title: Study 4- RNA-seq of male KOLF2.2J hiPSC-derived cortical brain organoids and extra-embryonic lineages homozygous null for six different transcription factors

summary: This study aimed to phenotype iPSC-derived cortical brain organoids that contain homozygous null alleles for transcription factors DMRTA1 and TBR1 and to phenotype iPSC-derived extra-embryonic cell lineages that contain homozygous null alleles for transcription factors OVOL1, PITX1, LMO2, and ZBTB7C. Null alleles were generated by the insertion of a premature termination codon with frameshift (PTC+1). This analysis provided insight into the impact of loss of function for these transcription factors in the respective cell lineages.

overall_design: The study involved the generation of RNA-seq on 28 samples, comprising 10 differentiated into cortical brain organoids (with dorsal forebrain patterning) and 18 differentiated into the trophoblast lineage, promoting the formation of a primitive syncytium. Three biological replicates were run for each gene KO line. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions.


title: Bulk RNA-seq study 4 and 5: 17 genes knocked out in extra-embryonic and cortical brain organoids

status: Public on Feb 03 2025

city: Hinxton

first: MorPhiC

last: Consortium

address: Wellcome Genome Campus

country: United Kingdom

institute: EMBL-EBI

type: Expression profiling by high throughput sequencing

summary: This study investigates the effects of knocking out 17 genes - DMRTA1, TBR1, OVOL1, PITX1, LMO2, ZBTB7C, HEY1, MBNL2, DLX4, DLX5, DLX6, HOPX, MAFF, TWIST2, MBNL3, SIX3 and NR2F1, by inserting premature termination codons (PTCs) in primitive syncytium and cortical brain organoid model systems derived from the KOLF2.2J iPSC line. Bulk RNA-seq analysis was performed to examine the transcriptional consequences of these gene knockouts, providing insights into their roles in brain development and cellular differentiation in organoid models

overall_design: Refer to individual Series


title: Heparan sulfate determines myofibroblast identity during alveolar morphogenesis (ATAC-seq)

status: Public on Feb 05 2025

city: Chengdu

first: Hua

last: He

state: Sichuan

address: 2222 Xinchuan Rd

country: China

institute: West China Second University Hospital Sichuan University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Postnatal respiration requires the bulk formation of alveoli that produces extensive surface area for gas diffusion from epithelium to the circulatory system. Alveolar morphogenesis initiates at late gestation or postnatal stage during mammalian development and is mediated by coordination among epithelial, mesenchymal and endothelial cells. Here we show that stromal cells produce Heparan Sulfate Glycosaminoglycan (HS-GAG) to maintain a niche for alveolar development by modulating both the biophysical and biochemical cues. HS is predominantly enriched in myofibroblasts in postnatal murine lungs, and genetic ablation of HS synthase gene Ext1 in fibroblast populations results in enlarged and simplified alveolar structures. HS is selectively required for the cell identity of a subset of alveolar myofibroblasts (PDGFRαhi αSMA+) residing in the distal alveolar region, which exhibit contractile properties and maintain WNT signaling activity to support normal proliferation and differentiation of alveolar epithelial cells. HS is essential to prevent precocious apoptosis of alveolar myofibroblasts. We show that these processes are dependent upon FGF/MAPK signaling and forced activation of MAPK/ERK signaling partially corrected alveolar simplification along with restoration of alveolar myofibroblast identity and AT2 cell proliferation in HS deficient mice. These data reveal HS as an essential orchestrator for developing alveolar niche critical for generation of gas exchange units.

overall_design: We performed ATAC-seq on FACS-sorted Rosa26-tdTomato+ lung mesenchymal cells from Ext1 flox/flox, Gli1CreER  mice and their littermate controls. Deletion was induced by treating mice with tamoxifen on P0 and P1, and lungs were harvested on postnatal day 8 (P8). ATAC libraries were constructed using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD502) and TruePrep® Index Kit V2 for Illumina (Vazyme, TD202).


title: Mapping and Quantification of Genome-wide DNA Damages by Catechol Estrogens Using Click probe-Seq and LC/MS2: Unraveling Endogenous Genotoxicity Beyond Receptor-mediated Signaling

city: Tainan

first: Trang

middle: Quynh

last: Do

state: Taiwan

address: No. 1, Daxue Rd, East District, Tainan City, 701

department: Chemistry

country: Taiwan

institute: National Cheng Kung University

summary: Genotoxic estrogen metabolites generate various endogenous DNA lesions but their carcinogenesis mechanisms have been overlooked by well-known cell proliferation pathways through estrogen receptor (ER). Genome-wide sequencing using click probe enrichment coupled with liquid chromatography-mass spectrometry (click probe-Seq/LCMS2) was developed to identify damaged genes and characterize global generation profiles of depurinating and stable adducts induced by 4-hydrolyxyl estradiol (4OHE2) in MCF-7 chromatin. Both data were combined to show guanine nucleobase in GC-rich transcription-relevant domain are main target sites. The damage abundance exhibited positive correlation with DNase hypersensitive sites, indicating 4OHE2 attacks chromatin exposure region beyond ER binding. Cell-based comparability studies indicated accumulated 4OHE2 caused suppressed transcription of target genes, in-effective damage repair, and decreased cell viability, differing from uncontrolled cell growth by extensive ER-signaling. Click probe-Seq/LCMS2 approach revealed the first chromatin damage map by endogenous metabolites, exposing a previously unexplored landscape in cancer research and being applicable to other genotoxic species.

overall_design: To study the DNA sequence where catechol estrogen DNA adducts are located, we treated extracted chromatin from MCF-7 cells with either catechol estrogen (experimental group) or acetonitrile (control/mock group). After treatment, the DNA was purified, and a biotin-labeled probe was introduced. This was followed by streptavidin bead pulldown. The pulled-down DNA sequences were then submitted for sequencing.

first: Quynh-Trang

first: Shun-Fen

last: Tzeng

first: Chih-Yen

last: Wang

first: Chih-Hsing

last: Wu

first: Husam

last: Kafeenah

first: Shu-Hui

last: Chen


title: Study 6- RNA-seq of female WIBJ2 hiPSC-derived cortical brain organoids and extra-embryonic lineages homozygous null for five different transcription factors

summary: This study aimed to phenotype iPSC-derived cortical brain organoids that contain homozygous null alleles for transcription factor PAX6 and to phenotype iPSC-derived extra-embryonic cell lineages that contain homozygous null alleles for transcription factors PPARG, EPAS1, GCM1, and GRHL1. Null alleles were generated by the insertion of a premature termination codon with frameshift (PTC+1). This analysis provided insight into the impact of loss of function for these transcription factors in the respective cell lineages.

overall_design: The study involved the generation of RNA-seq on 27 samples, comprising cells differentiated into cortical brain organoids (with dorsal forebrain patterning) and differentiated into the trophoblast lineage, promoting the formation of a primitive syncytium. Three biological replicates were run for each gene KO line. The hypoxia-inducible factor (EPAS1) was differentiated under both 20% oxygen and 3% (~ level present at peri-implantation) oxygen concentrations. Additionally, three biological replicates were run of wild-type WIBJ2 cells (the parental female cell line in which the KOs were generated) under the same differentiation conditions.

first: Juliana

middle: A

last: Diniz

first: Bill

last: Skarnes

first: Paul

last: Robson


title: RPB7/CTDP1 stabilizes and licenses Pol II termination to reinitiation [RNA-Seq]

city: Beijing

first: Xiong

last: Ji

address: Haidian District, Beijing Summer Palace Road No. 5

department: School of Life Sciences

institute: Peking University

summary: The regulations of transcription process have been extensively investigated, but how RNA Pol II terminates and reinitiates a new round of transcription remains elusive. Here, we found that RPB7 loss triggers the degradation of RNA Pol II. RPB7 degradation combing knock out of E3 ligase Cullin 3 led to the accumulation of phosphorylated Pol II in the soluble fractions and Pol II reinitiation failure. The stability of recycled Pol II is dependent on the loop-region and dimerization of RPB7 and the phosphorylation state of Pol II CTD. Site-specific cancer mutations in the RPB7 loop-region caused the dysregulations of specific gene expression. Moreover, the loss of RPB7 or CTD phosphatase subunit 1 (CTDP1) result in the defects in Pol II termination-reinitiation recycle. Together, our study demonstrates that RPB7/CTDP1 controls the dephosphorylation and stabilization of RNA Pol II to permit RNA Pol II termination to reinitiation recycle in mammalian cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data in RPB7 degron cells,  Cul3KO cells or RPB7 mutated cells upon RPB7 degradation.

first: Haonan

last: Zheng

first: Qiqin

last: Xu


title: Dolutegravir Disrupts Mouse Blood-Brain Barrier by Inducing Endoplasmic Reticulum Stress

status: Public on Feb 02 2025

city: Toronto

first: Chang

last: Huang

state: Ontario

address: 386 yonge st, unit4008, unit 4008

department: Pharmaceutical Sciences

country: Canada

institute: University of Toronto

summary: Dolutegravir (DTG) - based antiretroviral therapy is the contemporary first-line therapy to treat HIV infection. Despite its efficacy, mounting evidence has suggested a higher risk of neuropsychiatric adverse effect (NPAE) associated with DTG use with a limited understanding of the underlying mechanisms. Our laboratory has previously reported a toxic effect of DTG but not bictegravir (BTG) in disrupting the blood-brain barrier (BBB) integrity. The current study aimed to investigate the underlying mechanism of DTG toxicity. Primary cultures of mouse brain microvascular endothelial cells were treated with DTG and BTG at therapeutic relevant concentrations. RNA-sequencing, qPCR, western blot analysis and cell stress assays (Ca2+ flux, H2DCFDA, TMRE, MTT) were applied to assess the results. The Gene Ontology (GO) analysis revealed an enriched transcriptome signature of endoplasmic reticulum (ER) stress following DTG treatment. We demonstrated that therapeutic concentrations of DTG but not BTG activated the ER stress sensor proteins (PERK, IRE1, p-IRE1) and downstream ER stress markers (eIF2α, p-eIF2α, Hspa5, Atf4, Ddit3, Ppp1r15a, Xbp1, spliced-Xbp1). In addition, DTG treatment resulted in a transient Ca2+ flux, an aberrant mitochondrial membrane potential, and a significant increase in reactive oxygen species in treated cells. Furthermore, we found that prior treatment of ER sensor inhibitors significantly mitigated the DTG-induced downregulation of tight junction proteins (Zo-1, Ocln, Cldn5) and elevation of pro-inflammatory cytokines and chemokines (Il6, Il23a, Il12b, Cxcl1, Cxcl2). The current study provides valuable insights into DTG-mediated cellular toxicity mechanisms, which may serve as a potential explanation of DTG-associated NPAEs in the clinic.

