title: Clonally expanded targetable natural killer-like T cells seed the aged spinal cord to disrupt wound healing [scGEX and TCR]

city: London

first: Guiping

last: Kong

address: 55-96, Du Cane Road

department: Brain Sciences

institute: Imperial College London

summary: CD8+ T cells were found accumulated in the spinal cord preceding (aged) and following spinal cord injury. We used single cell TCR sequencing to analyze the clonality of T cells in the spinal cord and discovered that CD8+ T cells exhibit age and injury-dependent clonal expansion.

overall_design: Mouse spinal cord segments were collected from old-naive, old-28dpi and young-28dpi conditions. CD8+ and CD4+ T cells of the spinal cord were isolated by Fluorescence-activated Cell Sorting (FACS). Four biological replicates were included in each condition.

first: Luming

last: Zhou

first: Simone

last: Di Giovanni


title: Clonally expanded targetable natural killer-like T cells seed the aged spinal cord to disrupt wound healing

status: Public on Jan 12 2025

country: United Kingdom

type: Expression profiling by high throughput sequencing

summary: CD8+ T cells that were found accumulated in the spinal cord preceding (aged) and following a spinal cord injury disrupt wound healing. We isolated spinal cord cells of the aged mice who were treated with IgG or anti-CD8 monoclonal antibody following spinal cord injury and transcriptionally profiled them by doing single cell RNAseq to identify what cells were affected after depleting CD8+ T cells.

overall_design: Aged mouse spinal cord segments were collected from IgG or anti-CD8 antibody conditions 28 days after spinal cord injury. After, cells were isolated for single cell RNAseq. Two biological replicates were included in each condition.


title: Generation of expandable multipotential distal lung progenitors from human pluripotent stem cells that model idiopathic pulmonary fibrosis

status: Public on Jan 13 2025

city: New York

first: Hans-Willem

last: Snoeck

state: NY

address: 650 west 168th street

country: USA

institute: Columbia University Irving Medical Centre

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

overall_design: Refer to individual Series


title: Symmetric DNA Adenine Methylation Plays an Essential Role in Early-Diverging Fungi

city: Murcia

first: Carlos

last: Lax

address: Facultad de Biología

department: Genetics and Microbiology

country: Spain

institute: University of Murcia

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The discovery of N6-methyladenine (6mA), typically found in prokaryotic DNA, in eukaryotic genomes, has revolutionized epigenetics. Here, we show that symmetric 6mA is essential in the early diverging fungus Rhizopus microsporus, as the absence of the MT-A70 complex (MTA1c) responsible for this modification results in a lethal phenotype. 6mA is present in 70% of the genes, correlating with the presence of H3K4me3 and H2A.Z in open euchromatic regions. This modification is found predominantly in nucleosome linker regions, influencing the nucleosome positioning around the transcription start sites of highly expressed genes. Controlled downregulation of MTA1c results in a reduction of symmetric 6mA sites affecting nucleosome positioning and histone modifications, leading to altered gene expression, which is likely the cause of lethality. Our study highlights the indispensable role of the DNA 6mA in a multicellular organism and delineates the mechanisms through which this epigenetic mark regulates gene expression in a eukaryotic genome

overall_design: MNAse-seq experiments using R. microsporus WT and the UM67 mutant to analize nucleosome distribution across their genomes

first: Stephen

middle: J

last: Mondo

first: José

middle: F

last: Martínez

first: Anna

last: Muszewska

first: Leo

last: Baumgart

middle: A

last: Pérez-Ruiz

first: Pablo

last: Carrillo-Marín

first: Kurt

last: LaButti

last: Lipzen

first: Yu

last: Zhang

first: Jie

last: Huo

first: Vivian

last: Ng

first: Eusebio

last: Navarro

first: Teresa

middle: E

last: Pawlowska

first: Igor

middle: V

last: Grigoriev

first: Franciscos

last: Nicolás-Molina

first: Victoriano

last: Garre


title: Symmetric DNA Adenine Methylation Plays an Essential Role in Early-Diverging Fungi [RNA-seq]

last: Lax

overall_design: RNA-seq experiment to analize gene expression of R. microsporus ATCC 11559 growing with Zinc (20mg/L) and withouz Zinc (0mg/L)

first: Francisco


title: Cancer-associated fibroblast-specific expression of the matricellular protein CCN1 coordinates neovascularization and stroma deposition in melanoma metastasis

city: Saskatoon

first: Andrew

last: Leask

state: Saskatchewan

address: 105 Wiggins

department: College of Dentistry

country: Canada

institute: University of Saskatchewan

summary: Melanoma is the leading cause of skin cancer-related death. As prognosis of melanoma patients remains problematic, identification of new therapeutic targets remains essential. Matricellular proteins are non-structural extracellular matrix proteins. They are secreted into the tumor microenvironment to coordinate behavior among different cell types, yet their contribution to melanoma is under-investigated.  Examples of matricellular proteins include those comprising the CCN family. The CCN family member, CCN1, is highly pro-angiogenic. Herein, we show that, in human melanoma patients, although found in several tumor cell types, CCN1 is highly expressed by a subset of cancer-associated fibroblasts in melanoma patients and this expression correlates positively with expression of pro-angiogenic genes and progressive disease/resistance to anti-PD1 checkpoint inhibitors.  Consistent with these observations, in a syngeneic C57BL6 mouse model of melanoma, loss of CCN1 expression from Col1A2-Cre-fibroblasts impaired metastasis of subcutaneously-injected B16F10 tumor cells to lung, concomitant with disrupted neovascularization and collagen organization. Disruption of the extracellular matrix in the loss of CCN1 was validated using a novel artificial intelligence-based image analysis platform that revealed significantly decreased phenotypic fibrosis and composite morphometric collagen scores. As drug resistance is linked to matrix deposition and neo-angiogenesis, these data suggest that CCN1, due to its multifaceted role, may represent a novel therapeutic target for drug-resistant melanoma. Our data further emphasize the essential role that cancer-associated, Col1A2-Cre-fibroblasts and extracellular matrix remodeling play in coordinating behavior among different cell types within the tumor microenvironment

overall_design: Col1A2-Cre(ER)T/0; ROSA26mTmG mice (#007576, Jackson Laboratories) were used to specifically label Col1A2-Cre-fibroblasts with green fluorescent protein (GFP). At 3-week-old, mice were treated with tamoxifen (10 mg/mL) or corn oil (vehicle control) every day for 5 days. Six weeks later, mice were euthanized, and skin collected

first: James

last: Hutchenreuther

first: John

last: Nguyen

first: Katherine

last: Quesnel

first: Krista

middle: M

last: Vincent

first: Louis

last: Petitjean

first: Sophia

last: Bourgeois

first: Mark

last: Boyd

first: George

last: Bou-Gharios

first: Lynne-Marie

last: Postovit


title: Transcriptomic analysis of iPSC-derived endothelium reveals adaptations to high altitude hypoxia in energy metabolism and inflammation

city: Chicago

first: Anna

last: Di Rienzo

state: IL

department: Human Genetics

institute: University of Chicago

summary: Tibetan adaptation to high-altitude hypoxia remains a classic example of Darwinian selection in humans. Amongst Tibetans, alleles in the EPAS1 gene - whose protein product, Hif-2α, is a central regulator of the hypoxia response - have repeatedly been shown to carry some of the strongest signals of positive selection in humans and to influence several adaptive phenotypes. We recently showed that the selected haplotype at this locus spans a hypoxia-dependent enhancer (ENH5) that contributes to the regulation of EPAS1 in a variety of cell types. However, selective sweep signals alone may account for only part of the phenotypes that differentiate Tibetans from closely related lowlanders. Therefore, there is a pressing need to functionally probe adaptive alleles and their impact at the genome-wide level and across cell types to uncover the full range of beneficial traits. To cast a wider net, we established a library of induced pluripotent stem cells (iPSCs) derived from Tibetan and Han Chinese individuals, a robust model system allowing precise exploration of both locus-specific and genome-wide effects on transcriptional responses. We harness this system by differentiating the iPSC library into vascular endothelium and investigating the locus-specific effects of the ENH5 enhancer in this cellular context. In addition, we use it to explore Tibetan-specific transcriptome-wide responses and find evidence that energy metabolism and immune pathways have been shaped by natural selection in Tibetans. Finally, to aid with the interpretation of the transcriptional differences between populations, we test for polygenic adaptations as a complementary in silico approach for the identification of beneficial Tibetan phenotypes.

overall_design:
We developed a panel of sex-matched Tibetan and Han Chinese iPSCs using lymphoblastoid cell lines (LCLs) from 10 unrelated individuals of Tibetan origin and 10 Han Chinese (CHB) from the 1000 Genome Project.These 20 LCLs were reprogrammed into iPSCs in batches, balanced by population and sex and then successfully differentiated into vascular endothelium.
We used CRISPR-CAS9 editing to delete the region (chr2: 46578867 to 46579857; hg19) spanning the ENH5 enhancer region  of the iPSC line derived from Han individual CHB633  in order to create knockout cells carrying a low-altitude haplotype across the entire EPAS1 locus, but with complete loss of ENH5 activity. These DENH5 iPS cells (subsequently referred to as knockout, KO) and the original CHB633 (wild type, WT) iPS cells were differentiated into vascular endothelium in five independent replicates and cultured in hypoxic conditions (1% O2) for 48 hours.
In order to assess the impact of the ENH5 deletion in vascular endothelial cells, we performed single cell RNA-sequencing in a targeted 10,000 cells per KO and WT replicate. We assessed the expression of 25 endothelial markers (in the 10 UMAP clusters identified by Seurat) and identified two cell clusters as being robustly enriched in all endothelial markers. Transcript data from the cells in these two clusters were pooled to compare expression differences between KO and WT in a cell-type specific manner.
We utilized our complete panel of 20 cell lines to compare transcript levels between TAC and CHB iPSC-derived vascular endothelium under both normoxic and hypoxic conditions. Given the larger number of cell lines and treatment conditions, we performed bulk RNA-sequencing rather than single cell RNA-sequencing.

first: OA

last: Gray

first: DB

last: Witonsky

first: J

last: Jousma

first: DR

last: Sobreira

first: X

last: Van Alstyne

first: RT

last: Huang

first: Y

last: Fang

first: A


title: Clonally expanded targetable natural killer-like T cells seed the aged spinal cord to disrupt wound healing [scRNA-seq]

first: Guiping

last: Kong

summary: The wound healing ability of the central nervous system declines with aging. Spinal cord injury (SCI) has been increasingly affecting aged individuals, where functional impairment and mortality are highest. However, the aging-dependent mechanisms underpinning tissue damage and repair after SCI remain elusive, limiting effective interventions. Here, we hypothesized that changes to the molecular and cellular signatures of the local spinal cord tissue microenvironment across the lifespan might affect spinal wound healing, axonal injury responses and functional recovery. To test this hypothesis, we investigated the molecular and cellular signatures associated with the wound healing response after an experimental SCI in the mouse across the lifespan by conducting single-cell RNAseq (scRNAseq) experiments from the spinal cord of young and aged mice preceding and following a SCI.

overall_design: Young and aged mouse spinal cord segments were collected from conditions including naive, 14 days, and 28 days after spinal cord injury. After, cells were isolated for single cell RNAseq. Three biological replicates were included in each condition.

