title: Transcriptome analysis of tumors from genetically engineered mouse models carrying Cbfb and/or Pik3ca mutations

city: Bethesda

first: Jing

last: Huang

state: MD

institute: National Cancer Institute

summary: Tumors harvested from mice carrying positive MMTV-Cre transgene, homozygously or heterozygously deleted or wild type Cbfb allele, and heterozygous Pik3ca transgene were subject to RNA extraction. Extracted RNA with a RIN larger than 7 was subject to RNAseq. to generate the mice, breeder pairs of Gt(ROSA)26Sortm1(Pik3ca*H1047R)Egan/J (Stock No: 016977), Cbfbtm2.1Ddg/J (Stock No: 028550), and Tg(MMTV-Cre)4Mam/J (Stock No: 003553) strains were purchased from Jackson Laboratory. MMTV-Cre mice lines were genotyped using real time PCR [forward primer (FP): 5’-CCGGTTATTCAACTTGCACC-3' and reverse primer (RP) 5’-CTGCATTACCGGTCGATGCAAC- 3']. CBFB deletion was confirmed using real-time PCR with primers: FP: 5’-GCGCGCCAGTCACTTGTT-3' and RP: 5'- ATCCCACGAACCGAACCA-3'. Pik3ca mice were genotyped using primers; FP: 5'- AAAGTCGCTCTGAGTTGTTAT-3', RP1: 5'- GCGAAGAGTTTGTCCTCAACC -3' for mutant Pik3ca and RP2: 5'- GGAGCGGGAGAAATGGATATG -3' for wild type Pik3ca.

overall_design: Each RNAseq sample was from a tumor derived from a unique mouse. Bulk RNAseq was performed using RNA extracted from the tumor.

first: Maxwell

last: Lee


title: Rewiring of gene interactions during development of serially homologous appendages in male and female Drosophila

status: Public on Oct 21 2024

city: Durham

first: Javier

last: Figueras Jimenez

address: South Road

department: Biosciences

country: United Kingdom

institute: Durham University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Arthropod genitalia and walking legs are serially homologous appendages derived from a ventral appendage ‘ground state’ shaped by different Hox inputs. However, there has been little comparison between the downstream gene regulatory networks and how they build and pattern serially homologous appendages differently. In the pre-tarsal region of the developing leg and antennae of Drosophila, a combination of the transcription factors C15, Lim1 homeobox 1 (Lim1), and Al (Aristaless) are required for the development of the terminal structures, the tarsal claws and aristae, respectively, but the role and interactions among these factors as well as other genes known to regulate leg development was not known in the developing genitalia. Here, we explored the expression and function of C15, Lim1, and Al in male and female terminalia (genitalia and analia) development. We found that C15 represses male clasper bristle development but is required for promoting female epiproct bristles. Unlike in the antennae and leg discs, C15, Lim1, and Al are never all co-expressed in any male or female terminal structures, evidencing the interactions among these factors have changed among serially homologous appendages. However, we did infer interactions between C15 and other factors, including bab2 and Dll reflecting similarities between leg and genital development consistent with their homology. Finally, we identified a male-specific clasper enhancer of C15, probably regulated by the Hox gene Abdominal-B, which is not active in the female terminalia or the legs or antennae. This enhancer modularity of C15 may underpin tissue-specific regulatory logic, presumably programmed by different Hox inputs such as Abd-B in the genitalia, and that could have contributed to the diversification of these serially homologous structures from the ‘ground state’ ventral appendage as well as the rapid evolution of terminal morphology among species.

overall_design: Assay for Transposase-Accessible Chromatin (ATAC-seq) data was prepared from prepupae that were collected from D. melanogaster (Oregon R) and aged until 30 hAPF.

first: Amber

middle: M

last: Ridgway

first: Maria D

middle: S

last: Nunes

first: Alistair

middle: P

last: McGregor


title: Genetic Insights into the Rheological Strength of Candida albicans Biofilms

city: ANN ARBOR

first: Anuj

last: Kumar

state: MI

address: 1105 N. University Avenue, BSB Room 3210

department: MCDB

country: USA

institute: University of Michigan

type: Expression profiling by high throughput sequencing

summary: The principal opportunistic human fungal pathogen Candida albicans forms biofilms resistant to antifungal therapeutics. Biofilms are a class of soft matter with viscoelastic properties and response to flow, but little is known regarding the genes contributing to these rheological phenotypes in fungal biofilms. Here, we identify C. albicans genes with deletion phenotypes of altered biofilm viscoelasticity. We analyzed mutants deleted for genes contributing to cell wall structure or extracellular matrix (ECM) production, and we identified increased elastic moduli, indicative of higher viscoelasticity, in strains singly deleted for PMR1, KRE5, and ALG11. PMR1 encodes a secretory pathway calcium pump. KRE5 encodes a UDP-glucose:glycoprotein glucosyltransferase, and ALG11 encodes alpha-1,2-mannosyltransferase. These mutants form less biofilm ECM by weight relative to wild type when cultured on agar. For these strains, biofilm morphology is smooth, with reduced hyphal formation. The mutants exhibit decreased resistance to the antifungal agent fluconazole relative to wild type biofilm cultures. To identify intracellular changes underlying these altered rheological properties, we globally profiled transcript levels in the respective mutants. Genes encoding membrane proteins were enriched in the set of transcripts differentially abundant in the alg11 deletion mutant. RNA levels are altered for genes associated with translation in the pmr1 deletion mutant and protein catabolism in the kre5 deletion strain. Genes involved in lipid metabolism and filamentous development are differentially expressed in cells from alg11, kre5, and pmr1 deletion mutant biofilms. Collectively, the data indicate C. albicans biofilm rheology as a phenotype affected by ECM production and cell morphology, while identifying genes for the investigation of mechanisms underlying properties of fungal biofilm viscoelasticity.

overall_design: Wild-type and deletion mutant biofilms were grown from overnight cultures on polycarbonate membranes (nucleopore 0.8 micrometer pore size) placed on solid nutrient-limited Spider medium supplemented with uridine for seven days at 37 degrees C. Cell pellets were collected by centrifugation, and RNA was extracted from each sample using the RiboPure RNA purification kit according to suggested protocols and published methods. Approximately 10 to the seventh cells were lysed by agitation with cold Zirconia beads in a buffer containing SDS (1% final volume) and phenol:chloroform:isoamyl alcohol. The RNA was purified by ethanol washes and filtration using cartridges in the RiboPure kit. Prior to sequencing, approximately 3 micrograms of each RNA sample was treated with DNase I. The reaction was terminated with EDTA and incubation at 65 degrees C. The samples were subsequently precipitated with ethanol and washed with 70% cold ethanol. For sequencing, messenger RNA was purified from total RNA using poly-T oligonucleotide-attached magnetic beads. cDNA was generated using random hexamer primers for first-strand synthesis, and dUTP-priming for second-strand synthesis. The resulting cDNA library was quantified using fluorometry and real-time PCR. The size distribution of the synthesized cDNA was assessed using a bioanalyzer.

first: Joshua

last: Bauman


title: Conditions for the co-existence of promoter and gene-body condensates

city: Mainz

first: Jan

last: Padeken

address: Ackermannweg 4

country: Germany

institute: Institute of Molecular Biology

summary: In cells, transcription is tightly regulated on multiple layers. The condensation of the transcription machinery into distinct phases is hypothesized to spatio-temporally fine tune RNA polymerase II behaviour during two key stages, transcription initiation and the elongation of the nascent RNA transcripts. However, it has remained unclear whether these phases would mix when present at the same time or remain distinct chemical environments; either as multi-phase condensates or by forming entirely separate condensates. Here we combine particle-based multi-scale simulations and experiments in the model organism C. elegans to characterise the biophysical properties of RNA polymerase II condensates. Both simulations and the in vivo work describe a lower critical solution temperature (LCST) behaviour of RNA Polymerase II, with condensates dissolving at lower temperatures whereas higher temperatures promote condensate stability, which highlights that these condensates are physio-chemically distinct from heterochromatin condensates. The LCST behavior of CTD correlates with gradual shifts in the transcription program but is largely uncoupled from the classical stress response. Expanding the simulations we model how the degree of phosphorylation of the disordered C-terminal domain of RNA polymerase II (CTD), which is characteristic for each step of transcription, controls the existence and morphology of multi-phasic condensates. We show that the two phases putatively underpinning the initiation of transcription and transcription elongation constitute distinct chemical environments and are in agreement with RNA polymerase II condensates observed in C. elegans embryos by super resolution microscopy. Our analysis shows how depending on its post transcriptional modifications and its interaction partner a single protein can form multiple partially engulfed condensates, potentially promoting the selective recruitment of additional factors to these two phases.

overall_design: RNA was extracted from early wild-type (N2 Bristol) C. elegans embryos from a synchronized culture grown at 20°C and exposed to indicated temperatures for 2 h, using Trizol. Embryos were freeze cracked five times, then RNA was extracted with choloroform followed by isopropanol precipitation. Further purification was performed with the RNA Clean and Concentrator kit (Zymo). Libraries were produced using the Stranded Total RNA Prep Ligation with Ribo-Zero Plus Kit (Illumina). rRNA was depleted using the Ribo-Zero Plus kit supplemented with custom made C. elegans specific rDNA oligos (IDT). Libraries were profiled in a DNA 1000 Chip on a 2100 Bioanalyser (Agilent technologies) and quantified using the Qubit 1x dsDNA HS Assay Kit, in a Qubit 4.0 Fluorometer (Life technologies). Equimolar amounts of indexed libraries were pooled and sequenced on a NextSeq 2000 (Illumina).

