title: Reprogram-seq

city: South San Francisco

first: Tomasz

last: Włodarczyk

state: California

address: 1 DNA Way

institute: Genentech

summary: Transcription factors (TFs) and transcriptional coregulators represent an emerging and exciting class of targets. By quantifying target gene modulation, gene regulatory networks (GRNs) delineate disease biology and evaluate pharmacological agents targeting these regulators. However, none of the existing methods are explicitly designed to measure the effects of perturbations in which TF expression is decoupled from its activity. We present Epiregulon, a method that constructs GRNs from single-cell ATAC and RNA data for accurate prediction of TF activity. Our weight estimation, based on co-occurrence of TF expression and chromatin accessibility, avoids erroneous inflation of TF activity as seen with TF expression only approaches. Furthermore, our utilization of ChIP-seq data expands inference to include transcriptional coregulators lacking defined motifs. Our extensive network of regulators facilitates identification of cell-state specific interaction partners. Using Epiregulon, we uncover divergent cell fate transitions of prostate cancer cells driven by NKX2-1 and GATA6 expression. We accurately predicted the effects of AR inhibition across various drug modalities. Finally, Epiregulon was able to infer the context-dependent activity of a chromatin remodeler lacking a defined motif sequence and recapitulate the unique etiologies of prostate cancer. By mapping out the network of key regulators across a multitude of perturbations, Epiregulon can accelerate the discovery of new therapeutics targeting transcription factors.

overall_design: We separately introduced lentivirus encoding NKX2-1 and GATA6 into a prostate adenocarcinoma cell line LNCaP and tracked changes in transcriptomic and epigenomic profiles at the single cell level. We then performed single-cell multiome sequencing (ATACseq + scRNAseq).

first: Xiaosai

last: Yao

first: Russell

last: Xie


title: Histone Methyltransferase G9a in Primary Sensory Neurons Promotes Inflammatory Pain and Transcription of Trpa1 and Trpv1 via Bivalent Histone Modifications

status: Public on Jan 21 2025

city: Houston

first: Hui-Lin

last: Pan

state: TX

address: 1515 Holcombe Blvd

department: Anesthesiology and Perioperative Medicine

country: USA

institute: University of Texas MD Anderson Cancer Center

type: Expression profiling by high throughput sequencing

summary: This RNA-seq experiment was aimed to investigate genome-wide differential gene expression by Ehmt2 (G9a) knockout in dorsal root ganglion (DRG) neurons in mice treated with complete Freund's adjuvant(CFA) or vehicle. Ehmt2 ablation significantly reversed expression of 981 genes in the DRGs and among these genes 322 were downregulated and 659 genes are upregulated (P<0.05).

overall_design: Total RNA was isolated from DRGs in Ehmt2 (G9a) inducible conditional knockout (Avil-iCreERT2/AJwo /G9aflox/flox, G9a-iKO) and littermate control (WT, without Cre) mice. Both WT and G9a iKO mice were treated with vehicle or CFA and 14 days after, RNA from four groups were sequenced on illumina NovaSeq 6000 platform. Each group has two repeats. Differential gene expression was analyzed using Hisat2-FeatureCounts-Deseq2 pipeline.

first: Hui-lin

first: Krishna

last: Ghosh

first: Daozhong

last: Jin

first: Shao-Rui

last: Chen


title: An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX [RNA-seq re-analysis]

city: Manchester

first: Tim

middle: C

last: Somervaille

address: Wilmslow Rd

department: Manchester Institute

country: United Kingdom

institute: Cancer Research UK

summary: In acute myeloid leukaemia (AML), abnormal self-renewal and proliferation of a cell population defined as leukaemia stem cells (LSCs) results in the accumulation of undifferentiated blast cells in the bone marrow and blood. Gene expression analyses comparing LSCs to healthy haematopoietic stem cells (HSCs) revealed a significant upregulation of the transcription factor IRX3 in the LSC compartment, and functional experiments demonstrated that IRX3 contributes to a block in differentiation. Interestingly, IRX3 is minimally expressed in HSCs and not required for normal haematopoietic development. These discoveries highlight the necessity to investigate mechanisms underlying IRX3 misexpression in AML, as well as further investigate the mechanisms through which IRX3 expression contributes to the differentiation block.   To investigate the mechanism of IRX3 misexpression in AML, we applied long-range chromatin interaction analyses, genome editing, enhancer-function studies and diverse bioinformatics tools in both AML cell lines and primary patient samples. We characterise for the first time a cluster of bona-fide enhancers and its associated transcript (eRNA), and describe a model through which these regulatory elements contribute to IRX3 expression. The cluster is composed of four modules with IRX3-regulatory activity in AML cells. Moreover, transcripts arise from the E2 module and aids in the function of the super-enhancer by falicitating a chromatin loop with the IRX3 promoter and maintaining high levels of H3K27ac at both the enhancer modules and IRX3 promoter.

overall_design:
Fujioka cells were a gift from Dr. Vaskar Saha (Children’s Cancer Group, Manchester Cancer Research Centre). Cells were verified by STR analysis and confirmed to be mycoplasma-free.
Please note that GSM5292521-GSM5292528 (in GSE174351) samples have been re-analyzed for the current study.


title: An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX  [MeDIP-seq re-analyzed]

type: Methylation profiling by high throughput sequencing

overall_design:
Fujioka cells were a gift from Dr. Vaskar Saha (Children’s Cancer Group, Manchester Cancer Research Centre). Cells were verified by STR analysis and confirmed to be mycoplasma-free. Primary human AML samples  were obtained from the MCRC Tissue Biobank; their collection was approved by the South Manchester Research Ethics Committee. Biobank samples were screened for the expression level of IRX3.

Please note that GSM5292613-GSM5292618 (in GSE174354) samples have been re-analyzed for the current study.


title: Drug treatment

overall_design: We treated LNCap clone FGC, VCAP, DU145 and H660 cells with DMSO and 3 drugs: Enzalutamide,  ARV-110 and SMARCA2.4_1. We then performed single-cell multiome sequencing (ATACseq + scRNAseq).

first: Diana

last: Wu


title: An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX  [ChIP-seq re-analysis]

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Transcription factors are key determinants in myeloid identity and differentiation fates and when mis- expressed or mutated, they cause a differentiation block, the cardinal feature of acute myeloid leukaemia (AML). The Forkhead family transcription factor FOXC1, is de-repressed in a lineage- inappropriate manner in approximately 30% of cases of HOXAhigh AML with functional consequences and prognostic significance. The mechanisms by which FOXC1 is mis-expressed remain to be determined and they presumably belong to a network of transcription factors and chromatin proteins that represent attractive therapeutic targets in AML. We performed an integrative omics analysis using AML primary samples and discovered an active distal enhancer at the FOXC1 topologically associated domain (TAD) that is more acetylated, accessible and differentially interacts with FOXC1, positively regulating its expression in FOXC1high AML. Moreover, through integrated next generation sequencing and functional analysis, we identified SOX4 as one of the upstream regulators of FOXC1 in leukaemia, where it is broadly expressed and associated with a high level of differentiation block. SOX4 binds at multiple sites across the FOXC1 TAD and directly controls its expression. Genome wide, SOX4 is preferentially bound at sites of accessible and acetylated chromatin at both promoter regions and active enhancers of transcription factors genes that include FOXC1, where it acts as a transcription activator. Our studies reveal a set of regulatory elements that control FOXC1 expression and highlight the role of SOX4 as a transcriptional activator that promotes differentiation block in AML.

overall_design:
Primary human AML cells were obtained from the Manchester Cancer Research Centre Tissue Biobank (approved by the South Manchester Research Ethics Committee). The Biobank archive was searched for samples from patients with different levels of FOXC1 expression.

Please note that GSM5292586-GSM5292594 (in GSE174352) samples have been re-analyzed for the current study.


title: Epigenetics modifications involved in fetal growth restriction due to maternal smoking during pregnancy

city: Zaragoza

first: Ana

last: Gascón-Catalán

address: Domingo Miral s/n

country: Spain

institute: Universidad de Zaragoza

summary: To study the impact of smoking during pregnancy and its role in intrauterine growth restriction via epigenetic changes

overall_design: The sample comprised umbilical cord blood from newborns of smoking and non-smoking mothers.  Difference in  DNA methylation has been compared

first: Eva

last: Barrio

first: Diego

last: Lerma-Puertas

first: Jose

middle: I

last: Labarta


title: An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX

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

overall_design: Refer to individual Series


title: An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX [4C-seq]

overall_design: Fujioka cells were a gift from Dr. Vaskar Saha (Children’s Cancer Group, Manchester Cancer Research Centre). Cells were verified by STR analysis and confirmed to be mycoplasma-free. Primary human AML samples  were obtained from the MCRC Tissue Biobank; their collection was approved by the South Manchester Research Ethics Committee. Biobank samples were screened for the expression level of IRX3.


title: Perivascular adipose tissue plasticity in response to angiotensin II

city: Ann Arbor

first: Haocheng

last: Lu

state: Michigan

address: 2800 plymouth road

department: Internal Medicine

institute: University of Michigan

summary: Most blood vessels are surrounded with perivascular adipose tissue (PVAT), which is a unique adipose tissue that plays critical roles in vascular physiology and pathophysiology. PVAT displays regional differences that impact vascular homeostasis. Angiotensin II (Ang II) is the main biological active effector of the renin-angiotensin-aldosterone system (RAAS), which has been extensively studied in the vascular biology. However, the effects of Ang II on PVAT are limited explored and remain to be elucidated. In this study, we systematically investigated the regional heterogeneity of three portions of aortic PVAT, i.e., ascending thoracic aortic PVAT (ATA-PVAT), descending thoracic aortic PVAT (DTA-PVAT), and abdominal aortic PVAT (AA-PVAT), and their responses to 7-day Ang II infusion using RNA sequencing. We found that AA-PVAT is clearly distinguished from both ATA-PVAT and DTA-PVAT, with significantly down-regulated oxidative phosphorylation and up-regulated inflammatory response pathways. Furthermore, AA-PVAT expressed lower levels of brown adipocyte marker genes, such as Ucp1, Cidea, Cox8b, Dio2 and Pgc1α, but expressed higher levels of proinflammatory genes, such as Ccl2, Il1β and Tnfα, and components of the RAAS, including Agt, Ace and Agtr1α. Ang II infusion significantly down-regulated oxidative phosphorylation in all aortic PVAT and significantly up-regulated inflammatory response specifically in ATA-PVAT and DTA-PVAT. Moreover, ATA-PVAT was most responsive to Ang II induced inflammation. We further used a mitoNEET (a.k.a. CDGSH iron sulfur domain 1 protein) transgenic mouse model that exhibited a more brown adipose tissue (BAT)-like phenotype in aortic PVAT, as indicated by elevated expression levels of brown adipocyte marker genes, and confirmed that a more BAT-like phenotype of aortic PVAT could counterbalance Ang II induced inflammatory and oxidative effects.