first: Luming

last: Zhou

first: Simone

last: Di Giovanni


title: An early precursor CD8 T cell that adapts to acute or chronic viral infection [scRNA-seq]

status: Public on Jan 11 2025

city: Atlanta

first: Donald

last: McGuire

state: GA

address: 1462 Clifton Road room 412

institute: Emory University School of Medicine

summary: This study examines the origin and differentiation of stem-like CD8+ T cells that are essential for sustained T cell immunity in chronic viral infections and cancer and also play a key role in PD-1 directed immunotherapy. These PD-1+ TCF-1+ TOX+ stem-like CD8+ T cells, also referred to as precursors of exhausted T cells, have a distinct program that allows them to adapt to chronic antigen stimulation. Using the mouse model of chronic LCMV infection we found that virus specific stem-like CD8+ T cells are generated early (day 5) during chronic infection suggesting that this crucial fate commitment occurs irrespective of infection outcome. Indeed, we found that nearly identical populations of stem-like CD8+ T cells were generated early after acute or chronic LCMV infection and that antigen was essential for maintaining the stem-like phenotype. We next performed reciprocal adoptive transfer experiments to determine the fate of these early stem-like CD8+ T cells after viral clearance versus persistence. Following transfer of day 5 stem-like CD8+ T cells from chronically infected into acutely infected mice, these cells downregulated canonical markers of the chronic stem-like CD8+ T cells and expressed markers (CD127 and CD62L) associated with central memory CD8+ T cells. Reciprocally, when day 5 stem-like cells from acutely infected mice were transferred into chronically infected mice these CD8+ T cells functioned like chronic resource cells and responded effectively to PD-1 therapy. These findings highlight the ability of these early PD-1+ TCF-1+ TOX+ stem-like CD8+ T cells to adapt their differentiation trajectory to either an acute or chronic viral infection. Most importantly, our study shows that the host is prepared a priori to deal with a potential chronic infection.

overall_design:
Samples 1-2: Endogenous, LCMV-specific GP33+ CD8+ T cells from days 5 or >45 after chronic LCMV infection.
Samples 3-4: LCMV-specific GP33+ P14 Stem-like, TCF7-YFP+ Tim-3-) were sorted from LCMV Armstrong infected mice on day 5 and were transferred into day 5 chronic LCMV infected mice. On day 15 post-transfer, stem-like donor P14 cells as well as endogenous GP33+ from the same recipient mice were isolated from the spleen and analyzed via scRNA-seq.

first: Daniel

middle: T

last: McManus

first: Rajesh

last: Valanparambil

first: Christopher

middle: B

last: Medina

middle: D

last: Scharer

first: Ewelina

last: Sobierajska

first: Yinghong

last: Hu

middle: Y

last: Chang

first: Andreas

last: Wieland

first: Judong

last: Lee

first: Tahseen

middle: H

last: Nasti

first: Masao

last: Hashimoto

first: James

middle: L

last: Ross

first: Nataliya

last: Prokhnevska

first: Maria

last: Cardenas

first: Amanda

last: Gill

first: Elisa

middle: C

last: Clark

first: Kathleen

last: Abadie

first: Arjun

last: Kumar

first: Jonathan

last: Kaye

first: Byron

last: Au-Yeung

first: Hao

last: Kueh

first: Haydn

last: Kissick

first: Rafi

last: Ahmed


title: Spatiotemporal Transcriptomics of Hyacinthus orientalis L. cultivars

city: Shatin

first: Kwan-Ho

last: WONG

address: The Chinese University of Hong Kong, Ma Liu Shui

department: School of Life Sciences

country: Hong Kong

institute: The Chinese University of Hong Kong

summary: This study aims to provide referrable transcriptomic data for the exploration of floral pigment biosynthesis in hyacinth cultivars. This dataset consists of 189 RNA-Seq libraries prepared for seven cultivars at different developmental stages and perianth partitions, generated 1256.8 gigabytes data in total. After removal of adaptor trimming and quality trimming, the qualified sequences were adopted for de novo assembly of mRNA. The gene expressions were quantified as read counts and then normalized into FPKM. This study provides the first profiling on the gene expression of hyacinth, contributing to the new-generation molecular breeding in producing noval hyacinth cultivars.

overall_design: In this study, we applied top-down approach in exploring the underlying genotypes on the selected cultivars (phenotypes) with different flower colours. Seven hyacinth cultivars with distinct flower colours were selected for transcriptome profiling, namely ‘Jan Bos’ (JB) in red, ‘Pink Pearl’ (PP) in pink, ‘Gipsy Queen’ (GQ) in orange, ‘City of Haarlem’ (CH) in yellow, ‘China Pink’ (CP) in pinkish white, ‘Delft Blue’ (DB) in blue and ‘Peter Stuyvesant’ (PS) in dark purple. Three consecutive developmental stages were targeted, namely the first stage with green flower buds (B), the second stage with coloured flower buds (C) and the third stage with flowers in full anthesis (A). Three perianth partitions of each flower were collected separately, namely the outer perianth lobes (o), the inner perianth lobes (n), and the perianth tube (t). The perianth partitions of a single flower were dissected with sterile blade, with removal of androecium and gynoecium using autoclaved forceps. Biological triplicates of each partitions at different timepoints of each cultivars were taken.

last: Wong

first: Hoi-Yan

last: Wu

first: Wood-Yee

last: Shum

first: Ho-Lam

last: Hui

first: Pang-Chui

last: Shaw

first: Tai-Wai

last: Lau


title: An early precursor CD8 T cell that adapts to acute or chronic viral infection [bulk RNA-seq]

first: Donald

last: McGuire

overall_design:
Samples 1-15: Endogenous, LCMV-specific GP33+ stem-like cells or Tim3+ TD CD8+ T cells from days 5 or >45 after chronic LCMV infection.
Samples 16-30: LCMV-specific GP33+ Stem-like or Tim-3+ TD cells were sorted from LCMV Armstrong infected mice or LCMV clone 13 infected mice on day 5.

first: Daniel

last: McManus

first: Rajesh

last: Valanparambil

first: Christopher

last: Medina

last: Scharer

first: Ewelina

last: Sobierajska

first: Yinghong

last: Hu

last: Chang

first: Andreas

last: Wieland

first: Judong

last: Lee

first: Tahseen

last: Nasti

first: Masao

last: Hashimoto

first: James

last: Ross

first: Nataliya

last: Prokhnevska

last: Cardenas


title: Multi-omic spatial atlas shows Deleted inMalignantBrain Tumours 1 (DMBT1) glycoprotein is lost in colonic dysplasia II

city: Nashville

first: Nicholas

last: Markham

state: TN

address: 2213 Garland Ave

institute: VUMC

summary: Colorectal cancer(CRC) is responsible for 50,000 deaths annually in the United States. Emerging evidence supports a causal role for pro-carcinogenic bacteria in the colonic microbiome. We previously showed toxigenic C. difficile from human CRC-associated bacterial biofilms accelerates tumourigenesis in ApcMin/+ mice, both in specific pathogen-free mice and in gnotobiotic mice colonised with a defined consortium of bacteria. To further understand host-microbe interactions during colonic tumourigenesis, we combined single-cell RNA-sequencing (scRNA-seq), spatial transcriptomics, and immunofluorescence to define the molecular spatial organization of colonic dysplasia in our consortium model with or without C. difficile. Our data show a striking bipartite regulation of Deleted in Malignant Brain Tumours 1 (DMBT1) in the inflamed versus dysplastic colon. From scRNA-seq, differential gene expression analysis of normal absorptive colonocytes at 2 weeks post-inoculation showed DMBT1 upregulated by C. difficile compared to colonocytes from mice without C. difficile exposure. In contrast, our spatial transcriptomic analysis showed DMBT1 dramatically downregulated in dysplastic foci compared with normal-adjacent tissue. We further integrated our datasets to generate custom colonic dysplasia scores and ligand-receptor mapping. Validation with immunofluorescence showed DMBT1 protein downregulated in dysplastic foci from three mouse models of colonic tumourigenesis. Finally, we demonstrated downregulation of DMBT1 mRNA and protein in adenomatous dysplasia from human samples. Together, our data reveal cell type-specific regulation of DMBT1, a potential mechanistic link between bacteria and colonic tumourigenesis.

overall_design: C57Bl/6 mice with the ApcMin/+ (Δ716 Apc) genotype were bred germ-free. At age 8-10 weeks, adult males in the C. difficile experimental group were inoculated with a single gavage of the 29-member bacterial consortium described by Drewes et al, (2022 Cancer Discovery) along with 104 spores of C. difficile strain Cd_3728T. The control group was given a single gavage of the same consortium but without C. difficile (n = 3 mice/group). Two weeks after inoculation, all mice were euthanised humanely for tissue harvesting and scRNA-seq.

first: Emily

last: Green

first: Julia

last: Drewes

middle: O


title: TUT1 promotes PDAC progression through Alternative Splicing [RNA-seq]

city: Beijing

first: wei

middle: zi

last: guo

state: Beijing

address: ZhongGuanCun

country: China

institute: tsinghua university

summary: Pancreatic ductal adenocarcinoma (PDAC) is a malignant cancer with high lethality rate. In this study, we identify that terminal Uridylyl Transferase 1 (TUT1), a specific terminal uridylyltransferase for U6 snRNA, is required for survival of PDAC, but not for that of normal adult pancreas. Mechanistically, TUT1 uridylylation activity promotes tri-snRNP assembly though facilitating the binding of LSM protein to U6 snRNA. TUT1 deletion leads to global alternative splicing (AS) changes which in turn triggers PDAC cell cycle dysregulation. Here, we reveal a novel function of TUT1 in regulating AS by modulating tri-snRNP levels and identify TUT1 as a potential therapeutic target for the treatment of PDAC.

overall_design:
Examination of transcriptome of TUT1 knockdown in mouse pancreatic ductal adenocarcinoma cells.
Examination of transcriptome of TUT1 knockout in mouse healthy pancreas.

first: Ziwei

last: Guo

first: Charles

last: David

first: Mo

last: Chen


title: TUT1 promotes PDAC progression through Alternative Splicing [3‘-seq]

overall_design: Examination of transcriptome of TUT1 knockdown in mouse pancreatic ductal adenocarcinoma cells.


title: Timing of exercise differentially impacts adipose tissue gain in male adolescent rats

status: Public on Jan 10 2025

city: Barcelona

first: Luis

middle: P

last: Iniguez

state: Barcelona

address: 88 Dr. Aiguader

department: Computational Biology and Health Genomics

institute: Centre for Genomic Regulation

summary: Circadian rhythms of metabolic, hormonal, and behavioral fluctuations and their alterations can impact health. An important gap in knowledge in the field is whether the time of the day of exercise and the age of onset of exercise exert distinct effects at the level of whole-body adipose tissue and body composition. The goal of the present study was to determine how exercise at different times of the day during adolescence impacts the adipose tissue transcriptome and content in a rodent model. Rats were subjected to one of four conditions during their adolescence: early active phase control or exercise (EAC or EAE; ZT13), and late active phase control or exercise (LAC or LAE; ZT23). The effects of exercise timing were assessed at the level of subcutaneous and visceral adipose tissue transcriptome, body composition, hypothalamic expression of orexigenic and anorexigenic genes, blood serum markers and 24-hour core body temperature patterns. We found that late active phase exercise (ZT23) greatly upregulated pathways of lipid synthesis, glycolysis and NADH shuttles in LAE rats, compared to LAC or EAE. Conversely, LAE rats showed notably lower content of adipose tissue. In addition, LAE rats showed notably increased serum FGF21 levels and other serum markers of glucose metabolism impairment compared to other groups. Finally, LAE rats showed higher post-exercise core body temperature compared to other groups. Our results thus indicate that our exercise protocol induced an unusual effect characterized by enhanced lipid synthesis but reduced adipose tissue content in late active-phase but not early active phase exercise during adolescence.