first: Arya

last: Changiarath

first: David

last: Flores-Solis

first: Jasper

middle: J

last: Michels

first: Rosa

middle: H

last: Rodriguez

first: Sonya

last: Hanson

first: Markus

last: Zweckstetter

first: Friederike

last: Schmid

first: Lukas

last: Stelzl


title: Expression data from glioblastoma derived xenograft and original glioblastoma tumors

city: Lausanne

first: Pierre

last: Bady

address: Quartier UNIL-Sorge

department: Swiss Institute of Bioinformatics

country: Switzerland

institute: CHUV / BCF / SIB

summary: Amplification of the epidermal growth factor receptor (EGFR, A0 for non-amplified and A1 for amplified) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we report expression data for 33 samples including 6 GBM derived xenografts (3 controls and 3 treated by tyrosine kinase inhibitor gefitinib) and  27 glioblastoma tumors (11 controls and 16 treated by tyrosine kinase inhibitor gefitinib). Note that T0, T1 and T2 treatment types mean that control, Gefitinib treatment and Gefitinib treatment (but not according to protocol schedule), respectively.

overall_design: 33 samples including 6 (3 controls and 3 treated by gefitinib) GBM derived xenografts and  27 (11 controls and 16 treated by gefitinib) glioblastoma tumors

first: Monika

middle: E

last: Hegi


title: Impact of Ketogenic diet on aging associated gene expression in the brain.

city: Columbus

first: Matthew

last: Stratton

state: OH

address: 473 W 12th Ave

institute: Ohio State University

summary: The Ketogenic Diet (KD) was shown to extend lifespan and preserve memory in aged mice. We carried out RNA-seq on brains of mice aged 13-26 months on standard Carbohydrate-rich control Diet (CD) vs KD, to better understand KD's mechanism for memory preservation.

overall_design: At 12 months of age, mice were randomized to control or ketogenic diet. Animals were harvested at 13 and 26 months of age.

first: Matt

first: Jon

last: Ramsey


title: A marker chromosome in psychosis identifies glycine decarboxylase (GLDC) as a novel negative regulator of excitatory synaptic activity in the hippocampus

city: Boston

first: Rachita

last: Yadav

state: MA

address: 185 Cambridge street

department: Neurology

institute: MGH

summary: The biological significance of a small supernumerary marker chromosome generating a 9p24.1 duplication/triplication including a triplication of the GLDC gene encoding glycine decarboxylase in two patients with psychosis is unclear. Performing functional genomic fine-mapping using a series of copy number variant mouse models, we identify that additional copies of Gldc reduce extracellular glycine levels as determined by optical FRET in dentate gyrus (DG) but not in CA1, suppressed long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduced the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displayed prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference deficits.  Our results thus provide a link between a genomic copy number variation, biochemical, cellular and behavioral phenotypes, demonstrating that GLDC negatively regulates excitatory neurotransmission, supporting the view that extra copies of GLDC in the genome may contribute significantly to the development of neuropsychiatric disorders.

overall_design: bulk RNAseq based transcriptomic analyses of two tissues (hippocampus (HPC) and prefrontal cortex (mPFC)) from 9p24.1 deletion, duplication, and triplication as well as control mouse models

first: Maltesh

last: Kambali

first: Yan

last: Li

first: Petr

last: Unichenko

first: Jessica

last: Feria

first: Jing

last: Liu

first: Patrick

last: McGuiness

first: Johanna

middle: G

last: Cobb

first: Muxiao

last: Wang

first: Rajasekar

last: Nagarajan

first: Jinrui

last: Lyu

first: Vanessa

last: Vongsouthi

first: Colin

last: Jackson

first: Elif

last: Engin

first: Joseph

middle: T

last: Coyle

first: Michael

last: Talkowski

first: Gregg

last: Homanics

first: Vadim

middle: Y

last: Bolshakov

first: Christian

last: Henneberger

first: Uwe

last: Rudolph


title: Cell-cycle deregulation in diffuse large B-cell lymphoma promotes critical dependence on the cyclin-G associated kinase

city: miami

first: Jonathan

last: Schatz

state: Florida

address: 1580 NW 10th Ave 1st floor

department: Department of Medicine

institute: university of miami

summary: Diffuse large B cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma and the most frequently diagnosed hematologic malignancy in the United States. DLBCL exhibits significant molecular and clinical heterogeneity, and at least a third of patients are left uncured with standard frontline chemoimmunotherapy. As such, there is a critical need to identify novel targeted therapies to improve outcomes. We therefore conducted a phenotypic screen of kinase inhibitors against DLBCL cell lines and non-malignant controls. We identified the cyclin G-associated kinase (GAK) as a tumor-selective, readily druggable target whose inhibition killed DLBCL cell lines, while sparing non-malignant blood cells. Upon investigation of GAK’s cellular function, we discovered that inhibition results in G2/M-phase cell cycle arrest. Immunofluorescent confocal microscopy revealed significant chromosome misalignment and spindle distortion in DLBCL cells following GAK-inhibition, disrupting progression through mitosis. Analysis of RNA-seq data from clinical samples showed increased GAK expression associates strongly with RB1 deficiency in DLBCL cases, suggesting dependency on GAK for proper mitotic progression linked to retinoblastoma associated protein (RB) loss of function, a common DLBCL driver. In cell-cycle analyses and under microscopy, RB-deficient DLBCL cells treated with an exquisitely selective GAK inhibitor showed complete arrest at G2/M, pronounced distortion of mitotic spindles, and widespread chromosomal damage. Finally, in vivo studies of DLBCL xenograft-bearing NSG mice achieved a dramatic tumor-burden reduction in response to targeted GAK inhibition. These results reveal a novel cell cycle kinase suitable for therapeutic exploitation in DLBCL patients and linked to the common, undruggable biomarker of RB loss of function.

overall_design: DLBCL cell lines U2932 and Ly1 were used for RNA sequencing experiments. Cells were treated in triplicate with either DMSO (vehicle control) or SGC-GAK-1 at a concentration of 1 µM for 12 and 24 hours.

first: Olivia

middle: B

last: Lightfuss

first: Preet

last: Kumar

first: Lingxiao

last: Li

first: Toral

last: Shastri

first: Austin

middle: D

last: Newsam

first: Paola

last: Manara

first: Abdessamad

last: Alaoui

first: Diego

last: Parets

first: Pierre

last: Younes

first: Dhanvantri

last: Chahar

first: Artavazd

last: Arumov

first: Caroline

middle: A

last: Coughlin

first: Indigo

last: Williams

first: Ola

last: Landgren

first: Stephan

last: Schürer

first: Vance

last: Lemmon

first: Francesco

last: Maura

first: Daniel

last: Bilbao

first: Yangbo

last: Feng

first: Hassan

last: Al-Ali


title: Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [RNA-seq]

city: Poznań

first: Arkadiusz

last: Kajdasz

state: wielkopolskie

address: ul. Z. Noskowskiego 12/14

country: Poland

institute: Institute of Bioorganic Chemistry PAS

summary: Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry (SOCE). We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests, staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA-sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum, and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer (IPL),  ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer, which includes amacrine cells among others, showed a significant decrease in the number of GABAergic neurons. The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas.

overall_design: AB and double knockout for stim2 - (stim2a;stim2b)-/- zebrafish 5dpf larvae were used to isolate the total RNA from their eyes. The experimental setup involved 3 replicates per each condition (stim2 KO vs control) consisting of 35 eyes per repetition.

first: Sofiia

last: Baranykova

first: Rishi

middle: K

last: Gupta

first: Iga

last: Wasilewska

first: Matylda

last: Macias

first: Aleksandra

last: Szybińska

first: Tomasz

last: Węgierski

first: Karim

last: Nahia

first: Shamba

last: Mondal

first: Cecilia

middle: L

last: Winata

first: Jacek

last: Kuźnicki

first: Łukasz

last: Majewski


title: RNA Sequencing of C. elegans control and mutant worms after 24 hour P. aeruginosa infection

status: Public on Oct 22 2024

city: Portland

first: Supender

last: Kaur

state: Oregon

address: 3181 SW Sam Jackson Park Road L220

department: Molecular Microbiology and Immuno

institute: Oregon health and science university

summary:
Purpose: After infection there was dendrite degeneration in the mutant animals . To decipher the mechanism responsible and the changes in the gene expression of mutant worms
Methods: Control and mutant worms were exposed at young adult stage to P.aeruginosa PA14 infection for 24 hours. Total RNA was extracted and RNA sequencing was done following standard protocols
Results:  Detailed analysis of the transcriptomic data show that  immune genes were significantly upregulated.
Conclusions: This study showed the role of pathogenic infection in the life span modulation

overall_design: C. elegans RNA seq of young adults of control and mutant worms after 24 hour P.aeruginosa PA14 infection in triplicate, using Illumina platform

first: Alejandro

last: Aballay


title: Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [scRNA-seq]

summary: Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry (SOCE). We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests, staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA-sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum, and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer (IPL),  ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer, which includes amacrine cells among others, showed a significant decrease in the number of GABAergic neurons.The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas.

overall_design: Tg(HuC:GCaMP5G) zebrafish line and double knockout for stim2 expressing GCaMP5G under the same promoter [(stim2a;stim2b)-/- ; Tg(HuC:GCaMP5G)] were used. Single-cell suspensions were obtained by enzymatic and mechanical digestion of 5 days post fertilization (dpf) larva heads without eyes isolated from two fish lines: stim2 KO (2 replicates - 30 larvae per each sample) and Tg (control, 2 replicates- 30 larvae per each sample). The cells were sorted based on GCaMP5G fluorescence to obtain the cells of neuronal origin.


title: CXCL9 links skin inflammation and fibrosis via CXCR3-dependent upregulation of collagen 1a1 in fibroblasts

city: Worcester

first: Jillian

last: Richmond

address: 364 Plantation Street

department: Dermatology

institute: University of Massachusetts Medical School

summary: We performed a comparative transcriptomics study of murine immune, fibrotic and skin gene expression profiles in subcutaneous bleomycin injection to determine whether this model mirrors human morphea (localized scleroderma).

overall_design: Whole transcriptome profiling was performed on mouse skin injected subcutaneously with bleomycin or PBS vehicle control for 5, 9, 12, 16, 21 and 28 days.

