title: Med14 phosphorylation shapes genomic response to GLP-1 agonist [ChIP-seq]

city: La Jolla

first: Sam

last: Van de Velde

state: CA

address: 10010 North Torrey Pines Road

department: PBL

institute: Salk Institute

summary: Under feeding conditions, increases in circulating glucose concentrations trigger the release of glucagon-like peptide (GLP-1) from intestinal L cells. GLP-1 promotes insulin secretion and pancreatic beta cell viability in part via triggering of the beta cell GLP-1 receptor and subsequent induction of the cAMP signaling pathway, leading to the protein kinase A (PKA) mediated phosphorylation of CREB and induction of CREB target genes. By contrast with the acute effects of this pathway on immediate early CREB target genes, which attenuate the cAMP-CREB response, sustained exposure of beta cells to GLP-1 agonist (exenatide-4; Ex-4) or adenyl cyclase activator (Forskolin; FSK) stimulates the expression of beta cell specific CREB target genes with delayed kinetics. In a proteomic screen for transcriptional co-regulators that mediate the long-term effects of GLP-1, we identified Med14, a backbone subunit of the Mediator complex. Exposure to either Ex-4 or FSK stimulates Med14 phosphorylation at Ser983, corresponding to a conserved PKA recognition site (RRXS) that is located within an intrinsically disordered region of Med14. Phosphorylation of Med14 is essential for maintenance of enhancers that drive induction of beta cell-specific and diabetes-linked genes.  Mutation of Med14 at Ser983 to alanine decreased beta cell numbers and repressed growth factor signaling in primary mouse islets. Our work reveals how phosphorylation of a general transcription factor in response to GLP-1 analogs triggers a broad genomic response with salutary effects on beta cell function.

overall_design: ChIP-seq of CREB, CRTC2, H3K27Ac, PolIIpS2 and FLAG-Med19 (tagged in the genome) in control and forskolin (FSK) treated INS-1 cells.


title: Engineering Bi-directional Chemically-modulated Synthetic Condensates for Cellular Control

status: Public on Jun 15 2025

city: Shanghai

first: Qi

last: Zhang

state: Shanghai

address: Dongfang Load 1678

country: China

institute: Shanghai Jiaotong University

type: Expression profiling by high throughput sequencing

summary: Biomolecular condensates are membraneless compartments involved in a wide range of cellular processes. Despite their fundamental role in the spatiotemporal regulation of cellular functions, tools for precisely manipulating phase-separated condensates remain limited, and effective methods for discovering and functionalizing tunable phase separation modules from natural proteins are lacking. Here we present a rational engineering approach for androgen receptor (AR) and its clinically used drugs to create a chemical genetic platform, ARDrop, enabling condensates formation and dissolution. This platform is applied to a diverse set of proteins to achieve intended cellular functions, ensuring robust and long-lasting functionality through stable liquid-like properties. Our work develops a powerful toolkit for reversible manipulation of condensates that can be used for dissection of complicated cell signaling, laying the foundation for engineering designer condensates for synthetic biology applications.

overall_design: Bica is used to induce phase separation of ARDrop-ALK, thereby activating the downstream ALK signaling pathway.

first: Ni

last: Zhen


title: miRNA expression data of plasmablastic lymphoma and control group

city: Badalona

first: Miriam

last: Verdú-Bou

state: Barcelona

address: Ctra de Can Ruti, Camí de les Escoles, s/n

country: Spain

institute: Josep Carreras Leukaemia Research Institute

summary:
Plasmablastic lymphoma has been scarcely studied because of its rarity. Transcriptomic studies are crucial to unveil the biology of this lymphoma. In particular, miRNA expression will help to understand the lymphomagenesis of plasmablastic lymphoma.
We used microarrays to depict the miRNA expression landscape of plasmablastic lymphoma and to identify miRNAs differentially expressed compared to a control group.

overall_design: RNA was extracted from formalin-fixed paraffin-embedded sections of 66 plasmablastic lymphoma tumors and 14 reactive lymph nodes, as a control group. From total RNA we performed miRNA expression studies using GeneChipTM miRNA 4.0 Array of Affymetrix. miRNA expression profile was compared to the control group to identify miRNAs differentially expressed. We performed an enrichment analysis from miRNAs differentially expressed to identify in which processes these miRNAs are involved.

last: Verdu-Bou

first: Jose-Tomas

last: Navarro


title: Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes [RNA-Seq]

status: Public on Jun 16 2025

city: Helsinki

first: Divyesh

last: Patel

address: University of Helsinki

department: Applied Tumor Genomics Program, Research Programs Unit, Faculty of Medicine,

country: Finland

institute: University of Helsinki

summary: Derepression of transposable elements (TE) by epigenetic therapy leads to the activation of immune response in cancer cells. However, the molecular mechanism of TE regulation by distinct chromatin modifier enzymes (CME) in context of p53 is still elusive. Here, we used FDA-approved epigenetic drugs to systematically inhibit distinct CMEs in p53 wild-type and p53-mutant colorectal, esophageal, and prostate cancer cells. We show that distinct TE subfamilies are derepressed by inhibition of different CMEs in cell type-specific manner. Co-inhibition of DNMT and HDAC (DNMTi-HDACi) had the most consistent effect across cancer types. Loss of p53 results in stronger TE activation and TE-chimeric transcript expression and this effect is largely mediated by the non-genomic actions of p53. Robust immune response elicited by DNMTi-HDACi is due to induced inverted repeat Alu expression concomitant with reduced ADAR1-mediated Alu RNA editing. Collectively, our systematic analyses provide insights for rational use of epigenetic therapies in distinct cancers.

overall_design: Transcription factor binding  was determined by ChIP-seq in GP5d and OE19 cells. Gene expression profiling for GP5d, OE19, LNCAP-1FP, GP5d p53-KO and LNCAP-1Fo p53-KO cells (DMSO treated, DNMT inhibited, HDAC inhibited, SETDB1 inhibited and DNMT-HDAC co-inhibited, DNMT-SETDB1 co-inhibited, HDAC-SETDB1 co-inhibited) was performed by RNA-seq.

first: Ville

last: Tiusanen

first: Karttunen

last: Karttunen

first: Päivi

last: Pihlajamaa

first: Biswajyoti

last: Sahu


title: CDKN1B (p27kip1) enhances drug tolerant persister CTCs by restricting polyploidy following mitotic inhibitors.

status: Public on Jun 14 2025

city: Boston

first: Ben

middle: S.

last: Wittner

state: MA

address: 149 13th Street

department: Center for Cancer Research

country: USA

institute: Massachusetts General Hospital

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

overall_design: Refer to individual Series


title: CDKN1B (p27kip1) enhances drug tolerant persister CTCs by restricting polyploidy following mitotic inhibitors [docetaxelRNAseq]

summary: The mitotic inhibitor docetaxel (DTX) is often used to treat endocrine-refractory metastatic breast cancer, but initial responses are mitigated as patients eventually have disease progression. Using a cohort of ex vivo cultures of circulating tumor cells (CTCs) from patients with heavily pretreated breast cancer (n=18), we find two distinct patterns of DTX susceptibility, independent of clinical treatment history. In CTCs cultured from some patients, treatment with a single dose of DTX results in complete cell killing, associated with accumulation of non-viable polyploid (≥8N) cells arising from endomitosis. In others, a transient viable drug-tolerant persister (DTP) population emerges, ultimately enabling renewed proliferation of CTCs with preserved parental cell ploidy and DTX sensitivity. In these CTC cultures, efficient cell cycle exit generates a ≤4N drug-tolerant state dependent on CDKN1B (p27Kip1). Exposure to DTX triggers stabilization of CDKN1B through AKT-mediated phosphorylation at serine 10. Suppression of CDKN1B reduces the number of persister CTCs, increases ≥8N mitotic cells and abrogates regrowth after DTX exposure. Thus, CDKN1B-mediated suppression of endomitosis contributes to a reversible persister state following mitotic inhibitors in patient-derived treatment refractory breast cancer cells.

overall_design: Cell cultured derived from breast cancer circulating tumor cells were treated in vitro with the mitosis inhibitor chemotherapeutic agent docetaxel for 4 and 10 days.

first: Elad

last: Horwitz

middle: S

first: Shyamala

last: Maheswaran

first: Daniel

middle: A

last: Haber


title: CDKN1B (p27kip1) enhances drug tolerant persister CTCs by restricting polyploidy following mitotic inhibitors [scRNAseq]

overall_design: Circulating tumor cells derived cultures from four different patient donors were exposed to 10nM docetaxel and subjected to single-cell RNA sequencing.


title: SLNCR-E2F1 oncogenic complexes promote melanoma formation

status: Public on Jun 17 2025

city: Brighton

first: Leinal

last: Sejour

address: 63 Nonantum St.

department: Vlachos - Non Coding RNA Lab

institute: BIDMC

summary: The long non-coding RNA SLNCR and the transcription factor E2F1 both have oncogenic activities in melanoma. Here we show that melanoma metastasis depends upon the formation of an oncogenic SLNCR–E2F1 complex. Unbiased computational analyses of patient samples revealed worse overall survival only in melanoma patients overexpressing both SLNCR and E2F1. Blocking SLNCR–E2F1 complex formation without reducing the levels of either SLNCR or E2F1 reversed melanoma invasion ex vivo and melanoma extravasation in mice. We discovered that E2F1 bound to sequences in SLNCR that are the RNA analogs of the E2F consensus DNA-binding element, suggesting that RNA and DNA binding engages the same domain in E2F1. Binding competition assays demonstrated that E2F1 preferentially bound to RNA compared to its cognate DNA element and that RNA efficiently competed with the consensus DNA-binding element for E2F1 binding. Molecular dynamics simulations indicated increased thermodynamic stability of E2F1–DNA interactions as compared to E2F1–RNA interactions. However, molecular dynamics simulations also indicated greater number of RNA-amino acid contacts within the E2F1 DNA binding domain (E2F1DBD) than DNA. This suggests better kinetic binding properties of RNA to the E2F1DBD as compared to DNA and may explain how RNA efficiently competed with DNA for E2F1DBD binding. E2F1 often binds to DNA as a heterodimer with DP1. Molecular dynamics simulations suggested that RNA binding to E2F1 may prevent E2F1–DP1 heterodimerization by masking key amino acids required for DP1 binding. These data reveal the SLNCR-driven oncogenic activities of E2F1 and identify the SLNCR–E2F1 complex as a target for future melanoma therapy.

overall_design: To identify the genes regulated by SLNCR in A375 cells and characterize the transcription factors enriched in the differentially expressed genes we transfected a SLNCR  overexpression plasmid or control vector into A375 cells n = 3 biological replicates for each condition and performed RNA-Seq.

first: Kushani

last: Shah

first: Ioannis

last: Vlachos

first: Carl

middle: D

last: Novina


title: Mechanism of Topoisomerase 2 inhibitor tolerance uncovers a fundamental histone H1 biogenesis pathway

status: Public on Jun 13 2025

first: William

last: Gittens

state: I am not in the U.S. or Canada

address: Science Park Road

department: Genome Damage and Stability Centre

country: United Kingdom

institute: University of Sussex

summary: Topoisomerase 2 (TOP2) inhibitors (TOP2i) are mainstay cancer chemotherapeutic agents that undermine genome integrity by stabilizing TOP2-DNA complexes. Nevertheless, their clinical deployment is associated with toxicity, acquired resistance and secondary malignancies, necessitating the development of improved treatment strategies. From genome-scale CRISPR-Cas9 screens, we discovered that the uncharacterized human protein CRAMP1 and H1 linker histones display synthetic lethal interactions with TOP2i. Although loss of CRAMP1 selectively hypersensitizes cells to TOP2i but not other genotoxic agents, we show that CRAMP1 does not function as a conventional repair factor for TOP2-DNA complexes. Instead, CRAMP1 defines an H1-specific histone biogenesis factor, and we demonstrate that reduced histone H1 abundance underlies TOP2i hypersensitivity in CRAMP1-deficient cells. Mechanistically, CRAMP1 acts as a transcription factor for both replicative and non-replicative H1 genes, driven by its concurrent targeting to histone gene loci and H1-specific promoter motifs. We establish that defective H1 biogenesis induces TOP2i hypersensitization via a novel paradigm uncoupled from DNA repair, increasing cellular demand for TOP2 activity through extensive formation of novel TOP2 substrates due to chromatin decompaction, thereby lowering the threshold for TOP2i-induced cytotoxicity. Collectively, through revealing CRAMP1-dependent H1 synthesis as an essential prerequisite for TOP2i resistance, our findings elucidate the mechanistic basis of linker histone biogenesis in human cells and provide a framework for delineating H1 functions in chromatin biology.