overall_design: RNA-seq profiling of primary cultures of mouse brain microvascular endothelial cells treated by dolutegravir 5000 ng/ml for 24 h


title: Regulatory variation controlling architectural pleiotropy in maize

city: St. Louis

first: Andrea

last: Eveland

state: Missouri

address: 975 North Warson Road

country: USA

institute: Donald Danforth Plant Science Center

summary: Plant architecture is central to yield and has been at the core of crop domestication and improvement. In cereals, inflorescence branching and leaf angle are important traits that contribute to planting density and yield potential. Several classical maize mutants show disruptions in both traits, suggesting a core regulatory network underlies pleiotropy between them. Here, we investigate regulatory modules that contribute to architectural pleiotropy between tassel branch number (TBN) and leaf angle (LA) in maize by defining transcriptional networks that function in lateral organ boundaries to promote development of these morphologically distinct organs. Using a set of nine mutants with specific developmental defects in one or both traits, we generated dynamic, context-specific gene regulatory maps that describe ligule and tassel branch development at the molecular level. Mutants introgressed into B73 and control plants were grown in environmentally controlled chambers and precisely-staged tassel primordia were hand-dissected at two stages: right before and after first primary branches initiated. Two stages capturing early development of the ligular region, including the shoot apical meristem, were also collected from mutants with LA defects. RNA-seq was performed on 140 samples and integrated into gene regulatory and co-expression networks, which were extended to include publicly available transcription factor occupancy maps for important developmental regulators, chromatin accessibility maps and natural variation to help prioritize novel genes and regulatory elements underlying diversity in LA and tassel branching phenotypes. We also used these transcriptional networks to guide multi-trait genome-wide association studies (GWAS) based on three years of field phenotyping TBN and LA traits in over 500 diverse maize lines. Various network-assisted GWAS approaches were used to identify polymorphisms in candidate genes that associate with these architecture traits and the pleiotropy between them. Our data provide novel insight into regulatory mechanisms controlling architectural pleiotropy that can be used for targeted crop improvement.

overall_design: We use a set of 9 maize mutants (lg1, lg2, ra1, ra2, fun, Wab1-R, wab-rev, bin2-RNAi, bri1-RNAi) to generate 140 poly-A+ RNA-seq libraries from tassel primordia, apex2 and apex1 tissues.

first: Edoardo

last: Bertolini

first: Brian

middle: R

last: Rice

first: Jiani

last: Yang

first: Max

last: Braud

first: Sarah

last: Hake

first: Josh

last: Strable

first: Alexander

middle: E

last: Lipka

middle: L


title: Dissecting the evolving transcriptional landscape of the remyelinating spinal cord

city: Washington

first: George

middle: S

last: Melchor

state: DC

address: 3700 O St NW

department: Biology

institute: Georgetown University

summary: Demyelination, or the loss of myelin in the central nervous system (CNS) is a hallmark of multiple sclerosis (MS) and occurs in various forms of CNS injury and neurodegenerative diseases. The replenishing of myelin, termed remyelination, also occurs spontaneously following demyelination. The lysolecithin-induced focal demyelination model enables the investigation into the mechanisms of spontaneous remyelination, providing insight into the molecular basis underlying an evolving remyelinating microenvironment. Here, we present a detailed analysis using high-resolution single-nucleus RNA sequencing to investigate gene expression dynamics across all cell types involved in the remyelination process. We examine three specific time points following focal demyelinating injury. By delineating activation states within the heterogeneous cell populations of demyelinated lesions, we highlight changes in gene expression within subclusters of each cell type from the early stages of injury response to the initiation and maintenance of remyelination. Our findings reveal how shifts in microglial, astrocytic and fibroblast activities within lesions are associated with efficient oligodendrocyte differentiation during remyelination.

overall_design: Focal demyeination in the spinal cord, mediated by the injection of 1% lysophosphatidylcholine (LPC, lysolecithin) in the ventral white matter, was performed in 8-12 week old female and male mice. Lesioned tissue, visualized by the injection of 1% neutral red solution (500µL, intraperitoneal) 2 hours preceding euthanasia, was collected at 5, 10, and 20 days post lesion. Rostral, non-lesioned tissue was collected at 5, 10, and 20dpl as an internal control. As another contro, naive, unlesioned spinal cord white matter tissue from a separate cohort of mice was also collected. Single nuclei were isolated from tissue samples and analyzed via snRNAseq.

first: Jeffrey

middle: K


first: MorPhiC

last: Consortium

summary: Bulk RNAseq of differentiation timecourse of embryoid bodies from KOLF2.2J AAVS1 inducible CRISPRi lines

overall_design: Aggregates were generated using StemCell Tech aggrewell media on day 0 and then aggregates were moved into suspension dishes on day 1 using DMEM+20% KSR media. EBs remained in DMEM+20% KSR throughout experiment.

first: Aaron

last: Huebner

first: Ashir

last: Borah

first: Luke

last: Gilbert


title: Bulk RNA seq timecourse of KOLF2.2J hiPSC after Auxin-induced protein degradation of specific gene products

summary: This study investigates the impact of 2 gene products (RBBP4, CHD1) on transcriptional programming in growing hiPSC at 0, 6 & 24 hours after acute protein depletion. Crispr editing was used to add an auxin-dependent degron to the C-termini of gene products expressed from the endogeneous loci and to insert an auxin-responsive degradation E3 ligase to the AAVS locus. Rapid (<30 min), specific target protein degradation in KOLF2.2J hiPSCs was induced by a synthetic auxin derivative (5-Ad-IAA) and bulk RNA-seq was performed at timepoints to define early (6 hr) and later (24 hr) downstream transcriptional dependencies.

overall_design: Crispr editing was used to introduce an auxin dependent E3 ligase (atOsTir1) into the AAVS locus of the parental KOLF2.2J hiPSC line.    Cells were subsequently Crispr-edited to create independent clones with an auxin-specific degron added to the C-terminus of individual target gene products expressed from endogenous loci. Rapid degradation of target proteins was induced by treatment with the synthetic auxin derivative, 5-Ad-IAA, and protein loss was confirmed by Western blotting. Control cells were treated with DMSO. Bulk RNA-seq was performed in duplicate at 0 hr, 6hr, and 24 hr to evaluate transcriptional responses.

first: Mazhar

last: Adli

first: Tao

last: Bai

first: De

last: Xing

first: Ozlem

last: Neyisci

first: Hengqiang

last: Zhao

first: Xintong

last: Chen


title: Tex10 in day2 embryonic bodies (EBs)

status: Public on Feb 01 2025

city: New York city

first: Feifei

last: Yuan

state: NY

institute: Columbia University

summary: Testis-specific transcript 10 (Tex10) is highly expressed in the testis, embryonic stem cells (ESCs), and primordial germ cells (PGCs). We previously generated a Tex10 knockout mouse model demonstrating its critical roles in ESC pluripotency and preimplantation development. Here, using conditional knockout mice and dTAG-degron ESCs, we show Tex10 is required for spermatogenesis and ESC-to-PGCLC differentiation. Specifically, Tex10-null spermatocytes arrest at metaphase I, compromising round spermatid formation. Tex10 depletion and overexpression compromise and enhance ESC-to-PGCLC differentiation, respectively. Mechanistically, bulk and single-cell RNA sequencing reveals that Tex10 depletion downregulates genes involved in pluripotency, PGC development, and spermatogenesis while upregulating genes promoting somatic programs. Chromatin occupancy study reveals that Tex10 binds to H3K4me3-marked promoters of Psmd3 and Psmd7, negative regulators of Wnt signaling, and activates their expression, thereby restraining Wnt signaling. Our study identifies Tex10 as a previously unappreciated factor in spermatogenesis and PGC development, offering potential therapeutic insights for treating male infertility.

overall_design: Embryonic bodies (EBs) were treated with DMSO or dTAG13 for two days and dissociated with 0.05% Trypsin-EDTA to generate single-cell suspension for single-cell sequencing.

first: Jianlong

last: Wang


title: hMeDIP-seq of mouse cerebellum using nanopore sequencing

city: Bath

first: Adele

last: Murrell

address: Claverton Down

department: Life Sciences Department

institute: University of Bath

type: Methylation profiling by high throughput sequencing

summary: 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are modified versions of cytosine in DNA with roles in regulating gene expression. Using whole genomic DNA from mouse cerebellum, we benchmark 5mC and 5hmC detection by Oxford Nanopore Technologies sequencing against other standard techniques. In addition, we assess the ability of duplex base-calling to study strand asymmetric modification. Nanopore detection of 5mC and 5hmC is accurate relative to compared techniques and opens new means of studying these modifications. Strand asymmetric modification is widespread across the genome but reduced at imprinting control regions and CTCF binding sites in mouse cerebellum. Here we demonstrate the unique ability of nanopore sequencing to improve the resolution and detail of cytosine modification mapping.

overall_design: DNA from mouse cerebellum was sheared to a target of 6 kb using a Covaris g-TUBE (Covaris, 520079). An aliquot was sequenced as a control on an R10.4.1 MinION flow cell (Oxford Nanopore Technologies, FLO-MIN114) using Ligation Sequencing (SQK-LSK114) Kit 14 following the associated protocol. Sheared DNA was sonicated in triplicate using a Bioruptor® Pico sonication device (Diagenode, B0106001) in a 1.5 mL Bioruptor® Pico Microtube with cap (Diagenode, C30010016) to target fragment lengths < 500bp. A 2200 TapeStation system (Agilent, G2991A) with D1000 ScreenTapes (Agilent, 5067-5582) and associated reagents (Agilent, 5067-5583) was used to assess the fragment size distribution of each replicate (mean 362 bp). 5hmC immunoprecipitation was performed based on Nestor and Meehan23. 1 μg sonicated dsDNA was incubated overnight at 4°C rotating in IP buffer (10 mM Na-phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100) with 2 μL of either whole serum α-5hmC antibody (ActiveMotif, 39769) or IgG-purified α-5hmC antibody (ActiveMotif, 39791). Samples were then incubated with 40 μL equilibrated Dynabeads Protein G (Invitrogen, 10003D) for 1 hour while rotating at 4°C and washed three times in ice-cold IP buffer for 5 minutes at room temperature. Bound DNA was eluted in digestion buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS) and 200 ng proteinase K (ThermoFisher Scientific, EO0491) for 3 hours at 50 °C on a shaker at 800 rpm. Immunoprecipitated DNA was purified using the QIAQuick PCR Purification kit (QIAGEN, 28104) following manufacturer instructions and eluted into 50 μL elution buffer before sequencing. The eluate was prepared for sequencing following the MinION protocol SQK-LSK114. Libraries were loaded onto three R10.4.1 MinION flow cells and sequenced, generating 154-382 Mb of sequence data per flow cell.

first: Dominic

middle: O

last: Halliwell

first: Floris

last: Honig

first: Stefan

last: Bagby

first: Sandipan

last: Roy


title: Pluripotency factor Tex10 finetunes Wnt signaling for spermatogenesis and primordial germ cell development

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


title: Study 2- RNA-seq of male KOLF2.2J hiPSC-derived trophoblast cell lines homozygous null for seven different transcription factors

first: MorPhiC

last: Consortium

summary: This study aims to phenotype iPSC-derived trophoblast lines that contain homozygous null alleles for transcription factors expressed within the extra-embryonic lineage, with a focus on differentiation toward either primitive syncytium or extra-embryonic mesenchymal cells, depending on the TF being analyzed. Null alleles were generated using three distinct genetic engineering approaches: full protein coding region deletion (KO), critical exon deletion (CE), and insertion of a premature termination codon with frameshift (PTC+1). RNA-seq data was generated after 6 days of initiating differentiation from iPSCs. The study included the homozygous null alleles for the following transcription factors: MEIS1, MXD1, MEIS2, RUNX1, MEF2C, NCOA3, and BHLHE40. This analysis provided insight into the differences between the various CRISPR-Cas9-based approaches and the impact of loss of function for these transcription factors in trophoblast lineage differentiation.

