title: Tracing the evolution of single-cell 3D genomes in Kras-driven cancers

first: S

last: Agabiti

summary: Although three-dimensional (3D) genome structures are altered in cancer cells, little is known about how these changes evolve and diversify during cancer progression. Leveraging genome-wide chromatin tracing to visualize 3D genome folding directly in tissues, we generated 3D genome cancer atlases of oncogenic Kras-driven murine lung and pancreatic adenocarcinoma. Our data reveal stereotypical, non-monotonic, and stage-specific alterations in 3D genome folding compaction, heterogeneity, and compartmentalization as cancers progress from normal to preinvasive and ultimately to invasive tumors, discovering a potential structural bottleneck in early tumor progression. Remarkably, 3D genome architectures distinguish morphologic cancer states in single cells, despite considerable cell-to-cell heterogeneity. Gene-level analyses of evolutionary changes in 3D genome compartmentalization not only showed that compartment-associated genes are more homogeneously regulated but also elucidated prognostic and dependency genes in lung adenocarcinoma and a previously unappreciated role for polycomb-group protein Rnf2 in 3D genome regulation. Our results demonstrate the utility of mapping the single-cell cancer 3D genome in tissues and illuminate its potential to identify new diagnostic, prognostic, and therapeutic biomarkers in cancer.

overall_design: Tumor-bearing lungs from MADM-Lung mice were dissected out and GFP+ tumors selected,  Biologic replicates include 12.

first: Miao

last: Liu

first: Shengyan

last: Jin

first: Sherry

first: Tyler

last: Jensen

first: Tianqi

first: Jonathan

last: Radda

first: Christian

last: Ruiz

first: Gabriel

last: Baldissera

first: Moein

last: Rajaei

first: Fang-Yong

last: Li

first: Jeffrey

last: Townsend

first: Mandar

last: Muzumdar

first: Siyuan

last: Wang


title: snRNAseq of mouse thick ascending limb cells

status: Public on May 24 2025

city: Los Angeles

middle: William

last: Nelson

state: CA

address: 1333 San Pablo St.

department: Medicine

country: USA

institute: University of Southern California

type: Expression profiling by high throughput sequencing

summary: Kidney thick ascending limb (TAL) cells display a remarkable amount of regional and functional heterogeneity. We applied single-nucleus RNA-seq to map the molecular heterogeneity of enriched Slc12a1+ cells.

overall_design: Kidney thick ascending limb (TAL) cells from Slc12a1-CreERT2-INTACT mice were isolated by Fluorescence-activated nuclei sorting (FANS) according to the presence or absence of GFP signal and analyzed using snRNAseq.

middle: W

first: Jessica

last: Bahena-Lopez

first: Xiao-Tong

last: Su

first: David

middle: H

last: Ellison


title: Notch-dependent functional conversion of RUNX factors regulates the initiation of T-lineage program

status: Public on May 07 2025

city: minatoku toranomon

first: Tatsuma

last: Matsuda

address: tokyo

country: Japan

institute: toranomon hospital

summary: Runt-related transcription (RUNX) factors play a key role in T cell development. At the T-lineage commitment checkpoint, RUNX1 undergoes dynamic partner switching, resulting in its redeployment. Here, we investigated the functional differences in RUNX factors between the lymphoid progenitor (LP)- and Notch-stimulated earliest T progenitor stages (Phase 1). We identified CCCTC-binding factor (CTCF) as an LP-specific RUNX1-interacting partner, with LP-specific RUNX1 binding genomic sites significantly enriched for CTCF consensus motifs and co-occupied by CTCF. On Notch stimulation, Notch1 intracellular domain (Notch1-IC) directly interacts with RUNX1 and recruits the RUNX1/Mediator/p300 transcriptional activation complex to Notch-regulated T-signature gene loci. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and their binding partners revealed that the RUNX1/CTCF complex in LP negatively regulates T-signature gene expression, whereas the RUNX1/Mediator/p300 complex in Phase 1 promotes it. Our findings highlight the crucial role of Notch-mediated functional conversion of RUNX factors, including protein complex re-organization and genomic redeployment in initiating T-lineage program.

overall_design: The cell suspension obtained from the resected tumor specimens was processed according to 10×Genomics specifications, using the 10×Chromium Next GEM single-cell 3ʹ reagent kit and library construction kit. The cell suspension was processed in the Chromium Controller to create gel beads in emulsion (GEMs), incubated at a specific temperature to generate cDNA containing barcodes and Unique Molecular Identifiers (UMIs). Subsequently, the cDNA was washed, amplified, and its quality assessed, then enzymatically fragmented and size-selected, followed by adapter ligation and indexed PCR amplification. Finally, after quantification and quality assessment of the library, sequencing was performed on the NovaSeq 6000.

first: T

last: Tanaka

last: Hosokawa


title: Immune cell type divergence in a basal chordate

status: Public on May 23 2025

city: Boston

first: Tal

middle: Deborah

last: Scully

state: MA

address: 200 Longwood Ave

department: Systems Biology

institute: Harvard Medical School

summary: Evolutionary adaptations often occur at the level of cell types and cellular function. Innate immune cells are a promising system for studying cell type evolution, as they are widespread across metazoans, have several conserved functions, and are under selective pressure from pathogens. However, molecular characterizations of invertebrate immune cells are limited, and it remains unclear whether invertebrate immune cell types are homologous to those in vertebrates. Here we use single-cell RNA sequencing, in situ hybridization, and live reporters to define the identity of blood cell states from a basal chordate, Ciona robusta. We find evidence that C. robusta circulating blood contains a differentiation hierarchy that gives rise to at least eight major morphotypes, constituting approximately half of mature blood cell states. The mature cell states include phagocytes, as well as cells variously expressing vanadium-binding proteins, carbonic anhydrases, pattern recognition receptors, cytokines, and complement factors. Despite the expression of homologs to vertebrate immune components, extensive divergence between tunicate and vertebrate immune cells obscures cell state homology. Altogether, this work modernizes blood cell classifications in C. robusta and extends the known repertoire of immune cells within chordates.

overall_design:
Circulating blood cells were collected from adult Ciona robusta animals, then run directly and analyzed with scRNA-seq. Two libraries were collected in this study, and they were combined with one library from a previous study for analysis (reanalysis of Sample GSM8869531 from Series GSE292926). This combined data (combined_processed_data.h5ad, an H5ad Hierarchical Data Format file) was reanalyzed by removing likley doublets, normalizing across all libraries, identifying of highly variable genes, log normalizing, z-scoring, performing PCA on highly variable genes, batch correcting using BBKNN, Leiden clustering, and UMAP embedding. Additionally, cells from all samples were assigned to SNP profiles using Cellsnp-lite and Vireo. In the combined_processed_data.h5ad, adata.obs['library'] indicates which cell barcodes are associated with each library: 230518_1 and 230518_2 are from this GEO Series (Cr_blood_230518_1 and Cr_blood_230518_2, respectively), and 220428_1 is the reanalyzed Sample GSM8869531 from Series GSE292926.

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************

middle: D

first: C. J.

last: Pickett

first: Nicolas

middle: A

last: Gort-Freitas

first: Bradley

last: Davidson

first: Allon

middle: M

last: Klein


title: A novel human heart-derived natural adeno-associated virus capsid combines cardiospecificity with cardiotropism in vivo

city: Heidelberg

first: Shubhada

last: Kulkarni

address: In Neuenheimer Feld 669

country: Germany

institute: UKHD

summary: Adeno-associated virus serotype 9 (AAV9) is currently considered the benchmark vector for clinical cardiac gene transfer applications but can entail severe hepatotoxicity due to its strong hepatotropism. Against this background, we report the discovery of a novel AAV capsid (cap) isolated from human myocardium that uniquely combines cardiospecificity with cardiotropism in vivo and bears potential to impact future cardiac gene therapy developments.

overall_design: snRNA-Seq profile of left venctricle from a male C57BL/6N mouse 2 weeks after transduction with AAVC7.

first: Julia

last: Ritterhoff

first: Patrick

last: Most


title: Integrated single-cell multiome analysis resolves muscle fiber-type gene regulatory circuitry modulated by endurance exercise

status: Public on May 05 2025

city: New York

first: Elena

last: Zaslavsky

state: NY

address: 1 Gustave L. Levy Pl

institute: Icahn School of Medicine at Mount Sinai

summary: Endurance exercise is an important health modifier. We studied cell-type specific adaptations of human skeletal muscle to acute endurance exercise using single-nucleus multiome sequencing in human vastus lateralis samples collected before and 3.5 hours after 40 min exercise at 70% VO2 max in four subjects, as well as in matched time of day samples from two supine resting circadian controls. High quality same cell RNAseq and ATACseq data were obtained from 37,154 nuclei comprising 14 cell types. Among muscle fiber types, both shared and fiber-type specific regulatory programs were identified. Comprehensive single cell regulatory circuit analysis identified distinct fiber type programs involving 272 TFs regulating 1579 genes at specific regulated chromatin sites, with fast fibers showing more robust circuit responses. These data and comprehensive circuit reconstruction provide a high resolution map of the molecular adaptations to exercise at single cell resolution.

overall_design: Multiomic (RNA+ATAC) sequencing of PBMCs from 4 exercise response samples and 2 circadian control samples

first: Aliza

middle: B

last: Rubenstein


title: Skeletal muscle Rev-erbs regulates metabolic diurnal rhythm

status: Public on May 04 2025

city: HOUSTON

first: Jidong

last: Liu

state: TX

address: One Baylor Plaza

institute: Baylor College of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Rev-erb plays a pivotal role in coordinating circadian rhythms and in regulating metabolism. However, The regulation of muscle metabolism by Rev-erbs is still not fully understood. In this study, we explored the mechanisms through which Rev-erb regulates skeletal muscle by using ChIP-seq.

overall_design: Skeletal muscle was harvested at ZT10 and ZT22, we performed ChIP-seq analysis with skeletal muscle tissue to investigate Rev-erbα binding sites.

first: Fang

last: Xiao

first: Abhinav

last: Choubey

first: Zheng

last: Sun


title: Cortex Folding by Combined Progenitor Expansion and Adhesion-Controlled Neuronal Migration

status: Public on May 20 2025

city: Martinsried

first: Tobias

last: Straub

address: Großhadener Str. 9

department: Biomedical Center, Bioinformatics

institute: LMU Munich

summary: Folding of the mammalian cerebral cortex into sulcal fissures and gyral peaks is the result of complex processes that are incompletely understood. Previously we showed that genetic deletion of Flrt1/3 adhesion molecules causes folding of the smooth mouse cortex into sulci resulting from increased lateral dispersion and faster neuron migration, without progenitor expansion. Here, we find that combining the Flrt1/3 double knockout with an additional genetic deletion that causes progenitor expansion, greatly enhances cortex folding. Expansion of intermediate progenitors by deletion of Cep83 results in enhanced formation of sulci. Expansion of apical progenitors by deletion of Fgf10 results in enhanced formation of gyri. Single cell transcriptomics and simulations suggest that changes in adhesive properties of cortical neurons, their proportions and densities in the cortical plate, combined with lateral dispersion during their radial migration are important folding parameters. These results identify key developmental mechanisms that cooperate to promote cortical gyrification

overall_design: Embryonic E15.5 of rostral-medial region of whole neocortex where potential area of folding were collected for analysis of scRNA data

first: Seung

last: Chun

first: D

middle: S

last: Diaz Almeida

first: Mihail

last: Ivilinov Todorov

last: Ruff

first: Wei

last: Shao

first: Jianjun

last: Yang

first: Gönül

last: Seyit-Bremer

first: Yi-Ru

last: Shen

first: Ali

last: Ertürk

first: Daniel

last: del Toro

first: Songhai

last: Shi

first: Rüdiger


title: Transcriptome analysis of Rev-erbs skeletal muscle specific knockout muscles and WT muscles

summary: We used Rev-erbs skeletal muscle specific knockout mouse model to illustrate the Rev-erbs function in skeletal muscle. In this study, transcriptome sequencing was used to detect the mRNA expression profiles of KO muscles and WT muscles.