first: Heidi

last: Jacobe

first: Kathryn

last: Torok

first: Henry

middle: W

last: Chen

first: Giffin

last: Werner


title: Profiling of PRC1 and PRC2 occupancy reveals the preponderant role of PRC1 in regulating the limb development program [RNA-seq]

city: Montréal

first: Fanny

last: GUERARD-MILLET

state: Quebec

address: 110 AVENUE DES PINS OUEST

country: Canada

institute: IRCM

summary: The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissue-specific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through post-translational modifications of histone tails. A large body of work has revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a  PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1). Moreover, PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidences pointed to cPRC1 and vPRC1 sharing a significant subset of their targets. In turn, this raises the question of PRC2/cPRC1 contribution to gene regulation. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, consistent with the fact that the majority of PRC2-bound loci can also be bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of loci retaining CBX2 binding and even few loci gaining CBX2 binding. Importantly, these loci corresponds to developmental genes and include most genes known as playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings explain why PRC2 inactivation affects rather moderately the limb genetic program and questions the specific functional role of cPRC1/PRC2 in gene regulation.

overall_design: RNA-seq analysis in proximal and distal limb tissue at E12.5

first: Claudia

last: GENTILE

first: Alexandre

last: MAYRAN


title: Ageing-associated long non-coding RNA extends lifespan and reduces translation in non-dividing cells

city: London

first: Shajahan

last: Anver

address: Gower Street London

department: Genetics, Evolution and Environment

institute: University College London

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

overall_design: Refer to individual Series


title: Oncogenic Kras G12D activation in the non-hematopoietic bone marrow microenvironment causes myelodyplastic syndrom in mice

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

summary:
The bone marrow microenvironment is key player in the regulation and maintenance of hematopoiesis and is also involved in the pathogenesis of hematologic malignancies. We investigated the effect of KrasG12D expression in the non-hematopoietic bone marrow microenvironment. Therefore, we generated chimeric mice which express oncogenic Kras only in the non-hematopoietic system. Within 6-8 weeks, these mice developed a myelodysplastic syndrome characterized by cytopenia, myeloid expansion and signs of disturbed differentiation and marked dysplasia within the myeloid lineage.
Gene expression analysis of KrasG12D expressing non-hematopoietic cells of the bone marrow microenvironment revealed a significant upregulation of multiple genes and pathways including proinflammatory signaling such as upregulation of the NLRP3 inflammasome and related genes.

overall_design: Rosa26Cre;LSL-KrasG12D mice were lethally irradiated and transplanted with bone marrow harvested from wild type mice. Single allele littermates were used as control. When the mice were fully engrafted, tamoxifen was administered to induce Cre-mediated KrasG12D expression in the non-hematopoietic tissues. Blood and bone marrow analyisis was performed 6-8 weeks after tamoxifen administration. In order to analyze gene expression changes in KrasG12D expressing bone marrow niche cells, Rosa26Cre;LSL-KrasG12D mice were treated with tamoxifen to ubiquitously induce KrasG12D expression. After 5.5 weeks, osteoblasts, mesenchymal stem and progenitor cells, endothelial cells as well as CXCL12 abundant reticular cells were isolated using fluorescence-activated cell sorting according to published cell surface marker characteristics. RNA was extracted for microarray based gene expression analysis.

first: Lena

last: Osswald

first: Robert

last: Zeiser

first: Melanie

last: Boerries


title: Ageing-associated long non-coding RNA extends lifespan and reduces translation in non-dividing cells [aal1_ChIRP-RNAseq]

summary: Genomes pervasively produce long non-coding RNAs (lncRNAs) of largely unknown functions. Some of these lncRNAs have been implicated in roles related to ageing and associated diseases. Here we characterize aal1 (ageing-associated lncRNA 1) which is induced in non-dividing, ageing cells of fission yeast. Deletion of aal1 shortens the chronological lifespan of non-dividing cells, while ectopic overexpression of aal1 from a plasmid prolongs their lifespan, indicating that this lncRNA acts in trans. We find that aal1 genetically interacts with coding genes functioning in processes related to protein translation, and aal1 overexpression leads to repression of ribosomal protein genes and inhibition of cell growth. The aal1 RNA localizes to the cytoplasm and associates with ribosomes. Notably, aal1 deletion or overexpression is sufficient to increase or decrease the cellular ribosome content, respectively. We identify the mRNA rpl1901, encoding a ribosomal protein, as a binding target of aal1. The expression of rpl1901 is moderately repressed by aal1, and such moderate repression is critical and sufficient to extend the chronological lifespan. Remarkably, expression of the yeast aal1 lncRNA in the fly gut triggers a significant extension of the lifespan in flies. Based on these findings, we propose that the aal1 RNA can reduce the ribosome content by decreasing the levels of the Rpl1901 ribosomal protein, thus attenuating protein translation and promoting longevity. Although the aal1 lncRNA itself is not conserved, its effects in the fly raise the possibility that other organisms feature related mechanisms involving conserved ribosome-associated processes to control ageing.

overall_design: To determine aal1 interacting mRNAs/RNAs we used biotinylated antisense aal1 probes to pull down aal1 and associated RNAs. Wildtype cells (wt), cells ectopically overexpressing aal1 in a plasmid (aal1OE) and aal1 deleted cells with CRISPR-Cas9 (aal1D) were grown in minimal medium (EMMG) until Day 6 after entering stationary phase before harvesting.


title: Deciphering RpoD-family sigma factors transcriptional regulatory network highlights their multifaceted roles under heat shock conditions in Salmonella Typhimurium [RNA-seq]

status: Public on Oct 20 2024

city: Ulsan

first: Donghyuk

last: Kim

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

department: Department of Chemical Engineering

country: South Korea

institute: UNIST

summary: We present genome-wide RpoD-family sigma factors (RpoD, RpoS, and RpoH) binding profiles and transcriptome profiles in Salmonella Typhimurium 14028s under both control (37°C) and sublethal heat shock conditions (42°C). These genome-scale experimental data show how three RpoD-family sigma factors simultaneously coordinate many types of cellular processes to orchestrate the overall response of S. Typhimurium to heat stress.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: [RNA-seq] S. Typhimurium 14028s WT and rpoH-8myc tagged cell were grown to mid-exponential phage, cultured in M9 minimal 0.2% glucose with a biological replicate under both control (37°C) and sublethal heat shock conditions (42°C).

first: Joon

last: Park

first: Minchang

last: Jang


title: The Human Milk Oligosaccharide 3’Sialyllactose Promotes Inflammation Resolution and Reduces Atherosclerosis Development in Mice

city: Oxford

first: Adam

last: Cribbs

address: Windmill Road

department: NDORMS

institute: University of Oxford

summary: Macrophages contribute to the induction and resolution of inflammation and play a central role in the chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3’sialyllactose (3’SL) as a natural inhibitor of TLR4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3’SL attenuates a selected set of inflammatory gene expression and promotes activity of LXR and SREBP. These acute anti-inflammatory effects of 3’SL were associated with reduced histone H3K27 acetylation at a subset of LPS-inducible enhancers distinguished by preferential enrichment for CTCF, IRF2, BCL6, and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3’SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3’SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.

overall_design: RNA-seq of Bone marrow-derived macrophages untreated or treated with PAM3CSK4 or PAM3CSK4 and 3SL

first: Philip

last: Gordts


title: Single cell RNA-seq analysis of endothelial cells during diabetic atherosclerosis

city: Ann Arbor

first: Guizhen

last: Zhao

state: Michigan

address: 2800 Plymouth Road

department: Internal Medicine

summary: Single-cell RNA sequencing (scRNA-seq) was performed on the CD31-mircrobeads enriched endothelial cells isolated from aorta and heart from eight-week-old, male Klf11f/f-Ldlr-/- and Klf11ECKO-Ldlr-/- (Klf11flf-Tie2Cre-Ldlr-/-) mice fed a diabetogenic high-fat diet with 0.15% cholesterol (DDC) diet for 12 weeks. This study report the changes of EC subpopulations, fractions and transcriptomics during diabetic atherosclerosis.

overall_design: Examination of the transcriptomic profiles of endothelial cells isolated from aorta and heart pooled  from three Klf11f/f-Ldlr-/- or Klf11ECKO-Ldlr-/- mice fed a diabetogenic diet with 0.15% cholesterol (DDC) diet for 12 weeks

first: Jifeng

last: Zhang

first: Y


title: Comparative Analysis of Transcriptional RNA Expression in HEK293T Cells Transfected with the H3N2 Subtype Canine Influenza Viruses M1/M2 and Canine RIG-I

city: guangzhou

first: Ye

last: Shaotang

address: No. 483, Wushan Road, Tianhe District, Guangzhou

country: China

institute: South China Agricultural University

summary: Our previous study revealed that influenza virus matrix protein 1 (M1)and matrix 2 (M2) interacted with canine RIG-I. However, the influence of these interactions remains in the mist. To explore these cellular transcriptome modulation induced by the interaction between CIV M1/M2 and canine RIG-I, here we carried out the comparative transcriptional RNA analysis of the overexpression of CIV M1/M2 along with canine RIG-I in HEK 293T cells.

overall_design: RNA sequencing of HEK293T cells with 5 different treatment (mock, transfection with CIV M1/M2, transfection with CIV M1/M2 and canine RIG-I, respectively), in triplicate

first: Shaotang

last: Ye

first: Shoujun

last: Li


title: ZFPL1 drives hepatocellular carcinoma through CLDN3 in WNT signaling pathway

status: Public on Oct 19 2024

city: Shenyang

first: Wensu

last: Liu

address: 77 Puhe Road, Shenbei District

institute: China Medical University

summary: Massive studies have been applied in exploring the factors driving pathogenesis, progression and metastasis of hepatocellular carcinoma. However these studies were inefficient in disclosing the fundamental mechanism which promotes hepatocellular carcinoma. Zinc and zinc-finger proteins have been important in extensive biological processes for human. Supervised machine learning using bootstrapping algorithm on GEO and TCGA transcriptome data for hepatocellular carcinoma identified zinc-finger like protein ZFPL1 as potential hepatocellular carcinoma driver. Further studies validated ZFPL1 significantly promoted progression and metastasis of hepatocellular carcinoma. We performed RNA-seq on si-ZFPL1 xenograft tissue and identified CLDN3 as potential target gene for ZFPL1. Further experiments confirmed interaction between ZFPL1 and WNT signaling pathway markers. Conclusively, these studies indicated the effect and mechanism of ZFPL1 on promoting progression and metastasis of hepatocellular carcinoma and might gap the bridge between zinc-finger like proteins and hepatocellular carcinoma.