overall_design: The study involved the generation of RNA-seq on 87 samples. Four of the transcription factors were analyzed in the primitive syncytium differentiation scheme and three in the extra-embryonic mesenchymal cell scheme. Three biological replicates were run for each genetic engineering approach for each gene. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions. This comprehensive design allowed for a robust comparison of genetic engineering methods and the effects of transcription factor manipulation on cell fate decisions within the extra-embryonic lineage.

first: Juliana

last: Diniz

first: Bill

last: Skarnes

first: Paul

last: Robson


title: Nivolumab to restore T-cell fitness in CAR-T refractory multiple myeloma

city: Boston

first: Johannes

last: Waldschmidt

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

summary: We here applied single-cell RNA sequencing of circulating T-cells from primary MM patients to perform transcriptional profiling and assess T-cell fitness in the context of emerging resistance to CAR-T cell therapy. Response to PD-1 inhibition after CAR-T was dictated by the fitness state of non-CAR T cells

overall_design:
A total of 1,047 single cells from four patients before and after checkpoint inhibitor salvage treatment were examined by full-length single-cell RNA sequencing. Quality filtering resulting in the retention of 1,007 single cells. Due to consistency with available pre-and post timepoints the heatmap shown in Figure 1E was generated only for MM1 and MM2 with a total of 164 retained CD3+ T-cells. ***Raw data not submitted to GEO due to patient privacy concerns.***

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
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first: Johannes M

first: Noori

last: Sotudeh

first: Sankalp

last: Arora

first: Tushara

last: Vijaykumar

first: Praveen

last: Anand

first: Hannah

last: Stuart

first: Julia

last: Frede

first: Timothy

last: Campbell

first: Shari

last: Kaiser

first: Xirong

last: Zheng

first: Nikhil

last: Munshi

first: Kenneth C

last: Anderson

first: Hermann

last: Einsele

first: Andrew J

last: Yee

first: Birgit

last: Knoechel

first: Jens G

last: Lohr

first: Noopur S

last: Raje


title: CITEseq analysis of human CSF cells in Multiple sclerosis

city: Bethesda

first: Shinji

last: Ashdia

state: MD

address: 10 Center Drive

department: NIAID

institute: NIH

summary: In CSF bulk RNA sequence, patients with MS (pwMS) exhibited higher T cell receprot (TCR) clonality and specificity to EBV lytic proteins compared to controls. TCRs from pwMS showed greater clustering coefficient (i.e. TCR similarity) in complementarity-determining regions 3 (CDR3), particularly those predicted to target EBV antigens. Importantly, TCR clustering coefficient was associated with the expression of cytotoxic CD8 module genes in WGCNA, such as CD8A, GZMH, GZMK, and NKG7 and with interferon signaling module. scRNA-seq revealed a subpopulation of GZMK+GZMH+ double positive (DP) CD8 T cells expressing genes related to identified cytotoxicity and interferon signaling modules. These DP CD8 T cells had significantly higher predicted specificity for EBV proteins than other CD8 cells.

overall_design: CSF was collected on ice and processed according to a written standard operating procedure by investigators blinded to diagnoses, clinical, and imaging outcomes. Aliquots were assigned alphanumeric identifiers and centrifuged for 10 minutes at 300g at 4°C within 30 minutes of collection. The pelleted CSF cells were processed for single cell RNA sequencing. Cells were labelled with 79 Totalseq B antibodies (Biolegend, USA) and washed with PBS with 1% Bovine Serum Albumin (Thermo Fisher Scientific, USA). Labeled cells were fixed by using fixed RNA feature barcode kit (10x Genomics, USA) and stored at -80 degree.

last: Ashida

first: Francisco

last: Otaizo-Carrasquero

first: Paul

last: Gardina

first: Amir

last: Shamisa


title: Post-transcriptional monoadenylation by TENT2 terminates human RNA polymerase III transcript 3' end processing and promotes 7SL RNA biogenesis [global_RNAseq]

status: Public on Jan 31 2025

city: La Jolla

first: Cody

last: Ocheltree

state: CA

address: 9500 Gilman Drive

institute: UCSD

type: Non-coding RNA profiling by high throughput sequencing

summary: Small non-coding RNAs (sncRNAs) are subject to a variety of 3' end trimming and tailing activities during biogenesis, which dictate whether they undergo maturation or are instead subjected to degradation. To investigate the dynamics of human sncRNA 3' end processing at a global level we performed genome-wide 3' end sequencing of nascently-transcribed and steady state sncRNAs. This revealed widespread post-transcriptional adenylation of nascent sncRNAs, which came in two distinct varieties. One is characterized by oligoadenylation, which is transient, promoted by TENT4A/4B polymerases, and observed primarily on unstable sno- and scaRNAs that are not fully processed at their 3' ends. The other is characterized by monoadenylation, which is broadly catalyzed by TENT2 and stably accumulates at the 3’-end  of many RNA Polymerase-III-transcribed (Pol-III) RNAs as well as a subset of small nuclear RNAs. This monoadenylation event terminates 3' end uridylation/deuridylation dynamics characteristic of nascent Pol-III RNAs and, in the case of 7SL RNAs, prevents their accumulation with nuclear La protein and promotes their biogenesis towards assembly into cytoplasmic signal recognition particles.

overall_design: Total small RNA (100-500 bp) and small RNAs nascently-labeled with EU for 2 hours were extracted and libraries were prepared in biological triplicate for 3'-end global sequencing of small RNAs.

first: Tim

last: Nicholson-Shaw

first: Jens

last: Lykke-Andersen


title: Study 3- RNA-seq of male KOLF2.2J hiPSC-derived cortical brain organoids homozygous null for six different transcription factors

first: MorPhiC

last: Consortium

summary: This study aims to phenotype iPSC-derived cortical brain organoids that contain homozygous null alleles for transcription factors expressed in the early neuroectoderm. Null alleles were generated using three distinct genetic engineering approaches: full coding region deletion (KO), critical exon deletion (CE), and insertion of a premature termination codon with frameshift (PTC+1). RNA-seq data was generated at the indicated day post initiation of differentiation from hiPSCs. The study included the homozygous null alleles for the following transcription factors: LHX9, RFX4, PAX6, OTX1, LHX5, and FEZF2. This analysis provided insight into the differences between the various CRISPR-Cas9-based approaches and the impact of loss of function for these transcription factors in cortical brain organoid differentiation.

overall_design: The study involved the generation of RNA-seq on 179 samples at different time points of differentiation to cortical brain organoids. At least three different time points were selected for each line and the chosen time points were selected based on the known initiation of transcription of each respective transcription factor in wildtype cells within this differentiation scheme. Three biological replicates were run for each genetic engineering approach for each gene. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions. This comprehensive design allowed for a robust comparison of genetic engineering methods and the effects of transcription factor manipulation on brain organoid formation.

first: Juliana

last: Diniz

first: Bill

last: Skarnes

last: Robson


title: Deciphering CRC-Hepatocyte Interactions: A Multiomic Platform for Interrogation of Metabolic Crosstalk in the Liver-Tumor Microenvironment

city: Minneapolis

first: Stephen

last: Jameson

state: MN

address: Wallin Medical Biosciences Bldg, MMC 641

department: Lab Medicine and Pathology

institute: University of Minnesota

summary: Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to adapt to and exploit their microenvironment for sustained growth. The liver is a common site of metastasis, but the interactions between tumor cells and hepatocytes remain poorly understood. In the context of liver metastasis, these interactions play a crucial role in promoting tumor survival and progression. This study leverages multiomics coverage of the microenvironment via liquid chromatography and high-resolution, high-mass accuracy mass spectrometry-based untargeted metabolomics, stable isotope tracing, and RNA sequencing to uncover the metabolic impact of co-localized primary hepatocytes and a colon adenocarcinoma cell line, SW480, using a 2D co-culture model. Metabolic profiling revealed disrupted Warburg metabolism with an 80% decrease in glucose consumption and 94% decrease in lactate production by hepatocyte-SW480 co-cultures relative to SW480 control cultures. Decreased glucose consumption was coupled with alterations in glutamine and ketone body metabolism, suggesting a possible fuel switch upon co-culturing. Further, integrated multiomic analysis indicates disruptions in metabolic pathways, including nucleoside biosynthesis, amino acids, and TCA cycle, correlate with altered SW480 transcriptional profiles and highlight the importance of oxygen homeostasis in tumor adaptation. Finally, these findings were replicated in 3-dimensional microtissue organoids supporting a bioinformatic approach for studying metabolic crosstalk and discovery of potential therapeutic targets in preclinical models of the tumor microenvironment.

overall_design: This accession data consists of 2D culture and 3D organoids of SW480 cells with and without 3T3-J2 cells and/or Hepatocytes.  2D culture data is three replicates of SW480 cells, three replicates of SW480+3T3-J2 cells, and three replicates of SW480+3T3-J2 cells+Hepatocytes. 3D organoid data is three replicates of SW480+3T3-J2 cells and three replicates of SW480+J2 cells+Hepatocytes.

first: Lyndsay

last: Reese

first: Yinjie

last: Qiu

first: Braedan

last: McCluskey


title: Senescent-like microglia limit remyelination through the senescence associated secretory phenotype

city: Worcester

first: Violeta

last: Duran Laforet

address: 365 Plantation St

department: Neurobiology

institute: University of Massachusetts Chan Medical School

summary: The capacity to regenerate myelin in the central nervous system (CNS) diminishes with age. This decline is particularly evident in multiple sclerosis (MS), which has been suggested to exhibit features of accelerated biological aging. Whether cellular senescence, a hallmark of aging, contributes to remyelination impairment remains unknown. Here, we show that senescent cells (SCs) accumulate within demyelinated lesions after injury, and their elimination enhances remyelination in young mice but not in aged mice. In young mice, we observed the upregulation of senescence-associated transcripts primarily in microglia after demyelination, followed by their reduction during remyelination. However, in aged mice, senescence-associated factors persisted within lesions, correlating with inefficient remyelination. We found that SC elimination enhanced remyelination in young mice but was ineffective in aged mice. Proteomic analysis of senescence associated secretory phenotype (SASP) revealed elevated levels of CCL11/Eotaxin-1 in lesions, which was found to inhibit efficient oligodendrocyte maturation. These results suggest therapeutic targeting of SASP components, such as CCL11, may improve remyelination in aging and MS.

overall_design: 3 months old and 18 months old mice were used. Focal demyelitation was induced by  injecting 1% of L-α-Lysophosphatidylcholine (LPC) in PBS between the T11-12 vertebrae into the ventral white matter columns of the mouse spinal cord. To identify and dissect out the lesion, 500ul 1% neutral red dye in PBS was injected intraperitoneally 2 hours prior to cardiac perfusion with ice cold RNA-ase free 1X PBS. Spinal cords were flash frozen in isopentane and embedded in OCT on dry ice.

