title: IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself [RNA-Seq]

city: Los Angeles

first: Aleksandr

last: Gorin

state: CA

address: 611 Charles E Young Dr E 570C Boyer Hall

institute: UCLA

summary: Macrophages, as key sentinel cells of the innate immune system, can retain memory of prior stimulus exposure. Interferon gamma (IFNγ) plays a central role in maintaining trained immunity in vivo and can induce potent memory in macrophages. Such memory is associated with the formation of de novo enhancers that alter gene expression responses to subsequent stimuli. However, how such enhancers are maintained after cytokine exposure remains unclear. We report that durable IFNγ-induced enhancers can last for days after cytokine washout, yet the underlying persistence mechanism is not cell-intrinsic. IFNγ-treated macrophages continue to exhibit JAK/STAT signaling days after cytokine removal. Blocking IFNγ signaling with a JAK inhibitor or anti-IFNγ neutralizing antibodies after cytokine removal is sufficient to reverse IFNγ-induced enhancers and erase the potentiated state of the treated macrophages. Our findings suggest that epigenetic changes in macrophages do not inherently encode innate immune memory or a “potentiated” macrophage state, but that rather epigenetic changes are themselves dependent on ongoing cytokine signaling. These findings suggest new possibilities for pharmacologic interventions to reverse aberrantly trained immune states associated with pathology.

overall_design: RNAseq to identify LPS-induced genes potentiated by IFNy preatreatment and determine effect of continued JAK/STAT signaling on potentiated gene expression.

first: Vyas

last: Koduvayur

first: Noa

last: Harriott

first: Quen

last: Cheng

first: Alexander

last: Hoffmann


title: IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself [ATAC-Seq]

status: Public on Dec 22 2025

country: USA

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ATACseq was performed to identify chromatin opening induced by interferon gamma in human macrophages and assay the necessity of continued JAK/STAT signaling on chromatin accessibility after interferon withdrawal.


title: Diversity and immune dynamics of choroid plexus macrophages are shaped by distinct developmental origins [scRNA-seq Tgfbr2_cKO]

city: Saint Louis

first: Alina

last: Ulezko Antonova

state: MO

address: 660 S. Euclid Ave

department: Path/Immuno

institute: WUSM

type: Expression profiling by high throughput sequencing

summary: The choroid plexus (ChP) forms a critical interface between the central nervous system and systemic circulation. Despite its importance, the identity and dynamics of resident macrophages remain incompletely understood. Using single-cell RNA sequencing, flow cytometry, lineage tracing, and imaging, we identified three biologically distinct macrophage populations in the homeostatic ChP, defined by expression of CD163, MHCII, or CD9. These subsets arise from different combinations of primitive and definitive hematopoietic waves, occupy distinct anatomical niches, and differentially rely on CSF1 and IL34 for survival. TGFβ signaling is required to maintain their phenotype, and deletion of Tgfbr2 in these cells induces phenotypic reprogramming and disrupts the ChP epithelial barrier. During neuroinflammation, ChP macrophages initiate IFN-I responses and secrete chemokines for CD8 T cell recruitment, while microglia mainly express chemokines for B cells. Finally, we identify phenotypically conserved macrophage subsets in the human ChP, suggesting evolutionary preservation of their phenotypic and functional features.

overall_design: 10x Genomics single-cell RNA sequencing of CD45+ cells sorted from the choroid plexus of Tgfbr2-flox and Tgfbr2-flox : Lyz2-Cre mice

first: Khai

last: Nguyen

first: Simone

last: Brioschi

first: Marco

last: Colonna


title: Diversity and immune dynamics of choroid plexus macrophages are shaped by distinct developmental origins [CD45_TotalSeq_ICV_injection]

overall_design: 10x Genomics single-cell RNA sequencing of CD45+ cells sorted from the choroid plexus C57BL6 mice after ICV injection of CD45-Hashtag


title: Tractable mouse TNBC models capture the heterogeneous tumor immune microenvironment and adaptation to PD-L1 blockade

city: Zwijnaarde

first: VIB

last: Single Cell Core

address: Technologiepark-Zwijnaarde 71

department: VIB Single cell core

country: Belgium

institute: VIB

summary: Triple-negative breast cancer (TNBC) patients exhibit variable responses to programmed death (PD)-ligand (L)1 blockade, largely determined by the ‘hot’ versus ‘cold’ state of the tumor immune microenvironment (TIME). We here characterized 9 mouse TNBC models, relying on intraductal mammary gland inoculation of established mouse TNBC cell lines, with a heterogeneous TIME to study anti-PD-L1 resistance mechanisms. Complementary in vitro and in vivo screening classified the 4T1-hot-based model, a highly inflamed control through its immunogenic luciferase tag expression compared to the untagged 4T1-cold-based model, as displaying the ‘hottest’ TIME. However, both 4T1-based counterparts did not respond to anti-PD-L1, which was attributed to their immunosuppressive myeloid cell content as well as upregulation of cancer-associated fibroblasts in the 4T1-hot and high PD-L1-expressing CXCL10+ tumor-associated macrophages in 4T1-cold primary tumors. These anti-PD-L1 adaptation mechanisms across TIME states as captured by mouse TNBC models highlight specific cellular targets for future studies.

overall_design: 9 Different TNBC models were characterized on different levels, with as a first step genomic characterization of the 9 selected TNBC celllines (4T1-hot, 4T1-cold, EMT6, D2A1, 66CL4, PY230, AT3, MVT1 and MET1) in vitro using single-cell RNA-sequencing. Creating a single-cell ATLAS of these 9 cell lines, analyzing transcriptomic differences between these cell lines identifying their heterogeneity based on their transcriptome on the in vitro level. The scRNAseq was performed in collaboration with the VIB Single-cell core.

first: Robbe

last: Salembier

first: Caro

last: De Haes

first: Kristel

last: Demeyere

first: Steven

last: Van Laere

first: Lucie

last: Cervenkova

first: Nicolaas

last: Van Renne

first: Christos

last: Sotiriou

first: Evelyne

last: Meyer

first: Jonas

last: Steenbrugge


city: Merelbeke

state: East-Flanders

address: Salisburylaan 133

department: Department of Veterinary and Biosciences

institute: Ugent

overall_design: RNA was isolated from 4T1-hot and 4T1-cold primary tumors at 3 w p.i. using Rneasy Mini Kit (QiAgen, Valencia, CA, USA). All analyses were done on 4 samples  or 3 samples per treatment group (IgG control- or anti-PD-L1-treated) for 4T1-cold and 4T1-hot TNBC models respectively. Bulk RNA sequencing was done in collaboration with Azenta Life Sciences and performed on Illumina Novaseq system (paired end, length of 150 bp), aiming for 10 million reads per sample.


title: IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself [CUT&Tag]

first: Aleksandr

last: Gorin

overall_design: H3K4me1 CUT&Tag was performed to enhancer marks induced by interferon gamma in human macrophages and assay the necessity of continued JAK/STAT signaling on the persistence of chromatin marks after interferon withdrawal.

first: Vyas

last: Koduvayur

first: Noa

last: Harriott


title: Single-cell analysis of adult mouse testis exposed to cigarette smoke

city: Shanghai

first: Nengzhuang

last: WANG

address: Changhai Road NO.168

country: China

institute: Changhai hospital

summary: Smoking is a global public health issue, which can cause damage to testicular tissue and affect the entire process of spermatogenesis. We established a passive smoking model in mice exposed to cigarette smoke to investigate the single-cell transcriptome data of passively smoking mice, revealing a series of changes in the testicular damage and spermatogenesis process caused by smoking. Through measurements of mouse body weight and testicular weight, we found that, compared to the control group, both the testicular weight and body weight of the treatment group were significantly reduced, while the organ coefficient was significantly increased. The study on the impact of smoking on mouse sperm showed that smoking significantly damaged the spermatogenic process in mice. Pathological assessments of testicular damage and experimental results on cell apoptosis indicated that smoking significantly increased the proportion of apoptotic cells in mouse testicular tissue. Single-cell RNA sequencing (scRNA-seq) experiments using the 10x Genomics platform demonstrated that smoking had a time-dependent impact on the proportion of various cell types in mouse testes. Differentially expressed genes and Gene Ontology (GO) analysis results indicated that smoking affected the formation of the cytoskeleton and microtubules in various cells, as well as flagella formation during spermatogenesis; smoking induced apoptosis in multiple cell types within mouse testes; smoking affected the exocrine functions of various cells; and smoking influenced mitochondrial energy metabolism in multiple cell types within mouse testes. GSEA results also suggested that smoking could induce apoptosis in various cells of the mouse testes. The time-dependent changes in the proportion of reproductive cells we discovered may be due to the combined effects of multiple biological processes. Thus, we will continue to pay attention to and conduct more comprehensive research.

first: Lina

last: Qin

first: Yun

last: Peng

last: Wang


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [NACB-9mo-m-012]

status: Public on Dec 23 2025

city: La Jolla

first: Joseph

middle: R

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

type: Methylation profiling by high throughput sequencing

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

overall_design: Apply snmC-seq3 to mouse brain regions to identify aging effects on epigenomics

first: Wei

last: Tian

first: Anna

last: Bartlett

first: Jordan

last: Altshul

first: Rosa

last: Gomez Castanon

last: Nery

first: Jacinta

middle: D

last: Lucero

first: Julia

middle: K

last: Osteen

first: Huaming

last: Chen

first: M

middle: M

last: Behrens


title: Gene expression profile at single cell level of whole skin samples from human skin

city: Tel Aviv

first: Ofer

last: Sarig

address: 6 weitzman street

country: Israel

institute: TASMC

summary: We used single cell RNA sequencing (scRNA-seq) to analyze the diversity and differential gene expression between atopic dermatitis (AD) to healthy controls

overall_design: 4 mm skin punch biopsies were dissociated and analyzed using scRNAseq.


title: Archetypal Analysis of Deceased Donor Kidneys: A Molecular Approach for Posttransplant Outcomes

city: Praha 4

first: Petra

last: Hruba

address: Videnska 1958

department: Transplant Laboratory

country: Czech Republic

institute: Institute for Clinical and Experimental Medicine

summary: This prospective, single-center study (NCT06171438) included 276 kidneys from 174 deceased donors. Donor procurement biopsies were performed on the backtable and part of the biopsy was stored for future RNA extraction and molecular profiles analysis using Affymetrix microarrays. Three molecular classifiers (the logistic regression model, the Poisson regression and the extreme gradient boosting) with 10-fold cross validation were employed to identify donor categories based on clinical variables (age, BMI, hypertension, marginal kidney) and histological biopsy scores (the vascular fibrous intimal thickening (cv), interstitial fibrosis (ci) and tubular atrophy (ct) and arteriolar hyaline thickening (ah). Archetypal analysis was applied to determine molecular phenotypes and their association with short- and long-term transplant outcomes. Three donor molecular archetypes were identified. Marginal archetype was characterized by older age (median 68 years), extensive chronic morphological changes (IFTA>0 in 66%, ah>1 in 59%, cv>1 in 36%), and the highest Kidney Donor Risk Index (KDRI). Ideal archetype  was associated with young age (median 42 years), low KDRI, minimal chronic histological changes (IFTA>0 in 39%, ah>1 in 33%, cv>1 in 13%) and the highest posttransplant eGFR at 12 months (p<0.001). Intermediate archetype 3 was associated with older age (median 63 years) however better eGFR at 12 months follow-up despite similar chronic histological changes (IFTA>0 in 73%, ah>1 in 63%, cv>1 in 56%). Molecular profiling outperforms histology in procurement biopsies and enables the identification of ECD kidneys with better posttransplant kidney allograft function.

