagic/mitochondrial genes by the menin, we established control and MEN1 knockdown A549 cells using shRNA. Then, we performed gene expression profiling analysis using RNA-Seq data from both control and MEN1 knockdown A549 cells, each with three biological replicates. These two groups are labeled as shNC and shMEN1, respectively.

first: Jun-Bo

last: Yuan

first: Gui-Xin

last: Gu

first: Bang-Ming


title: Gene expression in liver of mice fed a high-fat, high-sucrose diet

city: Tokyo

first: Nobuhito

last: Goda

address: TWIns 02C218, 2-2 Wakamatsu-cho, Shinjuku-ku

country: Japan

institute: Waseda University, Faculty of Science and Engineering

summary: We have employed whole genome microarray expression profiling in the liver of mice fed a high-fat, high-sucrose diet for 15 weeks to search genes with N-terminal signal sequences whose expression is altered concomitantly with changes in islet size. At 15 weeks, this analysis revealed a significant >4-fold upregulation in 118 genes and downregulation by >75% in 22 genes encoding putative secreted factors in liver of mice fed a HFHS, although few changes (<2-fold upregulated or >one-half downregulated) were observed at 10 weeks. Expression of four genes (NRG1, GPNMB, SERPINB1 and HGF) from this signature was quantified in the different RNA samples by real-time PCR, confirming low variability between mice.

overall_design: Gene expression in mouse liver was measured at 0, 5, 10, and 15 weeks after a high-fat, high-sucrose diet. Four mice at each time point were treated with a high-fat, high-sucrose diet for 0, 5, 10, or 15 weeks.

first: Takatomo

last: Arai


title: Global gene expression profiles of freshly isolated control and treatment naive pediatric IBD colonic biopsies

city: Dublin

first: Patrick

last: Walsh

address: Trinity Centre for Health Sciences,, Trinity College Dublin, the University of Dublin,, St. James’s Hospital

department: Clinical Medicine

country: Ireland

institute: Trinity College Dublin

summary: This dataset provides a comparative analysis of global gene expression changes between rectal biopsies harvested from pediatric control non IBD (n=9) and treatment naive ulcerative colitis (n=5) and Crohn's Disease (n=9) patients.

overall_design: Pinch rectal biopsies were stored in RNAlater immediately after harvesting and subsequently analyzed for gene expression profiles by RNAseq.

first: Séamus

last: Hussey


title: Profiling of microRNAs in extracellular vesicles derived from human hepatoma cell line HepG2

status: Public on Jan 13 2025

city: Columbus

first: David

last: Brigstock

state: Ohio

address: 700 Children's Drive

institute: Nationwide Children's Hospital

type: Non-coding RNA profiling by high throughput sequencing

summary: Previously we have shown that HepG2 cell-derived extracellular vesicles (EVs) exhibit anti-fibrotic effects in human hepatic stellate cells in vitro and during toxin-induced liver injury in mice. The mechanisms by which HepG2 EVs ameliorate liver fibrosis have not been fully investigated but microRNAs are among the EV payload components that are delivered to recepient cells to regulate their functionsl properties. In this study, EVs were isolated by differential centrifugation of culture supernatants from HepG2 cells that ahd been maintained in serum-free conditions. The EVs were characterized by Nanosight Tracking Analysis and Western blot. Three different batches of HepG2 EVs were used to isolate total RNAs by QIAGEN miRNeasy kit, and approximately 200 ng of RNAs were subjected to small RNA-seq. A total of 205 miRNAs were identified, with miR-191-5p, miR-148a-3p, miR-320a, miR-423-5p, and miR-483-5p ranked as the top five most abundant miRNAs.

overall_design: HepG2 cells were cultured with serum-free medium for 48 hr for supernatant collection and EV isolation. Three batches of HepG2 EVs were used to isolate RNA, which was subjected to small RNA-seq.

first: Xinlei

last: Li

first: Ruju

last: Chen

first: Sherri

last: Kemper

first: Zhaohui

last: Xu


title: Multiplex, multimodal mapping of variant functional effects in secreted proteins

city: Seattle

first: Nicholas

middle: Anthony

last: Popp

email: poppn@uw.edu, poppn13@gmail.com

state: Washington

address: 3720 15th Avenue NE, S-320

department: Genome Sciences

institute: University of Washington School of Medicine

summary: Despite widespread advances in DNA sequencing in the past decade, the functional consequences of most rare genetic variants remain poorly understood, severely limiting our ability to connect variants to their consequences on protein function, identify biochemical mechanisms by which variation causes disease, and interpret variant pathogenicity. Multiplexed Assays of Variant Effect (MAVEs), which can measure the function of tens of thousands variants, are beginning to address this problem. However, existing MAVEs cannot be applied to the approximately 10% of human genes encoding secreted proteins, about a quarter of which are associated with disease. We developed a flexible and scalable human cell surface display method, Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP), that can simultaneously measure the functional effects of tens of thousands of variants in secreted proteins. We used MultiSTEP to study the consequences of missense variation in coagulation factor IX (FIX), a vitamin K-dependent plasma serine protease where variation can cause FIX deficiency and the bleeding disorder hemophilia B. We used a panel of antibodies to detect FIX secretion or FIX post-translational modification, measuring a total of 45,024 effects for 9,007 variants. 43.8% of all possible F9 missense variants impact FIX secretion, post-translational modification or both. We also identify new signals of functional constraint on secretion including within the signal peptide, folded domains, and for nearly all variants that caused gain or loss of cysteine. FIX secretion scores correlate strongly with FIX levels in patient plasma and also reveal that most F9 missense variants causing severe hemophilia do so by profoundly impacting secretion. We integrate the secretion and post-translational modification data to develop a F9 variant classifier that can identify loss of function variants with high specificity. We use the resulting classifications to reinterpret and upgrade 62 of 97 F9 variants of uncertain significance (VUS) in the MyLifeOurFuture hemophilia genotyping project to likely pathogenic. Lastly, we show that MultiSTEP can be applied to a wide variety of secreted proteins, ranging from small signaling proteins like insulin to large proteins like factor VIII. Thus, we establish a multiplexed, multimodal, and generalizable method for systematically assessing variant effects for secreted proteins at scale, paving the way for improved understanding of biochemical mechanisms of disease and clinical variant interpretation.

overall_design: Three barcoded single amino acid variant libraries of FIX variants spanning its entire sequence were recombined into an engineered, polyclonal Freestyle 293F line containing a lentivirally-introduced Bxb1 recombination site. FIX was engineered to be displayed on the surface of Freestyle 293F cells after recombination by C-terminal fusion of a flexible linker, eptiope tag, and single pass transmembrane domain. Cells were then stained with antibodies directed towards either FIX protein or the epitope tag and sorted using flow cytometry for Alexa Fluor 647 or Alexa Fluor 488 expression. Genomic DNA was then extracted from sorted cells, the barcodes were amplified by PCR, and the barcodes were sequenced by Illumina sequencing. Barcodes were associated back to corresponding FIX variants by comparison with a barcode-variant map created by sequencing the variant library plasmids using PacBio HiFi long-read sequencing.

first: Rachel

last: Powell

first: Melinda

middle: K

last: Wheelock

first: Brendan

last: Zapp

first: Kristen

last: Holmes

first: Kathryn

last: Sheldon

first: Shelley

middle: N

last: Fletcher

first: Xiaoping

last: Wu

first: Shawn

last: Fayer

first: Alan

middle: F

last: Rubin

first: Kerry

middle: W

last: Lannert

first: John

middle: P

last: Sheehan

first: Jill

last: Johnsen

first: Douglas

last: Fowler

first: Malvika

last: Tejura


title: Exploiting a metabolic vulnerability in brain tumour stem cells using a brain-penetrant drug with safe profile

city: Pessac

first: Ahmad

last: Sharanek

address: Allée Geoffroy St Hilaire

country: France

institute: BRIC INSERM U1312

summary: Glioblastoma (GB) remains one of the most treatment refractory and fatal tumour in humans. GB contains a population of self-renewing stem cells, the brain tumour stem cells (BTSC) that are highly resistant to therapy and are at the origin of tumour relapse. Here, we report, for the first time, that mubritinib potently impairs stemness and growth of patient-derived BTSCs harboring different oncogenic mutations. Mechanistically, by employing bioenergetic assays and rescue experiments, we provide compelling evidence that mubritinib acts on complex I of the electron transport chain to impair BTSC stemness pathways, self-renewal and proliferation. Global gene expression profiling revealed that mubritinib alters the proliferative, neural-progenitor-like, and the cell-cycling state signatures. We employed in vivo pharmacokinetic assays to establish that mubritinib crosses the blood-brain barrier. Using preclinical models of patient-derived and syngeneic murine orthotopic xenografts, we demonstrated that mubritinib delays GB tumourigenesis, and expands lifespan of animals. Interestingly, its combination with radiotherapy offers survival advantage to animals. Strikingly, thorough toxicological and behavioral studies revealed that mubritinib does not induce any damage to normal cells and has a well-tolerated and safe profile. Our work warrants further exploration of this drug in in-human clinical trials for better management of GB tumours.

overall_design: To investigate the effect in vitro of Mubritinib treatment on brain tumor stem cells. Three biological replicates by condition (Control, Mubritinib) were obtained.

