title: Deep profiling deconstructs features associated with memory CD8+ T cell tissue residence

last: Masopust

department: Microbiology and Immunology

summary: Tissue resident memory CD8+ T cells (Trm) control infections and cancer and are defined by their lack of recirculation. Because migration is difficult to assess, residence is usually inferred by putative residence-defining phenotypic and gene signature proxies. We assessed the validity and universality of residence proxies by integrating mouse parabiosis, multi-organ sampling, intravascular staining, acute and chronic infection models, dirty mice, and single-cell multi-omics. We report that memory T cells integrate a constellation of inputs— location, stimulation history, antigen persistence, and environment— resulting in myriad differentiation states. Thus, current Trm-defining methodologies have implicit limitations, and a universal residence-specific signature may not exist. However, we define genes and phenotypes that more robustly correlate with tissue residence across the broad range of conditions that we tested. This study reveals broad adaptability of T cells to diverse stimulatory and environmental inputs and provides practical recommendations for evaluating Trm cells.

overall_design:
Memory P14 CD8+ T cell populations specific to lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) were created for RNA-seq and transcriptome analyses. First, 5 x 10^4 naïve CD45.1+ P14 CD8+ T cells were adoptively transferred into naïve C57BL/6J mice followed by i.p infection with 2x10^5 PFU LCMV Arm. At >30 days p.i., i.v. antibody labeling was performed with anti-CD8a 3 minutes prior to euthanizing the mice and removal of tissues. We then created single cell suspensions from the excised tissues. Cells were stained with anti-CD8b, anti-CD45.1, anti-CD44, anti-CD69, anti-CD62L, and Ghost Dye Red 780 (Tonbo Biosciences) flow antibodies. Subsequently, LCMV Arm specific Tcm (CD8b+CD45.1+CD69-CD62L+), Tem (CD8b+CD45.1+CD69-CD62L-), and Trm (CD8b+CD45.1+CD69+CD62L-) populations were sorted from cell suspensions of pooled secondary lymphoid organs (SLO) with a FACSAria II (BD Biosciences). Extravascular Trm (identified as i.v. antibody negative CD8b+CD45.1+CD69+CD62L- P14 cells) were sorted from single-cell suspensions originating from the small intestine (SI) epithelial and lamina propria compartments, as well as the female reproductive tract (FRT). Extravascular LCMV Arm specific CD69+CD62L- P14 cells in the SI and FRT tissues were presumed to be Trm based on prior parabiosis studies.
Bystander activated (CD8+CD45.1+CD69+) P14 cells were sorted from pooled SLO of LCMV Arm immune mice 48 hours after i.v. VSV-NJ infection. Recently stimulated (CD8+CD45.1+CD69+) P14 cells were sorted from pooled SLO cell suspensions from mice 4 days after LCMV Arm infection. Endogenous CD69+ H-2Db/gp33 tetramer+CD8+ T cells were sorted from pooled SLO of mice 50 days after LCMV Cl13 infection in CD4 T cell depleted mice. Naïve (CD44 low) P14 CD8+ T cells were sorted from pooled SLO cell suspensions from CD45.1+/Thy1.2+ P14 transgenic mice.
After sorting to obtain cell populations of interest, RNA isolation, integrity assessment, and sequencing was performed. Sequencing libraries were prepared with the Clontech SMARTer Stranded Total RNA-seq Kit v2 (Pico Input Mammalian Kit). 50 base pair single-end sequences were then generated from the with the HiSeq 2500 Illumina.

first: Milcah

last: Scott

first: Zoë

last: Steier

first: Mark

last: Pierson

last: Stolley

first: Stephen

middle: D

last: O’Flanagan

first: Andrew

last: Soerens

first: Sathi

last: Wijeyesinghe

first: Lalit

last: Beura

first: Gayathri

last: Dileepan

first: Brandon

last: Burbach

last: Künzli

first: Clare

middle: F

last: Quarnstrom

first: Olivia

last: Ghirardelli Smith

first: Eyob

last: Weyu

first: Sara

last: Hamilton

first: Vaiva

last: Vezys

first: Alex

last: Shalek


title: The epigenetic reader Phf2 is positioned in the genome by cohesin [ChIP-seq]

status: Public on Dec 20 2024

city: Vienna

first: Roman

middle: R

last: Stocsits

address: Campus-Vienna-Biocenter 1

country: Austria

institute: IMP - Research Institute of Molecular Pathology

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Eukaryotic genomes are assembled into chromatin by histones and extruded into loops by cohesin. These mechanisms control important genomic functions, but whether histones and cohesin cooperate in genome regulation is poorly understood. Here we identify Phf2, a member of the Jumonji-C family of histone demethylases, as a cohesin-interacting protein. Phf2 binds to H3K4me3 nucleosomes at active transcription start sites (TSSs) but also co-localizes with cohesin. Cohesin depletion reduces Phf2 binding at sites lacking H3K4me3, and depletion of Wapl and CTCF re-positions Phf2 together with cohesin in the genome, resulting in the accumulation of both proteins in vermicelli and cohesin islands. Conversely, Phf2 depletion reduces cohesin binding at TSSs lacking CTCF, decreases the number of short cohesin loops, but increases the length of heterochromatic B compartments. These results suggest that Phf2 is an ‘epigenetic reader’, which is translocated through the genome by cohesin-mediated DNA loop extrusion, and which recruits cohesin to active TSSs and limits the size of B compartments. These findings reveal an unexpected degree of cooperativity between epigenetic and architectural mechanisms of eukaryotic genome regulation.

overall_design: The objective of this experiment was to investigate the role of cohesin in positioning the epigenetic reader Phf2 by performing ChIP-seq on wild-type (WT) and mutant (Smc3 KO, Phf2 KO, Wapl KO, and CTCF/Wapl dKO) mouse embryonic fibroblast cells (MEFs). For the conditional deletion of floxed Phf2, Wapl, Smc3, or CTCF alleles, we used either the inducible CreERT2 recombinase expressed from the Rosa26 locus (CreER) or Adeno-Cre virus. Each condition was performed in biological duplicates, triplicates, or tetraplicates. ChIP-seq was conducted using Phf2/Smc3 antibodies, and libraries were sequenced on an Illumina platform. The goal is to assess differences in Phf2/cohesin binding patterns between WT and mutant samples, particularly at active TSSs lacking CTCF binding sites.

first: Wen

last: Tang

first: Lorenzo

last: Costantino

first: Gordana

last: Wutz

first: Jan-Michael

last: Peters


title: Gene expression profile at single cell level of immune cells from the dura mater of naïve C57BL6/N mice and during different phases of EAE.

city: Freiburg

first: Lukas

last: Amann

address: Breisacher Straße 64

department: Institute of Neuropathology

country: Germany

institute: University Medical Center Freiburg

type: Expression profiling by high throughput sequencing

summary: While macrophages in the meningeal compartments of the central nervous system (CNS) has been comprehensively characterized under steady state, studying their contribution to physiological and pathological processes has been severely hampered by the lack of specific targeting tools in vivo. Recent findings have shown that the dural sinus and its adjacent lymphatic vessels act as a neuroimmune interface. Notably, the cellular and functional heterogeneity of extrasinusoidal dural macrophages outside this immune hub is currently unclear. Therefore, we comprehensively characterized these cells using single-cell transcriptomics, fate mapping, confocal imaging, clonal analysis and transgenic mouse lines. Extrasinusoidal dural macrophages were clearly distinct from leptomeningeal and CNS parenchymal macrophages in terms of their origin, expansion kinetics and transcriptional profiles. Lastly, functional studies demonstrated that during autoimmune neuroinflammation, extrasinusoidal dural macrophages perform efferocytosis of granulocytes. Our results highlight a previously unappreciated myeloid cell diversity and provide insights into the brain’s innate immune system.

overall_design: Dura mater was microdissected into sinusoidal and extrasinusoidal compartments. CD45+ immune cells from each compartment were pooled from two three mice per replicate. Two replicates (naïve) or one replicate (onset, peak and chronic) were analyzed by scRNAseq.

first: Marco

last: Prinz

last: Sankowski


title: Aberrant BCAT1 expression augments mTOR activity and accelerates disease progression in Chronic Lymphocytic leukemia

status: Public on Dec 23 2024

city: Ann Arbor

first: Karan

last: Bedi

state: MI

address: 1600 Huron Pkwy

department: Biostatistics

country: USA

institute: University of Michigan

summary: We performed gene expression profiling of mRNA/cDNA isolated from N = 117 flow sorted CLL. We detected aberrant expression of the metabolic enzyme branched chain amino acid transferase (BCAT1) in CLL with del17p/TP53mut. Through extensive validation, we confirmed the highly preferential expression of BCAT1 in CLL with del17p/TP53mut (66%) or trisomy 12 (77%). BCAT1 was not expressed in B cells isolated from normal human lymph nodes. The products of the bidirectional BCAT1 reaction, including leucine, acetyl-CoA, and alpha-ketoglutarate are known activators of MTOR. We measured an ~two-fold higher MTOR activity via normalized p-S6K levels in primary CLL with BCAT1 high versus absent expression before and after sIgM crosslinking. Through steady state metabolomics and heavy isotope metabolic tracing in primary CLL cells, we demonstrate that CLL cells are avid consumers of branched chain amino acids (BCAAs) and that BCAT1 in CLL engages in bidirectional substrate reactions. Of additional interest, CLL with aberrant BCAT1 expression were less sensitive to Venetoclax-induced apoptosis. Biologically, three CLL-derived cell lines with disruption of BCAT1 had substantially reduced growth ex vivo. Clinically, the expression of any detectable BCAT1 protein in CLL independently associated with shorter median survival (125 months versus 296 months; p < 0.0001), even after exclusion of del17p/TP53mut cases.

overall_design: We analyzed expression array data from 117 CLL samples.

first: Sami

middle: N

last: Malek


title: Phenotypic Screens Identify SCAF1 as Critical Activator of RNAPII Elongation and Global Transcription [CUT&RUN]

city: Kiel

first: Elmar

last: Wolf

state: Schleswig-Holstein

address: Rudolf-Höber-Str. 1

department: Biochemical Institute

institute: Christian-Albrechts-University Kiel

summary: Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used an siRNA-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1, using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery, is synthetically lethal.

overall_design: SCAF1-AID cells were treated with or without auxin 6 hours, depletion of SCAF1

first: Pranjali

last: Bhandare

first: Ashwin

last: Narain

first: Julia

last: Hofstetter-Hadry

first: Teresa

last: Rummel

last: Wenzel

first: Christina

last: Schülein-Völk

first: Stephanie

last: Lamer

first: Ursula

last: Eilers

first: Andreas

last: Schlosser

first: Martin

first: Florian

last: Erhard


title: Phenotypic Screens Identify SCAF1 as Critical Activator of RNAPII Elongation and Global Transcription [ChIP-seq]


title: Phenotypic Screens Identify SCAF1 as Critical Activator of RNAPII Elongation and Global Transcription [4sU-seq]

overall_design: U2OS SCAF1-AID cells were treated with or without auxin 6 hours, depletion of SCAF1


title: Single cell approaches define forebrain neural stem cell niches and identify microglial ligands that enhance precursor-mediated remyelination (MERFISH)

city: Vancouver

first: Freda

middle: D.

