title: Osteopontin links acquired resistance to hypoxia-inducing antiangiogenics with refractoriness to anti-PD-L1 agents in breast cancer [III]

city: Madrid

first: Jose Luis

last: Ruiz-Sepulveda

state: Madrid

address: C/ Melchor Fernández Almagro, 3

department: Translational Research

institute: CNIO

summary: Resistance to antiangiogenics is a major challenge in cancer therapy. These agents can either normalize or exacerbate tumor vascular abnormality and hypoxia. The mechanisms of resistance remain unclear in the latter setting. By integrating data from mouse models and clinical trials, we show that hypoxia-inducing anti-VEGF therapy upregulates programmed cell death ligand 1 (PD-L1), yet fails to sensitize tumors to PD-L1 blockade. Mechanistically, early hypoxic stress triggers epithelial osteopontin (Spp1) production, which recruits monocytes and skews macrophages toward M2 states, suppressing T-cell cytotoxicity. Pharmacologic Spp1 depletion impedes the development of hypoxia, M2 infiltration, restores T-cell activity and enables synergy between antiangiogenics and anti-PD-L1. Genetic dissection – tumor-epithelial Spp1 knockout grafts and bone-marrow chimeras generated by lethal irradiation and reconstitution with Spp1-/- or wild-type hematopoietic donors – show that myeloid Spp1 contributes only marginally compared with epithelial Spp1. These findings identify Spp1 as a central mediator of resistance to hypoxia-inducing antiangiogenics, contribute to a comprehensive model of antiangiogenic resistance, and support Spp1-targeted strategies to personalize immuno/antiangiogenic therapy according to tumor hypoxia.

overall_design: To investigate the effects of antiangiogenic mediated hypoxia on breast tumors, we trated 7-weeks old MMTV-PyMT spontaneous breast tumor mouse models with 5mg/kg anti-VEGF-A antibody clone B20-4.1.1 twice per week or isotype IgG2a. After 6 weeks of treatment mice where sacrified and tumor samples dissociated into single cell suspension. Debris and dead cells were excluded by sorting DAPI- cells in BD FACS ARIA. Subsequently, cell multiplexing oligo labeling was conducted using the 3’CellPlex Kit Set A (10xGenomics, cat no. 1000261), following manufacturer’s instructions. Next, a secondary sort of DAPI- cells was executed with the BD FACS ARIA to further enrich for viable cells. Multiplexed-single-cell suspensions sourced from three distinct tumors were processed together into one sample, which was drop encapsulated with Chromium device (10xGenomics). The generated libraries were later sequenced using Illumina Nextseq 550 (with v2.5 reagent kits) and post-processed by the Cell Ranger Software (10xGenomics) to obtain a single-cell RNA-seq expression matrix for each sample.

first: José

last: Ruiz-Sepúlveda

first: María

last: Bueno

first: Silvana

last: Mouron

first: Verónica

last: Jiménez-Renard

first: Manuel

last: Muñoz

last: Moradiellos

first: Leonardo

last: Garma

first: Luis

last: García-Jimeno

first: Adam

last: Watson

first: Ghassan

last: Mouneimne

first: Solip

last: Park

first: Rebeca

last: Jimeno

first: Miguel

last: Quintela-Fandino


title: Osteopontin links acquired resistance to hypoxia-inducing antiangiogenics with refractoriness to anti-PD-L1 agents in breast cancer

status: Public on May 05 2026

country: Spain

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series

middle: L

middle: J


title: Osteopontin links acquired resistance to hypoxia-inducing antiangiogenics with refractoriness to anti-PD-L1 agents in breast cancer [I]

overall_design: To investigate the effects oh antiangiogenic mediated hypoxia on tumor immune compartment we trated 7-weeks old MMTV-PyMT spontaneous breast tumor mouse models  with 5mg/kg anti-VEGF-A antibody clone B20-4.1.1 twice per week. After 6 weeks of treatment mice where sacrfied and breast tumors extracted. Intratumoral CD45+ immune cells were FACS-Sorted and RNA extracted to perform 3'RNA-seq experiment.


title: Stage-Specific RNA Turnover Drives Small RNA Dynamics in Arabidopsis – Colletotrichum Interactions

city: Davis

first: Patricia

last: Baldrich Gonzalez

state: CA

address: 451 Health Sciences Dr., Davis, CA 95616

department: Genome Center

country: USA

institute: University of California - Davis

type: Non-coding RNA profiling by high throughput sequencing

summary: Small RNAs (sRNAs) are key regulators of plant defense and have been implicated in cross-kingdom interactions with pathogens. The hemibiotrophic fungus Colletotrichum higginsianum infects Arabidopsis thaliana through three stages: appressorial penetration, biotrophy, and necrotrophy. However, the dynamics of fungal and plant sRNA populations across these three stages has not been elucidated. Using high-throughput sequencing, we profiled sRNAs from A. thaliana and C. higginsianum during in planta appressorium (PA), biotrophic (BP), and necrotrophic (NP) phases, and compared them to fungal mycelia (MY) and in vitro appressoria (VA). Our analyses revealed stage-specific patterns in sRNA accumulation in both the plant and the pathogen. In C. higginsianum, sRNAs were dominated by 29 nt species in PA, BP, MY, and VA, but shifted to 18 nt in NP, consistent with RNA degradation during host cell death. In A. thaliana, sRNAs transitioned from 30-33-nt in PA/BP to a 21 nt dominant peak in NP. Also, TE-derived siRNAs and other regulatory sRNAs (miRNAs, ncRNA, snoRNAs and snRNAs) declined during NP. A total of 62 host miRNAs showed differential accumulation, including core plant developmental regulators active across infection stages, and stage-specific miRNAs such as miR396, miR170/171, miR472, and miR858b. tRFs displayed opposite trends in host and pathogen: fungal tRFs declined in NP, while host tRFs increased. These tRFs contrasting trends may reflect differences in RNA processing or degradation between host and pathogen. Our results provide new insights into RNA-mediated plant-fungal interactions.

sample_organism:
Arabidopsis thaliana
Colletotrichum higginsianum IMI 349063

overall_design: 3 replicates of small RNA seq from total RNA are included in this study.