overall_design: RNA seq from subcutaneous or visceral adipose tissue (condition 1) from sedentary or exercised rats (condition 2) at zeitgeber time 13 or 23 (condition 3)

first: Yevheniy

last: Kutsenko

first: Luis P

last: Iñiguez

first: Alberto

last: Barreda

last: Pardo-Marin

first: Angel

last: Toval

first: Daniel

last: Garrigos

first: Marta

last: Martinez-Morga

first: Sergio

last: Pujante

first: Bruno

last: Ribeiro Do Couto

first: Kuey Y

last: Tseng

first: Joaquín J

last: Ceron

last: Garaulet

first: Marta B

last: Wisniewska

first: Manuel

last: Irimia

first: Jose L

last: Ferran


title: Neural Stem Cell Relay from B1 to B2 cells in the adult mouse Ventricular-Subventricular Zone

city: San Francisco

first: Arantxa

last: Cebrian Silla

state: California

address: 35 Medical center Way

department: Neurological Surgery

institute: UCSF

summary: Neurogenesis and gliogenesis continue in the Ventricular-Subventricular Zone (V-SVZ) of the adult rodent brain. B1 cells are radial glia-derived astroglial cells that function as primary progenitors or neural stem cells (NSCs) in the V-SVZ.  B1 cells, which have an apical contact with the ventricle, decline during early postnatal life, yet neurogenesis continues into adulthood. We found that a second population of V-SVZ astroglial cells (B2 cells), that do not contact the ventricle, function as NSCs in the adult brain. B2 cell numbers increase postnatally, remain constant in 12-month-old mice, and decrease by 18 months. Transcriptomic analysis revealed differences between B1 and B2 cells and shows that like B1 cells, B2 cells can be quiescent or activated.  Transplantation and lineage tracing of B2 cells demonstrate their function as primary progenitors for adult neurogenesis. This study reveals that NSC function is relayed from B1 to B2 progenitors to maintain adult neurogenesis.

overall_design: 10x Technology single cell sample preparation and multiplexing. Samples from mice injected with Ad:Cre:GFP were prepared as following:  15-24 hours after Ad:Cre:GFP intraventricular injections, mice (Experiment 1(ACSAAB01): n=14, P27-30, Experiment 2 (ACSAAB02): n=12, P27-p30) received intraperitoneal administration of 2.5% Avertin followed by decapitation. Brains were extracted and 0.5 mm slices were obtained with a mouse brain slicer (Steel Brain Matrix - Coronal 0.5mm, Alto). The V-SVZ lateral wall and dorsal wedge were microdissected and considered separate samples. To analyze the transcriptomic profile of aged mice we also microdissected the V-SVZ of two non-infected hGFAP:GFP 1 year old mice. The wedge region was not included. All V-SVZ micro-dissections were performed in L-15 medium on ice and the tissue was transferred to Papain-EBSS (LK003150, Worthington). Tissue was digested for 30 min at 37ºC in a thermomixer at 900 RPM. Mechanical dissociation with a P1000 pipette tip (20 seconds), then with a  fire-polished pasteur pipette for 3 min.  Cells were centrifuged for 5 min, 300 RCF at room temp, and the pellet was resuspended with DNAase/ovomucoid inhibitor according to the manufacturer's protocol (Worthington). Cells were incubated in red blood cell lysis buffer (420301, Biolegend) 3-4 min at 4ºC. For MULTI-seq barcoding, cells were suspended with Anchor:Barcode solution for 5 minutes at 4ºC. A Co-Anchor solution was added and incubated for 5 min87. Samples were combined and filtered with a FlowMi 40µm filter (BAH136800040-50EA, Sigma). To remove myelin, the cell suspension was incubated with Myelin Removal Beads (130-096-733, Miltenyi Biotec) (6μl/brain) for 15 min at 2-8°C. Cells were washed with 0.5% BSA-PBS and transferred to MACS columns. The effluent was collected, and the cell density was counted. Cells were loaded into a 10x Genomics Chromium Single Cell Controller. We used the 10x Genomics Chromium Single Cell 3’ Library & Gel Bead Kit v3.1 to generate cDNA libraries for sequencing according to the manufacturer's protocols. MULTI-Seq barcode libraries were prepared according to MULTI-Seq (McGinnis et al. 2019)  protocols. We confirmed that isolated cells from wedge dissections did not contain apical cells (GFP+ and/or  TdTomato+ cells) under an epifluorescence microscope. Libraries were sequenced in an Illumina NovaSeq 6000. Alignment was performed on STAR v2.7.11 and included both intronic and exonic mappings (--soloFeatures Gene GeneFull Velocyto). We used an optimized mouse reference genome 88 edited to include GFP and Cre genes. We also included a WPRE sequence to serve as a proxy for TdTomato expression.

last: Cebrian-Silla

first: Marcos

last: Assis Nascimento

first: Walter

last: Mancia

first: Susana

last: Gonzalez-Granero

first: Ricardo

last: Romero-Rodriguez

first: Kirsten

last: Obernier

first: David

last: M Steffen

last: A Lim

first: Jose Manuel

last: Garcia-Verdugo

first: Arturo

last: Alvarez-Buylla


title: EZH2 INHIBITION ENHANCES T CELL IMMUNOTHERAPIES BY INDUCING LYMPHOMA IMMUNOGENICITY AND IMPROVING T CELL FUNCTION (CUT&RUN)

first: Matt

last: Teater

address: 445 E 69th St

institute: Weill Cornell Medical College

summary: While T cell immunotherapies have demonstrated effectiveness in a subset of patients with diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), they are currently administered without guidance regarding whether the underlying biology or immunology is conducive to a durable response. Moreover, mechanisms explaining CAR-T resistance in B cell lymphomas remain largely unknown, and suitable systems to study lymphomas are currently unavailable, making it infeasible. To address these unmet needs, we developed syngeneic animal models reflecting the genetics, epigenetics, and immunology of human FL and DLBCL. We show that treatment of these models with EZH2 inhibitors reprogrammed them to re-express T cell engagement genes and rendered them highly immunogenic. Notably, EZH2 inhibitors did not exhibit deleterious effects on T cells. Instead, they promoted a memory CAR CD8 phenotype and reduced exhaustion, resulting in a superior elimination of lymphoma B cells and decreased tumor burden. Intravital 2-photon imaging of transformed FL bearing mice showed that EZH2 inhibition increased the recruitment of CAR-T into the tumor microenvironment and enhanced the quality of interactions with lymphoma cells, leading to increased killing. Therefore, inhibition of EZH2 enhances CAR-T cell activity through direct effects on CAR-T cells, in addition to rendering lymphoma B cells immunogenic. This offers an innovative approach for enhancing the prognosis of patients with refractory B cell lymphomas, and is currently under evaluation in a clinical trial (NCT05934838)

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: CUT&RUN in tFL cell line model, PDX samples

first: Yusuke

last: Isshiki

first: Cem

last: Meydan

first: Ari

last: Melnick


title: Spatial analysis of young and old murine lungs exposed to ozone and fibrogenic stress [spatial_ozone]

city: Salt Lake City

first: Alessandro

last: Venosa

state: UT

department: Pharmacology and Toxicology

institute: University of Utah

summary: Ozone (O3) is a ubiquitous pollutant known to produce acute and transient inflammation through oxidative injury and inflammation. These effects are exacerbated in susceptible populations, such as the elderly and those exhibiting genetic mutations in central nodes of pulmonary function. To comprehend the impact of these predisposing factors, the present study examines structural, mechanical, and immunological responses to single acute O3 exposure (0.8 ppm, 3h) in young (12 week old) and old (80 week old) mice. We therefore examined the impact of ozone in  a surfactant competent and mutant murine line. The latter models a clinically relevant mutation in the gene encoding for the alveolar epithelial type 2 specific surfactant protein C. Ozone exposure produced focal injury localized at the terminal bronchiole-to-alveolar junctions and enlarged alveoli in aged SP-C mutant lungs. This histological feature was used to select regions of interest for spatial transcriptomics.  Paraffin sections of 4 mice were prepared: a young/12wk SP-C wild type air control, a young/12wk SP-C wild type ozone (0.8 ppm, 3h), a young/12wk SP-C mutant ozone, and an old/80wk ozone. Immunofluorescence for DNA, CD45, CD68 was performed to identify macrophages (CD45+CD68+) and non-immune ("other", DNA+CD45-) cells within healthy (air control only), peri-injured and injured regions of the lung. A total of 24 regions (680um x 700um in size) were selected from CD68+ macrophages and CD45- non immune/"other" cells. RNA sequencing was then performed. Principal component and pathway analysis revealed spatial heterogeneity in response to ozone, as well as a senescence signature in the aging lungs.  These results illustrate regional and age dependent heterogeneity in immune and non-immune cells of the lung during an acute exposure to air pollution.

overall_design: 4 sections were prepared: young/12wk SP-C wild type air control, a young/12wk SP-C wild type ozone (0.8 ppm, 3h), a young/12wk SP-C mutant ozone, and an old/80wk ozone - spatial analysis of CD45+CD68+ macrophages and DNA+CD45- non-immune ("other") cells.

first: Jessica

last: Noll


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB  [SLAM seq in J1 ES]

city: Gosselies

first: Martin

last: Bizet

address: Rue Adrienne Bolland, 8

country: Belgium

institute: Université Libre de Bruxelles

summary: Marking of DNA, histones, and RNA is central to gene expression regulation in development and disease. Recent evidence links m6A, installed on RNA by the METTL3-METTL14 methyltransferase complex, to histone modifications, but the link between m6A and DNA methylation remains scarcely explored. This study shows that METTL3-METTL14 recruits the DNA methyltransferase DNMT1 to chromatin for gene-body methylation. We identify a set of genes whose expression is fine-tuned by both gene-body 5mC, which promotes transcription, and m6A, which destabilizes transcripts. We demonstrate that METTL3-METTL14-dependent 5mC and m6A are both essential for the differentiation of embryonic stems cells to embryoid bodies and that the upregulation of key differentiation genes during early differentiation depends on the dynamic balance between increased 5mC and decreased m6A. Our findings add a surprising new dimension to our understanding of how epigenetics and epitranscriptomics combine to regulate gene expression and impact development, most likely among other biological processes.