last: Miller

state: BC

department: Michael Smith Laboratories, Department of Medical Genetics

country: Canada

institute: University of British Columbia

summary: Here we used single cell RNA-sequencing and single cell spatial transcriptomics to characterize the forebrain neural stem cell (NSC) niche under homeostatic and injury conditions. We define the dorsal and lateral ventricular-subventricular zones (V-SVZ) as two distinct neighborhoods, and show that following white matter injury, dorsal NSCs are locally activated to make oligodendrocytes for remyelination. This activation is coincident with a robust increase in transcriptionally-distinct microglia in the dorsal V-SVZ niche. We modeled ligand-receptor interactions within this changing niche and identified two remyelination-associated microglial ligands, IGF1 and OSM, that promote precursor proliferation and oligodendrogenesis in culture. Infusion of either ligand into the lateral ventricles also enhanced oligodendrogenesis, even in the lateral V-SVZ, where NSCs normally make neuroblasts. These data support a model where gliogenesis versus neurogenesis is determined by the local NSC neighborhood and where injury-induced niche alterations promote NSC activation, local oligodendrogenesis, and likely contribute to myelin repair.

overall_design: Multiplexed in situ hybridization-based spatial transcriptomics using the MERSCOPE MERFISH platform was used to analyze coronal adult (P100) CD-1 mouse forebrain sections containing the V-SVZ and corpus callosum, with a 300 gene probeset that included marker genes for relevant cell types, ligands and receptors. We analyzed a region of interest that included the V-SVZ and corpus callosum from four different brains from mice which had been exposed to either a control or cuprizone-rapamycin (cup-rap) protocol (2 control, and 2 cup-rap). Replicates are indicated at the end of each sample name (i.e. Adult_Control_Forebrain_MERFISH_Biol1, Adult_CupRap_Forebrain_MERFISH_Biol1 ).

first: Ashleigh

last: Willis

first: Danielle

last: Jeong

first: Yunlong

last: Liu

first: Marissa

middle: A

last: Lithopoulos

first: Scott

last: Yuzwa

first: Paul

middle: W

last: Frankland

first: David

last: Kaplan


title: PRDM16 cooperates with LHX2 to shape the human brain

status: Public on Dec 22 2024

city: Rehovot

first: Tsviya

last: Olender

address: 234 Herzl St.

department: Molecular Genetics

country: Israel

institute: The Weizmann Institute

summary: In the developing mouse brain, PRDM16 exhibits robust expression in radial glia (RG) progenitors, serving as one of the conserved core RG genes shared between humans and mice. Much is known regarding the functions of PRDM16 in the developing mouse brain, yet its roles in the developing human brain are less explored. Our study was motivated by detecting a patient with a de novo nonsense mutation in PRDM16 exhibiting lissencephaly and microcephaly features. We introduced the mutation in human stem cells in both homozygous and heterozygous manner and generated human cortical organoids. The organoids differed in cell cycle parameters, and RNA-seq demonstrated changes in cell adhesion and WNT-signaling, which were confirmed by immunostaining. We further generated ChIP-seq data from human fetuses and compared the results to ChIP-seq data previously obtained from mice and differentially expressed genes in humans and mice (REFS he and baizabal papers). The top detected motif matched LHX2, so we compared our data with recently acquired LHX2 ChIP-seq data from the mouse. Our studies show highly conserved genes and pathways suggesting that PRDM16 and LHX2 complex and collaborate in the developing mouse and human brains. The multifaceted nature of PRDM16 and its intricate involvement in transcriptional regulation and various developmental processes highlight its importance in understanding neurodevelopmental mechanisms.

overall_design: Samples are forebrain organoids from day 32, from WIBR3 control line and PRDM16-KO line. 3 biological replecates were used

first: Varun

last: Suresh

first: Bidisha

last: Bhattacharya

first: Rami

middle: Y

last: Tshuva

first: Miri

last: Gotthold

first: Tsivya

first: Saurabh

last: Pradhan

first: Mahima

last: Bose

first: Bruria

middle: B

last: Zeev

first: Richard

middle: S

last: Smith

first: Shuba

last: Tole

first: Sanjeev

last: Galande

first: Corey

last: Harwell

first: Manuel

last: Baizabal

first: Orly

last: Reiner


title: A prenatal window for enhancing spatial resolution of cortical barrel maps

city: San Juan de Alicante

first: Guillermina

last: López-Bendito

state: Alicante

address: Av Ramon y Cajal s/n, Campus de San Juan

department: Instituto Neurociencias CSIC-UMH

country: Spain

institute: Universidad Miguel Hernández (UMH)

summary: Precise mapping of peripheral inputs onto cortical areas is essential for accurate sensory processing. In the mouse primary somatosensory cortex, mystacial whiskers correspond to large, well-defined barrels, while upper lip whiskers form smaller, less distinct barrels. These differences are traditionally attributed to variations in whisker input type and receptor density, but prenatal activity and transcriptional programs also impact somatosensory map development independently of sensory experience. Here, we demonstrate that prenatal ablation of mystacial whiskers leads to a remapping of cortical territories, enhancing the functional and anatomical definition of upper lip whisker barrels. This reorganization occurs without altering peripheral receptor types. Instead, thalamic neurons that receive upper lip inputs adopt a mystacial-like transcriptional profile. Our findings unveil a regulated prenatal mechanism in the thalamus that ensures sufficient cortical barrel size and spatial resolution for sensory processing, irrespective of peripheral receptor type or density, highlighting a critical developmental process in sensory mapping.

overall_design:
Bulk RNA sequencing was performed on the postero-medial barrel subfield (PMBSF) and antero-lateral barrel subfield (ALBSF) territories of the thalamic ventroposterior medial (VPM) nucleus in control and embWPC P0 pups. The PMBSF corresponds to the whisker pad input-receiving VPM (wpVPM), while the ALBSF corresponds to the upper-lip input-receiving VPM (ulVPM).
The bulk tissue was microdissected (five brains were pooled for each sample) and the total RNA was extracted as in the previous publication (Moreno-Juan et al., 2017).
Library construction and sequencing were performed at Novogene Co. Ltd. Genomics core facility (Cambridge, UK). The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Libraries were pooled and sequenced in 2x150bp paired-end mode on a S4 flowcell in the Illumina Novaseq6000 platform. A minimum of 40 million reads were generated from each library.
The RNA-seq analysis were performed as previously described (Herrero-Navarro et. al. 2021) with minor modifications: quality control of the raw data was performed with FastQC (v.0.11.9). RNA-seq reads were mapped to the Mouse genome (GRCm39) using STAR (v2.7.9a) and SAM/BAM files were further processed using SAMtools (v1.15). Aligned reads were counted and assigned to genes using Ensembl release 104 gene annotation and FeatureCounts, Subread (v2.0.1). Normalization of read counts and differential expression analyses were performed using DESeq2 (v1.32), Bioconductor (v3.15) in the R statistical computing and graphics platform (v4.2.2 “Innocent and Trusting”).

first: Lorenzo

last: Puche-Aroca

first: Mar

last: Aníbal-Martínez


title: Single cell approaches define forebrain neural stem cell niches and identify microglial ligands that enhance precursor-mediated remyelination (scRNA-Seq)

first: Freda

last: Miller

overall_design: High-throughput single cell transcriptomic profiles of the adult mouse brain (V-SVZ) cells, from CD-1 and lineage tracing NesCreERT2;EYFP animals exposed to either 6 weeks of cuprizone-rapamycin or vehicle control with 3 weeks recovery, generated by single cell RNA sequencing using the 10X Genomics platform.

first: Ashleigh

last: Willis

first: Danielle

last: Jeong

first: Yunlong

last: Liu

first: Marissa

last: Lithopoulos

first: Scott

last: Yuzwa

first: Paul

last: Frankland

first: David

last: Kaplan


title: Spatiotemporal single-cell architecture of gene expression in the Caenorhabditis elegans germ cells [smRNA-seq]

status: Public on Dec 21 2024

city: hangzhou

first: li

middle: li

last: li

state: zhejiang

address: 600 Dunyu Road

country: China

institute: westlake university

type: Non-coding RNA profiling by high throughput sequencing

summary: Spermatogenesis is an intricate and tightly controlled process, encompassing various layers of gene expression regulation. Despite our current understanding, the developmental trajectory and its regulatory mechanisms dictating spermatogenesis remain elusive. In this study, we have generated single-cell gene expression profiles for C. elegans sperm cells and constructed gene-regulated networks alongside the developmental trajectories of these cells. Our findings indicate that each pre- and post-developmental stage is closely linked by co-expressed genes, while simultaneously being uniquely identified by the combined expression of specific gene families. To illustrate the applicability of this exhaustive gene expression catalog, we used gene-regulated networks to uncover potential transcription factors for (1) the expression of genes in the phosphorylation pathway, identifying NHR-23-to-phosphatase regulation for the meiotic cell division process, and (2) the expression of constituent components of small RNA pathways, identifying ELT-1-to-Argonaute protein regulation for siRNA maintenance and sperm activation. We expect that this sperm cell-specific gene expression directory will prompt investigations into the underlying mechanisms determining anatomy, differentiation, and function across the reproductive system. Finally, our expression data can be explored using the web application CelegansGermAtlas (https://scgerm-atlas.sjtu.edu.cn/website/#/home).

overall_design: To investigate whether the suppression of elt-1 expression could result in the loss of 26G RNAs.

first: Lili

last: Li

first: Xuanxuan

last: Guo

first: Rao

last: Di

first: Junchao

last: Xue


title: Protein arginine methyltransferase 7 is linked to schizophrenia by regulating the function of neural progenitor cell

status: Public on Dec 19 2024

city: Changsha

first: Ting

last: Shen

address: Tongzhipo Road 172

institute: Central South University

summary: Schizophrenia (SCZ) is a severe mental disorder with strong heritability and complex inheritance, which affects about 1% of populations worldwide. In this study, we prioritized protein arginine methyltransferase 7 (PRMT7) for SCZ susceptibility. Next, we explored the cellular and molecular infrastructures conferring PRMT7 to SCZ risk. Down-regulation of PRMT7 in neural progenitor cells (NPCs) caused decreased proliferation and increased neuronal differentiation. Additionally, the differentiated neurons derived from these NPCs displayed longer neurites compared to controls. Conversely, Over-expression of PRMT7 led to enhanced NPC proliferation and reduced neuronal differentiation. Moreover, in 3D cerebral organoids, the similar NPC phenotypic changes were observed following PRMT7 depletion. Mechanistically, the expression of genes related to cell cycle and neuronal functions were under regulation of PRMT7 via depositing H4R3me2s on their promoter regions. Disease enrichment analysis revealed that these PRMT7-regulated genes were more strongly associated with SCZ compared to other mental disorders. In summary, this study uncovers that PRMT7 is a functional gene at 16q22.1 contributing to the etiology of SCZ by impacting the proliferation and differentiation of NPCs as an epigenetic regulator.

overall_design: To uncover the mechanism of PRMT7 contributing to SCZ risk, we establisded NPC model with PRMT7 overexpression.