overall_design: CC-seq-v2 maps of the genome-wide distribution of Top2ccs in CRAMP1 WT U2OS, CRAMP1-KO U2OS, and CRAMP1-KO U2OS complemented with GFP-CRAMP1. The experiment was conducted in quadruplicate.

middle: H

first: Andreas

last: Ingham

first: Niels

last: Mailand


title: ChIP and ATAC-seq data related to Rapid transcriptional response to a dynamic morphogen by time integration

city: Evanston

first: Shelby

last: Blythe

state: IL

address: Northwestern University, Hogan Hall, 2205 Tech Drive

department: Molecular Biosciences

institute: Northwestern University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: This study on blastoderm stage Drosophila embryos seeks to measure accessible chromatin and the enrichment on chromatin of transcription factors associated with BMP signaling (phospho-Mad, Medea) in either wild-type conditions, or in embryos mutant for the homeodomain transcription factor zerknullt (zen). The associated manuscript demonstrates that zen is a critical transcription co-activator that enables rapid responses to BMP signaling.

overall_design: Two measurement types were performed. 1) Chromatin IP (ChIP) was performed for either phospho-Mad or GFP-tagged Medea (or negative controls) in wild type embryos. GFP-tagged Medea was ChIPped in zen mutant embryos. 2) Chromatin accessibility was measured by ATAC-seq on single wild-type or zen mutant blastoderm stage embryos.

first: Susanna

middle: E

last: Brantley

first: Anna

last: Chao

first: Stefano

last: Di Talia


title: CRAMP1-dependent histone H1 biogenesis is essential for topoisomerase II inhibitor tolerance [CUT&RUN]

city: Copenhagen

address: Blegdamsvej 3B

country: Denmark

institute: Novo Nordisk Foundation Center for Protein Research

summary: Topoisomerase II (TOP2) inhibitors (TOP2i) are mainstay chemotherapeutic agents that undermine genome integrity by stabilizing TOP2-DNA complexes accompanied by DNA damage formation. Here, we reveal the uncharacterized protein CRAMP1 and H1 linker histones as key effectors of TOP2i tolerance in human cells. We demonstrate that CRAMP1 defines a dedicated histone H1 biogenesis factor stimulating transcription of both replicative and non-replicative H1 genes, driven by its concurrent targeting to histone gene loci and H1-specific promoter motifs. CRAMP1 promotes TOP2i tolerance by maintaining H1 supply, involving a novel mechanism uncoupled from TOP2i-induced DNA damage whereby reducing the H1 pool triggers unscheduled TOP2 substrate formation in low accessibility chromatin states. This amplifies total demand for TOP2 activity, lowering the threshold for TOP2i-mediated exhaustion of TOP2. Our discoveries elucidate the mechanistic basis of histone H1 biogenesis in human cells, opening opportunities for selectively manipulating linker but not core histone supply and targeting cancer-associated H1 deficiency.

overall_design: Cut under target and Relase using nuclease (CUT&RUN) assay of GFP-CRAMP1 in U2OS cells where CRAMP1 was unmutated or mutated for SANT or GB-N domain. 3 replicates are included for each condition.

first: Louise

last: Morlot

first: Ignacio

last: Alonso de Vega


title: CDKN1B (p27kip1) enhances drug tolerant persister CTCs by restricting polyploidy following mitotic inhibitors [CTClineRNAseq]

first: Ben

last: Wittner

overall_design: Breast Cancer circulating tumor cells derived cultures from 18 different patients were subjected to bulk RNA-seq at basal growth conditions.


title: CRAMP1-dependent histone H1 biogenesis is essential for topoisomerase II inhibitor tolerance [RNA-seq]

overall_design: Assessing the transcriptional changes using total RNA-sequencing in WT vs CRAMP1-KO cells (experiment 1) and in cells where CRAMP1 was KO and rescued with either the unmutated protein or CRAMP1 mutated for SANT or GB-N domain (experiment 2, containing also WT and CRAMP1-KO cells).


title: The histone H3 lysine 36 demethylase KDM2A/FBXL11 is essential for germ cell development in male mice [RNA-Seq]

city: Basel

first: Antoine

last: Peters

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute

summary: KDM2A/FBXL11 is a Jumonji-domain containing lysine demethylase catalyzing the removal of mono- and di-methyl modifications of histone H3 lysine 36. While Kdm2a is required for mouse embryogenesis, its role in adult physiology is unknown. Using conditional deletion approaches, we demonstrate that Kdm2a deficiency causes testicular atrophy and male infertility. Though spermatogonial stem cells were unaffected, proliferating and differentiating spermatogonia suffered from delayed cell cycle progression and apoptosis, which correlated with upregulated expression of several cell cycle inhibitors. Loss of Kdm2a in spermatocytes disrupted progression through meiotic prophase, as shown by impaired chromosome synapsis and  processing  of  meiotic  double  strand  breaks,  and  by  altered  chromatin  states.  Correspondingly, Kdm2a mutant spermatocytes failed to activate numerous genes controlling these processes. RNA-sequencing analyses on purified spermatogonia and spermatocytes showed that during normal spermatogonial differentiation over 700 genes undergo repression, which is controlled by KDM2A. CpG-rich promoter genes upregulated in Kdm2a deficient cells are marked by Polycomb Repressive Complexes (PRC) and associated modifications in wildtype male germ cells, suggesting that KDM2A is required for PRC-mediated repression. Our study thus identifies critical roles for KDM2A in coordinating gene expression programs during spermatogonial differentiation and meiosis, which are essential for male germ cell development.

overall_design: Gene expression profiling analysis using data obtained from RNA-seq from FACS sorted populations enriched for Undiferentaited Spermatogonia (SgU) , Differentiated Spermatogonia (SgD) and Zygotene Spermatocytes(ScLZ) from ctr mice and iKO mice

first: Grigorios

last: Fanourgakis


title: CRAMP1-dependent histone H1 biogenesis is essential for topoisomerase II inhibitor tolerance [ATAC-seq]

overall_design: Assay for transposable accessible chromatin (ATAC-seq) in WT and CRAMP1-KO cells. 3 replicates are included for each condition.


title: Phenotypic and transcriptomic characterisation of the human B cell response to a novel Ebola vaccine regimen (Ad26.ZEBOV, MVA-BN-Filo)

city: Oxford

first: Daniel

last: O'Connor

state: Oxfordshire

address: Churchill Hospital

department: Paediatrics

institute: University of Oxford

summary: Ebola virus disease (EVD) outbreaks are increasing in frequency — threatening health and wellbeing of affected communities. Early and effective public health measures to manage outbreaks rely on health care and frontline workers; consequently, protecting these at high-risk groups is a key pillar of EVD vaccination strategies. IgG specific to the viral glycoprotein is used as the correlate of protection in recent vaccine licensure. Here, we use contemporary cellular and transcriptomic analyses to dissect B cell responses to the heterologous Ad26.ZEBOV, MVA-BN-Filo Ebola vaccine. We reveal robust plasma cell and persistent B cell memory responses following vaccination. Using machine-learning, trained on blood gene expression post-vaccination, we were able predict the magnitude of antibody responses. Moreover, we described a B cell receptor CDRH3 sequence which was exclusively seen post-vaccination that is similar to known Ebola GP-binding antibody sequences. Single cell analysis was used to further characterise changes to plasma cell frequency, subclass usage and CDRH3 properties, following vaccination. These results demonstrate that early immune responses —captured in blood using systems immunology approaches — are delineative and predictive of B cell responses to vaccination.

overall_design: Venous peripheral blood (up to 3ml) was collected directly in TempusTM RNA tube immediately pre-vaccination, then 10 days post-first vaccine and 7 days post-second vaccine. RNA was extractedusing theTempus™spin RNAisolationkit(Invitrogen, USA).Eluted RNA was quantifiedusingQubit™ RNA BR Assay Kit(Invitrogen, USA) and quality control was conducted usingthe Agilent RNA ScreenTape assay on the 4200 TapeStation System (Agilent, USA). RNA was ribodepleted and globin depleted usingRibo-ZeroTM Gold rRNA removal Kit (Illumina, USA). RNA was converted to cDNA, second-strand cDNA synthesis incorporated dUTP. The cDNA was end-repaired, A-tailed and adapter-ligated and prior to amplification, samples underwent uridine digestion. The prepared libraries were size selected, multiplexed and quality controlled before 75bp paired-end sequencing (HiSeq4000). Sequencing was conducted at the Wellcome Trust Centre for Human Genetics (Oxford, UK). The sequencing data were aligned against the whole human (Homo sapiens) genome build GRCh38 (https://ccb.jhu.edu/software/hisat2/index.shtml), using STAR (version 2.6.1d). Gene features were counted using HTSeq (version 0.11.1), using human gene annotation general transfer format version GRCh38.92 (www.ensembl.org).


title: Effect of pharmacological inhibition of SMYD3 on gene expression in patient-derived CRC-SCs

city: Milan

first: Loris

last: De Cecco

address: via Venezian 1

country: Italy

institute: IRCSS Istituto Nazionale Tumori

summary: Cancer stem cells (CSC) are responsible for colorectal cancer (CRC) chemoresistance, recurrence, and metastasis. Therefore, it has become crucial finding critical molecular stemness targets that are essential for tumor growth. Here, we performed an extensive in vitro and in vivo molecular and functional characterization revealing the pivotal role of SMYD3 in CSC biology. Specifically, SMYD3 interacts with and methylates c-MYC at K158 and K163, modulating its transcriptional activity implicated in stemness and colorectal malignancy. Together, all the in vitro data collected, suggest that SMYD3 pharmacological inhibition affects clonogenic and self-renewal potential of patient-derived CRC-SCs and organoids by altering their molecular signature. Moreover, we showed that SMYD3 stable knock-out or its pharmacological inhibition drastically reduced CRC tumorigenicity in vivo, and reduced the metastatic potential of CRC-SCs. Thus, our findings identify SMYD3 as a promising therapeutic target, acting directly on c-MYC, with potential implications for countering CRC-SCs proliferation, chemoresistance, and metastatic dissemination.

overall_design: To investigate the function of SMYD3 in the regulation of stemness molecular signature, we performed a pharmacological inhibition of SMYD3 by the administration of EM127 the covalent SMYD3 inhibitor. We then performed gene expression profiling analysis using data obtained from RNA-seq of patient derived CRC-SCs treated or not with EM127 for 24 hours and we performed a comparative gene expression profiling analysis of RNA-seq data.

first: Vittoria

last: Disciglio


title: Plasma miRNA profiling in patients with hepetocellular carcinoma and liver cirrhosis

status: Public on Jun 12 2025

city: Hong Kong

first: Mengsu

last: Yang

address: City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Kowloon

country: Hong Kong

institute: City University of Hong Kong

summary: To evaluate the potential of circulating microRNAs as biomarkers for early hepetocellular carcinoma (HCC) screening, plasma miRNA profiles were compared between patients with HCC and liver cirrhosis (LC).