overall_design: Skeletal muscle was harvested at ZT0 and ZT12, we performed RNA-seq analysis with skeletal muscle tissue to investigate mRNA expression among skeletal muscle specific Rev-erbs KO and WT mice.


title: 2-hydroxyglutarate mediates whitening of brown adipocytes coupled to nuclear softening upon mitochondrial dysfunction

status: Public on May 14 2025

city: Cologne

first: Aleksandra

last: Trifunovic

address: Joseph-Stelzmann-Str. 26

institute: CECAD research center

summary: Mitochondria play a crucial role in regulating cellular homeostasis in response to intrinsic and extrinsic cues by changing cellular metabolism to meet these challenges. However, the molecular underpinnings of this regulation and the complete spectrum of these physiological outcomes remain largely unexplored. In this study, we elucidate the mechanisms driving the whitening phenotype in brown adipose tissue (BAT) deficient in the mitochondrial matrix protease CLPP. We find that CLPP-deficient BAT shows aberrant accumulation of lipid droplets, which occurs independently of defects in oxygen consumption and fatty acid oxidation. Our results indicate that mitochondrial dysfunction due to CLPP deficiency leads to the build-up of the oncometabolite D-2hydroxyglutarate (D-2HG), which in turn promotes lipid droplet enlargement. We further demonstrate that D-2HG influences gene expression and decreases nuclear stiffness by modifying epigenetic signatures. We propose that lipid accumulation and altered nuclear stiffness regulated through 2-HG are unique stress responses to mitochondrial dysfunction.

overall_design: ChIP Seq of histone modification H3K4me3 in primary brown adipose tissue cells (mBA cells). Three conditions were used, WT cells, WT cells treated with 2 ydrxyglutarate and CLPP KO cells.

first: Harshita

last: Kaul


title: Imaging-based drug screening combined with moleuclar profiling identifies signatures and drivers of therapy resistance in pediatric AML [ATAC-seq]

status: Public on May 02 2025

city: Vienna

first: Kaan

last: Boztug

state: Vienna

address: Zimmermannplatz 10

country: Austria

institute: St. Anna Children's Cancer Research Institute

summary: Despite recent advances in the understanding of genomic landscape of pediatric acute myeloid leukemia (pedAML), targeted treatments are only available for selected genomic alterations and the functional link between genotype and outcome remains partially elusive. Functional precision medicine approaches to investigate treatment resistance and patient risk have not been applied systematically for pedAML. We established an advanced functional screening platform combining high-content imaging and deep-learning-based phenotyping. In 45 pedAML patients, we identify BCL2 and FLT3 inhibitors and standard chemotherapy as major drivers of the chemosensitivity landscape, reveal substantial differential sensitivities between risk groups, and may effectively predict individual measurable residual disease and patient risk. Integration with genomic and epigenomic data uncovers a chemotherapy-resistant primitive state vulnerable to combined BCL2 and MDM2 inhibition and HDAC inhibition. Overall, we identify early present signatures of therapy resistance across genetic subgroups and prioritize targeted treatments for these functionally and epigenetically defined patient subsets.

overall_design: 44 ATAC-seq samples from isolated blasts of pedAML patients sampled at diagnosis

first: Ben

last: Haladik

first: Margarita

last: Maurer-Granofszky

first: Peter

last: Zoescher

first: Raul

last: Jimenez-Heredia

first: Alexandra

last: Frohne

first: Anna

last: Segarra-Roca

first: Chloe

last: Casey

first: Felix

last: Kartnig

first: Sarah

last: Giuliani

first: Christina

last: Rashkova

last: Repiscak

first: Michael

middle: N

last: Dworzak

first: Giulio

last: Superti-Furga


title: Imaging-based drug screening combined with moleuclar profiling identifies signatures and drivers of therapy resistance in pediatric AML

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

overall_design: Refer to individual Series


title: DNA bendability regulates transcription factor binding to nucleosomes

status: Public on May 01 2025

first: Luca

last: Mariani

state: ma

address: 77 Avenue Pasteur

first: Martha

middle: L

last: Bulyk


title: DNA bendability regulates transcription factor binding to nucleosomes [PIONEAR-seq and SELEX-seq]

summary: Cell fates are controlled by ‘pioneers’, sequence-specific transcription factors (TFs) that bind recognition motifs on nucleosomes (‘pioneer binding’). Pioneers occupy a minority of their recognition sequences in the genome, suggesting that the sequence context regulates their binding. Here, we developed PIONEAR-seq, a high-throughput biochemical assay to characterize pioneer binding to nucleosomes. We used PIONEAR-seq to assay 11 human TFs for binding to nucleosomes based on Widom 601 versus genomic sequences. We found that pioneer binding, while mediated primarily by TFs' recognition motifs, senses the broader nucleosome sequence context and that TFs previously found to be dyad or periodic binders on nucleosomes assembled on synthetic sequences exhibit exclusively end binding to nucleosomes based on genomic sequences. We propose a model where the nucleosome exploits the local bendability of the DNA sequence to position pioneer binding, revealing another cis-regulatory layer in eukaryotes.

overall_design: SELEX followed by sequencing for the following full-length human Transcription Factors: ASCL1, CEBPB, FOXA1, GATA1, GATA4, KLF4, OCT4, PAX7, PU.1, RUNX2, SOX2 and ZNF143. The DNA ligand libraries for SELEX were based on 4 DNA sequences of 147 bases (ALBN1, CX3CR1, NRCAM, W601) known to position nucleotide either in vivo or in vitro. These sequences were tiled with random 20-mers at several position, to select for preferences in motif binding and motif positioning. The libraries were either left as free DNA for the SELEX-seq assay, or assembled on nucleosome core particles (NCP) for the PIONEAR-seq assay. The TFs were tagged with GST, which was used for the pulldown of the TF and its associated DNA (eventually assembled on NCP in the case of PIONEAR-seq). After the pulldown, the DNA ligands were amplified by PCR and the selection process was repeated 3 further times. After 4 rounds of selection, the DNA ligands were prepared for Illumina sequencing. W601, also known as Widom 601, is a 147-bp DNA sequence of synthetic origin with the highest reported affinity to the histone octamer (PMID: 9514715 ). ALBN1 is a 147-bp DNA sequence based on the genomic sequence of the nucleosome N1 within the ALB enhancer (PMID: 31303471). CX3CR1 is a 147-bp sequence from the CX3 chemokine receptor 1 locus that have been proposed to be a candidate nucleosome target for pioneer factors in hematopoietic differentation (PMID: 31303471). NRCAM is a 147-bp genomic nucleosomal sequence from the Neuron-Glia-CAM- related cell adhesion molecule locus that gets digestable by Mnase upon neuronal reprogramming (PMID: 31303471)

first: Xiao

last: Liu

first: Kwangwoon

last: Lee

first: Stephen

last: Gisselbrecht

first: Philip

last: Cole


title: Lactate influx promotes immunoevasion in pancreatic cancer by activating autophagy of pancreatic stellate cells

status: Public on Apr 30 2025

city: WuHan

first: ZHUO

last: WENFENG

address: 湖北省武汉市硚口区航空路13号

country: China

institute: The Huazhong University of Science and Technology

summary: It has been reported that lactate plays as a bridging signaling molecule to coordinates tumor cell and tumor stroma. As pancreatic stellate cells (PSC) is the most majority and functional cells in tumor microenvironment (TME) of pancreatic cancer (PC), whether the lactate involving the crosstalk between PSC and pancreatic acinar cells (PACs) in tumorigenesis of PC has not been defined. The present study showed that lactate transporter, monocarboxylate transporter 1 (MCT1) was predominantly expressed in pancreatic stellate cells (PSC), and correlates with worse prognosis in PC patients. Moreover, we revealed lactate induced autophagy-mediated activation of PSC, while knockout MCT1 in PSC  (PSC-MCT1-/- mice) obviously inhibited the proliferation and metastasis of  PC in vivo. Meanwhile, results of single-cell sequence demonstrated that the PD-1 expression in CD8+T cells was dramatically increased. Mechanistically, the MCT1-mediated influx of lactate activated PSC by inducing lactylation of lysine residues K356 and K781 on Vps34, a key regulator for autophagy. Moreover, the PSC-derived CXCL9/CXCL10 upregulated PD-1 expression via activating CXCR3/STAT3 pathway in CD8+ T. Consistently, the growth of pancreatic tumor in situ was significantly inhibited in mice treated with MCT1 inhibitor AZD3965, which was further suppressed by combination with PD-1 antibody. In conclusion, our study implicates lactate functions as TME transducer mediating activation of PSC and formation of immunosuppressive TME, and indicates the synergistic effect of targeting PSC activation in immune checkpoint therapy in PC.

overall_design: Tumors formed by orthotopic pancreatic injection of KPC cells in FLOX/MCT1-/- mice were harvested and prepared for single-cell homogenization. Subsequently, the samples were analyzed using high-throughput expression profiling systems.

first: Wenfeng

last: Zhuo

first: Gang

last: Zhao

first: Zhu

last: Zeng

first: Yuhang

last: Hu

first: Shengbo

last: Han


title: Site-specific phosphorylation of ERα determines sex-dependent metabolic, reproductive, and body compositional phenotypes in mice

city: New Orleans

first: Binghao

last: Zou

address: 1430 Tulane Ave

institute: Tulane University

summary: Estrogen receptor alpha (ERα) phosphorylation modulates receptor activity and downstream signaling. Previous studies identified human phosphorylation sites, serine-167 (S167) and serine-212 (S212) as critical regulators of ERα-mediated gene expression. Serine(S) to alanine(A) phospho knock-in mouse models of the corresponding murine sites, S171 and S216, were generated to assess physiological impact. Compared to WT mice, ERα protein levels in tissues were altered in a sex- and phosphorylation site-dependent manner. S216A males exhibited reduced adipose and increased lean mass. S216A females exhibited decreased femur bone density whereas S171A females exhibited increased bone density. S216A females were subfertile with fewer pups/litter. Single-cell and spatial transcriptomics revealed markedly altered E2-regulated uterine gene expression in both models. S216A mice exhibited reduced plasma metabolites in several key metabolic pathways. This study identifies critical roles for site-specific ERα phosphorylation in modulating receptor level/activity and gene expression that has profound impact on murine body composition, fertility, and metabolism.

overall_design: This study employed NanoString CosMx spatial transcriptomics to analyze formalin-fixed, paraffin-embedded (FFPE) uterine sections from ovariectomized mice treated with either vehicle or estradiol (E2). A 1000-plex gene panel was used to capture spatial gene expression. Machine learning-based cell segmentation enabled the identification of individual cells. Data normalization, principal component analysis (PCA), and uniform manifold approximation and projection (UMAP) clustering delineated distinct cell populations. Differential gene expression analysis and module scoring revealed cell type-specific transcriptional changes between wild-type and ERα phosphorylation mutants (S171A and S216A).