overall_design: We used Trizol Reagent (Invitrogen Life Technologies) to extract total RNA in control group (n=3) and siZFPL1 group (n=3). The sequencing library was prepared using the TruSeq RNA sample preparation kit (Illumina, USA) and sequenced at the Hiseq platform (Illumina) of Shanghai Personal Biotechnology Co. LTD. (Shanghai, China). After screening for CutAdapt-based 3'end adapter sequences or reads with average quality score lower than Q20, the high-quality sequences were compared with the reference genome (Assembly GRCm38). The processed data were then normalized and derepeated. Finally fold change and p-value for Students’ t-test were calculated for control and siZFPL1 groups.

first: Zhuo

last: Wang

first: Xiaobei

last: Zhou

first: Zhanyou


title: Zhenqi Granules Attenuates the Inflammatory Response of Pulmonary Epithelial Cells by Inhibiting Endoplasmic Reticulum Stress in MRSA Infection

city: Beijing

first: Lulu

address: 16 Dongzhimen South St, Dongcheng District, Beijing

institute: Chinese Academy of Traditional Chinese Medicine

summary: The severe inflammatory response of pulmonary epithelial cells is the main pathological feature of pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). Zhenqi granules (ZQ), a compound herbal formula composed of Astragalus membranaceus and Ligustrum lucidum, has potential therapeutic effects on this inflammatory response, but its mechanism of action is still unclear. The study aims to explore the effect and mechanism of ZQ on the inflammatory response induced by establishing an in vitro model of MRSA-infected human pulmonary bronchial epithelial cells (MRSA-BEAS-2B). ZQ (10 mg/mL) had no inhibitory effect on MRSA or its biofilm, but it reduced the levels of LDH and c-di-AMP in MRSA-BEAS-2B cells. The mechanism is attributed to the downregulation of endoplasmic reticulum-related protein pathways, specifically involving a decrease in endoplasmic reticulum stress-related proteins such as STING and ERAdP; a decrease in inflammation pathway-related factors such as NF-ĸB, IFR3, NLRP3, and TLR4; and an increase in the STING antagonist RECON. These effects resulted in a reduction in the proinflammatory cytokines IFN-β, IL-1β, IL-6, and IL-18, thereby inhibiting pulmonary inflammation. ZQ attenuates the inflammatory response of pulmonary epithelial cells by inhibiting c-di-AMP-related endoplasmic reticulum stress during MRSA infection. This study provides a basis for the clinical application of ZQ and improves the development of treatment strategies for pulmonary MRSA infection.

overall_design: To study the effect of ZQ on the transcriptome of MRSA-BEAS-2B cells, we established a Control group and a 10 mg/mL ZQ group. BEAS-2B cells in both groups were pretreated with medium and ZQ for 24 hours, respectively, then collected after 2 hours of stimulation with MRSA, following an infection ratio of 1:1. Three replicate samples were set up for each group for RNA-seq assay.


title: Expression data from H2A.Z isoforms single KO cells

city: Sendai

first: Masahiko

last: Harata

address: 468-1 Aramaki Aza Aoba, Aoba-ku

department: Graduate School of Agricultural Science

country: Japan

institute: Tohoku University

summary: The histone variant H2A.Z is evolutionarily conserved from yeast to vertebrates. H2A.Z regulates gene expression when localized to promoter region. Recently, we identified two genes encoding H2A.Z, H2A.Z-1 and H2A.Z-2 in vertebrate genome. However, it is not clear that both H2A.Z-1 and H2A.Z-2 were required for the function of H2A.Z in gene regulation. To address this issue, we generated the H2A.Z-1 and H2A.Z-2 single knockout cells in chicken DT40 cells.We used microarrays to analysis the alternation of gene expression profiles in H2A.Z-1 KO and H2A.Z-2 KO cells and identify classes of up or down regulated gene by H2A.Z-1 and H2A.Z-2.

overall_design: In H2A.Z-2 KO cells, H2A.Z-2 gene is knocked out constitutively. However, in H2A.Z-1 KO cells, H2A.Z-1 gene is conditionally knocked out by tetracycline. The expression of H2A.Z-1 transgene is suppressed completely when H2A.Z-1  KO cells were cultured with tetracycline for three days. For this reason, we prepared the total RNA of H2A.Z-1 KO cells and H2A.Z-2 KO cells treated with tetracycline for three days and hybridization on Affymetrix microarrays.

first: Ryo

last: Matsuda

first: Masayuki

last: Kusakabe

first: Tetsuya

last: Hori

first: Hiroyuki

last: Oku

first: Tatsuo

last: Fukagawa


title: LncRNA EIF1AX-AS1 promotes endometrial cancer cell apoptosis via interacting with  PCBP1 to facilitate EIF1AX mRNA destabilization

city: Fuzhou

first: Chengyu

last: Lv

address: Xuefu North Road, Minhou County, Fuzhou

institute: Fujian Medical University

summary: RNA-sequencing analysis was utilized to investigated the differentially expressed genes of Endometrial cancer tissues compared to adjacent noncancerous tissues

overall_design: RNA-sequencing analysis was carried out to compare the transcriptome profiling between the Endometrial cancer tissues compared to adjacent noncancerous tissues.Pathological grade of Endometrial cancer: endometrioid adenocarcinoma

first: Shie


title: Multi-Omics Analysis and Molecular Subtypes Provide Potential Therapeutic Implications in Fumarate Hydratase-deficient Renal Cell Carcinoma

city: Sichuan

first: Hao

last: Zeng

address: Department of Urology, Third Inpatient Building, West China Hospital, Sichuan University, Chengdu,

institute: West China Hospital, Sichuan University

summary: Conducting an in-depth exploration of the multi-omics landscape of Fumarate Hydratase (FH)-deficient renal cell carcinoma (RCC), the study aimed to unravel the intricate molecular mechanisms underlying this rare and aggressive form of renal cancer. Analyzing 126 patients with FH-deficient RCC, we identified distinct variant spectrum of FH gene, with truncating mutation correlated with adverse prognostic factors and a worse response to immune checkpoint blockade. The identification of three molecular subtypes with diverse clinical/genomic characteristics and varying response to systemic treatment further enriched the understanding of the heterogeneous tumor microenvironment (particularly immune-related) in FH-deficient RCC, and suggested potential therapeutic targets. These findings offer a basis for molecular stratification, shedding light on the tailored therapeutic strategies in improving treatment response of FH-deficient RCC.

overall_design: To comprehensively explore the molecular landscape and biological subtypes of FH-deficient RCC, offering insights for novel therapies, we performed RNA-seq on primary tumors of FH-deficient RCC from 56 patients and 37 paired adjacent normal kidney tissues to conduct gene expression profiling analysis.


title: JOSD2 inhibits angiotensin II-induced vascular remodeling by deubiquitinating and stabilizing SMAD7

status: Public on Oct 18 2024

city: Wenzhou

first: Guang

last: Liang

address: Chashan University Town Ouhai District, Wenzhou City Zhejiang Province, PRC

institute: Wenzhou medical university

summary: Increased level of angiotensin II (Ang II) plays a central role in the development of hypertensive vascular remodeling. Here, we identify the deubiquitinating enzyme JOSD2 as a protective factor and investigate its molecular mechanism in Ang II-induced vascular remodeling. Firstly, we found that JOSD2 was up-regulated in aortic smooth muscle cells but not endothelial cells of Ang II-challenged mouse vascular tissues. Whole-body knockout of JOSD2 significantly deteriorated Ang II-induced vascular remodeling in mice. Conversely, Ang II-induced vascular remodeling was reversed by VSMC-specific JOSD2 overexpression. In vitro, JOSD2 deficiency aggravated the fibrosis, proliferation, and migration induced by Ang II in vascular smooth muscle cells (VSMCs), while these changes were reversed by JOSD2 overexpression. RNA-seq analysis showed that the protective effects of JOSD2 in VSMCs were related to TGFβ-SMAD pathway. Furthermore, the LC-MS/MS analysis identified SMAD7, a negative regulator in TGFβ-SMAD pathway, as the substrate of JOSD2. JOSD2 specifically bound to the MH1 domain of SMAD7 to removed K48-linked Ub chains of SMAD7 at lysine 220 to sustain SMAD7 stability. Taken together, our finding reveals that JOSD2-SMAD7 axis is critical for relieving Ang II-induced vascular remodeling and JOSD2 maybe a novel and potential therapeutic target for hypertensive vascular remodeling.

overall_design: we performed RNA sequencing analysis on the aortas of three WT+Ang II group mice and three KO+Ang II mice.