overall_design: Wild type (WT) C57BL/6J mice were from the Jackson Laboratory (stock number 000664), the mitoNEET transgenic mice (mitoNEET-Tg) in a C57BL/6J background were constructed to express human mitoNEET driven by the mouse Ucp1 promoter, as previously described [24]. The mice were housed in ventilated cage at 22°C with 12 h/12 h light/dark cycle and were maintained on a standard chow diet and tap water ad libitum. For Ang II infusion experiment, 8- to 12-week-old mice were subcutaneously implanted with osmotic mini-pumps (Alzet, Model #2004) carrying Ang II (1000 ng/kg/min, BACHEM) or saline (as control) for 7 days. Then the mice were sacrificed and the aortic PVAT were dissected.

first: Zhenguo

last: Wang

first: Minerva

last: Garcia-Barrio

first: Yanhong

last: Guo

first: Jifeng

last: Zhang

first: Lin

last: Chang

first: Y

middle: E


title: Krüppel-Like Factor 5 regulates CFTR expression through repression by maintaining chromatin architecture coupled with direct enhancer activation

city: Cleveland

first: Ann

last: Harris

state: Ohio

address: 2109 Adelbert Rd Building

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

overall_design: Analysis of the 3D chromatin architecture with 4C-seq at the CFTR and chr7.11p13 locus to determine interaction frequencies between elements in response to depletion or ablation of KLF5

first: Alekh

last: Paranjapye

first: Monali

last: NandyMazumdar


title: An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX [RNA-Seq]

overall_design: Fujioka cells were a gift from Dr. Vaskar Saha (Children’s Cancer Group, Manchester Cancer Research Centre). Cells were verified by STR analysis and confirmed to be mycoplasma-free.


title: Uncovering Biomarkers for Chronic Toxoplasmosis Detection Highlights Alternative Pathways Shaping Parasite Dormancy

status: Public on Jan 20 2025

city: La Tronche

first: Alexandre

last: Bougdour

state: Isère

address: Domaine de la Merci, Campus santé

department: Prevention and Therapy of Chronic diseases

country: France

institute: Centre de Recherche Inserm U1209 / CNRS UMR5309 / UGA

summary: Toxoplasma gondii, a protozoan parasite, causes toxoplasmosis, a widespread zoonotic and food-borne infection that poses significant risks, particularly in opportunistic and congenital cases. This obligate intracellular parasite is highly prevalent worldwide, largely due to its versatile life cycle, involving multiple hosts and transmission routes, and its ability to establish chronic infections. The presence of this neurotropic parasite in the brain poses a reactivation risk in immunocompromised individuals and might be associated with a higher likelihood of developing mental disorders. However, the role of the dormant bradyzoite stage in the pathophysiology of the disease is underexplored, mainly due to the lack of non-invasive detection methods and serologic tests targeting bradyzoite- or cyst-specific antigens. In this study, we performed an unbiased screening of the bradyzoite proteome and identified the Bradyzoite Serological Marker (BSM) as an additional serological biomarker, alongside the cyst-associated BCLA, to detect chronic stages in vivo. BSM and BCLA show high sensitivity and specificity in identifying cyst-bearing mice. However, in humans, these markers exhibit only moderate concordance, with a 30% positivity rate among individuals with prior immunity, suggesting variability in immune responses and complexities in diagnosing chronic toxoplasmosis. Importantly, bradyzoite serology helps differentiate recent from past infections by the teratogenic parasite Toxoplasma gondii, with BCLA improving the accuracy of pergestational infection diagnosis. Exploring the regulatory mechanisms controlling the expression of these markers revealed that the chromatin modifiers MORC and HDAC3 exert epistatic control over BFD1, a key regulator of bradyzoite development. While BFD1 governs a specific subset of bradyzoite markers, including BCLA, a sub-transcriptome comprising BSM relies on MORC/HDAC3 independently of BFD1. This complex gene regulation highlights the challenge in understanding Toxoplasma persistence, while offering new opportunities for improved serological diagnosis of congenital and chronic toxoplasmosis, particularly in individuals with mental health conditions or a risk of toxoplasmic reactivation.

overall_design: We report transcriptome of Toxoplasma gondii tachyzoites using Illumina RNA sequencing using the following methods: Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA quantity and quality were measured by NanoDrop 2000 (Thermo Scientific). For each condition, RNAs were prepared from three biological replicates. RNA integrity was assessed by standard non-denaturing 1.2% TBE agarose gel electrophoresis. RNA sequencing was performed following standard Illumina protocols, by Novogene (Cambridge, United Kingdom). Briefly, RNA quantity, integrity, and purity were determined using the Agilent 5400 Fragment Analyzer System (Agilent Technologies, Palo Alto, California, USA). The RQN ranged from 7.8 to 10 for all samples, which was considered sufficient. Messenger RNAs (mRNA) were purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. The samples were sequenced on the Illumina NovaSeq platform (2 x 150 bp, strand-specific sequencing) and generated ~40 million paired-end reads for each sample. The quality of the raw sequencing reads was assessed using FastQC (www.bioinformatics. babraham.ac.uk/projects/fastqc/) and MultiQC. For the expression data quantification and normalization, the FASTQ reads were aligned to the ToxoDB-49 build of the T. gondii ME49 genome using Subread version 2.0.1 with the following options ‘subread-align -d 50 -D 600 --sortReadsByCoordinates’. Read  counts  for  each  gene  were  calculated  using featureCounts from the Subread package.

first: Marie

middle: G

last: Robert

first: Christopher

last: Swale

first: Belen

last: Pachano

first: Léa

last: Dépéry

first: Valeria

last: Bellini

first: Céline

last: Dard

first: Dominique

last: Cannella

first: Charlotte

last: Corrao

first: Lucid

last: Belmudes

first: Yohann

last: Couté

first: Hervé

last: Pelloux

first: Emmanuelle

last: Chapey

first: Martine

last: Wallon

first: Marie-Pierre

last: Brenier-Pinchart

first: Mohamed-Ali

last: Hakimi


title: Qualifying a human Liver-Chip for predictive toxicology: Performance assesment and economic implications

city: Boston

first: Lorna

last: Ewart

state: MA

address: 27 Drydock Avenue

institute: Emulate Inc.

summary: We assess RNA expression in human primary hepatocytes in Liver-Chips on Day 3 and Day 5. The hepatocytes were co-cultured in Liver-Chip model with Kupffer cells, Liver sinusoidal endothelial cells (LSECs) and stellate cells .

overall_design: Global RNA expression profiles of Liver-Chips Day3 (n=4); Global RNA expression profiles of Liver-Chips Day5 (n=4); Generated by deep sequencing using Illumina HiSeq.

first: Dimitris

middle: V

last: Manatakis


title: Nuclear speckle specific hnRNP DL maintains speckle structure and regulates RNA splicing during brain aging

city: Hefei

first: Qingyang

last: Zhang

state: Anhui

address: No.443, HuangShan Road Shushan District

department: Division of Life Sciences and Medicine

country: China

institute: University of Science and Technology of China for School of Life Sciences

summary: We report the high-throughput sequencing for differential alternative splicing analyzation in hnRNP DL knockout and control N2a cells

overall_design: Examination of RNA alternative splicing patterns  in hnRNP DL knockout and control N2a cells

first: Qiang

last: Liu

first: Juan


title: H3K4 methylation-promoted transcriptional memory supports faithful zygotic genome activation and development [RNA-seq-Morpholino]

city: Munich

first: Eva

last: Hoermanseder

state: Bavaria

address: Feodor-Lynen-Str. 21

department: Institute of Epigenetics and Stem Cells

country: Germany

institute: Helmholtz Center Munich

summary: Accurately established transcriptional programs are paramount for successful embryonic development. At zygotic genome activation, gene expression is initiated for the first time in the life of an embryo. Pioneer transcription factors present in the embryo are essential for this process, however, the role of active chromatin modifications is less clear. It is unknown if active chromatin modifications established in the gamete are propagated in the embryo as epigenetic memory to support zygotic genome activation and development. Here, we provide evidence that in Xenopus laevis, H3K4 methylation contributes to epigenetic memory of active chromatin states, which is required for faithful zygotic genome activation and successful embryonic development. We find that chromatin configurations of promoters displaying high H3K4me3 intensity and breadth, alongside DNA hypomethylation and increased GC content, are maintained from the gametes to the embryo across multiple cell divisions and a transcriptionally quiescent phase in early development. We show that this maintenance of H3K4 methylation is essential for precise zygotic genome activation. Finally, we demonstrate that Kmt2b and Cxxc1 facilitate transcription-independent maintenance of H3K4me3 and proper zygotic gene expression. In summary, our study establishes a role for H3K4 methylation for memory of active chromatin states in embryos and reveals its importance for successful embryonic development.

overall_design: RNA-seq profiling of H3K4-demethylated Xenopus laevis embryos at ZGA stages after injection of Cxxc1, Kmt2b or control asMOs (anti-sense morpholinos) into 1-cell embryos.

first: Meghana

middle: S

last: Oak

first: Marco

last: Stock

first: Matthias

last: Mezes

first: Tobias

last: Straub

first: Antony

middle: M

last: Hynes-Allen

first: Jelle

last: van den Ameele

first: Ignasi

last: Forne

first: Andreas

last: Ettinger

first: Axel

last: Imhof

first: Antonio

last: Scialdone

last: Hörmanseder


title: H3K4 methylation-promoted transcriptional memory supports faithful zygotic genome activation and development [RNA-seq-Auxin]

overall_design: RNA-seq profiling of H4K4me3-depleted Xenopus laevis embryos at ZGA stages using Kdm5b-wt and H3.3-K4M mRNA mutant overexpression. Kdm5b-ci (catalytically inactive) plus H3.3-wt were overexpressed as the respective control conditions. Treatments were performed either in a developmental "window" from zygote to ZGA, or "persistently" from zygote onwards.


title: Resolution of a human super-enhancer by targeted genome randomisation [RNA-seq]

city: Hinxton

first: Pierre

last: Murat

address: Wellcome Trust Genome Campus

institute: Wellcome Sanger Institute

summary: Human gene expression is controlled from distance via enhancers, which can form longer ‘super-enhancer’-regions of intense regulatory activity. Whether super-enhancers constitute a separate regulatory paradigm remains unclear, largely due to the difficulty of dissecting the contributions and interactions of individual elements within their natural chromosomal context. To address this challenge, we developed enhancer scrambling, a high-throughput strategy to generate stochastic inversions and deletions of targeted enhancer regions by combining CRISPR prime editing insertion of symmetrical loxP sites with Cre recombinase-induced rearrangements. We applied our approach to dissect a distal super-enhancer of the OTX2 gene, generating up to 134 alternative regulatory configurations in a single experiment, and establishing how they drive gene expression and chromatin accessibility, as well as the individual contributions of its elements to this activity. Surprisingly, the presence of the sequence containing a single DNase I hypersensitive site predominantly controls OTX2 expression. Our findings highlight that enhancer-driven regulation of some highly expressed, cell-type-specific genes can rely on an individual element within a cluster of non-interacting, dispensable components, and suggest a simple functional core to a subset of super-enhancers. The targeted randomisation method to scramble enhancers can scale to resolve many super-enhancers and human gene regulatory landscapes.