overall_design: Whole-genome mRNA profiling was performed with Affymetrix microarrays (PrimeView Human Gene Expression, Applied Biosystems)  in 276  wedge donor kidney procurement biopsies performed at backtable, prospectively collected between April 2021 and November 2022.  The primary aim of the study was to create a molecular classifier of the donor kidney quality useful for post-transplant outcome prediction.

first: Jiri

last: Klema

first: Eva

last: Girmanova

last: Mrazova

first: Lucie

last: Capkova

first: Katarina

last: Jakubov

last: Fronek

first: Roman

last: Keleman

first: Ludek

last: Voska

first: Martina

last: Mackova

first: Konrad

last: Famulski

first: Philip

middle: F

last: Halloran

first: Ondrej

last: Viklicky


first: Robbe

last: Salembier

overall_design: RNA was isolated from primary tumors of all TNBC mouse models (D2A1, EMT6, 4T1-cold, AT3, 4T1-hot, 66CL4, PY230, MET1 and MVT1) at 2 w p.i and healthy mammary glands from Matrigel-inoculated BALB/C, C57BL/6 and FVB/N mice using Rneasy Mini Kit (QiAgen, Valencia, CA, USA). All analyses were done on 3 samples for all primary tumors and healthy mammary glands, except n=4 for PY230 and AT3 primary tumors. Bulk RNA sequencing was done in collaboration with Azenta Life Sciences and performed on Illumina Novaseq system (paired end, length of 150 bp), aiming for 10 million reads per sample. Differential expression of 2 w primary tumors was obtained for each model comparing to their respective healthy control mammary gland (BALB/c vs 4T1-hot, 4T1-cold, EMT6, D2A1 and 66CL4 tumors, C57BL/6 vs Py230 and AT3 tumors, FVB/N vs MVT1 and MET1 tumors). Identifying hallmarks of cancer for each model and comparing these models to eachother based on log2-fold changes in expressed genes using selection of different gene sets.

first: Caro

last: De Haes

first: Kristel

last: Demeyere

first: Steven

last: Van Laere

last: Cervenkova

first: Nicolaas

last: Van Renne

first: Christos

last: Sotiriou

first: Evelyne

last: Meyer

first: Jonas

last: Steenbrugge


title: E2F1 methylation by SETD6 disrupts BRD4-E2F1 binding and modulates gene regulation in prostate cancer cells [ChIP-seq]

city: Stuttgart

first: Albert

last: Jeltsch

address: Allmandring 31

department: Institute of Biochemistry and Technical Biochemistry

country: Germany

institute: University Stuttgart

summary: The SETD6 protein lysine methyltransferase has a role in the regulation of various cellular processes including cancer initiation and progression. It methylates several cellular proteins including K117 of the E2F1 transcription factor, but the functional consequences of this methylation event are unknown. In this study, the role of SETD6 mediated E2F1 methylation on the progression of prostate cancer was investigated. We identified distinct sets of genes that are bound and upregulated by methylated and unmethylated E2F1. Genes upregulated by methylated E2F1 lead to the negative regulation of cell migration,  while genes upregulated by unmethylated E2F1 have roles in cell proliferation and apoptosis. BRD4 is known to bind E2F1 acetylated at K117 and K120. Here, we demonstrate that methylation of E2F1 at K117 prevents E2F1-BRD4 binding of in vitro and in cells establishing the molecular mechanism of the differential gene regulation by methylated and unmethylated E2F1 that depends on SETD6 activity.

overall_design: The DU145 human prostate cancer cell line was used in the experiment. SETD6 knock-out cells were generated using the CRISPR/Cas9 system. Flag-E2F1 was stably integrated and constitutively expressed.

first: Gizem

middle: T

last: Ulu

first: Margarita

last: Kublanovsky

first: Michal

last: Feldman

first: Sara

last: Weirich

first: Dan

last: Levy


title: SlMBD5 Promotes Seed Germination by Epigenetically Repressing the Gibberellin Catabolism Gene SlGA2ox4 in Tomato [RNA-Seq]

city: Shenzhen

first: Yu

last: Ma

address: 1088 Xueyuan Bivd, Nanshan District

institute: Southern University of Science and Technology

summary: Seed germination represents a critical developmental transition in spermatophytes. Gibberellins (GAs) are essential phytohormones regulating seed germination. GA2-oxidases (GA2ox) play a key role in modulating GA levels by converting active GAs to inactive forms, critically influencing seed development and germination. How seed germination is epigenetically regulated by DNA methylation is poorly understood. Here, we identified the tomato methyl-CpG-binding domain (MBD) protein SlMBD5 as a regulator of seed germination. The slmbd5 mutant exhibits delayed germination and reduced GA4/GA7 levels, which can be rescued by exogenous GA4+7 application. Transcriptomic and biochemical analyses revealed that SlMBD5 represses the GA catabolism gene SlGA2ox4 by directly binding to its hypermethylated promoter. Furthermore, we show that SlMBD5 interacts with the histone methylation reader SlEBS, forming a functional complex that promotes the transcriptional repression of SlGA2ox4. Consistent with the model that SIMBD5 promotes seed germination through its repression of SlGA2ox4, the slmbd5/slga2ox4 double mutant shows partially restored germination. This study thus reveals an SlMBD5-SlEBS module that epigenetically regulates GA homeostasis to modulate seed germination in tomato.

overall_design: RNA-seq data of all seed, endosperm and embryo for wide-type and two slmbd5 mutant alleles

first: Zhaobo

last: Lang


title: Characterizing the expression, binding and function properties of Methyl-CpG-binding Domain (MBDs) genes in tomato [DAP-Seq]

summary: Genomic DNA methylation is an epigenetic mark, which plays important roles in plant growth and development, and gene stability. In Arabidopsis thaliana, studies have demonstrated that the methyl-CpG-binding domain (MBD) proteins AtMBD5, AtMBD6 and AtMBD7 can specifically bind methylated CpG dinucleotides and AtMBD7 is involved in active DNA demethylation to limit DNA hypermethylation and gene silencing. Yet, the features of MBDs in tomato are not well known. In tomato, 17 putative MBD proteins were identified. Here, we found that 9 MBDs are constitute expressed in different tissues and only SlMBD2, SlMBD10, SlMBD10L3 are highly expressed in tomato fruit. In addition, we detected that SlMBD5 and SlMBD7 can bind specifically on symmetrically mCG sites. While, SlMBD13L can bind in different cytosine contexts depending on the 5 different MBD domains in this protein. To gain insight into MBDs binding capacity in vivo, ChIP-sequencing was performed on MBD5, it showed that SlMBD5 preferentially binds to regions that are enriched with mCG and are close to genes. Knockout of MBD5 reduces seeds germinated speed via increase the expression of GA depredated genes, SlGAox4, resulting the faster metabolism of bioactive GAs (GA4, GA7) to inactive GAs. Therefore, our data suggesting that SlMBD5 is essential for tomato seeds germination. Together, our work provides an evidence of the essential function of MBDs in tomato plant growth and development, which would be helpful in further studies of these genes in tomato.

overall_design: DAP-Seq


title: Feeder-free culture of naive human PSCs preserving embryonic, extraembryonic and blastoid potential

city: Padua

first: Graziano

last: Martello

address: viale Colombo 3

department: Department of Biology

country: Italy

institute: University of Padua

summary: Conventional human pluripotent stem cells (hPSCs) are widely used to study early embryonic development, generate somatic cells, and model diseases, with differentiation potential aligned to a post-implantation epiblast identity. In the past decade, naive hPSCs, representing a pre-implantation stage, have been derived, exhibiting broader differentiation potential, including embryonic and extraembryonic lineages such as trophectoderm, primitive endoderm, and extraembryonic mesoderm. Naive hPSCs can also self-organize into blastocyst-like structures called blastoids. However, their culture typically relies on mouse embryonic fibroblasts (MEFs), which are variable, resource-intensive, and can contaminate analyses. We report the long-term maintenance of naive hPSCs in a feeder-free, serum-coated system. Growth rate, clonogenicity, and gene expression profiles on serum coating were comparable to MEF-based cultures, but serum coating minimised fibroblast contamination. Naive hPSCs cultured on serum exhibited faster exit from pluripotency, more efficient germ layer specification, and retained trophectoderm potential with high blastoid formation efficiency. Exome sequencing revealed fewer mutations in serum-cultured cells, and mass spectrometry identified extracellular matrix proteins like vitronectin, fibronectin, and collagens in the serum coating. Overall, serum coating offers a scalable, cost-effective alternative for naive hPSC culture, maintaining developmental potential, reducing DNA mutations, and eliminating MEF-related confounding factors. We believe serum coating will expand the use of naive hPSCs to large-scale studies and mechanistic insights of developmental and disease modelling.

overall_design: RNA-seq profiling of naive human embryonic stem cells cultured on serum (fetal bovine serum) coating and after differentiation of these cells in various conditions (N2B27, E8, mTeSR, XAV and FAX) for up to 8 days on Geltrex.

first: Giada

last: Rossignoli

first: Michael

last: Oberhuemer

first: Paolo

last: Martini

first: Leeb

last: Martin


title: E2F1 methylation by SETD6 disrupts BRD4-E2F1 binding and modulates gene regulation in prostate cancer cells

overall_design: The DU145 human prostate cancer cell line was used in experiment.  SETD6 knock-out cells was generated by CRISPR/Cas9 system. Cells had stable expression of Flag-E2F1 WT and E2F1 K117R.


title: SlMBD5 Promotes Seed Germination by Epigenetically Repressing the Gibberellin Catabolism Gene SlGA2ox4 in Tomato [ChIP-Seq]

overall_design: ChIP-seq


title: Effect of pancreatic cancer derived extracellular vesicles on bone marrow drived macrophages

city: Washington

first: Amrita

last: Cheema

state: DC

address: 3900 Reservoir Road NW

department: Oncology

institute: Georgetown University Medical Centre

type: Non-coding RNA profiling by high throughput sequencing

summary: Our results show that PaCa cell derived EVs were taken up by the macrophage in a time-dependent manner that resulted in an immunosuppressive phenotype characterized by higher expression of CD206 and PD-L1 and higher secretion of immunosuppressive cytokines including TGF-beta, IL-10 and GM-CSF.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: EVs were isolated from the conditioned medium that was used to culture one established pancreatic cancer cell (PANC-1), one PDX cell line (PPCL68) and a normal (non-tumorigenic) established pancreatic epithelial cell line (hTERT-HPNE), using size exclusion chromatography. Macrophages were generated from the bone marrow cells of C57/BL6 mice cultured in vitro using macrophage colony stimulating factor (M-CSF). The macrophages were seeded into 24 well plates and co-cultured with isolated PaCa or normal cell derived EVs at the concentration of 10 ug/mL EVs for 48h.