first: Jing

last: Yu

first: Bin

last: Xie

first: Cuiping

last: Huang

first: Jingjie

last: Cui

first: Kefu

first: Chunyu

first: Chao

last: Chen


title: Splicing and gene expression changes in cell lines treated with splicing switching oligonucleotide targeting TRA2B

city: Farmington

first: Marina

last: Yurieva

state: Connecticut

address: 10 Discovery dr

department: Computational Sciences

institute: Jackson Laboratory

summary:
RNA-sequencing data from MDA-MB231 breast cancer cells, U87MG glioblastoma cells, and mouse breast cancer PDX models treated with antisense oligonucleotides targeting exon 2 of TRA2B. Additionally, RNA-sequencing data from MDA-MB231 breast cancer cells and U87MG glioblastoma cells treated with siRNAs targeting TRA2B.
RNA-sequencing data from MDA-MB231 breast cancer cells nad U87MG glioblastoma cells treated with antisense oligonucleotides targeting exon 2 of TRA2B.

overall_design:
We examined the differential gene expression and splicing changes in cell lines (MDA-MB231 breast cancer cells and U87MG glioblastoma cells) treated with ASO-1570 (targeting TRA2B) vs a control ASO at different doses (50nM and 200nM). Additionally, we performed similar experiments in breast cancer PDX models treated with ASO-1570 (targeting TRA2B) vs a control ASO at 50mg/kg. Finally, we provide RNA-sequencing data of cell lines (MDA-MB231 breast cancer cells and U87MG glioblastoma cells) treated with siRNAs targeting TRA2B and respective negative controls.
We examined the differential gene expression and splicing changes in cell lines treated with 200nM ASO-1570 (targeting tRA2B) vs a 200nM control ASO.

first: Olga

last: Anczukow

first: Nathan

last: Leclair

first: SungHee

last: Park

first: Mattia

last: Brugiolo

first: Maeva

last: Devoucoux

first: Laura

last: Urbanski

first: Brittany

last: Angarola


title: To identify HDAC3 occupancy in the genomes of ME49 and ΔXII-5 strains

city: wuhan

first: xue

last: lilan

state: 请选择...

address: shizishan

institute: huazhong agriculture university

summary: Toxoplasma gondii is a ubiquitous protozoan parasite with a complex life cycle containing multiple developmental stages. The parasites have distinct gene expression patterns at different stages to enable stage specific life activities, but the underlying regulatory mechanisms are largely unknown. In this study, we identified a nuclear complex that controls the expression of developmentally regulated genes, especially merozoite specific genes. This complex consists of the AP2 family transcription factor AP2Ⅻ-5, the epigenetic factors MORC and HDAC3, as well as a novel AP2Ⅻ-5 interacting protein 1 (AIP1) that stabilizes this complex. At the tachyzoite stage when the parasites proliferate rapidly by asexual endodyogeny, AP2Ⅻ-5 binds to the promoter regions of developmentally activated genes and recruits MORC and HDAC3 to suppress their expression. When sexual commitment and merozoite development is triggered, the abundance of AP2Ⅻ-5 decreases and its suppression on target genes is relieved. In contrast to MORC and HDAC3, which regulate Toxoplasma development but are also essential for tachyzoite growth, AP2Ⅻ-5 and AIP1 are dispensable for tachyzoite proliferation in vitro. These data suggest that while MORC and HDAC3 have broad regulatory activities, forming a complex with AP2Ⅻ-5 and AIP1 enables them to specifically regulate gene expression during development.

overall_design: To determine the HDAC3 binding sites in the genomes of ME49 and ΔⅫ-5 strains using CUT&Tag.

first: Lilan

first: Bang


title: Gene expression analysis of SRBD1-deficient U2OS cells

city: Nashville

first: David

last: Cortez

state: TN

address: 2215 Garland Avenue

department: Biochemistry

institute: Vanderbilt University School of Medicine

summary: SRBD1 (S1 RNA-binding domain-containing protein 1) is a protein of unknown function. Genome-wide association studies have linked SRBD1 variants to an increased risk of glaucoma. SRBD1 has sequence and predicted structure similarity to histone chaperones and proteins that regulate transcription. The aim of this study was to determine if inactivating SRBD1 causes changes in gene expression.

overall_design: U2OS cells were transfected with non-targeting control or SRBD1 siRNAs. RNAseq was performed using cells were harvested 24 hours or 48 hours after transfection with the SRBD1 siRNA or 48 hours after tranfection with the non-targeting siRNA.

first: Sarah

last: Wessel

first: Courtney

last: Lovejoy


title: The epigenetic reader Phf2 is positioned in the genome by cohesin

first: Roman

last: Stocsits

overall_design: This study investigates the role of Phf2 in gene regulation by performing RNA-Seq analysis on samples with and without Phf2 expression. Experimental samples included wild-type and Phf2 KO in mouse embryonic fibroblast cells (MEFs), allowing us to compare transcriptional profiles and identify genes potentially regulated by Phf2. Total RNA was extracted from 2 replicates for each condition, followed by library preparation and sequencing to provide insights into the differential gene expression patterns associated with Phf2 function.

first: Wen

last: Tang

first: Lorenzo

last: Costantino

first: Gordana

last: Wutz

first: Jan-Michael

last: Peters


title: Gene expression profilling of day 30 cerebral organoids at the single cell level

city: Toronto

first: yacine

last: touahri

address: 2075 Bayview Ave

institute: Sunnybrook Research Institute

summary: To characterize cerebral organoids generated using STEMdiff Dorsal Forebrain Organoid Differentiation Kit were flash frozen on Day 30, we performed single cell RNA sequencing

overall_design: Human embryonic stem cells (hESCs) (H1/WA01) were used to generate cerebral organoids using STEMdiff Dorsal Forebrain Organoid Differentiation Kit and scRNAseq was performed at day 30, with each sample consists of 5 pooled organoids

first: Lakshmy

last: Vasan

first: Yacine

last: Touahri

first: Chao

last: wong

first: Carol

last: Schuurmans


title: Tralokinumab treatment of Atopic dermatitis induces a progressive transcriptomic response

first: Rashmi

last: Tandon

address: Rosalind-Franklin-Strasse 9

institute: UKSH

summary: Atopic dermatitis (AD) is characterized by a complex epidermal barrier deficiency and exaggerated immune responses dominated by type-2-mechanisms with variable contributions of additional immune axes. Interleukin (IL)-13 is overexpressed in AD skin and a key driver of both barrier dysfunction and inflammation. We here prospectively studied the effects of IL-13 inhibition with tralokinumab on cutaneous transcriptome profiles using RNA sequencing of biopsies from 16 moderate-to-severe AD patients obtained at baseline, week 2 and week 16. Tralokinumab therapy induced early and delayed expression changes, and progressively shifted the transcriptomic profile of lesional towards non-lesional skin by modulating both genes associated with keratinocyte proliferation and differentiation, itch signaling, and downstream inflammatory responses. At week 16, 751 genes were still significantly dysregulated as compared to healthy control skin, reinforcing the need for long-term immunomodulatory therapy of moderate to severe AD to achieve deep responses. The study was registered with ClinicalTrials.gov (NCT04556461)

overall_design: Skin biopies were obtained at baseline, week 2 and week 16 from patients with moderate-to-severe Atopic dermatitis received and were subjected to Bulk RNA- sequencing.

first: Inken

last: Harder

first: Dora

last: Stölzl

first: Matthias

last: Hübenthal

first: Nicole

last: Sander

first: Jan

last: Hartmann

first: Ina

last: Suhrkamp

first: Melina

last: Fonfara

first: Sascha

last: Gerdes

first: Stephan

last: Weidinger


title: RNA sequencing data of dural macrophages isolated from brains of Cx3cr1-GFP mice at different ages.

first: Lukas

last: Amann

overall_design: Dura macrophages were isolated from 3-5 individual mice per age and used for the generation of bulk transcriptomics data. RNA expression levels between the different and ages were compared. Microglia, meningeal macrophages (mMΦ) and perivascular macrophages (pvMΦ) were isolated from the same animals and data has been deposited before (GSE194432).

first: Marco

last: Prinz

first: Gianni

last: Monaco

first: Takahiro

last: Masuda


title: Identification of antigen-presenting cell-T cell interactions driving immune responses to food

city: New York

first: Tiago

last: Castro

state: New York

address: 1230 York Avenue

institute: The Rockefeller University

summary: The intestinal immune system must concomitantly tolerate food and commensals and protect against pathogens. Antigen-presenting cells (APCs) orchestrate these immune responses by presenting luminal antigens to CD4+ T cells and inducing their differentiation into regulatory (pTreg) or inflammatory (Th) subsets. Here, we used LIPSTIC to identify APCs that presented dietary antigens under tolerizing and inflammatory conditions. We show that helminth infections disrupted tolerance proportionally to the reduction in ratio between tolerogenic, including migratory cDC1s and Rorγt+ APCs, and inflammatory APCs, represented primarily by cDC2 subsets. However, the inflammatory subset of cDC2s expanded by helminth infection did not present dietary antigens, thus avoiding diet-specific TH2 cell differentiation. Our data uncover cellular mechanisms by which tolerance to food is induced and can be disrupted by infection.

overall_design:
DCs from duodenal gut-draining lymph nodes (D-gLNs) of mice under steady-state and helminth-infected conditions were analyzed by bulk RNA-seq.
Single-cell RNA-seq (SmartSeq2) was performed on individual DCs from D-gLNs of steady-state and helminth-infected mice.
DCs from the lamina propria of the duodenum in steady-state and helminth-infected mice were analyzed using 10x Genomics single-cell RNA-seq.

first: Maria

middle: C

last: Canesso

first: Tiago Bruno

first: Sandra

last: Nakandakari-Higa

first: Ainsley

last: Lockhart

first: Julia

last: Luehr

first: Juliana

last: Bortolatto

first: Roham

last: Parsa

first: Daria

last: Esterházy

first: Mengze

last: Lyu

first: Tian-Tian

last: Liu

first: Kenneth M

last: Murphy

first: Gregory

last: Sonnenberg

first: Bernardo

last: Reis

first: Gabriel

last: Victora

first: Daniel

last: Mucida


title: microRNA control of stem cell reconstitution and growth in root regeneration

city: Rosario

middle: Lidia

last: Baulies

state: Santa Fe

address: Ocampo y Esmeralda s/n

country: Argentina

institute: Instituto de Biología Molecular y Celular de Rosario

summary: Plants display a remarkable regeneration capacity, which allows them to replace damaged or lost cells, tissues and organs, and thus recover from a broad spectrum of injuries. Even lost stem cells can be regenerated from non-stem cells after competence acquisition, highlighting the enormous plasticity of plant cells. However, the molecular mechanisms underlying this process are still poorly understood. In the root, the highly conserved microRNA miR396 and its targets, the GROWTH REGULATING FACTORs (GRFs), control the transition from stem cells to proliferative cells. miR396 promotes stem cell activity by repressing the GRFs, which in turn promote cell proliferation. Here we show that the miR396-GRF regulatory module guides stem cell reconstitution after root tip excision, playing a dual role: while miR396 promotes competence, the GRFs control regeneration speed. Moreover, plants with ectopic miR396 expression have defined stem cell niches before the excision, but do not reconstitute them afterwards, remaining in an open state despite continuing to grow. We propose that this phenomenon is caused by dispersed stem cell activity, which supports growth after root tip excision without reconstituting the organized stem cell niche typical of Arabidopsis roots.