first: Audrey

last: Burban

last: Charanek

first: Mathieu

last: Larroquette

first: Nathalie

last: Nicot

first: Barbara

last: Klink

first: Johanna

last: Galvis

first: Benjamin

last: Dartigues

first: Macha

last: Nikolski

first: Thomas

last: Daubon


title: A central role for disordered renal metabolism in myotonic dystrophy type 1

first: Preeti

last: Kumari

address: 13 street

department: Neurology

institute: Massachusetts General Hospital

summary: Chronic kidney disease (CKD) and the genetic disorder myotonic dystrophy type 1 (DM1) each are associated with progressive muscle wasting, whole-body insulin resistance, and impaired systemic metabolism.  However, CKD is undocumented in DM1 and the molecular pathogenesis driving DM1 is unknown to involve the kidney.  Here we use urinary extracellular vesicles (EVs), RNA sequencing, droplet digital PCR, and predictive modeling to identify downregulation of metabolism transcripts Phosphoenolpyruvate carboxykinase-1, 4-Hydroxyphenylpyruvate dioxygenase, Dihydropyrimidinase, Glutathione S-transferase alpha-1, Aminoacylase-1, and Electron transfer flavoprotein B in DM1.  Expression of these genes localizes to the kidney, especially the proximal tubule, and correlates with muscle strength and function.  In DM1 autopsy kidney tissue, characteristic ribonuclear inclusions are evident throughout the nephron.  We show that urinary organic acids and acylglycines are elevated in DM1, and correspond to enzyme deficits of downregulated genes.  Our study identifies a previously unrecognized site of DM1 molecular pathogenesis and highlights the potential of urinary EVs as biomarkers of renal and metabolic disturbance in these individuals.

overall_design: RNA-Seq profiling of urinary Extracellular RNA of Myotonic dystrophy and Duchenne Muscular Dystrophy

first: Lauren

last: Sullivan

first: Zhaozhi

last: Li

first: Parker

last: Conquest

first: Elizabeth

last: Cornforth

first: Rojashree

last: Jayakumar

first: Ningyan

last: Hu

first: Alexander

last: Sizemore

first: Brigham

last: Mckee

first: Robert

last: Kitchen

first: Paloma

last: González-Pérez

first: Constance

last: Linville

first: Karla

last: Castro

first: Hilda

last: Gutierrez

first: Soleil

last: Samaan

first: Elise

last: Townsend

first: Basil

last: Darras

first: Seward

last: Rutkove

first: Paula

last: Clemens

first: Araya

last: Puwanant

first: Sudeshna

last: Das

first: Thurman

last: Wheeler


title: Antibody-dependent DENV infection induces distinct transcriptomic responses in infected and bystander human macrophages

city: Syracuse

first: Adam

middle: Tully

last: Waickman

state: New York

address: 766 Irving Avenue

institute: SUNY Upstate Medical University

summary: Antibody-dependent enhancement has been thought to induce differential cellular responses to infection compared to conventional receptor mediated DENV entry. However, the precise mechanisms underlying these responses remain poorly understood. In this work we applied single cell RNA sequencing (scRNA seq) to characterize the transcriptomic profiles of human macrophages infected in enhancing, and non-enhancing infection conditions.

overall_design: Monocyte-derived macrophages were generated from the peripheral blood mononuclear cells of a healthy human donor. Cells were cultured in one of three experimental conditions: control, inoculated with DENV2 alone, or inoculated with DENV2 and monoclonal IgG known to induce enhancing conditions.

first: Céline

middle: S

last: Hardy


title: Transcripts identified by microarray analysis that were deregulated in  P0+18h cultures of murine ureters upon treatment with Cyclopamine.

city: Hannover

first: Mark-Oliver

last: Trowe

address: Carl-Neuberg-Str. 1

department: Molekularbiologie

country: Germany

institute: Medizinische Hochschule Hannover

summary: Evaluation of the transcriptional changes in murine P0+18h ureters upon treatment with Cyclopamine.

overall_design: Ureters of newborn NMRI pups (P0)  were dissected and cultured for 18h in medium containing 10 µM Cyclopamine or in medium without Cyclopamine. Total RNA from these ureters was analyzed by microarray analysis.

first: Anja

last: Beckers

first: Andreas

last: Kispert


title: Parasitic plasmids are anchored to inactive regions of eukaryotic chromosomes through a nucleosome signal [RNA-seq]

city: Paris

first: Axel

last: Cournac

address: 28 rue du docteur Roux

institute: Institut Pasteur

summary: Natural plasmids are common in prokaryotes, but few have been documented in eukaryotes. The natural 2µ plasmid present in the yeast Saccharomyces cerevisiae is one of these best-characterized exceptions. This highly stable genetic element has coexisted with its host for millions of years, faithfully segregating at each cell division through a mechanism that remains unclear. Using proximity ligation methods (such as Hi-C, Micro-C) to map the contacts between 2µ plasmid and yeast chromosomes under dozens of different biological conditions, we found that the plasmid is tethered preferentially to regions with low transcriptional activity, often corresponding to long, inactive genes. These contacts do not depend on common chromosome-structuring factors, such as members of the structural maintenance of chromosome complexes (SMC) but depend on a nucleosome-encoded signal associated with RNA Pol II depletion. They appear stable throughout the cell cycle and can be established within minutes. This chromosome hitchhiking strategy may extend beyond the 2µ plasmid/S. cerevisiae pair, as suggested by the binding pattern of the natural eukaryotic plasmid Ddp5 along silent chromosome regions of the amoeba Dictyostelium discoideum.

sample_organism:
Lachancea waltii
Lachancea fermentati

platform_organism:
Lachancea waltii
Lachancea fermentati

overall_design: We explored the anchoring of parasitic plasmids with main chromosomes by performing Hi-C on various hosts and in different conditions for S.cerevisiae.

first: Fabien

last: Girard

first: Romain

last: Koszul


title: Parasitic plasmids are anchored to inactive regions of eukaryotic chromosomes through a nucleosome signal [Hi-C]

sample_organism:
Lachancea waltii
Saccharomyces cerevisiae
Lachancea fermentati
Dictyostelium discoideum


title: Tumor microenvironment acidosis favors pancreatic cancer stem cell properties and in vivo metastasis

city: Copenhagen

first: Yifan

last: Dai

address: Ole Maaløes Vej 5

department: Department of Biology

country: Denmark

institute: Copenhagen University

summary: The acidic tumor microenvironment favors cancer aggressiveness via incompletely understood pathways. Here, we asked whether acidic environments select for cancer stem cell (CSC) properties. Bulk RNA-seq of Panc-1 human pancreatic cancer cells adapted to extracellular pH 6.5 revealed upregulation of CSC markers including CD44, EpCam, Nestin and aldehyde dehydrogenases, and CSC pathway enrichment. We therefore assessed CSC characteristics of acid-adapted (AA) and non-adapted (Ctrl) PaTu8988s and MiaPaca-2 pancreatic cancer cells. Compared to Ctrl, AA cells exhibited increased ALDH- and β-catenin activity and pancreatosphere-forming efficiency, classical CSC characteristics. Panc-1, PaTu8988s and MiaPaCa-2 AA cells differed in CSC marker expression, and AA cells did not exhibit typical flow cytometric CSC populations. However, single-nucleus sequencing identified the acid adaptation-induced emergence of a population with clear CSC characteristics. Finally, in an orthotopic mouse model, AA Panc-1 cells drove strongly increased aggressiveness and liver metastasis compared to Ctrl cells. We conclude that acid-adaptation of pancreatic cancer cells leads to enrichment of a CSC phenotype with unusual traits, providing new insight into how acidic tumor microenvironments favor cancer aggressiveness.

overall_design: Isogenic human Panc-1 cells were randomly split into control and acid adaptation groups with three replicates in each group. AA samples were cultured under acidic conditions (pH 6.7) for 4 weeks, while Ctrl samples were maintained at constant physiological pH of 7.6 for the same time. At the end of the adaptation, nuclei from each cell group were extracted and subjected to single cell RNA sequencing using the 10x Genomics snRNA-seq workflow. One sample in each group was harvested twice, resulting in two technical AA and technical Ctrl replicates.

first: Michala

middle: G

last: Rolver

first: Juan

middle: C

last: Roda

first: Mette

last: Flinck

first: Renata

last: Ialchina

first: Julie

last: Hindkær

first: Rigmor

last: Dyhr

first: August

last: Bodilsen

first: Nanditha

last: Prasad

first: Jonathan

last: Baldan

first: Jiayi

last: Yao

first: Albin

last: Sandelin

first: Luis

last: Arnes

first: Stine

last: Pedersen


title: The anti-DNA methylation mark histone H3 lysine 4 methylation recruits DNA demethylases in Arabidopsis [WGBS, BS-PCR-seq]

first: Zhenhui

last: Zhong

address: 610 Charles E Young Dr East

department: Department of Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

type: Methylation profiling by high throughput sequencing

summary: Patterning of DNA methylation in eukaryotic genomes is shaped by de novo methylation, maintenance mechanisms, and demethylation pathways, but how these processes interface with epigenetic modification of histones is poorly understood. We found that the targeting of histone H3 lysine four trimethylation (H3K4me3) with the catalytic domain of the SDG2 histone methyltransferase potently erased DNA methylation and gene silencing at FWA and other genes, and also erased CG DNA methylation in the transcribed region of genes.  This methylation erasure was completely blocked in the ros1 dml2 dml3 triple mutant which eliminates DNA demethylation enzymes, showing that H3K4me3 promotes the active removal of DNA methylation rather than preventing CG methylation maintenance.  We also found that targeting the removal of H3K4me3 with the novel protein TRBIP1, which acts in part by recruiting the H3K4me3 demethylase JMJ14, causes potent gene silencing.  Furthermore, using a CRISPR-based system to simultaneously target both TRBIP1 and a CG specific DNA methyltransferase, we observed more potent establishment of DNA methylation and gene silencing than targeting the DNA methyltransferase by itself.  These results highlight H3K4me3 as a potent anti-DNA methylation mark, and reveal potent and specific tools for epigenome engineering.