first: Sirui

last: Shen


title: GENERATION OF MOUSE NEOCORTICAL ORGANOIDS TO MODEL THE IMPACT OF CIS-REGULATORY VARIATION ON CORTICAL NEUROGENESIS ACROSS EVOLUTIONARY TIMESCALES

city: New York

first: Daniel

last: Medina Cano

email: medinacd@mskcc.org, dmcdmc156@gmail.com

state: New York

address: 411E 67th St

department: Developmental Biology

institute: MSKCC

sample_organism:
Mus
Mus musculus

platform_organism:
Mus
Mus musculus


title: Investigating alterations in bovine embryonic pluripotency through scRNA-seq and establishing robust Formative stem cell lines

first: Jianyong

last: Han

address: 2rd, Yuanmingyuan West Road

department: State Key Laboratory of Agrobiotechnology

institute: China Agricultural University

summary: Bovine, as one of the most significant domestic animals providing humans with milk and meat, while also serving as bioreactors for producing valuable proteins, pose challenges in establishing embryo-derived stable pluripotent stem cells (PSCs) due to the unclear relationship between embryonic epiblast development and maintenance of stem cell pluripotency and self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, E14) to track pluripotency changes and their signal pathway dependence using modified single-cell transcription sequencing technology. Based on the remarkable similarity of the dependence of WNT/β-catenin, LIF/STAT3, TGFβ/Smads, and FGF/ERK signaling pathways between bovine and porcine during early embryonic lineages development, we have successfully established bovine embryonic epiblast stem cells (bEpiSCs) using porcine pre-gastrulation epiblast stem cell culture system 3i/LAF. The generated bEpiSCs exhibited consistent pluripotency gene expression levels and maintained clonal morphology, normal karyotype, and proliferative capacity for over 110 passages. Moreover, their high-efficiency teratoma formation potential as well as their ability to differentiate into various cell lineages were demonstrated. At the transcriptome level, bEpiSCs displayed similarities with bovine Formative epiblast cells. We also assessed the application potential of bEpiSCs in targeted differentiation towards muscle cells and as donor cells for somatic cell nuclear transfer (SCNT). In summary, this study establish stable Formative bEpiSCs which hold promise for advancements in cell-cultured meat production, gene-edited cloned cattle generation, and animal breeding.

overall_design:
To investigate the dynamic changes of early bovine embryo development and the characteristics of bovine embryonic stem cells, we constructed the bovine early embryonic single cell dataset, the bovine embryonic stem cell single cell dataset and the transcriptome dataset

The STRT-seq samples are multplexed so each library contains cells of different developmental stages. Please see the file cell_meta.xlsx available at the foot of this record to find the developmental stage for each cell ID and cell barcode.

first: Minglei

last: Zhi

first: Dengfeng

last: Gao


title: Environmentally relevant concentrations of individual per- and polyfluoroalkyl substances (PFAS) and a PFAS mixture impact proliferation and gene transcription in a human myometrial cell line.

city: Omaha

first: Kendra

last: Clark

state: Nebraska

address: 560 South 45th street

institute: University of Nebraska Medical Center

summary: Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants of concern due to their persistence and potential adverse health effects.  Epidemiological studies have linked PFAS with an increased risk of uterine diseases including fibroids however, the mechanisms involved remain to be elucidated.  This study investigated the effects of individual PFAS, including long-chain “legacy” PFAS [perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS)] and short-chain “alternative” PFAS compounds [undecafluoro-2-methyl-3-oxahexanoic acid (GENX/HFPO-DA), perfluorobutanesulfonic acid (PFBS)], as well as a mixture of these chemicals on the function and transcriptome of an immortalized human myometrial cell line (UT-TERT).  UT-TERT cells exposed to individual PFAS displayed increased cell viability and proliferation.  Flow cytometry analysis revealed that PFOS and the PFAS mixture altered cell cycle progression.  Migration assays demonstrated that PFOS and the PFAS mixture significantly enhanced UT-TERT cell migration.  Gap junction intercellular communication (GJIC) was impaired following PFOA, PFBS, and PFAS mixture exposure, indicating potential disruptions in cell-to-cell communication within the uterine environment.  Transcriptomic analysis using RNA-seq identified substantial changes in gene expression after exposure to environmentally relevant levels of individual PFAS and PFAS mixture.  Pathway analysis revealed common molecular pathways associated with PFAS exposure, including Cell-to-Cell Signaling, Lipid Metabolism, and Cell Death and Survival, while other pathways were unique to specific PFAS.  These findings highlight the multifaceted effects of PFAS on myometrial cells, providing insights into the potential mechanisms underlying PFAS-associated health risks.  Further research is necessary to elucidate the long-term implications of PFAS exposure on uterine function and overall reproductive health.

overall_design: To determine the impacts of PFAS and PFAS mixture on human mymoetrial cells, cells were treated with various concentraions of individual PFAS and a PFAS mixture and myometrial cell function endpoints were analyzed including cell proliferation, migration/invasion, gap junction activity, and transcriptome.

first: Jitu

last: George


title: Marginal zone B cells receive robust Notch2 signals within the B cell follicle in the absense of S1pr1 signaling.

city: Philadelphia

first: Brian

middle: Thomas

last: Gaudette

state: PA

address: 3620 Hamilton Walk, Pathology and Laboratory Medicine

department: Pathology and Lab Medicine

institute: University of Pennsylvania

summary: Marginal zone (MZ) B cells have innate-like response characteristics due to a unique ability receive and integrate Notch2 signals in the spleen. There is evidence showing MZ B cells receive these signals both in the marginal sinus and the B cell follicle. The relative strength and conservation of these Notch signals is not yet clear. Therefore, we generated mice in which S1pr1 is deleted in mature B cells. These mice have MZ B cells by phenotype but these cells are retained in the follicle and unable to transit to the marginal zone. In this experiment we examined the impact of Notch2 signaling in these cells and cre expressing control animals using in vivo anti-Notch2 antibody blockade followed by RNA-sequencing.

overall_design: S1pr1-floxed mice were mated to Mb1cre mice to conditionally delete S1pr1 in B cells. S1pr1 conditional knockout (S1pr1FL/FL.Mb1Cre/+ mice and S1pr1+/+.Mb1Cre/+  control animals were treated with anti-Notch2 blocking antibodies or isotype control antibodies for 48 or 96 hours prior to sacrifice. Marginal zone B cells (CD19+, CD93-, CD1d+, CD23-) and follicular B cells (CD19+, CD93-, CD1d-, CD23+) were purified by FACS sorting and RNA-sequencing library generation.

first: Anneka

last: Allman

first: David

first: Ivan

last: Maillard


title: Chemically Engineered Antibodies for Autophagy-based Receptor Degradation

city: Shanghai

first: Li

last: Cheng

state: Shanghai

address: 500 Quxi Road, Shanghai 200011

department: Department of Rehabilitation Medicine

institute: The Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine

summary: To assess whether AUTAB treatment induces damage to other membrane-structured organelles, as well as activates stress signaling pathways, we analyzed the changes in mRNA expression levels in AUTAB-treated cells through RNA sequencing. This approach allowed us to determine the activation of relevant stress-related signaling pathways within the cells.

overall_design: We analyzed the changes in mRNA expression levels in AUTAB-treated cells through RNA sequencing.

first: Binghua


title: Polycomb Repressive Complexes occupancy reveals PRC2- independent PRC1 critical role in the control of limb development [ChIP-seq]

city: Montréal

first: Fanny

last: GUERARD-MILLET

address: 110 AVENUE DES PINS OUEST

institute: IRCM

summary: The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissuespecific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through posttranslational modifications of histone tails. A large body of work revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1), in which PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidence pointed to cPRC1 and vPRC1 sharing a significant subset of targets. How the functional interplay between PRC2, cPRC1 and vPRC1 contributes to gene regulation remains partially understood. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, as the majority of PRC2- bound loci are bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of genes retaining CBX2 occupancy as well as a few genes with apparent gain of CBX2 binding. Importantly, among these genes, 56 of them correspond to developmental genes known for playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings emphasize the primary role of PRC2-independent PCR1 function in regulating developmental genes and questions the role of PRC2/cPRC1 in controlling developmental programs.

overall_design: ChIP analysis of SUZ12, H3K27me3, RING1B, H2AK119ub, RYBP, CBX2, CBX8, H3K27ac in proximal and distal limb tissue at E12.5

first: Claudia

last: GENTILE

first: Racheal

last: PAUL

first: Christian

last: POITRAS


title: Profiling of PRC1 and PRC2 occupancy reveals the preponderant role of PRC1 in regulating the limb development program

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

overall_design: Refer to individual Series


title: Gene editing of colocalized NCF1 loci is associated with homologous recombination and chromosomal rearrangements

first: Geoffroy

last: Andrieux

address: Breisacherstr 153


title: Interleukin-2 receptor signaling acts as a checkpoint that shapes the distribution of regulatory T cell subsets (scRNA-Seq)

city: Miami

first: Thomas

middle: R

last: Malek

state: FL

address: 1600 NW 10th Avenue

department: Microbiology/Immunology

institute: University of Miami

summary: Regulatory T cells (Tregs) require IL-2 for survival in the periphery, yet how IL-2 shapes Treg heterogeneity remains poorly defined. Here we found that inhibition of IL-2R signaling in post-thymic Tregs leads to a preferential early loss of central Tregs (cTregs). Gene expression of cTregs was more dependent on IL-2R signaling than effector Tregs (eTregs). Unexpectedly, ablation of IL-2R signaling in cTregs resulted in increased proliferation, expression of eTreg genes, and enhanced capacity to develop into eTregs. These findings indicate that physiological amounts of IL-2 act as a checkpoint to maintain cTreg homeostasis and tolerance while restraining their development into eTregs. Nevertheless, direct evaluation of eTregs revealed that loss of IL-2R signaling alters the distribution of eTreg subsets, with increased IFNgR1+ eTregs and CXCR5+ PD-1+ T follicular regulatory (TFR) cells but decreased intestinal RORgt+ TR17 cells. Thus, IL-2R signaling also shapes the development of specialized eTregs subsets.

overall_design: Treg-specific CD25 knockout was induced with tamoxifen and splenocytes were harvested 10 days later. Tregs were FACS purified from the spleen using viability, CD4, GFP and tdTtomato reporters. CD25iKO Tregs were gated on CD25lo cells. Purified cells were submittied for scRNAseq. Two pooled spleens were used for each sample.

first: Acacia

middle: N

last: Shouse

first: Alejandro

middle: V

last: Villarino


title: Interleukin-2 receptor signaling acts as a checkpoint that shapes the distribution of regulatory T cell subsets (RNA-Seq)

overall_design: Treg-specific CD25 knockout was induced with tamoxifen and splenocytes were harvested 10 days later. Tregs were FACS purified from the spleen using viability, CD4, GFP and tdTtomato reporters. cTregs were gated on CD62Lhi and eTregs were gated on CD62Llo. CD25iKO Tregs were gated on CD25lo cells. Each biological replicate includes pooled spleen and lymph nodes from 2-3 mice.