first: Philip

last: Gross

last: Durán-Laforet

first: Dorothy

middle: P

last: Schafer

first: Jeffrey

last: Huang


title: Senescent microglia limit remyelination through the senescence associated secretory phenotype

first: Phillip

middle: Steven

state: District Of Columbia

overall_design: 3 months old and 18 months old mice were used. Focal demyelitation was induced by  injecting 1% of L-α-Lysophosphatidylcholine (LPC) in PBS between the T11-12 vertebrae into the ventral white matter columns of the mouse spinal cord. To identify and dissect out the lesion, 500ul 1% neutral red dye in PBS was injected intraperitoneally 2 hours prior to cardiac perfusion with ice cold RNA-ase free 1X PBS. Spinal cords were flash frozen on dry ice.


title: Adipocyte-specific Steap4 deficiency reduced thermogenesis and energy expenditure in mice

city: Cleveland

first: Lizi

last: Zhang

state: Ohio

address: 2109 Adelbert Rd

department: Biochemistry

institute: Case Western Reserve University

summary: Steap4, highly expressed in adipose tissue, is associated with metabolic homeostasis. Dysregulated adipose and mitochondrial metabolism contribute to obesity, highlighting the need to understand their interplay. Whether and how Steap4 influences mitochondrial function, adipocytes, and energy expenditure remains unclear. Adipocyte-specific Steap4-deficient mice exhibited increased fat mass and severe insulin resistance in our high-fat diet model. Mass spectrometry identified two classes of Steap4 interactomes: mitochondrial proteins and proteins involved in splicing. RNA-seq analysis of white adipose tissue demonstrated that Steap4 deficiency altered RNA splicing patterns with enriched mitochondrial functions. Indeed, Steap4 deficiency impaired respiratory chain complex activity, causing mitochondrial dysfunction in white adipose tissue. Consistently, brown adipocyte-specific Steap4 deficiency impaired mitochondrial function, increased brown fat whitening, reduced energy expenditure, and exacerbated insulin resistance in a high-fat model. Overall, our study highlights Steap4’s critical role in modulating adipocyte mitochondrial function, thereby controlling thermogenesis, energy expenditure, and adiposity.

overall_design: To further characterize the impact of Steap4 on HFD-induced mitochondrial dysfunction in adipose tissue, we performed RNA-seq on iWAT collected from HFD-fed Steap4f/f and Steap4AKO mice.


title: Study 1- RNA-seq of male KOLF2.2J hiPSC-derived trophoblast cell lines homozygous null for seven different transcription factors

status: Public on Jan 30 2025

summary: This study aims to phenotype iPSC-derived trophoblast lines that contain homozygous null alleles for transcription factors expressed within the extra-embryonic lineage, with a focus on differentiation toward either primitive syncytium or extra-embryonic mesenchymal cells, depending on the TF being analyzed. Null alleles were generated using three distinct genetic engineering approaches: full protein coding region deletion (KO), critical exon deletion (CE), and insertion of a premature termination codon with frameshift (PTC+1). RNA-seq data was generated after 6 days of initiating differentiation from iPSCs. The study included the homozygous null alleles for the following transcription factors: GRHL1, POU2F3, EPAS1, FOSB, GCM1, PPARG, and ISL1. Notably, the hypoxia-inducible factor EPAS1 was evaluated under both 20% oxygen and 3% (~ level present at peri-implantation) oxygen concentrations. This analysis provided insight into the differences between the various CRISPR-Cas9-based approaches and the impact of loss of function for these transcription factors in trophoblast lineage differentiation.

overall_design: The study involved the generation of RNA-seq on 95 samples. Five of the transcription factors were analyzed in the primitive syncytium differentiation scheme and two in the extra-embryonic mesenchymal cell scheme. Three biological replicates were run for each genetic engineering approach for each gene. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions and under both 20% and, in the case of EPAS1, 3% oxygen concentrations. This comprehensive design allowed for a robust comparison of genetic engineering methods and the effects of transcription factor manipulation on cell fate decisions within the extra-embryonic lineage.

first: Paul


title: Study 1 and Study 2 - RNA-seq of male KOLF2.2J hiPSC-derived trophoblast cell lines homozygous null for 14 different transcription factors

summary:
This SuperSeries is composed of the SubSeries listed below.
This study aims to phenotype iPSC-derived trophoblast lines that contain homozygous null alleles for transcription factors expressed within the extra-embryonic lineage, with a focus on differentiation toward either primitive syncytium or extra-embryonic mesenchymal cells, depending on the TF being analyzed. Null alleles were generated using three distinct genetic engineering approaches: full protein coding region deletion (KO), critical exon deletion (CE), and insertion of a premature termination codon with frameshift (PTC+1). RNA-seq data was generated after 6 days of initiating differentiation from iPSCs. The study included the homozygous null alleles for the following transcription factors: GRHL1, POU2F3, EPAS1, FOSB, GCM1, PPARG, and ISL1, MEIS1, MXD1, MEIS2, RUNX1, MEF2C, NCOA3, and BHLHE40. Notably, the hypoxia-inducible factor EPAS1 was evaluated under both 20% oxygen and 3% (~ level present at peri-implantation) oxygen concentrations. This analysis provided insight into the differences between the various CRISPR-Cas9-based approaches and the impact of loss of function for these transcription factors in trophoblast lineage differentiation.

overall_design: Refer to individual Series


title: GPR34 senses myelin debris to promote neuroinflammation and pathology

city: Hefei

first: XiqQiong

last: Wang

state: Anhui

address: School of Life Sciences, No. 443 Huangshan Road

institute: University Of Science And Technology Of China

summary: Sterile neuroinflammation is a major driver of multiple neurological diseases. Myelin  debris, which is released from the damaged myelin sheaths during demyelination, can  act as an inflammatory stimulus to promote sterile inflammation and neurological pathologies, but the mechanism is poorly understood. Here, we show that lysophosphatidylserine (LysoPS)-GPR34 axis plays a critical role in microglia-mediated myelin debris sensing and the subsequent neuroinflammation. Myelin debris-induced microglia activation and proinflammatory cytokine expression relied on its lipid component LysoPS. Both myelin debris and LysoPS promoted microglia to produce IL-1and IL-6 via GPR34 and its downstream PI3K-AKT and ERK signaling. In vivo, LysoPS production and GPR34 signaling were found to be essential for neuroinflammation and pathologies in the mouse models of multiple sclerosis and stroke. Importantly, pharmacologic blockade of GPR34 showed potential therapeutic effects on these disease models. Thus, our results identify GPR34 as a key receptor to sense myelin debris and promote neuroinflammation, and suggest it as a potential target for demyelination-associated diseases.

overall_design: Examination of LysoPS effection on Primary microglia.

first: Rongbin

last: Zhou

first: Zonghui

first: Xiaqiong

first: Bolong

last: Lin


title: Sub-chronic administration of AM6545 enhances cognitive performance and induces hippocampal synaptic plasticity changes in naïve mice

city: Barcelona

first: Andrés

last: Ozaita

address: Dr Aiguader, 88

department: Department of Medicine and Life Sciences

country: Spain

institute: Universitat Pompeu Fabra

summary: Using RNA sequencing, we perform gene expression profile of three female mice treated with vehicle and four female mice sub-chronically treated with AM6545 at a dose of 1 mg/kg. To evaluate hippocampal modifications promoted by peripheral CB1R modulation, we analyze at transcript level hippocampal synaptoneurosomes fractions.

overall_design: mRNA profiles of hippocampal synaptoneurosomes from wild-type female mice after sub-chronic AM6545 or vehicle administration.

first: Araceli

last: Bergadà-Martínez

first: Lucia

last: de los Reyes-Ramirez


title: Microbiota-indole-3-propionic acid-heart axis mediates the protection of leflunomide against αPD1-induced cardiotoxicity in mice

city: Wuhan

first: Rong

address: Zhang Zhidong Road

institute: Renmin Hospital of Wuhan University

summary: Anti-programmed death 1 (αPD1) immune checkpoint blockade is used in combination for cancer treatment but associated with cardiovascular toxicity. Finding drugs that suppress cardiovascular toxicity but not compromise the efficacy of αPD1-mediated immunotherapy is of great importance. Leflunomide (Lef) can suppress the growth of several tumor and mitigate cardiac remodeling in mice. However, the specific role of Lef in αPD1-induced cardiotoxicity remains unclear.

overall_design: Using a novel melanoma mouse model, we assessed the protective effect of Lef on αPD1-induced cardiotoxicity. Fecal microbiota transplantation (FMT), 16S rRNA gene sequencing, metabolomics analysis and RNA sequencing were performed to reveal the underlying mechanisms.


title: Transcriptional changes of Tex10 in purified primordial gem cell-like cells (PGCLCs) at day2 and day6

first: Feifei

last: Yuan

overall_design: PGCLCs from day2 and day6 embryonic bodies (EBs, D2EBs vs. dD2EBs, D2EV vs. D2OE and D6EV vs. D6OE) were sorted by FASC with PE anti-mouse/rat CD61 and Alexa Fluorβ 647 anti-mouse/human SSEA1 antibodies.Total RNA from PGCLCs was extracted for RNA-sequencing.

first: Jianlong


title: Transcriptional changes of spermatogenic cells by Tex10 deficiency

overall_design: Spermatogenic cells were sorted from 8-week-old wildtype and Tex10f/-; Stra8iCre mice. Total RNA from Spermatogenic cells was extracted for RNA-sequencing.


title: Visium Spatial Transcriptomics of the Human Hypothalamus

status: Public on Jan 29 2025

city: Cambridge

first: Georgina

last: Dowsett

address: Box 289

institute: Institute of Metabolic Science

summary: The hypothalamus is a brain region that plays a key role in coordinating fundamental biological functions. However, our understanding of the underlying cellular components and neurocircuitries have, until recently, emerged primarily from rodent studies. Here, we combine single-nucleus sequencing of 433,369 human hypothalamic cells with spatial transcriptomics, generating a comprehensive spatio-cellular transcriptional map of the hypothalamus, the ‘HYPOMAP’. Whilst conservation of neuronal cell types between humans and mice, based on transcriptomic identity, is generally high, there are notable exceptions. Specifically, there are significant disparities in the identity of POMC neurons and in the expression levels of GPCRs between the two species that carry direct implications for currently approved obesity treatments. Out of the 452 hypothalamic cell types, we find 291 neuronal clusters are significantly enriched for expression of BMI GWAS genes. This enrichment is driven by 426 ‘effector’ genes. Rare deleterious variants in 6 of these, MC4R, PCSK1, POMC, CALCR, BSN and CORO1A, associate with BMI at population level, and CORO1A has not previously been linked to BMI. Thus, HYPOMAP provides a detailed atlas of the human hypothalamus in a spatial context and serves as an important resource to identify novel druggable targets for treating a wide range of conditions, including reproductive, circadian, and metabolic disorders.

overall_design: Visium Spatial transcriptomics was performed on 9 sections of human hypothalamus from 7 donors, to provide spatial localisation of transcript expression across the hypothalamus. Sections were taken across the hypothalamus spanning regions in the anterior, mid and posterior hypothalamus to capture nuclei throughout the hypothalamus.