overall_design: Total RNA extracted from 50 HCC patients and 40 LC patients were subjected to microarray analysis.

first: Kelvin Ka Ki

last: Ng


title: Cathepsin L-Dependent Positive Selection Shapes Clonal Composition and Functional Fitness of CD4 T Cells [TCR]

city: Martinsried

first: Tobias

last: Straub

address: Großhadener Str. 9

department: Biomedical Center, Bioinformatics

country: Germany

institute: LMU Munich

summary: The physiological significance of thymic positive selection and its reliance on a single stromal cell type, cortical thymic epithelial cells (cTECs), remain poorly understood. Cathepsin L (Ctsl) has been implicated in generating MHCII-bound peptides in cTECs for efficient CD4 T cell differentiation. Here, we addressed whether Ctsl-deficiency impacts the CD4 compartment not only numerically, but also qualitatively. When Ctsl is absent, many TCRs disappear from the repertoire due to a genuine defect in positive selection, while some allegedly ‘Ctsl-independent’ TCRs are retained. We demonstrate that these clones experience weaker positively selecting signals in absence of Ctsl, which suffice for further maturation, yet imprint aberrant responsiveness to agonist stimulation and impaired homeostatic behavior. Hence, the Ctsl-generated peptidome in cTECs engenders TCR-pMHCII interactions at the upper end of the permissible affinity spectrum to optimize both CD4 T cell diversity and functionality.

overall_design: Bulk TCRα sequencing of mature CD4SP thymocytes selected in Ctsl-deficient (CtslΔTEC) and -sufficient (control) mice. Each biological replicate consists of pooled cells coming from 2-3 donor mice.

first: Elisabetta

last: Petrozziello

first: Amina

last: Sayed

first: João

last: Freitas

first: Christine

last: Federle

first: Jelena

last: Nedjic

first: Sarina

last: Ravens

middle: M

last: Schulz

first: Dietmar

last: Zehn

first: Marc

last: Schidt-Supprian

first: Immo

last: Prinz

first: Martijn

last: Verdoes

first: Jan

last: Kisielow

first: Thomas

last: Reinheckel

first: Stephen

middle: R

last: Daley

first: Ludger

last: Klein


title: Single-Cell RNA Sequencing of Bone Marrow ILC2p and CHILPs, and Lung and Mesenteric Lymph Node ILC2s

city: Denver

first: Richard

middle: Lee

last: Reinhardt

state: Colorado

address: 1400 Jackson Street, K817A

department: Biomedical Research

institute: National Jewish Health

summary: To explore potential tissue origins of iILC2 during helminth infection, we performed single-cell RNA sequencing (scRNA-seq) of ILC2 and ILC progenitors using the 10x Genomics platform. Intestine draining mesenteric lymph nodes (Mes LN), lung, and bone marrow (BM) were collected 5 days post infection (dpi) with Nippostrongylus brasiliensis.

overall_design: ILC2 and ILC progenitors were enriched by lineage depletion prior to cell sorting. BM cells were sorted for progenitor cell markers CD45+Lin- CD127+ a4b7+ Flt3-. These markers account for both the common helper-like ILC progenitors (CHILP) and ILC2 progenitors (ILC2p). Although CHILP are upstream of ILC2p, they were included in our sorting strategy due to their similarity to the migratory lung iILC2 population in terms of markers CD90- Arg1- ST2- a4b7+.22 Mes LN and lung cells were sorted for conventional ILC2 markers CD45+ Lin- CD127+ and KLRG1low/+.

first: Richard Lee

first: Uryan Isik

last: Can


title: The histone H3 lysine 36 demethylase KDM2A/FBXL11 is essential for germ cell development in male mice

first: Antoine

last: Peters

type: Methylation profiling by high throughput sequencing


title: Accounting for differences between Infinium MethylationEPIC v2 and v1 in DNA methylation-based tools

city: Vancouver

first: Michael

last: Kobor

state: BC

address: 117-2194 Health Sciences Mall

department: Faculty of Medicine

country: Canada

institute: University of British Columbia

type: Methylation profiling by genome tiling array

summary: The recently launched Illumina Infinium MethylationEPIC v2.0 (EPICv2), successor of MethylationEPIC v1.0 (EPICv1), retains a majority of probes in EPICv1, while expanding coverage of regulatory elements.  The concordance between the two EPIC versions in DNA methylation-based tools has not yet been investigated. To address this, DNA methylation was profiled on both versions using matched blood samples across four cohorts spanning early to late adulthood. High concordance between versions at the array level but variable agreement at the individual probe level was noted. A significant contribution of EPIC version to DNA methylation variation was observed, though it was to a smaller extent compared to sample relatedness and cell type composition. Modest but significant differences in DNA methylation-based estimates between versions were observed, irrespective of the data preprocessing method used. Adjustments for EPIC version or calculation of estimates separately for each version largely mitigated these version-specific discordances. This work emphasizes the importance of accounting for EPIC version differences in research scenarios, especially in meta-analyses and longitudinal studies that require data harmonization across versions.

overall_design: DNA was extracted from samples, bisulfite converted using EZ-96 DNA Methylation kits (Zymo Research, Irvine, CA), hybridized to the MethylationEPIC BeadChip Infinium microarray v1.0 B5 (EPICv1) and Infinium MethylationEPIC v2.0 (EPICv2) arrays, and scanned with the Illumina iScan 2000 to obtain IDAT files that capture raw DNA methylation intensities.

first: Beryl

middle: C

last: Zhuang

first: Marcia

last: Jude

first: Chaini

last: Konwar

first: Natan

last: Yusupov

first: Calen

middle: P

last: Ryan

first: Hannah-Ruth

last: Engelbrecht

first: Joanne

last: Whitehead

first: Alexandra

last: Halberstam

first: Julia

middle: L

last: MacIsaac

first: Kristy

last: Dever

first: Toan

middle: K

last: Tran

first: Kim

last: Korinek

first: Zachary

last: Zimmer

first: Nanette

last: Lee

middle: W

last: McDade

first: Christopher

last: Kuzawa

last: Huffman

first: Daniel

last: Belsky

first: Elisabeth

middle: B

last: Binder

first: Darina

last: Czamara

first: Keegan

last: Korthauer


title: Dissection of the immune landscape in Psoriatic Arthritis patients

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

summary: Despite significant advancements in psoriatic arthritis (PsA) treatment modalities, a considerable proportion of patients continue to experience persistent joint inflammation and functional impairment, unresponsive to the armamentarium of biological disease-modifying antirheumatic drugs (bDMARDs) or Janus kinase (JAK) inhibitors. The etiology of resistance in these cases is complex, likely involving intricate immunological and genetic factors which are currently mostly unknown. Identifying novel biomarkers and targets for refractory disease is urgently needed. Here, utilizing single-cell RNA-sequencing of synovial fluid from 41 PsA patients and 9 Osteoarthritis (OA) controls we map the comprehensive immune landscape of PsA. Our analysis reveals major perturbations within the myeloid compartment, identifying distinct cell subsets and altered gene programs in PsA patients. Notably, within the myeloid compartment, refractory patients exhibit elevated expression of interferon- gamma induced gene programs linked to the immunoproteasome and MHC class I antigen presentation, at both the gene and protein level. These results suggest a potential interaction of the immunoproteasome and MHC class I antigen presentation in refractory PsA patients with sustained inflammation. Our research imparts critical insights into PsA pathogenesis, paving the way for both molecular level patient classification and potentially new therapeutic interventions for currently treatment resistant patients.

overall_design: Patients diagnosed with psoriatic arthritis according to the Classification Criteria for Psoriatic Arthritis (CASPAR criteria) (51), who had a personal history of psoriasis and active knee involvement were recruited to the study from the rheumatology departments at Sourasky Medical Center in Israel between June 2018 until April 2023. A control group of nine patients with active knee osteoarthritis was also recruited, either from the rheumatology department or the orthopedics surgery department at Sourasky Medical center. Additionally, seven healthy volunteers were recruited to provide blood samples. The study was approved by the Institutional Review Boards of Weizmann Institution of Science, Rehovot, Israel. All participants provided a written informed consent (in accordance with the Helsinki declaration) prior to sample collection. PsA patients were followed for a minimum of six months from the time of sampling (range) with disease activity assessed using the Disease Activity in Psoriatic Arthritis (DAPSA) score (16) calculated at both the sampling time and during follow up visits. Patients were categorized into four groups based on their clinical response to treatment (assessed using the DAPSA score). (For treatment-naïve patients, we considered the response to the first bDMARD treatment administered following the initial sampling. In case of patients with longitudinal samples (n=6), we included the samples from the last visit. ): Responders, achieved remission during follow up visits (DAPSA ≤ 4); Partial responders, exhibit low disease activity during follow up visits (4 ˂ DAPSA ≤ 14); Non-responders, displayed moderate to high disease during follow up visits (DAPSA ˃14); Refractory, displayed moderate to high disease (DAPSA>14) following three or more sequential bDMARDs/tsDMARD (specifically JAKi) with at least two different mode of action.

first: Reut

last: Tzemach

first: Chamutal

last: Gur

first: Shuang-Yin

last: Wang

first: Eyal

last: David

first: Mor

last: Zada

first: Meirav

last: Shmueli

first: Kfir

last: Mazuz

first: Truong

last: Phan

first: Fadi

last: Sheban

first: Vika

last: Furer

first: Arik

last: Polachek

first: Smadar

last: Gertel

first: Nimrod

last: Snir

first: Sara

last: Pel

first: Sharon

last: Nevo

first: Yifat

last: Merbl

first: Daphna

last: Paran

first: Ori

last: Elkayam


title: Divergent Evolution of Malignant Subclones: A Balance Between Induced Aggressiveness and Intrinsic Drug Resistance in cutaneous T-cell lymphoma

first: Terkild

middle: Brink

last: Buus

address: Blegdamsvej 3

department: ISIM

institute: University of Copenhagen

summary: Patients with leukemic cutaneous T-cell lymphoma (L-CTCL) have a poor prognosis due to development of drug-resistance and severe bacterial infections. Here, we use multimodal single-cell T-cell receptor and cellular indexing of transcriptomes and epitope sequencing (scTCR+CITE-seq) to show that most L-CTCL patients harbor multiple genetically distinct subclones that express an identical clonal antigen receptor but display distinct phenotypes and functional properties. These co-existing malignant subclones exhibit differences in tissue homing, metabolism, and cytokine expression, and respond differently to extrinsic factors like Staphylococcus (S.) aureus and cancer drugs. Indeed, while S. aureus toxins selectively enhance activation and proliferation of certain subclones, these responsive subclones are also the most intrinsically sensitive to cancer drugs when the stimuli are removed

overall_design:
Whole transcriptome (GEX), T-cell receptor alpha-beta (TCRαβ), and surface protein (antibody derived tags; ADT) expression profiles of dissociated lesional skin separated into dermis and epidermis and matched peripheral blood mononuclear cells (PBMCs) from 23 leukemic cutaneous T-cell lympoma (L-CTCL) patients was analyzed by scTCR+CITE-seq. Also includes PBMCs cultured in the presence or absence of Staphylococcus aureus enterotoxins or the anti-cancer drug Romidepsin at several time points between 4 and 144 hours. For some patients only PBMCs were analyzed.