first: Weiqiang

last: Lin

first: Muralidharan

last: Anbalagan

first: Brian

middle: G

last: Rowan


title: Single-cell multi-omic and multi-region atlas of the Aging Mouse Brain

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: Aging is the primary risk factor for many neurodegenerative diseases, primarily attributed to progressive changes in cellular epigenomes. Despite this known association, the complexity of the mammalian brain complicates the identification of brain regions and cell types that are most susceptible to aging effects. While alterations in gene expression have been observed in aging neural cells, comprehension of the epigenetic regulatory mechanisms underlying the transcriptomic changes remains largely elusive.  To unveil these complexities in a comprehensive manner, we utilized single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq) to produce extensive datasets comprising 132,551 methylomes and 72,666 chromatin conformation-methylome joint profiles from eight distinct brain regions of C57BL/6J mice aged 2, 9, and 18 months. These regions include the anterior hippocampus (AHC), posterior hippocampus (PHC), frontal cortex (FC), amygdala (AMY), nucleus accumbens (NACB), PAG/PCG, entorhinal cortex (ENT), and caudate putamen (CP). We utilized this dataset to investigate age-related changes in DNA methylomes and transcription factor motifs potentially affected by methylation alterations. We also examined methylation changes in transposable elements, observing locus-specific retrotransposon activation. At the chromatin conformation level, we analyzed aging-related changes in compartmentalization, topologically associating domains (TADs), and chromatin loops. Furthermore, we explored regional heterogeneity during aging within identical cell types and validated our findings using the MERFISH dataset.

overall_design: C57BL/6J (males and females) mice of different ages were purchased from Jackson Laboratories and maintained in the Salk animal barrier facility on 12-hour dark-light cycles with food ad-libitum for up to 4 weeks (Housing condition: Temperature: 21-23 °C, relative humidity: 61-63%). Brains were extracted, sliced, and dissected in an ice-cold dissection buffer as previously described (Liu et. al. 2021). Females were synchronized for their estrous cycle by exposing them to male bedding for three days, and collected during diestrus. For snmC-seq3 and snm3C-seq samples, brains were sliced coronally at 600 μm intervals from the frontal pole across the whole brain, yielding 18 slices, and dissected brain regions were obtained according to the Allen Brain Reference Atlas Common Coordinate Framework version 3 (CCFv3) as previously described (Liu et. al. 2023). For nuclei isolation, each region was dissected from 2-7 animals and pooled to make 2-3 biological replicates. No statistical methods were used to predetermine sample size. We empirically determined to use two to three biological experiments for all single-cell epigenomic experiments to achieve minimum reproducibility for this large-scale project. Blinding and randomization is not performed during handling the tissue samples. Additionally, all dissected regions were digitally registered into CCFv3 using ITK-SNAP60 (v4.0.0) at a 25 μm resolution.

first: Qiurui

first: Wei

last: Tian

first: Anna

last: Bartlett

last: Nery

first: Rosa

last: Gomez Castanon

first: Julia

last: Osteen

first: Jordan

last: Altshul

first: Nicholas

last: Johnson

first: Mia

last: Kenworthy

first: Cynthia

last: Valadon


title: Multi-omic analysis identifies erythroid cells as the major population in mouse placentas expressing genes for antigen presentation in MHC class II, chemokines, and antibacterial immune response

city: Novosibirsk

first: Roman

middle: Yurievich

last: Perik-Zavodskii

state: Novosibirsk region

address: Yadrincevskaya 14

country: Russia

institute: "Research Institute of Fundamental and Clinical Immunology" (RIFCI)

type:
Expression profiling by array
Other


title: Enhanced CLIP-Seq (eCLIP-Seq) of mouse Pxr in LS174T cells

status: Public on Apr 28 2025

city: Pittsburgh

first: Min

last: Zhang

state: PA

address: Salk Hall, 3501 Terrace St

institute: University of Pittsburgh

summary: eCLIP-seq identified mouse Pxr as an RNA-binding protein to interact with mature mRNAs

overall_design: Enhanced UV crosslinking and immunoprecipitation followed by sequencing (eCLIP-Seq) of mouse Pxr in LS174T cells

first: Xiaofei

last: Wang

first: Zehua

first: Sihan

last: Li

first: Yifei

first: Da

last: Yang


title: Mouse liver assembloids model periportal architecture and biliary fibrosis [bulk RNA-seq]

status: Public on Apr 29 2025

city: Dresden

first: Meritxell

last: Huch

address: Pfotenhauerstrasse 108

institute: Max Planck Institute of Molecular Cell Biology and Genetics

summary: Modelling liver disease requires in vitro systems that replicate disease progression1,2. Current tissue-derived organoids fail to reproduce the complex cellular composition and tissue architecture observed in vivo3. Here, we describe a multicellular organoid system composed of adult hepatocytes, cholangiocytes and mesenchymal cells that recapitulates the architecture of the liver periportal region and, when manipulated, models aspects of cholestatic injury and biliary fibrosis. We first generate reproducible hepatocyte organoids with functional bile canaliculi network that retain morphological features of in vivo tissue. By combining these with cholangiocytes and portal fibroblasts, we generate assembloids that mimic the cellular interactions of the periportal region. Assembloids are functional, consistently draining bile from bile canaliculi into the bile duct. Strikingly, manipulating the relative number of portal mesenchymal cells is sufficient to induce a fibrotic-like state, independently of an immune compartment. By generating chimeric assembloids of mutant and wild-type cells, or after gene knockdown, we show proof-of-concept that our system is amenable to investigating gene function and cell-autonomous mechanisms. Taken together, we demonstrate that liver assembloids represent a suitable in vitro system to study bile canaliculi formation, bile drainage, and how different cell types contribute to cholestatic disease and biliary fibrosis, in an all-in-one model.

overall_design: RNAseq of periportal assembloids cultured with homeostatic or fibrotic ratio of mesenchyme for 1 or 2.5 weeks and hepatocyte organoids grown in different media (MM or HM-Wnt media) cultured for 1 or 2 weeks

last: Dowbaj

first: Armin

last: Niksic

first: Cedric

last: Landerer

first: Fabian

last: Rost


title: DNA bendability regulates transcription factor binding to nucleosomes [MNase-seq]

first: Luca

last: Mariani

overall_design: MNase digestion followed by sequencing for the DNA libraries used for PIONEAR-seq. These libraries are based on 4 DNA sequences of 147 bases (ALBN1, CX3CR1, NRCAM, W601) known to position nucleosome either in vivo or in vitro. W601, also known as Widom 601, is a 147-bp DNA sequence of synthetic origin with the highest reported affinity to the histone octamer (PMID: 9514715 ). ALBN1 is a 147-bp DNA sequence based on the genomic sequence of the nucleosome N1 within the ALB enhancer (PMID: 31303471). CX3CR1 is a 147-bp sequence from the CX3 chemokine receptor 1 locus that have been proposed to be a candidate nucleosome target for pioneer factors in hematopoietic differentation (PMID: 31303471). NRCAM is a 147-bp genomic nucleosomal sequence from the Neuron-Glia-CAM- related cell adhesion molecule locus that gets digestable by MNase upon neuronal reprogramming (PMID: 31303471).  These sequences were tiled with random 20-mers at several position. The resulting DNA libraries were pooled together, assembled on nucleosome core particles (NCPs), digested with MNase and sequenced (Libraries: D1-WANC-1-Mnase, H1-WANC-2-Mnase). As controls, the libraries were sequenced also without the digestion of MNase(Libraries: C1-WANC-1-Input, G1-WANC-2-Input). In addition, MNase-seq was also perofmed on NCP samples based on single 147-bp sequences based on the NRCAM template (Libraries: F3-203-1-MNase, G3-203-2-MNase), eventually modified to include CEBPB specific binding sites (Libraries: E2-337-2-MNase, F2-338-1-MNase).

first: Xiao

last: Liu

first: Kwangwoon

last: Lee

first: Stephen

last: Gisselbrecht

first: Philip

last: Cole

first: Martha

last: Bulyk


title: Enhanced CLIP-Seq (eCLIP-Seq) of PXR in different cell types

summary: eCLIP-seq identified PXR as an RNA-binding protein to interact with mature mRNA while at its “inactivated” form in cell cytoplasm.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Enhanced UV crosslinking and immunoprecipitation followed by sequencing (eCLIP-Seq) of PXR in different cell types.


title: Effect of PXR on gene expression in LS174T cells

summary: To investigate the role of PXR in regulating RNA expression level and stability in LS174T cells

overall_design: Total RNA-seq of wildtype and PXR knockout LS174T cells treated by rifampicin and actinomycin D


title: Fluid shear stress activates a targetable mechano-metastatic cascade to promote medulloblastoma metastasis

status: Public on Apr 25 2025

city: Toronto

first: Qi

state: ON

address: 686 Bay St

department: Developmental & Stem Cell Biology

country: Canada

institute: The Hospital for Sick Children

summary: Biofluidflow generates fluid shear stress(FSS), a mechanical force widely present in tissue microenvironment. How brain tumor growth alters the conduit of biofluid, thereby impacting FSS-regulated cancer progression is unknown. Dissemination of medulloblastoma(MB) cells into the cerebrospinal fluid(CSF) initiates metastasis within the central nervous system. By simulating CSF dynamics based on magnetic resonance imaging of MB patients, we discovered that FSS is elevated at  the cervicomedullary junction. MB-relevant FSS promotes metastasis along mouse spinal cord. Mechanistically, FSS induces metastatic cell behaviours, including weakened cell-substrate adhesion, increased motility, cell clustering, and plasma membrane localization of glucose transporter 1 (GLUT1) to enhance glucose uptake. FSS is perceived by mechanosensitive ion channel PIEZO2, which drives actomyosin contractility-dependent GLUT1 recruitment at the plasma membrane. Genetic targeting  of PIEZO2 or pharmacologic inhibition of GLUT1 mitigates metastasis. Collectively, these findings define a targetable FSS-activated mechano-metastatic cascade for  the treatment of MB metastasis.

overall_design: The study included two cell lines, ONS76 and DAOY, each cultured under control conditions or exposed to artificial fluid shear stress (FSS). For every combination of cell line and experimental condition (control or FSS), three biological replicates were generated.

first: Hyun-Kee

last: Min

first: Hongyu

last: zhao

first: Xi

last: Huang


title: Human gastric multi-regional assembloids for functional parietal maturation

status: Public on Apr 23 2025

city: Bologna

first: Camilla

last: Luni

address: Via Terracini 28

department: DICAM

country: Italy

institute: University of Bologna

summary: Patient-derived human organoids have the remarkable capacity to self-organise into more complex structures. However, to what extent the gastric organoids can recapitulate the human stomach functions remains unexplored. Here, we report how gastric region-specific organoids can self-assemble into complex multi-regional assembloids showing levels of differentiation unattainable with simpler models. The assembloid shows preserved fundus, body and antrum regionality and gastric-specific crosstalk pathways arise. Remarkably, the increased complexity and cross communication between the different gastric regions, allows for the arise of the elusive parietal cell type, responsible for the production of gastric acid, the essential function for food digestion. We showed functional response to drugs targeting ATPase H+/K+ and further maturation of the assembloid when transplanted in vivo. This advanced in vitro model, using patient-derived tissue-specific progenitors, successfully recapitulates the structural and functional characteristics of the human stomach, offering a promising strategy for studying developmental processes, tissue interactions, and disease mechanisms that were previously challenging to attain.