last: Baldrich

first: Richard

last: O'Connell

first: Blake

middle: C

last: Meyers


title: Differential Expression of microRNAs after Induction of Endometriosis in Baboons

status: Public on May 04 2026

city: New Haven

first: Ramanaiah

last: Mamillapalli

state: CT

address: 310 Cedar Street

department: OBGYN

institute: Yale University

summary: Endometriosis is an inflammatory chronic gynecological condition that affects 10-15% of reproductive-age women which causes pelvic pain and infertility. The disease is characterized by the growth of endometrial tissue outside the uterine cavity. Currently, early diagnosis of endometriosis is difficult due to non-specific symptoms and the absence of reliable biomarkers. Understanding these early molecular alterations could support the development of diagnostic tools for timely detection and treatment. The objective of this study was to identify changes in circulating microRNA (miRNA) profiles during the initial stages of endometriosis using a longitudinal non-human primate baboon model. Endometriosis was induced in female baboons by laparoscopic inoculation of menstrual endometrial tissue and plasma was collected at various timepoints. Plasma miRNAs at 3, 6, 9, and 15 months were analyzed using RNA sequencing. A total of 2,731 miRNAs were detected, 76 of which showed significant differences at least at one time point. There are four, three, and one miRNA exclusively differentially expressed at 3-, 6- and 9-month timepoints respectively while several (68) miRNAs are significantly expressed at 15 months. Four miRNAs (miR-210-3p, miR-448, miR-1260a/1260b, and miR-1298-5p) exhibited significant alterations across all time points. Of these, miR-210-3p increased, while the others decreased significantly. Hierarchical clustering revealed separation between the early (pre-inoculation and three months) and late (nine and 15 months) stages, indicating progressive divergence of miRNA profiles. These findings identify candidate miRNAs that may provide insight into early pathophysiological mechanisms and represent candidate circulating biomarkers associated with temporal molecular changes following disease induction, warranting further validation.

overall_design: Endometriosis was induced in Baboons by inoculating menustrual fluid tissue into peritoneal cavity by laparoscopy surgery, confirmed the lesion development by laparoscopic and histological evaluation. Blood was drawn on at preincoculation, month 3, 6, 9 and 15th and serum was collected and processed for tRFs differential expression (RNA-sequence analysis) across all time-ppoints while proinoculation time point taken as basal expression of these tRFs.

first: Hanna

last: Surmann

first: Niraj

last: Joshi

first: Gregory

last: Burns

first: Asgerally

middle: T

last: Fazleabas

first: Hugh

middle: S

last: Taylor


title: Probing RNA-protein interactions in the early embryo

city: Worcester

first: Oliver

last: Rando

state: MA

address: 364 Plantation St.

department: BMB

institute: UMass Chan Medical School

summary: The union of two germ cells to form a zygote, and subsequent early embryo development, are marked by radical remodeling of virtually every major class of biomolecules as the specialized germline states give way to the rapid and active growth that marks early development. In recent years, advances in ultra-low input genome-wide methods have enabled systematic analyses of mRNA abundance, and of chromatin organization, throughout early development in a variety of model systems. Here, we extend these efforts to the study of RNA binding protein (RBP) function in early mouse embryos, adapting REMORA based on fusing an RNA-editing enzyme to an RBP of interest – for use in early embryos. We benchmark our approach for several well-studied RBPs, successfully recovering expected features of their RNA cargos, and overall surveyed the RNA cargos for 17 RBPs of interest for early gene regulation. Analysis of changes in mRNA metabolism following knockdowns of the RBPs surveyed here allowed us to define direct regulatory functions for a subset of RBPs in the early mammalian embryo, including an unanticipated role for the RNA export factor Alyref in control of 3’ polyadenylation sites. Together, our data provide a proof of concept resource for systematically exploring RBP functions in mammalian embryogenesis.

overall_design: Smart-seq3 RNA sequencing of 2-cell and 4-cell stage mouse embryos, with A-to-I RNA editing or siRNA mediated gene knockdown, mRNA and/or siRNA microinjected into zygotes.

first: Bora

last: Pablo

first: Rando

last: Oliver


title: Effect of grape pomace skin colonic ferments on  differentiated monolayers of Caco-2 cells

city: Macul

first: Omar

last: Porras

state: Santiago

address: El libano 5524

department: INTA

country: Chile

institute: University of Chile

summary: Dietary fibre usually comes associated with phenolic compounds either trapped in a network formed by indigestible polysaccharides or, in some cases, bound to this matrix by covalent bonds. This natural association protects some phenolic compounds during their journey through the gastrointestinal tract, reaching the colon, where most polysaccharides undergo fermentation, weakening the matrix and promoting the release of these compounds to exert benefits by modulating intestinal function. Unfortunately, there are still difficulties in investigating the impact of the human microbiota on the release of phenolic compounds from the indigestible fraction and on their effects on the intestinal epithelium.   In this study, grape pomace skin was subjected to in vitro digestion to obtain an indigestible fraction, which was further incorporated into SHIME bioreactors with human colonic microbiota. Throughout these stages, we monitored the release of phenolic compounds using HPLC-DAD. We determined the impact of a phenolic-rich indigestible fraction on the fermentative capacity of human microbiota by measuring the short-chain fatty acids (SCFA) levels and the effect of the resulting colonic ferments on the barrier properties of differentiated monolayers of Caco-2 cells by monitoring the transepithelial electrical resistance and the transcriptomic profile by RNA sequencing.

overall_design: RNA-seq profiling of Caco-2 cells exposed to colonic ferments of grape pomace skin

first: Miltha

last: Hidalgo

first: Rodrigo

last: Pulgar

first: Francisca

last: Vera-Tamargo

first: Adriano

last: Costa de Camargo

first: Alina

last: Concepción-Alvarez

first: Bárbara

last: Railef

first: Carolina

last: Añazco

first: Elva

last: González

first: Igor

last: Pacheco

first: Jorge

last: Meneses-Pacheco

first: Karen

last: Fuenzalida

first: Macarena

last: Moreno

first: Martina

last: Oyarzún

first: Raquel

last: Bridi

first: Vanessa

last: Rubio


title: NOVEL HEXOKINASE-2 ASSOCIATED INFLAMMATORY PROCESSES IN THE GLIOBLASTOMA TUMOUR MICROENVIRONMENT

city: Wolverhampton

first: Christopher

last: Edwards

address: Wulfruna Street

country: United Kingdom

institute: University of Wolverhampton

summary: Deregulation of cellular metabolism is a hallmark of cancer, in which tumour cells upregulate glycolysis and preferentially ferment glucose to lactate (rather than pyruvate) in the 'Warburg effect'. Tumour-specific upregulation of hexokinase 2 (HK2), which catalyses the first rate-limiting step of glycolysis, has been established as key to this Warburg metabolism. Interrogating the impact of HK2 expression inhibition upon the glioblastoma transcriptome has the potential to uncover a number of novel canonical/non-canonical relationships that could serve as future co-therapeutic targets. To investigate this, we employed RNAseq to compare the expression profiles of glioblastoma cell cultures transfected with either HK2-targeted or non-targeting negative-control siRNA, thereby identifying genes differentially expressed specifically following HK2KD. Gene ontology category enrichment (GOSeq, Enrichment map) and STRING protein-protein interaction analysis of genes identified to be differentially expressed post HK2 knockdown (HK2KD) highlighted potential non-canonical roles for HK2 in regulating inflammatory, immune and angiogenesis-related processes within the glioblastoma tumour microenvironment.