overall_design: To investigate the genes that can be upregulated by SDG2-ZF, RNA-seq of SDG2-ZF in both Col-0 background and rdd mutant background versus Col-0 and rdd mutant were performed. Chromatin immunoprecipitation sequencing (ChIP-seq) of myc-ROS1, Histone H3K4me3, H3K14ad, H2A.Z and ect was performed in the SDG2-ZF. Whole genome  bisulfite sequencing (WGBS)  was also performed to evaluate the genoe wide DNA methylation level of SDG2-ZF, TRBIP1-dCAS9-MQ1v, SunTag-TRBIP1-MQ1v, SunTag-StykC-TRBIP1-MQ1v and ect. Bifulfite PCR sequencing (BS-PCR-seq) was also performed to measure the DNA methylation level of Arabidopsis FWA gene promoter.

first: Ming

last: Wang

first: Steven

last: Jacobsen


title: Parasitic plasmids are anchored to inactive regions of eukaryotic chromosomes through a nucleosome signal [ChIP-seq]

first: Axel

last: Cournac

first: Fabien

last: Girard

first: Romain

last: Koszul


title: Parasitic plasmids are anchored to inactive regions of eukaryotic chromosomes through a nucleosome signal [shotgun_sequencing]

summary:
Natural plasmids are common in prokaryotes, but few have been documented in eukaryotes. The natural 2µ plasmid present in the yeast Saccharomyces cerevisiae is one of these best-characterized exceptions. This highly stable genetic element has coexisted with its host for millions of years, faithfully segregating at each cell division through a mechanism that remains unclear. Using proximity ligation methods (such as Hi-C, Micro-C) to map the contacts between 2µ plasmid and yeast chromosomes under dozens of different biological conditions, we found that the plasmid is tethered preferentially to regions with low transcriptional activity, often corresponding to long, inactive genes. These contacts do not depend on common chromosome-structuring factors, such as members of the structural maintenance of chromosome complexes (SMC) but depend on a nucleosome-encoded signal associated with RNA Pol II depletion. They appear stable throughout the cell cycle and can be established within minutes. This chromosome hitchhiking strategy may extend beyond the 2µ plasmid/S. cerevisiae pair, as suggested by the binding pattern of the natural eukaryotic plasmid Ddp5 along silent chromosome regions of the amoeba Dictyostelium discoideum.

This SuperSeries is composed of the SubSeries listed below.

platform_organism:
Lachancea waltii
Saccharomyces cerevisiae
Lachancea fermentati
Dictyostelium discoideum

overall_design: Refer to individual Series.

first: Agnès

last: Thierry

first: Léa

last: Meneu

first: Pauline

last: Larrous


title: Histone H3 lysine 4 methylation recruits DNA demethylases to enforce gene expression in Arabidopsis

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

overall_design: Refer to individual Series


title: The anti-DNA methylation mark histone H3 lysine 4 methylation recruits DNA demethylases in Arabidopsis [ChIP-seq]


title: Small RNAs in the testis and ovary of mice before and after sexual maturation

status: Public on Jan 12 2025

city: Chengdu

first: XingYu

state: Sichuan

address: No.211 Huimin Road

institute: Sichuan Agricultural University

summary: For a considerable period, reproductive health, fertility, and reproductive-related diseases have posed challenges to human well-being, as well as to the conservation of endangered species and the advancement of animal husbandry. PANDORA-seq, a recently introduced sequencing technique, demonstrates heightened sensitivity towards highly modified small RNAs like tsRNA and rsRNA. In this research endeavor, we leveraged PANDORA-seq to capture the small RNA expression profiles of mouse testes and ovaries pre- and post-sexual maturation. Our investigation successfully pinpointed an array of abundantly expressed small RNAs across various tissues, encompassing tsRNA, rsRNA, piRNA, miRNA, snoRNA, and ysRNA. Furthermore, we conducted detailed expression feature analyses specifically focusing on tsRNA, rsRNA, piRNA, and miRNA. These datasets stand poised to serve as invaluable resources for delving into the postnatal development of testes and ovaries, shedding light on the epigenetic mechanisms underpinning germ cell production and differentiation.

overall_design: This study utilized 12 mice (C57BL/6J strain, purchased from CHENGDUDASHUO). The mice were evenly divided into four groups based on birth time and gender: 3-week-old male mice, 4-week-old female mice, 10-week-old male mice, and female mice.To investigate the distribution patterns and dynamic changes of sncRNAs in the testes and ovaries of mice before and after sexual maturation, we performed PANDORA-seq analysis and annotated small RNA sequences using SPORTS1.1 software on testicular and ovarian tissues collected from pre- and post-sexually matured C57/BL6 mice.

first: Mailin

last: Gan

first: Xingyu

first: Jianfeng

last: Ma

first: Lei

last: Chen


title: The anti-DNA methylation mark histone H3 lysine 4 methylation recruits DNA demethylases in Arabidopsis [RNA-seq]


title: Murine sperm and liver tissue from Control and obese mice.

status: Public on Jan 11 2025

city: L'Hospitalet

first: Josep

last: Jimenez-Chillaron

state: Barcelona

address: c/ Feixa Llarga s/n, Pavello de Govern, 4a planta, Biophysics Unit

department: Physiological Sciences

institute: University of Barcelona

summary:
DNA methylation profile in sperm samples from 4-month-old Control mice (C), and mice that developed childhood obesisty through litter size reduction (SL).
DNA methylation profile in sperm of the first-generation offspring of C and SL mice. Sperm samples were colected at 4 months of age.
DNA methylation profile in the liver of the second-generation offspring of C and SL mice. Liver samples were collected at postnatalñ day 7.5.

overall_design:
Two condition experiment, Control (C) vs obese mice (SL) across 3 generations.

>>>Submitter states that raw data are not available due to file corruption.<<<

first: Flavio

last: Palmieri


title: Transcripts identified by microarray analysis that were deregulated in  P0+18h cultures of murine ureters upon treatment with IWP-2.

first: Mark-Oliver

last: Trowe

summary: Evaluation of the transcriptional changes in murine P0+18h ureters upon treatment with IWP-2.

overall_design: Ureters of newborn NMRI pups (P0)  were dissected and cultured for 18h in medium containing 5 µM IWP-2 or in medium without IWP-2. Total RNA from these ureters was analyzed by microarray analysis.

first: Anja

last: Beckers

first: Andreas

last: Kispert


title: Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 [microC]

status: Public on Jan 10 2025

city: Tianjin

first: Ke

last: Zhao

address: Qixiangtai Road, No.22

institute: Tianjin Medical University

summary: The majority of genetic loci linked to polygenic complex traits are found in non-coding regions of the human genome. These loci often exhibit complex gene regulatory relationships and linkage disequilibrium (LD) configurations, making it challenging to accurately identify causal variants and their target genes. We used multiplexed single-cell CRISPR interference and activation perturbations to investigate cis-regulatory element (CRE) and target gene expression relationships within tight LD in the endogenous chromatin context. We demonstrated the prevalence of multiple causality in perfect LD (pLD) for independent expression quantitative trait locus (eQTL) and uncovered fine-grained genetic effects on gene expression within pLD, which are difficult to decipher using traditional eQTL fine-mapping or existing computational methods. We found that over one third of the causal CREs lack classical epigenetic markers, and we functionally validated one of these hidden regulatory mechanisms. Leveraging Multiome single-cell epigenetic and sequence perturbations, we highlighted the regulatory plasticity of the human genome. Our study will guide the exploration of missing causal mechanisms in molecular trait formation and disease development.

overall_design: Nine scRNA-seq datasets under varying multiplicity-of-infection (MOI) conditions, three single-cell multiome datasets (ATAC + Gene Expression) from modified HAP1 clones(dCas9-KRAB-blast, dCAS9-VP64-blast and Cas9-blast stably expressed HAP1 clones), four ATAC-seq datasets from wild-type and CRISPR-edited HAP1 cells(64bp deletion, 17bp deletion and CRISPRi around rs73156934-tagged cis-regulate element(CRE)), six CUT&TAG histone modification datasets and 1 Micro-C dataset from wild-type HAP1 cells, and three 4C-seq datasets for validating CRE-gene regulatory relationships in HAP1 cells.