overall_design: Protoplasts from cut (5 days after cut) or uncut (control) roots, from wild-type and miR396-overexpressing plants, were isolated per quadruplicate, and scRNA-seq was performed.

middle: L

first: Ramiro

middle: E

last: Rodríguez

first: Franco

last: Lazzara

first: Daniela

last: Liebsch

first: XinAi

last: Zhao

first: Jian

last: Zeng

first: Lotte

last: Bald

first: Carla

last: Schommer

first: Jan

middle: U

last: Lohmann

first: Javier

last: Palatnik


title: Tumor-derived colorectal cancer organoids induce a unique Treg cell population through direct modulation of CD4+ T cell differentiation.

city: Utrecht

first: Sonia

last: Aristin Revilla

state: Utrecht

address: Heidelberglaan 100

department: CMM

country: Netherlands

institute: UMC Utrecht

summary: To investigate the capacity for CRC tumor-organoids to influence Treg cell fate specification in vitro, we established a transwell co-culture model utilizing CD4+ T cells together with CRC tumor-organoids in which we study murine tumor-organoid induced Treg (mTO-iTreg) cell differentiation. As control, murine CD4+ T cells were cultured in transwell inserts and treated with TGFβ to induce Treg (TGFβ-iTreg) cells. To define the transcriptional identity of in vitro mTO-iTreg cells we performed bulk RNA-sequencing. RNA-sequencing analysis identified distinct transcriptional profiles between CRC organoid-induced Treg cells and TGFβ-induced Treg cells, with upregulation of key functional signature genes linked to CRC Treg cells in vivo. High expression of genes upregulated in CRC organoid-induced Treg cells correlates with shorter progression free intervaland overall survival of CRC patients, highlighting their prognostic potential.

overall_design: We then performed RNA sequencing of 3 different cells at different time points. Firstly, Foxp3 eGFP- CD4+ T cells (n=3) were isolated on day 0, prior to co-culture establishment; secondly, CD4+ Foxp3 eGFP+ TGFβ-iTreg cells (n=3) were isolated on day 5 of culture; and thirdly, CD4+ Foxp3 eGFP+ mTO-iTreg cells (n=3) were isolated from the co-culture on day 5. Diferential gene expression analysis of RNAseq data for Foxp3 eGFP- CD4+ T cells, CD4+ Foxp3 eGFP+ TGFβ-iTreg cells and CD4+ Foxp3 eGFP+ mTO-iTreg cells was performed.

first: Cynthia

last: Frederiks

first: Stefan

last: Prekovic

first: Enric

last: Mocholi

first: Onno

last: Kranenburg

first: Paul

middle: J

last: Coffer


title: Age-related TFEB Downregulation in Proximal Tubules Causes Systemic Metabolic Disorders with Occasional ApolipoproteinA4-related Amyloidosis

city: Suita

first: Jun

last: Nakamura

state: Osaka

address: Box D11, 2-2 Yamada-oka

department: Nephrology

country: Japan

institute: the University of Osaka Graduate School of Medicine

summary: We generated proximal tubular epithelial cell-specific Tfeb-deficient mice and bred them for up to 24 months and found that Tfeb deficiency in the proximal tubules causes metabolic disorders, which occasionally lead to apolipoproteinA4 (APOA4) amyloidosis, with age. To elucidate the mechanism of the metabolic disturbances, we performed scRNA-seq analyses of the kidneys.

overall_design: Renal cortex samples dissected from kidneys were isolated into single cells and analyzed using scRNA-seq.

first: Takeshi

last: Yamamoto


title: Single cell RNA sequencing analysis of P63+ lung progenitor cells isolated from IPF patients

city: Shanghai

first: Wei

last: Zuo

state: Shanghai

address: No. 500 Zhennan Road, Putuo District, Shanghai

institute: University

summary: To gain molecular insights into the identity composition of the cells selectively grown in the culture system, we performed single-cell sequencing on the primary cell.  Unsupervised clustering of the cells uncovered 3 distinct subpopulations:C1, C2 and C3. Altogether the data indicated that the current cell isolation and expansion system could efficiently enrich P63+ lung progenitor cells from IPF patients.

overall_design: After removing feeder cells through gradient digestion, the primary cells isolated by the pharmaceutical-grade cell cloning culture system were digested to prepare a single-cell suspension. Single cells were captured and barcoded in 10× Chromium Controller (10× Genomics).

first: Yu


title: Enhanced conversion of T cells into CAR T cells by modulation of the MAPK/ERK pathway

city: Langen

first: Christian J.

last: Buchholz

state: Hessen

address: Paul-Ehrlich-Strasse 51-59

institute: Paul Ehrlich Institut

summary: Delivery of chimeric antigen receptors (CARs) to T cells is usually mediated by lentiviral vectors (LVs), which can have broad tropism or be T-cell targeted. To better understand the molecular events during CAR T cell generation, T cell transduction with four different LVs was followed by single-cell multi-omics analysis, distinguishing between transduced T cells and T cells with vector signal but no CAR. We found that only a fraction of the T cells that had encountered vectors converted into CAR T cells. Single-cell transcriptome data revealed that interferon-stimulated genes were upregulated in non-transduced cells, whereas ERK2 phosphatases were upregulated in CAR T cells. This expression pattern was evident in CAR T cells from healthy donors and patients. The role of these genes in T cell transduction was confirmed by chemical inhibitors. These data provide molecular insights into T cell transduction with implications for improving CAR T cell generation.

overall_design:
Activated PBMCs from two healthy donors were transduced with lentiviral vectors (LVs). lentiviral vectors pseudotyped with the vesicular stomatitis virus G (VSV-G) protein (VSV-LV), T-cell targeted vectors pseudotyped with paramyxoviral envelope proteins and using CD3, CD4, or CD8 as entry receptors were used for transduction and after 72 hours incubation cells were stained with AbSeqs and sample multiplexing tags and were analyzed with single-cell multi-omics using BD Rhapsody T cell targeted gene panel.
(SampleTag11_hs: Donor 1_CD4-LV, SampleTag12_hs: Donor 1_CD8-LV, SampleTag09_hs: Donor 2_CD4-LV, SampleTag10_hs: Donor 2_CD8-LV).
(SampleTag12_hs: Donor 1_CD3-LV, SampleTag11_hs: Donor 1_VSV-LV).
(SampleTag09_hs: Donor 2_CD3-LV, SampleTag10_hs: Donor 2_VSV-LV)

first: Elham

last: Adabi

first: Filippos

middle: T

last: Charitidis

first: Frederic

last: Thalheimer

first: Mar

last: Guaza-Lasheras

first: Colin

last: Clarke

first: Christian


title: Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs

city: New Haven

first: Carson

last: Thoreen

address: 333 Cedar Street

department: Cellular and Molecular Physiology

institute: Yale University

summary: We report an efficient, high-throughput method to optimize translation initiation from modified mRNAs. Using direct analysis of ribosome targeting, DART, we identify 5′ untranslated sequences that mediate 250-fold effects on ribosome recruitment, show that incorporation of m1Ψ significantly affects activity, and identify more active 5′ UTRs that surpass current mRNA vaccines. DART is broadly applicable to dissect translation initiation mechanisms of human genes and optimize mRNA regulatory sequences for desired protein outputs

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
An RNA pool containing > 35,000 human 5′UTRs were obtained through in vitro transcription of a synthesized DNA oligonucleotide library.
The RNA pool was allowed to translate in vitro in HeLa cell extract with the presence of cycloheximide. Initiating Ribosomes were frozen at start codons. 80S-associated RNAs were isolated using sucrose gradient.
We analysis the 5′UTRs in the fraction associated with 80S and in the total IVT input. The ratio indicates the initiation efficciency.
DMS-MapSeq was used to analysis the secondary structure of RNAs in the solution.

first: Cole

last: Lewis

first: Li

middle: H

last: Xie

first: Shivani

middle: M

last: Bhandarkar

first: Danni

last: Jin

first: Kyrillos

last: Abdallah

first: Austin

last: Draycott

first: Yixuan

last: Chen

first: Wendy

middle: V

last: Gilbert


title: Dynamic Control of RNA-DNA Hybrid Formation Orchestrates DNA2 Activation at Stalled Forks by RNAPII and DDX39A [ChIP-seq]

city: Hangzhou

first: Dong

last: Fang

state: Zhejiang

address: 866 Yuhangtang Road

department: Life Sciences Institute

institute: Zhejiang University

summary: The accurate processing of stalled forks by the DNA2 nuclease is pivotal for replication fork restart, as excessive degradation poses a threat to genomic stability. However, the regulation of DNA2 activity at stalled forks remains elusive. Here, we demonstrate that, upon replication stress, RNA polymerase II (RNAPII) is recruited to stalled forks, actively promoting the transient formation of RNA-DNA hybrids. Furthermore, we provide evidence that DDX39A, functioning as an RNA-DNA resolver, unwinds these hybrids, allowing DNA2 access to stalled forks. This orchestrated process facilitates controlled DNA2-dependent stalled fork processing and restart. Nevertheless, premature removal of RNA-DNA hybrids at stalled forks leads to DNA2-dependent excessive degradation of nascent DNA. Finally, we reveal that loss of DDX39A enhances the protection of stalled forks in BRCA1/2-deficient cells, consequently conferring chemoresistance within this specific cellular context. Our results suggest that the dynamic regulation of RNA-DNA hybrid formation at stalled forks by RNAPII and DDX39A precisely governs the timing of DNA2 activation, contributing to stalled fork processing and restart, ultimately promoting genome stability.

overall_design: DNA replication fork sequencing in WT and DDX39A KO HeLa cell line with or whithout hydroxyurea treatment.

first: Lizhi

last: Song

first: Yanjun

last: Zhang

first: Haonan

last: Fan


title: Organ-specific Microenvironments Drive Divergent T Cell Evolution in Acute Graft-Versus-Host Disease

city: Boston

first: Rene

last: Bermea

state: MA

address: 55 Fruit St

department: Pulmonary and Critical Care Medicine

institute: Massachusetts General Hospital

summary: Tissue-specific T cell immune responses play a critical role in maintaining organ health, but can also drive immune pathology during both auto- and alloimmunity. The mechanisms controlling intra-tissue T cell programming remain unclear. Here, we leverage a non-human primate model of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HCT) to probe the biological underpinnings of tissue-specific alloimmune disease, using a comprehensive systems immunology approach including multiparameter flow cytometry, population-based transcriptional profiling, and multiplexed single-cell RNA- and TCR- sequencing. Transcriptional profiling revealed substantial biological differences between T cells, infiltrating the lung and liver during aGVHD. These included enrichment for transcriptional pathways controlling extracellular matrix remodeling and chemotaxis in the lung, and enrichment for transcriptional pathways linked to nucleic acid metabolism and proliferation in the liver. Single cell RNA- and TCR-sequencing substantiated divergent organ-specific transcriptional programing of tissue-infiltrating T cells, which was linked to clonal expansion, with expanded clones progressively enriched for CX3CR1-expressing CD8 effector T cells in lung, and EOMES-expressing CD8 effector-memory T cells in liver. This divergent evolution of T cells was maintained even for T cells sharing the same TCRs, indicating its independence from antigen-specificity. Together, these results provide new insights into the principal role that tissue microenvironment-derived signals play in local T cell transcriptional programming during alloimmune-mediated clonal expansion, and which suggest potential opportunities to develop 64 tissue-specific therapeutics to curtail pathogenic immunity after transplant.