overall_design: RNAseq profiling of glioblastoma cell lines (U87-MG) and glioblastoma patient derived cell cultures (IN859, IN2045) that received HK2KD or non-targeting negative control.

first: Edwards

last: Christopher

first: Taylor

last: Richards

first: Katherine

last: Karakoula


title: Transcription factor 21 deletion from podocyte precursors as a model for congenital nephrotic syndrome

city: Chicago

first: Yalu

last: Zhou

state: IL

address: 303 E Superior St

department: Department of Medicine

institute: Northwestern University

summary: Here, we establish a mouse model of congenital nephrotic syndrome (NS) by genetic deletion of transcription factor 21 (Tcf21), a gene required for normal podocyte development, selectively from early podocyte precursors. Single cell transcriptomics of kidneys from these mice capture glomerular and tubulointerstitial changes that occur early in the course of NS and provide additional insights into: 1) the regulation of podocyte development and function by TCF21, 2) the effects of podocyte injury on other glomerular cells, and 3) the tubulointerstitial changes that occur in response to the glomerular dysfunction and associated massive proteinuria of podocytopathies.

overall_design: Tcf21 was deleted specifically from podocyte precursors via a Pax8 driven Cre recombinase. 2 male mice without Tcf21 expression in podocytes (Tcf21-KO mice) along with 2 control male mice (1 Cre positive control and 1 Cre negative control) were euthanized between 2-3 weeks of age and kidneys were harvested and digested into a single cell suspension for scRNAseq.

first: Vidhi

last: Dalal

first: Susan

last: Quaggin


title: mTOR inhibition enhances the antitumor efficacy of RAF dimer-MEK blockade by inhibiting the ATF4-MTHFD2 pathway

city: Montreal

first: Sonia

middle: Victoria

last: del Rincon

state: QC

address: E-537, 3755 Chemin de la Côte-Ste-Catherine

country: Canada

institute: McGill University

summary: BRAF V600 inhibitors are clinically approved for the treatment of BRAFV600-mutant melanoma in combination with a MEK inhibitor, but are ineffective in other melanoma subtypes. Moreover, pan-RAF inhibitors, such as belvarafenib, when combined with MEK inhibitors (cobimetinib), have promising but limited efficacy in non-BRAF-mutant melanomas. Here, we report that the mTOR inhibitor sapanisertib improves the efficacy of combined belvarafenib and cobimetinib therapy in NRAS, NF1, and KIT-mutant melanomas. Mechanistically, sapanisertib combined with belvarafenib and cobimetinib suppressed ATF4 expression and its target gene MTHFD2 while inducing DNA damage, revealing a previously underappreciated role of the ATF4-MTHFD2 axis in DNA damage repair and drug response. Human and murine models resistant to combined belvarafenib and cobimetinib exhibited elevated levels of ATF4 and MTHFD2 and were sensitive to sapanisertib. This study provides promising treatment opportunities for patients with non-BRAF-mutant melanomas, or those who relapse following belvarafenib and cobimetinib combination therapy.

overall_design: We treated NRAS-mutant WM3406 melanoma cell lines with belvarafenib (25 nM), cobimetinib (50 nM), and the mTOR inhibitor sapanisertib (INK128, 25 nM) and we performed RNA sequencing analysis after 24 h.

first: Feiyang

last: Cai

first: Fan

last: Huang

first: Christophe

last: Goncalves

first: Harinee

last: Srikannan

first: Natascha

last: Gagnon

first: Elizabeth

middle: M

last: Guettler

first: Léa

last: Mukeba-Harchies

first: Jennifer

last: Maxwell

first: Jie

last: Su

first: Krikor

last: Bijian

first: Fabrice

last: Journe

first: April

last: Rose

first: Alexandre

last: Orthwein

middle: V

last: del Rincón

first: Wilson

middle: H

last: Miller Jr.


title: Cytotoxic Tissue-Resident NK Cells Infiltrate and Control Solid Epithelial Tumor Growth [CITE-Seq]

city: STANFORD

first: Imran

middle: Ahammad

last: Mohammad

address: lokey stem cell research building, 2165, 265 Campus Drive, Palo Alto, CA 94305

department: OHNS

institute: Stanford University

summary: Human tissue-resident natural killer (trNK) cells – broadly defined by markers of tissue-residency, such as CD49a (integrin alpha1, ITGA1) and CD103 (integrin alpha E, ITGAE) – are increasingly recognized for their immunoregulatory role in host control of infection, malignancy, and autoimmunity. Although the importance of transforming growth factor beta (TGFβ) in trNK cell differentiation has been demonstrated, the context in which the differentiation of CD49a+CD103+ trNK cells occurs can result in either an immunosuppressive phenotype or a highly cytotoxic one. To understand this dichotomy better, we utilized a multiomics approach to molecularly characterize these cells and identified a highly cytotoxic trNK (ctrNK) cell phenotype, characterized by the expression of CD39.

overall_design: CITE seq: CTRL, HNSCC-induced, and TGF-β induced NK cells were collected and stained with Fixable Viability Dye eFluor 780 (eBioscience; Cat# 65-0865-18). Human TruStain FcX (BioLegend) was used for Fc blocking for 15 minutes at 4°C. NK cell surface proteins were stained using TotalSeq B oligonucleotide-labeled antibodies (0.5 µg of each in 50 µL, BioLegend) targeting CD56, CD16, CD49a, CD103, and CD39. Cell hashing with hashtag oligonucleotides (HTO) was performed for multiplexing different conditions. After antibody incubation for 25 minutes at 4°C, cells were washed once to remove unbound antibodies.