title: The Diversity of Methylation Patterns in Serous Borderline Ovarian Tumors and Serous Ovarian Carcinomas

city: Warsaw

first: Lukasz

middle: Michal

last: Szafron

address: Roentgena 5

institute: The Maria Sklodowska-Curie National Research Institute of Oncology

summary: Background: Changes in DNA methylation patterns are a pivotal mechanism of carcinogenesis. In some tumors, aberrant methylation precedes genetic changes, while gene expression may be more frequently modified due to methylation alterations than by mutations. Methods: Herein, 128 serous ovarian tumors were analyzed, including borderline ovarian tumors (BOTS) with (BOT.V600E) and without (BOT) the BRAF V600E mutation, low-grade (lg), and high-grade (hg) ovarian cancers (OvCa). The methylome of the samples was profiled with Infinium MethylationEPIC microarrays. Results: The biggest number of differentially methylated (DM) CpGs and regions (DMRs) was found between lgOvCa and hgOvCa. By contrast, the BOT.V600E tumors had the lowest number of DM CpGs and DMRs compared to all other groups and, in relation to BOT, their genome was strongly downmethylated. Remarkably, the ten most significant DMRs, discriminating BOT from lgOvCa, encompassed the MHC region on chromosome 6. We also identified hundreds of DMRs, being of potential use as predictive biomarkers in BOTS and hgOvCa. DMRs with the best discriminative capabilities overlapped the following genes: BAIAP3, IL34, WNT10A, NEU1, SLC44A4, and HMOX1, TCN2, PES1, RP1-56J10.8, ABR, NCAM1, RP11-629G13.1, AC006372.4, NPTXR in BOTS and hgOvCa, respectively. Conclusions: The global genome-wide hypomethylation positively correlates with the increasing aggressiveness of ovarian tumors. We also assume that the immune system may play a pivotal role in the transition from BOTS to lgOvCa. Given that the BOT.V600E tumors had the lowest number of DM CpGs and DMRs compared to all other groups, when methylome is considered, such tumors might be placed in-between BOT and OvCa.

overall_design: In this project, methylomes of 128 serous ovarian tumors have been analyzed using Infinium MethylationEPIC microarrays (Illumina). The cohort consisted of 10 formalin-fixed paraffin-embedded (FFPE) borderline ovarian tumors without the BRAF V600E mutation (BOT), 7 FFPE borderline ovarian tumors with the BRAF V600E mutation (BOT.V600E), 4 snap-frozen BOT, 4 snap-frozen BOT.V600E, 2 FFPE low-grade ovarian cancers (lgOvCa), 5 snap-frozen lgOvCa, 4 FFPE high-grade ovarian cancers (OvCa), and 92 snap-frozen OvCa.

first: Laura

first: Roksana

last: Iwanicka-Nowicka

first: Piotr

last: Sobiczewski

first: Marta

last: Koblowska

first: Agnieszka

last: Dansonka-Mieszkowska

first: Jolanta

last: Kupryjanczyk

middle: M


title: Light induced targeting enables proteomics on endogenous condensates

city: Freiburg

first: Choongman

last: Lee

address: Stuebeweg 51

institute: Max Planck Institute of Immunobiology and Epigenetics

summary: Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass-spectrometry and other proteomics profiling. Here we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates.

overall_design: To test the effect of LiTEC on transcription, global RNA-seq was performed with and without blue-light exposure (30 min, ~3.42 mW/cm^2) on wildtype mESCs and LiTEC mESCs. To check how transcription changes over time, RNAs were extracted on 0 hour, 1 hour and 4 hour after 30 min of blue light exposure (or without blue light exposure)

last: Arrigoni

first: Ward

last: Deboutte

first: Ibrahim

last: Cisse


title: Deciphering RpoD-family sigma factors transcriptional regulatory network highlights their multifaceted roles under heat shock conditions in Salmonella Typhimurium [ChIP-exo]

city: Ulsan

first: Donghyuk

last: Kim

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

institute: UNIST

summary: We present genome-wide RpoD-family sigma factors (RpoD, RpoS, and RpoH) binding profiles and transcriptome profiles in Salmonella Typhimurium 14028s under both control (37°C) and sublethal heat shock conditions (42°C). These genome-scale experimental data show how three RpoD-family sigma factors simultaneously coordinate many types of cellular processes to orchestrate the overall response of S. Typhimurium to heat stress.

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: [ChIP-mini] S. Typhimurium 14028s WT and rpoH-8myc tagged cell were grown to mid-exponential phage, cultured in M9 minimal 0.2% glucose with a biological replicate under both control (37°C) and sublethal heat shock conditions (42°C).

first: Joon

last: Park

first: Minchang

last: Jang


title: A noncoding variant confers pancreatic differentiation defect

city: WuHan

first: chenchao

last: yan

state: Hubei Province

address: Donghu Road, No. 115

institute: WuHan university

summary: GWAS provided many β cell function-associated single nucleotide polymorphisms (SNPs) without clearly pathogenic mechanism. Stepwise differentiation of pancreatic β cells provide the promise to study developmental genetic disorders. Here, we focused on SNP rs6048205 (A/G) located in downstream noncoding region of FOXA2, which was annotated with fast glucose and β cell function risk. We introduced the mutation into human pluripotent stem cells, directed pancreatic differentiation revealed that risk G mutation reduce the percentage of PDX1+NKX6-1+ progenitor cells at pancreatic progenitor 2 stage (PP2), and then defect the function of β cells in-vitro. Mechanistically, risk allele G variant could alter the upstream factor RXRA binding to ectopically promote the expression of FOXA2, which is an important transcription factor in pancreatic development.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for human PDX1+/NKX6-1+ pancreatic progenitor cells with different genotypes (A/A, G/G, FOXA2-control, FOXA2 conditional overexpressed during pancreatic progenitor cells).

first: Yinglei

last: Li

first: Chenchao

last: Yan

first: Wei

last: Jiang


title: Profiling of PRC1 and PRC2 occupancy reveals the preponderant role of PRC1 in regulating the limb development program [ChIP-seq]

first: Fanny

last: GUERARD-MILLET

summary: The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissue-specific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through post-translational modifications of histone tails. A large body of work has revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a  PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1). Moreover, PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidences pointed to cPRC1 and vPRC1 sharing a significant subset of their targets. In turn, this raises the question of PRC2/cPRC1 contribution to gene regulation. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, consistent with the fact that the majority of PRC2-bound loci can also be bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of loci retaining CBX2 binding and even few loci gaining CBX2 binding. Importantly, these loci corresponds to developmental genes and include most genes known as playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings explain why PRC2 inactivation affects rather moderately the limb genetic program and questions the specific functional role of cPRC1/PRC2 in gene regulation.

overall_design: ChIP analysis of H3K27me3, CBX2, H2AK119ub and RYBP in proximal and distal limb tissue at E12.5

first: Claudia

last: GENTILE


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

city: Leiden

first: Vincent

last: van Unen

state: ZH

address: Albinusdreef 2

department: Immunology

country: Netherlands

institute: Leiden University

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

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

first: Qinghui

last: Mu

first: Calvin

middle: J

last: Kuo


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

city: Ljubljana

first: Mojca

last: Pavlin

address: Vrazov Trg 2

country: Slovenia

institute: University of Ljubljana,

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

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

first: Tadeja

last: Snedec


title: The KMT2 complex protein ASH2L controls meiosis through silencing transposon

city: shanghai

first: Haorui

last: Luo

address: 320 Yue Yang Road

institute: Shanghai Institute of Biochemistry and Cell Biology

summary: Meiosis is precisely controlled by complex gene expression programs and involves epigenetic reprogramming and silencing of transposable elements. ASH2L (Absent, small or homeotic 2-like) is a core component of KMT2 complexes conferring H3K4 trimethylation, and plays key roles in mouse embryonic development, neurogenesis, and liver function. However, its role in meiosis remains elusive. Here, we demonstrate an essential role of Ash2l for meiosis. Using a germ cell-specific Ash2l knockout mouse model, we found that Ash2l deficiency leads to meiotic arrest and sterility in both sexes. Ash2l-deficient spermatocytes exhibit failures in chromosomal synapsis, blockades in meiotic recombination, and increased apoptosis.  Mechanistically, we found that the Ash2l deficiency results in a global loss of H3K4me3 in promoter regions and significantly decreases expression of thousands of genes, including those for H3K9 di-methylation, DNA methylation and transposon repression, crucial for meiotic prophase I progression. Furthermore, we observed that Ash2l mutant spermatocytes display hypomethylation at transposon sites and ectopic expression of LINE1-ORF1P. Our findings thus reveal a previously unappreciated role of the ASH2L-dependent H3K4me3 modification and provide clues to the molecular mechanisms in epigenetic disorders underlying male infertility.

overall_design: We isolated A4 spermatogonia and leptotene/zygotene spermatocytes from  wild-type and Ash2l-gKO mice, and conducted RNA-seq and H3K4me3 ChIP-seq analyses. Additionally, NOMe-seq was performed on zygotene spermatocytes.


title: Transcritome provides insights into predicting the heterosis level

city: Nanjing

first: Ma

last: Ce

address: weigang

department: nanjingnongyedaxue

institute: Nanjing

summary: we compared the change of transcritome between the parents and their F1 hybrids showing different heterosis level and obtained the potential index for predicting the heterosis level between rice parental lines.

overall_design: Examination of the profiles of transcription  in young seedlings, young panicles and filling panicles of 4 parents and their 5 hybrids. R998 ,R308, WFB and RFB are the parental lines, and other lines are F1 hybrids. Their relationship is WY998 (WFB X R998), WY308 (WFB X R308), RY998 (RFB X R998), RY308 (RFB X R308), WR (WFB X RFB).