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

first: Niels

last: Ødum


title: IT-scC&T-seq streamlines scalable, parallel profiling of protein-DNA interactions in single cells

status: Public on Jun 11 2025

first: Jingchun

last: Ma

state: Hong Kong

address: Sassoon Rd 21, Laboratory Block L3-53

department: School of Biomedical Sciences, LSK Faculty of Medicine

institute: The University of Hong Kong

summary: Single-cell profiling protein-chromatin interactions is often constrained by complex workflows, high cost, or dependence on specialised equipment. We present indexed tagmentation-based single-cell CUT&Tag-sequencing (IT-scC&T-seq), a modular, plate-based strategy using three-round combinatorial barcoding. IT-scC&T-seq robustly profiles histone modifications and transcription factors with high specificity and throughput, supporting simultaneous analysis of multiple samples and epitopes. Notably, it enables sensitive single-cell mapping of lamina-associated domains, low-abundance chromatin features previously difficult to resolve. Applied to adult mouse mammary gland, the method reveals cell-type-specific chromatin landscapes and lineage-regulatory dynamics. Together, IT-scC&T-seq provides a scalable, cost-effective, and broadly accessible approach for high-resolution chromatin profiling.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Indexed tagmentation-based single-cell CUT&Tag-sequencing (IT-scC&T-seq) of (1) H3K4me3 in mixed HEK293T and B6 mouse embyronic stem cells; (2) H3K4me3, H3K27me3, RNA-PolII and CTCF in K562; (3) LMNB1 in mixed H1 human embyronic stem cells, K562, HEK293T, and H1-derived MSCs; (4) multiple epitope (H3K4me3, H3K36me3, H3K27me3 and CTCF) across MSCs, H1-ESCs, and HEK293T; (5) H3K4me3 and H3K27ac in 10-week female WT mice mammary gland.

first: Wei

last: Jin


title: RNA expression profiles of BLID shRNA vs. control (scramble) shRNA transduced breast cancer cells

city: Washington

first: Usha

last: Kasid

state: DC

address: 3870 Reservoir Rd, NW

institute: Georgetown University Medical Center

summary: Downregulation of BLID in breast cancer has been correlated with poor disease-free survival and overall survival.  We used RNA arrays to study the effects of BLID knockdown on gene expression profiles and to identify downstream effectors of BLID in breast cancer cells.

overall_design: The Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays (HG-U133 Plus 2.0) were used for transcriptomics analysis of MDA-MB-231, LCC9 and MCF7 breast cancer cells stably transfected with BLID Mission shRNA lentiviral clone (SHCLNV,TRCN0000254447) or control shRNA particles (SHC202V, Mission@TRC2 pLKO.5-puro). Transcriptomics data were obtained from four RNA arrays per treatment group (BLID shRNA knockdown vs. control shRNA) in each cell line.

first: Rong

last: Hu

first: Rency

last: Varghese

first: James

last: Li

first: Xiaojun

last: Zou

first: Habtom

last: Ressom

first: Robert

last: Clarke


title: miRNA expression profiles of BLID shRNA vs. control (Scramble) shRNA transduced breast cancer cells

summary: Downregulation of BLID in breast cancer has been correlated with poor disease-free survival and overall survival.  We used miRNA arrays to study the effects of BLID knockdown on miRNA expression profiles in breast cancer cells.

overall_design: The Affymetrix GeneChip miRNA 3.0 arrays were used for miRNomics analysis of MDA-MB-231, LCC9 and MCF7 breast cancer cells stably transfected with BLID Mission shRNA lentiviral clone (SHCLNV,TRCN0000254447) or control shRNA particles (SHC202V, Mission@TRC2 pLKO.5-puro). MicroRNA profiles were obtained from three microRNA arrays per treatment group (BLID shRNA knockdown vs. control shRNA) in each cell line.


title: In situ pharmacological induction of pancreatic beta-cell regeneration by THR-123, a cyclic peptide with BMP-7-like activity

city: Miami

first: Juan

last: Domínguez-Bendala

state: FL

address: 1450 NW 10th Ave

department: Diabetes Research Institute

institute: University of Miami Miller School of Medicine

summary: The demonstration that BMP signaling activates progenitor-like populations within pancreatic ducts supports the potential use of BMP receptor agonists to induce islet regeneration in situ. In this context, we tested the ability of THR-123, a cyclic peptide with BMP-7-like activity, to restore b-cell mass in diabetic mice. Here we show that treatment with THR-123 restores normoglycemia through the rapid formation of new BrdU-labeled islets in the vicinity of ducts. Neogenic islets, unlike those from non-diabetic controls, feature an extensive intrainsular network of ductal tissue. The earlier stages of THR-123-induced b-cell neogenesis were reproduced in live pancreatic slices, an organotypic model that allowed us to visualize ductal cells transitioning to glucose-responsive insulin-expressing cells in real time. scRNAseq analyses further confirmed that this transition occurs through a hybrid ducto-acinar stage similar to that previously reported in humans. These results pave the way to the design of pharmacological strategies to treat insulin-dependent diabetes.

overall_design:
We conducted scRNAseq analysis of murine pancreatic slices generated from either non-diabetic or alloxan-induced diabetic mice. mPSs (n=6) from non-diabetic mice were sequenced after 5 days of culture (positive control). Slices from diabetic mice were either treated with THR-123 for 5 days (experimental group) or left untreated for the same period (alloxan, negative control) prior to sequencing. The experiment was repeated three times, and cells pooled for each of the three groups. In total, ~32,000 single cells were analyzed using the 10X Genomics 5’ Single Cell platform. Libraries were filtered for quality control and subjected to unsupervised clustering, integration and differential gene expression analysis using Seurat v4.1.1. We analyzed individual datasets as well as the integration of the alloxan (negative) control and the THR-123 (experimental) datasets to perform dynamic RNA velocity studies.
additional grants 1R01DK130846-01 and U01DK120393: Juan Dominguez-Bendala and Ricardo L Pastori

first: Silvia

last: Álvarez-Cubela

first: Isabella

last: Altilio

first: Mayur

last: Doke

first: Dagmar

last: Klein

first: Ricardo

last: L Pastori


title: PHGDH acts as an RNA-binding protein to stabilize PRKCD mRNA and promote hepatocellular carcinoma progression

city: chongqing

first: bin

last: cheng

address: No. 1, Medical College Road, Yuzhong District, Chongqing

department: Department of Infectious Diseases, Key Laboratory of Molecular Biology for Infectious Diseases

institute: chongqing medical university

summary: Overexpression of PHGDH in MHCC-97H cells, enrichment of PHGDH-bound RNA by antibody, and identification of target genes using high-throughput sequencing

overall_design: One group of MHCC-97H cells overexpressed PHGDH, and the other group served as a control. Collect cells for RIP sequencing

first: Bin

last: Cheng

first: Shi

first: Pai

last: Peng

first: Kai

last: Wang

first: Ni

last: Tang

first: Ailong

last: Huang


title: Cardiomyocyte OTUD1 drives diabetic cardiomyopathy via directly deubiquitinating AMPKα2 to inhibit AMPK activity and induce mitochondrial dysfunction

city: hangzhou city

first: liu

middle: guo

last: xuan

address: No. 182 Tianmushan Road, Hangzhou City, Zhejiang Province

institute: Hangzhou Medical College

summary: To analyze the mRNA profile of DUBs in diabetic heart, we performed a scRNA-seq of  heart cells from the wild type (WT) mice and db/db mice, a typical T2DM model mouse.

overall_design: wild type (WT) mouse (n=1) and  typical T2DM model mouse (n=1)

first: Guang

last: Liang

first: Xue

last: Han


title: Single-nucleus transcriptomic analysis of the amygdala of mice in acute and chronic neuropathic pain

city: Philadelphia

first: Gregory

last: Corder

address: 125 S. 31st Street

institute: University of Pennsyvannia

summary: Single-nucleus RNA sequencing was used to profile transcriptional differences in mice in uninjured or neuropathic chronic pain conditions and in response to multiple sensory stimuli to identify cell subtype-specific changes in the transcriptome due to pain

overall_design: We performed the 10x Genomics 3' gene expression assay (v3.1). Nuclei were isolated from the right amygdala of adult male mice that were either uninjured or 3 weeks following a left side spared nerve injury to induce chronic neuropathic pain. Uninjured mice were split into groups of 3 biological replicates that received either no stimulus or a light touch to the left hindpaw or a noxious hot water droplet to the left hindpaw. Mice with a spared nerve injury were split into two groups of 3 biological replicates that received either no stimulus or a light touch to the left (injured) hindpaw to induce chronic neuropathic pain. Nuclei capture and library preparation was perormed using 10x genomics.

first: Wojick

last: Jessica

first: Alekh

last: Paranjapye

first: Erica

last: Korb

first: Richard

last: Crist

first: Benjamin

last: Reiner


title: Uncovering Cell Type-Specific Phenotypes Using a Novel Human In vitro Model of Transthyretin Amyloid Cardiomyopathy

city: Pamplona

first: Asier

last: Ullate-Agote

state: Navarra

address: Av. de Pío XII, 55

department: Hemato-Oncology

institute: CIMA

summary: Transthyretin amyloid cardiomyopathy (ATTR-CM) is characterized by the misfolding of transthyretin (TTR), fibrillogenesis, and progressive amyloid fibril deposition in the myocardium, leading to cardiac dysfunction with dismal prognosis. In ATTR-CM, either destabilizing mutations (variant TTR, ATTRv) or ageing-associated processes (wild-type TTR, ATTRwt) lead to the formation of TTR amyloid fibrils. Due to a lack of representative disease models, ATTR-CM disease mechanisms are largely unknown, thereby limiting disease understanding and therapeutic discovery. Methods and Results: Here, we report a novel in vitro ATTR-CM model which uncovers cell type-specific disease phenotypes by exposing the three major human cardiac cell types to TTR fibrils, thereby providing novel insights into the cellular mechanisms of ATTR-CM disease. Human recombinant TTR proteins (WT, V122I, V30M) and respective fibrils were generated and characterized using Thioflavin T, Amytracker, Congo red and dot blot analyses. Seeding human induced pluripotent stem cell-derived-cardiomyocytes (hiPSC-CMs) and endothelial cells (ECs) on TTR fibrils resulted in reduced cell viability. Confocal microscopy revealed extracellular localization of TTR fibrils to hiPSC-CMs, leading to sarcomere disruption, altered calcium handling and disrupted electromechanical coupling, while ECs showed a reduced migration capacity with aberrant cell morphology. hiPSC-fibroblasts (hiPSC-FBs) were largely unaffected by TTR fibrils, presenting normal viability, but showing enhanced localization with TTR fibrils. Our model shows that WT and variant TTR fibrils lead to cell type-specific phenotypes, providing novel insights into the underlying cellular disease mechanisms of ATTR-CM, thereby facilitating the identification of novel therapeutic targets and biomarkers.

overall_design: Bulk RNA-seq for hiPSC derived cardiomyocytes (hiPSC-CMs), HUVEC endothelial cells (Ecs) and hiPSC derived fibroblasts (hiPSC-FBs) exposed to 2D seeded TTR fibrils derived from WT and variant TTR (V30M and V122I) for a 4-day (hiPSC-CMs) or 2-day (ECs, hiPSC-FBs)incubation period

first: Jiabin

last: Qin

first: Alain

last: van Mil

first: Olalla

last: Iglesias-Garcia


title: BAHCC1 promotes gene expression in neuronal cells by antagonizing SIN3A-HDAC1

first: Igor

last: Ulitsky

address: Hertzl St.

summary: Chromatin modifications play a key role in the regulation of gene expression during development and adult physiology. Histone acetylation, and in particular H3K27ac is associated with increased activity of gene regulatory elements. The regulation of the machinery that deposits or strips this modification remains poorly understood. In particular, the SIN3A-HDAC1 complex possesses a histone deacetylation activity, yet it commonly resides at active regulatory regions. Here, we study BAHCC1, a large chromatin-associated protein essential for viability, and recently reported to play a largely repressive role. We show that in cells of the neuronal lineage, BAHCC1 is most associated with regulatory elements marked with H3K27ac. BAHCC1 interacts and co-occupies shared genomic regions with the SIN3A scaffold protein and its perturbations lead to decreased acetylation and decreased expression of the proximal genes in a neuronal cell line and in primary cortical neurons. The regulated genes are enriched for those functioning in neurogenesis and cell migration. We propose a model in which BAHCC1 antagonizes SIN3A histone deacetylation and positively regulates the expression of genes important in the neuronal lineage.