overall_design: RNA-seq profiling of WT and engineered HAP1 clones. Engineered HAP1 clones were generated by inducing the deletion of R4 and R5 super-enhancer domains of the OTX2 gene.

first: Jonas

last: Koeppel

first: Gareth

last: Girling

first: Elin

last: Madli Peets

first: Mélanie

last: Gouley

first: Valentin

last: Rebernig

first: Anant

last: Maheshwari

first: Jacob

last: Hepkema

first: Juliane

last: Weller

first: Jenie

middle: H

last: Johnkingsly Jebaraj

first: Ronnie

last: Crawford

first: Fabio

last: Liberante

first: Leopold

last: Parts


title: Resolution of a human super-enhancer by targeted genome randomisation [Amplicon-seq]

overall_design: Amplicon-seq profiling of engineered HAP1 clones. Engineered HAP1 clones were generated by inducing deletions at TFBS within the R5 super-enhancer domains of the OTX2 gene.


title: ER-Localized Deadenylase PNLDC1 Suppresses Colorectal Cancer Progression by Targeting the mRNA decay of TUBB4B

city: Hangzhou

first: Lidan

last: Hu

address: No.268 Kaixuan Road

department: School of Medicine

institute: Zhejiang University

summary: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, highlighting the urgent need for novel therapeutic strategies. In this study, we find that PNLDC1, an endoplasmic reticulum (ER)-localized deadenylase, lower expresses in 91 clinical CRC tissues. Using both in vitro and in vivo models, we identify PNLDC1 as a key tumor suppressor by modulating cell cycle progression in CRC. RNA-immunoprecipitation and sequencing analyses reveal that PNLDC1 binds cell cycle-associated mRNAs, with TUBB4B emerging as a critical target. PNLDC1 downregulates TUBB4B mRNA, whose regulated degradation is essential for PNLDC1’s tumor-suppressive function. Restoration of TUBB4B expression counteracts PNLDC1-mediated suppression of CRC cell proliferation, underscoring its pivotal role in CRC pathogenesis. Importantly, we demonstrate that the TUBB4B inhibitor, mebendazole, effectively suppresses the enhanced proliferation in lower-expression PNLDC1 models, including cell, mouse xenografts, and patient-derived tumor-like cell clusters, positioning it as a promising therapeutic agent. These findings establish PNLDC1 as a valuable biomarker and therapeutic target, paving the way for developing novel CRC treatments.

overall_design: RIP assays were performed following established protocols54. 10 million cells were collected and lysed in RIP buffer (20 mM Tris-HCl pH=7.4, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% NP-40, 100U RNase inhibitor, 1×cocktail), and lysates were incubated with 6 μg anti-PNLDC1 antibody-coated beads (Proteintech, 25559-1-AP) overnight at 4°C. Immunocomplexes were washed with washing buffer for 3 times (20 mM Tris-HCl pH=7.4, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA), treated with proteinase K, and RNA was extracted using phenol/chloroform/isoamyl alcohol (125:24:1). Clean reads were aligned to the hg38 genome using bowtie2 (v2.5.1). Both input and RIP samples were prepared for next-generation sequencing.

first: Lexin

last: Liu

first: Yuelin

last: Guan

first: Yuzhou

last: Qin

first: Zhichao

last: Ma

first: Xiang

last: Yan

first: Guiping

last: Chen

first: Xue

last: Li

first: Lei

last: Sun

first: Wei

last: Yu

first: Zihao

last: Xu

first: Zhongkai

last: Cao

first: Fan

first: Tong

last: Zhou

first: Ziyou

last: Luo

first: Xiangjun

first: Yongbin


title: Non-invasive diagnosis of non-melanoma skin cancer subtypes predicted by microRNAs recovered from adhesive  tape discs

city: Salt Lake City

first: Doug

last: Grossman

state: Utah

address: Huntsman Cancer Institute, 2000 Circle of Hope

department: Dermatology

institute: Huntsman Cancer Institute

type: Non-coding RNA profiling by high throughput sequencing

summary: We investigated whether a tape-stripping approach could be used for analysis of microRNA (miR) expression as a non-invasive method for prediction of non-melanoma skin cancer subtypes. From direct miR sequencing of RNA recovered from adhesive tape discs applied to skin lesions prior to biopsy, we determined rank lists of miRs to predict superficial or invasive BCC or SCC. Expression of these miRs was then assessed by digital PCR of disc-derived miR from independent validation cohorts of BCC (n=24 Sup, n=38 Inv) and SCC (n=45 SCC-in-situ, n=14 Inv). Expression ratios or products of >2 miRs were superior to individual miRs in discriminating Sup from Inv BCC and SCC subtypes. Moreover, several miR combinations distinguished Sup BCC and Inv SCC from a cohort of benign keratoses that can clinically resemble these cancers.

overall_design:
Cohort identification and RNA preparation for sequencing
Prior to biopsy of lesions, an adhesive disc (D-Squame D101, Clinical and Derm) was firmly applied to the lesion multiple times with rotation to ensure the entire adhesive surface had made physical contact. Discs were immediately stored at -20 oC and then at -80 oC until processing. Whole RNA was isolated from the discs using a miRNeasy Micro Kit (217084, Qiagen) and the RNA eluate was stored at -80 oC until sequencing.
Analysis of miR sequencing
The human GRCh38 genome and gene annotation files were downloaded from Ensembl release 106 and a reference database was created using STAR version 2.7.9a (Dobin et al., 2013). A custom Perl script (smallRNA_pe_umi_extractor.pl from https://github.com/HuntsmanCancerInstitute/UMIScripts) was used to trim adapters and extract the Unique Molecular Index (UMI) from the second read of a paired-end Qiagen small RNA library with 76x51 base pair reads. The trimmed reads were aligned to the reference database using STAR options optimized for shorter miRNA reads (>=16 bases matched to the genome, <= 5% mismatches over mapped length, splicing switched off). A second Perl script (bam_umi_dedup.pl) removed PCR duplicates from the aligned BAM file and the mapped reads were assigned to annotated genes in Ensembl and to miRNAs in miRbase release 22.1 (Kozomara et al., 2019) using featureCounts version 1.6.3 (Liao et al., 2014). Differentially expressed miRNA in miRbase were identified using a 5% false discovery rate with DESeq2 version 1.36.0 (Love et al., 2014). Principal components analysis was run using the regularized log values from the top 500 variable miRNAs and ellipses were added representing the 90% confidence interval from a multivariate t-distribution.
Statistical Method for Feature Selection
Features (i.e. miRs) were identified as described previously (Fastner et al., 2024). Briefly, binomial lasso regression, as implemented in the R (version 4.3.2) package “glmnet” was used to select a group of features that separated subtypes (e.g. superficial from invasive BCC). Ten-fold cross validation was used to select the penalty parameter for the lasso regression. Low expression miRs with an adjusted mean count of less than 10 were removed from consideration.
qRT-PCR assays
RNA isolated from discs was converted to cDNA using a TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, A28007) and stored at -80 oC until used. Samples were read using QuantStudio™ Absolute Q™ Digital PCR System (Thermo Fisher Scientific) and analyzed using QuantStudio™ Absolute Q™ Digital PCR Software (version 6.3, Applied Biosystems) according to the manufacturer’s recommendations. Samples were diluted 1:100 in 0.1X TE buffer (Ambion, AM9849) and digital PCR was performed using a MAP16 Digital PCR kit (Ambion, A52688) according to the manufacturer’s instructions. Briefly, each reaction consisted of 2 L Absolute Q DNA Digital PCR 5X mix, 0.5 L 20X TaqMan Advanced miRNA probe (Applied Biosystems, A25576) and 7.5 L cDNA, with 15ul of Isolation Buffer added over each sample prior to sealing the wells with the provided gasket strips. Data was analyzed using Quant Studio Absolute Q digital PCR software, version 6. Threshold bars were set using the manual option and results reported as number of copies per L. Normalized expression values were obtained copy number for an individual miR by that for the invariant miR-16-5p. Combined expression scores were obtained from the products and/or quotients (based on whether miRs were “up” or “down,” respectively) of normalized expression scores of multiple miRs. Pearson correlations of normalized digital PCR values with normalized regularized log sequencing values was determined using R version 4.3.2. ROC curves and other analyses were generated using Prism software.

first: Hafeez

last: Rahman

first: David

middle: W

last: Mwelwa

first: Scott

middle: R

last: Florell

first: Abigail

first: Carly

middle: A

last: Becker

first: Chris

middle: J

last: Stubben

first: Kenneth

last: Boucher

first: Dekker

last: Deacon

first: Robert

middle: L

last: Judson-Torres

first: Douglas


title: Resolution of a human super-enhancer by targeted genome randomisation [ONT]

first: Pierre

last: Murat

overall_design: Oxford nanopore long-read sequencing from scrambled HAP1 cell lines and heterogeneous cell populations.

first: Jonas

last: Koeppel

first: Gareth

last: Girling

first: Elin

last: Madli Peets

first: Mélanie

last: Gouley

first: Valentin

last: Rebernig

first: Anant

last: Maheshwari

first: Jacob

last: Hepkema

first: Juliane

last: Weller

first: Jenie

last: Johnkingsly Jebaraj

first: Ronnie

last: Crawford

first: Fabio

last: Liberante

first: Leopold

last: Parts


title: H3K4 methylation-promoted transcriptional memory supports faithful zygotic genome activation and development [DamID-seq]

first: Eva

last: Hoermanseder

overall_design: Chromatin Accessibility profiling using Targeted DamID (CATaDa) sequencing performed on pre-ZGA (4.5hpf/256-cell) stage Xenopus laevis embryos. Dam mRNA construct was injected into 1-cell stage embryos and embryos were collected at the stage of interest.

first: Meghana

last: Oak

first: Marco

last: Stock

first: Matthias

last: Mezes

first: Tobias

last: Straub

first: Antony

last: Hynes-Allen

first: Jelle

last: van den Ameele

first: Ignasi

last: Forne

first: Andreas

last: Ettinger

first: Axel

last: Imhof

first: Antonio

last: Scialdone

last: Hörmanseder


title: Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle

first: Mary

middle: T

last: Couvillion

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Mitochondria play a critical role in cellular metabolism primarily through hosting the oxidative phosphorylation (OXPHOS) machinery that is encoded by mitochondrial DNA (mtDNA) and nuclear DNA, with each gene expression system being regulated by a distinct set of mechanisms. In this study, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to gain insights into the principles underlying the regulation of mtDNA-encoded protein abundance. Our analysis revealed a unique balance between the rapid turnover and high accumulation of mt-mRNA, leading to a 700-fold higher transcriptional output compared to nuclear-encoded OXPHOS genes. Additionally, we observed that mt-mRNA processing and its association to the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. These data resulted in a model of mtDNA expression that is predictive across cell lines, revealing that differential turnover and translation efficiencies are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome,  French–Canadian type, we found that the responsible nuclear-encoded gene, LRPPRC, disrupts OXPHOS biogenesis predominantly through altering mt-mRNA stability.  Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle for decoding the regulatory principles of mtDNA expression.