first: Chongyun

last: Fu

first: Feng

last: Wang


title: B1b cell homeostasis is maintained by CD40 in atherosclerosis

city: Amsterdam

first: Esther

last: Lutgens

address: Meibergdreef 15

department: Medical Biochemistry

institute: Amsterdam UMC, location AMC

summary:
Background: The co-stimulatory CD40-CD40L dyad is an important driver of atherosclerosis. CD40 exerts divergent, cell-specific roles, which differentially impact atherogenesis.
Objectives: We here investigate the role of the most prominent CD40-expressing cell-type, the B-cell, in atherosclerosis.
Methods: B-cell subset specific CD40-expression of patients with mild and severe coronary artery disease (CAD) was determined by mass-cytometry and correlated to CAD severity. Underlying mechanisms were elucidated using B-cell CD40-deficient CD19Cre-CD40flfl-ApoE-/-(CD40BKO) mice and control littermates (CD40BWT).
Results: Patients with severe CAD had similar CD40 expression levels on most B-cell subsets but showed a profound decrease in CD40 on putative B1 cells. This was associated with increased atherosclerotic plaque burden and decreased plaque fibrosis. Likewise, CD40BKO mice exhibited an increased plaque area and more advanced staging of atherosclerosis. Absence of CD40 on B-cells caused a decrease in immunoglobulin (Ig) producing cells, including germinal center-, plasma-, but especially B1 cells, thereby reducing levels of (anti-ox/MDA-LDL) IgG and protective (anti-ox/MDA-LDL) IgM. Transcriptomics analysis revealed that the absence of CD40 on B1b cells caused altered gene expression pathways related to lipid uptake (Cd36, Ldlr), cellular stress and metabolism (Nr4a1, Hif1a), and cell death (Naip5/6, Ccnd2). Indeed, in vitro analysis showed that B1bCD40KO cells took up excessive amounts of ac/oxLDL, exhibited defective BCR signaling, and were prone to apoptosis, especially in hyperlipidemic conditions. Transfer of wild-type B1b cells in CD40BKO mice prevented the increase in atherosclerosis.
Conclusions: Patients with severe CAD had similar CD40 expression levels on most B-cell subsets but showed a profound decrease in CD40 on putative B1 cells. This was associated with increased atherosclerotic plaque burden and decreased plaque fibrosis. Likewise, CD40BKO mice exhibited an increased plaque area and more advanced staging of atherosclerosis. Absence of CD40 on B-cells caused a decrease in immunoglobulin (Ig) producing cells, including germinal center-, plasma-, but especially B1 cells, thereby reducing levels of (anti-ox/MDA-LDL) IgG and protective (anti-ox/MDA-LDL) IgM. Transcriptomics analysis revealed that the absence of CD40 on B1b cells caused altered gene expression pathways related to lipid uptake (Cd36, Ldlr), cellular stress and metabolism (Nr4a1, Hif1a), and cell death (Naip5/6, Ccnd2). Indeed, in vitro analysis showed that B1bCD40KO cells took up excessive amounts of ac/oxLDL, exhibited defective BCR signaling, and were prone to apoptosis, especially in hyperlipidemic conditions. Transfer of wild-type B1b cells in CD40BKO mice prevented the increase in atherosclerosis.

overall_design: Comparison of gene expression in aortas (n=5 per genotype) and B1b cells (n=3 per genotype) from wild-type mice and B cell-specific CD40-deficient mice

first: Myrthe

middle: E

last: Reiche

first: Tanyaporn

last: Pattarabajinard

first: Pascal

last: Kusters

first: Aditi

last: Upadhye

first: Kikkie

last: Poels

first: Amber

last: Driessen

last: van Tiel

first: Hung-Jen

last: Chen

last: den Toom

first: Cindy

middle: P

last: van Roomen

first: Emma

last: van Rooijen

first: Angela

last: Taylor

first: Stephen

last: Malin

first: Christoph

last: Binder

first: Norbert

last: Gerdes

first: Christian

last: Weber

first: Dorothee

last: Atzler

first: Coleen

last: McNamara


title: Gene expression profile at single cell level of clinical mild to moderate COPD

city: Hangzhou

first: Baihui

last: lv

state: Zhejiang

address: Yuhangtang Road, Hangzhou City, Zhejiang Province

institute: Zhejiang University

summary: The research included a cohort of 11 patients who were diagnosed with mild or moderate COPD, as well as a control group of 7 individuals who were matched for age.

overall_design: Expression profiling by high throughput sequencing

last: Lv

first: Zhouyang

last: Li

first: Zhihua


title: Exploring the genetic changes caused by vaccine treatment on MC38 tumor tissue

city: Beijing

address: Zhongguancun street

institute: NCNST

summary: Investigating the tumor immune microenvironment and tumor cell proliferation in the vaccine group, which could reflefct the effect of vaccine.

overall_design: Briefly, healthy C57BL6N mice were subcutaneously immunized on days -22, -15, -8, and -1 with Lipo-adjuvant, or iPM nanovax. Mice were subcutaneously inoculated with 5 × 104 MC38 cells on day 0. Twenty-eight days later, the animals were sacrificed, and RNA-seq were assessed in vitro.

first: Ruifang

last: Zhao

first: Guangjun

last: Nie


title: GENERATION OF MOUSE NEOCORTICAL ORGANOIDS TO MODEL THE IMPACT OF CIS-REGULATORY VARIATION ON CORTICAL NEUROGENESIS ACROSS EVOLUTIONARY TIMESCALES  (bulk RNA-Seq)

city: New York

first: Daniel

last: Medina Cano

address: 411E 67th St

institute: MSKCC

summary: Natural selection has shaped the gene regulatory networks that orchestrate the development of the neocortex, leading to diverse neocortical structure and function across mammals, but the molecular and cellular mechanisms driving phenotypic changes have proven difficult to characterize. Here, we develop a reproducible protocol to generate cortical organoids from mouse epiblast stem cells that enable in depth mechanistic studies of cortical developmental in vitro. Cortical organoids develop with similar kinetics to the mouse cortex in vivo, recapitulate the cellular diversity present in the embryonic neocortex, and undergo relatively rapid maturation compared to human organoids. We generated cortical organoids from F1 hybrid epiblast stem cell lines from crosses between standard laboratory mice (C57Bl/6J) and four wild-derived inbred mouse strains from distinct sub-species that span ~1 million years of evolutionary divergence. Using scRNA-seq and allele-specific expression analysis we identified hundreds of genes that exhibit differential cis-regulation during cortical neurogenesis. These experimental methods and cellular resources represent a powerful new platform for investigating gene regulatory mechanisms across evolutionary timescales.

overall_design:
To investigate how natural selection has shaped development across mouse subspecies, we generated F1 hybrid epiblast stem cell lines from crosses between standard laboratory mice (C57Bl/6J) and four wild-derived inbred mouse strains from distinct sub-species that span ~1 million years of evolutionary divergence.
We performed bulk RNA-seq on F1 EpiSCs (CastB6, MolfB6, PwkB6 and SpretB6) to compare efficacy of allelic mapping using 10x scRNA_seq VS bulk RNA_seq

last: Medina-Cano

first: Mohammed

last: Islam

first: Thomas

last: Vierbuchen


title: BGN promotes epithelial migration in mammary gland development and breast cancer metastasis through activation of WNT signaling

first: Rongze

last: Ma

address: zhongke road

institute: Shanghaitech University

summary: This study investigates the role of Biglycan (BGN) in promoting epithelial migration during mammary gland development and breast cancer metastasis. Using in vitro and in vivo models, including a 3D organoid migration assay and single-cell RNA sequencing, we demonstrate that BGN enhances the interaction between WNT3A and FZD6, activating the WNT signaling pathway. This activation promotes epithelial cell migration and breast cancer metastasis. The findings provide new insights into breast cancer metastasis mechanisms and identify BGN as a potential therapeutic target.

overall_design: The study utilized RNA sequencing (RNA-seq) to analyze gene expression changes associated with BGN protein stimulation in breast cancer and mammary epithelial cells. Samples were collected from two conditions: control group and BGN protein stimulation group. Sequencing was performed on an Illumina platform. The analysis focused on identifying differentially expressed genes and pathway activations, particularly the WNT signaling pathway. Additional in vitro assays using 3D organoid models were conducted to validate the role of BGN in epithelial migration and tumor metastasis.

first: Zhixuan

last: Deng

first: Jing

last: Chen

first: Pengfei

last: Lu


title: Assessing the impact of GXYLT2 knockdown on the expression levels of genes associated with malignant characteristics, including proliferation, invasion, and stemness, in HGC-27 gastric cancer cells.

first: Yang

middle: jia

last: le

address: No. 501, Haike Road, Pudong New Area

institute: Chinese Academy of Sciences University

summary: Gastric cancer ranks among the top ten most prevalent tumors worldwide, characterized by substantial heterogeneity and diverse morphological manifestations. Although postoperative histopathological examination serves as the cornerstone for determining Lauren classification, there exists a notable time lag in acquiring Lauren grade through this method. Furthermore, the invasiveness of detection techniques and limited tissue specimens markedly compromise the diagnostic precision of Lauren classification. Moreover, distinct molecular expression profiles exist between intestinal and diffuse types, yet the lack of molecular markers hampers the identification of these subtypes and their underlying mechanisms. Hence, our objective is to unearth biomarkers capable of distinguishing between intestinal and diffuse types and elucidate their mechanistic underpinnings, thereby refining the accuracy and applicability of Lauren classification. This pursuit holds promise for informing prognosis and optimizing treatment strategies for gastric cancer patients. Glycosylation emerges as a pivotal player in gastric cancer pathogenesis. Leveraging bioinformatics analyses on publicly available gastric cancer databases, we identified, at the transcriptomic level, elevated expression of the glycosylation-associated gene GXYLT2 in the diffuse subtype compared to the intestinal subtype. Subsequently, we corroborated these findings at both cellular and clinical tissue levels. Notably, GXYLT2 knockdown attenuated the proliferative, invasive, and sphere-forming capacities of gastric cancer cells in vitro. RNA-seq analysis and cellular validation unveiled that GXYLT2 depletion operates through inhibition of the Wnt/β-catenin signaling pathway. Encouragingly, in vivo validation demonstrated that GXYLT2 knockdown impedes gastric cancer growth. In conclusion, our study positions GXYLT2 as a potential biomarker capable of discerning between intestinal and diffuse subtypes, with prognostic implications in gastric cancer. Furthermore, we elucidate its role in gastric cancer progression via modulation of the Wnt/β-catenin signaling pathway.