first: Baldev

last: Singh

first: Pankaj

last: Gaur

first: Pritha

last: Bose

first: Yanjun

last: Zhang

first: Yaoxiang

last: Li

first: Zihao

first: Jeyalakshmi

last: Kandhavelu

first: William

last: Klotzbier

first: Meth

last: Jayatilake

first: Shivani

last: Bansal

first: Mohd

last: Farhan

first: Sunain

last: Deol

first: Partha

middle: P

last: Banerjee

first: Keith

last: Unger

first: Seema

last: Gupta

first: Vivek

last: Verma


first: Graziano

last: Martello

summary: Conventional human pluripotent stem cells (hPSCs) are widely used to study early embryonic development, generate somatic cells, and model diseases, with differentiation potential aligned to a post-implantation epiblast identity. In the past decade, naive hPSCs, representing a pre-implantation stage, have been derived, exhibiting broader differentiation potential, including embryonic and extraembryonic lineages such as trophectoderm, primitive endoderm, and extraembryonic mesoderm. Naive hPSCs can also self-organize into blastocyst-like structures called blastoids. However, their culture typically relies on mouse embryonic fibroblasts (MEFs), which are variable, resource-intensive, and can contaminate analyses. We report the long-term maintenance of naive hPSCs in a feeder-free, serum-coated system. Growth rate, clonogenicity, and gene expression profiles on serum coating were comparable to MEF-based cultures, but serum coating minimised fibroblast contamination. Naive hPSCs cultured on serum exhibited faster exit from pluripotency, more efficient germ layer specification, and retained trophectoderm potential with high blastoid formation efficiency. Exome sequencing revealed fewer mutations in serum-cultured cells, and mass spectrometry identified extracellular matrix proteins like vitronectin, fibronectin, and collagens in the serum coating. Overall, serum coating offers a scalable, cost-effective alternative for naive hPSC culture, maintaining developmental potential, reducing DNA mutations, and eliminating MEF-related confounding factors. We believe serum coating will expand the use of naive hPSCs to large-scale studies and mechanistic insights into developmental and disease modelling.

overall_design: scRNA-seq of blastoids derived from feeder-free H9 human embryonic stem cells cultured in PXGL on serum-coated plates. Blastoids were subsequently derived by culture in Aggrewells in blastoid induction medium for 120h.

first: Ida

last: Brun

first: Anne

last: Wenzel

first: Karlien

last: Van Nerum

first: Jan

last: Zylicz


title: Deletion of Y Chromosome Ssty2 Repetitions produced alterations in Mice Sex Ratio

city: Madrid

first: Alfonso

last: Gutiérrez-Adán

address: Av. Puerta de Hierro 28

department: Animal Reproduction

country: Spain

institute: INIA

summary: Animal facilities often have surplus unused male mice. To reduce unnecessary mouse sacrifices, we proposed innocuously preselecting sex sperm using CRISPR/Cas9. Specifically, we aimed to significantly decrease the number of copies of the multicopy Ssty2 gene family (repeated over 220 times on the Y chromosome), which plays a pivotal role in regulating X and Y gene expression during sperm differentiation. Transgenic lines were created by microinjecting Cas9 RNA and specific Ssty2 sgRNAs into B6CBAF1 zygotes. Pup genotyping of Ssty2 copies was conducted via qPCR. Epididymal sperm samples were obtained from 2 to 6-month KO and WT males, and after a swim-out, sperm motility was evaluated using the Integrated Semen Analysis System. Motile sperm were categorized by K-means clustering of CASA kinetic parameters. To uncover the impact of Ssty2 depletion on sex-ratio segregation, we selected a line with a substantial decrease in Ssty2 copies, to fewer than 50. Consequently, 70 to 80% of pups were female upon in vivo fertilization, remaining consistent in IVF, but with a normal sex ratio in ICSI. However, we noted a reduction in litter size, declining from an average of 8.5 to 6.4 pups per litter. Testicle size in KO mice exhibited a slight enlargement, with a minor decrease in sperm concentration compared to controls (non-significant). However, over 80% of KO sperm displayed abnormal morphology, showing head, neck, and midpiece defects. Sperm motility comparison revealed a shift, with 60.6% of KO sperm static vs. 35.9% in WT controls, displaying KO sperm as slow and non-progressive motility patterns. In conclusion, by targeting the Ssty2 multicopy gene, we produced CRISPR-mediated KO mice that presented preferential fertilization of sperm carrying the X chromosome via in vivo and IVF.

overall_design: Differential gene expression analysis in mice Ssty2 knockout testis compared to wild-type testis

first: María

last: Maroto

first: Paula

last: Navarrete-López

first: Raul

last: Fernández-González

first: Eva

last: Pericuesta

first: Vladimir

last: González


title: Identification of Macrophage-Dependent Fcer1g+ Mesenchymal Cells as Key Regulators of Scar Formation in Wound Healing

city: Madison

first: jianhong

last: ou

state: WI

address: 330 N Orchard Street

institute: Morgridge institute for research

summary: Scarring is a common outcome of wound healing in adult skin, often leading to fibrotic tissue that lacks the functional and aesthetic properties of normal, uninjured skin. To effectively reduce and prevent scar formation, understanding the cellular players involved in fibrosis is crucial. Among these, macrophages and mesenchymal cells have received significant attention due to their vital roles in both tissue regeneration and fibrosis. Research has shown that depleting macrophages during the early stages of wound healing reduces the number of myofibroblasts in the wound bed and delays wound closure, but results in reduced scar formation, suggesting that macrophages contribute to fibrosis by regulating mesenchymal cells. However, there remains a need for unbiased, high-throughput approaches to precisely investigate the mechanisms through which macrophages regulate and induce mesenchymal subtypes in scar formation. In this study, we performed single-cell RNA sequencing to investigate changes in mesenchymal cell subpopulations during adult skin wound healing in a conditional macrophage depletion mouse model, macrophage Fas-induced apoptosis (MaFIA).

overall_design: To identify mesenchymal cell subtypes regulated by acute-phase macrophages during wound healing, we performed single-cell RNA sequencing (scRNA-seq) on 3 day-post-injury skin cells isolated from the wounds of MaFIA mice, with or without macrophage depletion.

first: Xinyi

last: Ma

first: Ergang

last: Wang

first: Vijitha

last: Puviindran

first: Jianhong

last: Ou


title: Feeder-free culture of naive human pluripotent stem cells retaining embryonic, extraembryonic and blastoid generation potential [RNA-Seq]

overall_design: Expression profiling by high throughput sequencing of naive human pluripotent stem cells cultutred on feeders (incative mouse embryonic fibroblasts) or serum (foetal bovine serum) coating.

first: Giada

last: Rossignoli

first: Michael

last: Oberhuemer

first: Paolo

last: Martini

first: Leeb

last: Martin


title: SMAD nucleocytoplasmic shuttling relies on nucleoporin RanBP2 and VIM-AS1 long non-coding RNA during TGF-β signaling

city: Uppsala

first: Mohamad

middle: Moustafa

last: Ali

state: Uppsala län

address: husargatan 3, Biomedical Center

department: IMBIM

country: Sweden

institute: Uppsala University

summary: Activation of the transforming growth factor β (TGF-β) signaling leads to established hallmarks of cancer such as the epithelial-to-mesenchymal transition (EMT), initiating cancer dissemination and increasing chemoresistance. TGF-β signal by binding to its type I and II receptors, leading to parallel activation of SMAD proteins, ubiquitin ligases and protein kinases. Here, using a panel of tumor cells we show that TGF-β activation induces the expression of the mammalian nuclear long non-coding RNA (lncRNA) VIM-AS1 (Vimentin antisense RNA 1) variant 2 (v.2) via the formation of a complex among the transcriptional factors GATA6-SMAD-SPI1. Furthermore, transcriptomic analysis indicated VIM-AS1 enhancing TGF-β signaling and EMT, as further validated by functional gain or loss assays. Mechanistically, the TGF-β-induced VIM-AS1 v.2 enhanced SMAD nucleocytoplasmic shuttling by interacting with the N-terminus domain of the nucleoporin RanBP2 (Nup358), whereby acting as a scaffold molecule, facilitates the binding of RanBP2 with SMAD2/3 and its further nuclear import. Finally, VIM-AS1 increases invasion and motility of tumor cells and emerges as a promising therapeutic target to sensitize cancer cells to chemotherapeutic drugs. Hence, we delineated a novel signaling mechanism for VIM-AS1 v.2 facilitating TGF-β response in tumor cells via SMAD nucleocytoplasmic shuttling.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for A549 lung adenocarcinoma cells trasfected with scrambled siRNA in the presence or absebce of TGFβ stimulation in comparison to cells transfected with siRNAs against the long noncoding RNA VIM-AS1 in the presence or absence of TGFβ stimulation.

first: Dorival

last: Rodrigues-Junior

first: Carl-Henrik

last: Heldin

first: Aristidis

last: Moustakas


title: Characterizing the expression, binding and function properties of Methyl-CpG-binding Domain (MBDs) genes in tomato [BS-Seq]

first: Yu

overall_design: Bisulfite sequencing (BS-Seq) data of 35day-leaf for wide-type and two slmbd5 mutant alleles

first: Zhaobo

last: Lang


first: Graziano

last: Martello

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

overall_design: Refer to individual Series


title: Single cell and isoform-specific translational profiling of the mouse brain [Bulk RNA-seq]

first: Brian

last: Yee

address: 9500 Gilman Dr

department: Health

institute: UCSD

summary: The brain displays the richest repertoire of post-transcriptional mechanisms regulating mRNA translation1–12. Among these, alternative isoform expression has been shown to drive cell type specificity and, when disrupted, is strongly linked to neurological disorders13–18. However, genome-wide measurements of mRNA translation with isoform-sensitivity at single-cell resolution have not been achieved. To address this, we deployed Ribo-STAMP (Surveying Ribosomal Targets by APOBEC-Mediated Profiling) coupled with short- and long-read single-cell RNA-sequencing in the brain19. We generated the first isoform-sensitive single cell translatomes of the mouse hippocampus, discovering 797 alternative isoforms across 325 genes, with CA1 exhibiting the most differentially expressed and CA3 the most differentially translated isoforms. We defined high and low translational states in CA1 and CA3 neurons, with ribosomal, metabolic, and synaptic genes enriched in high states. Surprisingly, we found that CA3 neurons exhibited higher basal translation compared to CA1, as confirmed by metabolic labeling of newly synthesized proteins and immunohistochemistry of mRNA translation factors. This easily adoptable platform will expand our understanding of how isoform-specific translation drives brain physiology and disease.

overall_design: To determine if RPS2-STAMP accurately reports ribosome occupancy on mRNAs in neurons, we benchmarked it against enhanced cross-linking and immunoprecipitation (eCLIP) of ribosomal proteins.