overall_design:
To determine whether gastric stem cells have the ability to retain the identity of the region of origin, we isolated mucosal biopsies from the antrum, body and fundus regions of four paediatric patients.
The organoids were derived according to previously established protocol (Giobbe et al., Nat. Comm., 2021) and cultured in gastric-specific medium.
We designed a system to allow the self-aggregation of human gastric organoids in more complex multi-regional structures defined as assembloids. Briefly, full grown organoids were released from Matrigel, resuspended in collagen I gel, and cultured in floating conditions. We firstly defined the self-assembly conditions for each region (single-region assembloid, SRA) and then multi-regional, in the order fundus-body-antrum (multi-regional assembloid, MRA).
Last, we implanted subcutaneously in immunodeficient NSG mice the MRA (implanted MRA).
We also characterized native tissues (mucosal controls).

first: Brendan

middle: C

last: Jones

first: Giada

last: Benedetti

first: Giuseppe

last: Calà

first: Ramin

last: Amiri

first: Lucinda

last: Tullie

first: Roberto

last: Lutman

first: Jahangir

last: Sufi

first: Lucy

last: Holland

first: Daniyal

middle: J

last: Jafree

first: Monika

last: Balys

first: Glenn

last: Anderson

first: Ian

last: Simcock

first: Owen

last: Arthurs

first: Simon

last: Eaton

first: Nicola

last: Elvassore

first: Vivian

first: Christopher

last: Tape

first: Kelsey

first: Giovanni

last: Giobbe

first: Paolo

last: De Coppi


title: A strategy for challenging tumorous bone regeneration by utilizing borosilicate bioactive glass synchronously boosting osteosarcoma combating and osteogenesis promotion of moderate magnetic hyperthermia

status: Public on Apr 20 2025

city: Shenzhen

first: Mengke

last: Fan

address: 1068 Xueyuan Avenue, Xili Shenzhen University City,

institute: Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences

summary: To investigate the relevant biological mechanism involved in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when cultured with BSG extracts under moderate hyperthermia (42℃, 5%CO2）or regular  atmosphere (37℃, 5%CO2 ), the high-throughput RNA sequencing was conducted.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for hBMSCs when cultured with different groups (NC,MSC-HOT, MSC-3BSG, MSC-3BSGHOT). n = 3

first: Chunyu

last: Liu

first: Yueyao

last: Zhang

first: Jinlei

first: Wei

last: Chen

first: Xu

last: Cui

first: Haobo

last: Pan


summary: To investigate the relevant biological mechanism involved in the inhibition of 143B cells when cultured with injectable magnetothermal bioactive systems (IMBSs) under alternating magnetic field (AMF), the high-throughput RNA sequencing was conducted.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 143B cells  when cultured with different groups (NC, BSG. M-BSG, M-BSG-Hot). n = 3


title: Distinct Mammary Stem Cells Orchestrate the Long-term Homeostasis of Adult Mammary Gland

status: Public on Apr 18 2025

city: HangZhou

first: Xiaoqin

state: ZheJiang

address: Shilongshan Road No.18, Cloud Town, Xihu District

institute: Westlake University

summary: The murine mammary gland is sustained by distinct pools of stem cells that are limited in space and time, exhibiting diverse multipotency and activity. However, the specific identities of these stem cells and their contributions to mammary gland homeostasis are not well understood. In this study, we investigated the spatial heterogeneity of the mammary gland at the single-cell transcription level. Our findings reveal that mammary basal cells exhibit spatially distinct populations and characteristics, which can be further divided based on the expression of Cd34 and Cd200 markers. Notably, Cd34-Cd200+ basal cells demonstrate strong long-term self-renewal ability and possess the highest stem cell frequency, while Cd34+Cd200- basal cells show reduced stem cell potency. Through lineage tracing experiments using their signature genes, we discovered that Bcl11b+ cells were enriched in the Cd34-Cd200+ population and exhibited bipotency even in the postnatal mammary gland, with an increasing contribution to mammary epithelia observed during long-term tracing and after multiple rounds of pregnancies. Conversely, lineage tracing of Sema3a+ cells, enriched in the Cd34+Cd200- population, predominantly revealed their unipotent nature and significant contribution during alveologenesis. Notably, the Bcl11b+ cells displayed a slow response to pregnancy but contributed to long-term mammary homeostasis, in contrast to the rapid response observed in Sema3a+ cells. Importantly, depletion of Bcl11b in Krt14+ mammary basal cells resulted in reduced bipotency of mammary stem cells and impaired their long-term contribution to the mammary gland. Overall, our study identifies distinct bipotent and unipotent populations of mammary basal cells with different dynamic properties that play critical roles in maintaining postnatal mammary homeostasis. These findings are crucial for advancing our understanding of breast health and breast cancer research.

overall_design: Mammary tissues were selected from nipple, duct and TEB. Cells were sorted into 96-cell plate with Fluorescence-activated cell sorted(FACS). After sequencing, differences were generated among nipple, duct and TEB.

first: Chunhui

last: Wang

first: Huiru

last: Bai

first: Shang

last: Cai

first: Meizhen

last: Lin


title: Open-Field Blast Injury Disrupts Corneal Gene Expression Linked to Ion Transport, Sensory Perception, and Neural Signaling

status: Public on Apr 16 2025

city: Columbia

first: Rajiv

last: Mohan

state: Missouri

address: 1520 East Rollins University of Missouri

department: Department of Veterinary Medicine and Surgery

institute: University of Missouri

summary: This study aimed to investigate the transcriptomic alterations in the cornea following open-field blast injury (OFBI) for the first time via RNA sequencing (RNA-Seq) with the objective of identifying key molecular signatures and pathways underlying OFBI-induced corneal pathology.

overall_design: A total of 64 C57BL/6J mice (32 OFBI-exposed, 32 naïve controls) were used in the study. OFBI was induced using 350 g of Composition C-4 explosive at a standoff distance of 3 m. RNA was extracted from pooled corneas, and RNA-Seq libraries were prepared and sequenced on an Illumina NovaSeq 6000 platform.

first: Rajnish

last: Kumar

first: Nishant

last: Sinha

first: Suneel

last: Gupta

first: Ratnakar

last: Tripathi


title: Upstream open reading frame translation enhances immunogenic peptide presentation in mitotically arrested cancer cells

status: Public on Apr 24 2025

city: Heidelberg

first: Andres

last: Elia

state: Baden-Württemberg

address: Im Neuenheimer Feld 223

institute: DKFZ

summary: Mitosis is a critical phase of the cell cycle and a vulnerable point where cancer cells can be effectively disrupted, leading to cell death and inhibition of tumor growth. However, challenges such as drug resistance remain significant in clinical applications. During mitosis, mRNA translation is generally downregulated, while non-canonical translation of specific transcripts proceeds. Here, we demonstrate that mitotic cancer cells redistribute ribosomes toward the 5' untranslated region (5' UTR) and the start of the coding sequence (CDS), enhancing the translation of thousands of upstream open reading frames (uORFs) and upstream overlapping open reading frames (uoORFs). This mitotic induction of uORF/uoORF enriches the presentation of immunopeptides at the surface of cancer cells following treatment with mitotic inhibitors. Functional assays indicate the potential of such neoepitopes to provoke cancer-cell killing by T cells. Altogether, our findings highlight the therapeutic potential of targeting uORF/uoORF-derived neoepitopes in combination with mitotic inhibitors to enhance immune recognition and tumor cell elimination.

overall_design: Cancer cells were either treated with different mitosis-arresting drugs or DMSO. Ribosomes were extracted and subjected to RiboSeq library preparation.

first: Alexander

last: Kowar

first: Jonas P

last: Becker

first: Rossella

last: Del Pizzo

first: Zhiwei

last: Tang

first: Julien

last: Champagne

first: Pierre-Rene

last: Körner

first: Jasmine

last: Montengegro Navarro

first: Fiona Megan

last: Tilghman

first: Hanan

last: Sakeer

first: Angelika B

last: Riemer

first: Reuven

last: Agami

first: Fabricio

last: Loayza-Puch


title: IgM and IgG1 B cell receptors differentially impacts B cell fates and strongly dictates the pathogenesis of mature B cell lymphomas

city: Chicago

first: Vipul

last: Shukla

state: Illinois

address: 303 E. Superior Street

department: Department of Cell and Developmental Biology

institute: Northwestern University

summary: Majority of B cell malignancies originate in the germinal centers (GCs), where B cells undergo activation to mutationally diversify their antibody genes and switch from the default Immunoglobulin M (IgM) or IgD B cell receptor (BCR) to IgG, IgA or IgE BCR isotypes. Similar to normal B cells, malignant B cells rely on signals emanating from the BCR for their survival and growth. While studies have primarily focused on elucidating the functions of IgM-mediated signaling in lymphoma pathogenesis, GC-derived lymphomas also express IgG and IgA BCR isotypes that greatly differ from IgM in their signaling potential. How these distinct non-IgM BCRs contribute to the pathogenesis of B cell malignancies is not well understood. Interestingly, B cell lymphoma patients expressing IgG BCR isotypes show better prognosis and survival than those expressing IgM BCR isotype. Consistent with this, we show that Diffuse Large B Cell Lymphoma (DLBCL) patients with higher IgG1 expression are associated with lower stages of disease than those with higher IgM expression. To directly test the roles of distinct BCR isotypes, we developed isogenic lymphoma cells that either express IgM or IgG1 BCRs. Interestingly, the IgG1-switched lymphoma cells fail to form tumors in mice whereas their IgM counterparts establish aggressive tumors. Moreover, co-transplantation of IgG1 and IgM expressing human and mouse lymphoma cells leads to emergence of tumors expressing IgM (and not IgG1), demonstrating that IgM BCRs are strongly favored over IgG1. Mechanistically, IgG1 expressing lymphoma cells and non-malignant activated B cells have reduced survival which is associated with a dysfunctional mitochondrial state triggered by stronger calcium responses in IgG1 in comparison to IgM cells. Genetic reversal of IgG1 to IgM or pharmacological dampening of the calcium signaling is sufficient to correct the mitochondrial defects and rescue the survival of IgG1 B cells. Our findings demonstrate that distinct BCR isotypes are inherently unique in their activities and can differentially impact B cell lymphoma pathogenesis.

overall_design: To investigate the changes of gene expression in IgM and IgG1 cells treated with or without anti-kappa antibody

first: Rebecca

last: Southern

last: Schmitz

first: Daniela

last: Samaniego-Castruita

first: Isabella

last: Han

first: Prathyaya

last: Ramesh

first: Carlos

last: Montes

first: Taek-Chin

last: Cheong

first: Roberto

last: Chiarle

first: Daniel

last: Balsells

first: Ajay

last: Abraham

first: Robert

last: Rickert

first: Ashima


title: Bronchopulmonary Dysplasia with Pulmonary Hypertension Associates with Loss of Semaphorin Signaling and Functional Decrease in FOXF1 Expression

status: Public on Apr 14 2025

city: Nashville

first: Jennifer

last: Sucre

state: TN

address: 2215 B Garland Ave, 1125 Light Hall

department: Division of Neonatology, Department of Pediatrics

institute: Vanderbilt University Medical Center

summary: Lung injury in preterm infants leads to lifelong structural and functional respiratory deficits, with a risk for bronchopulmonary dysplasia (BPD). In its most severe form BPD is accompanied by pulmonary hypertension (PH).  While impaired alveologenesis and vasculogenesis in BPD are well-described, the molecular mechanisms driving these phenotypes and the cellular dynamics associated with evolving BPD and BPD+PH in humans are not well-described. We performed single-cell RNA sequencing on preterm infant lungs in early stages of BPD, BPD+PH and term infants. Analysis of the endothelium revealed an aberrant capillary cell-state primarily in BPD+PH marked by ANKRD1 expression and a transcriptomic signature not previously described in previously analyzed human lung disease, a finding validated in an expanded repository of human infant lung tissue. Predictive signaling analysis identified deficits in the semaphorin guidance-cue signaling pathway and decreased expression of pro-angiogenic transcription factor FOXF1 within the alveolar parenchyma of the human neonatal lung samples with BPD and BPD+PH. We observed similar trends in the in decreased semaphoring signaling in a murine BPD mouse model and in analysis of ACDMPV single-nuclear sequencing data. These transcriptional deficits in semaphorin signaling common to BPD+PH and ACDMPV suggest a mechanistic link between the developmental phenotype in BPD and ACDMPV, providing a foundation for further exploration of the role of semaphorins in normal alveolar development. Further, these data fill a critical gap in currently available transcriptomic analysis of the normal and diseased lung across the lifespan as searchable atlas of early human lung development and injury.