overall_design: TimeLapse-seq and TimeLapse-seq-mitoribosome IP data for HeLa S3, HEK293T, and LRPPRC KO (HEK293T) cells using both 50 uM and 500 uM 4sU. Also includes HeLa RNA-seq, mitochondrial polysome RNA-seq, and K562 cytosolic and mitochondrial ribosome profiling

first: Erik

last: McShane

first: Robert

last: Ietswaart

first: Gyan

last: Prakash

first: Brendan

last: Smalec

first: Iliana

last: Soto

first: Autum

last: Baxter-Koenigs

first: Karine

last: Choquet

first: Stirling

last: Churchman


title: H3K4 methylation-promoted transcriptional memory supports faithful zygotic genome activation and development [MBD-seq]

first: Eva

last: Hoermanseder

overall_design: Profiling of DNA methylation in pre-ZGA (4.5hpf/256-cell) stage embryos using MBD-sequencing, performed by affinity purification of methylated genomic DNA to the MethylCap protein (H6-GST-MBD).

first: Meghana

last: Oak

first: Marco

last: Stock

first: Matthias

last: Mezes

last: Straub


title: Transcription inhibition in early mouse embryos impacts transcriptome at 4-cell stage

city: Wuhan

first: Li-quan

last: Zhou

state: Hubei

address: 13 Hangkong road

department: Tongji Medical College

institute: Huazhong University of Science and Technology

summary: Mouse zygotes were treated with DRB to inhibit transcription activity, and early embryos were collected at 4-cell stage to examine transcriptome alteration by RNA-seq.

overall_design: We treated mouse zygotes 6 h post-fertilization using KSOM medium supplemented with 20 μM of DRB (5,6 - Dichloro - 1 - β - D - ribofuranosylbenzimidazole), a transcription inhibitor. Following a 20-hour incubation period with DRB, the embryos were meticulously washed to ensure complete removal of the inhibitor and then transferred to fresh KSOM medium. The culturing process was extended for an additional 26 hours, and subsequently, 4-cell embryos from both groups were collected and subjected to transcriptome sequencing using RNA-seq to examine the alterations in the transcriptome.

first: Ying

last: Yin

first: Liquan


title: Modeling human gastrulation and secondary yolk sac formation with BMP4 signaling center

city: Hang Zhou

first: Tao

last: cheng

state: Zhe Jiang

address: 886 Yuhangtang Road

department: Institute of genetics

summary: Purpose: Characterization of cell types in BMP4 induced human gastruloids. Methods: BMP4 induced human gastruloids were harvested on days 2, 4, 6, 8, 10, and 15, and then dissociated into single cells. Libraries were constructed with  a Chromium Single Cell 3′v3 Reagent Kit (10x Genomics) according to the manufacturer’s protocol for 10000 cell recovery.Sequencing was performed with Illumina Novaseq 6000 according to the manufacturer’s instructions (Illumina). Results: Definiitve endoderm, yolk sac endoderm,  hematopoiesis cell fates were detected in BMP4 induced human gastruloids. Conclusions: BMP4 induced the formation of human gastruloids with a yolk sac-like cavity structure.

overall_design: In vitro BMP4 induced human embryoids  from hESCs at different developmental stages were harvested  for scRNA-seq.

last: Huang

last: Cheng

first: Jiewen

last: Sun

first: Chenyi

last: Chen


title: Study on the toxicity and action mechanism of diquat on Caenorhabditis elegans

city: Haikou, China

first: Bing

last: Wang

address: No. 58 Renmin Avenue, Haikou City, Hainan Province

institute: Hainan university

summary: Diquat (DQ), a contact herbicide extensively utilized in both agricultural and non-agricultural domains, exhibits a high correlation with neuronal disorders. Nevertheless, the toxicity and underlying mechanisms associated with exposure to environmental concentrations of DQ remain ambiguous. Here, we report a dose-dependent cellular neurotoxicity of DQ in C. elegans. Firstly, DQ significantly compromised the development and brood size of worms, shortened the lifespan, and caused epidermal abnormalities. An unbiased transcriptomic analysis disclosed several pathways related to cell death and peroxisome homeostasis underlying this organismal-level toxicity. Moreover, the exposure of DQ to C. elegans led to a notable increase of embryonic cell death. Concurrently, DQ exposure specifically caused the loss of dopamine neurons but not two types of neurons in adulthood, which is in accordance with DQ-induced muscle-related defects such as pharyngeal pumping, body bends, and head thrashes.

overall_design: Wild-type N2 Caenorhabditis elegans were exposed to 0 and 100.00 μg/L DQ dibromide monohydrate solution (Tanmo Quality Inspection Technology Co., Jiangsu, China) at a constant temperature of 20°C for 72 hours. Samples were collected for transcriptional level analysis, with the untreated 0 μg/L DQ-exposed C. elegans used as the control. In the document, control-1, control-2, and control-3 represent the three replicates for the 0 μg/L DQ treatment, while treatment-1, treatment-2, and treatment-3 represent the three replicates for the 100.00 μg/L DQ treatment.


title: Ezh2 Delays Activation of Differentiation Genes During Normal Cerebellar Granule Neuron Development and in Medulloblastoma [RNA-seq]

city: Kingston

first: James

last: Purzner

state: Ontario

address: 76 Stuart Street

department: Surgery

country: Canada

institute: Queen's University

summary: This study explores the role of Ezh2 in delaying the activation of differentiation genes during the development of cerebellar granule neurons (GNPs) and in SHH subtype medulloblastoma. To investigate the epigenetic landscape, we performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) on GNPs and medulloblastoma cells derived from Ptch1 heterozygous mice. We profiled histone modifications and key regulatory proteins, including H3K27me3, H3K27ac, H3K36me3, H3K4me3, H3K4me1, H2Aubi119, Ring1b, and Pol2S5. Additionally, we conducted ATAC-seq to assess chromatin accessibility and MIRA-seq to study DNA methylation.  This dataset aims to compare the epigenetic state of normal granule neuron progenitors with that of medulloblastoma cells, providing insight into how epigenetic mechanisms contribute to neuronal differentiation. We found that the inhibition of Ezh2-mediated H3K27 methylation represses differentiation in both GNPs and medulloblastoma cells.

overall_design:
To identify genes that were increasing during GNP differentiation we performed RNAseq at E15, P1, P7 and P14.  We also performed RNAseq on medulloblastomas derived from Ptch heterozygous mice.
To characterize differentiation genes we performed ChIP-seq for H3K27me3, H3K27ac, H3K36me3, H3K4me3, H3K4me1, H2Aubi119, Ring1b, and Pol2S5 in purifiend GNPs.  We also performed MIRA-seq and ATACseq on the same cells.
To compare the epigenetic landscape of P7 granule neuron progenitors (GNPs) with medulloblastomas derived from Ptch heterozygous mice, we performed ChIP-seq for H3K27me3.

first: Alexander

last: Brown

first: Teresa

first: Lauren

last: Ellis

first: Sara

last: Broski

first: Ulrike

last: Litzenburger

first: Kaytlin

last: Andrews

first: Aryaman

last: Sharma

first: Xin

first: Michael

middle: D

last: Taylor

first: Yoon-Jae

last: Cho

first: Margaret

last: Fuller

first: Matthew

middle: P

last: Scott


title: Transcriptional profiling of liver obtained from a mouse model for metabolic dysfunction-associated steatotic liver disease

city: Xi'an

first: Wenhui

last: Yan

state: Shaanxi

address: No. 76 Yanta West Road

institute: Xi'an Jiaotong  University

summary: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent hepatic disorder worldwide and closely associated with type 2 diabetes mellitus (T2DM). The pathogenesis of MASLD is multifaceted and its treatment options  are very limited. Consequently, it is urgent to identify novel therapeutic targets and develop effective treatment strategies. RNA sequencing analysis was performed to understand the transcriptomic changes in a mouse model of MASLD.

overall_design: Male C57BL/6 weighing 16-18 g were fed a chow diet or high-fat diet (HFD, consists of 60% fat, 20% carbohydrate, and 20% protein) for 8 weeks. Food and drinking water were provided ad libitum. After that, the HFD-fed mice were subjected to an intraperitoneal injection with 60 mg/kg body weight streptozotocin for 3 consecutive days while the chow diet-fed mice were injected with equivalent volume of citrate buffer. Fasting blood glucose (FBG) levels were measured one week post-STZ injection, and T2DM was assured when FBG levels were in the range of 15-25 mmol/L. Six weeks later, the control and model mice were sacrificed to isolate the liver tissues. Triplicate samples from livers of control and T2DM mice were harvested for RNA sequencing.

first: Lina


title: Ezh2 Delays Activation of Differentiation Genes During Normal Cerebellar Granule Neuron Development and in Medulloblastoma [ChIP-seq]

overall_design:
To characterize differentiation genes we performed ChIP-seq for H3K27me3, H3K27ac, H3K36me3, H3K4me3, H3K4me1, H2Aubi119, Ring1b, and Pol2S5 in purifiend GNPs.  We also performed MIRA-seq and ATACseq on the same cells.
To compare the epigenetic landscape of P7 granule neuron progenitors (GNPs) with medulloblastomas derived from Ptch heterozygous mice, we performed ChIP-seq for H3K27me3.


title: Endogenous degron alleles enable tunable control of EWSR1-FLI1 oncoprotein expression

city: Dallas

first: Laura

last: Banaszynski

address: 5323 Harry Hines Blvd

department: Green Center for Reproductive Biology Sciences

institute: UT Southwestern Medical Center

summary: Pediatric malignancies frequently harbor chromosomal translocations that induce expression of fusion oncoproteins. The EWSR1-FLI1 fusion oncoprotein acts as a neomorphic transcription factor and is the dominant genetic driver of Ewing’s sarcoma. However, interrogation of the mechanisms by which EWSR1-FLI1 drives tumorigenesis has been limited by a lack of model systems to precisely and selectively control its expression in patient-derived cell lines and xenografts. Here, we report the generation of a panel of patient-derived EWS cell lines in which inducible protein degrons have been engineered into the endogenous EWSR1-FLI1 locus. These systems enable precise control of EWSR1-FLI1 expression and the timing of oncoprotein depletion. We establish that complete suppression of EWSR1-FLI1 leads to a reversible cell cycle arrest at the G1-S phase transition, and we identify a core set of transcripts downstream of EWSR1-FLI1 across multiple cell lines and degron systems. Additionally, depletion of EWSR1-FLI1 using this system potently suppresses tumor growth in xenograft models validating efforts to directly target EWSR1-FLI1 in Ewing’s sarcoma.

overall_design: CUT&RUN analysis of FLI in a human Ewing Sarcoma cell line (A673). EWS-FLI was either untagged or endogenously tagged with an AID degron. Degron lines were either treated with DMSO or IAA before FLI CUT&RUN.