first: Sison

middle: L

last: Samantha

first: Yeo

middle: W

last: Eugene


title: Extracellular matrix protein 1 shapes metabolic and spatial dynamics of the kidney fibrotic microenvironment

city: Pittsburgh

first: Shuchang

last: Liu

state: PA

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: The fibrotic kidney microenvironment is a battlefield shaped by cellular crosstalk, ECM remodeling, metabolic shifts, and spatial heterogeneity, all fueling fibrosis. In late-stage kidney disease, myofibroblast-produced ECM dominates organ function by altering bioenergetics and metabolite signaling. However, the role of early-activated matrix proteins remains unclear. This study identifies four expression patterns of core matrisome proteins, with Ecm1 emerging as a key early player in kidney remodeling. Ecm1 knockout in mice leads to spontaneous kidney fibrosis and early death, yet serum Ecm1 levels rise in CKD patients. Targeting Ecm1 via adeno-associated virus-mediated knockdown or fibroblast-specific deletion substantially reduces kidney fibrosis. Mechanistically, proteomics and spatial transcriptomics show that fibroblast-Ecm1 ablation disrupts Hippo-Yap signaling via integrin α2β1 and RhoC in tubules, enhancing OXPHOS in tubular cells and promoting repair, as validated across pharmacological, in vivo, ex vivo, and in vitro models. Notably, diminished Hippo-Yap in fibroblasts silenced their aberrant activation without crippling their own OXPHOS. This selective ECM-mitochondrial crosstalk uncovers a mechano-metabolic pathway where mitochondrial shifts drive defense against kidney fibrosis.

overall_design: Heterozygous Ecm1 mice (Product #: S-KO-01828) were obtained from Cyagen (Santa Clara, CA). Male and female Ecm1 heterozygous mice were mated to generate Ecm1-wild type (WT) and Ecm1-knockout (KO) offspring. Homozygous ECM1-floxed mice (Product #: S-CKO-02130) were also acquired from Cyagen (Santa Clara, CA). Col1α2-CreER mice (Strain #: 029567) and Pdgfrβ-P2A-CreERT2 mice (Strain #: 030201) were obtained from the Jackson Laboratory (Bar Harbor, ME). By crossing Ecm1-floxed mice with Col1α2-CreER or Pdgfrβ-P2A-CreERT2 transgenic mice, conditional knockout mice were generated where Ecm1 gene was specifically deleted in kidney Col1α2+ or Pdgfrβ+ fibroblasts (genotype: ECM1fl/fl, Cre ±) were created. These conditional knockouts were bred with homozygous Ecm1-floxed mice (genotype: ECM1fl/fl), yielding litters with 50% Col1α2-Ecm1-/- or Pdgfrβ-Ecm1-/- mice and 50% control mice (Col1α2-Ecm1+/+ or Pdgfrβ-Ecm1+/+). Kidney ECM1 WT slide and ECM1 KO slide were pooled into one spatial transcritpomics imaging for analysis.

first: Yuan

last: Gui

first: Wenxue

last: Li

first: Jia-Jun

first: Yuanyuan

first: Cameron

last: Jones

first: Riddhi

last: Bansal

first: Samantha Mae

last: Mallari

first: Henry Wells

last: Shaffer

first: Saiya

last: Zhu

first: Edward

last: Malakh

first: Yanbao

last: Yu

first: Haiyan

last: Fu

first: Tracy

last: Tang

first: Silvia

first: Yansheng

first: Dong

last: Zhou


title: Single cell and isoform-specific translational profiling of the mouse brain [Ribo-STAMP]

first: Brian

overall_design: To evaluate Ribo-STAMP's sensitivity in detecting rapid translational changes on the order of minutes to hours, we treated neurons with Brain-Derived Neurotrophic Factor (BDNF) after Ribo-STAMP expression.

first: Sison

first: Yeo


title: Combined epigenetic therapy with FACT and BET inhibitors remodels chromatin and disrupts oncogenic transcription in Diffuse Midline Glioma [RNA-seq]

city: Kensington

first: Holly

last: Holliday

address: Lowy Cancer Research Centre

country: Australia

institute: Children's Cancer Institute

summary: Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be a key therapeutic strategy against this highly aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition revealed potent and synergistic cytotoxicity across a range of DMG cultures, with H3K27M-mutant cells demonstrating heightened sensitivity. These results were recapitulated in vivo, significantly extending survival in three independent orthotopic PDX models of DMG. Mechanistically, we show that CBL0137 treatment decreased chromatin accessibility, synergizing with BET inhibition to disrupt transcription, silencing several key oncogenes including MYC, PDGFRA and MDM4, alongside alterations to the splicing landscape. Combined, these data highlight the therapeutic promise of simultaneously targeting FACT and BRD4 in DMG, proposing a novel strategy for combating this devastating pediatric brain tumor.

overall_design: Two H3.3 K27M DMG cell lines (HSJD-DIPG007 and SU-DIPGVI) were treated with CBL0137 (1µM for DIPG7 and 2µM for DIPGVI) or JQ1 (1µM) as single agents and in combination for 4h. Vehicle cotrol cells were treated with DMSO at the same concentration. RNA was then extracted and sequenced. RNA is matched from ATAC-seq experiments.

first: Nisitha

last: Jayatilleke

first: Chelsea

last: Mayoh

first: Xinyi

last: Guo


title: Diversity and immune dynamics of choroid plexus macrophages are shaped by distinct developmental origins [bulk RNA-seq]

first: Alina

last: Ulezko Antonova

address: 660 S. Euclid Ave

institute: WUSM

summary: The choroid plexus, responsible for cerebrospinal fluid production, is vital for brain functions. This organ serves as a crucial interface between the central nervous system and systemic circulation and thus faces constant immunological challenges. While resident macrophages within the choroid plexus provide immunosurveillance, their biological characteristics and immune dynamics remain poorly understood. In this study, we extensively examined the phenotype of choroid plexus macrophages using high-throughput single-cell RNA sequencing, flow cytometry, and imaging techniques. Our data unveiled three distinct macrophage populations within the homeostatic choroid plexus expressing either CD163, MHCII, or CD9. These macrophage subsets exhibited differential input from primitive and definitive hematopoietic waves, different spatial location and relied on distinct factors for survival. Upon neuroinflammation, these cells orchestrated IFN responses producing chemokines for effector CD8 T cells, while microglia predominantly expressed chemokines for B cells. Finally, we present evidence of phenotypically conserved macrophage subsets in the human choroid plexus.

overall_design: Bulk RNA-seq of the total RNA extracted from the choroid plexus, dissected from lateral and fourth ventricles of the mouse brain, to assess cytokine expression.

first: Siling

last: Du

first: Marco

last: Colonna

first: Simone

last: Brioschi


title: Combined epigenetic therapy with FACT and BET inhibitors remodels chromatin and disrupts oncogenic transcription in Diffuse Midline Glioma [ATAC-seq]

overall_design: Two H3.3 K27M DMG cell lines (HSJD-DIPG007 and SU-DIPGVI) were treated with CBL0137 (1µM for DIPG7 and 2µM for DIPGVI) or JQ1 (1µM) as single agents and in combination for 4h. Vehicle cotrol cells were treated with DMSO at the same concentration. ATAC-seq was then performed to measure chromatin accessibility

first: Samuel

middle: E

last: Ross


title: Combined epigenetic therapy with FACT and BET inhibitors remodels chromatin and disrupts oncogenic transcription in Diffuse Midline Glioma [CUT&RUN]

summary: Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be key therapeutic strategies against this highly aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition revealed potent and synergistic cytotoxicity across a range of DMG cultures. These results were recapitulated in vivo, significantly extending survival in three independent orthotopic PDX models of DMG. Mechanistically, we show that CBL0137 treatment decreased chromatin accessibility, synergizing with BET inhibition to cause broad transcriptional collapse, silencing several key oncogenes including MYC, PDGFRA, MDM4 and SOX2, as well as causing alterations to the splicing landscape. Notably, this combination also elicited immune-related effects, including activation of the interferon response and antigen presentation mechanisms in DMG cells and induction of an activated state in macrophages and T cells, as demonstrated in an immunocompetent setting with spatial transcriptomics. Altogether, our data highlights the therapeutic promise of simultaneously targeting FACT and BET proteins in DMG, offering a dual tumor-intrinsic and immune-mediated strategy for combating this devastating pediatric brain tumor.

overall_design: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) for SPT16, BRD4, H3K27M, H3K27me3 and H3K4me3 in 3 DMG cell lines (HSJD-DIPG007, SU-DIPGVI, VUMC-DIPG10). IgG was used as a negative control.


title: Diversity and immune dynamics of choroid plexus macrophages are shaped by distinct developmental origins


title: VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING (RNA-Seq).

city: Cambridge

first: Niek

last: Wit

state: Not US or Canada

address: Puddicombe Way

department: Department of Medicine

country: United Kingdom

institute: University of Cambridge

summary: Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and may reactivate anti-tumour immune surveillance.

overall_design: Total RNA was extracted from cell lines. Library preparation was undertaken with a NEBNext Ultra II RNA Library Preparation kit for polyA transcript selection. These libraries were sequenced on a Illumina NovaSeq 6000 platform

first: James

last: Bertlin

last: Nathan


title: cFGF23 alleviates kidney inflammatory gene signatures induced by sustained FGF23 signaling

city: Zurich

first: Ganesh

last: Pathare

state: Zürich

address: Winterthurerstrasse 190

department: Institute of Anatomy

country: Switzerland

institute: University of Zurich

summary: RNA-seq was performed on the kidneys of Hyp-Duk mice (a model for lifelong elevated FGF23 levels) and Hyp-Duk mice harboring adeno-associated virus expressing cFGF23 (AAV-cFGF23). The kidneys of Hyp-Duk mice showed significantly elevated sustained-ERK signaling and concomitant key gene signatures of inflammation, which were significantly alleviated in the AAV-cFGF23 treated Hyp-Duk mice.

overall_design: We included 5 mice in each group, with three groups in total: WT, Hyp-Duk, cFGF23-Hyp-Duk

first: Giuseppe

last: Ronzitti

first: Claire

last: Bardet


title: Diversity and immune dynamics of choroid plexus macrophages are shaped by distinct developmental origins [scRNA-seq steady state]

city: Saint Louis

overall_design: scRNA-seq of FACS sorted choroid plexus CD45+ cells.


title: VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING (CRISPR).

overall_design: Cas9 containing cells were transduced with the Toronto KO v3 sgRNA library at an MOI of ~0.3, and after 27 hours selected with puromycin for 7 days. Every two or three days cells were pooled, counted and passaged. After 17 population doublings, genomic DNA was extracted. Lenti viral inserts with the sgRNAs were PCR amplified and sequenced.


title: Single cell and isoform-specific translational profiling of the mouse brain [ribostamp]

first: Brian

last: Yee

overall_design: To optimize Ribo-STAMP for expression in brain cells, we compared RPL10a, RPL22 and RPS2 NT (deposited at GSE275091) Ribo-STAMP constructs in mouse cortical neurons.

first: Samantha

last: Sison

first: Eugene

last: Yeo


title: Feeder-free culture of naive human pluripotent stem cells retaining embryonic, extraembryonic and blastoid generation potential [Exome-Seq]

first: Graziano

last: Martello

overall_design: Exome sequencing of naive human pluripotent stem cells cultutred on feeders (incative mouse embryonic fibroblasts) or serum (foetal bovine serum) coating.