overall_design: Cryopreserved NHP cells were flow cytometrically sorted for Live, CD45+ CD3+, and CD14/CD20-cells. The cells were then immediately processed for single cell sequencing using the Next GEM 5’ v2 Gel Beads and Single Cell 5’ v2 Sample Index Plate TT. Gene expression alignment and TCR reconstruction was performed using Cellranger “multi” and “aggregate” pipelines (10X Genomics) to integrate both gene expression (GEX) and VDJ libraries. For the reference transcriptome, we first filtered Ensembl’s gtf file for v107 of the Macaca mulatta gene annotation using Cellranger’s “mkgtf” command. We provide the raw fastq files following sequencing in this repository. We also provide the individual sample outputs from Cellranger “multi” as well as the .hdf5 files used in our analysis pipeline as processed data.

first: Kayleigh

last: Ingersoll Omdahl

first: Ryan

last: Fleming

first: Kyle

last: Kimler

first: James

last: Kaminski

first: Lida

middle: P

last: Hariri

first: Amy

last: Ly

first: Xianliang

last: Rui

first: Lorenzo

last: Cagnin

first: Jennifer

last: Lane

first: Ulrike

last: Gerdemann

first: Bruce

last: Blazar

first: Victor

last: Tkachev

first: Leslie

last: Kean


title: Transcriptomics of Tgfb signaling

city: Rancagua

first: Nicolas

last: Santander

address: Libertador Bernardo O'Higgins 611

country: Chile

institute: Universidad de O´Higgins

summary: Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia progenitor-expressed integrin beta 8 activates microglia-expressed TGFB1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary domains of developmentally arrested “dysmature” microglia and homeostatic microglia that persist into adulthood. In the absence of autocrine TGFB1 signaling, we find that microglia adopt a similar reactive microglial phenotype, leading to astrogliosis and neuromotor symptoms almost identical to Itgb8 mutant mice. By comparing mice with genetic deletions in critical components downstream of Itgb8, we show that non-canonical (Smad-independent) signaling partially suppresses the dysmature microglia phenotype, associated neuromotor dysfunction and expression of disease-associated genes, providing compelling evidence for the adoption of microglial develomental signaling pathways in the context of injury or disease.

overall_design: We inactivated key members of Tgfb signaling in microglia (with Cx3cr1Cre) or neural progenitors (with NestinCre) and collected brain or FACS-isolated microglia. Total RNA from these samples was used for RNA-Seq.

first: Gabriel

last: McKinsey

first: Thomas

last: Arnold


title: Replication stress, microcephalic primordial dwarfism, and compromised immunity in ATRIP deficient patients.

city: Gent

first: Evi

last: Duthoo

address: Molenaarsstraat 148

country: Belgium

institute: Ghent University

summary: Ataxia telangiectasia and Rad3-related (ATR) kinase and its interacting protein ATRIP orchestrate the replication stress response. Homozygous splice variants in the ATRIP gene, resulting in ATRIP deficiency, were identified in two patients of independent ancestry with microcephaly, primordial dwarfism, and recurrent infections. The c.829+5G>T patient exhibited lymphopenia, poor vaccine responses, autoimmune features with hemolytic anemia, and neutropenia. Immunophenotyping revealed reduced CD16+/CD56dim NK cells and absent naïve T cells, MAIT cells, and iNKT cells. Lymphocytic defects were characterized by TCR oligoclonality, abnormal class switch recombination, and impaired T cell proliferation. ATRIP deficiency resulted in low-grade ATR activation but impaired CHK1 phosphorylation under genotoxic stress. ATRIP-deficient cells inadequately regulated DNA replication, leading to chromosomal instability, compromised cell cycle control, and impaired cell viability. CRISPR-SelectTIME confirmed reduced cell fitness for both variants. This study establishes ATRIP deficiency as a monogenic cause of microcephalic primordial dwarfism, highlights ATRIP's critical role in protecting immune cells from replication stress, and offers new insights into its canonical functions.

overall_design: Approximately 10 million PBMCs were isolated each for one ATRIP-deficient patient and three sex/age matched controls (human). Additionally, approximately 4000 viable CD19+ B cells were sorted for each sample by FACS. In sequencing lane 1, approximately 6000 viable PBMCs each from both the ATRIP-deficient patient and healthy control 1 were hashed (total approximately 12000 cells) and loaded for CITE-sequencing and TCR-sequencing. In lane 2, approximately 6000 viable PBMCs each from both healthy control 2 and healthy control 3 were hashed (total approximately 12000 cells) and loaded for CITE-sequencing and TCR-sequencing. In sequencing lane 3, approximately 3000 CD19+ B cells were loaded for each of the samples (total approximately 12000 cells). Due to concerns of underloading of sequencing lane 3, an additional 4000 B-cells were sorted per sample, and loaded onto sequencing lane 4 (approximately 12000 cells).

first: Elien

last: Beyls

first: Lynn

last: Backers

first: Thorkell

last: Gudjónsson

first: Peiquan

last: Huang

first: Leander

last: Jonckheere

first: Sebastian

last: Riemann

first: Bram

last: Parton

first: Likun

last: Du

first: Veronique

last: Debacker

first: Marieke

last: De Bruyne

first: Levi

last: Hoste

first: Ans

last: Baeyens

first: Anne

last: Vral

first: Eva

last: Van Braeckel

first: Jens

last: Staal

first: Geert

last: Mortier

first: Tessa

last: Kerre

first: Qiang

last: Pan-Hammarström

first: Claus

last: Sørensen

first: Filomeen

last: Haerynck

first: Simon

last: Tavernier


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [PROX1 CUT&RUN Liver/MEF/Hep3B]

status: Public on Dec 18 2024

city: Heidelberg

first: Moritz

last: Mall

address: Im Neuenheimer Feld 280

institute: German Cancer Research Center (DKFZ)

summary: Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. While transcription master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether plasticity is actively suppressed by lineage-specific repressors. Here, we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong. We validated hepatocyte-specific candidates using reprogramming, revealing that Prospero homeobox protein 1 (PROX1) enhanced hepatocyte identity by direct repression of alternative fate master regulators. In mice, Prox1 was required for efficient hepatocyte regeneration after injury and was sufficient to prevent liver tumorigenesis. In line with patient data, Prox1 depletion caused hepatocyte fate loss in vivo and enabled the transition of hepatocellular carcinoma to cholangiocarcinoma. Conversely, overexpression promoted cholangiocarcinoma to hepatocellular carcinoma transdifferentiation. Our findings provide evidence for PROX1 as a hepatocyte-specific safeguard and support a model where cell type-specific repressors actively suppress plasticity throughout life to safeguard lineage identity and thus prevent disease.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: CUT&RUN of MEFs and Hep3B cells upon Prox1 ovexpression, both harvested after two days of Prox1 ovexpression; along with CUT&RUN of endogenous Prox1 from primary mouse liver.

first: Bryce

last: Lim

first: Aryan

last: Kamal

first: Borja

last: Gomez Ramos

first: Juan

last: Adrian-Segarra

first: Judith

last: Zaugg


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [PROX1 OE Hep3B RNA-seq]

overall_design: RNA-seq of Hep3B human HCC cell lines ± PROX1 overexpression, harvested after 7 days.


title: Dynamic Control of RNA-DNA Hybrid Formation Orchestrates DNA2 Activation at Stalled Forks by RNAPII and DDX39A [RNA-seq]

first: Dong

last: Fang

overall_design: RNA replication fork sequencing in WT and DDX39A KO HeLa cell line with or whithout hydroxyurea treatment.

first: Lizhi

last: Song

first: Yanjun

last: Zhang

first: Haonan

last: Fan


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [PROX1 OE Hep3B ATAC-seq]

overall_design: ATAC-seq of Hep3B human HCC cell lines ± PROX1 overexpression, harvested after two days.


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 Late OE CCA RNA-seq]

overall_design: RNA-seq of CCA liver tumour nodules induced by hydrodynamic tail vein injection (HDTVI) to induce Akt and NICD overexpression, ± dox-induced overexpression of Prox1 from week 3 to 4.


title: The epigenetic reader Phf2 is positioned in the genome by cohesin [HiC]

city: Vienna

first: Roman

middle: R

last: Stocsits

address: Campus-Vienna-Biocenter 1

institute: IMP - Research Institute of Molecular Pathology

summary: Eukaryotic genomes are assembled into chromatin by histones and extruded into loops by cohesin. These mechanisms control important genomic functions, but whether histones and cohesin cooperate in genome regulation is poorly understood. Here we identify Phf2, a member of the Jumonji-C family of histone demethylases, as a cohesin-interacting protein. Phf2 binds to H3K4me3 nucleosomes at active transcription start sites (TSSs) but also co-localizes with cohesin. Cohesin depletion reduces Phf2 binding at sites lacking H3K4me3, and depletion of Wapl and CTCF re-positions Phf2 together with cohesin in the genome, resulting in the accumulation of both proteins in vermicelli and cohesin islands. Conversely, Phf2 depletion reduces cohesin binding at TSSs lacking CTCF, decreases the number of short cohesin loops, but increases the length of heterochromatic B compartments. These results suggest that Phf2 is an ‘epigenetic reader’, which is translocated through the genome by cohesin-mediated DNA loop extrusion, and which recruits cohesin to active TSSs and limits the size of B compartments. These findings reveal an unexpected degree of cooperativity between epigenetic and architectural mechanisms of eukaryotic genome regulation.

overall_design: The objective of this experiment was to investigate how cohesin affects chromatin structure by positioning the epigenetic reader Phf2, using Hi-C on wild-type (WT) and mutant (Phf2 KO and Wapl KO) mouse embryonic fibroblast cells (MEFs). For the conditional deletion of floxed Phf2 and Wapl alleles, we used the inducible CreERT2 recombinase expressed from the Rosa26 locus (CreER). Each condition was performed in biological duplicates, and libraries were sequenced on an Illumina platform. The goal is to assess how Phf2 and cohesin impact chromatin structure. These findings reveal an unexpected level of cooperativity between epigenetic and architectural mechanisms in eukaryotic genome regulation.

first: Wen

last: Tang

first: Lorenzo

last: Costantino

first: Gordana

last: Wutz

first: Jan-Michael

last: Peters


title: A novel nuclear protein complex controlling the expression of developmentally regulated genes in Toxoplasma gondii [RNA-seq]

first: xue

last: lilan

address: shizishan

institute: huazhong agriculture university

summary: To investigate the transcriptional effects of TgAP2XII-5 on type II Toxoplasma gondii (ME49) in the tachyzoite and bradyzoite stages. Also, to study the transcriptional role of TgAIP1 on ME49 tachyzoites.