first: Yao

last: Zhou

first: Mulin

last: JunLi


title: Transcriptome response of the white-rot fungus Trametes versicolor to hybrid poplar exhibiting unique lignin chemistry

city: Charleston

first: Thomas

last: Canam

state: IL

address: 600 Lincoln Avenue, Life Sciences Building 2070

department: Biological Sciences

institute: Eastern Illinois University

summary: Background: Production of biofuels and bioproducts from lignocellulosic material is limited due to the complexity of the cell wall structure. This necessitates the use of physical, chemical, and/or physico-chemical pretreatment technologies, which adds significant capital, operational, and environmental costs. Biological pretreatment strategies have the potential to mitigate these expenses by harnessing the innate ability of specialized bacteria and fungi to deconstruct lignocellulose. White-rot fungi (e.g. Trametes versicolor) have been shown to be effective at biological pretreatment of lignocellulose, yet it was uncertain if these fungi are feedstock agnostic or are able to sense subtle changes in cell wall chemistry. Results: The present study examined the transcriptome response by Trametes versicolor to transgenic hybrid poplar (Populus tremula × alba) lines with altered syringyl (S) and guaiacyl (G) lignin. Specifically, the transcriptional response of the fungus to wild-type wood was compared to that from the wood of six transgenic lines within three lignin phenotypes, LSX (low S with hydroxy-G), LSHG (low S with high G), and HS (high S), with 350 transcripts showing significant differences among the samples. The transcriptome of T. versicolor varied according to the lignin phenotype of the wood, with the LSX wood resulting in the most substantial changes in T. versicolor transcript abundance. Specifically, the LSX wood led to 50 upregulated and 48 downregulated transcripts from WT at the 2-fold or greater threshold. For example, transcripts for the lignin peroxidases LiP3 and LiP10 were downregulated (approximately 12X and 31X lower, respectively) by the fungus on LSX wood compared to wild-type wood. LSX wood also resulted in approximately 11X lower transcript numbers of endo-β-1,4-glucanase yet led to an increase in expression of certain hemicellulases, further highlighting the altered deconstruction strategy by the fungus on this wood type. Conclusions: Overall, the results of this study demonstrated that T. versicolor was able to respond to transgenic poplar wood with the same genetic background, which has important implications for biological pretreatment strategies involving feedstocks that are genetically modified or have considerable natural variations in cell wall chemistry.

overall_design: Trametes versicolor grown on discs of wood from four different phenotypic categories (wild-type, high syringyl lignin, low syringyl with high guaiacyl, and low syringyl with hydroxy-guaiacyl). RNA extracted for RNA-Seq analysis after 3 weeks of growth.

first: Anbarah

last: Alzabaidi

first: Noor

last: Alabbasi

first: Richard

last: Meilan

first: Scott

last: Meiners


title: Disabling leading and lagging strand histone transmission results in parental histones loss and reduced cell plasticity and viability [repliATAC-seq]

last: Groth

address: Blegdamsvej 3B

institute: Novo Nordisk Foundation Center for Protein Research

summary: In the process of DNA replication, the first steps in restoring the chromatin landscape involve parental histone recycling and new histone deposition. Disrupting histone recycling to either the leading or lagging strand induces asymmetric histone inheritance, impacting epigenome maintenance and cellular identity. However, the order and kinetics of these effects remain elusive. Here, we employ inducible mutants to dissect the early and late consequences of impaired histone recycling. Simultaneous disruption of both leading (POLE4) and lagging strand (MCM2-2A) recycling pathways impairs transmission of parental histones to newly synthesized DNA, with release of some parental histones to the soluble pool. Subsequently, H3K27me3 accumulates aberrantly during chromatin restoration in a manner preceding gene expression changes. Loss of histone inheritance and the ensuing chromatin restoration defects alter gene expression in embryonic stem cells, challenges differentiation programs and cell viability. Our findings demonstrate the importance of efficient transmission of histone-based information during DNA replication for maintaining chromatin landscapes, differentiation potential, and cellular viability.

overall_design: repliATAC-seq POLE4-dTAG (#555) and POLE4-dTAG/MCM2-2A (#581) mouse embryonic stem cells (DMSO and dTAG treatment). Nascent chromatin samples were collected after 2 hours of DMSO or dTAG treatment.

first: Leonie

last: Kollenstart

first: Alva

last: Biran

first: Nicolas

last: Alcaraz

first: Nazaret

last: Reverón-Gómez

first: Victor

last: Solis-Mezarino

first: Moritz

last: Völker-Albert

first: Fion

last: Jenkinson

first: Valentin

last: Flury


title: Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1

last: Zhao


city: Lund

first: Johan

last: Staaf

address: Medicon Village

country: Sweden

institute: SCIBLU - Swegene Centre for Integrative Biology at Lund University

summary: DNA methylation profiling of ERpHER2n primary tumors (surgically resected) to target epigenetic regulation of FGFR4 in primary resectable (early-stage) ER-positive and HER2-negative tumors.

overall_design:
DNA methylation of tumor DNA was performed using Illumina MethylationEPIC V1 beadchips on 499 tumors according to manufacturer's instructions. No replicates.
Note from submitter:  Due to Swedish law we are not allowed to openly deposit (like in GEO) any data that can be considered personal patient information. The EPIC platform contains non CpGs, therefore we cannot deposit IDAT files.

last: Vallon-Christersson


title: Disabling leading and lagging strand histone transmission results in parental histones loss and reduced cell plasticity and viability [SCAR-seq]

first: Anja

overall_design: SCAR-seq (Sister Chromatids After Replication) measuring histone partition in nascent chromatin of H4K20me2, H4K20me0 in POLE4-dTAG (1 clone) and POLE4-dTAG/MCM2-2A (2 clones)  mouse embryonic stem cells (DMSO and dTAG treatment,  2 replicates for histone partition scores, includes stranded input controls).


title: Disabling leading and lagging strand histone transmission results in parental histones loss and reduced cell plasticity and viability [RNA-Seq]

overall_design: RNA-seq of POLE4-dTAG (1 clone) and POLE4-dTAG/MCM2-2A (1 clone) mouse embryonic stem cells (DMSO and dTAG treatment). Samples were taken after 24 hours of DMSO and dTAG treatment in self-renewing conditions (FBS+LIF, T0) and after addition of retinoic acid (-LIF, T12, T24, T60).


title: Disabling leading and lagging strand histone transmission results in parental histones loss and reduced cell plasticity and viability [ChOR-seq]

overall_design: ChOR-seq of POLE4-dTAG (1 clone, #555) and POLE4-dTAG/MCM2-2A (2 clones, #557 and #581) mouse embryonic stem cells (DMSO and dTAG treatment). Nascent chromatin samples were collected after 2 hours of DMSO or dTAG treatment.


title: Transcriptional changes upon overexpression of seven candidate miRNAs in human saphenous vein endothelial cells. [HSVEC]

city: Edinburgh

first: Julie

last: Rodor

address: 47 Little France Crescent

department: Centre for Cardiovascular Science

country: United Kingdom

institute: UNIVERSITY OF EDINBURGH

summary: Proliferation of vascular smooth muscle cells (vSMCs) following injury is a crucial factor contributing to pathological vascular remodelling. MicroRNAs (miRNAs) are powerful gene regulators and attractive therapeutics agents. Here, we aim to systemically identify and characterise miRNAs with therapeutic potential in targeting aberrant vSMC proliferation. We performed a high-throughput in vitro screen using a library of 2042 human miRNA-mimics for their impact on VSMC proliferation and identified seven novel antiproliferative miRNAs i.e miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b.Overexpression of these 7 miRNAs affects proliferation of vSMCs from different vascular beds. Focusing on vein graft failure, a condition in which miRNA based therapeutics can be applied to the graft ex-vivo, we showed that these miRNAs reduced human saphenous vein SMC (HSVSMC) proliferation without inducing apoptosis or senescence, and five of them also significantly decreased migration.In contrast to HSVSMC, miRNA overexpression on saphenous vein endothelial cells (ECs) led to no or lower decrease of proliferation for the seven miRNAs. We performed RNA-seq on HSVEC overexpressing the seven miRNAs and showed a limited response of ECs to the miRNA overexpression.

overall_design: After inducing quiescence by culturing the cells in 0.2% FBS for 12h, human saphenous vein endothelial cells were transfected with miRNA mimics of the 7 miRNA candidates for six hours (miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b or mimic Control) then cultured in 10% FBS for 48h to promote a proliferative phenotype. Three biological replicates were performed with each replicate corresponding to cells derived from different patients. RNA sequencing was performed on HSVSMCs treated with each mimic miRNA, a control mimic or untransfected cells.

first: Andrew

middle: H

last: Baker

first: Jean

last: Iyinikkel


title: Gene expression profile of skin-derived migratory DC subsets of papain-immunized mice

status: Public on Jan 09 2025

city: Newark

first: Yosuke

last: Kumamoto

state: NJ

address: 205 South Orange Ave

department: Center for Immunity and Inflammation

institute: Rutgers New Jersey Medical School

summary: CD301b+ DCs are requried for the differentiation of Th2 cells. Our data suggests that CD301b+ DCs express genes related to CD40 pathway at higher levels than other DC subsets. We isolated MHCIIhi CD11c+ migratory DCs from the skin draining LNs of naïve and papain-immunized mice and characterized their transcriptome.

overall_design: B6 mice were immunized with OVA + papain in both rear footpads. Popliteal lymph nodes were harvested from 1 day post-immunization or naïve mice. Cells were stained with CITE-seq antibodies and Live MHCIIhi CD11c+ B220- cells were isolated and used for further processing and sequencing.

first: Jihad

last: El-Fenej

first: Seema

last: Husain

first: Mainul

last: Hoque


title: Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2

status: Public on Jan 08 2025

city: Princeton

first: Yuri

last: Pritykin

address: Lewis-Sigler Institute for Integrative Genomics, Princeton University

institute: Princeton University

summary: We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA-seq profiling of C57BL/6 and 129S1/SvlmJ F1 cross cells and A549 cells to investigate the profile of several gRNA libraries.

first: Henri

last: Schmidt

first: Minsi

last: Zhang

first: Dimitar

last: Chakarov

first: Vineet

last: Bansal

first: Haralambos

last: Mourelatos

first: Francisco

last: Sánchez-Rivera

first: Scott

last: Lowe

first: Andrea

last: Ventura

first: Christina

last: Leslie


title: Disabling leading and lagging strand histone transmission results in parental histones loss and reduced cell plasticity and viability

first: Anja

last: Groth


title: Differential Impacts of CD8+ T-cells landscapes on Gastric Cancer Immune Microenvironment

city: Fukuoka

first: Naoki

last: Katayama

address: 3-1-1 Maidashi, Higashi-ku

institute: Kyushu University

summary: The present data suggest that the infiltration of inactive CD8+ T cells with low clonality is induced by chemotaxis in the HIGH group, possibly leading to poor prognosis of GC patients.

overall_design:
We performed scRNA-seq analysis from 34 gastric cancers and 7 adjacent normal gastric mucosae.