first: John

middle: B

last: Sunwoo

middle: A

last: Foltz


title: Cytotoxic Tissue-Resident NK Cells Infiltrate and Control Solid Epithelial Tumor Growth [ATAC-Seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: 50,000 sorted NK cells from different conditions  (CTRL, HNSCC-induced double negative, HNSCC-induced double positive, and TGF-β induced double positive NK cells) were processed using the Diagenode ATAC-seq kit.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [mESC Bisulfite-seq]

city: Hong Kong

first: Jian

last: Yan

state: Hong Kong Special Administrative Region

address: 83 Tat Chee Avenue, Kowloon

department: Department of Biomedical Sciences

country: China

institute: City University of Hong Kong

type: Methylation profiling by high throughput sequencing

summary: CCCTC-binding factor (CTCF) is an evolutionarily conserved transcription factor with diverse regulatory roles. Around stable CTCF binding sites, nucleosomes are highly ordered and DNA methylation is reduced, yet the mechanisms establishing and maintaining this epigenetic environment remain unclear. Utilizing an innovative eBioID method, we identified that virtually all subunits of the nucleosome remodeling and deacetylase (NuRD) complex are associated with CTCF in cells. Further analyses revealed that the NuRD complex is necessary for the chromatin binding of CTCF, and emerges as a novel regulator of the genome architecture. Additionally, we discovered that MBD3-NuRD facilitates the recruitment of TET demethylases to CTCF sites, initiating local DNA demethylation essential for the activation of adjacent gene expression. Embryonic stem cells deficient in the NuRD complex showed impaired lineage commitment. In summary, this study elucidates a mechanism illustrating the interplay between CTCF binding and the epigenome including 3D genome organization and DNA methylation, with the NuRD complex serving as an indispensable mediator.

overall_design: CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRDComplex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESCWT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and CHI-SEQ, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development. In this SubSeries, we performed BS-Seq on mESC WT, SKO (sextuple knockout for Dnmt1/3a/3b and Tet1/2/3), and TKO (triple knockout for Tet1/2/3) cells to analyze genome-wide methylation levels across these three cell types.

first: Xi

last: Wang

first: Wenju

last: Sun

last: Wu


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [mESC_BPK25_HiC batch 2]

overall_design: CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRDComplex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESCWT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and CHI-SEQ, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development. In this SubSeries, we performed Hi-C experiments after mESC treatment with BPK25 to evaluate the effect of NuRD complex degradation on the three-dimensional structure of the genome.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [mESC ChIP-seq]

overall_design: CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRDComplex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESCWT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and CHI-SEQ, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development. In this subset, we carried out CTCF ChIP-Seq in three types of mESC cells (WT/SKO/TKO) to analyze the effect of DNA methylation on CTCF binding.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [HEK293T ChIP-seq]

overall_design: CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRDComplex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESCWT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and CHI-SEQ, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development. In this subset, we used DMSO, IAA and BPK25 to treat HEK293T cells and then conducted ChIP-Seq experiments of CTCF, MBD2/3 and TET1 to analyze the effects of degradation of CTCF and NuRD complex on the binding sites of these proteins.


title: NPAS3-regulated astrocyte mitochondria bioenergetics is required for cognition

city: Buffalo

first: Henry

middle: G.

last: Withers

state: New York

address: Elm and Carlton Streets

department: Biostatistics and Bioinformatics

institute: Roswell Park Comprehensive Cancer Center

summary: The basic helix-loop-helix (bHLH) transcription factor neuronal PAS domain protein 3 (NPAS3) provides transcriptional regulation of metabolic pathways, and despite its name is highly expressed in astrocytes. NPAS3 variants have been associated with cognitive dysfunction in several neuropsychiatric conditions, but the underlying brain cell type-specific mechanisms remain obscure. Here, we report that NPAS3 is a key regulator of mitochondria bioenergetics in astrocytes. Selective deletion of Npas3 in mature murine astrocytes decreases expression of Slc25a18 and its protein product, mitochondrial glutamate carrier 2, leading to reduced oxidative phosphorylation and lactate production in astrocytes. This deficit subsequently causes reduced excitability, dendritic spine density, and dendritic arborization of medial prefrontal cortex pyramidal neurons. Mice with Npas3-deficient cortical astrocytes exhibit impaired trace fear conditioning, which is rescued by lactate treatment. Thus, the present study demonstrates a mechanistic link between NPAS3-dependent astrocyte mitochondria bioenergetics and cognitive function and provides insights for glia-targeting treatment of cognitive dysfunction in neuropsychiatric disease.

overall_design: To investigate the role of Npas3 in the regulation of energy metabolism in astrocytes, we utilized mouse model of constitutive knockout of Npas3. We then performed gene expression profiling analysis using data obtained from RNAseq of forebrain tissue from heterozygous Npas3+/- and wildtype Npas3+/+ mice at embryonic day 16-17.

first: Mikhail

last: Pletnikov

first: Kateryna

last: Murlanova

first: Juhyun

last: Kim

first: Frederick

last: Nucifora Jr

first: Russell

last: Margolis

first: Dong

middle: W

middle: G


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [BPK25 Hi-C]

overall_design:
CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.
In this SubSeries, we performed Hi-C experiments after mESC treatment with BPK25 to evaluate the effect of NuRD complex degradation on the three-dimensional structure of the genome.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [HEK293T_siNURD_TET1_ChIP_Seq]

overall_design:
CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.
In this SubSeries, we used siRNA to knock down NuRD complex components such as CHD3/4/5 and MBD2/3 in HEK293T cells, respectively, and then performed ChIP-Seq with TET1 antibodies to evaluate the effect of knocking down different components of the NuRD complex on TET1 binding sites.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [CTCF_MBD2_HT_Me_CAP_SELEX]

overall_design:
CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.
In this subset, CTCF and MBD2 proteins were expressed and purified in vitro, and HT-SELEX and Methyl-SELEX experiments were carried out on the two proteins respectively. And we have developed a new CAP-Methyl-SELEX method by combining the published CAP-HT-SELEX and Methyl-SELEX techniques.The CAP-HT-SELEX and CAP-Methyl-SELEX experiments were carried out on CTCF-MBD2. Three replicates were designed for all experiments, and four cycles were performed for each. By analyzing the proportion of dinucleotides (especially CG) in the SELEX sequencing data with the number of cycles, the methylation preference of individual CTCF, MBD2 and their dimers was inferred.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [HEK293T_siNURD_CTCF_ChIP_Seq]

overall_design:
CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.
In this SubSeries, we used siRNA to knock down NuRD complex components such as CHD3/4/5 and MBD2/3 in HEK293T cells, respectively, and then performed ChIP-Seq with CTCF antibodies to evaluate the effect of knocking down different components of the NuRD complex on CTCF binding sites.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [mESC_d0246_DMSO_BPK25_IAA_RNA_Seq]

overall_design:
CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.
In this SubSeries, we treated mESC during induced differentiation with DMSO, BPK25 and IAA respectively, resulting in the degration of CTCF or NuRD complex during differentiation. Then, RNA was collected at d0246 days for RNA-SEQ sequencing. Finally, the expression profiles of the same treatment at different time points and different treatment groups at the same time points were compared.