overall_design: Ribodepleted total RNA from ES cells wild type, knocked out for Dnmt1 and Mettl3i treated were analysed for expression levels using RNA-seq technology

first: Giuseppe

last: Quarto

first: Andrea

last: Li Greci

first: Penning

last: Audrey

first: Irina

last: Primac

first: Frédéric

last: Murisier

first: Liliana

last: Garcia-Martinez

first: Rodrigo

last: L. Borges

first: Qingzeng

last: Gao

first: Pradeepkumar

last: Cingaram

first: Emilie

last: Calonne

first: Bouchra

last: Hassabi

first: Celine

last: Hubert

first: Adèle

last: Herpoel

first: Pascale

last: Putmans

first: Frédérique

last: Mies

last: Jérôme

first: Louis

last: Van Der Linden

first: Gaurav

last: Dube

first: Kumar

last: Pankaj

first: Romuald

last: Soin

first: Jie

last: Lan

first: Morgane

last: Paque

first: Arnaud

last: Blomme

first: Pierre

last: Close

first: Pierre-Olivier

last: Esteve

first: Kristin

last: M. Riching

first: Elizabeth

last: Caine

first: Cyril

last: Gueydan

first: Daniels

last: Danette

first: Pradhan

last: Sriharsa

first: Ramin

last: Shiekhattar

first: Yael

last: David

first: Morey

last: Lluis

first: Jana

last: Jeschke

first: Deplus

last: Rachel

first: Evelyne

last: Collignon

first: François

last: Fuks


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB [RNAseq in Mettl14KO and rescued in E14TG2a ES/EB]

first: Martin

last: Bizet

overall_design: Ribodepleted total RNA from ES and EB cells wild type, rescued and knocked out for Mettl14 were analysed for expression levels using RNA-seq technology

first: Giuseppe

last: Quarto

first: Andrea

last: Li Greci

first: Penning

last: Audrey

first: Irina

last: Primac

first: Frédéric

last: Murisier

first: Liliana

last: Garcia-Martinez

first: Rodrigo

last: L. Borges

first: Qingzeng

last: Gao

first: Pradeepkumar

last: Cingaram

first: Emilie

last: Calonne

first: Bouchra

last: Hassabi

first: Celine

last: Hubert

first: Adèle

last: Herpoel

first: Pascale

last: Putmans

first: Frédérique

last: Mies

last: Jérôme

first: Louis

last: Van Der Linden

first: Gaurav

last: Dube

first: Kumar

last: Pankaj

first: Romuald

last: Soin

first: Jie

last: Lan


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB  [Polysome seq in HeLa]

first: Martin

last: Bizet

overall_design: Ribodepleted total RNA from HeLa cells wild type and knocked out for METTL3 were analysed for expression levels using RNA-seq technology

first: Giuseppe

last: Quarto

first: Andrea

last: Li Greci

first: Penning

last: Audrey

first: Irina

last: Primac

last: Murisier

first: Morgane

last: Paque

first: Arnaud

last: Blomme

first: Pierre

last: Close

first: Pierre-Olivier

last: Esteve

first: Kristin

last: M. Riching

first: Elizabeth

last: Caine

first: Cyril

last: Gueydan

first: Daniels

last: Danette

first: Pradhan

last: Sriharsa

first: Ramin

last: Shiekhattar

first: Yael

last: David

first: Morey

last: Lluis

first: Jana

last: Jeschke

first: Deplus

last: Rachel

first: Evelyne

last: Collignon

first: François

last: Fuks


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB  [ActinoD in E14TG2a ES]

first: Martin

last: Bizet

overall_design: Ribodepleted total RNA from mouse ES cells wild type and knocked out for Mettl14 were analysed for expression levels using RNA-seq technology

first: Giuseppe

last: Quarto

first: Andrea

last: Li Greci

first: Penning

last: Audrey

first: Irina

last: Primac

first: Frédéric

last: Murisier

first: Liliana

last: Garcia-Martinez

first: Rodrigo

last: L. Borges

first: Qingzeng

last: Gao

first: Pradeepkumar

last: Cingaram

first: Emilie

last: Calonne

first: Bouchra

last: Hassabi

first: Celine

last: Hubert

first: Adèle

last: Herpoel

first: Pascale

last: Putmans

first: Frédérique

last: Mies

last: Jérôme

first: Louis

last: Van Der Linden

first: Gaurav

last: Dube

first: Kumar

last: Pankaj

first: Romuald

last: Soin

first: Jie

last: Lan


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa and EB  [5mC in RNAi-treated HeLa]

first: Martin

last: Bizet

overall_design: Total genomic DNA from HeLa cells RNAi treated against DNMT1, DNMT3A, DNMT3B, SETD2 and control were analysed for 5mC changes using Infinium 850k

first: Giuseppe

last: Quarto

first: Andrea

last: Li Greci

first: Penning

last: Audrey

first: Irina

last: Primac

last: Murisier

first: Morgane

last: Paque

first: Arnaud

last: Blomme

first: Pierre

last: Close

first: Pierre-Olivier

last: Esteve

first: Kristin

last: M. Riching

first: Elizabeth

last: Caine

first: Cyril

last: Gueydan

first: Daniels

last: Danette

first: Pradhan

last: Sriharsa

first: Ramin

last: Shiekhattar

first: Yael

last: David

first: Morey

last: Lluis

first: Jana

last: Jeschke

first: Deplus

last: Rachel

first: Evelyne

last: Collignon

first: François

last: Fuks


title: The impact of IL-33 on Srebf2-dependent cholesterol gene expression in ST2+ eVAT Tregs

first: Chris

last: Scharer

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

summary: Tregs are highly enriched in the eVAT of male mice. We show that ST2+ VAT Tregs preferentially upregulate and depend on Srebf2-mediated cholesterol metabolism compared their spleen Treg or ST2- VAT Treg counterparts. However, it is unclear whether this increased reliance on cholesterol metabolism is a consequence elevated IL-33 signaling in ST2+ Tregs. Therefore, the goal of experiment is to determine whether IL-33 signaling influences Srebf2-dependent cholesterol gene expression in ST2+ eVAT Tregs in vivo.

overall_design: Foxp3-YFPcre Srebf2 wt/wt or Srebf2 flox/flox mice were given 2 doses of either PBS or 2ug recombinant IL-33 (3 days apart) the final week before sacrifice. 1000 ST2+ Tregs were isolated and sort-purified for bulk-RNA sequencing.

first: Christopher

first: Chaoran

last: Li

first: Cody

last: Elkins


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB  [PROseq in E14TG2a ES/EB]

first: Martin

last: Bizet

overall_design: Ribodepleted total RNA from ES cells wild type were analysed at various time points for expression levels using RNA-seq technology

first: Giuseppe

last: Quarto

first: Andrea

last: Li Greci

first: Penning

last: Audrey

first: Irina

last: Primac

first: Frédéric

last: Murisier

first: Liliana

last: Garcia-Martinez

first: Rodrigo

last: L. Borges

first: Qingzeng

last: Gao

first: Pradeepkumar

last: Cingaram

first: Emilie

last: Calonne

first: Bouchra

last: Hassabi

first: Celine

last: Hubert

first: Adèle

last: Herpoel

first: Pascale

last: Putmans

first: Frédérique

last: Mies

last: Jérôme

first: Louis

last: Van Der Linden

first: Gaurav

last: Dube

first: Kumar

last: Pankaj

first: Romuald

last: Soin

first: Jie

last: Lan

first: Morgane

last: Paque

first: Arnaud

last: Blomme

first: Pierre

last: Close

first: Pierre-Olivier


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB  [ActinoD in HeLa]

first: Martin

last: Bizet

first: Giuseppe

last: Quarto

first: Andrea

last: Li Greci

first: Penning

last: Audrey

first: Irina

last: Primac

first: Frédéric

last: Murisier

first: Liliana

last: Garcia-Martinez

first: Rodrigo

last: L. Borges

first: Qingzeng

last: Gao

last: Esteve

first: Kristin

last: M. Riching

first: Elizabeth

last: Caine

first: Cyril

last: Gueydan

first: Daniels

last: Danette

first: Pradhan

last: Sriharsa

first: Ramin

last: Shiekhattar

first: Yael

last: David

first: Morey

last: Lluis

first: Jana

last: Jeschke

first: Deplus

last: Rachel

first: Evelyne

last: Collignon

first: François

last: Fuks


title: Gene expression in primed, coculture with mouse ESC, coculture-derived naïve-like state, and chemically reset (cR) naïve human H9 ESCs.

city: Cincinnati

first: Kentaro

last: Iwasawa

state: OH

address: 3333 BURNET AVENUE

department: Division of Gastroenterology, Hepatology and Nutrition

institute: Cincinnati Children's Hospital Medical Center

summary: We compared the global transcriptome between primed, coculture, coculture-derived naïve-like state human ESCs together with the cR naïve cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for human ESCs cultured in the different protocols.

first: Yosuke

last: Yoneyama

first: Takanori

last: Takebe


title: Identification of the subventricular tegmental nucleus as brainstem reward center

city: Budapest

first: <Up loaded by Bioinformatics core  facility>

last: <PI shown in the article>

address: Szigony st 43.

country: Hungary

institute: HUN-REN Institute of Experimental Medicine

summary: Rewards are essential for motivation, decision-making, memory, and mental health. We identified the subventricular tegmental nucleus (SVTg) as a brainstem reward center. In mice, reward and its prediction activate the SVTg, and SVTg stimulation leads to place preference, reduced anxiety, and accumbal dopamine release. Mice self-stimulate the SVTg, which can also be activated directly by the neocortex, resulting in effective inhibition of the lateral habenula, a region associated with depression. This mechanism may also explain why SVTg suppression induces aversion and increases fear. The translational relevance of these findings is supported by evidence in the rat, monkey, and human brainstem, establishing SVTg as a key hub for reward processing, emotional valence, and motivation.

overall_design: Single cell RNA-sequencing on virally labelled SVTg GABAergic neurons collected from vGAT-Cre mice injected with AAVretro-EF1a-DIO-mCherry into bilateral LHb. The GN029 cell was excluded from further analysis because it did not express either the Cre enzyme or the mCherry gene.

first: Krisztián

last: Zichó

first: Boldizsár

last: Balog

first: Réka

middle: Z

last: Sebestény

first: János

last: Brunner

first: Virág

last: Takács

first: Albert

last: Barth

first: Charlotte

last: Seng

first: Áron

last: Orosz

first: Manó

last: Aliczki

first: Hunor

last: Sebők

first: Éva

last: Mikics

first: Csaba

last: Földy

last: Szabadics

first: Gábor

last: Nyiri


title: The impact of Srebf2 deletion on the transcriptional phenotype of visceral adipose tissue (VAT) regulatory T (Treg) cells

first: Chris

last: Scharer

summary: Regulatory T (Treg) cells are highly enriched in the visceral adipose tissue (VAT) to maintain metabolic homeostasis, but they are lost during obesity. VAT Treg cells exhibit enhanced Srebf2-dependent cholesterol metabolism compared lymphoid tissue Tregs at steady state, but this metabolic pathway is disrupted during obesity. Therefore, the goal of this study is to determine the impact of Treg-specific Srebf2 deletion on the transcriptional phenotype of bulk Foxp3+ VAT or splenic Tregs.

overall_design: Live CD3+ CD4+ Foxp3+ Tregs were purified by FACS sorting from the VAT or spleen of male 25-week-old Foxp3 YFP-cre Srebf2-wt/wt or Srebf2-flox/flox mice for bulk-RNA sequencing.

first: Christopher

first: Chaoran

last: Li


title: RBBP6 anchors pre-mRNA 3’ end processing to nuclear speckles for efficient gene expression

city: Irvine

first: Yoseop

last: Yoon

state: CA

address: 1002 Health Sciences Rd

institute: University of California Irvine

summary: Pre-mRNA 3′ processing is an integral step in mRNA biogenesis. However, where this process occurs in the nucleus remains unknown. Here, we demonstrate that nuclear speckles (NSs), membraneless organelles enriched with splicing factors, are major sites for pre-mRNA 3′ processing in human cells. We show that the essential pre-mRNA 3′ processing factor RBBP6 associates strongly with NSs via its C-terminal intrinsically disordered region (IDR). Importantly, although the conserved N-terminal domain (NTD) of RBBP6 is sufficient for pre-mRNA 3′ processing in vitro, its IDR-mediated association with NSs is required for efficient pre-mRNA 3′ processing in cells. Through proximity labeling analyses, we provide evidence that pre-mRNA 3′ processing for over 50% of genes occurs near NSs. We propose that NSs serve as hubs for RNA polymerase II transcription, pre-mRNA splicing, and 3′ processing, thereby enhancing the efficiency and coordination of different gene expression steps.