first: Giada

last: Rossignoli

first: Chiara

last: Colantuono

first: Davide

last: Cacchiarelli


title: Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

city: Chicago

first: Ali

last: Shilatifard

state: IL

address: 320 E Superior St

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University Feinberg School of Medicine

summary: Negative elongation factor (NELF) is a transcriptional regulator that is primarily known for its role in the promoter-proximal pausing of RNA polymerase II (RNAPII) [1-2]. We have previously demonstrated that acute depletion of the NELF subunit NELF-C causes genome-wide RNAPII pausing at a second site associated with the +1 nucleosome [3]. Here, we show that acute depletion of the NELF subunit NELF-C results in a reversible cellular senescence phenotype that includes impaired growth, robustly detectable β-galactosidase activity, and upregulated expression of senescence-associated genes. We also identify a small set of genes at which NELF-C depletion induces RNAPII release rather than second site pausing. We identify Elongin A (ELOA) as a transcription factor mediating the impacts of NELF depletion, and we demonstrate that the primate-specific ELOA homolog ELOA3 can also play this role. Mechanistically, ELOA mediates the impacts of NELF depletion at least in part by altering the kinetic coupling between the elongation and splicing of nascent transcripts, which results in intron retention and other splicing defects. Despite reports implicating splicing defects in aging-related phenomena [4-6] [7-9], mechanisms have remained elusive. The findings presented here indicate that the crosstalk between splicing factors, ELOA, and NELF may be critical to understanding cellular senescence and aging

overall_design: TT-seq in NELFC-AID and ELOA-KO DLD1 cells

first: Saeid

last: Parast

first: Marta

last: Iwanaszko

first: Yuki

last: Aoi


summary: Elongation factors control post-initiation steps of transcription by RNA polymerase II (RNAPII). We have established distinct mechanistic roles for the essential elongation factors PAF1, NELF, SPT5, and SPT6 via acute depletion of each individually in auxin-inducible degron lines. Here, we leverage these degron lines to explore the regulatory intersection of elongation control and mRNA processing. Integrating long- and short-read RNA-seq data to quantify transcript isoform usage at single-molecule resolution, we identify elongation factor-specific RNA processing regulons including a cellular senescence-enriched regulon shared by NELF and SPT6. We then show that long-term depletion of NELF or SPT6 results in reversible growth arrest following early upregulation of a small group of genes, which include the senescence-associated genes CDKN1A (p21) and CCN2. We perform genetic suppressor screens that implicate the elongation factor Elongin A (ELOA) in NELF or SPT6 depletion-induced growth arrest. ELOA KO suppresses NELF depletion-induced mRNA processing defects and the 3’ extension of RNAPII occupancy past transcription end sites (TES) at genes induced by NELF depletion. ELOA also occupies TES-proximal regions under normal conditions, and acute ELOA depletion results in a loss of RNAPII processivity at the 3’ end of genes, opposing the effects of NELF or SPT6 depletion. Finally, we demonstrate that genetic ELOA loss confers a growth advantage to aging human primary dermal fibroblasts. These findings establish the existence of novel ELOA-dependent mechanisms regulating transcription termination-coupled processes, and links these to the complex phenomena of cellular senescence and aging.

overall_design: polyA RNA-seq in NELFC-AID and ELOA-KO DLD1 cells


overall_design: ChIP-seq for Pol2, pSer2, PAF1, SPT6, ELOA, ELOA3 in NELFC-AID DLD-1 cells


title: Bulk RNA Sequencing of 3 DIPG Cases

city: New York

first: Lorenzo

last: Tomassoni

address: 1130 St Nicholas Avenue

institute: Columbia University

summary: Tumors of 3 DIPG Patients have been subjected to Bulk RNA Sequencing as part of a program at Rady Children's Hospital

overall_design: Tissue from 3 untreated DIPG Tumors was isolated and prepared for Bulk RNA-Sequencing

first: Ester

middle: C

last: Fernandez

first: Xu

last: Zhang

first: Junqiang

last: Wang

first: Aleksandar

last: Obradovic

first: Pasquale

last: Laise

first: Aaron

last: Griffin

first: Lukas

last: Vlahos

first: Hanna

last: Minns

first: Diana

middle: V

last: Morales

first: Christian

last: Simmons

first: Matthew

last: Gallitto

first: Hong-Jian

last: Wei

first: Timothy

middle: J

last: Martins

first: Pamela

middle: S

last: Becker

first: John

last: Crawford

first: Theophilos

last: Tzaridis

first: Robert

last: Wechsler-Reya

last: Garvin

first: Robyn

last: Gartrell

first: Luca

last: Szalontay

first: Stergios

last: Zacharoulis

first: Cheng-Chia

last: Wu

first: Zhiguo

first: Andrea

last: Califano

first: Jovana

last: Pavisic


title: CRISPR/Cas9-mediated validation of candidate Master Regulators Proteins in DMG/GBM Cell Lines

first: Lorenzo

last: Tomassoni

summary: Three GBM and three DMG cell lines were selected to be used in a function validation CRISPR screening to elucidate essentiality of canidate Master Regulator proteins

overall_design: A pooled CRISPR/Cas9-mediated KO screen was performed in three human DMG cell lines (SF8628, DIPG4 and DIPG17) and three human GBM cell lines (GBM22, KNS42 and GBM43)—using guide RNAs (sgRNAs) targeting 1,433 genes

first: Ester

last: Fernandez

first: Xu

last: Zhang

first: Junqiang

last: Wang

first: Aleksandar

last: Obradovic

first: Pasquale

last: Laise

first: Aaron

last: Griffin

first: Lukas

last: Vlahos

first: Hanna

last: Minns

first: James


title: Generation of Drug Perturbational Profiles in SF8628 and DIPG6 DMG Cell Lines

summary: 372 clinically relevant drugs, including FDA-approved and late-stage experimental oncology drugs were tested in both SF8628 and DIPG6 DMG Cell Lines

overall_design: RNA-seq profiles were then generated at 24 hours following treatment of each cell line with the 372 drugs—titrated at the lowest of their highest sublethal concentration (48h EC20) or max tolerated serum concentration from the literature—as well as DMSO as vehicle control, using the PLATE-Seq microfluidic automation platform

first: Diana

last: Morales

first: Christian

last: Simmons

first: Matthew

last: Gallitto

first: Hong-Jian

last: Wei

first: Timothy

last: Martins

first: Pamela

last: Becker

first: John

last: Crawford

first: Theophilos

last: Tzaridis

first: Robert

last: Wechsler-Reya

first: Robyn


title: Elucidating and Pharmacologic Targeting of Coexisting Diffuse Midline Subpopulation Dependencies

first: Lorenzo

last: Tomassoni

summary: Diffuse Midline Glioma (DMG) are universally fatal, primarily pediatric malignancies affecting the midline structures of the central nervous system. Despite decades of clinical trials, their treatment remains limited to palliative radiation therapy. A major challenge is the coexistence of molecularly distinct malignant cell states with potential orthogonal drug sensitivities. To address it, we leveraged established network-based methodologies to elucidate Master Regulator (MR) proteins representing mechanistic dependencies of seven coexisting subpopulations identified from tumor biopsies. We then leveraged perturbational profiles of 372 clinically relevant drugs to identify those capable of inverting the activity of subpopulation specific MRs and validated their specificity in vivo. While individual drugs predicted by the analysis to target individual subpopulations—such as avapritinib, larotrectinib, and ruxolitinib—produced only modest tumor growth reduction in orthotopic allograft models, they systemic co-administration induced highly significant survival extension.

overall_design: Single cells were isolated and sequenced from NOD.Cg Prkdcscid/J (NSG) subcutaneously injected with 500,000 patient-derived DMG cells (SU-DIPG-XVII) at five days following treatment with Etoposide, Dinaciclib, Avapritnib, Trametinib, Mocetinostat, Larotrectinib, Ruxolitnib, Napabucasin and Venetoclax

first: Ester

last: Fernandez

first: Xu

first: Junqiang

last: Garvin

last: Gartrell

first: Luca

last: Szalontay

first: Stergios

last: Zacharoulis

first: Cheng-Chia

last: Wu

first: Zhiguo

first: Andrea

last: Califano

first: Jovana

last: Pavisic


title: NAC controls nascent chain fate through tunnel sensing and chaperone action [Ribo]

city: Stanford

first: Jae Ho

last: Lee

address: 318 Campus Drive E200

department: Biology

institute: Stanford University

summary: The nascent polypeptide-associated complex (NAC) is a conserved ribosome-bound factor with essential yet incompletely understood roles in protein biogenesis1. Here, we show that NAC is a multifaceted regulator that coordinates translation elongation, cotranslational folding, and organelle targeting through distinct interactions with nascent polypeptides both inside and outside the ribosome exit tunnel. Using NAC-selective ribosome profiling in C. elegans, we identify thousands of sequence-specific NAC binding events across the nascent proteome, revealing broad cotranslational engagement with hydrophobic and helical motifs in cytosolic, nuclear, ER, and mitochondrial proteins. Unexpectedly, we discover an intra-tunnel sensing mode, where NAC engages ribosomes with extremely short nascent polypeptides inside the exit tunnel in a sequence-specific manner. Moreover, initial NAC interactions induce an early elongation slowdown that tunes ribosome flux and prevent ribosome collisions, linking NAC’s chaperone activity to kinetic control of translation. We propose NAC action protects aggregation-prone intermediates by shielding amphipathic helices, thus promoting cytonuclear folding. NAC also supports mitochondrial membrane protein biogenesis and ER targeting by early recognition of signal sequences and transmembrane domain. Our findings establish NAC as an early-acting, multifaceted orchestrator of cotranslational proteostasis, with distinct mechanisms of action on nascent chains depending on their sequence features and subcellular destinations.

overall_design: Selective-ribosome profiling of NAC and NAC(deltaN1-53), and translatome profiling of C. elegans upon NAC_KD

first: Jae

middle: H

first: Laurenz

last: Rabl

first: Martin

last: Gamerdinger

first: Fabian

last: Morales-Polanco

first: Elke

last: Deuerling

first: Judith

last: Frydman


title: Generation of Syngeneic Orthotopic DMG Murine Models

first: Lorenzo

summary: Diffuse Midline Glioma (DMG) syngeneic orthotopic murine mouse models were used as high fidelity model of human DMG tumors as they recapitulate the heterogeneity, cell states and OncoTarget/OncoTreat drug predictions of human DMG tumors.