overall_design: RNA-seq and CUT&RUN from N2a and mouse primary cortical neurons after perturbing Bahcc1 expression

first: Alan

last: Monziani


title: Distal Electrical Stimulation Enhances Neuromuscular Reinnervation and Satellite Cell Differentiation for Functional Recovery

city: Tainan

first: Chunwei

last: Lin

address: No. 1, Daxue Rd., East Dist.

country: Taiwan

institute: National Cheng Kung University

summary: Peripheral nerve injuries cause significant motor deficits, with limited treatment options for full recovery. Distal electrical stimulation (E-stim) shows potential in promoting neuromuscular reinnervation, although its mechanisms remain unclear. This study examines how distal E-stim influences neuromuscular junction reinnervation and Satellite cell activity in denervated muscles.

overall_design: The satellite cell cluster was identified using the Seurat package in R and subsequently subjected to gene set enrichment analysis for further insights.

first: Chun-Wei

first: Szu-Han

last: Chen

first: SiaoMuk

last: Cheng

first: Tzu-Chun

last: Chung

first: Wentai

last: Liu

first: Daw-Yang

last: Hwang

first: Song

last: Li

first: Sheng-Che

first: Yuan-Yu

last: Hsueh


title: Deciphering the role of CAPZA2 in neurodevelopmental disorders: Insights from mouse models

status: Public on Jun 10 2025

city: Beijing

first: Yong

email: yongcheng@muc.edu.cn, guomei153@163.com

address: Zhongguancun South St, Haidian District

institute: College of Life & Environmental Science

summary: Intellectual disability affects 1%-3% of the global population, with many unidentified genetic causes. This study investigates the role of CAPZA2, an actin cytoskeleton regulator, in neurodevelopmental disorders using CAPZA2 heterozygous knockout (CAPZA2+/−) and heterozygous point mutant (CAPZA2c.G776T/+) mice. CAPZA2+/- and CAPZA2c.G776T/+ mice demonstrated notable decreases in CAPZA2 expression within the hippocampus and prefrontal cortex (PFC), crucial for learning and memory. Interestingly, the reduction of CAPZA2 in CAPZA2c.G776T/+ mice is less than 50%. Behavioral assays revealed that CAPZA2+/- mice exhibited motor dysfunction and anxiety-like behaviors, along with impairments in both spatial and non-spatial memory, accompanied by deficits in social interactions. These phenotypic manifestations were also mirrored in the CAPZA2c.G776T/+ mice, thus validating the genotype-phenotype correlation. Morphological analyses of these gene-edited mice indicated structural synaptic impairments, with increased dendritic spine density, altered spine morphology in the hippocampus, and decreased dendritic complexity in the PFC. Single-cell RNA-seq analysis of hippocampal tissue in CAPZA2+/- mice showed widespread transcriptional dysregulation, affecting neurodevelopment and synaptic function genes. The altered PSD95 and glutamate receptor levels support these findings. These in-sights highlight the gene's essential role in brain development and function, with po-tential implications for understanding and treating neurodevelopmental disorders.

overall_design: This study performed single-cell RNA sequencing on hippocampal tissues from wild-type (WT) and CAPZA2 heterozygous knockout (CAPZA2+/-) mice to compare cell population proportions.

first: Mei

last: Guo

first: Liming

first: Xiao

last: Mao

first: Manyu

last: Xiao

first: Xiaobin

last: He

first: Xing

last: Pan

first: Yuewen

first: Wanying

last: Yi

first: Qibin

first: Xianglan

last: Piao

first: Hua

last: Wang

first: Yang

last: Du


title: Viral-Track integrated single-cell RNA-sequencing reveals HBV lymphotropism and immunosuppressive microenvironment in HBV-associated hepatocellular carcinoma

city: Wuhan

first: Tanzeel

last: Yousaf

state: Hubei

address: 185 Donghu Road

department: Taikang Medical School (School of Basic Medical Sciences)

institute: Wuhan University

summary: The tumor microenvironment (TME) is a crucial mediator of tumor progression and treatment response. Here, we compare the immune microenvironments of HBV and non-HBV-HCC and investigate the reason for the persistence of HBV infection in the liver. We combine the Viral-Track method with single-cell RNA sequencing and profile the transcriptomes of 71,466 cells from HBV and non-HBV-HCC patients. In addition to hepatocytes and macrophages, HBV transcripts were also detected in T and B cells using the Viral-Track method, confirming the HBV-lymphotropic nature for the first time in scRNA-sequencing data. HBV-HCC tumors have reduced levels of CD4+ Tregs, NK, macrophages, dendritic cells (DCs), and increased malignant hepatocytes compared with non-HBV tumors. Notably, we explicitly reported the enrichment of metallothioneins (MTs), particularly MT1G expression in HBV-related HCC TAMs, which was associated with a worse prognosis. HBV-tumor-infiltrated CD8+ T cells exhibited a cytotoxic dysfunctional T cell phenotype, characterized by upregulated MDK and CTLA4 expression and reduced IFN-γ production, unlike the non-HBV-HCC. Additionally, HBV-HCC exhibited immunosuppressive ligand-receptor interactions, whereas non-HBV-HCC exhibited antitumor ligand-receptor interactions. Our comprehensive understanding of the  HBV HCC ecosystem by viral-track integrated scRNA sequencing analysis provides deeper insights into immune evasion mechanisms and HBV lymphotropism associated with viral persistence.

overall_design:
This study examined the cellular diversity and immunosuppressive microenvironment in HBV and non-HBV-related hepatocellular carcinoma (HCC) tumors. Surgical specimens from 5 HBV-positive and 3 non-HBV patients underwent single-cell RNA sequencing (scRNA-seq) and Viral-Track analysis.Single-cell suspensions (700-1200 cells/µl) were prepared and converted into scRNA-seq libraries using the Chromium Single Cell Library Kit (10x Genomics). Both HBV and non-HBV samples from each patient were processed in parallel within the same thermal cycler for consistency.Sequencing libraries were generated and sequenced on the Illumina Novaseq platform. Data processing with Cell Ranger (v3.1.0) included demultiplexing, alignment, UMI counting, and filtering. Clean reads were aligned to the GRCh38 reference genome to quantify gene expression.The Seurat R package (v3.1.5) was used to filter, preprocess, and normalize the gene-barcode matrices for each case. The study compared HBV and non-HBV tumors to uncover differences in immune profiles and the tumor microenvironment.

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

first: Zhepeng

first: Wajeeha

last: Naz

first: Junwei

last: Sun


title: TRIM33 loss reduces Androgen Receptor transcriptional output and H2BK120 ubiquitination [RNA-seq]

city: Amsterdam

first: Wilbert

last: Zwart

state: Noord-Holland

address: Plesmanlaan 121

department: Oncogenomics

country: Netherlands

institute: Netherlands Cancer Institute

summary: The Androgen Receptor (AR) is a ligand-dependent transcription factor that drives prostate cancer development and progression. Although, a detailed effect on AR biology has been described for a number of interacting proteins, many AR coregulators remain to be characterized in relation to their distinct impact on AR function. Here, we describe TRIM33 as a conserved AR-interactor across multiple prostate cancer cell lines. We observed that TRIM33 and AR share overall chromatin interaction profiles, in which TRIM33 is involved in downstream responsive transcriptomic output. In contrast to prior reports, we show that TRIM33 does not impact AR protein stability, but instead propose a model in which TRIM33 facilitates maximal AR activity by interfering with H2BK120 ubiquitination levels.

overall_design: RNA-seq was performed in triplicate in LNCaP and 42D prostate cancer cells treated with 5nM R1881 for 6h or DMSO

first: Nils

last: Eickhoff

first: Andries

last: Bergman


title: TRIM33 loss reduces Androgen Receptor transcriptional output and H2BK120 ubiquitination [ChIP-seq]

overall_design: ChIP-seq for Androgen receptor (AR), TRIM24, TRIM33, H3K18ac, H3K9me3 and H2BK120ub were performed in triplicate in LNCaP prostate cancer cells (NT = non-targeting, C2 = TRIM33 monoclonal knockout) treated with 10nM R1881 or DMSO for 4h or 24h.


title: Non-canonical activating roles of RCoR2 sustain transcription in adrenergic neuroblastoma [RNA-Seq, 2]

city: Bologna

first: Giorgio

last: Milazzo

address: Via F.Selmi 3

department: FaBiT

institute: University of Bologna

summary: CoREST complexes (LSD1, HDAC1/2 and RCoR1/2/3) are pivotal in neurodevelopment and have long been recognized as transcriptional repressors across various cancers. However, distinct roles of RCoR factors remain underexplored. Here, we unveil non-canonical functions of RCoR2 in MYCN-amplified neuroblastoma (NB), underscoring its unique significance compared to its paralogues. This insight shifts the paradigm, highlighting RCoR2 as a key determinant of the NB chromatin landscape. Our findings demonstrate that RCoR2 is a super-enhancer driven gene, which, unlike RCoR1, acts as a positive regulator of gene expression as a partner of the adrenergic NB core regulatory circuitry (CRC). We propose a model in which RCoR2 facilitates interactions between CRC-bound enhancers and their associated transcription start sites, thereby sustaining the expression of genes critical for neuroblastoma cell survival. Thus, we identify RCoR2 as a critical vulnerability in high-risk neuroblastoma and a promising target for cancer therapeutics.

overall_design: To investigate the transcriptional roles of RCoR1 and RCoR2 we established neuroblastoma Kelly cell lines in which each target gene has been conditionally (dox-on) knocked down by shRNA. As a control, we also generated Kelly cells conditionally expressing a non-targeting shRNA (shCTRL). For each targeting and non-targeting shRNA, we then performed RNA-seq in non-treated conditions (NT) and treated conditions 72h.

first: Sara

last: Aloisi

first: Berkely

last: Gryder

first: Giovanni

last: Perini


title: Non-canonical activating roles of RCoR2 sustain transcription in adrenergic neuroblastoma [ChIP-seq 1]

overall_design:
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Pol2 and histone modifications H3K27ac and H3K4me2 in Kelly and LAN5 neuroblastoma cells.
ChIP-seq experiments were performed in Kelly and LAN5 cells conditionally (dox-on) expressing shRNA targeting RCOR1 or RCOR2.


title: Non-canonical activating roles of RCoR2 sustain transcription in adrenergic neuroblastoma [ChIP-Seq 2]

overall_design:
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for RCoR1, RCoR2, RCoR3 and LSD1 in Kelly neuroblastoma cells.
LSD1 ChIP-seq was performed from Kelly cells conditionally (dox-on) expressing an shRNA against RUNX1T1 under non-treated conditions.


title: Non-canonical activating roles of RCoR2 sustain transcription in adrenergic neuroblastoma [RNA-Seq]

overall_design: To investigate the transcriptional roles of RCoR1 and RCoR2 we established neuroblastoma Kelly cell lines in which each target gene has been conditionally (dox-on) knocked down by shRNA. As a control, we also generated Kelly cells conditionally expressing a non-targeting shRNA (shCTRL). For each targeting and non-targeting shRNA, we then performed RNA-seq in non-treated conditions (NT) and treated conditions (24 - 48h).


title: Non-canonical activating roles of RCoR2 sustain transcription in adrenergic neuroblastoma