overall_design: To study the role of RBBP6 and its localization to nuclear speckles in pre-mRNA 3' processing, we conducted the following experiments: 1. Depleted RBBP6 and performed 4sU-seq to detect transcription readthrough. 2. Depleted RBBP6 and rescued it with either full-length or domain-deleted RBBP6 variants, then performed PAS-seq to detect changes in polyadenylation sites (PAS). 3. Overexpressed RBBP6 variants and performed PAS-seq. 4. Depleted nuclear speckle scaffold proteins and performed PAS-seq

first: Elodie

last: Bournique

first: Lindsey

last: Soles

first: Hong

last: Yin

first: Yinyin

last: Zhuang

first: Xiangyang

last: Liu

first: Liang

first: Clinton

last: Yu

first: Marielle

last: Valdez

first: Lusong

last: Tian

first: Lan

last: Huang

first: Xiaoyu

last: Shi

first: Fangyuan

last: Ding

last: Tong

first: Remi

last: Buisson

first: Yongsheng


title: CRISPR/Cas9 mutagenesis screening identifies PRDM1 as a key regulator of the natural killer T cell central memory program and effector function

city: Houston

first: Antonino

last: Montalbano

state: TX

address: 1102 Bates Ave

institute: Baylor School of Medicine

summary: Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in regulation of NKT therapeutic activity have been identified. To discover regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that employs a guide (g)RNA library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (CAR.NKTs) were transduced with the gRNA library and exposed to CD1d+/GD2- leukemia or CD1d-/GD2+ neuroblastoma cells, respectively, over six challenge cycles in vitro. Quantification of gRNA abundance revealed enrichment of PRDM1-specific gRNAs in both NKTs and CAR.NKTs, a result that was validated through targeted PRDM1 knockout. Transcriptional, phenotypic, and functional analyses demonstrated that CAR.NKTs with PRDM1 knockout undergo central memory-like differentiation and resist exhaustion while downregulating granzyme B and mediating reduced cytotoxicity in vitro and moderate antitumor activity in an in vivo xenogeneic neuroblastoma model. In contrast, shRNA-mediated knockdown of PRDM1 expression preserved CAR.NKT effector function while promoting central memory differentiation, resulting in CAR.NKTs with potent in vivo antitumor activity. Thus, we identified PRDM1 as a regulator of NKT memory differentiation and effector function that can be exploited to improve the efficacy of next-generation NKT-based cancer immunotherapy.

overall_design: To determine how PRDM1 CRISPR KO impacts GD2.CAR NKT gene expression, we sequenced RNA isolated from PRDM1 KO GD2.CAR NKTs after one and six rounds of tumor challenge. To determine the impact of knocking down PRDM1 on the GD2.CAR NKT transcriptome, we defined the genome-wide expression profile of PRDM1 KD GD2.CAR NKTs and compared with that of scrambled shRNA control cells after one cycle or six cycles of serial tumor challenge.

first: Gengwen

first: Gabriel

last: Barragan

first: Hangjin

first: Claudia

last: Amador

first: Akshaya

last: Adaikkalavan

first: Xavier

last: Rios

first: Linjie

last: Guo

first: Janice

last: Drabek

first: Osmay

last: Pardias

first: Chunchao

last: Zhang

first: Yanchuan

first: Amy

middle: N

last: Courtney

first: Erica

middle: J

last: Di Pierro

first: Leonid

middle: S

last: Metelitsa


title: Obesity disrupts cellular cholesterol homeostasis to inhibit the homeostatic clonal expansion of ST2hi VAT Treg cells

first: Gregory

middle: K

last: Tharp

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

summary: Regulatory T (Treg) cells are highly enriched in the visceral adipose tissue (VAT) to maintain metabolic homeostasis, but they are lost during obesity. Currently, how VAT Treg cells rewire cellular metabolism to support their homeostatic clonal expansion, and whether this process is disrupted in obesity, is unclear. Here, we found that cholesterol metabolism was uniquely upregulated in VAT-, but not other non-lymphoid-tissue Treg cells. Disrupting cholesterol homeostasis (CH) by Treg-specific deletion of Srebf2 led to a specific loss of VAT Treg cells and enhanced obesity-induced systemic metabolic dysfunction. Mechanistically, Srebf2-mediated CH potentiated strong TCR signaling, which specifically promoted the clonal expansion of ST2hi, but not other, VAT Treg subsets. However, long-term high-fat-diet feeding disrupted VAT Treg CH and impaired ST2hi VAT Treg clonal expansion. Restoring Treg CH rescued VAT Treg accumulation in obese mice, suggesting CH modulation could be a potential option for Treg-targeted therapies in obesity-associated metabolic diseases.

overall_design: VAT Treg cells were isolated from 25-week-old male Foxp3YFP-cre Srebf2WT/WT (n=4) and Srebf2flox/flox (n=4) mice at steady state and from 23-27-week-old male Foxp3thy1.1 PpargTdt mice fed a normal chow diet (NCD) (n=5) and high fat diet (HFD) (n=5) for 22 weeks.

first: Cody

last: Elkins

first: Chengyu

last: Ye

first: Pulavendran

last: Sivasami

first: Roy

last: Mulpur

first: Pamela

last: Diaz-Saldana

last: Peng

first: Miaoer

last: Xu

first: Yeun-Po

last: Chiang

first: Samara

last: Moll

first: Dormarie

last: Rivera

first: Luisa

last: Cervantes-Barragan

first: Christopher

last: Scharer

first: Mandy

last: Ford

first: Haydn

last: Kissick

first: Chaoran

last: Li


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB [m6A in ES and EB and RNAseq in EB]

first: Martin

last: Bizet

type: Methylation profiling by high throughput sequencing

summary: The epigenetic marking of DNA, histones, and RNA is central for regulating gene expression in development and disease. On DNA, well-characterized 5-methylcytosine (5mC) controls the chromatin state. On mRNA, the most prevalent internal mark, N6-methyladenosine (m6A), installed by METTL3-METTL14 methyltransferases, regulates RNA metabolism. Recent evidences link RNA modifications with epigenetics, notably by connecting m6A to histone marks. The crosstalk between m6A and DNA methylation, however, remains poorly understood. Here we reveal a direct mechanistic link between m6A and DNA methylation. Our findings indicate a proximal co-occurrence of m6A and 5mC marks at gene body and this contributes to enhanced gene expression. We show that the m6A writer METTL3 regulates intragenic methylation and gene expression. Mechanistically, we evidence a direct binding of METTL3-METTL14 to the 5mC methyltransferase DNMT1 and reveal that chromatin-tethered METTL14 recruits DNMT1 to chromatin. We demonstrate that gene-body 5mC and mRNA-stability-enhancing effect of coding-sequence m6A contribute to increased gene expression. We further substantiate our findings in embryonic stem cells by showing that METTL3 regulates gene-body methylation and expression of key differentiation markers. Altogether, our work establishes a key layer of gene expression regulation, involving a direct mechanistic crosstalk between DNA methylation and RNA methylation machinery.

overall_design: PolyA enriched RNA from ES and EB cells wild type and knockout for METTL3 were analyzed for expression using RNAseq. Additionally, PolyA enriched RNA from EB wild type cells were analysed for m6A marks using m6A-seq technology.

first: Andrea

last: Li Greci

first: Giuseppe

last: Quarto

first: Irina

last: Primac

first: Pankaj

last: Kumar

first: Gaurav

last: Dube

first: Jana

last: Jeschke

first: François

last: Fuks


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB [5mC in ES and EB]

type: Methylation profiling by genome tiling array

overall_design: Total genomic DNA from mouse EB cells wild type and knocked out for METTL3 were analyzed for 5mC changes using Infinium MouseMethylation array


title: Multipotent neural stem cells originating from neuroepithelium exist outside of the mouse central nervous system

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary:
It has been widely accepted that mammalian neural stem cells (NSCs) exist only in the central nervous system. Here, we report that peripheral NSCs (pNSCs) exist outside of the CNS and can be isolated—from mouse embryonic limb, postnatal lung, tail, dorsal root ganglia, and adult lung tissues. pNSCs are from Sox1+ cells and originate from neuroepithelial cells. Many pNSCs that migrate out of the neural tube can differentiate into neurons and limited glia cells during embryo development. We found the existence of pNSCs provides previously unidentified insight of the mammalian nervous system development and presents an alternative potential strategy for neural regenerative therapy. Our study demonstrates that directly converted cells can be a promising source for generating transplantable assembloids.
Mice and Cells: RNA-seq : RNA quality was verified on an Agilent Bioanalyzer Nano Eukaryote chip. 1 mg of total RNA was used for poly-A selection with NEBNext PolyA mRNA magnetic isolation module and subsequent cDNA synthesis, Illumina Tru-seq adaptor ligation and library preparation were performed with NEBNext Ultra RNA Library Prep Kit (NEB) according to the manufacturer’s instructions. Library quality and concentration were determined using an Agilent Bioanalyzer DNA1000 chip and a Qubit fluorometer. Libraries were sequenced on Illumina NextSeq500 in single-end mode. HISAT24 was used to align the RNA‐seq reads for each of the samples to the mouse reference genome GRCm38, and Cufflinks to annotate them. The counts of aligned reads to each gene were calculated with HTSeq. In‐house software was used to merge the expression results into a single text file used in the downstream analysis in Matlab (MathWorks). We performed quantile normalization to equalize the data and stabilized them through the log2 transform of the data plus one. The heatmap of the most highly variable transcripts, the hierarchical clustering dendrograms (calculated using the unweighted pair group method with arithmetic mean and Euclidean distance measure), and the PCAs were performed using in‐home functions developed in Matlab (MathWorks).

overall_design: The data Tail-pNSC (pNSC), is performed with 2 replicates. The data Brain-NSC (NSC), is performed with 2 replicates. The data Tail (Tail), is performed with 2 replicates. The data Lung-pNSC (pNSC), is performed with 2 replicates. The data Lung (Lung), is performed with 2 replicates. The data Sox2-GFP brain NSCs (NSC), is performed with 3 replicates. The data Sox2-GFP lung pNSCs (pNSC), is performed with 3 replicates. The data Wnt1Cre-mTmG Tail pNSCs (pNSC), is performed with 3 replicates. The data DRG NCSCs (NSC), is performed with 3 replicates. The data Wnt1Cre-mTmG brain NSCs (NSC), is performed with 3 replicates. The data Wnt1Cre-mTmG DRG pNSCs (pNSC), is performed with 3 replicates.

first: Dong

last: Han

first: Daniela

last: Gerovska

first: Hans

middle: R

last: Schöler


title: Gene expression profiles at single cell level of CD45+ tumor-infiltrating immune cells in intra- and extra-cranial tumors

city: Lausanne

first: Nadine

last: Fournier

state: Vaud

address: Agora Cancer Research Center, Bugon 25A

country: Switzerland

institute: Agora Cancer Research Center & Swiss Institute of Bioinformatics

summary: Tumor cells and the anatomical location determine the composition and functionality of the non-malignant tumor microenvironment (TME). To determine the influence of the anatomical location, we have compared the transcriptome of CD45+ tumor-infiltrating immune cells isolated from intra- and extra-cranial tumors, respectively. The same tumor cell line was used to generate both tumor models.

overall_design: Fluorescence-activated cell sorting was used to isolate CD45+ immune cells from intra- and extra-cranial tumors established through orthotopic injection of a breast cancer (BC) brain metastasis (BrM) cell line either in the brain or the mammary fat pad (MFP), and analyzed using scRNAseq.