first: Ryan

last: O'Hara

first: Purbita

last: Saha

first: David

last: McFadden


title: A Multi-Kinase Inhibitor Screen Identifies Inhibitors Preserving Stem-Cell-Like Chimeric Antigen Receptor T Cells

city: Chapel Hill

first: Mark

last: Woodcock

state: NC

address: 170 Manning Drive

institute: University of North Carolina at Chapel Hill

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

overall_design: Refer to individual Series


title: A Multi-Kinase Inhibitor Screen Identifies Inhibitors Preserving Stem-Cell-Like Chimeric Antigen Receptor T Cells [RNA-Seq]

summary: Chimeric antigen receptor T cells (CAR-Ts) with T-stem cell (TSCM)-like phenotypic characteristics promote sustained antitumor effects. We performed an unbiased and automated high-throughput screen of a kinase-focused compound set to identify kinase inhibitors (KIs) that preserve human TSCM-like CAR-Ts. We identified three KIs UNC10225387B, UNC10225263A and UNC10112761A that combined in vitro increased the frequency of CD45RA+CCR7+TCF1high TSCM-like CAR-Ts from both healthy donors and cancer patients. KIs-treated CAR-Ts showed enhanced antitumor effects both in vitro and in vivo. The KI cocktail maintains TSCM-like phenotype preferentially in CAR-Ts originating from naïve T cells, and promotes dynamic transcriptomic changes without arresting T-cell activation or modulating the chromatin organization. Specific kinases ITK, ADCK3, MAP3K4 and CDK13 targeted by the KI cocktail in a dose-dependent manner are directly associated with the preservation of TSCM-like CAR-Ts. While individual knockdown of these kinases or their combination enriched in TSCM-like CAR-Ts, only CAR-Ts generated in the presence of the KI cocktail show robust expansion and differentiation upon stimulation with tumor cells. Overall, transient pharmacologic inhibition of strategically targeted kinases maintains stem-like features of CAR-Ts and improves their antitumor activity.

overall_design: Human T-cells were isolated from collected PBMCs from two healthy donors, maintained with anti-CD3 and anti-CD28 antibodies and transduced with a CD19-directed CAR via retroviral vector prior to expansion using IL-7 and IL-15.  These cells were exposed to either DMSO or a collection of kinase inhibitors (KIs).  Samples were collected prior to exposure (baseline), and at 1, 3 and 7 days post-exposure before being sequenced via RNA and/or ATAC-seq.

first: Feifei

last: Song

first: Ourania

last: Tsahouridis

first: Simone

last: Stucchi

first: Tara

last: Walhart

first: Sophie

last: Mendell

first: Brian

last: Hardy

last: Axtman

first: Shiva

middle: K

last: Guduru

first: Thomas

last: Gilbert

first: Lee

last: Graves

last: Herring

first: Barbara

last: Savoldo

first: Xingcong

last: Ma

first: Justin

last: Milner

first: Anastasia

last: Ivanova

first: Kenneth

last: Pearce

first: Yang

last: Xu

first: Gianpietro

last: Dotti


title: A traditional Chinese medicine decoction developed based on the theory of “Yang transforming into Qi with Yin constituting Form” suppresses hepatic tumorigenesis in mouse models

city: DaLian

first: aiguo

last: Wang

address: DaLian

institute: Dalian Medical University

summary:
Background
Also the traditional Chinese medicine (TCM) theory of "Yang transforming into Qi with Yin constituting Form" was recognized as a potential mechanism underlying the tumorigenesis. The decoction based on this theory and the underlying molecular mechanisms remained exploration.
Methods
A TCM decoction was prepared based on "Yang transforming into Qi with Yin constituting Form". Its anti-tumorigenesis effect was evaluated on mouse models of hepatocellular carcinoma (HCC) (H-rasG12V transgenic mice (Ras-Tg) and Diethylnitrosamine (DEN)-induced HCC mice) by liver/body weight, number and size of tumors, histopathological changes and serum biochemistry. RNA sequencing was utilized to check differentially expressed genes (DEGs) and gene ontology (GO) and KEGG were used for bioinformatics analysis. The concerned biological processes were further confirmed by RT-qPCR and metabolite detection kits.
Results
The TCM decoction significantly suppressed both Ras oncogene- and DEN-induced hepatic tumorigenesis evidenced by reduced serum ALT and AST levels, liver weight, liver/body weight ratio, hepatic tumor numbers and sizes. RNA sequencing showed 659 up-regulated genes and 222 down-regulated genes in comparison of liver tissues between TCM-treated and untreated Rag-Tg (n=5). GO entries and KEGG analysis revealed multiple biological processes and pathways involving the hepatic tumorigenesis suppressing effective of the TCM. Further molecular examination confirmed that activation of retinal metabolism, reduction of ROS and electrophilic levels by inhibiting the H2O2 production and promoting GSH production, and inhibition of expression of inflammation factors play pivotal roles in the anti-tumorigenesis effects of TCM.
Conclusion
A TCM decoction based on "Yang transforming into Qi with Yin constituting Form" showed effective anti-hepatic tumorigenesis functions and its regulation functions on retinal, ROS and inflammation processes play important roles. The present study provides valuable clue for clinical therapy on tumorigenesis.

overall_design: Firstly, develop and prepare traditional Chinese medicine decoctions, and confirm the anti liver tumor occurrence or development effect of TCM by feeding Ras oncogene and DEN induced HCC mouse models. Perform bioinformatics analysis to investigate potential mechanisms by sequencing RNA from peri-tumor and tumor tissues.

first: Lili

last: Dong

first: Lingxin

last: Kong

first: Shuangshuang

last: Gao

first: Yaxin

first: Jun

last: Chen

first: Aiguo


title: A Multi-Kinase Inhibitor Screen Identifies Inhibitors Preserving Stem-Cell-Like Chimeric Antigen Receptor T Cells [ATAC-Seq]

first: Mark

last: Woodcock

first: Feifei

last: Song

first: Ourania

last: Tsahouridis

first: Simone

last: Stucchi

first: Tara

last: Walhart

first: Sophie

last: Mendell

first: Brian

last: Hardy

first: Matthew

last: Axtman

first: Shiva

first: Laura


title: The profiling of methylation and acetylation modification of histone H3 in Intrahepatic cholangiocarcinoma cells

first: Fei

last: Xiong

address: Jiefang Road No.1095

summary: Abnormal distribution of histone modifications has been confirmed to influence the development and progression of neoplastic diseases. Methylation modifications of histone H3 include H3K9me3, H3K27me3 and H3K4me3. Acetylation modifications of histone H3 include H3K9ac and H3K27ac. Among these modifications in the promoter region, H3K9me3 and H3K27me3 serve as inhibiting factors for transcription, while H3K4me3, H3K9ac and H3K27ac are important promoting factors for transcription. To make sure the distribution of these histone modifications in intrahepatic cholangiocarcinoma (ICCA) cells, CUT & Tag was performed. We found that these modifications were located in the promoter or non-promoter regions (e.g., intergenic, exon and intron regions) of key tumor-associated genes, indicating the potential role of chromatin remodeling complexes in ICCA diagnosis and treatment.

overall_design: This dataset includes six CUT & Tag samples. Immunoprecipitation was performed with antibodies for different histone modifications (H3K9me3, H3K9ac, H3K27me3, H3K27ac and H3K4me3). The immunoprecipitation of control group was performed by anti-IgG antibodies.

first: Bing

first: Yong-jun


title: Transcriptome sequencing of phaseolus vulgaris pods and grains

first: Yunxiu

last: Liu

address: No. 258 Xueyuan Street, Xiasha Higher Education Park

institute: China Jiliang University

summary: A total of 18 samples were selected from 4 periods during the growth of phaseolus vulgaris pods and grains, and total RNA was extracted respectively for transcriptome sequencing and functional detection of the corresponding genes, which laid a certain data foundation for subsequent studies.

overall_design:
Firstly, 18 samples were selected from four periods of phaseolus vulgaris pod and grain respectively.
RNA was extracted from different samples.
RNA quality detection, sequencing and data processing were performed.


title: The distribution of HP1α in the genome of intrahepatic cholangiocarcinoma cells

summary: Heterochromatin Protein 1α (HP1α, encoded by the CBX5 gene) is one of the most important HP1 family members and is a nonhistone chromosomal proteins involved in transcriptional silencing via heterochromatin formation and structural maintenance. Many studies have shown that the distribution of HP1α on polytene chromosomes is not restricted to the chromocenters or telomeres. HP1α binds to chromatin mainly through direct interactions with modified histones, especially trimethylated H3K9 (H3K9me3), through the Chromo domain, and by interactions with other proteins through the Chromo shadow domain. In cancerous lesions, HP1α is involved in the regulation of malignant behaviors, such as cell proliferation and cell cycle progression. To study the distribution of HP1α in the genome of intrahepatic cholangiocarcinoma cells, CUT & Tag was performed. We found that HP1α was significantly distributed in the promoter region of proliferation-related encoding genes. No peaks were detected in the normal control sample.

overall_design: Immunoprecipitation was performed with anti-HA antibodies. This dataset includes two Cut & Tag samples. One is HUCCT1 cell sample that treated with HP1α overexpression vector and the other is HUCCT1 cell sample that treated with normal control vector.


title: Transcriptome analysis for the effect of Trichostatin A on intrahepatic cholangiocarcinoma cells

summary: Trichostatin A (TSA) is a commonly used antifungal drug characterized by inhibiting the activity of histone deacetylases (HDACs). As an antineoplastic agent, the pharmacological actions of TSA includes: 1. Inhibiting the proliferation of tumor cells directly; 2. Promoting the antitumor effect of immune cells; 3. Enhancing the efficacy of other antitumor drugs. The efficacy of TSA in the treatment of intrahepatic cholangiocarcinoma (ICCA) is still unknown and needs further verification. Therefore, in this study, ICCA cell line HUCCT1 was treated by TSA. The total RNA samples were used for transcriptome analysis to find the potential target of HDACs.

overall_design: This dataset included two groups of samples. Every group contained three repetitions (rep). TSA was dissolved in DMSO. HUCCT1 cells were treated by TSA (0.5 μM) for 24h. Another group of HUCCT cells underwent the same treatment with DMSO.  The total RNA was extracted and used for RNA-seq.


title: Epigenetic and transcriptional regulation of mouse oocytes and early embryos

first: Li-quan

last: Zhou

summary: In this study, we performed ChIP-seq of RNA polymerase II, phosphorylated RNA polymerase II and related epigenetic modifications to elucidate transcriptional activities of mouse oocytes and preimplantation embryos. Through analyzing epigenetic alterations of transgenically-modified growing oocytes, early embryos from aged female, and early embryos treated with drugs disturbing proteostasis, we identified critical roles of proteostasis in transcriptional regulation of mouse oocytes and early embryos.

overall_design: We collected genetically-modified growing oocytes from P14 ovaries, early embryos cultured in KSOM with indicated drugs, or early embryos from aged female mice, removed zona pellucida, and performed ChIP-seq with optimized CUT&Tag protocol to elucidate how epigenetic modifications and transcriptional events crosstalk with each other.

first: Ying

last: Yin


title: Transcriptome analysis of oocytes and early embryos to demonstrate trancriptional regulation during mouse oocyte-to-embryo transition

summary: In this study, we performed RNA-seq to elucidate how transcriptional activities of mouse oocytes and preimplantation embryos are regulated to control oocyte-to-embryo transition. Through analyzing transcriptome alterations of transgenically-modified growing oocytes, early embryos from aged female, early embryos treated with drugs disturbing proteostasis, and embryonic stem cells treated with drugs disturbing proteostasis, we identified critical roles of proteostasis in transcriptional regulation of mouse oocytes and early embryos.