overall_design: To examine the cellular dynamics and mediators of neonatal lung injury, single-cell RNA sequencing was performed on human neonatal lungs of varying stages of lung injury and chronic lung disease: 1 acute preterm infant with lung injury, 2 BPD samples at term corrected gestational age (CGA), 2 BPD+PH at term CGA, and 2 term infants in the first 3 weeks of life.

first: Shawyon

middle: P

last: Shirazi

first: Nicholas

last: Negretti

first: Christopher

last: Jetter

first: Alexandria

last: Sharkey

first: Shriya

last: Garg

first: Meghan

last: Kapp

first: Devan

last: Wilkins

first: Gabrielle

last: Fortier

first: Saahithi

last: Mallapragada

middle: E

last: Banovich

middle: V

last: Wright

first: David

last: Frank

first: Jonathan

last: Kropski


title: RNA sequencing of B. subtilis overexpressing GFPnb, XynA and PrgA

status: Public on Apr 17 2025

city: Amsterdam

first: Mariah

last: Kes

address: De Boelelaan 1108

department: A-LIFE

country: Netherlands

institute: Vrije Universiteit Amsterdam

summary: Bacillus subtilis is widely used for industrial protein production due to its high capacity and GRAS status. Despite extensive efforts to optimize the host, bottlenecks still limit its full production potential, particularly for secreted heterologous proteins. In this study, we employ RNA sequencing as an unbiased approach to investigate B. subtilis responses to the overexpression of three distinct secreted proteins: the native xylanase XynA, the protein glutaminase PrgA from C. proteolyticum, and a model nanobody targeting GFP. Our analyses reveal that most transcriptome changes occur regardless of the overexpressed construct. However, when comparing different expression systems, constitutive versus inducible, we observe that many transcriptional regulations are reversed. This highlights the importance of performing transcriptome analyses under conditions that closely mimic the industrial fermentation setting to optimize protein production.

overall_design: RNA-seq profiling of Bacillus subtilis BWB143 cells harboring plasmids encoding GFPnb, XynA and PrgA under either PamyQ (constitutive) or Pxyl (xylose-inducible).

first: Biwen

last: Wang

first: Joen

last: Luirink

first: Leendert

last: Hamoen


title: Programmed cell death-1 receptor deficiency enhances CD30+ T regulatory cell function in Melanoma [Murine NanoString CosMx SMI]

status: Public on Apr 09 2025

city: Newcastle Upon Tyne

first: Jing Xuan

last: Lim

address: Framlington Place

department: Bioscience Institute

country: United Kingdom

institute: Newcastle University

summary: Regulatory T cells (Tregs) are vital for immune suppression rendering their function through multiple pathways including the expression of co-inhibitory receptors. The role of co-receptors namely PD1 in Treg function remains controversial. We demonstrate that PD1 is a master checkpoint in Tregs controlling several aspects of Treg biology in tumor immunity. Our data demonstrates that PD1 controls the expression and function of a unique co-inhibitory receptor network that dictates Treg function. We found that CD30 plays a central role within this network in driving Treg suppressive function within the tumor microenvironment (TME). Mechanistically, PD1 deficiency unleashed enhanced STAT5 signaling in Tregs, which drove CD30 expression. Our work highlights a new role for PD1 as a checkpoint that negatively controls CD30 expression in Tregs thereby dampening their function. Understanding the landscape changes that PD1 renders to Treg function can enable the use of combination therapies for better treatment outcomes in cancer.

overall_design: Tumors were harvested from B16 tumor from B6 Rag1-/- mice when tumor size reached 800mm3. Tumors were fixed with 10% neutral-buffered formalin, embedded in paraffin and mounted onto slides before being subjected to CoxMx SMI analysis. CosMxTM technology [CosMxTM Mouse Universal Cell Characterization RNA Panel (1000-plex); Nanostring, USA] was applied to 4 FFPE samples: 2 tumors with WT Teff and WT Tregs, 2 tumors with WT Teff and PD1KO Tregs with a mean of 29 fields of view (FOVs) per sample. The following cell surface markers were used for morphology visualization: PanCK, CD68, CD229/B2M, CD45 antibodies and DAPI.

first: Shoba

last: Amarnath

first: Tegan

last: McTaggart


title: RNASeq analysis of skin from mice with keratinocyte-specific loss of MCPIP3.

city: Krakow

first: Agata

last: Lichawska-Cieslar

address: Gronostajowa 7

department: Faculty of Biochemistry, Biophysics and Biotechnology

country: Poland

institute: Jagiellonian University

summary: Mice with keratinocyte-specific deletion of MCPIP3 (MCPIP3EKO) develop epidermal hyperplasia and abnormal differentiation. Loss of MCPIP3 function in murine keratinocytes leads to global changes of skin transcriptome. This study was funded by the National Science Centre grant number 2022/47/D/NZ3/01654 (to A. Lichawska-Cieslar).

overall_design: RNA-seq was performed to analyse transcriptome of mouse skin lysates from control and keratinocyte-specific MCPIP3 knockout mice.

first: Weronika

last: Szukala

first: Pawel

last: Pilat

first: Leopold

last: Eckhart

first: Jacek

last: Szepietowski

first: Jolanta

last: Jura


title: A Bacterial Artificial Chromosome system to uncover RNA Binding Protein functions in deep intronic regions [RNA-seq]

status: Public on Apr 03 2025

city: Frederick

first: Francesco

last: Tomassoni Ardori

state: MD

address: Building 560

institute: Mouse Cancer Genetics Program

summary: The processing of long introns in alternative splicing of pre-mRNAs is still not well understood. Using bacterial artificial chromosome (BAC) recombineering technology, we generated a Ntrk2-BAC minigene including endogenous 50 Kb introns flanking alternatively expressed exons generating two isoforms. We found that the RNA-binding protein RbFox1 regulates the expression of the minigene-encoded isoforms. Enhanced RNA Cross-Linking Immuno-Precipitation (eCLIP), revealed that RbFox1 binds clusters of (U)GCAUG motifs present in deep intronic regions. Deletion of these clusters completely abolished RbFox1-mediated Ntrk2-isoform expression regulation. Bioinformatic analysis showed (U)GCAUG-clusters are present in both mouse and human genes. Integrative data analysis from eCLIP and RNAseq experiments showed a global increase in RNA isoform modulation of genes with Rbfox1 eCLIP-peaks associated with these clusters. These data suggest that BACs are powerful tools to study RNA metabolism across large genomic fragments and clustered distal intronic Rbfox motifs are important determinants of RbFox1 function in the mammalian genome providing a target for identification of intronic pathogenic mutations.

overall_design: The mRNA-Seq was performed using the 77-5 clonal cell line stably expressing the Ntrk2-BAC minigene. In the RNA-Seq experiment, three samples (replicates) of 77-5 cells without any treatment were used as control while three samples (replicates) of 77-5 cells were treated with Doxycycline 0.5 mg/ml for 48 hours in order to induce Rbfox1 expression in the cells (tetracycline inducible system).

last: Tomassoni-Ardori

first: Mary

last: Palko

first: Lino

last: Tessarollo

first: Melissa

last: Galloux

first: Bao

last: Tran

first: Yongmei

last: Zhao

first: Jyoti

last: Shetty


title: Microenvironmental signals combine to induce non-additive molecular and phenotypic responses in mammary epithelial cells

status: Public on Apr 07 2025

city: Portland

first: Laura

last: Heiser

state: OR

address: 2730 S Moody Ave

department: Biomedical Engineering

institute: Oregon Health and Science University

summary: Cellular phenotypes are dictated not by single signals but by the integration of multiple extracellular cues, a process that remains poorly understood. Here, we systematically dissect how combinations of Oncostatin M, Transforming Growth Factor β1, and Epidermal Growth Factor shape the behavior of MCF10A mammary epithelial cells. Live-cell imaging revealed that ligand combinations drive emergent phenotypes absent in single-ligand contexts, pointing to the induction of novel molecular programs. Transcriptomic profiling uncovered synergistically regulated genes, while partial least-squares regression linked these transcriptional signatures to quantitative imaging-derived phenotypes and validated them across independent datasets. Functional analysis revealed CXCR2 signaling, upregulated through CREB activation, as a key driver of enhanced cell motility under combinatorial ligand treatment. Together, these findings establish a framework for uncovering how extracellular signals converge to modulate gene expression and phenotype, providing new insight into the principles governing complex epithelial cell behaviors.

overall_design: RNA-seq profiling of MCF10A cells treated with EGF, OSM, and TGFB individually and in combination, 24 hours after ligand treatment.

first: Ian

last: McLean

first: Sean

last: Gross

first: Jeremy

last: Copperman

first: Daniel

last: Derrick

first: Indranil

last: Paul

first: Andrew

last: Emili


title: A Bacterial Artificial Chromosome system to uncover RNA Binding Protein functions in deep intronic regions [eCLIP]

overall_design: eCLIP was performed using the 77-5 clonal cell line stably expressing the Ntrk2-BAC minigene and the 32-1 cell line stably expressing  the Ntrk2-BAC minigene with Cluster 1 and Cluster 2 deletion. In the eCLIP experiment, two samples (replicates) of 77-5 cells without any treatment were used as control while two samples of 77-5 cells and two samples of 32-1 cells (replicates) were treated with Doxycycline 0.5 mg/ml for 48 hours in order to induce Rbfox1 expression in the cells (tetracycline inducible system).

first: Kylie

last: Shen

first: Heather

last: Foster


title: Gut health in multiple joint osteoarthritis: a multimodal investigation in humans and pet dogs

status: Public on Apr 01 2025

city: Chapel Hill

first: Liubov

last: Arbeeva

state: North Carolina

address: 3300 Thurston Bldg., CB# 7280

institute: UNC

summary: The Gut health in multiple joint osteoarthritis (MJOA) study leverages data from parallel community-based cohorts in humans and in pet dogs to elucidate the role of altered microbiota in MJOA. One hundred Johnston County Health Study human participants were 35 to 70 years of age at enrollment (2022-2023), self-identified as Hispanic, White, or Black, and lived in Johnston County, North Carolina. Demographic, clinical information, multiple joint radiographs, and stool samples for microbiome profiling by 16S rRNA gene sequencing were obtained from all participants.  Similar data were collected from an independent group of pet dogs (N=115) from the local community, at the North Carolina State University (NCSU) College of Veterinary Medicine. The central hypothesis of the study is that intestinal permeability, with or without dysbiosis, is a major driver in the development and worsening of MJOA.