first: V

last: Wischnewski

first: J

middle: A

last: Joyce

first: N


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa and EB [m6A in HeLa]

summary: Epigenetic marking of the genome via modifications of DNA and histones is central to the regulation of gene expression. DNA methylation is a well-characterized modification involved in regulating chromatin states. RNA also undergoes modifications. The most prevalent internal modification of mRNA is N6-methyladenosine (m6A) installed co-transcriptionally by the METTL3-METTL14 methyltransferases. This process is an essential post-transcriptional mechanism of gene regulation. Recent evidence links m6A function to histone modifications. Here we reveal a direct connection between m6A and DNA methylation. We find that the DNA methyltransferase DNMT1 interacts both in vitro and in vivo with METTL14 within the METTL3-METTL14 complex. Using genome- and transcriptome-wide mapping, we show that methylated cytosine (5mC) tends to occur in DNA near positions corresponding to m6A sites in mRNAs, especially in gene bodies, this being associated with increased gene expression. This tight co-occurrence of 5mC and m6A appears to be favoured by a high m6A density. METTL3-depleted cells display strong intragenic DNA hypomethylation and reduced genome-wide binding of DNMT1 to DNA as evidenced by ChIP-Seq, this leading to reduced expression of the corresponding genes. We provide further mechanistic insights by showing in vitro that m6A impedes binding of DNMT1 to RNA. Accordingly, transcriptome-wide mapping of DNMT1 in vivo confirms that METTL3-mediated m6A formation interferes with DNMT1 binding to mRNA targets, causing increased gene expression. Together, our results reveal that METTL3-METTL14 favors intragenic DNA methylation by causing DNMT1 to be repelled from mRNA by m6A and targeted to gene bodies, thereby promoting transcriptional activation. They highlight a previously unrecognized layer of gene expression regulation, involving a direct mechanistic link between DNA methylation and RNA modification.

overall_design: PolyA enriched RNA from HeLa cells wild type were analyzed for m6A marks using m6A-seq technology

first: Jérôme

last: Martin

first: Liliana

last: Garcia-Martinez

first: Emilie

last: Calonne

first: Pierre-Olivier

last: Esteve

first: Elizabeth

last: Caine

first: Bouchra

last: Hassabi

first: Audrey

last: Penning

first: Jie

last: Lan

first: Danette

last: Daniels

first: Sriharsa

last: Pradhan

first: Rachel

last: Deplus

first: Lluis

last: Morey


first: Hyun-Woo

last: Jeong

state: NRW

department: Dept. Tissue Morphogenesis

institute: Max-Planck-Institute for Molecular Biomedicine

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

overall_design: Refer to individual Series


title: Targeting TUT1 Depletes Tri-snRNP Pools to Suppress Splicing and Inhibit Pancreatic Cancer Cell Survival

first: wei

last: guo


title: Prospective identification and characterization of multipotent peripheral neural stem cells out of mammalian central nervous system

summary: It has been widely accepted that mammalian neural stem cells (NSCs) only exist in central nervous system (CNS). Here, we challenge this concept and show that peripheral NSCs (pNSCs) could be isolated out of CNS from mouse embryonic limbs, postnatal tail and adult lung tissues. Derived-pNSCs express multiple NSC-specific markers, exhibit cell morphology, self-renewing capacity, genome-wide transcriptional profile, epigenetic features similar to those of brain control NSCs. Derived-pNSCs can differentiate into astrocytes, electrophysiologically functional neurons, and oligodendrocytes in vitro and in vivo, indicating multipotency. Lineage-tracing results reveal that pNSCs are progeny cells of neural epithelial cells, but not neural crest cells. Furthermore, single cell RNA-seq results identify a unique molecular feature of in vivo pNSCs which express NSCs and oligodendroglial markers. Our finding of pNSCs out of CNS expands the field of developmental neuroscience and presents an alternative potential strategy for neural regenerative therapy.

overall_design: Single cell RNA sequence of andult mouse brain and lung Nestin-GFP+ cells

first: Dong

last: Han

first: Ralf

last: Adams

first: Hans

last: Schöler


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa and EB [RNAseq in HeLa]

first: Martin

last: Bizet

overall_design: Ribodepleted total RNA from HeLa cells wild type and knocked out for METTL3 were analyzed for expression levels using RNA-seq technology

first: Andrea

last: Li Greci

first: Giuseppe

last: Quarto

first: Irina

last: Primac

first: Pankaj

last: Kumar

first: Gaurav

last: Dube

first: Jana

last: Jeschke

first: François

last: Fuks


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa and EB [5mC in HeLa]

overall_design: Total genomic DNA from HeLa cells wild type and knocked out for METTL3 were analyzed for 5mC changes using Infinium 850k

first: Jérôme

last: Martin

first: Liliana

last: Garcia-Martinez

first: Emilie

last: Calonne

first: Pierre-Olivier

last: Esteve

first: Elizabeth

last: Caine

first: Bouchra

last: Hassabi

first: Audrey

last: Penning

first: Jie

last: Lan

first: Danette

last: Daniels


title: EZH2 INHIBITION ENHANCES T CELL IMMUNOTHERAPIES BY INDUCING LYMPHOMA IMMUNOGENICITY AND IMPROVING T CELL FUNCTION (WGS)

city: New York

first: Matt

last: Teater

overall_design: We generated a murine B cell lymphoma cell line called "tFL-P6" that mimics the features of human transformed follicular lymphoma from a genetically engeneered mouse model (GEMM; R26BCL2+Ezh2Y641F). To investigate if the cell line has acquired additional mutations, we perfromed whole genome sequencing and compared mutations in tFL-P6, original tumor grown in the GEMM (P0), and germ line control isolated from the tail of the GEMM.

first: Yusuke

last: Isshiki

first: Cem

last: Meydan


title: Multipotent neural stem cells originating from neuroepithelium exist outside of the mouse central nervous system [scRNA-seq]

first: Hyun-Woo

last: Jeong

summary: It has been widely accepted that mammalian neural stem cells (NSCs) exist only in the central nervous system. Here, we report that peripheral NSCs (pNSCs) exist outside of the CNS and can be isolated—from mouse embryonic limb, postnatal lung, tail, dorsal root ganglia, and adult lung tissues. Derived pNSCs are distinguished from neural crest stem cells, express multiple NSC-specific markers and exhibit cell morphology, self-renewing capacity, genome-wide transcriptional profile, epigenetic features, and differentiation ability similar to those of control brain NSCs. pNSCs are from Sox1+ cells and originate from neuroepithelial cells. Single-cell RNA sequencing identifies that pNSCs in situ show similar molecular feature to brain NSCs. Furthermore, many pNSCs that migrate out of the neural tube can differentiate into neurons and limited glia cells during embryo development. Our finding of the existence of pNSCs provides previously unidentified insight of the mammalian nervous system development and presents an alternative potential strategy for neural regenerative therapy.

overall_design: Single cell RNA sequencing of Sox2-GFP+ lung cells, Sox1-GFP+ brain cells and Sox1-GFP+ tail cells

first: Dong

last: Han

first: Ralf

last: Adams

first: Hans

last: Schöler


title: The bacterial microbiome modulates the initiation of brain metastasis by impacting the gut-to-brain axis.

status: Public on Jan 09 2025

first: Nadine

last: Fournier

summary: Brain metastases (BrMs) are the most common brain tumors in patients and are associated with poor prognosis. Investigating the systemic and environmental factors regulating BrM biology represents an important strategy to develop effective treatments. Towards this goal, we explored the contribution of the gut microbiome to BrM development by using in vivo breast-BrM models under germ-free conditions or antibiotic treatment. This revealed a detrimental role of gut microbiota in fostering BrM initiation. We thus evaluated the impact of antibiotics and BrM outgrowth on the gut-brain axis. We found the bacterial genus Alistipes was differentially present under antibiotic treatment and BrM progression. In parallel, we quantified circulating metabolites, revealing kynurenic acid as a differentially abundant molecule which impaired the interaction between cancer cells and the brain vasculature in ex vivo functional assays. Together, these results illuminate the potential role of gut microbiota in modulating breast-BrM via the gut-to-brain axis.

overall_design: 16S rRNA gene sequencing of fecal samples from healthy or BrM-bearing mice treated with either of the two antibiotic formulations (4-ABX, 2-ABX) or controls for 14 days.

first: Matteo

last: Massara

first: Johanna

last: Joyce


title: Regulation of Mammalian Cellular Metabolism by Endogenous Cyanide Production [Glycine_RNA-Seq]

city: Fribourg

first: Karim

last: Zuhra

address: Chemin du Musée, 18

institute: University of Fribourg

summary: Small, gaseous molecules – such as nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) are produced as signalling molecules in mammalian cells. Here, we show that low concentrations of cyanide are generated endogenously in various mammalian tissues and cells. We detect cyanide in several cellular compartments of human cells and in various tissues and the blood of mice. Cyanide production is stimulated by glycine, occurs at the low pH of lysosomes and requires peroxidase activity. When generated at a specific rate, cyanide exerts stimulatory effects on mitochondrial bioenergetics, cell metabolism, and cell proliferation, but impairs cellular bioenergetics at high concentrations. Cyanide can modify cysteine residues via protein S-cyanylation, which is detectable basally in cells and mice, and increases in response to glycine. Low-dose cyanide supplementation exhibits cytoprotective effects in hypoxia and reoxygenation models in vitro and in vivo. Conversely, pathologically elevated cyanide production in nonketotic hyperglycinemia is detrimental to cells. Our findings indicate that cyanide should be considered part of the same group of endogenous mammalian regulatory gasotransmitters as NO, CO and H2S.

overall_design: The experimental design included N=3 independent replicates for Control group and N=3 independent replicates for Treated group. HepG2 cells were treated for 24h with vehicle (Control group) or Glycine 10 mM (Treatment group).


title: Dynamics in zebrafish development define transcriptomic specificity after angiogenesis inhibitor exposure (Sorafenib and Rotenone)

city: Leipzig

first: Paul

last: Michaelis

address: Permoserstr. 15

institute: Helmholtz-Centre for Environmental Research (UFZ)

summary: Time-series transcriptomic data of ZFE exposed to the angiogenesis inhibitor SU4312 with exposure start time points at 0 hpf and 24 hpf.

overall_design: Wild-type adult zebrafish (D. rerio, strain OBI/WIK, generation F3) were kept at 26°C in 20 L aquaria with 1-2 fish/L, fed twice a day and on a 14h:10h light dark cycle. Eggs were collected within 1h of spawning, fertilized and normally developed eggs were selected and incubated at 26°C. Eggs for the 24 hpf exposure were stored for at 26°C with 50 eggs per 100 mL of ISO-water, shaken at 75 rpm. Eggs were exposed at 2 hpf or at 24 hpf. 10 embryos were incubated in 4 mL of treatment or control solution for the desired exposure time in 6 mL covered GC vials. Incubation was done at 26°C in a 12h:12h light dark cycle and shaken at 75 rpm. Two vials were pooled as one replicate (total of 20 embryos). For the 0 hpf exposure, data points have 4 replicates and one time point was measured (96 hpf). Embryos were exposed to 2.4 μM Sorafenib and 25 nM Rotenone. For the 24 hpf exposure there 3 time points (36, 48 and 96 hpf), with three replicates per time point and concentration. Two concentrations were measured per substance: 1.3 and 2.4 μM for Sorafenib and 25 and 50 nM for Rotenone.

first: Julia

last: Nöth

first: Stefan

last: Scholz

first: Volker

last: Haake

first: Janet

last: Krüger

first: Lennart

last: Schüler

first: Wibke

last: Busch


title: ZIP-seq for functional characterizations of Z-DNA in rice

city: Nanjing

first: Zexue

last: He

state: Jiangsu Province

address: Xuanwu District,1 Weigang

department: Agricultural College

institute: Nanjing Agricultural University

summary: A Z-DNA is a left-handed DNA double helix functioning in various DNA templated events. However, antibody based high throughput methodologies for Z-DNA profiling are still missing in both humans and plants. We here developed the Z-DNA antibody-based IP-seq, termed ZIP-seq, for characterization of Z-DNA across the rice genome.