Please note that the re-analyzed samples included in the current study are listed in the Series data table below and the GC.CD8T.rds file contains their processed data.

first: Kenoki

last: Ohuchida


title: UFMylation maintains YAP stability to promote vascular endothelial cell senescence

city: hangzhou

first: yanan

last: liu

address: NO.2318, Yuhangtang Rd, Yuhang District, 311121, Hangzhou, Zhejiang, P.R.China

institute: Hangzhou Normal University

summary: Endothelial cell senescence is an accomplice for vascular aging, which leads to cardiovascular diseases (CVD). Evidences showed that Hippo- Yes-associated protein (YAP) signaling pathway plays an essential role in aging-associated CVD. However, the exact role of YAP protein in endothelial cell senescence remains not fully clear. Here, we reported that YAP was elevated in senescent human umbilical vein endothelial cells (HUVECs) and inhibition of YAP by either specific siRNAs or inhibitor Verteporfin could attenuate HUVECs senescence. In contrast, overexpression of YAP induces cell senescence. Besides, we found that UFMylation activity and YAP were both elevated in senescent cells. Mechanistically, we found that UFMylation, a newly identified ubiquitin-like modification with essential biological functions, maintains the stability of YAP and further found that YAP is a substrate for UFMylation. Importantly, we found that compound 8.5, an inhibitor of E1 of UFMylation, can attenuate cell senescence with reduced YAP protein and alleviate vascular aging and improve cardiac function in aged mice. Therefore, compound 8.5 may be a potential small molecule for delaying vascular aging. Together, our finding provides molecular mechanism by which UFMylation maintains YAP stability and exerts an important role in promoting cell senescence, and identified that a previously unrecognized UFMylation is a potential therapeutic target for anti-aging.

overall_design:
To investigate the downstream target genes of compound 8.5 in the arteries of aged  mice.
We have used RNA-Seq (sequencing) technology to compare the transcriptomic profiles of the aortas of aged mice and of aged mice treated with compound 8.5.

first: Yanan

last: Liu

first: Min

last: Zuo

first: Aiwei

last: Wu

first: Siting

last: Wang

first: Yongping

last: Bai

first: Junzhi

last: Zhou

first: Hu


title: Targeting signaling rewiring and resistant subpopulations in Philadelphia Chromosome-like Acute Lymphoblastic Leukemia [Bulk RNA-seq]

city: Philadelphia

first: Kai

last: Tan

state: PA

address: 3501 Civic Center Boulevard

department: Pediatrics

institute: Children's Hospital of Philadelphia

summary: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a common high-risk leukemia subtype defined by a kinase-activated gene expression pattern and chemoresistance, with low overall survival rates in both children and adults. Clinical responses of patients with Ph-like ALL to tyrosine kinase inhibitor (TKI)-based therapies are incomplete, suggesting partial oncogene addiction and highlighting the need to elucidate alternative biologic dependencies to cure patients. Herein, we report integrated bulk and single-cell multiomics studies of Ph-like ALL cells treated in vitro and in vivo with targeted agents to comprehensively define signaling adaptations and resistant subpopulations. Characterizing residual/resistant human ALL cells harvested from murine spleens revealed transcriptional regulatory network changes and kinase signaling adaptations. We identified key transcription factors including c-MYC and AP1 that mediate transcriptional rewiring associated with therapeutic escape, and demonstrate a specific MYC dependency in Ph-like ALL cells. We found a sub-population of leukemia blasts that have high senescence-associated stem cell-like features that is effectively eradicated only with combined STAT and BCL-2 inhibition. This is clinically significant since both conventional chemotherapy and ruxolitinib are ineffective against senescent stem-like blasts, which may mediate therapeutic tolerance and repopulate relapse. Our findings demonstrate that systems level discovery of key oncogenic dependencies and escape pathways can be exploited for co-targeting or sequential treatment to optimally eradicate blasts and cure this high-risk leukemia subtype.

overall_design: Bulk RNA-seq was performed on Ph-like ALL PDX models treated with control or ruxolitinib at 5 timepoints (3, 7, 28, 56 days).

first: Yang-Yang

last: Ding

first: Jonathan

last: Sussman

first: Sarah

last: Tasian


title: Chemically-defined and dynamic click hydrogels support hair cell differentiation in human inner ear organoids

city: Indianapolis

first: Matthew

last: Arkenberg

state: IN

address: 980 W Walnut St

department: Otolaryngology - Head and Neck Surgery

institute: Indiana University School of Medicine

summary: The mechanical properties in the inner ear microenvironment play a key role in its patterning during embryonic development. To recapitulate inner ear development in vitro, three-dimensional tissue engineering strategies including the application of representative tissue models and scaffolds are of increasing interest. Human inner ear organoids are a promising model to recapitulate developmental processes; however the current protocol requires Matrigel that contains ill-defined extracellular matrix components. Here, we implement an alternative, chemically-defined, dynamic hydrogel to support differentiation of human inner ear organoids. Specifically, thiol-norbornene and hydrazide-aldehyde click chemistries are used to fabricate inner ear organoid-laden, gelatin-based scaffolds. We identify optimal formulations to support hair cell development with comparable efficiency and fidelity to Matrigel-cultured organoids. These results suggest that the chemically-defined hydrogel may serve as a viable alternative to Matrigel for inner ear tissue engineering.

overall_design: Gene expression profiling of FACS sorted SOX2-ntdTomato+ cells from d40 inner ear organoids in Matrigel and dynamic GelNB-CH-based hydrogels

first: Mahboubeh

last: Jafarkhani

first: Chien-Chi

last: Lin

first: Eri

last: Hashino


title: Transcriptional changes upon overexpression of candidate miRNAs in human saphenous vein endothelial cells and human saphenous vein smooth muscle cells.

first: Julie

last: Rodor

overall_design: Refer to individual Series


title: Origins, diversity and clinical relevance of small intestinal  neuroendocrine tumors [methylation]

city: LYON

first: Benjamin

last: Gibert

address: 28 promenade Léa et Napoléon Bullukian

institute: CRCL – UMR 1052 / CNRS 5286

summary: Small intestine neuroendocrine tumors (siNETs) are rare bowel tumors arising from malignant enteroendocrine cells (EECs), which normally regulate digestion  throughout the intestine. Though infrequent, their incidence is rising through better diagnosis, fostering research into their origin and treatment. To date, siNETs are  considered to be a single entity and are clinically treated as such. Here, through the largest meta-omics analysis ever conducted on siNETs, we unveiled four distinct molecular  groups with strong clinical relevance and provide an invaluable resource to study their origin and clinical features. Genetic and DNA methylation profiles identified two groups  linked to distinct enteroendocrine differentiation patterns, another with a strong immune phenotype, and the last with mesenchymal properties. This latter subtype displayed the  worst prognosis and resistance to treatments. These data provide new insights into the origin and diversity of these rare diseases, in the hope of improving clinical research into their management.

overall_design: DNA methylation profiling of 200 siNETS samples using the Infinium MethylationEPIC BeadChip arrays (HM850K, Illumina)

first: Céline

last: Patte

first: Roxane

middle: M

last: Pommier

first: Anthony

last: Ferrari

first: Felicia

last: Chung

first: Maria

last: Ouzounova

first: Pauline

last: Moullé

first: Mathieu

last: Richaud

first: Rita

last: Khoueiry

first: Maëva

last: Hervieu

first: Silvia

last: Breusa

first: Nicolas

last: Rama

first: Laura

last: Gérard

first: Valérie

first: Gilles

last: Poncet

first: Vincent

last: Cahais

first: Anne-Sophie

last: Sertier

first: Anne

last: Boland

first: Delphine

last: Bacq-Daian

last: Ducarouge

first: Jean-François

last: Deleuze

first: Alain

last: Viari

first: Jean-Yves

last: Scoazec

first: Colette

last: Roche

first: Patrick

last: Mehlen

first: Thomas

last: Walter

first: Marion

last: Allio


title: Transcriptional changes upon overexpression of seven candidate miRNAs in human saphenous vein smooth muscle cells. [HSVSMC]

first: Julie

last: Rodor

summary: Proliferation of vascular smooth muscle cells (vSMCs) following injury is a crucial factor contributing to pathological vascular remodelling. MicroRNAs (miRNAs) are powerful gene regulators and attractive therapeutics agents. Here, we aim to systemically identify and characterise miRNAs with therapeutic potential in targeting aberrant vSMC proliferation. We performed a high-throughput in vitro screen using a library of 2042 human miRNA-mimics for their impact on VSMC proliferation and identified seven novel antiproliferative miRNAs i.e miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b.Overexpression of these 7 miRNAs affects proliferation of vSMCs from different vascular beds. Focusing on vein graft failure, a condition in which miRNA based therapeutics can be applied to the graft ex-vivo, we showed that these miRNAs reduced human saphenous vein SMC (HSVSMC) proliferation without inducing apoptosis or senescence, and five of them also significantly decreased migration.We performed RNA sequencing on HSVSMC overexpressing each of these 7 miRNAs. This analysis showed that each miRNA overexpression affects a core cell cycle gene network. However, this effect is mediated by distinct miRNA targets.