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

overall_design: Refer to individual Series

first: Berkley


title: Effect of the expression of HP1 proteins from different species (mouse, fruit fly, fission yeast) in mouse 3T3 cells

city: Toulouse

first: Fabian

last: Erdel

address: 169 Avenue Grunberg-Manago

department: MCD

country: France

institute: CNRS, CBI Toulouse

summary: HP1 proteins play a role in heterochromatin regulation and higher-order chromatin organization. The domain structure of HP1 is conserved, but the properties and functions of HP1 homologs from different organisms might vary. To address this issue, we expressed HP1 from different species in mouse 3T3 cells and assessed the impact on gene expression by RNA-seq.

overall_design: To test the effect of different HP1 homologs on gene expression, we established stable 3T3 cell lines with GFP-tagged HP1 from fission yeast, fruit fly and mouse under the control of a doxycycline-inducible promoter. We then performed total RNA-seq in doxycycline-induced (GFP-positive during sorting) and uninduced cells.

first: Sanâa

last: Bensaha

first: Michela

last: Anfossi


title: Cellular crosstalk mediated by TGF-β drives epithelial-mesenchymal transition in patient-derived multilineage biliary organoids [scRNA-seq]

city: Dallas

first: Jorge

middle: A.

last: Bezerra

state: Texas

address: 5323 Harry Hines Blvd.

department: Department of Pediatrics

institute: University of Texas Southwestern Medical Center

summary: The developmental integrity of bile duct epithelium and peribiliary glands (PBGs) plays a key role in maintaining bile flow and preventing liver diseases. We emplpoyed multilineage biliary organoids (MBOs) engineered from co-cultures of human liver epithelial cells, umbilical vein endothelial cells (HUVECs), and mesenchymal stem cells (MSCs) to model biliary development and pathogenesis of biliary atresia (BA). Self organization of these cells into MBOs recapitulated the dual compartmentalization of bile ducts with the epithelial layer and PBGs. Notably, BA patient-derived MBOs exhibited markers of epithelial-mesenchymal transition (EMT) and activated TGF-β/Activin-SMAD2/3 signaling pathways, suggesting a link to the delayed maturation of bile duct epithelium. Inhibition of TGF-β signaling suppressed EMT, promoted epithelial maturation, and improved organoid structure. In in vivo, TGF-β inhibitor treatment in a neonatal mouse model of BA reduced biliary injury and liver fibrosis. These data support the critical role of epithelial and mesenchymal cell interactions in bile duct development, give insight into molecular mechanisms of BA, and point to the therapeutic potential of targeting TGF-β signaling.

overall_design: MBOs generated by co-culturing HUVECs and human MSCs with dissociated epithelial organoid cells derived from liver biopsies of controls (n=4) and BA patients (n=4) were subjected to scRNA-seq after 14 days of culturing.

first: Ayabe

last: Hiroaki

first: DePasquale

last: Erica A K.

last: Li

first: Amarachintha

last: Surya P.

first: Nalluri

last: Shreya

first: Fox

last: Sejal R.

first: Mourya

last: Reena

first: Konishi

last: Ken-ichiro

first: Shivakumar

last: Pranavkumar

first: Bezerra

last: Jorge A.


title: Non-canonical activating roles of RCoR2 sustain transcription in adrenergic neuroblastoma [HiChIP]

first: Giorgio

last: Milazzo

overall_design:
HiChIP DNA-sequencing for H3K27ac and Pol2 in Kelly neuroblastoma cells.
HiChIP experiments were performed in Kelly cells conditionally (dox-on) expressing shRNA targeting RCOR2.

first: Sara

last: Aloisi


title: Non-canonical activating roles of RCoR2 sustain transcription in adrenergic neuroblastoma [ATAC-seq]

summary: CoREST complexes, composed of LSD1, HDAC1/2, and RCoR1/2/3, are pivotal in neurodevelopment and have long been recognized as transcriptional repressors across various cancers. However, distinct roles of the three RCoR factors remain underexplored. Here, we unveil non-canonical functions of RCoR2 in MYCN-amplified neuroblastoma (NB), underscoring its unique significance compared to its paralogues. This novel insight shifts the paradigm, highlighting RCoR2 as a key determinant of NB chromatin landscape.NB cell growth and tumorigenesis critically depend on RCoR1 and 2, with the RCOR2 gene exhibiting high histone acetylation levels and selective increased expression in NB. Unlike the well-known repressive roles of RCoR1, RNA-seq analyses demonstrate RCoR2 as a positive regulator of gene expression. RCoR2 predominantly occupies active promoters and regions of open chromatin marked by H3K27ac and Pol2, defining its distinct binding pattern compared to RCoR1, which primarily targets enhancers. Surprisingly, CoREST complexes co-occupy chromatin with NB core regulatory transcription factors (CRTFs), which positively drive NB-specific signatures. RCoR2, and not RCoR1, interacts with CRTFs, regulates their expression and its transcription is activated through a strong Super-enhancer. Collectively, these data indicate RCoR2 as a new component of the adrenergic NB core regulatory circuitry (CRC). Mechanistically, H3K27ac and H3K4me2 levels are not drastically altered upon RCoR2 reduction, suggesting RCoR2 is not the primary driver of CoREST complexes’ catalytic activity. Instead, HiChIP data indicate that RCoR2 mediates chromatin looping, facilitating enhancer-promoter interactions and maintaining 3D chromatin architecture to sustain oncogenic transcriptional programs. Taken together, our data underscore RCoR2 as a key oncogenic determinant in neuroblastoma due to its role in chromatin landscape. We propose a model in which RCoR2 facilitates interactions between CRTF-bound enhancers and their associated transcription start sites, thereby sustaining the expression of genes essential for neuroblastoma survival. Consequently, we reveal RCoR2 as a novel critical vulnerability in high-risk neuroblastoma and a promising target for cancer therapeutics.

overall_design: ATAC-seq in Kelly neuroblastoma cells conditionally (dox-on) expressing shRNA targeting RCOR1 or RCOR2.

first: Berkely

last: Gryder

first: Giovanni

last: Perini


title: EBNA Leader Protein is required to reshape chromatin architecture during B cell transformation by Epstein-Barr Virus (RNA-Seq)

first: Italo

last: Tempera

address: 3601 Spruce Street

institute: The Wistar Institute

summary: Epstein-Barr virus Nuclear Antigen Leader Protein (EBNA-LP) plays a pivotal role in the transformation of B cells by Epstein-Barr virus (EBV), functioning independently of EBNA2 to regulate chromatin architecture and gene expression. Our study reveals that EBNA-LP binds to chromatin regions distinct from EBNA2 and facilitates the formation of long-distance chromatin loops by interacting with the cellular factor YY1. This interaction reconfigures the three-dimensional structure of the host genome, enhancing the integrity of topologically associating domains (TADs) and promoting the interaction between enhancers and promoters within these domains. In EBV-infected B cells, EBNA-LP strengthens YY1-mediated chromatin loops within TADs, which helps maintain stable regulatory programs essential for B cell transformation. Notably, EBNA-LP is crucial for establishing EBV-induced enhancers, yet it is not required for their maintenance once formed. Additionally, our data suggest a compensatory increase in CTCF binding in the absence of EBNA-LP, leading to more promiscuous chromatin interactions between TADs and a reduced TAD insulation at their boundaries. These findings provide new insights into the molecular mechanisms by which EBNA-LP contributes to B cell transformation and highlight potential therapeutic targets for disrupting EBV-driven oncogenesis

overall_design: RNA-seq was used to detect differences in gene expression between EBNA-LP WT and KO cell lines

first: Davide

last: Maestri

first: Lisa

last: Caruso

first: Jana

last: Cable

first: Rachel

last: Sklutuis

first: Sarah

last: Preston-Alp

first: Robert

last: White

first: Micah

last: Luftig


title: EBNA Leader Protein is required to reshape chromatin architecture during B cell transformation by Epstein-Barr Virus


title: TRIM33 loss reduces Androgen Receptor transcriptional output and H2BK120 ubiquitination

first: Wilbert

last: Zwart

first: Nils

last: Eickhoff

first: Andries

last: Bergman


title: EBNA Leader Protein is required to reshape chromatin architecture during B cell transformation by Epstein-Barr Virus (HiC)

overall_design: In situ HiC was used to capture chromatin conformation in B cells before and after EBV-driven transformation and in EBNA-LP WT and KO cell lines


title: small RNA-seq of rice

status: Public on Jun 09 2025

city: Fuzhou

first: Jing

last: Zou

address: 15 shangxiadian road

institute: Fujian Agriculture and Forestry University

type: Non-coding RNA profiling by high throughput sequencing

summary: small RNA-seq of rice

overall_design: Using 500 ng total RNA or enriched small RNA200ng, the 3' splices and 5' splices were connected respectively and then reverse-transcribed using RT primer as primer, using the small RNAs attached to the 5' and 3' splices as templates, cDNA was synthesized under the action of reverse transcriptase. Universal Primer and PCR Primer were used as primers of the two strands respectively, and the first strand of reverse transcriptional cDNA was used as a template to amplify and enrich the fragments. Finally, the glue was cut to recover the small RNA fragments.


title: Transcriptional landscape of the cell cycle in a model thermoacidophilic archaeon reveals similarities to eukaryotes

city: paris

first: yuping

last: dia

address: 4 pl.jussieu

institute: sorbonne university

summary: The cell cycle is a series of events that occur from the moment of cell birth to subsequent cell division. In eukaryotes, the cell growth, genome replication, genome segregation and cytokinesis, are strictly coordinated, defining discrete cell cycle phases, whereas in bacteria, these key processes may occur concurrently. Thermoacidophilic archaea in the genus Saccharolobus follow a defined cell cycle program, with the first pre-replicative growth (G1) phase, followed by the chromosome replication (S) phase, the second growth (G2) phase, and rapid genome segregation (M) and cytokinesis (D) phases. However, whether other processes, such as metabolism, catabolism, protein translation, antiviral defense, etc., also occur at specific cell cycle phases, like in eukaryotes, or are active throughout the cell cycle, like in bacteria, remains unclear. To address these questions, we synchronized the cultures of S. islandicus and performed an in-depth transcriptomic analysis of samples enriched in cells undergoing the M-G1, S and G2 phases. Differential gene expression and consensus gene co-expression network analyses provided a holistic view of the unfolding of the S. islandicus cell cycle. In addition to the core transcriptome network, which is expressed throughout the cell cycle, we show that diverse metabolic pathways, protein synthesis, cell motility and even antiviral defense systems, are expressed in a cell cycle dependent fashion. Our data also refines the understanding of the processes previously known to be linked to cell cycle, such as DNA replication. We show that most DNA replication genes are expressed prior to the S phase, during the M-G1, whereas expression of the major chromatin genes, and accordingly, chromatinization are concomitant with replication. A machine learning-based classifier allowed defining sets of signature genes characteristic of each of the analyzed cell cycle phases, emphasizing transcriptional stratification of the phases. Signature genes are more conserved across Thermoproteota than non-signature genes and their peak of expression, especially for the M-G1 and G2 specific genes, matches that of their homologs in yeast. Collectively, our data expose the complexity of the S. islandicus cell cycle and suggest that it more closely resembles the cell cycle of eukaryotes than previously appreciated.

overall_design: Cells were plated on solid media and 15 single colonies were inoculated in liquid media. forming 15 cultures that represent completely independent biological replicates. The 15 biological replicates were grown in 3 different batches (5 cultures per batch), each batch was grown and synchronized at a different time (5 cultures at a time).

first: Miguel

middle: V

last: Gomez-Raya-Vilanova

first: Jérôme

last: Teulière

first: Sofia

last: Medvedeva

first: Yuping

last: Dai

first: Eduardo

last: Corel

first: Philippe

last: Lopez

first: François-Joseph

last: Lapointe

first: Debashish

last: Bhattacharya

first: Louis-Patrick

last: Haraoui

first: Elodie

last: Turc

first: Marc

last: Monot

first: Virginija

last: Cvirkaite-Krupovic

first: Eric

last: Bapteste

first: Mart

last: Krupovic


title: The genotype-integrated gene expression profile at the single-cell level of bone marrow CD34-positive hematopoietic stem and progenitor cells (HSPCs) from a patient with myelodysplastic neoplasms (MDS) harboring germline and somatic DDX41 variants.