overall_design: Pregerminated rice seeds of Nipponbare (Oryza sativa L., Japonica) were transferred into the soil and grew in a greenhouse at 28-30℃ with a 14h/10h light-dark cycle. Two-week old seedlings were harvested and quickly frozen in liquid nitrogen for storage at -80℃ until used

first: Wenli

last: Zhang

first: Ying

last: Yang


title: Dynamics in zebrafish development define transcriptomic specificity after angiogenesis inhibitor exposure (SU4312)

overall_design: Wild-type adult zebrafish (D. rerio, strain OBI/WIK, generation F3) were kept at 26°C in 20 L aquaria with 1-2 fish/L, fed twice a day and on a 14h:10h light dark cycle. Eggs were collected within 1h of spawning, fertilized and normally developed eggs were selected and incubated at 26°C. Eggs for the 24 hpf exposure were stored for at 26°C with 50 eggs per 100 mL of ISO-water, shaken at 75 rpm. Eggs were exposed at 2 hpf or at 24 hpf. 10 embryos were incubated in 4 mL of treatment or control solution for the desired exposure time in 6 mL covered GC vials. Incubation was done at 26°C in a 12h:12h light dark cycle and shaken at 75 rpm. Two vials were pooled as one replicate (total of 20 embryos); control data points have two replicates, treatment data points have one replicate.For the 0 hpf exposure there are 10 time points (3, 6, 12, 24, 27, 30, 36, 48, 72 and 96 hpf) and for the 24 hpf exposure there are 6 time points (27, 30, 36, 48, 72 and 96 hpf). In addition to controls, two concentrations were measured for each exposure scenario: 1 and 2 µM in the 0 hpf exposure and 2 and 5 µM in the 24 hpf exposure.


title: Regulation of Mammalian Cellular Metabolism by Endogenous Cyanide Production [TST_RNA-Seq]

overall_design: The experimental design included N=4 independent replicates for HepG2 WT group (Ctr), N=4 independent replicates for HepG2 cells silenced for thiosulfate sulfurtransferase (shTST) and N=4 independent replicates for HepG2 cells overexpressing thiosulfate sulfurtransferase (overTST).


title: Study of 5mC-DNA, m6A-RNA and RNA-expression in HeLa, ES and EB

first: Martin

last: Bizet


title: Perturbing LSD1 and WNT rewires transcription to synergistically induce AML differentiation (CUT&RUN)

city: Oxford

first: Yang

last: Shi

address: Old Road Campus Research Building off Roosevelt Drive

department: Nuffield Department of Medicine

institute: Ludwig Institute for Cancer Research, University of Oxford

summary: Impaired differentiation is a hallmark of myeloid malignancies1,2. Therapies that enable cells to circumvent the differentiation block, such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), are by and large curative in acute promyelocytic leukaemia3, but whether 'differentiation therapy' is a generalizable therapeutic approach for acute myeloid leukaemia (AML) and beyond remains incompletely understood. Here we demonstrate that simultaneous inhibition of the histone demethylase LSD1 (LSD1i) and the WNT pathway antagonist GSK3 kinase4 (GSK3i) robustly promotes therapeutic differentiation of established AML cell lines and primary human AML cells, as well as reducing tumour burden and significantly extending survival in a patient-derived xenograft mouse model. Mechanistically, this combination promotes differentiation by activating genes in the type I interferon pathway via inducing expression of transcription factors such as IRF7 (LSD1i) and the co-activator β-catenin (GSK3i), and their selective co-occupancy at targets such as STAT1, which is necessary for combination-induced differentiation. Combination treatment also suppresses the canonical, pro-oncogenic WNT pathway and cell cycle genes. Analysis of datasets from patients with AML suggests a correlation between the combination-induced transcription signature and better prognosis, highlighting clinical potential of this strategy. Collectively, this combination strategy rewires transcriptional programs to suppress stemness and to promote differentiation, which may have important therapeutic implications for AML and WNT-driven cancers beyond AML.

overall_design: THP-1 cell treated with DMSO, GSK-LSD1, LY2090314 and combination of both inhibitors and cells prepared for CUT&RUN for IRF7 and β-catenin.

first: Amir

last: Hosseini

first: Natalia

last: Sikora

first: Andrés

last: Blanco

first: Maria

last: Jurgensen Amar

first: Alan

last: Jiao


title: Perturbing LSD1 and WNT rewires transcription to synergistically induce AML differentiation


title: Perturbing LSD1 and WNT rewires transcription to synergistically induce AML differentiation (RNA-Seq)

overall_design:
Cells were plated in triplicates pre-added with Vehicle, GSK-LSD1, LY2090314 or combination of inhibitors. Colony forming unit assays were scored every 7 to 10 /days’ post seeding.
RNA-seq analysis was performed using 12 samples (3x each condition)
Comparative gene expression analysis was performed for samples treated with GSK-LSD1 or LY2090314 vs. vehicle in TPH1 cells.


title: An epigenetic-signaling pathway interplay re-wires transcriptional program to induce  therapeutic differentiation of AML

city: Philadelphia

first: Andres

state: PA

address: 380 S. University Avenue, Hill Pavillion Room 414

department: Biomedical Sciences

institute: University of Pennsylvania

summary: Differentiation therapy has proven to be curative for the rare acute myeloid leukemia (AML) subtype, the acute promyelocytic leukemia (APL). However, whether differentiation induction is a generalizable therapeutic approach for AML and beyond remains incompletely understood. In this study, we demonstrate that simultaneous inhibition of the histone demethylase LSD1 (LSD1i) and the Wnt pathway GSK3β kinase (GSK3βi) robustly promotes therapeutic differentiation of established AML cell lines and primary human AML cells, as well as reducing tumor burden in a xenograft mouse model. Mechanistically, this combination promotes differentiation by activating genes in the type I interferon pathway (IFN-I) via inducing expression of IRF7 (LSD1i) and b-catenin (GSK3i) and their selective co-occupancy at targets such as STAT1, which is necessary for combination-indued differentiation. Combination treatment also suppresses the canonical, pro-oncogenic Wnt pathway and cell cycle genes. Importantly, analysis of AML patient datasets suggests a correlation between the combination-induced transcription signature and better prognosis, highlighting clinical potential of this strategy. Collectively, our findings suggest that this combination strategy re-wires transcriptional programs to suppress stemness and to promote differentiation, which may have important therapeutic implications for AML and Wnt-driven cancers beyond AML.

overall_design: ER-HoxA9 cells were treated with GSK-LSD1, LY2090314, GSK-LSD1 + LY2090314, or Vehicle for 5 days and were harvested for bulk RNA-seq


title: Simultaneous perturbation of epigenetic and signaling pathways synergistically induces therapeutic differentiation of acute myeloid leukemia

summary: While most current therapies for acute myeloid leukemia (AML) suffer from limited efficacy and substantial toxicity, differentiation therapy has proven to be curative for the rare AML subtype, acute promyelocytic leukemia (APL). However, whether differentiation induction is a generalizable therapeutic approach for AML and beyond remains incompletely understood. In this study, we demonstrate that simultaneous inhibition of the histone demethylase LSD1 (LSD1i) and the Wnt pathway GSK3? kinase (GSK3?i) robustly promotes therapeutic differentiation of established AML cell lines and primary human AML cells, as well as reducing tumor burden in a xenograft mouse model. Mechanistically, this combination treatment induces selective co-occupancy of IRF7 and  -catenin at promoters of the type I interferon signaling pathway component genes such as STAT1 essential for AML differentiation, and key downstream target genes, and consequently their expression. Combination treatment also appears to suppress the canonical, pro-oncogenic Wnt pathway. Importantly, analysis of AML patient datasets suggests a correlation between the combination-induced transcription signature and better prognosis, highlighting clinical potential of this strategy. Collectively, our findings suggest that this combination strategy may potentially re-route transcriptional programs to favor differentiation, which may have important therapeutic implications for AML and Wnt-driven cancers beyond AML.

overall_design: ER-HoxA9 cells cultured with estradiol (E2) were treated with 50 nM GSK-LSD1, 100 nM LY2090314, 50 nM GSK-LSD1 and 100 nM LY2090314 simultaneously, or vehicle (DMSO) for 24 and 72 hours, at which point cells were harvested for ATAC-seq processing.


title: Generation of expandable multipotential distal lung progenitors from human pluripotent stem cells that model idiopathic pulmonary fibrosis [bulk RNA-Seq]

first: Hans-Willem

last: Snoeck

address: 650 west 168th street

institute: Columbia University Irving Medical Centre

summary: Human lungs contain distal respiratory airways (RAs) that are absent in rodents and contain unique cell populations that are more abundant in patients with lung injury and idiopathic pulmonary fibrosis (IPF). A deeper understanding of the potential of these cells requires an in vitro model. Here we report the generation from human pluripotent stem cells of expandable spheres (‘induced respiratory airway progenitors’ (iRAPs)) consisting of RA-associated cell types that could be differentiated into type 1 and type 2 alveolar epithelial cells in defined conditions and show defects that are hallmarks of IPF when mutant for HPS1, a gene associated with IPF in humans. These data indicate that alveolar epithelial cells can derive from distal airway progenitors and that their intrinsic dysfunction may be involved in aberrant, fibrotic repair in IPF. iRAPs thus provide a model to gain insight into human lung regeneration and into pathogenesis of IPF.

overall_design: To identify pathways of differentiation, and find association in our alveoloar type 1 model we performed RNA sequencing on transitional AT2-like cells, iRAPs and iAT1 samples.

first: Mikael

middle: G

last: Pezet

first: Juan

last: Torres

first: Tania

last: Thimraj

first: Ivana

last: Matkovic Leko

first: Nadine

last: Schrode

first: John

middle: W

last: Murray

first: Kristin

last: Beaumont


title: Role of PI3K in Atrial Myopathy: Insights from Transgenic Mouse Models and Identification of a Dysregulated PI3K Lipid Profile in Individuals with Atrial Fibrillation

city: Melbourne

first: Graham

middle: William

last: Magor

state: Victoria

address: 99 Commercial Rd

country: Australia

institute: ACBD

summary: In a serendipitous discovery, we identified atrial enlargement, fibrosis and thrombi in a subset of transgenic mice with reduced phosphoinositide 3-kinase (PI3K, class IA) in cardiac myocytes. Understanding mechanisms underlying atrial myopathy has important implications for understanding and preventing atrial fibrillation (AF). Prior work had shown that PI3K is an essential regulator of exercise-induced ventricular enlargement and protection, but the role in the atria was unknown. Further, while targeting IGF1-PI3K-Akt signaling has been considered a potential therapeutic strategy for the failing heart, growing evidence suggests fine-tuning IGF1-PI3K signaling would be necessary. Here, we undertook comprehensive physiological and molecular analyses in cardiac-specific transgenic mice with increased or decreased PI3K to assess the dose response impact of directly regulating PI3K. Elevated PI3K was associated with a dose-dependent increase in heart size, and preserved/enhanced function. In contrast, reduced PI3K led to cardiac dysfunction, fibrosis, arrhythmia, and increased susceptibility to atrial enlargement and thrombi. This phenotype was associated with dysregulation of a lipid species (GM3) that regulates the IGF1-PI3K pathway, cardiac stress and contractility genes. Proteomic profiling identified distinct signatures across atria with varying degrees of atrial dysfunction, enlargement, and presence of atrial thrombi. To assess the potential relevance in humans we assessed circulating PI3K-related lipids in plasma from athletes with/without AF. Dysregulation of GM3 and PI3K-related lipids were identified in athletes with AF. Collectively, this work advances our understanding of mechanisms underpinning atrial pathophysiology, offers new insights for therapeutic approaches targeting atrial myopathy and AF, and has identified potential new lipid markers for identifying individuals at risk of AF.