overall_design: After inducing quiescence by culturing the cells in 0.2% FBS for 48h, human saphenous vein smooth muscle cells were transfected with miRNA mimics of the 7 miRNA candidates for six hours (miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b or mimic Control) then cultured in the presence of IL1a and PDGF-BB for 48h to promote a pro-proliferative phenotype. Three biological replicates were performed with each replicate corresponding to cells derived from different patients. RNA sequencing was performed on HSVSMCs treated with each mimic miRNA, a control mimic or the transfection reagent only. We also performed RNA-seq on quiescent cells and cells treated with IL1a+ PDGF-BB.

first: Andrew

last: Baker

first: Jean

last: Iyinikkel


title: CA-125 as a Biomarker in Renal Medullary Carcinoma: Integrated Molecular Profiling, Functional Characterization, and Prospective Clinical Validation [RNA-Seq]

status: Public on Jan 07 2025

city: Houston

first: Amrit

last: Koirala

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: Purpose: Renal medullary carcinoma (RMC) is a highly aggressive malignancy defined by the loss of the SMARCB1 tumor suppressor. It mainly affects young individuals of African descent with sickle cell trait, and it is resistant to conventional therapies used for other renal cell carcinomas. This study aimed to identify potential biomarkers for early detection and disease monitoring of RMC. Experimental Design: Integrated profiling of primary untreated RMC tumor tissues and paired adjacent kidney controls was performed using RNA-sequencing (RNA-seq) and histone Chromatin Immunoprecipitation Sequencing (ChIP-seq). The expression of serum cancer antigen 125 (CA-125), was prospectively evaluated in 47 patients with RMC. Functional studies were conducted in RMC cell lines to assess the effects of SMARCB1 re-expression and MUC16 knockdown. Results: MUC16, encoding for CA-125, was identified as one of the top upregulated genes in RMC tissues, with concomitant enrichment of active histone marks H3K4me3 and H3K27ac at its promoter. Elevated serum CA-125 levels were found in 31 of 47 (66%) RMC patients and correlated significantly with metastatic tumor burden (p = 0.03). SMARCB1 re-expression significantly reduced MUC16 expression in RMC cell lines. Functional studies in RMC cell lines demonstrated that SMARCB1 re-expression significantly reduced MUC16 expression, and that MUC16 knockdown induced apoptosis and reduced cell proliferation. Conclusions: The correlation between serum CA-125 levels and metastatic burden suggests that CA-125 is a clinically relevant biomarker for RMC. These findings support further exploration of CA-125 for disease monitoring and targeted therapeutics in RMC.

overall_design: Cell lines derived from Renal medullary carcinoma (RMC) were transfected with either empty vector or SNF5 (SMARCB1) constructs and treated with doxycycline to express SNF5 (SMARCB1).

first: Pavlos

last: Msaouel

first: Cristian

last: Coarfa

first: Sandra

last: Grimm

first: Menuka

last: Karki


title: Interaction of bZIP transcription factor NRL with RNA-binding proteins implicates R-loops in photoreceptor gene regulation

city: Bethesda

first: Zachary

last: Batz

state: MD

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

summary: RNA-binding proteins (RBPs) perform diverse functions including the regulation of chromatin dynamics and the coupling of transcription with RNA processing. However, our understanding of their actions in mammalian neurons remains limited. Using affinity purification, yeast-two-hybrid and proximity ligation assays, we identified interactions of multiple RBPs with NRL, a Maf-family bZIP transcription factor critical for retinal rod photoreceptor development and function. In addition to splicing, many NRL-interacting RBPs are associated with R-loops, which form during transcription and increase during photoreceptor maturation. Focusing on DHX9 RNA helicase, we demonstrate that its expression is modulated by NRL and that the NRL-DHX9 interaction is positively influenced by R-loops. ssDRIP-Seq analysis reveals both stranded and unstranded R-loops at distinct genomic elements, characterized by active and inactive epigenetic signatures and enriched at neuronal genes. NRL binds to both types of R-loops, suggesting an epigenetically independent function. Our findings suggest additional functions of NRL during transcription and highlight complex interactions among transcription factors, RBPs, and R-loops in regulating photoreceptor gene expression in the mammalian retina.

overall_design: Single strand DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq) was performed in retinas from four adult mice (P28). Briefly, Genomic DNA from one retina was enzymatically digested, treated with RNase III with or without RNase H and immunoprecipitated with the DNA/RNA hybrid antibody S9.6.

first: Ximena

last: Corso-Díaz

first: Xulong

last: Liang

first: Kiam

last: Preston Jr

first: Bilguun

last: Tegshee

first: Milton

last: English

first: Jacob

last: Nellissery

first: Sharda

last: Yadav

first: Claire

last: Marchal

first: Anand

last: Swaroop


title: Loss of GRB2 results in nonreceptive endometrium and female infertility.

city: Columbia

first: Jae-Wook

last: Jeong

state: MO

address: 1030 Hitt Street

department: Obstetrics, Gynecology and Women's Health

institute: University of Missouri

summary: To understand the role of GRB2 in the uterus, we generated mice with conditional ablation of Grb2 in the PGR positive cells (Pgrcre/+Grb2f/f; Grb2d/d). Grb2d/d mice are infertile due to implantation failure. Although ovarian functions are normal, Grb2d/d mice had non-receptive endometrium due to dysregulation of progesterone and estrogen signaling. Our transcriptomic analysis identified deficiency of progesterone and FOXA2 signaling.

overall_design: We used RNA-sequencing to determine the differences in uterine gene expression resulting from deletion of endometrial Grb2.

first: Tae

last: Kim


title: The Colorado potato beetle gene expression atlas

city: Mainz

first: Shuqing

last: Xu

address: Hanns-Dieter-Hüsch-Weg 15

institute: University of Mainz

summary: The Colorado potato beetle (CPB) is a major pest of potato crops that has evolved resistance to more than 50 pesticides. For decades, CPB has been a model species for research on insecticide resistance, insect physiology, diapause, reproduction and evolution. Yet, the research progress in CPB is constrained by the lack of comprehensive genomic and transcriptomic information. Here, building on a recently established chromosome-level genome assembly, we built a gene expression atlas of the CPB using the transcriptomes of 61 samples representing major organs and developmental stages. By using both short and long reads, we improved the genome annotation and identified 6,623 more genes that were missed in previous annotations. We then established a web portal allowing the search and visualization of the gene expression atlas for the research community. The CPB atlas provides useful tools and comprehensive gene expression data, which will accelerate future research in both pest control and insect biology fields.

overall_design: We sequenced the transcriptomes of 12 tissues in adults (five from female, seven from male), five tissues in last instar larvae, as well as the entire body of each larval stage, and eggs.

first: Léonore

last: Wilhelm

first: Yangzi

last: Wang


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [bisulphite-seq]

status: Public on Jan 06 2025

city: Durham

first: Charles

last: Gersbach

state: North Carolina

address: 101 Science Dr.

department: Biomedical Engineering

institute: Duke University

summary: Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging
Stable polyclonal Tet1-dCas9 or VP64-dCas9-VP64 cell lines were established by transducing with lentivirus and selecting for transgene-expressing cassette with 1.5 ug/mL blasticidin for 5 days. Then, cells were transduced with gRNA lentivirus and selected for gRNA-expressing cassette with puromycin (1ug/mL) for 3 days.
For bisulphite sequencing of iPSCs and neurons, genomic DNA was extracted from cultured cells with the Qiagen DNEasy Blood and Tissue kit (Qiagen, 69504) or the Zymo QuickDNA Miniprep kit (Zymo D3024). gDNA was stored at -80C. 250ng of gDNA were input into the bisulphite conversion reaction. For bisulphite conversion and purification, we used the Zymo EZ DNA Methylation Gold kit as instructed (Zymo, D5005).
2uL of bisulphite-converted gDNA was used as input for PCR as described above, with an annealing temperature of 57C. PCR1 reactions were then cleaned with Ampure XP beads (Beckman, A63881) at a ratio of 1.8x. 1/10th of PCR1 was used as input for PCR2 with Q5 polymerase. PCR2 added i5 and i7 barcodes and P5 and P7 overhangs for dual-index Illumina short read sequencing. PCR2 products were purified with Ampure XP beads. Products were visualized via electrophoresis on an Agilent TapeStation 4200 and quantified with Qubit dsDNA HS assay kit (Invitrogen, Q32851) on a Qubit fluorometer (Invitrogen). Libraries were pooled and sequenced on an Illumina Miseq instrument with a Miseq v3 600 cycle kit (Illumina, MS-102-3003) with read lengths of 250x250.