first: G K C

first: S

last: Lundh

first: H

last: Solvang Nielsen

first: J

last: Juul

first: J C

last: Bruning

first: G S H

last: Yeo

first: B Y H

last: Lam


title: Caspase 9-induced apoptosis for efficient fetal cell ablation and disease modeling

city: Minato-ku

first: Kenji

last: Matsui

state: Tokyo

address: Nishi-Shimbashi 3-25-8

country: Japan

institute: The Jikei University School of Medicine

summary: Animal models for targeted cell ablation are invaluable in the field of biology and medicine. Specifically, fetal targeted cell ablation has the potential for organ regeneration and xenotransplantation. Previous methods involve conventional knockout models, which offers limited control over severity, or conditional ablation models, which use inducers that are harmful to fetuses. In this study, we apply the inducible caspase 9 (iC9) system to target mouse fetal nephron progenitor cells. It triggers specific, rapid, and efficient target cell ablation through the intrinsic apoptosis pathway by the administration of a safe, placental-permeable inducer. This feature allowed for precise adjustment of severity based on the timing of administration, enabling the creation of a viable severe kidney failure model. We also demonstrate that cells with low iC9 expression levels and those in the solid organ state are less susceptible to apoptosis induction. RNA-seq of wild type mouse fetal kidneys revealed that DNA damage response is enhanced after dissociation, suggesting that it is the underlying mechanism for the reduced susceptibility to apoptosis in solid-state cells compared to single-cell states. Apoptosis can still be triggered in these conditions by the combined inhibition of X-linked inhibitor of apoptosis protein. This approach will not only facilitate the study of mechanisms involved in induced cell death but also enhance its utility as a research tool, including for fetal organ transplantation models.

overall_design: We hypothesized that the single-cell state exhibits enhanced susceptibility to apoptosis. To investigate how gene expression related to apoptosis susceptibility differs before and after single-cell dissociation in fetal kidneys, we analyzed the transcriptomes between untreated wild-type fetal kidneys (pre-dissociation) and those enzymatically and manually dissociated into single-cell suspensions (post-dissociation) by bulk RNA-sequencing (n = 3 vs. 3).

first: Shuichiro

last: Yamanaka


title: A distinct follicular helper CD4 T cell programming characterizes neutralization activity in chronic HIV infection

city: Atlanta

first: Rafick-Pierre

last: Sekaly

state: GA

address: 1750 Haygood Drive

department: Pathology

institute: Emory University

summary: A subset of people living with HIV (PLWH) can generate broadly neutralizing antibodies (bNAbs), yet the cellular and molecular dynamics within the lymph nodes (LNs) that facilitate this process remain unclear. To investigate these mechanisms, we performed single-cell RNA sequencing (scRNA-seq) on CD45+ cells isolated from LNs of ART-naïve PLWH who produce bNAbs. Comparative analyses revealed distinct transcriptional profiles and cellular compositions between the two groups. Our data identified potential molecular mediators of TFH-B cell interactions that are unique to neutralizers. Together, these findings provide new insights into the germinal center dynamics and immune cell networks underlying bNAb development in PLWH.

overall_design: Lymph nodes from people living with HIV (PLWH) who produce broadly neutralizing antibodies (bNAbs) (n=4) and those who do not produce bNAbs (n=4) were collected. All CD45+ cells from the lymph nodes were isolated and analyzed using single-cell RNA sequencing (scRNA-seq). All PLWH were antiretroviral therapy (ART)-naive, and their bNAb repertoire was assessed prior to scRNA-seq.

first: Eirini

last: Moysi

first: Ashish

last: Sharma

first: Sijy

last: O’Dell

first: Spiros

last: Georgakis

first: Perla

middle: M

last: Estrada

first: Fernanda

last: Torres-Ruiz

first: Mauricio

middle: G

last: Navarro

first: Yara

last: Villalobos

first: Santiago

last: Rios

first: Gustavo

last: Reyes-Teran

first: Margaret

middle: H

last: Beddall

first: Sung-Hee

last: Ko

first: Frida

last: Belinky

first: Michail

last: Orfanakis

first: Laurence

last: de Leval

first: Ana

middle: B

last: Enriquez

first: Clarisa

last: Buckner

first: Susan

last: Moir

first: Nicole

last: Doria-Rose

first: Eli

last: Boritz

first: John

last: Mascola

first: Richard

last: Koup

first: Constantinos

last: Petrovas


title: Regulated somatic hypermutation enhances antibody affinity maturation

status: Public on Jan 27 2025

city: New York

first: Gabriela

last: Silva Santos

address: 1230 York Avenue

institute: The Rockefeller University

summary: Germinal centers (GCs) are microenvironments where B cells undergo affinity maturation through somatic hypermutation (SHM) and selection by T follicular helper (TFH) cells. While SHM introduces mutations at a fixed rate (~1x10⁻³ per base pair per division), most mutations are deleterious, particularly in high-affinity B cells undergoing many divisions. This study tests a theoretical model suggesting that high-affinity B cells optimize maturation by dividing more but mutating less per division. Data from mice immunized with SARS-CoV-2 or a model antigen support the model, showing that high-affinity B cells shorten their G0/G1 phases and reduce mutation rates, safeguarding their lineages and improving affinity maturation outcomes.

overall_design: Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 5′ v2 Reagent Kit (10x Genomics) according to manufacturer’s protocol. Libraries were loaded onto an Illumina NextSeq with the mid-Output Kit (150 paired end) for V-D-J analysis or onto an NOVAseq for pooled V-D-J and single  cell gene expression. Hashtag indexing was used to demultiplex the sequencing data and generate gene-barcode matrices, respectively.

first: Julia

last: Merkenschlager

first: Andrew

last: T Pyo

first: Dennis

last: Schaefer-Babajew

first: Melissa

last: Cipolla

first: Harald

last: Hartweger

first: Alexander

middle: D

last: Gitlin

first: Ned

last: Wingreen

first: Michel

middle: C

last: Nussenzweig


title: Identifying Compounds that Alleviate Acute Hypoxic Stress by Fast-tracking Cellular Adaptation

city: San Francisco

first: Li

last: Li

address: 1700 4th Street, MC 2540

institute: UCSF

summary: Severe acute hypoxic stress is a major contributor to the pathology of human diseases, including ischemic disorders. Current treatments focus on managing consequences of hypoxia, with few addressing cellular adaptation to low-oxygen environments. Here, we hypothesize that accelerating hypoxia adaptation could provide a strategy to alleviate acute hypoxic stress. We developed a high-content phenotypic screening platform to identify compounds that fast-track adaptation to hypoxic stress. Our platform captures a high-dimensional phenotypic hypoxia response trajectory consisting of normoxic, acutely stressed, and chronically adapted cell states. Leveraging this trajectory, we identify compounds that phenotypically shift cells from the acutely stressed towards the adapted state, revealing mTOR/PI3K or BET inhibition as strategies to induce this phenotypic shift. Importantly, our compound hits promote the survival of liver cells exposed to ischemia-like stress, and rescue cardiomyocytes from hypoxic stress. Our platform offers a general, target-agnostic strategy to accelerate cellular adaptation, applicable across various stress conditions.

overall_design: RNA-seq profiling of HepG2 cells under 3 oxygen conditions: Normoxia (21% oxygen), 24hr treatment of hypoxia (1% oxygen), and 6day treatment of hypoxia (1% oxygen).

first: Heinz

last: Hammerlindl

last: Shen

first: Feng

last: Bao

first: Sabrina

first: Steven

last: Altschuler

first: Lani

last: Wu


title: Transcriptomic analysis of RNA psi modification in colorectal cancer reveals an epitranscriptomic regulation in cellular senescence

status: Public on Jan 28 2025

city: Guangzhou

first: Yingguo

last: Gan

address: 26 Yuancun Erheng Road

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

summary: To identify the senescence level of PUS enzymes on colorectal cancer cells, we established WIDR cell lines in which DKC1 has been knocked down by shRNA. We then performed gene expression profiling analysis using data obtained from RNA-seq of DKC1 knockdown WIDR cells as well as negative control shRNA group.

overall_design: Compare negative control shRNA and DKC1 knockdown WIDR cancer cells using RNA-seq.

first: Mingzhe

last: Huang


title: Functional memory T cells are derived from exhausted clones and expanded by checkpoint blockade [scRNATCR_aPDL1]

city: Stanford

first: Colin

middle: J

last: Raposo

address: 240 Pasteur Drive, BMI 4600

institute: Stanford University

summary: Immune checkpoint blockade can facilitate tumor clearance by T cells, resulting in long term patient survival. However, the capacity of exhausted CD8+ T cells (Tex), present during chronic antigen exposure, to form memory after antigen clearance remains unclear. Here, we performed longitudinal single cell RNA/T cell receptor sequencing and ATAC-sequencing on antigen-specific T cells after the peripheral clearance of chronic lymphocytic choriomeningitis virus infection. These data revealed the formation of a robust population of memory CD8+ T cells that transcriptionally, epigenetically, and functionally resemble central memory T cells (Tcm) that form after clearance of acute infection. To lineage trace the origin and memory recall response of Tex-derived memory clones, we utilized T cell receptor sequencing over the course of primary infection and rechallenge. We show that chronic Tcm are a clonally distinct lineage of Tex derived from progenitor exhausted cells, persist long-term in the absence of antigen, and undergo rapid clonal expansion during rechallenge. Finally, we demonstrate that αPD-L1 immune checkpoint blockade selectively expands clones which form Tcm after clearance. Together, these data support the concept that chronically stimulated T cells form bona fide functional memory T cells through an analogous differentiation pathway to acutely stimulated T cells, which may have significant implications for enhancing immune memory to cancer through checkpoint blockade and vaccination.

overall_design: LCMV-Cl13-infected mice were treated six times with either aPDL1 or PBS from days 22 to 34. At 102 days post-infection, scRNA/TCR-seq was performed on GP33+ CD8+ T cells.

first: Kamir

last: Hiam-Galvez

first: Ansuman

middle: T

last: Satpathy


title: Functional memory T cells are derived from exhausted clones and expanded by checkpoint blockade [scRNATCR_rechallenge]

overall_design:
To track T cell clones during rechallenge, CD8+ T cells were isolated from the spleens of mice that cleared LCMV-Cl13 or LCMV-Arm infection. Half of the cells were used for bulk TCR sequencing prior to rechallenge. The remaining cells were transferred into new host mice, challenged with LCMV-Arm. At 8 days post-infection transferred T cells were sorted, and scRNA/TCR-seq was performed.