city: Kashiwa

first: BIN

last: WU

state: Chiba

address: #304, 6-6-2, Kashiwanoha

department: Research

country: Japan

institute: ImmunoGeneTeqs

summary: DDX41 is recognized as a major gene responsible for myeloid neoplasms associated with germline predisposition. Individuals typically inherit a germline pathogenic variant in one allele and later acquire a somatic variant—most commonly the p.R525H variant—in the other allele, leading to the development of myeloid neoplasms. In this study, we conducted single-cell RNA sequencing integrated with p.R525 site genotyping to investigate how acquiring the p.R525H variant influences gene expression changes in hematopoietic cells.

overall_design: CD34-positive cells were isolated from the bone marrow of an MDS patient carrying germline and somatic DDX41 variants using a magnetic bead-based method. These cells were subjected to scRNA-seq using the terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) method (PMID: 35760847). To distinguish cells with and without the somatic p.R525H variant, amplicons for genotyping DDX41 p.R525 sites were generated from amplified whole transcripts. The genotyping data were then integrated with the transcriptomic profiles for comprehensive analysis.

first: Hirotaka

last: Matsui

first: Shigeyuki

last: Shichino


title: EBNA Leader Protein is required to reshape chromatin architecture during B cell transformation by Epstein-Barr Virus (ChIP-Seq)

first: Italo

last: Tempera

overall_design: Chromatin Immunoprecipitation Sequencing (ChIP-seq) of the transcription factor YY1, RNA Pol II and the histone modification H3K27ac in LCL and Mutu1 cell lines

first: Davide

last: Maestri

first: Lisa

last: Caruso

first: Jana

last: Cable

first: Rachel

last: Sklutuis

first: Sarah

last: Preston-Alp

first: Robert

last: White

first: Micah

middle: A

last: Luftig


title: Role of DDX3X in translation regulation in acute ER stress

city: Baton Rouge

first: Anastasios

last: Vourekas

state: LA

address: 202 life sciences building

institute: Louisiana State University

summary: In eukaryotes, regulation of mRNA translation initiation greatly impacts gene expression, and is critical for cellular stress responses. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in 5´ UTR scanning and start codon decoding in non-stressed and stressed cells is still elusive. Here we show that DDX3X engages with thousands of mRNAs as part of the 48S scanning complex, simultaneously acting to promote or suppress translation of select mRNAs in non-stressed conditions, and switches this regulation in opposite directions to establish a stress response translational program upon acute ER stress. We find distinct DDX3X binding patterns of differentially regulated mRNAs, which lead us to identify N4-acetylation of cytidines surrounding the start codon as a crucial feature for DDX3X-mediated selective regulation. Our findings highlight a novel co-dependence between an RNA helicase and a post-transcriptional modification in regulating mRNA translation

overall_design:
We prepared 10%-50% sucrose gradients in polyallomer tubes for SW-41 rotor using the Gradient Master 108, and collected the polysome profiles using the Piston Fractionator with a TRIAX Flowcell (260 nm) (BioComp). 293FT cells were seeded in 100-mm culture dishes (3 dishes per polysome profile sample) and grown to 80% confluency. 1.5 h prior to collection, cell culture media was replaced with full media containing DMSO (non-stressed condition) or 400 nM Thapsigargin (acute ER stress). Cells were incubated for the last 5 min of the treatment with cycloheximide (CHX) at a final concentration of 100 μg/mL. Cells were then washed twice with ice cold phosphate-buffered saline supplemented with CHX and centrifuged at 4,500 X g for 5 min at 4° C. Cell pellets were lysed in 800 μL of Hypotonic buffer (5 mM Tris HCl pH 7.5, 2.5 mM MgCl2, 1.5 mM KCl, 0.5% Triton X, 0.5% sodium deoxycholate, 100 μg/mL CHX, 2 mM DTT, 200 units/mL RNase inhibitor, 1X protease inhibitor cocktail). Cells were resuspended in the hypotonic buffer either by pipetting or vortexing for 5 sec followed by centrifugation at 17,000 xg for 7 min at 4° C. The supernatant (post-nuclear lysate) OD was measured at 260 nm to determine the amount of lysate needed to be added to sucrose gradient. Samples of the post-nuclear lysates were also kept for western blots and input RNA extraction 500 mL from the top of the sucrose gradient was removed and the lysate volume per condition was adjusted so they contain the same OD between 10 to 20 at 260 nm and added to the top of the sucrose gradient. Each gradient was weighed and balanced before ultracentrifugation at 222,222 xg for 2 hours at 4° C while using low brake option. 14 fractions of 800 μL were collected.
Immediately after polysome fractions were collected, 400 μL of each fraction was mixed with equal volume TRIZOL LS (Thermo) and kept at -80C. RNA extraction was carried out by manufacturer’s instructions. RNA was treated with RQ1 DNase (Promega), followed by phenol extraction and ethanol precipitation. The concentration and the purity of RNA was measured using nanodrop and pure RNA with a 260/280 ratio within 1.8 - 2.0 was acquired. To prepare polyA mRNA RNA-Seq libraries, approximately 800 ng of high-quality total RNA was used per sample. The quality was determined with a Nanodrop spectrophotometer, and only RNA that had a OD260/280 of 1.8-2.0 was used. We followed the Illumina Stranded mRNA Prep, Ligation Reference Guide, Document # 1000000124518 V03 protocol without changes.

first: Abd-El-Monsif

last: Shawky

first: Allison

last: Scarboro

first: Kenneth

last: Avanzino

first: Constantine

last: Simintiras

first: Maria

last: Hatzoglou


title: Role of DDX3X in translation regulation in acute ER stress [mouse]

overall_design: Mouse Embryonic Fibroblasts were grown to ~80% confluency and treated with control (DMSO) or Thapsigargin (400 nM) containing full medium for 1 h to induce acute ER stress. The cells where then collected by trypsinization and kept in ice-cold HBSS during UV crosslinking (254 nm, 3x400 mJ/cm2). To ensure that the procedure is not inducing stress in the control cells, we checked levels of eIF2α-P and ATF4 by western blot, and performed DDX3X immunofluorescence to observe possible induction of stress granules. For the IP step our own custom rabbit polyclonal DDX3X antibody was used. The rest of the CLIP-Seq procedure was performed as previously described (Vourekas and Mourelatos, 2014).


title: Chromatin landscape of H3K9ac and its modifier GCN5 in the malaria parasite

city: Tampa

first: JUN

last: MIAO

state: Florida

address: 3720 Spectrum Boulevard, MDC84

department: Internal Medicine

institute: University of South Florida

summary: This study is to analyze the chromatin landscape of the active mark H3K9ac, an active mark catalyzed by GCN5, and GCN5 in the malaria parasite Plasmodium falciparum at the schizont stage by both ChIP-seq and CUT&Tag-seq for H3K9ac and CUT&Tag-seq for GCN5.

overall_design: In this study,  wild-type 3D7 parasites at the schizont stage were harvested for H3K9ac ChIP-seq and CUT&Tag-seq for H3K9ac and GCN5. For H3K9ac ChIP-seq was conducted by the standard methods. Briefly, Synchronized WT 3D7 parasites at the schizont stage were harvested and crosslinked with 1% paraformaldehyde and then neutralized by glycine (0.125 M). The fixed iRBCs were lysed with saponin (0.06% final concentration) and parasites were treated with a lysis buffer (10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 10 mM Hepes, pH 7.9, 1 × protease inhibitor) and then were homogenized using a douncer to free the nuclei. Pelleted nuclei were sonicated in a shearing buffer (0.1% SDS, 5 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1× protease inhibitor) using a rod bioruptor (Microson ultrasonic cell disruptor, Misonix, Inc. USA) at high power for 20 cycles of 30 sec ON/30 sec OFF, resulting in sheared chromatin of approximately 100–1000 bps. Fifty μl of input samples were set aside, and the remaining chromatin was diluted in an incubation buffer (0.01% SDS, 1.5% Triton X-100, 0.5 mM EDTA, 200 mM NaCl, 5 mM Tris-HCl, pH 8.1). The chromatin (75 μl/400 ng) was incubated with rabbit anti-H3K9ac (Diagenode pAb-004-050) followed by the addition of 20 μl of agarose beads. Beads were then washed with the following: buffer 1 (0.1% SDS, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris HCl, pH 8.1); buffer 2 (0.1% SDS, 1% Triton X-100, 500 mM NaCl, 2 mM EDTA, 20 mM Tris HCl, pH 8.1), buffer 3 (250 mM LiCl, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris HCl, pH 8.1) and finally twice with buffer 4 (10 mM EDTA, 10 mM Tris HCl, pH 8). The immunoprecipitated (IPed) chromatin was eluted with the elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature. The eluted chromatin and input samples were reverse crosslinked in 10% SDS, 1MNaHCO3, 5MNaCl, and 10% Triton X-100 at 45˚C overnight while shaking and purified by the phenol:chloroform method. The immunoprecipitated gDNA (~20 ng/sample) was then used to generate libraries using the KAPA Hyper prep kit for the Illumina sequencing platform according to the manufacturer's protocol (KAPA Biosystems). Pooled libraries were sequenced on the Illumina NextSeq 550 instrument using 150 bp paired-end sequencing and dual indexing.  CUT&Tag-seq for H3K9ac and GCN5 was conducted by the establshed methods (PMC6488672) with some modifications. Briefly, parasites at the schizont stage were harvested and fixed with 1% formaldehyde followed by saponin lysis. The parasite pellet was suspended in a nuclear extraction buffer. Ten μl of Activated Concanavalin A-coated magnetic beads (EpiCypher # 21-1401) were added to each sample (~0.5 × 106 nuclei). For H3K9ac CUT&Tag-seq, the bead-bound WT parasite nuclei were resuspended in 50 μl Antibody150 buffer containing 0.5 μg of rabbit anti-H3K9ac (Diagenode pAb-004-050) as the primary antibody and rabbit IgG (Cell Signaling Technology #2729S) serving as a control. For GCN5 CUT&Tag-seq, the bead-bound GCN5::GFP nuclei were incubated with 0.5 μg of goat anti-GFP (Novus biologicals NB100-1770) while WT 3D7 nuclei were used as control.  Following primary antibody incubation, nuclei were incubated with the secondary antibodies, donkey anti-goat IgG (Abcam ab6885). The nuclei were resuspened in Digitonin300 Buffer containing CUTANA pAG-Tn5 (EpiCypher #15-1017). Next, the nuclei were resuspended in Tagmentation buffer (10 mM MgCl2 in Digitonin300) at 37 °C for 1 h followed by washing with 50 µl TAPS buffer, quenching tagmentation with 5 µL SDS release buffer, incubating at 58ºC to release tagmented chromatin into the solution, and quenching SDS by adding 0.67% Triton-X. PCR with appropriate barcoded primers was performed using the CUTANA High Fidelity 2x PCR Master Mix (EpiCypher #15-1018) according to the manufacturer's recommendations. Amplified DNA libraries were captured by Kapa pure beads (Roche #KK8001) according to the manufacturer's recommendations, and 150 bp paired-end sequencing was performed on the NextSeq 550 platform.