overall_design: RNA-seq

first: Lilan

last: Xue

first: Jingwen

first: Bang

last: Shen


title: Gene expression profilling of NEUROG2-mCherry lineage in cerebral organoids

first: yacine

last: touahri

summary: To identify and purify NEUROG2-expressing cells and trace their short-term lineage, we engineered two NEUROG2-mCherry knock-in human embryonic stem cell (hESC) lines. Transcriptomic profiling of NEUROG2:mCherry knock-in hESC-derived cerebral organoids revealed an enrichment of neurogenic, oligodendrocyte precursor cell and extracellular matrix-associated gene transcripts in mCherry-high cells. Conversely, only neurogenic gene transcripts were enriched in mCherry-high cells from Neurog2:mCherry knock-in mouse cortices.

overall_design: We engineered two NEUROG2-mCherry knock-in human embryonic stem cell (hESC) lines and and compared Transcriptomic profiling of NEUROG2:mCherry positive and negative cells in hESC-derived cerebral organoids

first: Vorapin

last: Chinchalongporn

first: Carol

last: Schuurmans


title: PRMT5 promotes chemoresistance of breast cancer stem cells by regulating DNA repair gene splicing facilitating an enhanced DNA damage response

city: Birmingham

first: Kelly

last: Chiang

address: Vincent Drive

department: Institute of Cancer and Genomic Sciences

country: United Kingdom

institute: University of Birmingham

summary: Breast cancer stem cells (BCSCs) are a rare cell population that are responsible for tumour initiation, metastasis, chemoresistance and tumour relapse. Despite this, the mechanism by which BCSCs withstand genotoxic stress is largely unknown. Here, we uncover a pivotal role for the arginine methyltransferase PRMT5 in mediating BCSC chemoresistance by modulation of DNA repair efficiency. Notably, PRMT5 inhibitors synergise with cisplatin and radiotherapy, inducing defective DNA repair thereby potentiating BCSC apoptosis. Mechanistically, we demonstrate the crucial function of PRMT5 in ensuring splicing fidelity of DNA damage response genes, particularly those integral to the Fanconi Anaemia and homologous recombination pathways. A comparison of BCSCs and their bulk cell progeny identified shared (ATM, DDX11, EXO1, FAN1, SLX4) and unique (ATR, RAD17, RAD51D, RUVBL1) PRMT5-dependent alternative splicing events. Surprisingly, these events rarely repressed gene expression, suggesting that PRMT5 inhibition predominantly results in nuclear detention of intron-containing transcripts and the production of non-canonical isoforms with compromised protein function. This study thus reveals promising strategies to improve the therapeutic efficacy of PRMT5 inhibitors that promote effective BCSC eradication.

overall_design: MCF7 or AU565 breast cancer cells were treated with 0.5uM GSK591 for 4 days. Changes in gene expression and splicing were determined by RNA-seq. Each sample has multiple raw fastq files corresponding to number of lanes and/or runs per sample.

first: Clare

last: Davies

first: Roland

last: Arnold


title: Pseudovibriamides from Pseudovibrio marine sponge bacteria promote swarming motility via transcriptional modulation

city: CHICAGO

first: Yitao

last: Dai

state: IL

address: 900 S Ashland ave Room 3106, MBRB Building 919 M/C 870

institute: University of Illinois at Chicago

summary: Pseudovibrio a-Proteobacteria have been repeatedly isolated from marine sponges and proposed to be beneficial to the host. Bacterial motility is known to contribute to host colonization. We have previously identified pseudovibriamides A and B, produced in culture by Pseudovibrio brasiliensis Ab134, and shown that pseudovibriamide A promotes flagellar motility. Pseudovibriamides are encoded in a hybrid nonribosomal peptide synthetase-polyketide synthase gene cluster that also includes several accessory genes. Pseudovibriamide A is a linear heptapeptide and pseudovibriamide B is a nonadepsipeptide derived from pseudovibriamide A. Here we define the borders of the pseudovibriamides gene cluster, assign function to biosynthetic genes using reverse genetics and test the hypothesis that pseudovibriamides impact motility by modulating gene transcription. RNA-seq transcriptomic analyses of strains having different compositions of pseudovibriamides suggested that both pseudovibriamides A and B affect genes potentially involved in motility, and that a compensatory mechanism is at play in mutants that produce only pseudovibriamide A, resulting in comparable swarming motility as the wild type. The data gathered suggest that pseudovibriamides A and B have opposite roles in modulating a subset of genes, with pseudovibriamide B having a primary effect in gene activation, and pseudovibriamide A on inhibition. Finally, we observed many differentially expressed genes (up to 29% of the total gene number) indicating that pseudovibriamides have a global effect on transcription that goes beyond motility.

overall_design: To investiagte the impact of different compositions of pseudovibriamides on gene transcription, we extracted RNA from 24-h triplicate liquid cultures of the wild type (all pseudovibriamides are present), the ∆pppA mutant (only pseudovibriamide C is present), the ∆pppD mutant (only pseudovibriamide A is present) and the ∆pppE mutant (no pseudovibriamides are present). We then calculated gene expression levels using raw reads obtained from RNA-seq of each strain. Finally, we performed pairwise differential expression analysis between each mutant and the wild type, and between the ∆pppA mutant and the ∆pppD mutant using average TPM values.

first: Alessandra

last: Eustáquio


title: Dynamic Control of RNA-DNA Hybrid Formation Orchestrates DNA2 Activation at Stalled Forks by RNAPII and DDX39A.

first: Dong

last: Fang

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

overall_design: Refer to individual Series


title: Low fucosylation defines the glycocalyx of progenitor cells and melanocytes in the limbal epithelial stem cell niche

first: Pablo

last: Argueso

address: 150 Harrison Avenue

institute: Tufts Medicine

summary: It is widely recognized that the glycocalyx has significant implications in regulating the self-renewal and differentiation of adult stem cells. Yet, its composition remains poorly understood. Here, we show that the fucose-binding Aleuria aurantia lectin (AAL) binds differentially to basal cells in the stratified epithelium of the human limbus, hair follicle and meibomian gland duct. Using fluorescence-activated cell sorting in combination with single-cell transcriptomics, we find that most epithelial progenitor cells and melanocytes in the limbus display low AAL staining (AALlow) on their cell surface, an attribute that is gradually lost in epithelial cells as they differentiate into mature corneal cells. AALlow epithelial cells were enriched in putative limbal stem cell markers and displayed high proliferative potential. Further analyses revealed that AALlow epithelial cells had reduced expression of GDP-mannose-4,6-dehydratase, an enzyme catalyzing the first and regulatory steps in the de novo biosynthesis of GDP-fucose, and that inhibition of fucosylation using a small-molecule fucose analogue stimulated the proliferative potential of limbal epithelial cells ex vivo. These results provide critical insights into the composition of the glycocalyx in adult stem cells and could thereby have implications to the regeneration of the human limbal stem cell niche.

overall_design: Single cell transcriptional profiling was performed on AALlow and AALhigh subpopulations to identify the cell types present and the transcript levels of fucosylation-related genes.

first: Ashley

last: Woodward

first: Damien

last: Guindolet

first: Rafael

last: Martinez-Carrasco

first: Eric

last: Gabison

first: Robert

last: Lavker

last: Argüeso


title: Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [WGRS]

city: Beijing

first: Jiadong

last: Wang

address: 38 College Road, Haidian District

department: Department of Radiation Medicine

institute: Peking University Health Science Center

type: Genome variation profiling by high throughput sequencing

summary: Safeguarding replication fork stability in transcriptionally active regions, which require high DNA replication accuracy, is crucial for precise DNA replication and prevention of mutations. However, how cells ensure the stability of replication forks in these regions remains a critical challenge. Here, we discovered the pervasive existence of replication forks-associated RNA-DNA hybrids (RF-RDs) within transcriptionally active regions, where they act as a protective barrier against DNA2-mediated nascent DNA degradation and prevent replication fork collapse upon replication stress. Subsequently, the RNA helicase DDX39A dismantles RF-RDs, facilitating proper DNA2-mediated DNA resection and replication fork restart. Excessive dissolution of RF-RDs causes replication fork collapse and genomic instability, while insufficient dissolution of RF-RDs under replication stress increases fork stability, resulting in chemoresistance that can be reversed by eliminating RF-RDs. In summary, we elucidated the prevalence of RF-RDs at replication forks within transcriptionally active regions, revealed their pivotal role in safeguarding replication fork stability, and proposed that targeting RF-RDs holds promise for augmenting chemotherapeutic efficacy.

overall_design: Whole-genome resequencing (WGRS) to test the mutations caused by 5,6-dichloro-benzimidazole 1-β-D-ribofuranoside (DRB) treatment or DDX39A overexpression under replication fork stress.

first: Zhanzhan

last: Xu

first: Chen

last: Nie

first: Weibin


title: Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [REFORC]

overall_design: We developed a method called Replication Fork RNA Capture (REFORC) to exactly determine the RNA associated with nascent replication fork DNA, and prepared for cDNA library building and related high-throughput sequencing.


title: Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [RNA_seq]

overall_design: We performed RNA sequencing (RNA-seq) to test the transcriptome of normal HeLa cell.


title: Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [EdU_seq]

overall_design: EdU-seq to analyze the genomic location of replication fork with or without fork stress treatment in DDX39A knockdown or wild-type cells.


title: Carfilzomib inhibits the progression of hepatocellular cancer via upregulating GADD45a expression

city: Zhengzhou

first: wenzhi

last: guo

address: Zhengzhou, Henan Province

institute: The First Affiliated Hospital of Zhengzhou University

summary: Hepatocellular carcinoma (HCC) ranks among one of the most prevalent and lethal cancers affecting humans. Currently, there are limited effective treatments available for HCC. In the search for potential anti-HCC agents, we found that carfilzomib, a proteasome inhibitor, exerts anti-HCC activities. However, the underlying mechanisms of action are main unclear. In this study, we evaluated the effectiveness of carfilzomib against HCC capability and explored its underlying mechanisms. Cell Counting Kit 8 (CCK8), EdU (5-ethynyl-2-deoxyuridine) staining, and colony formation assays were employed to analyze the antiproliferative ability of carfilzomib on MHCC-97H and Huh7 cells. Additionally, flow cytometry was used to assess the effect on the cell cycle. Western blotting was utilized to examine the protein expression levels associated with cell cycle arrest. Furthermore, short hairpin RNA (shRNA) transfection was used to investigate the role of GADD45a (growth arrest and DNA damage 45a) on carfilzomib-induced cell cycle arrest. A xenograft tumor model using nude mouse was employed to evaluate the anti-HCC activity of carfilzomib in vivo. The finding demonstrated that carfilzomib inhibited the proliferation, invasion, and migration in both MHCC-97H and Huh7 cells. In addition, carfilzomib caused cell cycle arrest by suppressing the expression of cyclin A1, cyclin E1, cyclin-dependent kinases 2 (CDK2), and cyclin-dependent kinases 4 (CDK4). It also upregulated the expression of GADD45a, and inhibition of GADD45a through shRNA abolished carfilzomib-induced cell cycle arrest in HCC cells. Importantly, carfilzomib demonstrated its ability to inhibit the Huh7 cell growth in vivo. Our research has revealed, for the first time, that carfilzomib inhibited the progression of HCC cells by upregulating GADD45a expression. These findings suggest that carfilzomib could be a potential chemotherapeutic agent against HCC.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HCC cells treated with DMSO and carfilzomib

first: Mingxun

last: Chen

first: Wenzhi

last: Guo


title: Decoding the immune checkpoint signatures in human atherosclerosis: implications of type 2 diabetes and lipid-lowering intervention [human_PBMC_CITEseq]

city: New York

first: Michael

last: Gildea

address: 550 1st avenue

institute: NYU Langone Health

summary: Immune checkpoint inhibitor (ICI) therapies in cancer accelerate atherosclerosis progression. Here, we have charted the landscape of immune checkpoint gene expression and defined the network of disease-relevant interactions with single-cell resolution. We found that signaling through PD-1 and CTLA4 is driven by a population of dendritic cells enriched for FSCN1 that can be derived from peripheral blood cells following anti-PD-1 or -CTLA4 treatment ex vivo. Type 2 diabetes dampened plaque PD-1 and CTLA4 signaling, showing that cardiometabolic comorbidities elicit unique responses to ICIs. Lipid-lowering therapy equalized the intensity and direction of immune checkpoint interactions in human blood, while atherosclerotic mice subjected to a lipid-lowering diet displayed both increased co-inhibitory signaling and a downregulation of inflammatory transcriptional programs in plaques. Our findings underscore the potential of lipid-lowering therapies in stabilizing immune checkpoint interactions and reducing plaque inflammation, offering new insights on atherosclerosis and cardiovascular risks in cancer patients undergoing ICI treatments.