title: RNAP-seq: in vitro genome-scale transcription reveals preferential RNA polymerase pausing on Clostridioides difficile antisense DNA

city: Dallas

first: Ping

last: Shen

state: Texas

address: 5323 Harry Hines Blvd

department: Microbiology

institute: UT Southwestern Medical Center

summary: Mechanistic dissection of gene transcription and its regulation in diverse organisms remains a central challenge for molecular biology. In vivo analyses using high-throughput DNA sequencing readouts (e.g., RNA-seq, Term-seq, and NET-seq) have revealed that transcriptional pausing by RNA polymerase (RNAP) underpins the regulation of gene transcription. However, these in vivo methods are difficult to apply in non-model organisms and lack the power of bottom-up biochemical analysis that can deconvolute the confounding effects of the cellular environment. Here we report a method for unbiased transcription of entire genomes in vitro called RNAP-seq, which is capable of defining pause, arrest, and termination sites with single-nucleotide resolution and of defining the regulatory contributions of cis-acting RNA and DNA sequences and dissociable transcription factors with complete control over transcription conditions. Using RNAP-seq to compare transcription by recombinant RNAPs from Clostridioides difficile (Cdf) and Escherichia coli (Eco), we discovered that Cdf RNAP exhibits pausing signatures distinct from Eco RNAP. Cdf RNAP pauses more frequently and more strongly at T-rich sequences, particularly on antisense regions of genes. We also defined the action of Cdf NusG at genome scale, revealing its preferential action in riboswitch control regions. These findings demonstrate lineage-specific pausing features and suggest that genome composition and RNAP co-evolve to shape gene expression. RNAP-seq is broadly adaptable to diverse bacterial species and offers a powerful framework for uncovering fundamental principles of transcription that govern gene expression.

sample_organism:
Escherichia coli
Clostridioides difficile

overall_design: We performed in vitro RNAP-seq to map transcriptional pausing and termination across genomes. E. coli and C. difficile RNA polymerases were assayed on matched DNA templates. Pausing sites were identified using a multi-round Z-score–based approach, and terminator-associated sites were defined based on proximity to in vivo termination sites. Strand-specific coverage and 3′ end signals were generated.

first: Michael

last: Wolfe

first: Xinyun

last: Cao


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [mESC_d0246_CTCF_MBD3_ChIP_Seq]

overall_design:
CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.
In this SubSeries, we performed ChIP-Seq on mESC cells induced to differentiate d0246 days with antibodies to CTCF and MBD3 (one of NuRD components), respectively, to analyze the changes in the binding sites of CTCF and MBD3 during normal mESC differentiation.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [mESC_d0246_RNA_Seq]

overall_design:
CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.
In this SubSeries of our study, we performed RNA-Seq analysis on mESC at d0246 days, separately. By comparing the expression profiles at different time points during differentiation, we aimed to demonstrate the successful establishment of mESC differentiation lineages.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [hESC_H9_d049_RNA_Seq]

overall_design: CTCF is closely related to the three-dimensional structure of the genome, and its stable binding site shows a low level of DNA methylation. Through eBioID experiments for CTCF and TET1/2/3, we found that multiple components of NuRD Complex appeared simultaneously in the list of CTCF and TETs interacting proteins. We performed BS-Seq and CTCF ChIP-Seq on mESC WT/SKO/TKO cells and found that genomic methylation had no significant effect on CTCF binding. We also performed HiC sequencing in BPK25-treated mESCs, and combined with published mESC WT/CTCF-AID HiC data, analyzed and compared the effects of CTCF or NuRD complex degradation on the three-dimensional structure of the genome. We performed a series of treatments on HEK293T cells, including DMSO, IAA(degraded CTCF), BPK25(degraded NuRD complex), etc., and then conducted RNA-Seq, ChIP-Seq, and BS-Seq experiments respectively. Explore the dependencies between CTCF, NuRD complex, and TET1/2/3. In addition, to investigate the biological significance of NuRD complexes in reshaping the chromatin environment and maintaining genomic structure around CTCF binding sites, we performed differentiation experiments on human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). During differentiation, we specifically target CTCF or NuRD complexes for knockout. To assess the impact of these gene knockdowns, We analyzed genome-wide methylation levels, gene expression profiles, and changes in CTCF, MBD2/3, and TET1/2/3 binding sites by BS-Seq, RNA-Seq, and ChIP-Seq, respectively, to clarify the important role of MBD3-NuRD, TET, and CTCF interaction regulatory axis in stem cell differentiation and development.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [hESC_H9_d049_ChIP_Seq]


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [HEK293T_IAA72h_ChIP_Seq]


title: Uncovering Critical Lysines in Mammalian Histone H3 with High-Throughput CRISPR Prime Editing [RNA-seq]

status: Public on May 09 2026

city: London

first: Aliaksandra

last: Radzisheuskaya

address: 237 Fulham road

department: Cancer Biology

institute: The Institute of Cancer Research

summary: Histone proteins are central to regulating eukaryotic genomes, and their post-translational modifications play key roles in controlling gene expression, DNA repair and chromatin structure. Understanding the functions of individual histone modifications is a significant challenge in mammalian chromatin biology, primarily due to the multiple copies of histone genes found in mammalian genomes. In this study, we present a high-throughput CRISPR prime editing platform that allows for base-precise, combinatorial, and reversible mutagenesis of all canonical and non-canonical histone H3 genes within their native genomic context. Using this system, we systematically substituted each lysine residue in histone H3 with arginine and compared each mutation against synonymous lysine-to-lysine controls. This unbiased functional screen revealed a core set of lysines, including H3K4, H3K9, H3K14, H3K18, and H3K79, whose mutations impair cellular fitness in mouse embryonic stem cells, highlighting the critical roles of their post-translational modifications in chromatin function. In addition, our approach demonstrated that H3K56 acetylation, previously linked to genome stability in yeast and Drosophila, plays a conserved role in safeguarding the genome in mammalian cells. Extending this approach, we generated double histone H3 mutants to probe functional redundancy between several lysine residues. While individual substitutions caused minimal defects, specific combinations, most notably H3K27R+H3K36R, revealed impairments in stem cell self-renewal and distinct transcriptional consequences, uncovering regulatory crosstalk not evident from single-site perturbations. This study presents the first comprehensive functional map of histone H3 lysines in a mammalian system, establishing a broadly applicable platform for dissecting histone modifications with advanced precision.

overall_design: RNA-seq profiling to assess differential gene expression in mouse ESCs with the following genotypes: wild-type, H3K27R, H3K36R and H3K36R+H3K27R


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [HEK293T_BPK25_ChIP_Seq]


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [HEK293T_BPK25_BS_Seq]