overall_design: To elucidate transcriptome changes of mouse oocytes and early embryos, we collected genetically-modified growing oocytes from P14 ovaries, early embryos cultured in KSOM with indicated drugs, and early embryos from aged female mice, and performed low-Input RNA Sequencing. For transcriptome analysis of mouse embryonic stem cell AB2.2, we extracted total RNA, followed by polyA RNA capture for standard RNA-seq library preparation.


title: Deciphering transcriptional activities in mouse early embryo

summary: Through analyzing elongating RNA polymerase II in different stages of mouse early embryos, we identified transcriptional activities at various stages and characterized principles of transcriptional transition between adjacent stages.

overall_design: We collected oocytes and early embryos, removed zona pellucida and performed CUT&Tag experiment.

first: Yi-ran

last: Zhang

first: Ximiao

last: He


title: Identification of the IFN-stimulated genes in IFN-α2b-treated intrahepatic cholangiocarcinoma cells

summary: Interferon-α2b (IFN-α2b) is a highly active cytokine protein that belongs to the interferon-α (IFN-α) family. As a pleiotropic cytokine, IFN-α2b has beneficial antiviral, antitumour, antiparasitic, and immunomodulatory activities. In vivo and in vitro, IFN-α2b can activate the JAK-STAT pathway (also known as IFN-I pathway) and function by inducing the transcription of downstream IFN-stimulated genes (ISGs).To identify the ISGs in intrahepatic cholangiocarcinoma (ICCA) cells and evaluate the therapeutic value of IFN-α2b, ICCA cells were treated with recombinant IFN-α2b protein. Total RNA samples were extracted and used for RNA-seq. Certain ISGs (e.g., MXs, IFITs, OASs) were found to be significantly upregulated upon IFN-α2b treatment.

overall_design: This dataset included two groups of samples. Every group contained three repetitions (rep). IFN-α2b was dissolved in the double distilled water with 1% fetal bovine serum (FBS), which was recommended by the suppliers. HUCCT1 cells were treated by IFN-α2b (60 ng/mL) for 24h. Another group of HUCCT1 cells underwent the same treatment with the recommended solvent.  The total RNA was extracted and used for RNA-seq.

last: Wang


title: Imeglimin, unlike metformin, does not perturb differentiation of human induced pluripotent stem cells towards pancreatic β-like cells and rather enhances gain in β cell identity gene sets

city: Suita

first: Shugo

last: Sasaki

state: Osaka

address: 2-2 Yamadaoka, Suita-shi

department: Metabolic Medicine

country: Japan

institute: Osaka University

summary:
Aims/Introduction: Metformin treatment for hyperglycemia in pregnancy (HIP) beneficially improves maternal glucose metabolism and reduces perinatal complications. However, metformin could impede pancreatic β cell development via impaired mitochondrial function. A new anti-diabetes drug imeglimin, developed based on metformin, improves mitochondrial function. Here we examine the effect of imeglimin on β cell differentiation using human induced pluripotent stem cell (iPSC)-derived pancreatic islet-like spheroid (SC-islet) models.
Materials and Methods: Human iPSCs are differentiated into SC-islets by three-dimensional culture with and without imeglimin or metformin. Differentiation efficiencies of SC-islets were analyzed by flow cytometry, immunostaining, quantitative PCR, and insulin secretion assay. RNA sequencing and oxygen consumption rate were obtained for further characterization of SC-islets. SC-islets were cultured with proinflammatory cytokines, in part mimicking the uterus environment in HIP.
Results: Metformin perturbed SC-islet differentiation while imeglimin did not alter it. Furthermore, imeglimin enhanced the gene expressions of β cell lineage markers. Maintenance of mitochondrial function and optimization of TGF-β and Wnt signaling were considered potential mechanisms for augmented β cell maturation by imeglimin. In the presence of proinflammatory cytokines, imeglimin ameliorated β cell differentiation impaired by cytokines and metformin.
Conclusions: Imeglimin does not perturb differentiation of SC-islet cells and rather enhances gain in β cell identity gene sets in contrast to metformin. This may lead to the improvement of in vitro β cell differentiation protocols.

overall_design: mRNA profiles of human iPSC-derived β-like cell spheroids treated with and without compounds at differentiation day 22

first: Tasuku

last: Imada

first: Hiroki

last: Yamaguchi

first: Ayaka

last: Ueda

first: Dan

last: Kawamori

first: Naoto

last: Katakami

first: Iichiro

last: Shimomura


title: Characterization of nuclear tRNA-derived fragments in Arabidopsis infected with Pseudomonas syringae pv tomato

city: San Marcos

first: Hong-Gu

last: Kang

address: 601 University Dr

institute: Texas State University

summary: Characterization of tRNA-derived small RNAs in regulating defense genes in Arabidopsis responding to a bacterial pathogen

overall_design: We characterized chromatin regions associated with our tRNA-derived small RNAs. To this end, a ChAP-seq approach was utilized by using biotin-tagged RNA and streptavidin beads


title: Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell competition

status: Public on Jan 19 2025

city: New york

first: Xin

state: New york

department: Ophthalmology

institute: Columbia University

summary: Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but causes cell competition-mediated extrusion and apoptosis of only acinar progenitors. Csk mutants can be phenocopied by constitutively active Yap and rescued by the genetic ablation of Yap or Taz, indicating remarkable functional equivalence between Src and Yap signaling. Although Csk inactivation induces tyrosine phosphorylation of Yap, mutating these tyrosine residues in both Yap and Taz fails to alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Chemical genetics studies further demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas the deletion of either Crk/CrkL or Rac1/Rap1 rescues the Csk mutant phenotype. These results show that Csk controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to regulate cell competition.

overall_design: Whole lacrimal glands were isolated from P2 control and Csk conditional knockout (Csk(CKO)) mice and analyzed using scRNAseq.

first: Hannan

last: Abdul

first: Wang

last: Qian

first: Wu

last: Yihua

first: Makrides

last: Neoklis

first: Qu

last: Xiuxia

first: Mao

last: Junhao

first: Que

last: Jianwen

first: Cardoso

last: Wellington

first: Zhang

last: Xin


title: Single-cell transcriptomics and epigenomics point to CD58/CD59-CD2 interactions in controlling primary melanoma growth and immunity

city: Leipzig

first: Maria

last: Schmidt

address: Härtelstr. 16-18

institute: IZBI

summary: To uncover new molecules in melanoma immunological control, single-cell transcriptomic and epigenomic analyses are performed on human melanoma samples using the 10x Genomics technology. Melanoma samples were categorized as high, intermediate and low immune, based on the percentage of lymphocyte infiltration of the tumors.  Single-cell transcriptomic analysis identified a number of different cell populations. Melanoma cells were categorized into nine subclusters with developmental trajectories from melanocytic cells to de-differentiated neural crest-like cells. An emphasis is put on the interaction of melanoma cells with T cells, analyzing possible ligand and receptor interactions. Apart from HLA molecules, a number of new ligand receptor interactions were identified. Single-cell ATAC-seq was used to identify open chromatin regions that might be involved in the immunological control of melanoma.

overall_design: Analysis of primary melanoma lesions that were processed after surgical excision. None of the patients received any treatment in advance nor were any metastatic outgrowths observed in the patients. Additionally, benign melanocytic nevi were processed and analysed within the same pipeline.

first: Antonia

last: Stubenvoll

first: Clara

last: Schoeder

first: Johanna

last: Moeller

first: Chacon

last: Max Lingner

first: Carolyn

last: Schultz

first: Olga

last: Antoniadou

first: Henry

last: Loeffler-Wirth

first: Stephan

last: Bernhart

first: Beatrice

last: Thier

first: Annette

last: Paschen

first: Ulf

last: Anderegg

first: Jan C.

last: Simon

first: Mirjana

last: Ziemer

first: Hans

last: Binder

first: Manfred

last: Kunz


title: CAR-T cell therapy for chronic kidney disease

city: Jinan

first: Fan

last: Yi

address: Wenhua Road

institute: Shandong University

summary: Kidney fibrosis is a hallmark of chronic kidney disease (CKD) and a potential therapeutic target. However, clinical interventions and therapies targeting kidney fibrosis remain conceptual and practical challenges due to the complex origin, functional heterogeneity and regulation of scar-forming cells. Here, we used single cell sequencing technology to identify cell-surface antigens that are broadly and specifically expressed at high levels in kidney cell types that produce extracellular matrix (ECM) during CKD, and study the therapeutic effect of CART therapy targeting the major ECM-producing cell types on kidney fibrosis, which provides a comprehensive treatment strategy for multi-organ fibrosis and its cardiovascular complications in CKD mice.

overall_design: Mouse kidney cells treated with CART cells and each control group were isolated, and single-cell sequencing was used to investigate the effects of CART therapy on renal fibrosis targeting primarily ECM-producing cell types


title: Comparative Transcriptome  Analysis Reveals New Insight into Russeting of the Semi-Russeted ‘Cuiguan’ Pear Variety

city: yangzhou

first: Liu

middle: zi

last: yu

address: shuangqiao

institute: Yangzhou University

summary: We carried out the transcriptome analysis to identify the key genes involved in pear fruit semi-russet formation, by comparing the CK (russet) and bagging treated (green) ‘Cuiguan’ pear fruit skin 115 DAFB.

overall_design: Transcriptome  profiling of untreated and fruit bagging treated ‘Cuiguan’ pear fruit skin, insights into pear fruit semi-russet formation

first: Chunlei

last: Wang

first: Jing

last: Zhang


title: Effect of zinc on zerafish brain

city: Wuxi

last: Yu

address: Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China

summary: We evaluated the effect of zinc on zebrafish brain via single cell analysis

overall_design: Evaluate the effect of zinc on zebrafish brain via single cell analysis

first: Zhi-Shuai

last: Hou

first: HongRui

last: Luo

first: XueFan

last: Cui


title: To study the mechanism of BIP regulation gene in 143B cells using RNA-seq Analysis

city: shanghai

first: tao

last: zhang

address: 650 new songjiang road

institute: Department of Orthopedics,Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine

summary: RNA-seq has developed to study the gene regulation mechanism. The goals of this study are to investigate the regulated potential genes of BIP.

overall_design: We depleted BIP in 143B  using specific targeting shRNA, and then performed  RNA sequencing (RNA-seq).