overall_design: peripheral blood mononuclear cells were profiled via CITE-seq after treatment with immunce checkpoint inhibitors.

middle: A

first: Jose

middle: G

last: Duran

first: Dayasagar

last: Das

first: Kathryn

last: Moore

first: Chiara

last: Giannarelli


title: Precise RNA targeting with CRISPR-Cas13d

first: Sydney

last: Hart

address: 101 AVENUE OF THE AMERICAS NEW YORK, NY

institute: New York Genome Center

summary: The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR-Cas13. We show that collateral activity only occurs with high RfxCas13d expression. Using low-copy RfxCas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Further, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability.

overall_design: We engineered three human cell lines, HAP1, HEK293FT, and A375, to express the nuclear-localized RfxCas13d effector under doxycycline-inducible control. We also engineered HAP1 cells to express nuclear-localized High fidelity Cas13d under doxycycline-inducible control. Next, we transduced each cell line with one of 3 lentiviral gRNA libraries (Screen 1: RfxCas13d-expressing HAP1 and HEK293FT cells; Screen 2: RfxCas13d expressing A375 cells; or Screen 3: HfCas13d-expressing HAP1 cells) at a low multiplicity-of-infection to ensure each cell received only a single perturbation and induced Cas13 expression via doxycycline addition. We harvested genomic DNA from these cells at day 0 or 14 days post-Cas13 induction and computed changes in gRNA abundance via amplicon sequencing.

middle: K


title: Decoding the immune checkpoint signatures in human atherosclerosis: implications of type 2 diabetes and lipid-lowering intervention [human_carotid_plaque]

overall_design: CD45+ Immune cells were isolated from human carotid plaque tissue from patients undergoing carotid endarterectomy. Cells were profiled using scRNAseq.


title: Frequently arising ESX-1 associated phase variants influence Mycobacterium tuberculosis fitness in the presence of host and antibiotic pressures

city: Quincy

last: Luna

state: Massachusetts

address: 7 Seaport Dr, 322

institute: UMass Chan Medical School

summary: Mycobacterium tuberculosis (Mtb) exhibits an impressive ability to adapt to rapidly changing environments, despite its genome’s apparent stability. Recently, phase variation through indel formation in homopolymeric tracts (HT) has emerged as potentially important mechanism promoting adaptation in Mtb. This study examines the impact of common phase variants associated with the ESX1 Type VII secretion system, focusing on a highly variable HT upstream of the ESX1 regulatory factor, espR. By engineering this frequently observed indel into an isogenic background, we demonstrate a single basepair insertion in the espR 5’ UTR causes post-transcriptional upregulation of EspR protein abundance and corresponding alterations in the EspR regulon. Consequently, this mutation increases expression of ESX1 components in the espACD operon and enhances ESX1 substrate secretion.  We find that this indel specifically increases isoniazid resistance without impacting the effectiveness of other drugs tested. Furthermore, we show that commonly observed HT indels that regulate either espR translation or espACD transcription increase bacterial fitness in a mouse infection model. The presence of multiple ESX1 associated HTs provides a mechanism to combinatorially tune protein secretion, drug sensitivity, and host-pathogen interactions.  More broadly, these findings support emerging data that Mtb utilizes HT-mediated phase variation to strategically direct genetic variation to certain sites across the genome in order to adapt to changing pressures.

overall_design: RNAseq was performed on mid-log phase Mycobacterium tuberculosis H37Rv. Expression levels in ESX mutant backgrounds were compared to a wild type control


title: Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [EdUseq]

overall_design: EdU-seq to analyze the genomic location of replication fork with or without fork stress treatment in DDX39A knockdown, DDX39A over-expression or wild-type cells.


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 OE iHep Timecourse RNA-seq]

first: Moritz

last: Mall

overall_design: RNA-seq of MEFs ± Prox1 overexpression, harvested after 2, 7, 14, or 28 days of 4in1-driven reprogramming to induced hepatocytes.

first: Bryce

last: Lim

first: Aryan

last: Kamal

first: Juan

last: Adrian-Segarra

first: Judith

last: Zaugg


title: CCL2/CCR2/Erk signal induced by interaction between cancer cells and macrophages contributes to hepatocellular carcinoma progression

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

institute: Toray Industries,Inc.

summary:
Macrophages infiltrating into tumors are known as tumor-associated macrophages (TAMs) and play a central role in tumor progression. In immunohistochemistry, intra-tumoral CD68-positive macrophages correlated with poor prognosis and clinicopathological factors of patients with hepatocellular carcinoma (HCC). Next, we established an indirect co-culture assay between HCC cells and peripheral blood-derived macrophages. cDNA microarray analysis revealed that C-C motif chemokine ligand 2 (CCL2) was highly expressed in HCC cells co-cultured with macrophages. CCL2 bound to CCR2, known as a main receptor of CCL2, and promoted the proliferation and migration of HCC cells and macrophages. The Erk pathway as a downstream signal of the CCL2/CCR2 axis contributed to controlling these phenotypes of HCC cells and macrophages.
In the ELISA using serum samples, the serum CCL2 levels of patients with HCC were significantly elevated compared with healthy volunteer donors. The survival outcomes of patients with intra-tumoral CCL2 expression Positive based on immunohistochemistry tended to be worse, without statistical significance. Consequently, the CCL2 upregulated through the interactions between HCC cells and macrophages contributed to HCC progression and the CCL2/CCR2/Erk signal might be an effective candidate for the treatment target of HCC and macrophages.

overall_design: Peripheral blood CD14-positive monocytes (PBMos) were selectively collected from healthy volunteer donors using autoMACS Pro Separator. Thereafter, PBMos were incubated with 10 ng/mL recombinant human M-CSF and 1 ng/mL recombinant human GM-CSF for six days to induce macrophage (M0). For macrophage polarization to TAM-like macrophage, macrophages were washed and incubated with 50% HCC-conditioned medium for 24 hours.

first: N

last: Ishihara

first: YI

last: Koma


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 fusion OE iHep RNA-seq]

overall_design: RNA-seq of MEFs with overexpression of various Prox1 fusion proteins or controls, harvested after one week of 4in1-driven reprogramming to induced hepatocytes.


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 KO iHep RNA-seq]

overall_design: RNA-seq of MEFs ± Prox1 knockout, harvested after two weeks of 4in1-driven reprogramming to induced hepatocytes.


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 Late OE HCC RNA-seq]

overall_design: RNA-seq of HCC liver tumour nodules induced by hydrodynamic tail vein injection (HDTVI) to induce Myc overexpression and Tp53 knockout, ± dox-induced overexpression of Prox1 from day 14 to 16.

first: Borja

last: Gomez Ramos


title: Conserved patterns of transcriptional dysregulation, heterogeneity and cell states in clear cell kidney cancer [Bulk RNA-seq]

city: Oxford

first: David

middle: Robert

last: Mole

address: Roosevelt Drive, Headington

department: Nuffield Department of Medicine

institute: University of Oxford

summary: Clear cell kidney cancers (ccRCC) stem from proximal tubule cells in which the VHL gene has been disrupted, causing activation of HIF transcription factors. Although this is essential for cancer initiation, it not sufficient for development of aggressive tumors. To better understand transcriptional programs underpinning ccRCC progression, we perform single cell transcriptomics on 75 kidney tumor and patient-matched normal kidney biopsies. We describe tumor dysregulated transcriptional programs that are conserved across patients, deconvoluted by cell type. Leveraging recurrent intratumor heterogeneity in chromosome 14q loss, a metastasis-associated copy number alteration, we describe the ensuing transcriptional programs to reveal potential mechanisms priming cells for metastasis. Lastly, we describe co-existing cancer cell states consistently found in all patients, underpinned by distinct transcriptional regulators and whose transcriptional signatures are prognostic. These cancer cell states reflect heterogeneity found in the normal proximal tubule epithelium, strongly indicating that they arise from lineage plasticity.

overall_design: Single cell RNA-seq was performed on kidney tumor and tumor-adjecent normal kidney samples to understand transcriptional dysregulation and cell type/clonal diversity in ccRCC. Both freshly-biopsied samples and primary cultures were analyzed, the latter of which was subject to hypoxia and small-molecule intervention to study the HIF pathway. Bulk RNA-seq was performed on siRNA-transfected kidney cancer cells (RCC4) to disentangle HIF-1 and HIF-2 target genes, in order to test whether chromosome 14q loss (a metastasis-associated CNV) had a differential effect on HIF isoforms.

first: Olivia

last: Lombardi

first: Ran

last: Li

first: Faiz

last: Jabbar

first: Hannah

last: Evans

first: Silvia

last: Halim

first: Lisa

last: Browning


title: Conserved patterns of transcriptional dysregulation, heterogeneity and cell states in clear cell kidney cancer [scRNA-seq]

overall_design:
Single cell RNA-seq was performed on kidney tumor and tumor-adjecent normal kidney samples to understand transcriptional dysregulation and cell type/clonal diversity in ccRCC. Both freshly-biopsied samples and primary cultures were analyzed, the latter of which was subject to hypoxia and small-molecule intervention to study the HIF pathway. Bulk RNA-seq was performed on siRNA-transfected kidney cancer cells (RCC4) to disentangle HIF-1 and HIF-2 target genes, in order to test whether chromosome 14q loss (a metastasis-associated CNV) had a differential effect on HIF isoforms.

This study includes the reanalysis of 5 Samples from GSE200207 --GSM6022042, GSM6022043, GSM6022044, GSM6022045, and GSM6022046. The reanalyzed processed data from these Samples are in files Cultured.rds and Cultured_epithelial_T3_ccRCC_reclustered.rds.

***Raw data not provided due to patient privacy concerns***


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 OE MEF/4in1 ATAC-seq]

overall_design: ATAC-seq of MEFs ± Prox1 ovexpression, with and without reprogramming towards induced hepatocytes with 4in1, harvested after two days.