overall_design:
population based
Dog participants. Eligibility: at least 5 years old, weight >= 5kg. Exclusion criteria: any contraindication for undergoing sedation, neurological problems that significantly affect mobility, or any history of aggressive behavior.
Isolation of total DNA from stool samples and analysis by Illumina NextSeq 16S rRNA bacterial amplicon sequencing were performed in the UNC Microbiome Core Facility following standard Illumina sequencing protocols. Data sequencing output was converted to fastq format and demultiplexed using Illumina Bcl2Fastq 2.20.0 . The resulting paired-end reads were processed with the QIIME ver.2.2020.6  wrapper for DADA2 including merging paired ends, quality filtering, error correction, and chimera detection. Amplicon sequencing units from DADA2 were assigned taxonomic identifiers using the Silva database.

first: Amanda

last: Nelson

first: Duncan

last: Lascelles


title: Molecular characterization of endosomal self RNA Rmrp-engaged TLR3 dimerization to prime innate activation

city: 东城区

first: 张仕坤

last: Zhang

state: 北京市

address: 东单三条五号

institute: 协和医学院基础医学研究所

summary: In this study, we demonstrate that endosome-localized self RNA Rmrp directly binds to TLR3 and induces TLR3 dimerization in the early endosome but does not interact with endosome-localized TLR7, TLR8, TLR9 or cytoplasmic RNA sensor RIG-I under homeostatic conditions. Cryo-EM structure of Rmrp-TLR3 complex reveals a novel lapped conformation of TLR3 dimer engaged by Rmrp, which is distinct from the activation mechanism by dsRNA and the specific structural feature at the 3'-end of Rmrp is critical for its functional interaction with TLR3. Furthermore, K42 residue of TLR3 is essential for binding to Rmrp and subsequent dimerization. Rmrp dissociates from TLR3 following endosomal acidification, generating a matured TLR3 dimer which is primed for innate recognition and activation. Myeloid-cell deficiency of Rmrp reduces TLR3 dimerization and attenuates TLR3-mediated antiviral responses against influenza A both in vitro and in vivo.

overall_design: Two RNA-seq experiments were performed by extracting early endosome and lysosome RNAs from RAW264.7 cells, then analyzed using Illumina Xten. iCLIP experiment was performed using anti-HA antibody in RAW264.7 cells overexressing HA-TLR3, and then analyzed by Illumina HiSeq2500 SE50 sequencing

first: Shikun

first: Bo

last: Li

first: Lun

last: Liu

first: Dongsheng

last: Gong

first: Deyu

first: Fengjiang

first: Xiuna

last: Yang

first: Hua

last: Qin

first: Deling

last: Kong

first: Shuyang

first: Zihe

last: Rao

first: Xuetao

last: Cao


title: Cosmx Spatial Analysis of whole brain slices and ventral pallidum

status: Public on Mar 31 2025

city: Baltimore

first: Seth

last: Ament

address: 670 W Baltimore Street

department: Institute for Genome Sciences

institute: University of Maryland, Baltimore

summary: Pilot Cosmx experiment to explore and spatially atlas cell types present in the Striatum

overall_design: Mice: 1 Femal Whole Brain Section, 2 Male Whole Brain Sections, 1 female Ventral Pallidum section, 1 male Ventral Pallidum section. Standard Cosmx Mouse Brain Array

first: Rianne

last: Campbell

first: Mary Kay

last: Lobo

first: David

last: Dietz


title: Mechanistic Insights into Lead Acetate-Induced Neurotoxicity Using RNA-Seq Analysis

city: Kanagawa

first: Tsunehiko

last: Hongen

address: Matanocho

institute: university

summary: Lead acetate (Pb-acet) is a neurotoxicant that significantly impacts human brain development. This study used LUHMES cells, a human embryonic stem cell-derived neuronal model, to explore its molecular toxicity mechanisms. RNA sequencing (RNA-seq) showed distinct gene expression changes due to Pb-acet exposure. Principal Component Analysis (PCA) highlighted significant gene expression alterations at high toxicant concentrations (Ld10) compared to low concentrations (Ld1) and controls. Pb-acet disrupted pathways related to ribosome function and RNA splicing, and activated endoplasmic reticulum (ER) stress pathways, including PERK, ATF6, and IRE1. It also upregulated genes linked to ER-associated degradation (e.g., SVIP) and apoptosis (Bcl2 and CHOP), and disrupted the ubiquitin ligase complex, indicating impaired protein homeostasis. This study identifies ribosomal processes and ER stress pathways as potential biomarkers of Pb-acet exposure, demonstrating the utility of LUHMES cells in neurodevelopmental toxicity and environmental biomarker research.

overall_design: This investigation utilized RNA sequencing to elucidate neurotoxicity mechanisms in LUHMES cells . LUHMES cells were subjected to lead (II) acetate (Pb-acet) at low (Ld1) and high (Ld10) concentrations. Total RNA was extracted at multiple time points (3 and 24 hours) and analyzed using DESeq2 for differential expression. Pathway enrichment analysis (KEGG/GO) identified perturbations in oxidative phosphorylation, ribosome function, and endoplasmic reticulum stress, elucidating key biomarkers of neurotoxicity.

first: Tomohiro

last: Ito

first: XianYang

first: Hideko

last: Sone


title: Single-cell integration and multi-modal profiling reveals phenotypes and spatial organization of neutrophils in colorectal cancer

city: Zürich

first: Kristina

last: Handler

address: Winterthurerstrasse

department: Institute of Experimental Immunology

country: Switzerland

institute: University of Zürich

summary: The immune composition of the tumor microenvironment (TME) has a major impact on the therapeutic response and clinical outcome in patients with colorectal cancer (CRC). Here, we comprehensively characterize the TME at the single-cell level by first building a large-scale atlas that integrates 4.27 million single cells from 1,670 patient samples. We then complemented the atlas with single-cell profiles from four CRC cohorts with 266 patients, including cells with low mRNA content, spatial transcriptional profiles from 3.7 million cells, and protein profiles from 0.7 million cells. The analysis of the atlas allows refined tumor classification into four immune phenotypes: immune desert, B cell enriched, T cell enriched, and myeloid cell enriched subtypes. Within the myeloid compartment we uncover distinct subpopulations of neutrophils that acquire new functional properties in blood and in the TME, including anti-tumorigenic capabilities. Further, spatial multimodal single-cell profiling reveals that neutrophils are organized in clusters within distinct functional niches. Finally, using an orthotopic mouse model we show that cancer-derived systemic signals modify neutrophil production in the bone marrow, providing evidence for tumor-induced granulopoiesis. Our study provides a big data resource for the CRC and suggests novel therapeutic strategies targeting neutrophils.

overall_design:
Single cell RNA sequencing using the BD Rhapsody workflow of immune cells, with a special focus on neutrophils, from an orthotopic mouse colorectal cancer (CRC) model (AKPS) and non-tumor bearing controls.

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************

first: Valentin

last: Marteau

first: Niloofar

last: Nemati

first: Deeksha

last: Raju

first: Erika

last: Kvalem Soto

first: Georgios

last: Fotakis

first: Sandro

last: Carollo

first: Nina

last: Boeck

first: Alessia

last: Rossi

first: Alexander

last: Kirchmair

first: Alexandra

last: Scheiber

first: Arno

last: Amann

first: Andreas

last: Seeber

first: Elisabeth

last: Gasser

first: Steffen

last: Ormanns

first: Michael

last: Günther

first: Agnieszka

last: Martowicz

first: Zuzana

last: Loncova

first: Giorgia

last: Lamberti

first: Lena

last: Horvath

first: Hassan

last: Fazilaty

first: Tomas

last: Valenta

first: Gregor

last: Sturm

first: Dietmar

last: Rieder

first: Sieghart

last: Sopper

last: Pircher

first: Patrizia

last: Stoitzner

first: Paul

last: Ziegler

first: Markus

last: Tschurtschenthaler

first: Florian

last: Huemer

first: Daniel

last: Neureiter

first: Richard

last: Greil

first: Lukas

last: Weiss

first: Marieke

last: Ijsselsteijn

first: Noel

last: de Miranda

first: Dominik

last: Wolf

first: Isabelle

last: Arnold

first: Stefan

last: Salcher

first: Zlatko

last: Trajanoski


title: Inherent single-cell heterogeneity of the transcriptional response to hypoxia in cancer cells.

status: Public on Mar 29 2025

first: Małgorzata

last: Wilk

address: Winterthurerstrasse 190

summary: Hypoxia-inducible factor (HIF) is a master regulator of cancer cell adaptation to tumor hypoxia and is involved in cancer progression. Single-cell (sc) differences in the HIF response allow for tumor evolution and cause therapy resistance. These sc-differences are usually ascribed to tumor microenvironmental differences and/or clonal (epi)genetic variability. However, the sc-heterogeneity of the HIF response in otherwise identical clonal cells cultured under defined in vitro conditions has not yet been addressed. Therefore, we generated clonal HIF-1α- or HIF-2α-deficient cell lines and analyzed the sc-response to hypoxia. While HIF-1α and HIF-1 target mRNA sc-heterogeneity was slightly higher than global transcription or specific housekeeping mRNAs, HIF-2α and especially HIF-2 target mRNA sc-heterogeneity was extraordinary, and remained in independent subclones as well as following Isoform-specific HIFα ablation. Unexpectedly, neither HIF-2α mRNA nor nuclear protein levels correlated with target mRNA levels. Unsupervised but not supervised HIF target gene dimensionality reduction revealed the initial sample composition after scRNAseq, demonstrating that, owing to sc-heterogeneity, individual HIF target genes are not sufficient to unequivocally identify hypoxic cancer cells. In conclusion, the pronounced intrinsic sc-heterogeneity of the HIF response represents a hitherto unrecognized feature of cancer cells that impairs clinical HIF pathway-dependent cancer cell identification and targeting.

overall_design: There were 3 cell lines used in this experiment: MCF-7 HIF-1α knockout, MCF-7 HIF-2α knockout, and MCF-7 NTC (control cell line). MCF-7 cells were incubated under normoxic (control) or hypoxic conditions for 48 hours and hypoxically-induced gene expression on a single-cell level was investigated with the use of scRNA-sequencing.


title: Differentiation in the human urothelia is defined by distinct alternative transcript polyadenylation

city: Houston

first: Angela

middle: H

last: Ting

state: Tx

address: 1881 East Road

department: Epigenetics and Molecular Carcinogenesis

institute: The University of Texas MD Anderson Cancer Center

summary: Tissue epithelia comprise distinct cell states, yet the mechanisms that diversify their transcriptomes remain incompletely understood. The human ureter urothelium contains basal progenitors, intermediate cells, and terminally differentiated umbrella cells that maintain urinary tract barrier integrity. Although transcriptional differences are widely assumed to define these states, prior single-cell RNA sequencing (scRNA-seq) revealed highly similar global gene expression profiles. Here, we show that alternative cleavage and polyadenylation (APA) introduces a major layer of transcriptomic diversity during urothelial differentiation, largely independent of changes in total mRNA abundance. Analysis of 13,544 urothelial cells identified hundreds of differentiation-associated APA events. By single-cell imaging, we visualized APA events in the tissue, revealing their spatial specificity within the adult human ureter. Using a reporter assay, we demonstrated that the alternative 3′ UTRs in these differentiation-associated APA genes directly impacted protein expression levels. We discovered that key APA genes share conserved motifs in their 3′ UTRs, including transcription factor binding sites and Alu elements, suggesting a potential mechanism regulating poly(A) site selection. Our study establishes APA as a major source of urothelial transcriptome diversity.