first: Jun

last: Miao


title: Role of DDX3X in translation regulation in acute ER stress [human]

overall_design: HEK293FT were grown to ~80% confluency and treated with control (DMSO) or Thapsigargin (400 nM) containing full medium to induce acute ER stress. The cells where then collected by trypsinization and kept in ice-cold HBSS during UV crosslinking (254 nm, 3x400 mJ/cm2). To ensure that the procedure is not inducing stress in the control cells, we checked levels of eIF2α-P and ATF4 by western blot, and performed DDX3X immunofluorescence to observe possible induction of stress granules (Figure S1). For the IP step our own custom rabbit polyclonal DDX3X antibody was used. The rest of the CLIP-Seq procedure was performed as previously described (Vourekas and Mourelatos, 2014).


title: Transcriptomic changes in P. falciparum upon knockdown of PfGCN5

summary: This study is to analyze the overal transcriptomic alteration after knockdown of PfGCN5 by TetR-DOZI system. The transcriptomic changes of a parasite line (TetR-PfGCN5::GFP) after PfGCN5 knockdown (KD) by withdral of anhydrotetracycline (aTc) compared to its control (TetR-PfGCN5::GFP with aTc) were analyzed during asexual stage development fron ring, to schizont stage by RNAseq. Total RNA were harvested from four stage (ring, early trophozoite, late trophozoite, and schizont) using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Using an optimized data analysis workflow, we mapped about 5 million sequence reads per sample to the malaria parasite genome (pf3D7_V3.0.) and identified over 5 thousands transcripts with high mapping rate at the range between 80% and 95. Differential expression analysis showed that PfGCN5 KD profoundly changed the global transcription pattern. GO enrichment analysis indicated that PfGCN5 was involved in general and parasite-specific gene transcription. Collectively, transcriptomic analysis of PfGCN5 KD shows that PfPfGCN5 plays important role in global gene regulation .

overall_design: In this study, the transcriptomic changes of a parasite line (TetR-PfGCN5::GFP) after PfGCN5 knockdown (KD) by withdrawal of anhydrotetracycline (-aTc) compared to its control (+aTc) were analyzed during asexual stage development from Ring (R), Early trophozoite (ET), Late trophozoite (LT), and Schizont (S) stage by RNAseq in three biological replicates

first: Liwang

last: Cui


title: The transcription factor IRF4 regulates the differentiation and function of intestinal ILC3s

city: hangzhou

first: lie

last: wang

state: zhejiang

address: Zijingang Campus, Zhejiang University，Yuhangtang Road 866

department: immunology

institute: zhejiang universiry

summary: To investigate the role of IRF4 in the regulation of ILC3 development and function, we used the wildtype mice as control and carried out single-cell RNA sequencing  (BD Rhapsody) on  gut ILC3s.

overall_design: ILC3s from Irf4flox/floxRorccre and Irf4flox/flox  mice were isolated from the small intestine and analyzed by cleavage under targets and tagmentation (CUT&Tag).

first: Xianzhi

last: Gao

first: Lie

last: Wang


title: Cellular crosstalk mediated by TGF-β drives epithelial-mesenchymal transition in patient-derived multilineage biliary organoids [bulk RNA-seq]

first: Jorge

last: Bezerra

overall_design: MBOs generated by co-culturing HUVECs and human MSCs with dissociated epithelial organoid cells derived from liver biopsies of controls (n=3) and BA patients (n=3) were subjected to bulk RNA-seq after 14 days of culturing.

first: Hiroaki

last: Ayabe

first: Erica

last: DePasquale

first: Li

last: Yang

first: Surya

last: Amarachintha

first: Shreya

last: Nalluri

first: Sejal

last: Fox

first: Reena

last: Mourya

first: Ken-ichiro

last: Konishi

first: Pranavkumar

last: Shivakumar


title: The transcription factor IRF4 regulates the differentiation and function of intestinal ILC3s.

summary: To investigate the role of IRF4 in the regulation of ILC3 development and function, we used the IRF4flox/floxRorccre  mice and C57BL/6 mice(6-8 weeks-old ) to carry out single-cell RNA sequencing  (BD Rhapsody) on  gut ILC3s.

overall_design: ILC3s from   IRF4flox/floxRorccre mice (6-8 weeks-old ) and control C57BL/6 mice (6-8 weeks-old ) are isolated from the small intestine and analyzed by single-cell RNA sequencing technology (BD Rhapsody).


title: EBNA Leader Protein is required to reshape chromatin architecture during B cell transformation by Epstein-Barr Virus (HiChIP)

first: Italo

last: Tempera

overall_design: HiChIP was used to capture chromatin contacts mediated by YY1 in LCLs and in EBNA-LP WT and KO cell lines

first: Davide

last: Maestri

first: Lisa

last: Caruso

first: Jana

last: Cable

first: Rachel

last: Sklutuis

first: Sarah

last: Preston-Alp

first: Robert

last: White

first: Micah

last: Luftig


title: Telomeric Repeat-Containing RNA Increases in Aged Human Cells

city: Taipei

first: Hsueh-Ping

last: Chu

address: No. 1 Sec. 4 Roosevelt Road

department: Molecular and Cellular Biology

institute: National Taiwan University

summary: Telomeric repeat-containing RNA (TERRA), transcribed from subtelomeric regions towards telomeric ends, poses challenges in deciphering its complete sequences. Utilizing TERRA-capture RNA-seq and Oxford Nanopore direct RNA sequencing to acquire full-length TERRA, we annotate TERRA transcription regions in the human T2T-CHM13 reference genome. TERRA transcripts encompass hundreds to over a thousand nucleotides of telomeric repeats, predominantly originating from 61-29-37 bp repeat promoters enriched with H3K4me3, RNA pol II, CTCF, and R-loops. We develop a bioinformatics tool, TERRA-QUANT, for quantifying TERRA using RNA-seq datasets and find that TERRA increases with age in blood, brain, and fibroblasts. TERRA upregulation in aged leukocytes is confirmed by RT-qPCR. Single-cell RNA-seq analysis demonstrates TERRA expression across various cell types, with upregulation observed in neurons during human embryonic stem cell differentiation. Additionally, TERRA levels are elevated in brain cells in the early stage of Alzheimer’s disease. Our study provides evidence linking TERRA to human aging and diseases.

overall_design: TERRA was enriched using a C-rich telomeric antisense probe and subsequently subjected to Illumina pair-end or Nanopore RNA direct sequencing. For Illumina sequencing, first-strand cDNA was synthesized with a random hexamer (random group) or a C-rich telomeric primer (specific group), followed by library construction. In the case of Nanopore sequencing, the TERRA-enriched sample was polyadenylated and ligated to an adaptor for direct RNA sequencing.

first: Yu-Hung

last: Hsieh

first: Chin-Hua

last: Tai

first: Meng

middle: T

last: Yeh

first: Yu-Chen

last: Chen

first: Chien-Ping

last: Yen

first: Der-Sheng

last: Han

first: Po-Cheng

first: Hong-Jhih

last: Shen

first: Chan-Hsien

first: Hung-Chih

last: Kuo


title: Loss of Snhg5 disrupts cell-cycle regulation without altering cystogenesis in a mouse model of polycystic kidney disease.

status: Public on Jun 30 2025

city: Stony Brook

first: Karam

last: Aboudehen

state: NY

address: 101 Nicolls Rd, HSC T 15-112

department: Division of Nephrology & Hypertension

institute: Stony Brook University

summary: Long non-coding RNAs (lncRNAs) regulate diverse cellular pathways and are increasingly linked to human disease. Snhg5 is frequently described as a pathogenic lncRNA in many human diseases, including cancer. Our previous studies revealed that Snhg5 is one of the most upregulated lncRNAs in multiple mouse models of polycystic kidney disease (PKD). Yet its role in renal biology and in autosomal dominant PKD (ADPKD) is not known. To elucidate the role of Snhg5, we generated a global Snhg5-null mouse. Homozygous animals were viable and displayed normal kidney morphology and function. RNA-sequencing of Snhg5-null kidneys and renal epithelial cells revealed common alterations in gene expression linked to cell cycle progression and DNA replication. At the molecular level, Snhg5-null cells showed an increased mitotic fraction, coinciding with depletion of ARPC5—a core ARP2/3 subunit— suggesting that reduced ARPC5 may contribute to this phenotype. To determine whether Snhg5 upregulation is pathogenic in mouse PKD, we crossed Snhg5-null mice with a collecting duct-specific Pkd1-mutant mouse model. Loss of Snhg5 did not attenuate cyst formation; if anything, disease severity was mildly but not significantly exacerbated. These findings indicate that Snhg5 modulates mitotic control and is dispensable for kidney development and cystogenesis in collecting duct-derived cysts.

overall_design: RNA-sequencing profiling of in vitro wild-type and Snhg5 knockout mIMCD3 cells, and in vivo wild-type and Shng5 knockout mouse kidneys.

first: Stephen

last: D'Amico


title: Regulatory mechanism of host cell contact-dependent T3SS gene expression in Vibrio parahaemolyticus

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: Many pathogenic bacteria have mechanisms that regulate gene expression in response to the surrounding environment to establish an infection. One such mechanism is the regulation of gene expression in response to contact with host cells. Here, we show that Vibrio parahaemolyticus, a causative agent of foodborne gastroenteritis, has a host cell contact-dependent regulatory mechanism for virulence gene expression. T3SS2, an essential virulence determinant for acute gastroenteritis encoded by V. parahaemolyticus pathogenicity island (Vp-PAI), recognizes host cell contact by sensing high intracellular K+ levels and switches its secretory substrates. This switching of secretory substrates is regulated by proteins called gatekeepers. Mutants deficient in the gatekeeper gene lose the ability to switch secretory substrates and lock their secretory state into contact with the host cell. Transcriptome analysis of T3SS2 gatekeeper gene-deficient strains showed that the genes encoded by Vp-PAI were specifically upregulated, and this upregulation was dependent on T3SS2 secretory activity, implying the presence of a negative regulator secreted by T3SS2. Comparative proteomic analyses identified a previously unidentified T3SS2 secretory substrate, VtrN, that negatively regulates Vp-PAI gene transcription. Secretion of VtrN was promoted under conditions that mimic host cell contact as well as other T3SS2 effectors. vtrN gene deletion specifically upregulated Vp-PAI gene expression, but unlike gatekeeper gene deletion, it was independent of T3SS2 secretory activity. Furthermore, VtrN interacted with VtrB, a transcription factor essential for Vp-PAI-encoded gene expression and repressed its transcriptional activity. Thus, V. parahaemolyticus has a mechanism to upregulate virulence gene expression in response to host-cell contact by utilizing the host-cell recognition mechanism of T3SS2.

overall_design:
To investigate the effect of gatekeeper dysfunction (vpa1360 and vpa1359) on T3SS2 gene expression, the gene transcription profiles of the gatekeeper deletion mutants and parental strain were examined under a general culture condition (RIMD2210633_vpa1360 deletion mutant and RIMD2210633_vpa1359 deletion mutant vs RIMD2210633_WT). The experiment was repeated 3 times.
To investigate the function of a T3SS2 regulatory gene (vpa1369), the gene transcription profiles of the deletion mutant and parental strain were  compared when culture in a general condition (RIMD2210633_vpa1369 deletion mutant vs RIMD2210633). The experiment was repeated 3 times.

first: Toshio

last: Kodama

first: Sarunporn

last: Tandhavanant


title: EBNA Leader Protein is required to reshape chromatin architecture during B cell transformation by Epstein-Barr Virus (ATAC-seq)

city: Philadelphia

first: Italo

last: Tempera

overall_design: Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to assess chromatin accessibility differences in EBNA-LP WT and KO LCLs

first: Davide

last: Maestri

first: Lisa

last: Caruso

first: Jana

last: Cable

first: Rachel

last: Sklutuis

first: Sarah

last: Preston-Alp

first: Robert

first: Micah


title: Cellular crosstalk mediated by TGF-β drives epithelial-mesenchymal transition in patient-derived multilineage biliary organoids

first: Jorge

last: Bezerra

overall_design: Refer to individual Series