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [CTCF_Methyl_SELEX_data]


title: A massively parallel reporter assay of MECP2 cis-regulatory elements reveals genetic candidates for male-biased autism

status: Public on May 08 2026

city: Houston

first: Rebecca

middle: Meyer

last: Schuman

state: TX

address: 1250 Moursund Street, Suite 1350

institute: Zoghbi Lab at BCM

summary: Autism affects males four times more often than females, yet the basis of this sex bias remains unclear. One hypothesis is that hypomorphic variants in X-linked genes—genes where loss-of-function alleles cause syndromic neurodevelopmental disorders (NDDs) predominantly in females—produce milder, non-syndromic phenotypes in hemizygous males. We tested this by investigating cis-regulatory elements (CREs) of MECP2, a dosage-sensitive X-linked gene. Using a massively parallel reporter assay in human neurons, we mapped transcription factor binding sites within MECP2 CREs and tested autism-associated variants for functional impact. We identified two noncoding variants that change CRE activity, each with a male-biased phenotype. One of these, a promoter variant, disrupts NFY binding and reduces MECP2 expression by ~30%, a magnitude that produces autism-like phenotypes in mice. These findings suggest noncoding MECP2 variants can cause non-syndromic, male-biased autism, and provide a framework for uncovering regulatory variants in other X-linked NDD genes that may contribute to autism’s missing heritability.

overall_design: NFY-B CUT&Tag of iPSC-derived neurons after four weeks of differentiation. 100,000 nuclei and 0.5ug of antibody (Diagenode C15410241 and EpiCypher 13-0042) were used for each reaction.

last: Meyer-Schuman

first: Huda

last: Zoghbi


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [ChIP-Seq]

last: Yan


title: SMART-seq profiling of the ascending aorta and aortic arch from male and female Wnt1-Cre;Rosa26-mTmG mice

status: Public on May 03 2026

city: Chongqing

first: Qing

middle: Rex

last: Lyu

state: Chongqing

address: #118 Xingguang Avenue

department: Affiliated Chongqing General Hospital

institute: Chongqing University

summary: Vascular smooth muscle cells (VSMCs) in the ascending aorta and aortic arch arise from two distinct embryonic origins: the second heart field (SHF) and the cardiac neural crest (CNC). Whether these populations exhibit sex-dependent differences remains unknown. Here, we used Wnt1-Cre;Rosa26-mTmG mice to genetically label CNC-derived VSMCs with GFP, while non-CNC-derived cells expressed tdTomato. The ascending aorta and aortic arch were dissected from male and female mice, and single-cell suspensions were generated by enzymatic digestion. Cells were then sorted by fluorescence-activated cell sorting (FACS) based on GFP or tdTomato expression. RNA-expression profiles for each group were obtained using the Smart-seq platform. Thi study revealed the differential expression of VSMCs from different embryonic origin in both sexes.

overall_design: Single-cell suspensions were prepared from the ascending aorta and aortic arch of Wnt1-Cre;Rosa26-mTmG mice. Two male samples were pooled, while one female sample was processed separately. GFP-positive and tdTomato-positive cells were isolated from these suspensions by fluorescence-activated cell sorting (FACS), followed by SMART-seq analysis to profile gene expression in each sample.

first: Yanzhi

last: Wang

first: Cheng

last: Luo

first: Songmao


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [BS-seq]

first: Jian

summary: The currently available data of CTCF binding in mammalian cells are exclusively obtained with ChIP-seq. In order to fully retain the dynamic information of CTCF binding, we have developed a new method called GpC-Methylation Assisted Tracing (G-MAT) of a nuclear protein, which leverages DNA cytosine methylation to record the footprints of a protein within a given period of time.

overall_design: In principle, endogenous CTCF proteins are coupled with a cytosine methyltransferase M.CviPI cloned from Chlorella virus NYs-1 that covalently adds a methyl-group on carbon 5 (C5) of cytosine at the GpC dinucleotides that is un-erasable by any known demethylase. As a result, GpC adjacent to CTCF interacting sites can be permanently methylated, enabling genome-wide tracing the trajectory of CTCF footprints on chromatin using the whole genome bisulfite sequencing (WGBS). To enhance the methylation efficiency for improved signal-to-noise ratio in footprint detection, we took advantage of the SunTag system8 in fusion with endogenous CTCF proteins that recruited up to 10 copies of M.CviPI proteins fused with a single chain antibody fragment (scFv).

first: Xi

first: Wenju

last: Sun

last: Wu


title: Epithelial Edar is Responsible for Dental Anomalies in HED

city: Shanghai

first: Wenyu

last: Xu

address: 500 Quxi Road

department: Department of second dental center

institute: Shanghai ninth people's hospital

summary: In this study, we generated epithelial-specific Edar knockout mouse models and reported that the epithelial Edar is responsible for ectodermal dysplasia, determines tooth size and influences tooth morphology and number. Ko is from K14cre;Edarfl/fl mice and ctrl is from Edarfl/fl mice. Single-cell RNA sequencing revealed that impaired differentiation from preameloblasts to early ameloblasts may underlie the observed enamel defect. Taken together, these findings reveal a critical role of epithelial EDAR in tooth development and, for the first time, provide evidence that its downregulated expression can lead to enamel formation defects, offering new insights into the pathogenic mechanisms of EDAR-related HED.

overall_design: These cells were isolated from the dental germ of the the mandible first molar from Edarfl/fl and K14cre;Edarfl/fl  newborn C57black/B6 mice.


title: Pivotal Role of the NuRD Complex in Remodeling Chromatin Environment and Maintaining Genome Architecture at CTCF Sites [Hi-C]

summary: Interactions between nuclear proteins and chromatin are critical in transcriptional regulation. The state-of-the-art approaches provide merely snapshot information on their interactions, which impedes our understanding of the causality in the dynamic regulatory processes. Here, we developed a novel method termed G-MAT that profiles the footprints of a nuclear protein on chromatin and DNA methylome simultaneously in living mammalian cells. Utilizing G-MAT to transcription factor CTCF, we inferred that CTCF searches its binding sites in the genome through stochastic collision to the chromatin. Together with eBioID, which is an enhanced BioID method facilitated by the Suntag system, we found hundreds of proteins with distinct functions co-occupying CTCF binding sites on chromatin, including RNA splicing and chromatin remodeling. Intriguingly, two methyl-CpG binding domain (MBD) proteins were found as interacting partners of CTCF, largely leading CTCF to preferentially bound to methylated DNA segments, consistent with the co-occurrence of CTCF footprints and DNA hyper-methylation when analyzing the G-MAT data at the single-molecule resolution. MBD2, one of the MBD proteins shown to be associated with demethylases, could likely erase DNA methylation near CTCF binding sites, resulting in seemingly anti-correlations between CTCF binding and DNA methylation. Altogether, G-MAT has enabled us to investigate the causal relationship between CTCF target searching and DNA methylation at the single-molecule level, providing an important data resource and novel insights for advanced understanding of transcriptional regulation dynamics.