overall_design: We reanalyzed previously generated 10x 3' scRNA-seq data on 10 normal human ureters (GSE184111) to identify and quantify polyadenylation site usage information in basal, intermediate, and umbrella cells of the urothelium.

first: Ninh

last: Le

first: Surbhi

last: Sona

first: Byron

last: Lee


title: Preventing CpG hypermethylation in oocytes safeguards mouse development [Cut&Run]

status: Public on Mar 27 2025

city: Basel

first: Antoine

last: Peters

address: Maulbeerstrasse 66

institute: Friedrich Miescher Institute

summary: DNA methylation (DNAme) controls gene expression, genome stability and cell identity in many mammalian organisms. Genomes of most somatic cells and spermatozoa are largely methylated, apart from CpG-dense sequences including gene promoters1. In mouse oocytes, DNAme is mostly limited to transcribed regions2,3. The mechanisms restricting global DNAme acquisition in developing oocytes and the relevance thereof for regulating gene expression and embryo development after fertilization are unknown. Here we show that the histone H3 lysine 36 dimethyl (H3K36me2) demethylases KDM2A and KDM2B redundantly execute multiple chromatin functions during oocyte development, which are vital to pre- and post-implantation development. Firstly, by serving as recruitment factors of variant Polycomb Repressive Complex 1 (vPRC1), they control genome-wide H2A mono-ubiquitination deposition (H2AK119u1) and PRC1-dependent gene repression4. Secondly, as demethylases, KDM2A/KDM2B prevent global H3K36me2 accumulation, thereby impeding DNMT3A-catalyzed de novo DNAme within PRC1-controlled promoter, genic, and intergenic regions, and even at non-PRC1-controlled CpG-dense gene promoters5. Decisively, we demonstrate that aberrant Dnmt3a-dependent DNAme established in Kdm2a/Kdm2b double mutant oocytes represses transcription from maternal loci in two-cell embryos and impairs pre-implantation development. Hence, KDM2A/KDM2B are essential for defining the appropriate oocyte DNA methylome which in turn conveys competence for early embryonic development. Our research implies that the reprogramming capacity eminent to early embryos is insufficient to erase aberrant DNAme from maternal chromatin. Lastly, our work shows that early development is vulnerable to gene dosage haplo-insufficiency effects, possibly in a parental-specific manner.

overall_design:
To study the function of KDM2A and KDM2B in regulating genome wide H2AK119u1, H3K36me2 and H3K36me3 in oocytes, we conditionally altered their expression in growing oocytes in several ways.
First, by using Zp3-driven CRE recombinase-mediated excision, we removed exons encoding the histone demethylase JmjC domains of Kdm2a or Kdm2b, which fully abrogated expression of either protein.
We also generated mice expressing a KDM2B protein lacking its CxxC domain, unable to be recruited to CpG island regions of the mouse genome.
To address possible combinatorial roles of both proteins, we generated oocytes conditionally deficient for Kdm2a in combination with either Kdm2b mutation.
We profiled genome-wide histone modification marks (H2AK119u1, H3K36me2 and H3K36me3) by Cut &Run method in fully grown germinal vesicle oocytes of following genotypes: (1) Kdm2a-KO; Kdm2b-deltaCxxC, (2) Kdm2a-KO; Kdm2b-KO and (3) controls.

first: Yumiko

middle: K

last: Kawamura

first: Panagiotis

last: Papasaikas

first: Evgeniy

last: Ozonov


title: Interactions between the genome and the nuclear lamina are multivalent and cooperative (Hi-C)

first: Bas

last: van Steensel

address: Plesmanlaan 121

institute: Netherlands Cancer Institute

summary: Mammalian genomes contain hundreds of lamina-associated domains (LADs), which are large, often megabase-sized heterochromatin domains that are anchored to the nuclear lamina (NL). Even though LADs play a key role in the spatial organization of the genome and potently repress gene activity, it is not yet understood how their interactions with the NL are encoded in their DNA. Here, we investigated the principles that govern LAD-NL interactions by taking a "LAD scrambling" approach. This approach relies on 1) local "hopping" of a Sleeping Beauty transposon to randomly insert loxP sites within and around a LAD of choice; and 2) Cre-mediated recombination between the loxP sites. This approach is highly efficient, enabling us to establish a large collection of clonal cell lines with deletions and inversions up to ~2Mb in size, spanning LAD and their flanking inter-LAD sequences. Mapping of NL interactions in these clones revealed that a single LAD contacts the NL through multiple regions. These regions act cooperatively and redundantly; however, some have more affinity for the NL and can boost NL contacts of neighbouring sequences. We also observed a crosstalk between two neighbouring LADs, when close enough to each other. Finally, changes in the heterochromatin mark H3K9me3 only partially mirrored changes in NL contacts. They also correlated more with gene expression changes than NL contacts did. These principles of LAD - NL interactions provide insight into the overall architecture of the genome and the impact on gene regulation.

overall_design: Hi-C experiment were performed on parental (non-edited) and Cre-recombined clones.

first: Lise

last: Dauban

first: Marcel

last: de Haas

first: Mathias

last: Eder

first: Vinícius

last: Franceschini-Santos

first: Tom

last: van Schaik

first: Christ

last: Leemans

first: Koen

last: Rademaker

first: Moreno

last: Martinovic

first: J. Omar

last: Yañez-Cuna


title: Preventing CpG hypermethylation in oocytes safeguards mouse development [WGBS GVO]

overall_design:
To study the function of KDM2A and KDM2B in regulating genome wide DNA methylation in oocytes, we conditionally altered their expression in growing oocytes in several ways.
First, by using Zp3-driven CRE recombinase-mediated excision, we removed exons encoding the histone demethylase JmjC domains of Kdm2a or Kdm2b, which fully abrogated expression of either protein.
We also generated mice expressing a KDM2B protein lacking its CxxC domain, unable to be recruited to CpG island regions of the mouse genome.
To address possible combinatorial roles of both proteins, we generated oocytes conditionally deficient for Kdm2a in combination with either Kdm2b mutation.
We profiled genome-wide mCpG methylation in fully grown germinal vesicle oocytes of following genotypes: (1) Kdm2b-deltaCxxC, (2) Kdm2a-KO; Kdm2b-deltaCxxC, (3) Kdm2a-KO; Kdm2b-KO  and (4) controls.


title: TRIBAL/TRIBA1AL and TRIB1 suppression in differentiated HepaRG cells

status: Public on Mar 20 2025

city: Ottawa

first: Ruth

last: McPherson

state: Ontario

address: 40 Ruskin

institute: UOHI

summary: HepaRGs treated with antisense oligonucleotides targeting TRIBAL or TRIB1 (or Non-Target control) for 72 h

overall_design: HepaRGs were concurrently treated with ASOs for 72 h.  RNA was then isolated and reduction of the primary target (TRIBAL or TRIB1) was first confirmed by qRT-PCR.  RNA was then subjected to Clariom_D assays.

first: S

last: Soubeyrand

first: R


title: Effects of treatment with cyanidin-3-glucoside on mammary tumors arising from MMTV-PyVT mice

status: Public on Mar 24 2025

city: Taipei

first: Shih-Ping

last: Cheng

address: 92, Chung-Shan North Road, Section 2

department: Department of Surgery

country: Taiwan

institute: MacKay Memorial Hospital

summary: Cyanidin-3-glucoside (C3G) is one of the most prevalent anthocyanins among dietary polyphenols. Experimental studies suggest that C3G exhibits antioxidant, anti-inflammatory, and cytoprotective effects against various disorders induced by oxidative stress. We examined its in vivo anti-neoplastic properties using MMTV-PyVT transgenic mice, a well-validated model for the development and progression of multistage mammary carcinoma.

overall_design: MMTV-PyVT mice were treated with cyanidin-3-glucoside (C3G) or DMSO control once ductal cells began to exhibit hyperplasia. Mice were sacrificed when the tumor burden reached the predefined criteria, and mammary tumors were harvested for analysis.

first: Yuan-Ching

last: Chang


title: Bulk RNA sequencing for gene expression profileing of primary liver sinusoidal endothelial cells and iPSC-derived hepatic endoderm, septum mesenchymal cells, endothelial cells, and liver bud organoids

city: Tokyo

first: Norikazu

last: Saiki

address: 1-5-45 Yushima, Bunkyo-ku

department: Institute of Research

institute: Tokyo Medical and Dental University

summary: We performed bulk RNA-seq analysis to compare the expression of differentiation markers, angiocrine factor, and coagulation-associated genes and to analyze intercellular communication.

overall_design: Total RNAs for sequence library preparation were obtained from differentiated cells in iPSC-derived stepwise 2D differentiation, primary LSECs, and liver bud organoids dissociated into single cells.

first: Tamaki

last: Kono

first: Masaru

last: Koido

first: Takanori

last: Takebe


title: Hominoid-specific transposable elements reshaped neural crest migration in craniofacial development

status: Public on Mar 23 2025

city: London

first: Marco

last: Trizzino

state: UK

address: Imperial College Road

department: Life Sciences

institute: Imperial College London

summary: Craniofacial development is evolutionarily conserved, yet subtle changes in its regulatory network drive species-specific traits. Transposable elements (TEs) contribute to genome evolution, but their role in cranial neural crest cells (CNCCs) remains unclear. Here, we investigate the domestication of hominoid-specific TEs (LTR5Hs and SVAs) as enhancers during human CNCC specification, a process critical for vertebrate craniofacial development. Using human iPSC-derived CNCCs, we identified ~515 hominoid-specific TEs functioning as enhancers, including ~250 human-specific, predominantly LTR5Hs. These elements are enriched for CNCC coordinator motifs, are bound by the CNCC signature factor TWIST1, and their enhancer activity appears largely CNCC-specific. CRISPR-interference targeting ~75% of these active TEs led to widespread transcriptional dysregulation of genes involved in neural crest migration, and two orthogonal functional assays confirmed that CNCC migration is impaired upon TE repression. Finally, genes near human-specific TEs showed higher expression in human CNCCs compared to chimpanzee CNCCs, but TE repression restored gene expression to chimpanzee levels. These findings highlight how young TEs were domesticated to fine-tune CNCC regulatory networks, potentially contributing to lineage-specific craniofacial evolution.

overall_design:
RNA-seq of day 5 NCCs (5-day protocol) with and without gRNAs (2 replicates and 3 replicates respectively)
ChIP-seq of day 5 NCCs (5-day protocol) with and without gRNAs (1 replicate each)
ATAC-seq of day 5 NCCs (5-day protocol) with and without gRNAs (2 replicates each)
ATAC-seq of day 18 NCCs (18-day protocol) with and without gRNAs (2 replicates each)
ChIP-seq (H3k4me3) of day 5 NCCs (5-day protocol) with and without gRNAs (1 replicate each)

first: Laura

last: Deelen

first: Zoe

last: Mitchell

first: Martina

last: Demurtas

first: Beatriz

last: Garcia Del Valle

first: Andria

last: Koulle


title: Single-cell RNA sequencing data of human craniopharyngioma tumor tissue

city: minatoku toranomon

first: Tatsuma

last: Matsuda

address: tokyo

institute: toranomon hospital

summary: The craniopharyngioma tumor specimen was digested immediately after surgical resection, and a single-cell library was prepared using 10x Genomics. The obtained sequencing data were processed and analyzed using Cell Ranger.