overall_design: Target-locus-amplification of genomic DNA from dnPI3K transgenic mice to reveal transgene insertion site

first: Sebastian

last: Bass-Stringer

first: Yow

last: Tham

first: Gunes

last: Yildiz

first: Aya

last: Matsumoto

first: Helen

last: Kiriazis

first: Claudia

last: Harmawan

first: Celeste

middle: M

last: Tai

first: Roger

last: Chooi

first: Lauren

last: Bottrell

last: Ezeani

first: Daniel

last: Donner

first: Jenny

middle: Y

last: Ooi

first: Natalie

last: Mellett

first: Jieting

last: Luo

first: Emma

last: Masterman

first: Kristel

last: Janssens

first: Gavriel

last: Olshansky

first: Erin

last: Howden

first: Jonathon

last: Cross

first: Christoph

middle: E

last: Hagemeyer

first: Peter

last: Meikle

first: Ruby

last: Lin

first: Colleen

last: Thomas

first: Andrew

last: Perkins

first: Thomas

last: Marwick

first: Hiroshi

last: Kawakami

first: David

last: Greening

first: Kate

last: Weeks

first: André

last: La Gerche

first: Bianca

last: Bernardo

first: Julie

last: McMullen


title: Maintenance of p-eIF2 alpha levels by the eIF2B complex is vital for colorectal cancer

city: Berlin

first: Chiara

last: Giacomelli

address: Robert-Rössle-Str. 10

institute: Max Delbrück Center for Molecular Medicine

summary: Protein synthesis is an essential cellular process highly deregulated in multiple tumour types, where control of several translation factors is hijacked to benefit oncogenic growth. Here we show that colorectal cancer (CRC) is characterized  specifically by elevated levels of phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2alpha). In contrast to its canonical association with reduced translation rates, we reveal that CRC with high p-eIF2alpha has increased protein synthesis rates. Thus, we hypothesize that eIF2B, the sensor of p-eIF2 alpha, plays a central role in cells’ inability to relay this inhibitory signal. Using a combination of in cellulo biochemistry, phenotypic assays, and analysis of translation upon modulation of eIF2B subunits alpha and delta, the two eIF2B subunits responsible for sensing p-eIF2 alpha, we demonstrate that CRC cells require an intact eIF2B complex to sense p-eIF2 alpha. Crucially, we show that the alpha subunit of eIF2B is necessary to translate the oncogenic programs driven by APC loss, highlighting its central role in oncogenic transformation. To conclude, we demonstrate that whilst normal cells do not depend on eIF2B alpha, CRC cells require this eIF2B subunit for correct sensing of p-eIF2 alpha and regulation of their proteostasis, thus validating eIF2B alpha as a target for therapeutic intervention in CRC.

overall_design: To investigate the role of eIF2B in colorectal cancer, we depleted the 2B alpha (EIF2B1) and 2B delta (EIF2B4) subunits by inducible shRNA, comparing the effects to the induction of a non-targeting control (NTC/shCTRL). Ribo-seq and total cytoplasmic RNA (further mentioned only as Total RNA) sequencing were performed to address the effects on translation as well as transcript levels. This was performed in SW480, a well-established colorectal cancer cell line model. The experiment was performed in four independent experiment (replicates), carried out in separate weeks. Replicates are indicated by _1/_2/_3_c/_4. The replicate _3_c refers to appended sequencing results from two runs of the same library prep due to a partial failed first run of the library.

first: Ivana

last: Paskov-Skapic

first: Stefanie

last: Schimdt

first: Armin

last: Wiegering

first: Martin

last: Bushell


title: Using prognostic signatures and machine learning to identify core features associated with response to CDK4/6 inhibitor-based therapy in metastatic breast cancer

status: Public on Jan 08 2025

city: Buffalo

first: Jianxin

last: Wang

state: NY

address: 665 Elm Street

department: Molecular and Cellular Biology

institute: Roswell Park Comprehensive Cancer Center

summary: CDK4/6 inhibitors in combination with endocrine therapy are widely used to treat HR+/HER2- metastatic breast cancer leading to improved progression-free survival (PFS) compared to single agent endocrine therapy. Over 300 patients receiving standard-of-care CDK4/6 inhibitor combination therapy for metastatic disease were enrolled at a single institution. Clinical, pathological, and gene expression data were employed to define determinants for PFS duration. Visceral disease (HR 1.55, p=0.0013), prior endocrine therapy (HR 2.34, p<0.001), and the type of endocrine therapy (HR 2.16, p<0.001) were highly associated with PFS duration. Multiple pre-defined gene expression signatures were employed to determine association with response to CDK4/6 inhibitor-based therapy. Random survival forest was applied to define key gene expression and clinical features associated with PFS and develop a predictive model. The time to progression predicted by this model was related to the median PFS observed in PALOMA-2/3 and PEARL studies. Interrogating genes identified as highly significant across all studies indicated common enrichment of gene networks associated with cell cycle and estrogen receptor signaling. These findings indicate that there are common features from real-world use of CDK4/6 inhibitors that could be used to infer time to progression and better inform treatment.

overall_design:
Patient tissue samples were obtained during standard of care treatment from various clinical timepoints and were sent to HTG Molecular Diagnostics, Inc. for targeted sequencing using their HTG EdgeSeq Oncology Biomarker Panel (OBP) consisting of 2549 cancer-associated genes. Raw data was assembled from five batches of separate HTG runs. Batch effect removal was performed using the ComBat-seq function from the sva R package (v3.44.0). Data was then normalized on this batch effect-corrected raw read count matrix using the edgeR Bioconductor package (V3.38.4). Finally, a pseudo-count of 1 was added to each value of the normalized data matrix and then log2 transformed.

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the raw data files are unavailable from an out-of-business vendor.
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first: Agniewszka

last: Witkiewicz

first: Emily

last: Schultz

last: O'Connor

first: Tracey

first: Ellis

last: Levine

first: Erik

last: Knudsen


title: Dose-dependent sensitivity of human 3D chromatin to a heart disease-linked transcription factor

city: San Francisco

first: Benoit

middle: G.

last: Bruneau

address: 1650 Owens St

institute: Gladstone Institutes

summary: Dosage-sensitive transcription factors (TFs) underlie altered gene regulation in congenital heart disease (CHD), and cell-type specific gene regulation is linked to the reorganization of 3D chromatin during cellular differentiation. Here, we show dose-dependent regulation of chromatin organization by the congenital heart disease (CHD)-linked, lineage-restricted TF TBX5 in human cardiomyocyte differentiation. Genome organization, including compartments, topologically associated domains, and chromatin loops, are sensitive to reduced TBX5 dosage in a human model of CHD, with variations in response across individual cells. Regions normally bound by TBX5 were especially sensitive, while co-occupancy with CTCF partially protected TBX5-bound TAD boundaries and loop anchors. These results highlight the importance of lineage-restricted TF dosage in cell-type specific 3D chromatin dynamics, suggesting a new mechanism for TF-dependent disease.

overall_design: TBX5 ChIP-seq of cells from atrial cardiomyocyte differentation at D11, D20 and D45, CTCF ChIP-seq, CTCF ChIP-seq of cells from atrial cardiomyocyte differentation at D20 from WT (TBX5+/+), heterozygous (TBX5in/+) and null (TBX5in/del) WTC11

first: Zoe

middle: L

last: Grant

first: Shuzhen

last: Kuang

first: Shu

last: Zhang

first: Abraham

last: Horrillo

first: Kavitha

last: Rao

first: Vasumathi

last: Kameswaran

first: Carine

last: Joubran

first: Pik Ki

last: Lau

first: Keyi

last: Dong

first: Bing

last: Yang

first: Weronika

last: Bartosik

first: Nathan

last: Zemke

last: Ren

first: Irfan

last: Kathiriya

first: Katherine

last: Pollard

first: Benoit. G


title: Dose-dependent sensitivity of human 3D chromatin to a heart disease-linked transcription factor [RNA-Seq]

overall_design: RNA-seq of WTC11, cells from atrial cardiomyocyte differentation at D2, D4, D6, D11, D20 and D45, cells from ventricular cardiomyocyte differentation at D2, D4, D6, D10, D12, D15 and D30, each sample has three biological replicates


title: Dose-dependent sensitivity of human 3D chromatin to a heart disease-linked transcription factor [ChIP-Seq]


title: Deciphering the Role of IGFBP5 in Delaying Fibrosis and Sarcopenia in Aging Skeletal Muscle

city: Shanghai

first: Luze

last: Shi

address: Xinsongjiang Road 650

institute: Shanghai General Hospital

summary: D-galactose (D-gal) is a widely used chemical to induce cellular senescence. Cells undergoing senescence induced by D-gal  exhibit mitochondrial structural damage and a decline in energy metabolism, which are highly related to cellular aging Studies have confirmed that D-gal can induce senescence, fibrosis, and redox imbalance in skeletal muscle fibroblasts. Our results confirm that D-galactose effectively induces cellular senescence and skeletal muscle fibrosis in both cellular and animal models. The increase in senescence markers and fibrosis-related proteins, along with the observed decline in muscle strength and mass, are consistent with previous studies that highlight the role of D-galactose in modeling aging-associated pathologies. The observed lethargy and reduction in muscle fiber cross-sectional area further validate the model's relevance to sarcopenia research.

overall_design: RNA-seq profiling of NOR-10 induced by D-gal compared with the control group


title: Dose-dependent sensitivity of human 3D chromatin to a heart disease-linked transcription factor [Hi-C]

first: Benoit

last: Bruneau

overall_design: Hi-C 3.0 of WTC11, cells from atrial cardiomyocyte differentation at D2, D4, D6, D11, D20 and D45, cells from ventricular cardiomyocyte differentation at D2, D4, D6, D11 and D23, cells from atrial cardiomyocyte differentation at D20 from WT (TBX5+/+), heterozygous (TBX5in/+) and null (TBX5in/del) WTC11


title: Effect of pharmacologic inhibition of KPNB1 by Importazole on gene expression  of NB-4 cells

city: Chongqing

first: Zhe

last: Chen

address: 1 Yixueyuan Road, Yuzhong District

institute: Chongqing Medical University

summary: Acute myeloid leukemia (AML) is the most common malignancy of the hematologic system with refractory relapse and limited treatments. Nucleocytoplasmic trafficking of biomolecules mediated by Karyopherin proteins plays vital roles in AML promotion and development, but the underlying mechanism is not clearly elucidated. KPNB1, a key member of Karyopherin beta subunits, is highly expressed in multiple cancers. Here, we found that elevated expression of KPNB1 correlates with unfavorable outcomes in patients with AML, and KPNB1 knockdown induces growth blocked and apoptosis in AML cells in vitro. In addition, pharmacologic inhibition of KPNB1 reduces tumor burden and extends survival in an MLL-AF9 leukemia model by KPNB1-specific inhibitor importazole (IPZ) in vivo. Moreover, KPNB1 inhibition by IPZ sensitizes AML cell lines and patients' cells to venetoclax. At the molecular level, KPNB1 directly binds to and mediates the nuclear import of HMGB2, a modulator of DNA repair. The inhibition of KPNB1 blocks the nuclear import of HMGB2 and consequently induces DNA damage in AML cells. Overall, our work elicits that inhibition of KPNB1-mediated HMGB2 nuclear translocation disrupts DNA damage repair and validates a potential target for treating AML therapy.

overall_design: NB-4 cells were treated with vehicle (DMSO) or IPZ (5 μM) for 48 h. The cells were collected for RNA-seq analysis.

first: Yuxin

last: Xie