overall_design: To investigate the involvement of GXYLT2 in the malignant phenotype of gastric cancer, we generated a stable transfection strain of HGC-27 cells with shGXYLT2 and employed shRNA to suppress GXYLT2 expression. Subsequently, RNA-seq analysis was conducted on both the shC (control) and shGXYLT2 groups, followed by gene expression profiling of the resultant data.

first: Jiale

last: Yang


title: A cross-disease human microglial framework identifies disease-enriched subsets and tool compounds for microglial polarization.

first: John

last: Tuddenham

state: NY

institute: Columbia University

summary: Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. We use single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and central nervous system regions. We observe a central divide between oxidative and heterocyclic metabolism and identify subsets associated with antigen presentation, motility, and proliferation. Specific subsets are enriched in susceptibility genes for neurodegenerative diseases or the disease-associated microglial signature. We validate subtypes in situ with an RNAscope-immunofluorescence pipeline and leverage our dataset as a classification resource, finding that iPSC model systems recapitulate substantial in vivo heterogeneity. Finally, we identify and validate candidates for chemically inducing subtype-specific states in vitro, showing that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimer’s.

overall_design: Comparative bulk RNA-sequencing data of HMC cells treated with one of several drugs or DMSO control.

middle: F

first: Verena

last: Haage

first: Philip

last: De Jager


title: Transcriptome analysis reveals genes responsive to three low-temperature treatments in Arabidopsis thaliana

city: SAN LUIS POTOSI

first: Cesaré

last: Ovando-Vázquez

state: SAN LUIS POTOSI

address: Camino a la Presa de San José 2055. Lomas 4 sección

department: CNS

country: Mexico

institute: CONACyT IPICyT

summary: Cold stress impedes the growth and development of plants, restricts the geographical distribution of plant species, and impacts crop productivity. In this study, we analyzed the Arabidopsis thali-ana transcriptome to identify genes with differential expression (DEGs) in 14-day-old plantlets exposed to temperatures of 0°C, 4°C, and 10°C for 24 h, compared to the 22°C control group. We found 31 genes shared among three low temperatures, with 9 genes common to 0°C-4°C, 8 genes to 4°C-10°C, and 2 genes to 0°C-10°C among the top 50 cold-induced genes from each tempera-ture. Our data revealed that genes, such as galactinol synthase 3 (Gols3, At1g09350), CIR1 (At5g37260), DnaJ (At1g71000), and At5g05220 (unknown function), exhibited the highest ex-pressions at 0°C and 4°C during the 24, 48, and 72-h intervals. We also studied genes from the UDP-glycosyltransferase (UGT78) family, including At5g17030 (D3), At5g17040 (D4), At5g17050 (D2), and At1g30530 (D1), which showed increased expression at low temperatures compared to plantlets at 22°C for 24 h. Gene ontology analysis revealed that DEGs highly en-riched were found in biological processes including “RNA secondary structure unwinding” and “rRNA processing” induced at the three low temperatures, whereas processes related to photo-synthesis were repressed. Our findings indicated upregulation in the expression of four RNA hel-icases (RH13, RH48, RH32, and RH29), belonging to the “RNA secondary structure unwinding” category, mainly at 0°C and 4°C. This study provides valuable information on the molecular mechanisms that activate Arabidopsis thaliana in its early response to these three low tempera-tures.

overall_design: After growing for two weeks, 14-day-old plantlets were subjected to low temperatures of 0ºC, 4ºC, and 10ºC for 24h, 48h, and 72h, respectively. Control plants were maintained in a growth chamber at 22ºC. After the cold treatments, the plants were harvested, rapidly frozen in liquid nitrogen, and stored at -80°C for further analysis. Each temperature condition had three biological replicates (n=3), with each replicate comprising groups of 10 plantlets per Petri plate.


title: Tuning circadian rhythm of CAFs against cancer progression

city: Seoul

first: Han-Sung

last: Jung

address: 50, Yonsei-ro, Seodaemun-gu

department: Dental School

institute: Yonsei University

summary: Circadian disruption orchestrates various biological functions associated with carcinogenesis and tumor progression in cancer cells. Yet the influential role in the tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs), remains unveiled. Here, we report an uncovered signaling axis, BMAL1-LHX8, that elicits the transitioning of CAF-like properties via coordinating the tumor-supportive activity in ameloblastoma (AM) stromal. Both in the co-culture system and stroma-rich AM tumoroid model, CAFs augmented tumor growth, cell proliferation, and metabolic activities. Time-series RNA-sequencing analysis indicated the perturbation of circadian rhythm in the AM+CAF group and LHX8 was considerably involved. CRISPR/cas9 system-based loss-of-function study suggested that the BMAL1-LHX8 signaling axis was highly correlated with several tumor-supportive activities, including secretion of growth factors and ECM remodeling molecules. It also contributed to the resistance of BRAF inhibitor (PLX4032), which could be effectively suppressed by the clock regulator (GSK4112, an agonist of REV-ERB which suppresses BMAL1 expression). This phenomenon confirmed that GSK4112 elevated the anti-tumor efficacy of PLX4032 in the xenograft model. Collectively, these data underscore the role of the BMAL1-LHX8 axis in exacerbating the CAF-like transitioning in AM stromal and tuning the circadian rhythm via clock regulators. This research offering the experimental evidence for the development of chrono-chemotherapy and offering new possibilities for the cancer treatment in the future.

overall_design: Comparative gene expression profiling analysis of RNA-seq data among stroma-rich ameloblastoma tumoroids (ameloblastoma tumoroids enriched with normal fibroblasts or ameloblastoma tumoroids enriched with cancer-associated fibroblasts derived from ameloblastoma patients) at time points of 0,8,16,24,32,40,48 h after synchronized with dexamethasone.

first: Shujin

last: Li

first: HanSung


title: A dynamic subcutaneous adipose transcriptome of human insulin resistance identifies a molecular predisposition shared with visceral adipose tissue

city: NASHVILLE

first: Marisol

last: Ramirez

state: TENNESSEE

address: 2220 Pierce Avenue, 571 Preston Research Building

department: Center for Quantitative Sciences, Department of Biostatisticss

institute: Vanderbilt University Medical Center

summary: Subcutaneous adipose tissue (SAT) is classically viewed as a metabolic buffer for lipid deposition during positive caloric balance, while visceral adipose tissue (VAT) is viewed as the dominant contributor and prime mediator of insulin resistance (IR) and cardiometabolic disease risk. Nevertheless, a growing body of data suggests that similar morphologic and molecular changes may occur in SAT as in VAT during obesity. In addition, while pro-inflammatory immune changes within adipose are thought to drive IR there is increasing data implicating a role for adipocytes and stromal populations especially in humans. Here, we identified a transcriptional landscape of IR in SAT of 220 humans across the spectrum of obesity and IR states, highlighting a broad range of metabolic pathways central to IR. Using single cell and nucleus deconvolution and statistical learning techniques, we identified a 35-gene signature that (1) achieved high predictive accuracy for homeostatic model of IR (HOMA-IR) across BMI; (2) was expressed across a variety of non-immune cell populations (most prominently adipocytes and adipocyte stem and precursor cells [ASPCs]), with primarily “protective” IR associations for adipocyte transcripts and “deleterious” associations for macrophage transcripts; (3) displayed a high concordance between SAT and VAT (greater than non-IR associated genes). Multiple SAT genes exhibited dynamic expression 5-years after weight loss surgery and with insulin stimulation. Finally, using available expression quantitative trait loci in SAT and/or VAT, we demonstrate similar genetic effect sizes of SAT and VAT on type 2 diabetes and BMI, suggesting underlying similarities in genetic determinants of IR between adipose depots. These results implicate a dynamic transcriptional architecture of IR that resides in both immune and non-immune populations in SAT and that is shared with VAT, nuancing the current VAT-centric concept of IR in humans.

overall_design: The discovery cohort consisted of 76 samples collected from individuals with obesity and pre-diabetes by lipoaspiration from the subcutaneous adipose depot. These individuals had matching metadata including height, weight, and measures of insulin resistance.

first: Mona

last: Mashayekhi

last: Koethe

first: James

last: Luther

first: Nancy

last: Brown


title: RNA-seq analysis for MDA-MB-468 cells treated with SAHA and sonidedgib

city: Shanghai

first: Min

last: Huang

address: 555, Zuchongzhi Road

institute: Shanghai Institute of Materia Medica

summary: Epigenetic therapies that cause genome-wide epigenetic alterations, could trigger local interplay between different histone marks, leading to a switch of transcriptional outcome and cellular phenotypes. In human cancers with diverse oncogenic activation, how oncogenic pathways cooperate with epigenetic modifiers to regulate the histone mark interplay is poorly understood. We herein discover that the hedgehog (Hh) pathway reprograms the histone methylation landscape in triple-negative breast cancer (TNBC), which facilitates histone acetylation caused by histone deacetylase (HDAC) inhibitors and gives rise to new therapeutic vulnerability of combination therapies. Specifically, overexpression of zinc finger protein of the cerebellum 1 (ZIC1) in TNBC promotes Hh activation, facilitating the switch of H3K27 methylation (H3K27me) to acetylation (H3K27ac). The mutually exclusive relationship of H3K27me and H3Kac allows their functional interplay at oncogenic gene locus and switches therapeutic outcomes. Using multiple in vivo TNBC models including patient-derived xenograft, we show that Hh signaling-orchestrated H3K27me and H3Kac interplay tailors combination epigenetic drugs in treating TNBC.

first: Xiaomin

last: Wang

first: Jun

last: Xu

first: Ting


title: Splenic fibroblasts control marginal zone B-cell movement and function via two distinct Notch2-dependent regulatory programs

city: Philadelphia

first: Brian

last: Gaudette

address: 3620 Hamilton Walk, Pathology and Laboratory Medicine

institute: University of Pennsylvania

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

overall_design: Refer to individual Series