overall_design: The cell suspension obtained from the resected tumor specimens was processed according to 10×Genomics specifications, using the 10×Chromium Next GEM single-cell 3ʹ reagent kit and library construction kit. The cell suspension was processed in the Chromium Controller to create gel beads in emulsion (GEMs), incubated at a specific temperature to generate cDNA containing barcodes and Unique Molecular Identifiers (UMIs). Subsequently, the cDNA was washed, amplified, and its quality assessed, then enzymatically fragmented and size-selected, followed by adapter ligation and indexed PCR amplification. Finally, after quantification and quality assessment of the library, sequencing was performed on the NovaSeq 6000.

first: T

last: Tanaka


title: Expression data from adipose-derived stem cells differentiated into endothelial cells

status: Public on Mar 06 2025

city: Barcelona

first: Gemma

last: Arderiu

address: Avda. S. Antoni M. Claret 167

department: Cardiovascular area

country: Spain

institute: Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau IIB-Sant Pau

summary:
The development of obesity is associated with substantial modulation of adipose tissue (AT) structure. The plasticity of the AT is reflected by its remarkable ability to expand or to reduce in size throughout the adult lifespan. The expansion of AT is linked to the development of its vasculature. Adipose tissue-derived stem cells (ASCs) have the capacity to promote angiogenesis by their differentiation into endothelial cells (ECs).
We used microarrays to detail the global miRNA expression profile underlying cell differentiation and identified distinct classes of up-regulated and down-regulated genes during this process.

overall_design: Human ASCs from white AT were obtained by surgical procedures from lean and obese patients. ASCs from fat depots of lean and obese subjects were induced to differentiate into ECs. The expression of 2025 pre-miRNAs and 2578 mature miRNAs was assessed using microarrays technology (miRNA 4.0 GeneChips, Thermo Fisher Scientificf).

first: Lina

last: Badimon

first: Anna

last: Civit

first: Sandra

last: Camino

first: Olaya

last: García


title: Dynamic shift in trophoblast nucleos(t)ide metabolism, transport, and adenosine signaling during gestion and preterm birth

status: Public on Mar 08 2025

city: Hradec Kralove

first: Mohammed

last: Ali

address: Čechova 722/2, 50002 Hradec Králové 2

department: pharmacology and Toxicology

country: Czech Republic

institute: Charles university

summary:
This study investigates the role of nucleos(t)ide metabolism, transport, and adenosine signaling in placental development and spontaneous preterm birth (PTB). We analyzed gene expression in human placentas, primary trophoblasts, BeWo cells and rat placentas across different gestational stages to understand metabolic adaptations and stress responses
To understand the role of nucleos(t)ide metabolism (de novo synthesis and salvage patways), transport, and adenosine signaling in placental development and the adaptive response during spontaneous preterm birth (PTB), we analyzed gene expression in first-trimester and term human placentas, PTB placentas, primary human trophoblasts, and BeWo cells. Additionally, we examined rat placentas at different gestation days (GD12, GD15, and GD20) to provide developmental context and validate findings from human samples. Our results highlight the upregulation of nucleos(t)ide metabolism and adenosine signaling during placental growth and trophoblast differentiation (cytotrophoblast to syncytiotrophoblast), with further metabolic shifts in PTB placentas, particularly in pyrimidine de novo synthesis, purine salvage, and adenosine metabolism, suggesting an adaptive stress response. Additionally enhanced and also adenosine signaling and nucleoside transport was observed. This integrated approach provides novel insights into the metabolic regulation of placental function under both normal and pathological conditions

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: This study includes n = 10 for each human pregnancy stage first-trimester, term, and preterm birth (PBT) placentas. In the animal model, rat placentas (n = 5) were analyzed at gestation days (GD) 12, 15, and 20. For in vitro models, BeWo cells (n = 5) and primary trophoblast cells (n = 3) were examined. Each sample was run in technical triplicate to ensure reproducibility.  Gene expression data were processed using the ΔΔCt (delta-delta Ct) method. An interplate calibrator reaction was included in each run, analyzed in triplicates, and its mean value was subtracted from the Ct values in all analyses to generate ΔCt values. For normalization, the mean of the ΔCt values of the reference genes B2M, GAPDH, and YWHAZ was used. The normalized ΔCtNORM values were obtained by subtracting the ΔCt value of the target gene from the ΔCt value of the reference gene. These values were then re-scaled to the median of the reference group and presented as log₂ expression, corresponding to the difference between the ΔCtNORM of the sample and the median ΔCtNORM of the reference group.

first: Moahmmed

first: Mariia

last: Adler

first: Antonin

last: Libra

first: Ivan

last: Vokral

first: Rona

last: Karahoda

first: Eva

last: Cifkova

first: Miroslav

last: Lisa

first: Jakub

last: Tomek

first: Magdalena

last: Novotna

first: Frantisek

last: Staud

first: Lukas

last: Cerveny


title: Melanoma plasticity is regulated by mTOR-mediated crosstalk between MITF and TFE3

city: Iowa City

first: Jeremy

last: Chang

state: IA

address: 285 Newton Rd

department: Surgery

institute: University of Iowa

summary: Bulk RNAseq was performed to identify differences in gene expression in SKMEL28 cells as related to MITF and TFE3 expression.

overall_design:
TFE3KO cell lines were created in the D2-MITFKO SKMEL28 and A375 background via CRISPR-cas9 strategies
500k SKMEL28: WT, D2 MITFKO, D2 MITFKO/TFE3KO (clone 3 and clone 13) and A375: WT and TFE3KO cells were plated in triplicate for biologic replicates. RNA was extracted utilizing RNeasy Plus Kit (Qiagen).

first: Colin

last: Kenny


title: Using an RNA-binding antibody for tumor-directed immunogenic RNA delivery

status: Public on Mar 14 2025

city: New Haven

first: Haoting

last: Chen

state: CT

address: 333 Cedar St

institute: Yale University

summary: The inability to selectively deliver therapeutic RNAs within target cells currently hinders the development of novel treatments for cancer and other disorders. Here, we report that a tumor-targeting, cell-penetrating, and RNA-binding antibody, TMAB3, can form non-covalent antibody/RNA complexes that mediate highly specific and functional delivery of RNAs into tumors. We observed a robust anti-tumor efficacy of systemically administered 3p-hpRNA, an agonist of viral RNA sensor RIG-I, in complex with TMAB3 in animal models of pancreatic cancer, medulloblastoma, and melanoma. In pancreatic cancer models, treatment with TMAB3/3p-hpRNA tripled animal survival, decreased tumor growth, and specifically targeted malignant cells, with a 1500-fold difference in RNA delivery into tumor cells versus non-malignant cells within the tumor mass. Single-cell RNA-sequencing (scRNA-seq) and flow cytometry demonstrated that TMAB3/3p-hpRNA treatment elicited anti-tumoral immune responses against tumor cells. These studies establish that TMAB3/3p-hpRNA complexes can effectively deliver RNA payloads to malignant cells in hard-to-treat tumors to achieve anti-tumor efficacy, providing an antibody-based platform to advance RNA therapies for cancer.

overall_design: Mice with the KPC orthotopic model were treated with an RNA-binding antibody, TMAB3, to deliver an immunogenic RNA, 3p-hpRNA, into the tumor. The mice received three doses on days 10, 13, and 16 post-tumor implantation. Mice were sacrificed at 1 day (D+1) and 7 days (D+7) post-final treatment. Tumors were harvested and dissociated, and dead cells were discarded before scRNA-seq analysis

first: Elias

last: Quijano

first: Diana

last: Martinez-Saucedo

first: Robert

last: Tseng

first: Xinning

last: Shan

first: Luisa

middle: F

last: Escobar-Hoyos

first: Peter

last: Glazer


title: Preventing CpG hypermethylation in oocytes safeguards mouse development

first: Antoine

last: Peters

summary: Except for regulatory CpG-island sequences, genomes of most mammalian cells are widely DNA-methylated. In oocytes though, DNAme is largely confined to transcribed regions. The mechanisms restricting de novo DNAme in oocytes and the relevance thereof for zygotic genome activation and embryonic development are largely unknown. Here we show that KDM2A and KDM2B, two histone demethylases, prevent genome-wide accumulation of histone H3 lysine 36 di-methylation, thereby impeding DNMT3A-catalyzed DNAme. We demonstrate that aberrant DNAme at CpG-islands inherited from Kdm2a/Kdm2b double mutant oocytes represses gene transcription in two-cell embryos. Aberrant maternal DNAme impairs pre-implantation embryonic development which is suppressed by Dnmt3a deficiency during oogenesis. Hence, KDM2A/KDM2B are essential for confining the oocyte methylome, thereby conferring competence for early embryonic development. Our research implies that the reprogramming capacity eminent to early embryos is insufficient for erasing aberrant DNAme from maternal chromatin, and that early development is vulnerable to gene dosage haplo-insufficiency effects.

overall_design: Refer to individual Series


title: Description of Bacterial RNA Transcripts Detected in Mycobacterium tuberculosis – Infected Cells from Peripheral Human Granulomas using Single Cell RNA Sequencing

status: Public on Mar 04 2025

city: Salt Lake City

first: Philip

last: Moos

state: UT

department: Pharmacology & Toxicology

institute: University of Utah

summary: Next-gen sequencing has facilitated studies of bacterial genomes and uncovered pathogen variants associated with clinically relevant phenotypes such as antibiotic resistance. However, these studies are primarily focused on bacteria cultured from patient tissues, and thus, viable but non-cultivable bacteria are not assessed. This yields an incomplete genetic state of bacteria during infection. Nevertheless, it is possible to capture bacterial transcripts in libraries designed to amplify eukaryotic mRNA. These reads are often considered spurious or nuisance and are rarely investigated. Here we demonstrate that Mycobacterium tuberculosis (Mtb) ribosomal RNA sequences are detected in host human cells obtained from peripheral lymph node aspirates from patients infected with Mtb.

overall_design: Fine needle aspirates from periferal lymphadenopathies were collected from concenting patients arriving at the Port Moresby General Hospital (POMGH) Tuberculosis Clinic, Papua New Guinea, processed to collect the single cells, and fixed and sterilized with MeOH before returning to the University of Utah for processing for single-cell RNA sequencing using the 10X Genomics system.

first: Louis

last: Barrows


summary:
ATAC-seq performed to identify chromatin accessibility across the genome.
CUT&RUN-seq was performed to identify and compare transcription factor and histone mark occupancy across genome of WT and MITF and TFE3 KO cells.

overall_design:
SKMEL28 MITF-WT and D6 MITF-KO cells were grown in appropriate condiitons. Cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40: Sigma). Transposition was performed directly on nuclei using 25 μl tagmentation reaction mix (Tagment DNA Buffer #15027866, Tagment DNA Enzyme #15027865 from Illumina Tagment DNA kit #20034210). Tagged DNA was subjected to PCR amplification and library indexing, using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs #M0451S) with Nextera DNA CD Indexes (Illumina #20015882), according to the following program: 72°C for 5 minutes; 98°C for 30 seconds, 12 cycles of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. The PCR product was purified with 1.8 times the volume of Ampure XP beads (Beckman Coulter #A63881). Library quality was assessed using a BioAnalyzer 2100 High Sensitivity DNA Chip (Agilent Technologies). All DNA libraries that exhibited a nucleosome pattern were pooled and processed for 150 bp paired-end sequencing.
SK-MEL-28: WT, MITF-KO, MITF/TFE3-KO; A375: WT, TFE3-KO cells were seeded and grown in appropriate conditions per cell line. 200k cells were harvested by cell scraping and Cleavage Under Targets and Release Using Nuclease (CUT and RUN) was performed for analysis utilizing Anti-MITF, Anti-TFE3, Anti-H3K27me3, Anti-H3K27ac, Anti-H3K9ac, Anti-H3K4me.