first: Tao


title: Safety and Efficacy of a STAT3-Targeted Cyclic Oligonucleotide: From Murine Models to a Phase I Clinical Trial in Pet Cats with Oral Cancer

status: Public on Jan 18 2025

city: California

first: Natalia

middle: Faraj

last: Murad

state: CA

address: 500 Parnassus Ave

department: HemOnc

institute: University of California, San Francisco

summary: STAT3 is an oncogenic transcription factor that activates both cancer cell signaling and induces an immunosuppressive immune environment, thus making it an attractive therapeutic target. Transcription factors have been considered “undruggable” and no STAT3 inhibitor has advanced beyond early clinical testing. We previously reported positive pharmacodynamics of a STAT3 decoy oligonucleotide administered intratumorally in a Phase 0 trial in patients with head and neck cancer squamous cell carcinoma (HNSCC). Here, we describe the anti-tumor and immune effects in immunocompetent preclinical HNSCC murine models and the safety and efficacy of systemically administered cyclic STAT3 decoy (CS3D) in a clinical trial in pet cats with HNSCC. Responders (35% of the cohort) showed significant differences in selected peripheral blood immune parameters as well as upregulation of PD-1 expression in the tumors compared with non-responders. These findings support a clinical trial of the CS3D in patients with cancers characterized by STAT3 activation.

overall_design: RNA-seq profiling of oral cancer tumor biopsies from cats at Day 1 (pre-treatment) and on Day 8 prior to treatment with CS3D.

first: Jennifer

last: Grandis

first: Katherine

last: Skorupski

first: Ning

last: Cheng

first: Zhibin

first: Hua

last: Li

first: Liam

middle: C

last: Woerner

first: Yan

last: Zeng

first: Jamie

last: Shiah

first: Neil

last: Bhola

first: Kelly

last: Blum

first: Mi-Ok

last: Kim

first: Daniel

last: York

first: Robert

middle: B

last: Rebhun

middle: F

first: Adam

last: Olshen

first: Ellen

last: Sparger

last: Johnson


title: Single cell RNA-Seq of blood cells in an irradiated patient

city: suzhou

first: zhang

middle: shu

last: yu

address: No. 199, Ren'ai Road

department: soochow university

institute: Soochow University

summary: This study aims to explore the cell clusters and mRNA expression at the  single cell level in a irradiated patient. The patient was a nuclear accident patient exposed to an X-ray fluorescence spectrometer. In this study, the blood of irradiated patient was collected from a patient who was exposed to irradiated.

overall_design: The blood  was collected from patient who was a nuclear accident patient. ScRNA sequencing libraries were further generated by the 10x Chromium Single Cell 3′ Library & Single Cell 3’ v3 Gel Beads (10x Genomics). Briefly, gel beads with cell barcodes and blood cell suspension were wrapped in liquid droplets by a microfluidic platform (10x Genomics). A droplet containing single cells was collected and then lysed to generate Gel Bead-In Emulsions (GEMs).

first: Shuyu

last: Zhang

first: Tao

last: Yan

first: Daojiang

last: Yu


title: Effect of kncoking down CtBP and TRIM28 on gene expreession in MCF-7 cells

city: Zhuhai

first: lixin

last: Tai

address: Hengqin

institute: University of Macau

summary: Both TRIM28 and CtBP are important transcriptional repressors and always shown high expression in many types of cancers, in our current study, we found TRIM28 interacts with CtBP in breast cancer and normal cells. To elucidate the functions of interaction between TRIM28 and CtBP in breast cancer progression, we performed the RNAseq of knocking down TRIM28, CtBP and Ctrl(NC) genes in MCF-7 cell line.

overall_design: MCF-7 cells were transfected with TRIM28, CtBP1/2, Ctrl(NC) siRNA for 2days. RNA was extracted using the RNEasy Plus Micro kit (Qiagen, 74034). RNA-seq and analysis were performed by Novogene. Briefly, samples were sequenced using an Illumina HiSeq, with a 2 × 150 base pair configuration and 20 million paired-end reads was sequenced across nine samples.

first: Lixin

first: Lijun

last: Di


title: Transcriptional profiling of mammalian preimplantation embryos

city: München

first: Marlies

middle: E

last: Oomen

address: Feodor-Lynen-Strasse 21

institute: Helmholtz Zentrum München

summary: We developed an adapted version of Smart-seq2, which we call Smart-seq+5', to capture both internal transcript fragments, as well as 5' transcript information, in mammalian preimplantation stages on a single-embryo level. Focussing specifically on transposable elements, we find many conserved patterns and features shared between mouse, pig, cow, rabbit and rhesus.

sample_organism:
Macaca mulatta
Sus scrofa
Bos taurus
Oryctolagus cuniculus
Mus musculus

overall_design: Transcriptional profiling using an adapted version of the Smart-seq2 protocol during preimplantation stages of mouse, pig, cow, rabbit and rhesus

first: Diego

last: Rodriguez-Terrones

first: Maria-Elena

last: Torres-Padilla


title: NFE2-driven neutrophil polarization promotes pancreatic cancer liver metastasis progression

city: Beijing

first: haoran

last: Huang

state: Beijing Municipality

address: Peking Union Medical College Hospital (East), No.1 Shuaifuyuan,Wangfujing, Dongcheng District

institute: Peking Union Medical College Hospital

summary: Pancreatic cancer liver metastasis (PCLM) is an important factor leading to dismal prognosis. The adaptive remodeling of the tumor microenvironment (TME), especially the role of neutrophil in liver metastasis remains elusive. Here we combined single-cell sequencing combined with spatial transcriptomics on patients’ samples to characterize the landscape of PCLM, and explored the functional diversity possessed by neutrophils infiltrated in liver metastases. We identified the pivotal Neutrophils_S100A12 cluster capable of promoting metastatic progression by releasing neutrophil extracellular traps (NETs); spatially, Neutrophils_S100A12 cells are specifically distributed at the invasive front of the metastatic lesions. Mechanistically metastatic TME activates canonical TGF-β/SMAD3 signaling within neutrophil, resulting in NFE2-mediated phenotypic polarization; furthermore NFE2 induces NETs generation by up-regulating PADI4 expression. Interference with NFE2 activation in neutrophils prevents phenotypic conversion and reduces liver metastasis. Our data demonstrate that NFE2-mediated neutrophil polarization in PCLM is a potential target for anti-metastatic therapy.

overall_design: Three primary advanced pancreatic cancer (PC) tumor, three paired liver metastases (LM) were included in this cohort. Cells from tumor tissue were extracted for RNA-seq analysis.

first: Wenchao

last: Xu

first: Jianzhou

last: Liu

first: Qiaofei

first: Jia

first: Li

last: Zhou

first: Zhiyong

last: Liang

first: Haoran

first: Bowen

first: Gary Guishan

last: Xiao

first: Junchao

last: Guo


title: Effect of short-term glucocorticoid treatment on gene expression of cultured neonatal cardiomyocytes

city: Bologna

first: Gabriele

last: D'Uva

address: via Massarenti 9

country: Italy

institute: University of Bologna

summary: To investigate the direct effects of endogenous glucocorticoids on global gene expression of cardiomyocytes and to explore their potential impact on their proliferation and regenerative capacity, we performed whole-transcriptome RNA sequencing on primary neonatal murine cardiomyocytes cultured in vitro and treated with corticosterone. Corticosterone was administered over a short time frame to identify genes directly regulated by glucocorticoids.

overall_design: Gene expression profiling analysis of RNA-seq data of cardiomyocytes isolated from control mouse models at postnatal-day-1 (P1), cultured in vitro and treated with corticosterone (CORT 10^-6M for 30 minutes).


title: RNA-Seq of RAW 264.7 macrophages treated with Interferon gamma and Nitric Oxide Synthase Inhibitor LNMA

city: Bangalore

first: Avik

last: Chattopadhyay

state: Karnataka

address: Devasandra Layout

department: Department of Biochemistry

country: India

institute: Indian Institute of Science

summary: Interferon-gamma (IFN-γ) is a key regulator of immune responses. A hallmark of the IFN-γ response is inducible nitric oxide (NO) production, driven primarily by nitric oxide synthase 2 (NOS2). In this study, we investigated the influence of NO on the IFN-γ-induced transcriptomic and metabolic changes in the RAW 264.7 macrophage cell line. IFN-γ activation led to NO-dependent lactate production and lower cell survival. Bulk RNA sequencing analysis identified genes differentially expressed by IFN-γ that were either NO-independent or NO-dependent. Inhibition of NO modulated a minor subset of the transcriptome, notably affecting Klf6 (a tumor suppressor) and Zfp36 (a regulator of pro-inflammatory cytokines). Both Klf6 and Zfp36 correlatively showed high expression in most cancers.  The PPI network exhibited dense clustering with scale-free and small-world properties, identifying Stat1, Irf7, Irf1, Cxcl10, and Isg15 as top five hubs. The top IFN-γ signalling genes exhibited deficient expression in the brain but were highly expressed in lung, spleen, and EBV-transformed lymphocytes. Gene-disease associations linked the IFN-g-regulated genes to immunodeficiencies, chronic inflammatory responses and malignancies. Interestingly, IFN-γ upregulated genes were involved in nicotinamide metabolism, suggesting a transcriptional basis for modulation of metabolic pathways. This novel aspect was experimentally tested to show that IFN-γ induced NAD amounts. Importantly, the inhibition of purine nucleoside phosphorylase (using Forodesine hydrochloride) which is involved in the endogenous pathway for NAD generation, lowered IFN-γ induced nitrite and increased cell survival in vitro. Functionally, enriched nicotinamide metabolism by IFN-g may regulate inflammatory responses and the implications of our findings are discussed.

overall_design: RAW 264.7 macrophage cells were treated with IFN-γ under conditions with and without NO inhibition by NG-methyl-L-arginine (LNMA) for 6 hours, and transcriptomic changes were assessed using bulk RNA sequencing.

first: Sai

last: Shyam

first: Shreyasee

last: Das

first: Dipankar

last: Nandi


title: STUB1 Regulates Epithelial Function Through Mediating the EDAR/ NF-κB Pathway

city: Shanghai

first: Lan

last: Rong

address: Zhizaoju road No.639,Huangpu District,Shanghai,China

institute: Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine

summary: Besides ectodermal derivatives morphogenesis, the inactivation of the Ectodysplasin A(EDA)/Ectodysplasin A Receptor(EDAR) signaling pathway has also been demonstrated to impede the process of skin repair. EDAR, a member of the tumor necrosis factor receptor superfamily, plays a central role in this signaling pathway.However, the regulatory mechanism of EDAR and its target genes in wound healing remain unclear.As a membrane protein, the turnover of EDAR is more likely to be affected by ubiquitination, where an E3 Ubuitin ligase targets substrates for degradation. We performed a yeast two-hybrid assay to identify the potential E3 ligase. STIP1 homology and U-box-containing protein 1 (STUB1) was found to interact with EDAR and mediate its ubiquitination and degradation. To explore the function of STUB1 in keratinocytes, we constructed STUB1 knockout HaCaT cells, which showed accelerated healing and growth capabilities. Elevated protein level of EDAR and upregulated activation of downstream nuclear factor-κB signaling were detected in STUB1 knockout HaCaT cells. To find out why STUB1 knockout induced the functional alteration of HaCaT cells and which genes might be affected, an RNA-seq analysis was performed.