Pre-rechallenge TCR-seq can be found at doi.org/10.5281/zenodo.14648171


title: Targeting chemokine crosstalk between T-cells and macrophages to treat immune checkpoint inhibito-mediated myocarditis

first: Han

last: Zhu

address: 1291 Welch Road

institute: Stanford Cardiovascular Institute

summary: Immune checkpoint inhibitors (ICIs) are effective against many cancers but can also cause immune-related adverse events. Although rare, ICI-mediated myocarditis has a high fatality rate of up to 40% and is also associated with heart failure and life-threatening arrhythmias. We characterized a population of CXCL9+CXCL10+ macrophages and CXCR3hi CD8+ T-cells in the hearts of mice with myocarditis and elucidated chemokine crosstalk between the CXCR3hi CD8+ effector T-cells and the CXCL9+CXCL10+ macrophages in the heart. Depletion of macrophages or blockade of CXCR3 both resulted in significantly decreased immune cell infiltration in the hearts of mice. Similarly, CXCR3 blockade decreased immune cell infiltration into the heart,, thus posing CXCR3 and its ligands as an attractive therapeutic target. Additionally, an in vitro transwell assay showed that selective blockade of CXCR3 and its ligand CXCL10 significantly decreased CD8+ T-cell migration towards macrophages, implicating this interaction in T-cell cardiotropism towards cardiac macrophages. These findings were compared with cardiac biopsies from patients with ICI myocarditis that also demonstrated infiltrating CXCR3+ lymphocytes and CXCL9+/CXCL10+ macrophages. In both mouse cardiac immune cells and patient peripheral blood immune cells, T-cell receptor (TCR)-sequencing revealed expanded TCRs that correlated with CXCR3hi CD8+ T-cells.  The identification of a CXCR3-specific T-cell and macrophage interaction as important in the pathogenesis of ICI myocarditis offers a new potential target for a chemokine/chemokine receptor inhibition-focused form of therapy in this disease.

overall_design: Here, we used a genetic mouse model of PD-1 deletion combined with a novel pharmacological mouse model of anti-PD1 and anti-CTLA-4 monoclonal antibody treatment on a Murphy Roths Large (MRL) background to model ICI myocarditis. We performed single-cell RNA-sequencing (scRNAseq), single-cell TCR-sequencing (scTCRseq), and CITE-sequencing on immune cells isolated from MRL and MRL/Pdcd1-/- mice at serial timepoints 1, 2, 4 weeks of age. We depleted macrophages from MRL/Pdcd1-/- mice to study the role of macrophages in ICI myocarditis. Subsequently, we inhibited a key chemokine receptor, CXC chemokine receptor 3 (CXCR3), in MRL pharmacologic mouse to assess for reduction in myocarditis. Subsequently, we studied CXCL9/10-CXCR3 interactions between macrophages/T-cells in a transwell assay. Finally, we correlated the results in human single-cell multi-omics data from blood and heart biopsy data. We analyzed immune cell populations in time point data, clodronate macrophage depletion data, and CXCR3 inhibition in a novel pharmacologic mouse model. We study mechanistic relationships between T-cells and macrophages using a transwell assay and also analyzed patient cardiac biopsy and blood single cell data.

first: Yuhsin

middle: V


title: Functional memory T cells are derived from exhausted clones and expanded by checkpoint blockade [ATAC-Seq]

overall_design: ATAC-seq was performed on sorted populations of GP33+ T cell subsets from 176 days post-infection LCMV-Cl13, 28 days post-infection LCMV-Cl13, and 176 days post-infection LCMV-Arm.


title: PGLYRP-1 mediated intracellular peptidoglycan detection promotes mucosal protection

city: Dallas

first: Lei

last: Guo

state: TX

address: 5323 Harry Hines Blvd

department: Department of Population and Data Sciences

institute: University of Texas Southwestern Medical Center

summary: We identified PGLYRP-1 as a receptor for the disaccharide motif of lysine N-acetylglucosamine N-acetylmuramic tripeptide (GMTriP-K) with a newly constructed PGN microarray. Surprisingly, PGLYRP-1 was required for innate immune activation of macrophages by GMTriP-K but not N-acetylglucosamine N-acetylmuramic dipeptide (GMDiP) or muramyl dipeptide (MDP). In macrophages, intracellular PGLYRP-1 complexed with NOD2 and GEF-H1, both of which were required for GMTriP-K-regulated gene expression. PGLYRP-1 localized to the endoplasmic reticulum and interacted at the Golgi with NOD2 upon GMTriP-K stimulation.  PGLYRP-1 upregulation and its dependent gene expression signatures were induced in both mouse intestinal inflammation and human ulcerative colitis. Importantly, PGLYRP-1 activation by GMTriP-K resulted in innate immune activation and protection of mice from colitis.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Bone marrow derived macrophages were stimulated with peptidoglycan derivatives. Biopsies from healty, inactive and active ulcerative colitis patients

first: Hans-Christian

last: Reinecker


title: Single-cell profiling uncovers regulatory programs of pathogenic Th2 cells in allergic asthma

city: Vienna

first: Nicole

last: Boucheron

address: Lazarettgasse 19

country: Austria

institute: Institute of Immunology

summary: Lung pathogenic T helper type 2 (pTh2) cells are important drivers of allergic asthma, but fundamental questions remain regarding their regulation and heterogeneity. The differentiation and effector functions of immune cells are tightly regulated by epigenetic processes. Histone deacetylase 1 (HDAC1) is an important epigenetic regulator of T cells, however, its role in pTh2 cells is yet to be determined. Here we investigate immune regulation in allergic asthma by single-cell RNA sequencing (scRNA-seq) in mice challenged with house dust mite, in the presence and absence of HDAC1 function. Our analyses reveal two distinct subsets of lung pTh2 cells: pathogenic effector Th2 (peTh2) and pathogenic Th2 tissue-resident memory (Th2 Trm) cells. Both pTh2 cell subsets are highly proinflammatory and exhibit distinct transcriptional and phenotypic signatures as compared with other lung Th subsets. Based on our scRNA-seq analysis, we identify conditions to generate pTh2 cells in vitro and confirm that these in vitro generated pTh2 cells have a similar transcriptional profile as lung peTh2 cells. Using our new in vitro model, we demonstrate that the p38 mitogen-activated protein kinase pathway is critical for interleukin-5 (IL-5) and IL-13 expression in pTh2 cells. Our data further underline the importance of HDAC1 in limiting the pathogenicity of lung and in vitro pTh2 cells and in the formation of lung Th2 Trm cells. In summary, we have generated novel insights into pTh2 cell biology and established a new in vitro model for investigating pTh2 cells that will be useful for discovering molecular mechanisms involved in pTh2-mediated allergic asthma.

overall_design: To understand how pathogenic Th2 differentiation is established at a chromatin level, and how HDAC1 regulates this process, we isolated naive CD4+ T cells from HDAC1fl/fl and HDAC1fl/fl Cd4-Cre mice, and cultured these cells either under "conventional" Th2 conditions (anti-CD3/anti-CD28 with IL-4) or "pathogenic" Th2 conditons (anti-CD3/anti-CD28 with IL-4, anti-GITR agonist, TSLP).To have a time resolved chromatin accessibility profiling. the cells were harvested after 24 and 48 hours of culture and processed for assay for transposase-accessible chromatin using sequencing (ATAC-seq). For each condition, three experimental replicates were performed.

first: Matarr

last: Khan

first: Marlis

last: Alteneder

first: Johan

last: Henriksson

first: Benjamen

last: White

first: Michael

last: Schuster


title: Effect of YAP5SA expression on the transcriptome of proliferating and senescent cells

city: Seoul

first: Jaejin

last: Kim

address: Gwanak-ro 1

country: South Korea

institute: Seoul National University

summary: We have generated IMR90-hTERT cells carrying a Doxycyclin-inducible (Tet-On) vector expressing an N-terminally Flag tagged YAP5SA. To identify differentially expressed genes by YAP5SA expression during cellular senescence, we performed transcriptional profiling by RNA sequencing (RNA-seq) with or without YAP5SA expression (using doxycyclin), before and after senescence induction using a stress-induced senescence model (bleomycin-induced senescence).

overall_design: IMR90-hTERT cells carrying a Dox-inducible (Tet-On) vector expressing an N-terminally Flag tagged YAP5SA were grown with or without Dox and treated with bleomycin (10 ug/ml for 24 h) to induce senescence. Total RNA was isolated using the acid phenol extraction method. One microgram of high-quality RNA samples (RIN > 7.0) was used to construct RNA-seq libraries using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). RNA-seq was performed on an Illumina NovaSeq 6000 sequencer.

first: Joae

last: Joung

first: Yekang

last: Heo

first: Chanhee

last: Kang


title: CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis

city: Suzhou

first: Peng

last: Xu

state: Jiangsu

address: Office3625 Building703, RENAI ROAD No.199, INDUSTRIAL PARK DISTRICT

department: Suzhou Medical College

institute: Soochow University

summary:
Background: The three-dimensional chromatin niche provides a precise gene expression control of cell identity, differentiation, and disease development. CTCF is considered as the most essential transcription factor regulating chromatin architecture and gene expression. However, the genome-wide impact of CTCF on erythropoiesis has not yet been extensively investigated.
Results: Here, using a state-of-the-art human erythroid progenitor cell model (HUDEP-2 and HEL cell lines), we systematically investigated the effects of acute CTCF loss by an auxin-inducible degron system on transcriptional programs, chromatin accessibility, CTCF genome occupancy, and the 3D genome architecture. By integrating multi-omics datasets, we revealed that acute CTCF loss notably disrupted genome-wide chromatin accessibility and transcription network. Importantly, we identified that over thousands of chromatin accessibility regions were decreased while only a few hundred increased regions after depletion of CTCF in both HUDEP-2 and HEL lines, suggesting the role of CTCF in maintaining the proper chromatin openness in erythroid lineage. Moreover, CTCF depletion in the erythroid context significantly disrupted TAD boundary integrity and chromatin loops while not affecting the nuclear compartmentalization. Our study also reported that hundreds of erythroid lineage-specific genes were suppressed in both immature and mature stages, including metabolism-related target genes such as GOT2 and FXN. Notably, we identified a subset of genes increased their transcriptional level after the CTCF depletion, accompanied by the decreased chromatin accessibility regions that were enriched with the GATA motif. Specifically, we identified that CTCF repressed the master transcription factor GATA2 with distal decreased chromatin regions via multiple functional studies, including CTCF occupancy profiling and CRISPR/Cas9 editing of the CTCF binding site. These results suggest a suppressive role of CTCF in gene expression in erythroid lineage specification.
Conclusions: In summary, our results revealed a novel role of CTCF in regulating erythroid differentiation by maintaining its proper chromatin openness, which will undoubtedly extend our understanding of CTCF biology.

overall_design: The HEL cells used in the experiment were knock in cell lines of GFP-miniAID-CTCF constructed by ourselves. We treated the cell lines with drugs for 24 hours. The drugs used included DMSO and IAA. IAA was used for quick depletion CTCF protein.