Raw reads were trimmed using Trimmomatic to remove low quality reads and adapters with the following settings: HEADCROP: 25; ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10; TRAILING: 20; SLIDINGWINDOW:4:15; MINLEN:40
Bismark was used to create a bisulphite-converted reference genome from the hg19 genome build. Trimmed, paired-end reads were aligned to the genome (hg19) and analysed with Bismark version 0.22.3. The output coverage files were used to determine percentage of methylated cytosine for each CpG site in the amplicon.
Bismark coverage (cov) files were retrieved, and cov files were filtered for CpG sites located on chr15 within the target amplicon (24 CpG sites total) and compiled into a filtered methylation table for all samples. Methylation coverage and counts at CpG sites within the target amplicon were then compared between treatments.
Processed output files: methylation coverage tables for all samples in each group (combined Bismark .cov files)

first: Dahlia

last: Rohm


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [bisulphite-seq_neurons]

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging. Neurons were differentiated from iPSCs using an Ngn2 overexpression protocol.
Stable polyclonal gRNA cassette-expressing cell lines were established by transducing with lentivirus and selecting for transgene-expressing cassette with 1ug/mL puromycin for 3 days. Then, cells were nucleofected with Tet1v4 plasmid and enriched for nucleofected cells using cell sorting on reporter Thy1.1. iPSCs were differentiated into neurons 30 days post-nucleofection.
For bisulphite sequencing of iPSCs and neurons, genomic DNA was extracted from cultured cells with the Qiagen DNEasy Blood and Tissue kit (Qiagen, 69504) or the Zymo QuickDNA Miniprep kit (Zymo D3024). gDNA was stored at -80C. 250ng of gDNA were input into the bisulphite conversion reaction. For bisulphite conversion and purification, we used the Zymo EZ DNA Methylation Gold kit as instructed (Zymo, D5005).
2uL of bisulphite-converted gDNA was used as input for PCR as described above, with an annealing temperature of 57C. PCR1 reactions were then cleaned with Ampure XP beads (Beckman, A63881) at a ratio of 1.8x. 1/10th of PCR1 was used as input for PCR2 with Q5 polymerase. PCR2 added i5 and i7 barcodes and P5 and P7 overhangs for dual-index Illumina short read sequencing. PCR2 products were purified with Ampure XP beads. Products were visualized via electrophoresis on an Agilent TapeStation 4200 and quantified with Qubit dsDNA HS assay kit (Invitrogen, Q32851) on a Qubit fluorometer (Invitrogen). Libraries were pooled and sequenced on an Illumina Miseq instrument with a Miseq v3 600 cycle kit (Illumina, MS-102-3003) with read lengths of 250x250.

Raw reads were trimmed using Trimmomatic to remove low quality reads and adapters with the following settings: HEADCROP: 25; ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10; TRAILING: 20; SLIDINGWINDOW:4:15; MINLEN:40
Bismark was used to create a bisulphite-converted reference genome from the hg19 genome build. Trimmed, paired-end reads were aligned to the genome (hg19) and analysed with Bismark version 0.22.3. The output coverage files were used to determine percentage of methylated cytosine for each CpG site in the amplicon.
Bismark coverage (cov) files were retrieved, and cov files were filtered for CpG sites located on chr15 within the target amplicon (24 CpG sites total) and compiled into a filtered methylation table for all samples. Methylation coverage and counts at CpG sites within the target amplicon were then compared between treatments.
Processed output files: methylation coverage tables for all samples in each group (combined Bismark .cov files)


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [VP64-sublib]

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging
The screen was performed in triplicate with independent transductions. For each replicate, 1.7 x 106 matSNRPN-2A-GFP Tet1cdCas9 or VP64dCas9VP64 iPSCs were dissociated using Accutase (Stemcell Tech, 7920) and transduced in suspension in a matrigel-coated 10-cm dish in mTesR (Stemcell Tech 85850) supplemented with 10 μM Rock Inhibitor (Y-27632, Stemcell Tech, 72304). Cells were transduced at a MOI of 0.2 to obtain one gRNA per cell and ~580-fold coverage of the gRNA sub-library. The medium was changed to fresh mTesR without Rock Inhibitor 18-20 h after transduction. Antibiotic selection was started 30 h after transduction by adding 1 μg/mL puromycin (Sigma, P8833) directly to the plates without changing the medium. The cells were fed daily and passaged as necessary maintaining library coverage until harvest.
Cells were harvested for sorting 14 d after transduction of the gRNA sub-library. Cells were washed once with 1x PBS, dissociated using Accutase, filtered through a 30 μm CellTrics filter (Sysmex, 04-004-2326) and resuspended in FACS Buffer (0.5% BSA (Sigma, A7906), 2 mM EDTA (Sigma, E7889) in PBS). The highest and lowest 10% of cells were sorted based on GFP expression and 0.4 x 106 cells were sorted into each bin. Sorting was done with a SH800 FACS Cell Sorter (Sony Biotechnology).
After sorting, genomic DNA was harvested with the DNeasy Blood and Tissue Kit (Qiagen, 69506). The gRNA libraries were amplified from each gDNA sample across 100 μL PCR reactions using Q5 hot start polymerase (NEB, M0493) with 1 μg of gDNA per reaction. The PCR amplification was done according to the manufacturer’s instructions, using 25 cycles at an annealing temperature of 60 °C with the following primers 18:
Fwd: 5′-AATGATACGGCGACCACCGAGATCTACACAATTTCTTGGGTAGTTTGCAGTT
Rev: 5′-CAAGCAGAAGACGGCATACGAGAT-(6-bp index sequence)- GACTCGGTGCCACTTTTTCAA
The amplified libraries were purified with Agencourt AMPure XP beads (Beckman Coulter, A63881) using double size selection of 0.65× and then 1× the original volume. Each sample was quantified after purification with the Qubit dsDNA High Sensitivity assay kit (Thermo Fisher, Q32854). Samples were pooled and sequenced on a MiSeq (Illumina) with 21-bp paired-end sequencing using the following custom read and index primers:
Read1: 5′-GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG
Index: 5′-GCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC
Read2: 5’ - GTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAAC

FASTQ files were aligned to custom indexes (generated from the bowtie2-build function) using Bowtie 2. Counts for each gRNA were extracted and used for further analysis. All enrichment analysis was done with R. Individual gRNA enrichment was determined using the DESeq2 package to compare between high and low conditions for each screen.
Differentially-enriched gRNAs were gRNAs were identified via DESeq2 using an adjusted p-value cutoff of 0.05.


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [ATAC-seq]

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging.
Stable polyclonal Tet1-dCas9 or VP64-dCas9-VP64 cell lines were established by transducing with lentivirus and selecting for transgene-expressing cassette with 1.5 ug/mL blasticidin for 5 days. Then, cells were transduced with gRNA lentivirus and selected for gRNA-expressing cassette with puromycin (1ug/mL) for 3 days.
14 days after gRNA virus transduction, cells were harvested with Accutase, pelleted, and the ATAC-seq protocol from Corces et al. 2017 was followed with 45,000 cells per sample with the following alterations: KAPA polymerase (Roche, KK2101) was used in the PCR step, following reaction condition recommendations for KAPA polymerase. Libraries were sequenced with 2x25 paired-end reads with a coverage of 50 million reads per sample.
ATAC libraries were prepared according to Corces et al. 2017 Omni-ATAC protocol, with KAPA polymerase as the polymerase used for library PCR.

Raw reads were trimmed using Trimmomatic to remove adapters.
Trimmed reads were aligned to hg19 genome using Bowtie2. Reads mapping to ENCODE blacklisted regions were removed, and duplicated reads were removed with Picard MarkDuplicates. Peaks were called using MACS2. bamCoverage was used to generate rpkm-normalized bigwig files from deduplicated bam files for visualization.
Union peak set was generated from narrowPeak files of all samples for each effector (VP64 or Tet1). Counts files were generated using the featureCounts function.
Differential peak analysis was determined with DESeq2 on the feature counts files, where gene counts are fitted into negative binomial generalized linear models (GLMs) and Wald statistics to determine significantly differentially expressed genes, with an adjusted p-value threshold of p<0.01.


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [VP64screen]

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging.
Each screen was performed in triplicate with independent transductions. For each replicate, 24 x 106  SNRPN-2A-GFP VP64dCas9VP64 (maternal) or dCas9KRAB (paternal) iPSCs were dissociated using Accutase (Stemcell Tech, 7920) and transduced in suspension across five matrigel-coated 15-cm dishes in mTesR (Stemcell Tech 85850) supplemented with 10 μM Rock Inhibitor (Y-27632, Stemcell Tech, 72304). Cells were transduced at a MOI of 0.2 to obtain one gRNA per cell and ~430-fold coverage of the gRNA library. The medium was changed to fresh mTesR without Rock Inhibitor 18-20 h after transduction. Antibiotic selection was started 30 h after transduction by adding 1 μg/mL puromycin (Sigma, P8833) directly to the plates without changing the medium. The cells were fed daily and passaged as necessary maintaining library coverage until harvest. Cells were harvested for sorting 9 d after transduction of the gRNA library for all screens.
Cells were washed once with 1x PBS, dissociated using Accutase, filtered through a 30 μm CellTrics filter (Sysmex, 04-004-2326) and resuspended in FACS Buffer (0.5% BSA (Sigma, A7906), 2 mM EDTA (Sigma, E7889) in PBS). Before sorting, an aliquot of 4.8 x 106 cells were taken to represent a bulk unsorted population. The highest and lowest 10% of cells were sorted based on GFP expression and 4.8 x 106 cells were sorted into each bin. Sorting was done with a SH800 FACS Cell Sorter (Sony Biotechnology).