overall_design: Single-cells were isolated and sequenced from B6(Cg)-Tyrc-2J/J (B6) mice. An intracranial injection with 100,000 DIPG4423 cells was performed and tumor was isolated 3 weeks post-injection for single cell. Additionally, single cells were also isolated from normal brainstem (no tumor) of the same model

last: Zhang

last: Wang

first: Aleksandar

last: Obradovic

first: Pasquale

last: Laise

first: Aaron

last: Griffin

first: Lukas

last: Vlahos

first: Hanna

last: Minns

first: Diana

last: Morales

first: Christian

last: Simmons

first: Matthew

last: Galitto

first: Hong-Jian

last: Wei

first: Timothy

last: Martins

first: Pamela

last: Becker

first: John

last: Crawford

first: Theophilos

last: Tzaridis

first: Robert

first: James

first: Robyn


title: NSD2 targeting reverses lineage plasticity and drug resistance in advanced prostate cancer [snATAC-seq and multiome snATAC-seq]

first: Jia

middle: Jessie

last: Li

state: NY

department: Medicine

institute: Columbia University Irving Medical Center

summary: Treatment with highly potent androgen receptor signaling inhibitors (ARSIs), such as enzalutamide and abiraterone promotes lineage plasticity in metastatic castration-resistant prostate cancer (mCRPC), which results in intra-tumor heterogeneity and emergence of mCRPC subtypes. The histological transformation from adenocarcinoma to aggressive neuroendocrine prostate cancer (CRPC-NE) has been associated with a loss of dependency on lineage-survival signals, leading to targeted drug resistance. Epigenomic reprogramming might be a fundamental driver of lineage plasticity. To determine CRPC-NE vulnerabilities, we have performed image-based screening using a small library of antibodies targeting different histone marks on CRPC-NE organoids derived from genetically engineered mice. We find that the histone mark H3K36me2 and the histone methyltransferase NSD2 (also known as MMSET/WHSC1) play important roles in maintaining the state of CRPC-NE. Knockout of NSD2 or ablation of H3K36me2 using H3.3K36M oncohistone reverted CRPC-NE to CRPC-AR-like phenotype. Simultaneous profiling of the transcriptome and epigenome from single cells collected from control (sgCtrl) and NSD2 knockout (sgNSD2) organoids, or from empty vector (EV) and H3.3K36M transduced organoids confirms the lineage reversal of treatment-resistant cells and maps the re-establishment of canonical AR signaling dependency. Moreover, H3K36me2 or NSD2 depleted mouse and human CRPC-NE organoids responded to enzalutamide treatment in vitro and in vivo, suggesting a restoration of ARSI sensitivity. Most importantly, a small molecule inhibitor of NSD2, which is similar to KTX-1001 (NCT05651932), in combination with enzalutamide leads to growth suppression or apoptosis of mouse and human CRPC-NE organoids, organoids of other CRPC subtypes and xenografts in vitro and in vivo. In conclusion, inhibition of NSD2 reverses lineage plasticity and reverts the state of treatment-resistant cells back into an ARSI-sensitive state. Thus, we suggest that the combination of NSD2 inhibition with AR inhibition may represent a novel therapeutic approach for patients with CRPC-NE and other CRPC subtypes.

overall_design: The mouse NE tumors derived from NPp53 (Nkx3.1CreERT2/+; Ptenflox/flox; TrpP53flox/flox; Rosa26-EYFP) mice were cultured as organoids. To detect intercellular heterogeneity within organoids, I used single-cell RNA sequencing (scRNA-seq) to analyze lineage diversity of five mouse NE tumor organoids on passage 2. I also used single-nucleus ATAC sequencing (snATAC-seq) to profile the chromatin accessibilities of NPPO-1 organoids on passage 8, a multilineage NE tumor organoid line. The non-NE tumor cells from NPPO-1 organoids on passage 3 were isolated by fluorescence-activated cell sorting (FACS) according to a higher side scatter (SSC) value in non-NE compared to NE tumor cells. Some isolated non-NE tumor cells were analyzed by scRNA-seq to detect any contamination by NE tumor cells. Other isolated non-NE tumor cells were transduced with H2B-RFP lentivirus without antibiotic selection. H2B-RFP labeled tumor cells were either cultured alone or co-cultured with NE tumor cells to induce non-NE to NE conversion. To trace cell lineage of H2B-RFP-labeled cells, organoids from either monoculture or co-culture on passage 4 were analyzed by scRNA-seq (gene name: addgene26001). To quantitively compare the histone mark expression between NE and non-NE cells, epigenomic profiling using Cut&Tag was performed on 4 NE and 4 non-NE organoids derived from the same NPp53 background. To accurately quantify gene regulation, bulk RNA-seq was performed alongside epigenomic profiling on the organoids. To study the effects of NSD2 on NE lineage maintenance, we knockout of NSD2 on two mouse NE tumor organoids using CRISPR/Cas9. After antibiotic selection for 14 days, positive-transduced cells were isolated by FACS sorting according to the presence of RFP reporter signal. We used multiome single-nucleus ATAC and RNA sequencing (multiome snATAC + snRNA-seq) to analyze changes of cell lineage and state in sgNSD2 transfected NE tumor organoids in compared with sgControl (sgCtrl) organoids. To provide direct evidence of H3K36me2 histone modification in modulating NE lineage, mouse NE tumor organoids were transduced with lentivirus carrying the gene encoding the H3.3K36M oncohistone that can cause significant reduction in global levels of H3K36me2. After antibiotic selection for 14 days, cells positively transduced with H3.3K36M or empty vector (EV) control were analyzed using multiome snATAC + snRNA-seq to detect changes in cell lineage and state. In parallel, quantitative epigenomic profiling using Cut&Tag was performed on sgCtl and sgNSD2, or EV and H3.3K36M organoids to quantify global epigenomic changes. We have synthesized a small molecular inhibitor of NSD2 (NSD2i), which is similar to KTX-1001 (NCT05651932) in phase 1 clinical trial in patients with relapsed and refractory multiple myeloma. To evaluate if NSD2i recapitulates the effects of genetic interventions in reversal of lineage plasticity and restoring androgen receptor signaling inhibitor (ARSI) sensitivity, 1 µM NSD2i or DMSO was applied every other day on NPPO-1NE organoids cultured in a medium without DHT for 12 days. Organoids treated with either DMSO or 1 µM NSD2i were then collected for multiome snATAC + snRNA-seq analysis or Cut&Tag epigenomic profiling.

first: Alessandro

last: Vasciaveo

first: Michael

last: Shen


title: NSD2 targeting reverses lineage plasticity and drug resistance in advanced prostate cancer [scRNA-seq]


title: NAC controls nascent chain fate through tunnel sensing and chaperone action[RNA]

first: Jae Ho

last: Lee

overall_design: Transcriptome profiling of whole lysate, membrane-bound, and soluble fraction of C. elelgans upon NAC_KD.

first: Natalia

last: Barbosa

first: Jae

first: Fabian

last: Morales-Polanco

first: Martin

last: Gamerdinger

first: Elke

last: Deuerling

first: Judith

last: Frydman


title: NSD2 targeting reverses plasticity and drug resistance in prostate cancer


title: Diversity and immune dynamics of choroid plexus macrophages are shaped by distinct developmental origins [scRNA-seq LCMV]

first: Alina

last: Ulezko Antonova

first: Siling

last: Du

first: Marco

last: Colonna

first: Simone

last: Brioschi


title: Studies on genomic imprinting in preimplantation embryos of non-human primates [WGBS]

city: Beijing

first: Falong

last: Lu

address: 1 West Beichen Road Chaoyang District

institute: Institute of Genetics and Developmental Biology

summary: Genomic imprinting resulted by asymmetric epigenome of parents is a conserved phenomenon restricting biased expression of genes on two alleles. Disorder of imprinting can cause severe defects and diseases. In mouse and human, most of imprinted genes are expressed in species-specific and tissue-specific style. Here, rhesus monkeys as non-human primates are used to investigate primate genomic imprinting. We got a novel candidate canonical imprinted genes list in genome-wide by integrated analysis of allele-specific DNase I hypersensitive sites, differential expressed genes and differential DNA methylation. Further analysis revealed that both DNA methylation and H3K27me3 have an effect on the expression of imprinted genes. Consistent with human, analysis of H3K27me3 showed that H3K27me3 undergoes reprogramming in pre-implantation. Thus, our data provides a genome-wide view of genomic imprinting in non-human primates and sheds light on the further understanding of epigenetic programming process of monkey.

overall_design: We performed DNase-seq, RNA-seq, WGBS (whole genome bisulfite sequencing) and CUT&RUN (cleavage under targets and release using nuclease)  in androgenetic and gynogenetic embryos.

first: Jixiang

last: Zhang

first: Haifeng

last: Wan

first: Yali

last: Xie

first: Hu

last: Nie


title: Studies on genomic imprinting in preimplantation embryos of non-human primates [RNA-seq]

first: Lianyong

last: Qiao


title: Studies on genomic imprinting in preimplantation embryos of non-human primates

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

overall_design: Refer to individual Series


title: Studies on genomic imprinting in preimplantation embryos of non-human primates [CUT&RUN]


title: KDM2A and KDM2B cooperatively protect a subset of CpG Islands from DNA methylation [ChIP-seq]

summary: In the mammalian genome, most CpGs are methylated, however, regions of the genome known as CpG islands (CGIs) are largely unmethylated, which are closely associated with modulating gene expression. The KDM2 proteins (KDM2A and KDM2B) are recruited via their ZF-CxxC domains to CGIs and mediate demethylation of H3K36me2 at these regions. However, the mechanism whether and how KDM2 proteins protect CGIs from DNA methylation remains unknown. Here, we report that depletion of each or both KDM2 proteins, or mutation JmjC domains of KDM2 proteins, leads to a modest increase of DNA methylation at CGIs, but a significant increase at CGI shores. Depletion of both KDM2 proteins shows most obvious increase of DNA methylation, indicating that KDM2A and KDM2B cooperatively regulate DNA methylation at CGIs. We further show that DNA methylation in the absence of KDM2 proteins show more obvious increase at a subset of CpG Islands such as non-marked and H3K27me3-only CGIs than other CpG Islands like bivalent and H3K4me3-only CGIs. Taken together, our finding reveals the role for KDM2 proteins in regulating DNA methylation at CGIs, and uncovers the mechanism by which complex chromatin state including histone modifications and CGI binding proteins synergistically protect CGIs from DNA methylation.

overall_design: We performed ChIP-seq in WT, Kdm2a/Kdm2b DKO, Kdm2a/Kdm2b JmjC double mutation E14 cell (two replications).

first: Yuan

last: Liu

first: Ying

first: Yunji

last: Zhu


title: KDM2A and KDM2B cooperatively protect a subset of CpG Islands from DNA methylation


title: KDM2A and KDM2B cooperatively protect a subset of CpG Islands from DNA methylation [RRBS]

overall_design: We performed RRBS (Reduced Representation Bisulfite Sequencing) in WT, Kdm2a KO,Kdm2b KO, Kdm2a/Kdm2b DKO, Kdm2a/Kdm2b JmjC double mutation E14 cell (two replications).