title: Post-transcriptional monoadenylation by TENT2 terminates human RNA polymerase III transcript 3' end processing and promotes 7SL RNA biogenesis [gene_specific_RNAseq]

city: La Jolla

first: Cody

last: Ocheltree

address: 9500 Gilman Drive

institute: UCSD

summary: Small non-coding RNAs (sncRNAs) are subject to a variety of 3' end trimming and tailing activities during biogenesis, which dictate whether they undergo maturation or are instead subjected to degradation. To investigate the dynamics of human sncRNA 3' end processing at a global level we performed genome-wide 3' end sequencing of nascently-transcribed and steady state sncRNAs. This revealed widespread post-transcriptional adenylation of nascent sncRNAs, which came in two distinct varieties. One is characterized by oligoadenylation, which is transient, promoted by TENT4A/4B polymerases, and observed primarily on unstable sno- and scaRNAs that are not fully processed at their 3' ends. The other is characterized by monoadenylation, which is broadly catalyzed by TENT2 and stably accumulates at the 3’-end  of many RNA Polymerase-III-transcribed (Pol-III) RNAs as well as a subset of small nuclear RNAs. This monoadenylation event terminates 3' end uridylation/deuridylation dynamics characteristic of nascent Pol-III RNAs and, in the case of 7SL RNAs, prevents their accumulation with nuclear La protein and promotes their biogenesis towards assembly into cytoplasmic signal recognition particles.

overall_design: RNAs from control or TENT2-depleted conditions were gene-specifically PCR amplified for 3' end sequencing. RNAs associated with La and SRP54 during IP were PCR amplified for 3' end sequencing. RNAs labeled with s4U (two 3-hour pulses) in control or TENT2 KO conditions were PCR amplified for 3' end sequencing.

first: Blake

last: Skrable

first: Tim

last: Nicholson-Shaw

first: Jens

last: Lykke-Andersen


title: Inhibition of the STAT3/Fanconi anemia axis is synthetic lethal with PARP inhibitor in breast cancer

city: MARSEILLE

first: Christophe

last: Ginestier

address: 27 Bd Leï Roure

country: France

institute: CRCM

summary: The targeting of cancer stem cells (CSCs) has proven to be an effective approach for limiting tumor progression, thus necessitating the identification of new drugs with anti-CSC activity. Through a high-throughput drug repositioning screen, we identified the antibiotic Nifuroxazide (NIF) as a potent anti-CSC compound. Utilizing a click chemistry strategy, we demonstrated that NIF is a prodrug that is specifically bioactivated in breast CSCs. Mechanistically, NIF-induced CSC death is a result of a synergistic action that combines the generation of DNA interstrand crosslinks with the inhibition of the Fanconi anemia (FA) pathway activity. NIF treatment mimics FA-deficiency through the inhibition of STAT3, which we identified as a non-canonical transcription factor of FA-related genes. NIF induces a chemical HRDness in CSCs that (re)sensitizes breast cancers with innate or acquired resistance to PARP inhibitor (PARPi) in PDX models. Our results suggest that NIF may be useful in combination with PARPi for the treatment of breast tumors, regardless of their HRD status

overall_design:
SUM159 and SW620 cell lines were used in three independent biological replicates.  ALDHneg and ALDHbr cells were isolated by cell sorting for control condition of treated by NIF during 30 hours. Total RNA was extracted using the Maxwell RSC simply RNA Tissue Kit (AS1340)  and its quality was assessed by Tapestation (only samples with RIN score > 8 were considered for sequencing).

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

first: Celia

last: Rouault

first: Lucile

last: Bansard

first: Elena

last: Martínez-Balsalobre

first: Caroline

last: Bonnet

first: Julien

last: Wicinski

first: Emmanuelle

last: Charafe-Jauffret


title: CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis [ChIP-seq_CTCF]

summary: The three-dimensional chromatin niche provides a precise gene expression control of cell identity, differentiation, and disease development. CTCF is considered as the most essential transcription factor regulating chromatin architecture and gene expression.  However, the genome-wide impact of CTCF in erythropoiesis has not been extensively investigated yet. To further study the direct transcriptional regulatory role of CTCF, we established two endogenous C-terminus AID-CTCF system in t human erythroblast cell lines and systematically investigated the effects of acute CTCF loss by auxin-inducible degron system on transcriptional programs and chromatin accessibility. By integrating multi-omics datasets, we revealed that acute CTCF loss notably suppressed the subset of GATA1 downstream targets. Importantly, we revealed that acute CTCF loss notably disrupted genome-wide chromatin accessibility and transcription network. Importantly, we identified that over thousands of chromatin accessibility regions were decreased while only a few hundreds of increased regions after depletion of CTCF in both HUDEP-2 and HEL lines, suggesting the role of CTCF in maintaining the proper chromatin openness in erythroid lineage. Moreover, CTCF depletion in erythroid context significantly disrupted TAD boundary integrity and chromatin loops while not affecting the nuclear compartmentalization.  In summary, our results addressed a novel role of CTCF in regulating erythroid differentiation by maintaining its proper chromatin openness, which will undoubtedly extend our understanding of CTCF biology.

overall_design: The HUDEP-2 cells and HEL cells used in the experiment were our own GFP-miniAID-CTCF knock in cell line. We treated the cell line with drugs for 24 hours, and then conducted ChIP-seq experiments using CTCF antibodies.


title: Systematic inference of super-resolution cell spatial profiles from histology images

city: Beijing

first: Shao

last: Li

state: Beijing

address: Haidian district

institute: Tsinghua University

summary: Inferring cell spatial profiles from histology images is critical for cancer diagnosis and treatment in clinical settings. In this study, we report a weakly-supervised deep-learning method, HistoCell, to directly predict the super-resolution cell spatial profiles from histology images at the single-nucleus-level. Benchmark analysis demonstrates that HistoCell robustly achieves state-of-the-art performance in terms of cell type/states prediction solely from histology images across multiple cancer tissues. HistoCell can significantly enhance the deconvolution accuracy for the spatial transcriptomics data and enable accurate annotation of subtle cancer tissue architectures. Moreover, HistoCell is applied to de novo discovery of clinically relevant spatial organization indicators across diverse cancer types. HistoCell also enable image-based screening of cell populations that drives phenotype of interest and is applied to discover the cell population and corresponding spatial organization indicators associated with gastric malignant transformation risk, which is validated on independent longitudinal cohorts. Overall, HistoCell emerges as a powerful and versatile tool for cancer studies in histology image-only cohorts.

overall_design: Two gastric samples with both cancer and pre-cancerous lesions were used for this analysis.

last: Zhang

first: Chaofei

last: Gao

first: Zhiyuan

last: Yuan

first: Zhuoyu


title: CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis [ChIP-seq_GATA1]

summary: By using an unbiased genetic screen and proteome profiling in human erythroblast, we identified and verified KMT2D as an essential factor for human erythropoiesis.  Accordingly, we observed that KMT2D is indispensable for the activation of erythroid signature genes in a stage dependent manner. Moreover, to further study the direct transcriptional regulatory role of KMT2D, we established two endogenous N-terminus AID-KMT2D system in t human erythroblast cell lines and systematically investigated the effects of acute KMT2D loss by auxin-inducible degron system on transcriptional programs and chromatin accessibility. By integrating multi-omics datasets, we revealed that acute KMT2D loss notably suppressed the subset of GATA1 downstream targets. Importantly, we profiled the specific genome-wide occupancy of KMT2D by comparison between the presence and absence of KMT2D in AID model.  Strikingly, KMT2D occupies over hundreds of erythroid specific genes mostly on their promoters. More importantly, we revealed that the KMT2D co-localized with GATA1 in numerous erythroid genes and the occupancy of GATA1 significantly reduced after KMT2D degradation. These results suggest KMT2D is indispensable for the enhancer-binding of GATA1 in gene expression in erythroid lineage specification.

overall_design: The HUDEP-2 cells and HEL cells used in the experiment were our own GFP-miniAID-CTCF knock in cell line. We treated the cell line with drugs for 24 hours, and then conducted ChIP-seq experiments using GATA1 antibodies.


title: CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis [RNA-seq]

overall_design: The HEL and HUDEP2 cells used in the experiment were knock in cell lines of GFP-miniAID-CTCF constructed by ourselves. We treated the two cell lines with drugs for 0 hours, 6 hours and 12 hours,, with three replicates at each time point. The drugs used included DMSO and IAA.


title: Effect of in vitro co-culturing of Lactobacillus crispatus on gene expression of Enterococcus faecalis OG1RF

city: Gainesville

first: Ling Ning Kaylie

last: Lam

state: Florida

address: 1395 Center Dr

department: Oral Biology

institute: University of Florida College of Dentistry

summary: Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both Enterococcus faecalis and Lactobacillus crispatus are commensal residents of the vaginal tract, the molecular mechanisms that enable E. faecalis to outcompete L. crispatus, and consequently cause vaginal infections remains unknown. To begin to address this, we need to gain a better understanding of the competitive interactions between E. faecalis and L. crispatus. Here, we employed a RNAseq approach to identify adaptive genes and transcriptional networks that enable E. faecalis to compete with L. crispatus.

overall_design: Single species E. faecalis biofilms and Transwell co-cultured E. faecalis and L.crispatus biofilms are grown for 24 hrs in MRS media, and then RNA was isolated for RNA-seq differential gene expression analysis

first: Jose

last: Lemos


title: Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation [RNA-Seq]

first: Ming

last: Wang

address: #15 Datun Road, Chaoyang, Beijing 100101, China

department: Key Laboratory of RNA Biology

institute: Institute of Biophysics, Chinese Academy of Sciences

summary: The Piwi-interacting RNA (piRNA) pathway plays a crucial role in protecting animal germ cells by repressing transposons. However, the mechanism behind this RNA-mediated epigenetic regulation that leads to heterochromatin formation is not yet fully understood. Through RNA interference screens, we discovered Pcf11 and PNUTS, two conserved termination factors of RNA polymerase II (Pol II), as essential for piRNA-guided silencing. When Pcf11 is artificially tethered to a reporter, it leads to co-transcriptional repression and Pol II stalling, both of which are dependent on an alpha-helical region of Pcf11 capable of forming condensates when triggered by the C-terminal repeat domain (CTD) of Pol II. Interestingly, an intrinsically disordered region (IDR) derived from the Plant FCA can substitute for the alpha-helical region of Pcf11 in its silencing capacity and support animal development, demonstrating a causal relationship between phase separation and Pcf11’s function. Further exploration into how Pcf11 causes Pol II stalling revealed a direct interaction between Pcf11’s CTD-interaction domain (CID) and a CTR region of Spt5. The dephosphorylation of Spt5-CTR by PP1/PNUTS is essential for Pcf11’s recruitment during Pol II termination. It appears that the phosphorylated state of CTR inhibits Pcf11 condensate formation, while the phosphorylation of Pol II CTD at Threonine 4 enhances Pcf11’s ability to undergo phase separation. In conclusion, we propose a model in which Pcf11 regulates transcriptional termination by slowing down Pol II elongation, a process that is aided by phase separation with the unphosphorylated Spt5. The ability of Pcf11 to stall Pol II, utilized by the piRNA pathway to initiate heterochromatin formation, might represent a universal strategy for nascent RNA-mediated epigenetic control.

overall_design:
To validate Pcf11’s essential role in Panx-mediated silencing, we generated an shRNA line targeting a distinct region of the Pcf11 mRNA.
We then performed gene expression profiling analysis using data obtained from RNA-seq of Pcf11 and control knockdown.
Compare gene expression profiling analysis of RNA-seq data for control knockdown and Pcf11 knockdown.
To explore the functional significance of the α-helical domain in Pcf11, we utilized CRISPR/Cas9 to generate a loss-of-function mutant. We create transgenic flies expression either the wild-type Pcf11, a version lacking the α-helical domain (Pcf11∆-helical), a version with the α-helical domain replaced by the IDR from Arabidopsis FCA (Pcf11FCA)  and a version with α-helical domain replaced with the FUS-IDR (Pcf11FUS)
We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 developmental stages (L1, L2 and Adult).
Compare gene expression profiling analysis of RNA-seq data for wildtype rescue and its mutant (Pcf11∆-helical, Pcf11FUS, Pcf11FCA).