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis.

overall_design: Refer to individual Series


title: Bone Marrow-Derived NGFR-Positive Dendritic Cells Regulate Arterial Remodeling

city: Kanazawa

first: Shinichiro

last: Takashima

address: 13-1 Takara-machi

department: Cardiovascular Medicine

institute: Kanazawa University

summary: It has been proposed that bone marrow contributes to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is expressed in bone marrow stromal cells; it is also present in peripheral blood and ischemic coronary arteries. We hypothesized that bone marrow-derived NGFR-positive (NGFR+) cells regulate arterial remodeling. We found that human NGFR+ mononuclear cells (MNCs) in peripheral blood expressed markers for plasmacytoid dendritic cells (DCs) and were susceptible to apoptosis in response to proNGF secreted by activated arterial smooth muscle cells (SMCs). Bone marrow-specific depletion of NGFR+ cells increased neointimal formation following arterial ligation in mice. Bone marrow-derived NGFR+ cells accumulated in the neointima and underwent apoptosis. In contrast, in a bone marrow-specific NGFR-knockout model, SMCs occupied the neointima with augmented proliferation. NGFR+ cells in the neointima promoted mannose receptor C-type 1-positive antiinflammatory macrophage accumulation and secreted antiinflammatory IL-10, thereby inhibiting SMC proliferation in the neointima. In patients with acute coronary syndrome (ACS), NGFR+ peripheral MNCs increased after ACS onset. Multiple linear regression analysis showed that an insufficient increase in NGFR+ peripheral MNCs in ACS was an adjusted independent risk factor for 9-month intimal progression of a nontargeted lesion. Taken together, these observations imply that bone marrow-derived NGFR+ DCs are suppressors of arteriosclerosis.

overall_design: To explore the cellular characteristics of NGFR+ peripheral MNCs derived from bone marrow, human NGFR+ MNCs were isolated from three healthy volunteers. NGFR+ and NGFR-negative (NGFR-) MNCs were sorted using fluorescence-activated cell sorting. Comparative gene expression analysis of NGFR+, NGFR- MNCs, or unsorted MNCs was performed using data from RNA-seq.

first: Soichiro

last: Usui

first: Chiaki

last: Goten

first: Masayuki

last: Takamura


title: Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [DNA2_MRE11_ChIPseq]

first: Jiadong

last: Wang

overall_design: We performed MRE11 or DNA2 ChIP-seq to test the MRE11/DNA2 distribution under replication fork stress.

first: Zhanzhan

last: Xu

first: Chen

last: Nie

first: Weibin


title: Chemokine positioning determines specialized roles during transdendothelial trafficking of human Th cells with pathogenic features

status: Public on Dec 17 2024

city: Bethesda

first: Farhat

last: Parween

state: MD

address: 10 Center Drive

department: NIAID

institute: NIH

summary: Human Th17/type 17 cells uniformly express the chemokine receptor CCR6. However, the heterogeneity of this cell population and the precise functional role of CCR6 and other chemokine receptors in human Th17 cell extravasation have not been fully delineated. Here we show that CD4+CCR6+ cells co-expressing CCR2 have a pathogenic Th17 signature in the blood, can produce inflammatory cytokines without TCR activation, and show enhanced expression of pathogenicity- and activation-associated genes in the cerebrospinal fluid of patients with multiple sclerosis as compared to control subjects. In flow chambers with activated endothelial cell (EC) monolayers, CD4+CCR6+CCR2+ cells were highly efficient at transendothelial migration (TEM). Ligands for CCR5, CCR6 and CXCR3 localized to EC surfaces and mediated only arrest, whereas CCR2 ligands, although secreted by ECs, failed to bind to ECs and mediated only TEM. Conversely, expressing a chimeric CCR2 ligand engineered to bind glycosaminoglycans on ECs resulted in CCR2-mediated arrest but blocked TEM. Our data suggest T cell arrest requires chemokine bound to EC surfaces, whereas TEM requires a transendothelial chemokine gradient. We show that for human pathogenic-like type 17 T cells, these requirements are met in this system by using separate chemokine/chemokine receptor groups subspecialized to mediate arrest versus TEM.

overall_design:
RNASeq profiling of human CD4 T cells isolated from the blood of healthy donor and sorted by FACS based on CCR6 and CCR2 expression

CCR6highCCR2Pos memory CD4 T cells and total memory CD4 T cells after FACS sorting, were stimulated with PMA for three hours

first: Joshua

last: Farber


title: Salmonella infection accelerates potential maturation of the intestinal epithelium

city: Aachen

first: Mathias

last: Hornef

address: Pauwelstr. 30

department: Institute for Medical Microbiology

institute: University Hospital RWTH Aachen

summary: Postnatal establishment of enteric metabolic, host-microbial and immune homeostasis is the result of precisely timed and tightly regulated developmental and adaptive processes. Here, we show that infection with the invasive enteropathogen Salmonella Typhimurium results in a Myd88-dependent accelerated maturation of the neonatal epithelium with premature appearance of antimicrobial, metabolic, developmental, and regenerative features of the adult tissue.

overall_design: To study intestinal epithelial maturation during homeostatic conditions, we isolated intestinal epithelial cells from small intestinal tissue obtained from C57Bl/6J wild type specific pathogen free (SPF) newborn (5-day-old) and adult (8-week-old) mice, C57Bl/6J Myd88-/- newborn (5-day-old) mice and C57Bl/6N wild tyoe germ free (GF) and SPF newborn (5-day-old) mice. In addition, we isolated intestinal epithelial cells from newborn (5-day-old) and adult (8-week-old) mice that had been infected 4 days earlier with Salmonella Typhimurium. We then isolated RNA and performed 3'mRNA-Seq. Four biological replicates were used per group.

first: Aline

last: Dupont

first: Stefan

last: Schlößer

first: Anna-Lena

last: Ullrich

first: Matthias

last: Schmitz

first: Isabel

last: Richter

middle: W


title: Cooperation between the Hippo and MAPK pathway activation drives acquired resistance to TEAD inhibition [ChIP-Seq TEAD]

city: San Fransisco

first: Xiaosai

last: Yao

state: CA

address: 1 DNA Way

department: Oncology Bioinformatics

institute: Roche/Genentech

summary: TEAD transcription factors (TEAD1-4) serve as the primary effectors of the Hippo signaling pathway in various cancers. There has been significant progress in the development of therapeutic strategies aimed at disrupting the interaction of TEAD with its coactivators YAP/TAZ. However, targeted therapy leads to the emergence of resistance which poses a barrier to achieving complete cures. Currently, the underlying mechanism of resistance to TEAD inhibition in cancers remains unexplored. We uncover that upregulation of the AP-1 transcription factors, along with restored YAP/TEAD activity, drives resistance to GNE-7883, a pan-TEAD and allosteric TEAD inhibitor. Acute GNE-7883 treatment abrogates YAP binding and attenuates FOSL1 activity but compensation by increased MAPK pathway activity remains insufficient for cell survival. In contrast, TEAD inhibitor resistant cells are able to restore YAP and TEAD occupancy and acquire additional FOSL1 binding sites, leading to increased chromatin accessibility at AP-1 motifs. Resistant cells undergo transcriptional reprogramming to acquire a mesenchymal-like cell state and sustained MAPK activity. We uncover a dependence on the MAPK pathway in the TEAD inhibitor resistant cells, further highlighting the key role of MAPK pathway inhibitors, such as Cobimetinib and Belvarafenib to mitigate resistance mechanisms to TEAD inhibition in Hippo pathway dependent cancers. This study describes a clinically relevant interplay between the Hippo and MAPK pathway in cancers and offers a promising avenue to address TEAD inhibitor resistance in the clinic.

overall_design: To investigate the mechanism involved in TEAD acquired resistance, we developed TEADi resistant cell lines. We then performed gene expression profiling using data obtained from TEAD inhibitor sensitive cells incubated with DMSO or TEAD inhibitor GNE-7883 and TEAD inhibitor resistant cells incubated with DMSO or TEAD inhibitor GNE-7883.

first: Thijs

last: Hagenbeek

first: Sayantannee

last: Paul

first: Anwesha

last: Dey

first: Julien

last: Tremblay

first: Bence

last: Daniels


title: Cooperation between the Hippo and MAPK pathway activation drives acquired resistance to TEAD inhibition [CUT&RUN]


title: Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [gH2AX_ChIPseq]

first: Jiadong

last: Wang

overall_design: We performed γH2AX ChIP-seq to test the γH2AX distribution with or without DRB treatment under replication fork stress.

first: Zhanzhan


title: Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis [Prox1 KD HCC RNA-seq]

first: Moritz

last: Mall

address: Im Neuenheimer Feld 280

institute: German Cancer Research Center (DKFZ)

summary: Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. While transcription master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether plasticity is actively suppressed by lineage-specific repressors. Here, we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong. We validated hepatocyte-specific candidates using reprogramming, revealing that Prospero homeobox protein 1 (PROX1) enhanced hepatocyte identity by direct repression of alternative fate master regulators. In mice, Prox1 was required for efficient hepatocyte regeneration after injury and was sufficient to prevent liver tumorigenesis. In line with patient data, Prox1 depletion caused hepatocyte fate loss in vivo and enabled the transition of hepatocellular carcinoma to cholangiocarcinoma. Conversely, overexpression promoted cholangiocarcinoma to hepatocellular carcinoma transdifferentiation. Our findings provide evidence for PROX1 as a hepatocyte-specific safeguard and support a model where cell type-specific repressors actively suppress plasticity throughout life to safeguard lineage identity and thus prevent disease.

overall_design: RNA-seq of HCC liver tumour nodules induced by hydrodynamic tail vein injection (HDTVI) to induce Myc overexpression and Tp53 knockout, ± Prox1 knockdown, harvested at humane endpoint.

first: Bryce

last: Lim

first: Aryan

last: Kamal

first: Juan

last: Adrian-Segarra

first: Judith

last: Zaugg


title: Cooperation between the Hippo and MAPK pathway activation drives acquired resistance to TEAD inhibition [ATAC-seq]

overall_design: To investigate the mechanism involved in TEAD acquired resistance, we developed TEADi resistant cell lines. In order to map chromatin accessibility across our different experimental conditions, we perform ATAC-seq (Assay for Transposase-Accessible Chromatin ) from TEAD inhibitor sensitive cells incubated with DMSO or TEAD inhibitor GNE-7883 and TEAD inhibitor resistant cells incubated with TEAD inhibitor GNE-7883


title: Targeted degradation of oncogenic KRAS G12V triggers antitumor immunity in lung cancer.

first: Yuan

last: Hao

address: Laura and Isaac Perlmutter Cancer Center

summary: We performed bulk-RNA sequencing analysis from KRAS G12V mouse tumor nodules from different treatment.

overall_design: KRAS G12V mutant mice with lung tumors were treated with either vehicle or dTAGV-1. Tumor nodules were collected for sequencing.

first: Hua

last: Zhang


title: Cooperation between the Hippo and MAPK pathway activation drives acquired resistance to TEAD inhibition III

overall_design: To investigate the mechanism involved in TEAD acquired resistance, we performed a xenograft of metastatic cells (PATU-8902) silenced for the YAP/WWTR1 gene (shYAP/WWTR1) in a cohort of 6-8 weeks Nu/Nu nude female mice.