title: Studies on genomic imprinting in preimplantation embryos of non-human primates [DNAse-seq]


title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells from Old Individuals towards Senescent Fibroblasts

status: Public on Dec 21 2025

city: Ulm

first: Pallab

last: Maity

address: Meyerhofstrasse/James Franck Ring

department: Dermatology

institute: University of Ulm

summary: Senescent fibroblasts accumulate in the connective tissue of all organs and promote organ aging and aging-related diseases. The underlying mechanisms for the accumulation of senescent fibroblasts are poorly understood. Natural killer (NK) cells of innate immunity play a critical role in the removal of tissue resident senescent cells. We here show that NK cells from old adults and old mice fail to efficiently remove senescent fibroblasts due to severely reduced efficiency of aged NK cells to induce perforin mediated membrane disruptions and granzyme B induced targeted death of senescent fibroblasts. We demonstrate elevated activation of the small Cdc42 Rho GTPase in aged NK cells to be responsible for the disruption of the microtubular organisation of aged NK cells. A correct microtubular organisation of NK cells is essential for cellular polarity, degranulation of perforin and granzyme B into the synaptic cleft between NK cells and senescent fibroblasts, and for a proper function of energy metabolism. Attenuation of the elevated activity of Cdc42 in aged human NK cells with CASIN, a small molecule Cdc42 inhibitor, rebalances Cdc42 activity to a young level, restores a proper perforin and granzyme B release and attenuates reduced ATP levels and mitochondrial dysfunction in aged NK cells to result in a youthful cytotoxicity of aged NK cells against senescent cells. Collectively, we identified a previously unreported molecular mechanism underlying functional impairment of NK cells from older adults. In perspective, our data hold promise to develop novel strategies against age-related disorders driven by tissue-resident senescent fibroblasts.

overall_design: Total RNASeq profiling of natural killer (NK) cells from young and aged human donors


title: NSD2 targeting reverses lineage plasticity and drug resistance in advanced prostate cancer [multiome snRNA-seq]

first: Jia

last: Li

first: Alessandro

last: Vasciaveo

first: Michael

last: Shen


title: Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

first: Ali

last: Shilatifard

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

Elongation factors control post-initiation steps of transcription by RNA polymerase II (RNAPII). We have established distinct mechanistic roles for the essential elongation factors PAF1, NELF, SPT5, and SPT6 via acute depletion of each individually in auxin-inducible degron lines. Here, we leverage these degron lines to explore the regulatory intersection of elongation control and mRNA processing. Integrating long- and short-read RNA-seq data to quantify transcript isoform usage at single-molecule resolution, we identify elongation factor-specific RNA processing regulons including a cellular senescence-enriched regulon shared by NELF and SPT6. We then show that long-term depletion of NELF or SPT6 results in reversible growth arrest following early upregulation of a small group of genes, which include the senescence-associated genes CDKN1A (p21) and CCN2. We perform genetic suppressor screens that implicate the elongation factor Elongin A (ELOA) in NELF or SPT6 depletion-induced growth arrest. ELOA KO suppresses NELF depletion-induced mRNA processing defects and the 3’ extension of RNAPII occupancy past transcription end sites (TES) at genes induced by NELF depletion. ELOA also occupies TES-proximal regions under normal conditions, and acute ELOA depletion results in a loss of RNAPII processivity at the 3’ end of genes, opposing the effects of NELF or SPT6 depletion. Finally, we demonstrate that genetic ELOA loss confers a growth advantage to aging human primary dermal fibroblasts. These findings establish the existence of novel ELOA-dependent mechanisms regulating transcription termination-coupled processes, and links these to the complex phenomena of cellular senescence and aging.


title: Single-cell sequencing data of brain tissue in septic mice

city: Harbin

first: Qiye

last: Le

address: Baojian Subdistrict, Nangang District, Harbin City, Heilongjiang Province

institute: The Second Affiliated Hospital of Harbin Medical University

summary: To explore the specific molecular mechanisms underlying brain injury caused by sepsis, we conducted single-cell transcriptomic analysis on mouse brain tissues. In the experimental group, mice were administered 54 mg/kg of lipopolysaccharide (LPS) via intraperitoneal injection to establish a mouse model of sepsis-associated encephalopathy (SAE). In the control group, mice received an equivalent volume of phosphate-buffered saline (PBS).

overall_design: scRNA-seq: This experiment included three LPS-treated mouse samples and three PBS-treated mouse samples.

first: Jiayu

last: Song

first: Qile

last: Ye

first: Rong

last: Tang

first: Yuwen

last: Wang

first: Mengmeng

first: Rui

last: Zhao

first: Jie


title: Gemcitabine and nab-paclitaxel with or without the VDR agonist paricalcitol for metastatic pancreatic cancer: A randomized, multi-arm, phase I trial

city: Boston

first: Jonathan

middle: Andrew

last: Nowak

state: MA

address: 75 Francis St.

department: Pathology

institute: Brigham and Women's Hospital

summary: Vitamin D receptor (VDR) agonists promote quiescence of cancer-associated fibroblasts and improve efficacy of chemotherapy in preclinical models of pancreatic cancer. We conducted a phase I trial (NCT03520790) with primary endpoint of safety when the VDR agonist paricalcitol is given with first-line gemcitabine and albumin-bound paclitaxel (GA) in patients with metastatic pancreatic cancer. Secondary endpoints included pharmacodynamic analyses. Thirty-six patients were randomized to GA plus placebo, GA plus intravenous paricalcitol or GA plus oral paricalcitol with pre-treatment and on-treatment tumor biopsies. Paricalcitol was safely administered with GA, although 5 (42%) patients receiving oral paricalcitol had grade 2-4 hypercalcemia and required dose reduction. Nuclear VDR protein expression was heterogeneous across patients, and VDR was expressed in tumor, immune, and stromal cells. Compared to pre-treatment specimens, on-treatment biopsies had decreased proportion of αSMA+ fibroblasts, altered fibroblast VDR activation signature, and greater density and spatial colocalization of CD8+ T cells with tumor cells in the GA plus paricalcitol arms. VDR expression was predictive of tumor response in the GA plus paricalcitol arms. Paricalcitol can be safely administered with chemotherapy to patients with metastatic pancreatic cancer, and on-treatment biopsies indicated favorable modulation of the tumor microenvironment by paricalcitol as predicted by preclinical models.

overall_design: A total of 50 tissue slides (31 pre-treatment, 19 on-treatment) were profiled using the GeoMx Digital Spatial Profiler (DSP) and multiple regions of interest (ROI) were selected within areas containing tumor after hybridization with the Human Whole Transcriptome Atlas (WTA) probe mix.  Next, the ROIs were further segmented into areas of illumination (AOIs) using a customized contouring approach, in which cytokeratin is used as a reference to define three non-overlapping areas for ultraviolet light-directed barcode collection: center (tumor epithelial region), ring 1 (stromal region within 1-25 um of the tumor epithelial region), and ring 2 (stromal region within 26-50 um of the tumor epithelial region). The barcodes were then deposited into 96-well plates, subjected to library preparation, and sequencing using S1 flow cells on an Illumina NovaSeq instrument. Samples then underwent demultiplexing. Lastly, raw read counts were analyzed to generate AOI-level expression data. The data was then quality-controlled and third-quartile normalized, resulting in a dataset including counts from 18,412 probes from 676 areas of illumination.

first: Andressa

last: Dias Costa

first: Alexander

last: Jordan

first: Brian

last: Wolpin


title: The cytotoxicity of gomesin peptides is mediated by the glycosphingolipid pathway and lipid-cholesterol interactions

city: Camperdown

first: G.

middle: Greg

last: Neely

state: NSW

address: Charles Perkins Centre, D17.

institute: University of Sydney

summary: Gomesins (AgGom and HiGom) are therapeutically promising spider-derived peptides that target a specific phospholipid composition (3PC:1PS:1Chol) to disrupt melanoma cell membranes and induce cytotoxicity. Their antiproliferative properties are interrelated to lipid metabolism, particularly glycosphingolipid biosynthesis. We used lipidomics, CRISPR/Cas9 knockout screening, molecular and biophysical experiments, followed by xenograft melanoma animal studies to demonstrate that gomesins target the glycosphingolipid pathway, via inhibition of the ST3GAL5 gene. Notably, the addition of cholesterol reduced the cytotoxicity of gomesins, which may explain why melanoma cells with lower cholesterol levels than neonatal foreskin fibroblasts are more sensitive to gomesins. We propose that gomesins bind to melanoma CRAC domains, restricting intracellular cholesterol and thereby enhancing their cytotoxicity. In line with this hypothesis, cholesterol sequestration and the disruption of lipid raft microdomains enhanced the cytotoxic effects of gomesins in melanoma cells, whereas adding cholesterol in membrane permeability and proliferation assays reduced the effects of gomesin treatment. Taken together, this study highlights the specific role of cholesterol and the glycosphingolipid pathway in melanoma cells, paving the way for new strategies in targeted melanoma therapies.

overall_design: A whole genome CRISPR knockout screen was performed in MM96L cells transduced with the Toronto Knockout Library v3 (TKOv3) sgRNA library and treated with gomesin. Following selection, genomic DNA was extracted from both selected and untreated cells, sgRNA sequences were amplified by PCR and next generation sequencing used to quantify reads. Analysis was performed using MAGeCK.

first: Matthew

middle: A

last: Waller

first: Naomi

last: McKinnon

first: Felicity

last: Chung

first: Graham

middle: G


title: RNA sequencing of ex vivo microtumor models embedded in different matrices and constructed from either control or Arpc4-knockout cells.

city: Heidelberg

first: Yina

last: Qiao

institute: Heidelberg University Hospital

summary: The loss of Arpc4 resulted in reduced expression of other Arp2/3 complex components, rendering the Arp2/3 complex nonfunctional. The ex vivo microtumor(3D) models were composed of either control or Arpc4 knockout (KO) KC 8025 cells embedded in different matrices containing three condition:25% collagen I, 25% collagen I + 75% Matrigel, and 75% Matrigel for 5 days. To identify genes affected by Arpc4 knockout in this model, RNA-seq analysis was performed on bulk samples. 8025 cell line was derived from primary cells isolated from genetically engineered p48Cre/+; LSL-KrasG12D/+ (KC).

overall_design: The 8025 cell line were established from primary cells isolated from genetically engineered p48Cre/+; LSL-KrasG12D/+ (KC) mice and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. The cell line was transfected with the CRISPR/Cas9 p20-ARC Double Nickase Plasmid (Santa Cruz Biotechnology, USA) to target the Arpc4 gene, alongside a negative control plasmid (Santa Cruz Biotechnology, USA). Transfections were carried out using Lipofectamine 3000 (Thermo Fisher, USA) according to the manufacturer’s protocol. Forty-eight hours post-transfection, green fluorescent cells were sorted via fluorescence-activated cell sorting (FACS) and selected using puromycin (InvivoGen, Toulouse, France) at concentrations of 0.7 mg/mL. After 5–7 days of puromycin selection, cells were seeded into 96-well plates for clonal expansion. Arpc4 knockout (Arpc4ko) clones were validated via Western blot analysis. Ex vivo microtumor models (3D) were constructed using organoids derived fron 8025 control or Arpc4ko cells embedded in different matrices containing three condition:25% collagen I, 25% collagen I + 75% Matrigel, and 75% Matrigel for 5 days. Total RNA was extracted by adding TRIzol reagent (Thermo Fisher, USA) to the gel after removing the supernatant and dissolving the gel by thorough mixing. Chloroform (Thermo Fisher, USA) was then added, followed by vigorous vortexing for 15 seconds. After incubation at room temperature for 3 minutes, the mixture was centrifuged at 12,000×g for 15 minutes at 4°C. The upper aqueous phase was carefully transferred to a fresh tube, mixed with an equal volume of ≥95% ethanol, and purified using the Monarch Total RNA Miniprep Kit (New England Biolabs, Germany) according to the manufacturer’s protocol. RNA sequencing was performed by Biomarker Technologies(BMKGENE), Münster, Germany.