first: Weiwei

last: Liu

first: Lijun

last: Deng

first: Xuan

last: Ouyang

first: Yang

last: Yu


title: Establishment and characterization of persistent Pseudomonas aeruginosa infections in air-liquid interface cultures of human airway epithelial cells

city: Braunschweig

first: Matthias

last: Preusse

address: Inhoffenstr. 7

department: Molecular Bacteriology

country: Germany

institute: Helmholtz Center for Infection Research

summary: Bacteria exhibit distinct behaviors in laboratory settings compared to infection environments. The presence of host cells induces changes in bacterial activity, while pathogens trigger immune responses that shape the microenvironment. Studying infection dynamics by microscopy, cytokine screening, and Dual RNA sequencing in an air-liquid interface model, we found that prolonged Pseudomonas aeruginosa colonization of airway epithelium led to a pro-inflammatory response, consistent across P. aeruginosa strains, despite differences in the dynamics of this response. Concurrently, P. aeruginosa formed non-attached aggregates on the apical side of the cell layer and up-regulated genes involved in biofilm formation and virulence. Notably, there was remarkable resemblance between the P. aeruginosa transcriptional profile in our model and that previously reported upon host cell contact. Developing a platform that replicates host microenvironments is vital not only for gaining deeper insights into the interplay between host and pathogen, but also for evaluating therapeutic strategies in conditions that closely mirror clinical environments.

sample_organism:
Pseudomonas aeruginosa
Homo sapiens

platform_organism:
Pseudomonas aeruginosa
Homo sapiens

overall_design: Calu-3 cells grown at air-liquid interface were infected with Pseduomonas aeruginosa PAO1 and treated basolaterally with 100µg/ml tobramycin

first: Safaa

last: Bouheraoua

first: Sven

last: Cleeves

first: Mathias

last: Müsken

first: Peter

last: Braubach

first: Maximilian

last: Fuchs

first: Christine

last: Falk

first: Katherina

last: Sewald

first: Susanne

last: Häussler


title: Transcriptomic profiling of 30 serum EV Sample

city: Kalyani

first: Kartiki

middle: V.

last: Desai

state: West Bengal

address: N.S.S kalyani

country: India

institute: National Institute of Biomedical Genomics

summary: 26 breast cancer patient serum EV derived RNA and 4 control serum EV derived RNA was sequenced to explore the diagnostic potential of EV. EV RNA was isolated by EXOQUICK® Exosome isolation and RNA purification kit for serum/plasma (System Biosciences,CA) and was reverse transcribed to make amplified cDNA by SMART seq v4 ultra-low input RNA kit (Takara Bio Inc, USA). A sequencing library was made using 1 ng of sheared cDNA using The Low Input Library Prep Kit v2 (Takara Bio Inc, USA). DNA unique Dual index kit was used to combine libraries for sequencing (Takara Bio Inc, USA).  NOVASEQ 6000 was used for sequencing to generate 25 million paired end reads per sample.

overall_design: Comparison of gene expression profiles of serum EVs between Healthy control and breast cancer patients using RNA-seq data .

first: Desai

last: Kartiki

first: Gupta

last: Aritra


title: CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis [H2_ATAC]

first: Peng

last: Xu

summary: The three-dimensional chromatin niche provides a precise gene expression control of cell identity, differentiation, and disease development. CTCF is considered as the master transcription factor regulating chromatin architecture and gene expression. However, the genome-wide impact of CTCF has not been extensively investigated due to the lack of proper research tools. Here, using a state-of-the-art auxin-inducible degron cellular model built up in human erythroid progenitors, we investigated the effects of acute CTCF loss on transcriptional programs and chromatin accessibility during human erythroid maturation. By integrating multi-omics datasets, we revealed that CTCF acute loss significantly disrupted genome-wide chromatin accessibility and transcription network. We further identified several direct novel target genes of CTCF in erythroid progenitor cells, including one master transcription factor GATA2 in hematopoiesis. Acute depletion of CTCF protein or disrupting CTCF binding sites in the topologically associated domains (TAD) boundary region of the GATA2 locus results in a significant transcriptional increase of GATA2. In summary, our results addressed a novel role of CTCF in regulating erythroid differentiation, which will undoubtedly extend our understanding of CTCF biology.

overall_design: Integrative analysis HiC/ATAC/RNA/ChIP-seq in HUDEP2 with or without acute degradation of CTCF.

first: Li

last: Cheng

first: Beisi

first: Ruopeng

last: Feng

first: Yu

last: Yao

first: Judith

last: Hyle

first: Mitchell

last: Weiss

first: Chunliang

last: Li


title: Effect of myb107 mutation and temperature on seedcoat and endosperm gene expressions during mature green stage

city: Geneva

first: Lena

last: Hyvärinen

state: Geneva

address: 30, Quai Ernest-Ansermet

department: Plant Sciences

country: Switzerland

institute: University of Geneva

summary: Seed dormancy is an adaptive trait whereby germination is blocked under favourable conditions to avoid germination out of season. Over time mature dry seeds lose dormancy and gradually acquire the capacity to germinate. Dormancy levels, i.e. the time required to release dormancy of the newly produced dry seed, are influenced by the environment of the mother plant and particularly by cold temperatures, which increase dormancy levels. It is also known that the seed coat, a maternal dead tissue, is important to keep seeds dormant over time, probably by shielding the seed living tissues from atmospheric oxygen.   Biochemical and genetical evidence previously established that cold promotes polyester accumulation in the seed coat and mutant seeds deficient in polyester biosynthesis have low dormancy and viability. However, it is unclear which seed coat structures, such as apoplastic barriers, are remodeled or created de novo in response to cold during seed development.  Combining histological and genetical approaches, a previously uncharacterized apoplastic barrier located in the outer integument 1 (oi1) cell layer was discovered. This barrier is strongly reinforced by cold and this process requires a MYB107 transcription factor , specifically expressed in oi1 cells. myb107 mutants is lacking the MYB107 TF, lacking the barrier and are less dormant, thus providing direct evidence that this barrier promotes dormancy in Arabidopsis.

overall_design:
To investigate MYB107 downstream targets in outer integument 1 (oi1) barrier and thus investigate oi1 barrrier composition, we compared WT and myb107 mutant transcriptome to identify MYB107 downstream targets at 22°C and with cold (13°C) transfert.
To do so, we isolate RNA from WT and myb107 mutant at 11 Days After Pollination (DAP) grown at 22°C and with a 48h cold treatment. To have a better understanding of cold effect on WT, we also isolate RNA from WT with 6h cold treatment. Finally a 13DAP WT at 22°C was also included in the analysis to have an idea of the developmental effect on the transcriptome.

first: Hyvärinen

last: Lena

first: Fuchs

last: Christelle

first: Petit

last: Brice

first: Lopez-Molina

last: Luis


title: A long-distance inhibitory system regulates haustoria numbers in parasitic plants

status: Public on Jan 26 2025

city: Uppsala

first: Charles

last: Melnyk

address: Almas allé 5

country: Sweden

institute: SLU

summary: Transcriptomic response changes following infection in shoots of the parasitic plant Phtheirospermum japonicum. We performed RNAseq on shoots of Phtheirospermum japonicum infecting and not infecting at 10 days after addition of the host Arabidopsis thaliana. We investigated the systemic transcriptomic changes in the shoot following the establishment of haustoria in the roots of P.japonicum.

overall_design: Transcriptomic changes in the shoots of 17 days old Phtheirospermum japonicum that has been infecting 7 days old Arabidopsis thaliana (Col-0) for 10 days in vitro. Samples taken at 10 days after addition of the host. Shoots from infecting plants were compared to shoots from not-infecting plants grown in the same conditions.

first: Anna

last: Kokla

first: Martina

last: Leso

middle: W


title: Determining sex-specific gene expression differences in human chorion trophoblast cells

city: Manila

first: Ryan

last: Lintao

address: 547 Pedro Gil Street

department: College of Medicine

country: Philippines

institute: University of the Philippines Manila

summary: Given that we recently characterized CTCs as a distinct cell type from placental trophoblasts, we investigated in this study whether there is evidence for sexual dimorphism in CTCs. We also determined if differential response exists between male and female CTCs when they are treated with LPS, mimicking ascending infection that is associated with chorioamnionitis.

overall_design: Primary chorion trophoblast cells were isolated and immortalized from term caesarean section fetal membranes of 3 male and 3 female offspring. To see if differential response exists between male and female CTCs during infection, cells are treated with LPS. RNA-sequencing and cytokine measurement was done to check the response.

first: Ryan C

first: Lauren

last: Richardson

first: Daphne

last: Goncharov

first: Ramkumar

last: Menon


title: CTCF is selectively required for maintaining chromatin accessibility and gene expression in human erythropoiesis [H2_HiC]

first: Peng

last: Xu

first: Li

last: Cheng

first: Beisi

first: Ruopeng

last: Feng

first: Yu


title: Functional memory T cells are derived from exhausted clones and expanded by checkpoint blockade [scRNATCR_Atlas]

first: Colin

last: Raposo

summary: Immune checkpoint blockade can facilitate tumor clearance by T cells, resulting in long term patient survival. However, the capacity of exhausted CD8+ T cells (Tex), present during chronic antigen exposure, to form memory after antigen clearance remains unclear. Here, we performed longitudinal single cell RNA/T cell receptor sequencing and ATAC-sequencing on antigen-specific T cells after the peripheral clearance of chronic lymphocytic choriomeningitis virus infection. These data revealed the formation of a robust population of memory CD8+ T cells that transcriptionally, epigenetically, and functionally resemble central memory T cells (Tcm) that form after clearance of acute infection. To lineage trace the origin and memory recall response of Tex-derived memory clones, we utilized T cell receptor sequencing over the course of primary infection and rechallenge. We show that chronic Tcm are a clonally distinct lineage of Tex derived from progenitor exhausted cells, persist long-term in the absence of antigen, and undergo rapid clonal expansion during rechallenge. Finally, we demonstrate that αPD-L1 immune checkpoint blockade selectively expands clones which form Tcm after clearance. Together, these data support the concept that chronically stimulated T cells form bona fide functional memory T cells through an analogous differentiation pathway to acutely stimulated T cells, which may have significant implications for enhancing immune memory to cancer through checkpoint blockade and vaccination.

overall_design: scRNA/TCR-seq of GP33+ CD8+ T cells from at different time points following LCMV infection: post LCMV-Cl13 clearance (100+ days post-infection),  chronic LCMV-Cl13 infection (21–26 days post-infection), and post LCMV-Arm clearance. To enrich for memory cells, GP33+ CD8+ CD62L+/- T cells were sorted and sequecned separately in some samples.

first: Kamir

last: Hiam-Galvez

first: Ansuman

last: Satpathy