overall_design: In situ Hi-C of cultured HEK293T cells to identify chromatin long range interaction loops, Topologically associating domains, and Compartment A and B.


title: Transcriptomic profile of patient derived glioblastoma stem cells from recurrent and newly diagnosed background

city: Miami

first: Shanta

last: Dhar

state: Florida

address: 1011 NW 15th Street, Room 437

department: Biochemistry

institute: University of Miami

summary: Metabolic rewiring of neoplastic cells can fuel tumorigenicity of cancer stem cells contributing to tumor recurrence. One such cancer is glioblastoma for which tumor recurrence is universal. After first line therapy includes surgical resection, temozolomide chemotherapy, and radiotherapy. Tumorigenic fueling of glioblastoma stem cells (GSCs) which have invaded into distal brain parenchyma that are unresectable and/or resistant to standard chemo and radiation therapy promote recurrence. We wanted to focus on studying whether there is a correlaion between metabolic substrate utilization, stemness, and tumorigenicity of patient derived GSC populations. Leveraging the knowledge of metabolic substrate utilization by the GSCs and their stemness potential may be a promissory target to curb the aggressiveness of the GSCs. Thus we embark on a journey to investigate the transcriptomic profile of the patient derived GSCs from varied background. Considering the heterogeneity of the GSCs, it is of utmost important to study patient derived GSCs from varied bakground. Our pool of cells comprise of 2 recurrent patient derived GSCs and 2 newly diagnosed GSCs. Our long term goal being to design and develop alternative nanotherpaeutic approaches to target the otherwise hard-to-target GSCs in the brain, we concentrated on using patient derived cells to closely recapitulate the tumor characteristics whiich would have been difficult if conventional cell lines were used. Thereby, a complete understanding of the genomic profile of the GSCs from varied patient background would help us figure out the best possible target site for therpeutics against GSCs and as a result delay/stop the tumor recurrence.

overall_design: We performed HISAT2 based RNA sequencing analysis on patient derived glioblastoma stem cells (GSCs) from varied background. The pool of cells comprise of 2 patient derived GSCs from recurrent background and 2 patient derived GSCs from newly diagnosed background. We aim at understanding the differential genomic profile of the patient derived GSCs based on their metabolic and stemness features so that we can use this knowledge to design and develop non-ivasive and/or invasive therapeutic interventions to target this heterogenous cell population.

first: S

last: Sarkar

first: A

last: Ashokan

first: B

last: Surnar

first: D

last: Eichberg

first: T

middle: I

last: Slepak

first: M

middle: E

last: Ivan


title: Enhancer regulation of OSR2 in mandibular incisor agenesis susceptibility [RNA-Seq]

status: Public on May 02 2026

city: Nanjing

first: Liwen

last: Fan

address: Hanzhong Road 140

institute: Nanjing Medical University

summary: Mandibular incisors are the most frequently congenitally absent teeth in Asian populations, affecting mastication, speech, aesthetics, and oral and maxillofacial functions over the long term. However, the genetic etiology of mandibular incisor agenesis (MIA) remains obscure. In this study, we conducted the first genome-wide association study on MIA in a Chinese population and identified an enhancer variant rs71514987 associated with an increased risk of MIA (odds ratio = 2.18, 95% confidence interval = 1.83-2.58, P = 3.62×10-19). Functional characterization indicated that rs71514987 C allele reduced the binding affinity of GATA4 to the OSR2 promoter, leading to increased OSR2 expression via remote regulation. Notably, elevated OSR2 levels promoted cell proliferation but diminished cell differentiation, migration, and apoptosis capabilities in stem cells from human exfoliated eciduous teeth (SHED), as well as abnormal development of pharyngeal teeth in zebrafish models. Mechanistic experiments further revealed that OSR2 bound to the promoter region of CXCR4, promoting its transcription level in the GPCR pathway, thereby influencing odontogenic differentiation during tooth development. Our findings provide novel insights into the molecular etiology of MIA.

overall_design: Stem cells from human exfoliated deciduous teeth (SHED) at passages 2-4 were divided into two groups: (1) control SHED cells transduced with empty vector (control group), and (2) SHED cells with stable overexpression of OSR2 (OSR2-OE group). Each group contained three independent biological replicates (total n=6 samples). All cells were cultured in α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a 5% CO₂ incubator. After 48 hours of culture, total RNA was extracted using an RNA isolation kit (Vazyme, China) and reverse-transcribed into cDNA. Sequencing was performed on an Illumina NovaSeq 6000 platform.

first: Xinyu

last: Zhang

first: Ji

last: Mao

first: Barbara

last: Vona

first: Xinze

first: Mulong

last: Du

first: Lan

last: Ma

first: Yongchu

last: Pan


title: Promoter strength and position govern promoter competition via transcript-dependent insulation [Micro-C]

city: Stanford

first: Joanna

last: Wysocka

state: California

address: 265 Campus Drive

summary: Competition between promoters within a shared regulatory landscape has been implicated in development and disease, but the determinants of promoter competition remain unclear. Here, we introduce diverse promoters into defined genomic sites within the Sox2 locus and measure how these insertions attenuate endogenous Sox2 expression. We find that the level of reduction in endogenous Sox2 transcription is correlated with the strength of the inserted promoter. Transcription from the inserted promoter is required for competition, with longer transcript resulting in more competition. The inserted active promoter and its associated transcriptional unit function as an insulator, rendering competition position-dependent. Finally, we observe HUSH-mediated silencing of the inserted promoters, which counteracts competition. Together, our work uncovers the rules governing promoter competition, highlights its impact on tuning gene expression levels and genome evolution, and suggests that transcripts of sufficient level and length can mediate insulation independently of CTCF and cohesin.

overall_design: Micro-C with capture of Sox2 locus in mouse embryonic stem cells, with different promoter integrations.