FASTQ files were aligned to custom indexes (generated from the bowtie2-build function) using Bowtie 2. Counts for each gRNA were extracted and used for further analysis. All enrichment analysis was done with R. Individual gRNA enrichment was determined using the DESeq2 package to compare between high and low conditions for each screen. Only forward read (read 1) of paired-end sequenced sequencing files was used for data processing and enrichment analysis.
Differentially-enriched gRNAs were gRNAs were identified via DESeq2 using an adjusted p-value cutoff of 0.05.


title: Sox10 is required for systemic initiation of bone mineralization

city: Cincinnati

first: Lindsey

last: Barske

address: 3333 Burnet Ave

department: Human Genetics

institute: Cincinnati Children's Hospital

summary: Heterozygous variants in SOX10 cause syndromes affecting pigmentation, digestion, hearing, and neural development, primarily attributable to failed differentiation or loss of non-skeletal neural crest derivatives. We report here an additional novel requirement for Sox10 in bone mineralization. Neither crest- nor mesoderm-derived bones initiate mineralization on time in zebrafish sox10 mutants, despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment, resulting in severe calcium deficiency. As these ionocytes derive from ectoderm, not crest, we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNAseq revealed significantly elevated stanniocalcin (Stc1a), an anti-hypercalcemic hormone, in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros-derived glands that synthesize Stc1a interact with sox10+ cells, but these cells are missing in mutants. We conclude that sox10+ crest-derived cells non-autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization.

overall_design: Bulk RNA-seq was performed on pooled whole-body zebrafish wild-type and sox10 mutant larvae at 45 hpf, 4 dpf and 7 dpf.  Total of 18 samples. 45 hpf (3 replicates: controls n = 10, 20 & 25; mutants n = 10,15 & 15 embryos), 4 dpf (3 replicates: controls n = 10, 15 & 12; mutants n = 10,11 & 12 embryos) and 7 dpf (3 replicates: controls n = 12, 30 & 18; mutants = 18, 12 & 7 embryos)

first: Stefani

last: Gjorcheska

first: Sandhya

last: Paudel

first: Sarah

last: McLeod

first: David

last: Paulding

first: Louisa

last: Snape

first: Karen

last: Camargo Sosa

first: Cunming

last: Duan

first: Robert

last: Kelsh


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [CUT&Run]

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging.
Stable polyclonal Tet1-dCas9 or VP64-dCas9-VP64 cell lines were established by transducing with lentivirus and selecting for transgene-expressing cassette with 1.5 ug/mL blasticidin for 5 days. Then, cells were transduced with gRNA lentivirus and selected for gRNA-expressing cassette with puromycin (1ug/mL) for 3 days.
2 weeks after gRNA virus transduction, cells were harvested with Accutase, and cell concentration and cell number were assessed with a Countess II cell counter (Invitrogen) and Trypan Blue (Invitrogen).
Library preparation was completed using the CUT&RUN Library Prep Kit with primer set 1 (Epicypher 14-1001). Libraries were sequenced on an Illumina Nextseq 2000 with a coverage of at least 5 million reads per sample.

Sequencing data were processed using CUT&RUNTools 2.0, and peaks for H3K4me3 signal were annotated using MACS2 narrow peak calling.
Data were visualised in a genome viewer as cpm-normalized bigwig files.

Genome: hg19
processed file: cpm-normalized bigwig


title: A soil-grown wheat root atlas with validated cross-species cluster annotations allows to delineate conserved tissue specific marker genes [Spatial Transcriptomics]

city: Ghent

first: Bert

last: De Rybel

address: Technologiepark 71

department: Center for Plant Systems Biology

country: Belgium

institute: VIB

summary: Despite the broad use of single-cell and single-nucleus RNA sequencing in plant research, accurate cluster annotation in less studied plant species remains a major challenge due to the lack of validated marker genes. Here, using soil-grown wheat roots as a model, we generated a single-cell RNA-sequencing (scRNA-seq) atlas and annotated cluster identities in an unbiased way by transferring existing annotations from publicly available datasets in wheat, rice, maize and Arabidopsis. These cross-species orthology-based predictions were next validated using untargeted spatial transcriptomics. This information refined existing cluster annotations for different datasets across key plant model species. We then used the validated clusters to generate cell type-specific gene regulatory networks for root tissues of wheat and two other monocot crop species. By integrating all available data, including homeolog expression in wheat, we predicted reliable tissue-specific markers which are conserved across different species. In summary, we provided an annotated and validated single cell transcriptomic resource for soil-grown wheat root apical meristems and revealed conserved cell type-specific regulators and markers across species. These data expand upon previous root single cell atlas resources in crops, and will facilitate cell type annotation in non-model plant species in the future.

overall_design: Stereo-seq profiling was performed on a root cap region and regions more distal in soil-grown wheat roots.

first: Yuji

last: Ke

first: Vincent

last: Pujol

first: Jasper

last: Staut

first: Lotte

last: Pollaris

first: Ruth

last: Seurinck

first: Thomas

last: Eekhout

first: Marnik

last: Vuylsteke

first: Maria

last: Saura-Sanchez

first: Carolin

last: Grones

first: Michiel

last: Van Bel

first: Andrea

last: Ariani

first: Christophe

last: Liseron-Monfils

first: Klaas

last: Vandepoele

first: Yvan

last: Saeys


title: A soil-grown wheat root atlas with validated cross-species cluster annotations allows to delineate conserved tissue specific marker genes

overall_design: Single-cell RNA-seq experiments were performed on protoplasts extracted from soil-grown wheat roots.

middle: T


title: CA-125 as a Biomarker in Renal Medullary Carcinoma: Integrated Molecular Profiling, Functional Characterization, and Prospective Clinical Validation

first: Amrit

last: Koirala

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


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [Tet1]

first: Charles

last: Gersbach

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging
The screen was performed in triplicate with independent transductions. For each replicate, 1.7 x 106 matSNRPN-2A-GFP Tet1cdCas9 or VP64dCas9VP64 iPSCs were dissociated using Accutase (Stemcell Tech, 7920) and transduced in suspension in a matrigel-coated 10-cm dish in mTesR (Stemcell Tech 85850) supplemented with 10 μM Rock Inhibitor (Y-27632, Stemcell Tech, 72304). Cells were transduced at a MOI of 0.2 to obtain one gRNA per cell and ~580-fold coverage of the gRNA sub-library. The medium was changed to fresh mTesR without Rock Inhibitor 18-20 h after transduction. Antibiotic selection was started 30 h after transduction by adding 1 μg/mL puromycin (Sigma, P8833) directly to the plates without changing the medium. The cells were fed daily and passaged as necessary maintaining library coverage until harvest.
Cells were harvested for sorting 14 d after transduction of the gRNA sub-library. Cells were washed once with 1x PBS, dissociated using Accutase, filtered through a 30 μm CellTrics filter (Sysmex, 04-004-2326) and resuspended in FACS Buffer (0.5% BSA (Sigma, A7906), 2 mM EDTA (Sigma, E7889) in PBS). The highest and lowest 10% of cells were sorted based on GFP expression and 0.4 x 106 cells were sorted into each bin. Sorting was done with a SH800 FACS Cell Sorter (Sony Biotechnology).
After sorting, genomic DNA was harvested with the DNeasy Blood and Tissue Kit (Qiagen, 69506). The gRNA libraries were amplified from each gDNA sample across 100 μL PCR reactions using Q5 hot start polymerase (NEB, M0493) with 1 μg of gDNA per reaction. The PCR amplification was done according to the manufacturer’s instructions, using 25 cycles at an annealing temperature of 60 °C with the following primers 18:
Fwd: 5′-AATGATACGGCGACCACCGAGATCTACACAATTTCTTGGGTAGTTTGCAGTT
Rev: 5′-CAAGCAGAAGACGGCATACGAGAT-(6-bp index sequence)- GACTCGGTGCCACTTTTTCAA
The amplified libraries were purified with Agencourt AMPure XP beads (Beckman Coulter, A63881) using double size selection of 0.65× and then 1× the original volume. Each sample was quantified after purification with the Qubit dsDNA High Sensitivity assay kit (Thermo Fisher, Q32854). Samples were pooled and sequenced on a MiSeq (Illumina) with 21-bp paired-end sequencing using the following custom read and index primers:
Read1: 5′-GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG
Index: 5′-GCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC
Read2: 5’ - GTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAAC

FASTQ files were aligned to custom indexes (generated from the bowtie2-build function) using Bowtie 2. Counts for each gRNA were extracted and used for further analysis. All enrichment analysis was done with R. Individual gRNA enrichment was determined using the DESeq2 package to compare between high and low conditions for each screen.
Differentially-enriched gRNAs were identified via DESeq2 using an adjusted p-value cutoff of 0.05.

first: Dahlia

last: Rohm

middle: A