first: Xiufen

last: Yang

first: Yifeng

last: Sun

first: Christoph

last: Michalski

first: Bo

last: Kong


title: RNA-seq data of the kidneys of diabetic nephropathy rats treated with TSK

last: ZHAO

address: No. 2, Xiangjiang Road, Xiangfang District, Harbin City, Heilongjiang Province

institute: Department of Integrative Medicine (Clinical), The Third Affiliated Hospital of Heilongjiang University of Chinese Medicine.

summary: TSK (TangShenKang) is a compound traditional Chinese medicine. To explore the core corresponding program of this medicine acting on diabetic kidneys, RNA-seq was conducted on rats kidney tissues under different treatments in this study. Specifically, this experiment was divided into three groups (6 rats in each group) : Normal control group (Ctrl) , DN model group, treatment group.

overall_design: 6 rats in each group:  control group (Ctrl) (ordinary feed + solvent gavage), the DN model group (high-fat diet +STZ injection to induce DN), and the treatment group (DN + TSK) (TSK 500 mg/kg/d oral administration, treated for 4 weeks).

first: Ran

last: Wei

first: Nuan

last: Wen

first: Jian

last: Ma

first: Xicheng

last: Jiang

first: Huijun

last: Chen

first: Bingmei

first: Chuwen

last: Feng

first: Lei

last: Zheng

first: Shanshan

first: Wei

last: Zhou

first: Zeyu

last: Yu


title: TRIM40 Drives Pathological Cardiac Hypertrophy and Heart Failure through PKN2 Ubiquitination

city: Jilin City

first: Mengyang

last: Wang

state: Jilin

address: No. 3999, Huashan Road, Fengman District, Jilin City, Jilin Province

institute: Beihua university

summary: Pathological cardiac hypertrophy is a key predisposing factor for heart failure (HF). This study investigates the role of the E3 ubiquitin ligase Tripartite Motif-Containing 40 (TRIM40) in cardiac hypertrophy. Using TRIM40 knockout (TRIM40-/-) and cardiac-specific overexpressing mice, pathological hypertrophy was induced by angiotensin II (Ang II) infusion or transverse aortic constriction (TAC). Results demonstrated that TRIM40 expression was upregulated in hypertrophic hearts. TRIM40 deficiency attenuated cardiac hypertrophy and dysfunction, whereas its overexpression exacerbated pathological remodeling. Mechanistically, TRIM40 binds Protein Kinase N2 (PKN2) via its B-box domain, promoting K63-linked ubiquitination at cysteine 29 that enhances PKN2 phosphorylation at Ser815 and activates downstream signaling. Pharmacological inhibition of PKN2 attenuated cardiac remodeling induced by TRIM40 overexpression. These findings indicate that TRIM40 promotes cardiac hypertrophy through K63-linked ubiquitination and activation of PKN2, identifying TRIM40 as a potential therapeutic target for HF.

overall_design: We performed RNA sequencing analysis on the heart tissues of three control group mices and three Ang II-induced mices.

first: Risheng

last: Zhao


title: 3D fractal topography attenuates inflammation and confers resilience to glomerular podocytes

city: Toronto

first: Milica

last: Radisic

state: Ontario

address: 160 College St

country: Canada

institute: University of Toronto

summary: In vivo, podocytes wrap around a looping capillary forming interdigitations required to establish a barrier function in kidney filtration. Cultured human podocytes can mimic key in vivo disease-associated changes—such as junctional and cytoskeletal remodeling, but their intricate, self-similar 3D branching structure remains poorly understood and technically difficult to replicate in vitro, especially in the context of inflammatory injury. Here, we established 3D fractal hemispheres capturing key features of glomerular architecture using nanoscale two-photon polymerization (2PP), enabling a detailed investigation into the effects of 3D topographies with various fractal indices on podocyte morphology and function. Podocytes cultured on intermediate fractal hemispheres,  exhibited enhanced cytoskeletal branching, increased expression of slit diaphragm markers, and upregulation of nephrogenesis-related genes. To explore cellular responses to inflammatory stimuli by TNF-α and IL-6, we established a culture of fluorescently labelled podocytes within otherwise optically silent monolayer. The changes of single podocyte structure, fractal dimension and RNA sequencing revealed that podocytes grown on intermediate fractal hemispheres exhibited an enhanced immunosuppressive response, highlighting the potential of fractal topological structures in mitigating inflammation induced podocyte damage and kidney disease progression. A co-culture system with reporter THP-1-derived macrophages demonstrated a pronounced shift toward the M1 macrophage phenotype and heightened activation of inflammation pathways when podocytes were co-cultured on smooth hemispheres,  compared to those on intermediate fractal hemispheres. These findings suggest that fractal microenvironments may play a crucial role in modulating podocyte-macrophage interactions and inflammatory responses in kidney disease.

overall_design: To investigate the behavior of PODO/TERT256 human podocytes, cultured on 3D hemispheres with either smooth or fractal topography, in response to inflammation induced by cytokines (TNF-α and IL-6) or by THP-1-derived macrophages, we performed RNA sequencing of PODO/TERT256 human podocytes from the following groups: 1. Cells cultured on 3D hemispheres with smooth topography under the control condition 2. Cells cultured on 3D hemispheres with fractal topography under the control condition 3. Cells cultured on 3D hemispheres with smooth topography under the inflammation condition induced by cytokines (TNF-α and IL-6) 4. Cells cultured on 3D hemispheres with fractal topography under the inflammation condition induced by cytokines (TNF-α and IL-6) 5. Cells cultured on 3D hemispheres with smooth topography under the inflammation condition induced by THP-1-derived macrophages 6. Cells cultured on 3D hemispheres with fractal topography under the inflammation condition induced by THP-1-derived macrophages

first: Ying

first: Karl

last: Wagner

first: Shira

last: Landau

first: Chuan

last: Liu

first: Sargol

last: Okhovatian

first: David

middle: F

last: Bodenstein

first: Daniel

last: Vosoughi

first: Yimu

first: Karen

last: Shen

first: Qinghua

last: Wu


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [DHC-18mo-m-003]

city: La Jolla

first: Joseph

middle: R

last: Ecker

address: 10010 North Torrey Pines Road

institute: HHMI-Salk-Institute

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of both male and female mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

overall_design: Apply snmC-seq3 to mouse brain regions to identify aging effects on epigenomics

last: Tian

first: Anna

last: Bartlett

first: Jordan

last: Altshul

first: Rosa

last: Gomez Castanon

last: Nery

first: Jacinta

middle: D

last: Lucero

first: Julia

middle: K

last: Osteen

first: Huaming

first: M

middle: M

last: Behrens


title: Longitudinal monitoring of Type 1 Diabetes progression to disease onset

city: Ann Arbor

first: Lonnie

last: Shea

state: MI

address: 1600 Huron Parkway

department: Biomedical Engineering

institute: University of Michigan

summary: Preventing autoimmune type 1 diabetes (T1D) necessitates improved monitoring for disease progression prior to symptom onset. Current diagnostic methods assess circulating autoantibodies, C-peptide levels, or dysglycemia, yet these approaches fail to identify β cell destruction preceding glucose dysregulation. Herein, a subcutaneous microporous scaffold is employed as an immunological niche (IN), which provides a non-vital accessible tissue reflecting many immune changes occurring in the pancreas. RNA sequencing analysis of the IN successfully delineates at-risk from non-risk groups, as well as disease progressors from non-progressors at six weeks of age in the NOD mouse model. Within progressors, we identify disease five to seven weeks prior to disease onset. Collectively, disease occurring in a poorly accessible site can be identified early by sampling a distant non-vital tissue, indicating the systemic nature of the disease and informing the timing of disease modifying therapies to halt or delay the progression of T1D.

overall_design: RNA-seq profiling of microporous scaffolds implanted into NOD and NOR mice. Samples were taken from all mice at 6 weeks of age. Additional samples were taken at 7, 5, 3, and 1 week before symptom onset in NOD mice that progressed to hyperglycemia, as well as at the time of symptom onset. Samples were taken at 10, 14, 18, and 22 weeks of age for NOD mice that did not develop hyperglycemia and NOR controls.

first: Jessica

middle: L

last: King


title: Transfection experiments with several stimulators

city: Shanghai

first: Haiyang

last: Hu

address: 320 Yueyang Road

institute: PICB

summary: We conducted RNA-seq following transfection experiments with several stimulators

overall_design: Total RNAs  were isolated and sequenced by deep sequencing using Illumina HiSeq 4000

first: Mingju

last: Lyu


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [DHC-18mo-m-002]

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

first: Wei

last: Chen


title: Essential role of NONO-HOXA1-Wnt axis in cardiomyocyte differentiation

first: Feizhen

state: Shanghai

address: Zhangheng Road 825, Pudong

department: Intelligent Medicine Institute

institute: Fudan Univ, Shanghai, China

summary: NONO is recognized as a critical molecular scaffold involved in both transcriptional and posttranscriptional regulation. Mutations in NONO are frequently linked to congenital heart diseases (CHDs) in humans. However, the mechanisms by which NONO regulates cardiac development remain elusive. Here, we identified NONO as a pivotal dual-function regulator of cardiomyocyte differentiation in human induced pluripotent stem cells (hiPSCs). NONO deficiency in hiPSCs results in a distinct defect in early cardiomyocyte differentiation. Mechanistically, NONO interacts with HOXA1 and regulates the dynamic expression of key genes during early cardiomyocyte differentiation. ChIP-seq analysis reveals that NONO loss reduces HOXA1 occupancy at target genes, compromising its transcriptional regulation. Additionally, NONO and HOXA1 cooperatively activate the Wnt signaling. Taken together, these findings establish the NONO-HOXA1-Wnt axis as a key molecular mechanism in cardiomyocyte differentiation and provid insights into the etiology of CHDs associated with NONO mutations.

overall_design:
We performed ChIP-seq of NONO in Day2-differentiated cells. We performed ChIP-seq of HOXA1 in WT and NONO-KO Day2-differentiated cells overexpressing Dox-induced HOXA1-HA using anti-HA tag antibody, Input of ChIP-seq in Day2-differentiated cells, mRNA-seq of WT,  NONO-KO Day2, 5, 7, 9-differentiated cells and HOXA1-KO Day2-differentiated cells.
We performed reChIP-seq of NONO in Day2-differentiated cells overexpressing Dox-induced HOXA1-HA using anti-HA for primary ChIP and anti-NONO for re-ChIP. We performed ChIP-seq of HOXA1 in WT and NONO-KO Day2-differentiated cells using anti-H3K27AC antibody, Input of ChIP-seq in Day2-differentiated cells. We performed ATAC-seq in Day2-differentiated cells.

last: Sheng

first: Guoying

last: Huang

first: Zhiyu

last: Feng

first: Yuan

last: Gao