overall_design: Control and STUB1 knockout HaCaT cells

first: Xinke

last: Jiang

first: Rong

last: Lan

first: Wenyu

first: Chuanyin

last: Li

first: Feng

last: Wang


title: Free cholesterol primes the IFN-I response by facilitating the release of TLR9 from UNC93B1

city: Guangzhou

first: Jiancheng

last: Huang

state: Guangdong

address: NO.74, Zhongshan er Road

institute: Sun Yat-sen University

summary: Elevated cholesterol is positively associated with autoimmunity. However, it is unclear which mechanism directly link them. Here, we report that free cholesterol in the endolysosomal membrane regulates the IFN-I response in plasmacytoid dendritic cells (pDCs) by facilitating the release of TLR9 from UNC93B1. The cholesterol transporter NPC1 was highly expressed in pDCs, and conditional deletion of Npc1 via Cd11c-Cre (Npc1∆DC) impaired IFNα production by TLR9-stimulated pDCs. Npc1∆DC mice exhibited a weakened IFNα response to HSV-1 infection, and ameliorated anti-DNA autoantibodies and nephritis when crossed to B6.Sle1yaa transgenic lupus model. Mechanistically, through click chemistry-based proteome-wide screening, we found that the TLR chaperone UNC93B1 could bind to free cholesterol. The juxtamembrane cholesterol gradient, mediated by NPC1, promoted TLR9 release from UNC93B1. Increased membrane-bound cholesterol dramatically enhanced IFNα response by murine and human pDCs. Our results suggested that the participation of free cholesterol in TLR9 signaling could directly link elevated cholesterol with autoimmunity.

overall_design: To investigate the effect of NPC1 on immune reponses in vivo, we  performed gene expression profiling analysis obtained from RNA-seq of three independent samples of splenic immune cells from Npc1fl/fl and  Npc1∆DC mice at steady state.

first: Ling

first: Y

middle: Y

last: Cliff


title: Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs using dCasRx conjugated methyltransferase and demethylase

city: Changchun

first: Tao

last: Zhang

address: Xinzhu Road

institute: Jilin University

summary: 5-methylcytosine, an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification systems (termed ‘RCMS’), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localised dCasRx conjugated to either to a methyltransferase, NSUN2/NSUN6 or a demethylase, the catalytic domain of mouse Tet2 (Tet2 CD), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within tRNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.

overall_design: To investigate whether the RCMS activity leads to substantial non-specific methylation, we assessed the impact of the RCMS dCasRx-NSUN6 editor, guided by gRNA -150 nt targeting RPSA m5C234, on the overall m5C content using BSP-seq.We then performed  the m5C methylation levels in mRNA by the dCasRx-NSUN6  and dCasRx-dNSUN6 with the RPSA gRNA −150 nt or nontarget gRNA.Comparative m5C  methylation level profiling analysis of BSP-RNAseq in HEK293T cells( NT,NSUN6,dNSUN6）

last: zhang

first: Feiyu

last: zhao

first: jinze

last: li


title: Comprehensive analysis of H3K27me3 LOCKs under different DNA methylation contexts reveal epigenetic redistribution in tumorigenesis

city: Shenzhen

first: Yueyuan

last: Zheng

state: China

address: Zhenyuan Road, 628

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

summary: Histone modification H3K27me3 is crucial for normal development and is implicated in many diseases, including cancer. H3K27me3 peaks form clusters called Large Organized Chromatin Lysine Domains (LOCKs) that span several hundred kilobases. In this study, we identified H3K27me3 LOCKs in 109 normal samples from the Roadmap project and categorized them into long and short LOCKs. Long LOCKs are preferentially linked to developmental functions, while short LOCKs strongly repress gene expression and are enriched in poised promoters. By examining LOCK distribution in common PMDs (Partially Methylated Domains) and HMDs (Highly Methylated Domains), we discovered that long LOCKs are primarily located in short-PMDs (S-PMDs). Oncogenes within S-PMDs are often covered by long LOCKs, exerting the strongest repression. Analyzing LOCKs in esophageal squamous cell carcinoma (ESCC) and breast cancer (BRCA) cell lines and their normal counterparts, we observed a transition of long LOCK distribution from S-PMDs, to intermediate-PMDs (I-PMDs) and long-PMDs (L-PMDs). Interestingly, a substantial subset of tumor-gain long LOCKs within I-PMDs (23%-40%) and L-PMDs (28%-61%) in BRCA exhibit reduced H3K9me3 modification, indicating H3K27me3 compensates for H3K9me3 loss in tumor. Besides, genes upregulated upon short LOCK loss in tumors are frequently poised promoter genes in normal cells and are transcriptionally regulated by ETS1. These results highlight the role of H3K27me3 LOCKs in various epigenomic contexts and their involvement in cancer biology, revealing underlying epigenetic mechanisms.

overall_design: KYSE150 cell line was harvested and performed ChIP-Seq using ETS1 antibody (Cell Signaling Technology, 14069S).

first: Yuan

last: Liang

first: Benjamin

last: Ziman

first: De-Chen

last: Lin


title: Cleavage Under Targets and Tagmentation（CUT&Tag）analysis of ovarian cancer cells treated by Paeonol

city: Wuhan

first: Jingchun

last: Liu

state: HUBEI

address: Zhangzhidong road

institute: Renmin Hospital of Wuhan University

summary: Cells were harvested and aliquoted (500,000 cells). Cell nucleus was prepared and resuspended. Incubation of the cells was performed with ACSS2 and IgG primary antibodies throughout the night at 4℃. The cells were centrifuged and washed using Dig-Wash buffer. A dilution of the pA-Tn5 adapter complex was introduced into the cells, followed by incubation at RT for 1 h. Subsequently, the cells were subjected to incubation in Tagmentation buffer for 1 hour at 37℃, and the STOP buffer was eventually added. The DNA library was purified and amplified for sequencing, and CUT&Tag data analyses were performed.

overall_design: CUT&Tag analysis was performed on ovarian cancer cells (SKOV3 cells) after being treated by 2DG for 24 hours

first: Jiang

last: Yang

first: Li

last: Hong


title: Copper oxide nanoparticles treated MH-S cells

status: Public on Jan 17 2025

city: Chengdu

first: xiaojing

address: Sichuan University, No. 24 South Section 1, Yanshan Road, Chengdu, Sichuan Province, China

institute: Sichuan University

summary: The three groups of MH-S cells were control group, 20 μg/ml CuO NPs group, and 20 μg/ml CuO NPs + 10 μM TTM group. Total RNA of the  MH-S cells were extracted using cell RNA isolation kit (Vazyme Biotech Co., Ltd, China). The number of MH-S cell sample for each group were three (n = 3 cell samples/group). All samples were sent to Majorbio Biotech (Shanghai, China) for transcriptome sequencing.

overall_design: The three groups of MH-S cells were control group, 20 μg/ml CuO NPs group, and 20 μg/ml CuO NPs + 10 μM TTM group for 24 h.

first: Xiaojing

first: Donglei

last: Sun


title: Analysis of B cell neighborhoods via single cell spatial transcriptomics in head and neck cancer patients

city: Pittsburgh

first: Zhentao

state: PA

address: 5051 CENTRE AVE, HCC SUITE

institute: UMPC Hillman Cancer Center

summary: Head and neck squamous cell carcinoma tissue microarrays were stained via CD20 single plex IHC and then FOVs were selected for B cell neighborhoods (including tertiary lymphoid structures) in confirmed tumor areas via H&E evaluation by a pathologist.

overall_design: In this study, 1000 plex CoSMX via Nanostring was performed on 11 patients with multiple FOVs for B cell neighborhoods

first: Tullia

last: Bruno

first: Raja

last: Seethala


title: TaqMan Array Human Cytokine Network 96-well Plate for Caco-2 cell line

city: Pecs

first: Hairui

last: Ou

address: Rokus

country: Hungary

institute: University of Pecs

summary:
Peripheral blood was acquired from the Pécs Regional Blood Transfusion Center (Pécs, Hungary) and processed using Lymphocyte Separation Medium 1077 (Promocell, Heidelberg, Germany) in accordance with the manufacturer's instructions. Subsequently, the viable mononuclear cells were isolated, counted, and seeded into 6-well plates at a density of 1,000,000 cells per well.   The culture medium was specifically formulated using RPMI-1640 medium (Biowest, Nuaillé, France), enriched with 1% L-glutamine (Lonza, Basel, Switzerland), 2% penicillin/streptomycin (Lonza, Basel, Switzerland), and 10% exosome-depleted FBS (Thermo Fisher Scientific, Waltham, USA) to minimize EV interference from bovine serum, further supplemented with 100 ng/mL Peprotech M-CSF 300-25 (Gibco, Waltham, USA). The cells were cultured for 5 days in a 5% CO2 incubator at 37 °C, resulting in the differentiation of all monocytes into macrophages (M0) (Cao et al., 2019).
Differentiated macrophages were treated with MEV (Milk-derived extracellular vesicles) from healthy and mastitis groups separately to achieve the final concentration of 2×10^11 particles/mL. Additionally, untreated cell cultures with the same volume of PBS were maintained as negative controls. After 48 hours, macrophage supernatants were collected and stored at -80 °C (for a maximum of 3 months) for future experiments.

overall_design: Human colorectal adenocarcinoma Caco-2 cells were purchased from the American Type Culture Collection and were used from passages 52 to 58 following the supplier’s recommendations. Caco-2 cells were cultured in DMEM medium (Biosera, Cholet, France) with 10% exosome-depleted FBS (Thermo Fisher Scientific, Waltham, USA). Cells (30,000 per well) were seeded in 24 well plates one day before treatment. The supernatant from macrophages accounted for 1/3 of the total volume of the Caco-2 culture medium. The experimental groups were divided into negative control, healthy group and mastitis group.

first: Ou

last: Hairui

first: Kvell

last: Krisztian


title: TaqMan™ Array Human Inflammation 96-well Plate for Caco-2 cell line


title: Effects of p65 palmitoylation on gene expression when overexpressing vector or p65 WT or p65 2CS in  p65 knockout 293A cells combined with TNFα treatment

city: Charlestown

first: Xu

last: Wu

state: MA

institute: Massachusetts General Hospital

summary: Nuclear factor kappa B  (NF-κB) is a central regulator of inflammation, infection, and immunity pathways and is often dysregulated in autoimmune diseases, inflammatory disorders, and cancers. However, the mechanisms by which different stimuli, including cellular metabolism, modulate NF-κB activation remain unclear. Here, we report that  RelA ( p65), a major component of NF-κB,  is S-palmitoylated at cysteine 197 and 206 by directly reacting with palmitoyl-CoA. Autopalmitoylation of p65  promotes its nuclear localization and p65-p50 complex formation, thereby enhancing NF-κB transcriptional activities. Furthermore, p65 palmitoylation upregulates RelB expression and promotes non-canonical NF-κB complex formation, further amplifying pathway activation. We also demonstrate that a high-fat diet could enhance p65 palmitoylation and correlate with an increased cytokine production in mouse tissues. These results reveal that lipid metabolism might directly regulate NF-κB activity and suggest that targeting p65 auto-palmitoylation could be a potential therapeutic strategy for diseases with deregulated NF-κB pathway.

overall_design: RNA-seq profiling of vecotr or p65 WT or p65 2CS expreesiong HEK293A p65 knockout cells when treated with TNFα for 2h.

first: Jianan

last: Zhang