first: Mervenaz

last: Koska

first: Masahiro

last: Nagano

first: Tomek

last: Swigut

first: Alistair

last: Boettiger

first: Anders

last: Hansen


title: Pregnancy induced TRM cells protect against breast cancer [scRNAseq]

city: Clayton

first: Wei

last: Shi

state: Victoria

address: 15 Innovation Walk

department: Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute

country: Australia

institute: Monash University

summary: Age and parity are well-known factors that influence breast cancer development (1-3). Epidemiological studies have shown that women who have had multiple pregnancies are at a lower risk of developing breast cancer than women who have no history of pregnancy (4, 5). However, mechanisms that link parity and cancer protection are unclear. We discovered that CD8+ T cells with a tissue-resident memory (TRM) phenotype enriched in the mammary glands of parous women and mice compared to their virgin counterparts. Developmental kinetics revealed that these cells seeded the mammary gland during early gestation but persisted post lactation and was specific to the mammary glands. While these CD8 T cells expressed the TRM specific transcription factor Hobit, single cell transcriptomics revealed expansion of transcriptionally distinct canonical and non-canonical TRM populations, including a novel Hobit+ innate T cells (ITCs) in the parous mammary gland compared to their virgin counterparts. Furthermore, imaging analysis uncovered that the TRM like cells were associated with mammary epithelial cells and impairment in mammary gland branching during pregnancy significantly affected their differentiation or population expansion. In keeping with their association with mammary epithelial cells, all TRM subsets, including the Hobit+ITCs commonly required the cytokines TGF-β and IL-15 for their differentiation. Notably, depletion of CD103+ T cells exacerbated tumour growth in parous mice and lineage tracing experiments revealed that the canonical TRM like cells switched to an effector phenotype in tumours and contributed to tumour control. Together, we show for the first time that parity induces novel and heterogenous TRM like populations in the mammary gland that are central in providing protection against breast cancer.

overall_design: Live CD8 T cells (CD45+ TCRB+ CD8a+) were sorted from virgin and parous 4th mammary fat pad.

first: Ajithkumar

last: Vasanthakumar

first: Tabinda

last: Hussain

first: Liam

last: Neil

first: Bhupinder

last: Pal

first: Adelynn

last: Tang

first: David

last: Chisanga

first: Axel

last: Kallies


title: Control of naive T cell reactivity and peripheral tolerance by ascorbate and TET activity [RNAseq in vitro stimulation]

city: Memphis

first: Beisi

last: Xu

state: Tennessee

address: 262 Danny Thomas Pl

department: Center for Applied Bioinformatics

institute: St Jude Children's Research Hosipital

summary: Maintenance of T cell naïve (Tn) state and restricting effector T cell differentiation upon basal level stimulations would be critical for immune homeostasis and prevention of autoimmunity. To understand how Tn state is regulated by nutrient cues in a cell-intrinsic manner, here we perform an in vivo CRISPR screening to identify required solute carrier (SLC) proteins that transport metabolites and ions into T cells. Among SLC proteins revealed by this experiment, vitamin C transporter Slc23a2 appears to play a role. Conditional ablation of Slc23a2 in T cells by genetic approaches reduces intracellular vitamin C levels by approximately 80%, accompanied by spontaneous activation and differentiation of Tn cells, autoantibody production, and autoimmune pathology in select organs. Slc23a2-deficient Tn cells exhibit profound DNA hypermethylation, dysregulation of genes controlling signaling transduction and transcription, and enhanced differentiation of helper T cells upon stimulation. These results define a broad regulatory space of vitamin C as a cofactor of Tet enzymes in controlling active DNA demethylation. In agreement, conditional deletion of Tet genes impairs Tn state and increases helper T cell differentiation. Thus, our study reveals a cell-intrinsic mechanism by which micronutrient vitamin C via Slc23a2 maintains Tn state and self-tolerance by promoting Tet-mediated DNA demethylation.

overall_design: Bone marrow from Slc23a2fl/fl mice and Slc23a2fl/flCD4Cre mice were co-transferred with CD45.1+CD45.2- wild-type B6 mice into CD45.2+CD45.1+ recepient mice at 1:1 ratio. 12 weeks after bone marrow transfer, CD4Tnaive cells were sorted from spleen and pLNs from recipient mice and in vitro stimulated for 3hours with anti-CD3/CD28 antibodies

first: Xiaolei

last: Hao

first: Lu

last: Bai

first: Yongqiang

last: Feng


title: Promoter strength and position govern promoter competition via transcript-dependent insulation

first: Joanna

last: Wysocka

overall_design: ChIP-seq of RNA Polymerase II (Pol2) and cohesin subunit Rad21 on edited F123 mouse embyronic stem cells

first: Mervenaz

last: Koska


title: Conserved replication strategies facilitate the cross-species

status: Public on May 01 2026

city: Bochum

first: Leyla

last: Sirkinti

address: Universitätsstraße 150

department: Translational and Computational Infection Research

country: Germany

institute: Ruhr University

summary: A significant number of pandemics, endemics and even sporadic outbreaks trace back to zoonotic spillovers of emerging RNA viruses.  Especially, zoonotic infections with the distantly related hepatitis E virus (HEV, species Paslahepevirus balayani) from various animal origins, such as pigs, deer, wild boar, rabbits and camelids, are well-documented and account for 20 million acute hepatitis cases each year. The detection of HEV RNA in rats surrounding pig farms suggest that they serve as an additional reservoir and source of zoonotic HEV infection. Moreover, the rising number of zoonotic spillover events with the distantly related rat hepatitis E virus (ratHEV, species Rocahepevirus ratti) underscores the urgent need to elucidate the role of rats as a reservoir for HEV infection and in the transmission dynamics of viruses within the family Hepeviridae. Herein, we introduce a novel HEV reverse zoonosis model based on the infection of rat liver cells, which supports completion of the entire HEV replication cycle. RNA sequencing analyses unraveled distinct host responses to HEV infection characterized by minimal activation of innate immune responses. Importantly, host-directed antivirals targeting entry factors previously identified in human host cells effectively restricted HEV infection in rat liver cells. Furthermore, human-specific genomic rearrangements conferred increased fitness across human and rat hepatoma cells. In conclusion, our study indicates conserved viral replication mechanisms of HEV between rats and humans, providing valuable insights into the molecular mechanisms of zoonotic spillover across species.

overall_design: total RNA-seq of untreated primary rat hepatocytes of 3 donors of Wistar Han rats (species Rattus norvegicus forma domestica) and two passages of untreated McA-RH7777 rat hepatoma cells. Further samples were analyzed of McA-RH7777 cells inoculated with HEV 3 (strain Kernow-C1/p6 and 83-2-27) and DMSO, NIT008 or Baricitinib treated  or uninfected.

first: Nicola

last: Frericks

first: Daniel

last: Todt

first: Eike

last: Steinmann


title: PD-1 regulates latent effector differentiation of thymic cytotoxic CD8+ T cells [spatial transcriptomics]

city: Rochester

first: Haidong

last: Dong

state: MN

address: 200 First Street SW

institute: Mayo Clinic

summary: Durable T cell immunity against cancer depends on the continual replenishment of effector CD8⁺ T cells. Thymic output has been associated with favorable prognosis in cancer patients across a range of ages, suggesting that the thymus is an important source for replenishing T cells capable of controlling cancer progression. However, whether CD8⁺ T cells acquire effector potential within the thymus, and how thymic output of effector CD8⁺ T cells contribute to peripheral tumor immunity, remain unclear. In this study, we discover that thymic single-positive (SP) CD8⁺ T cells undergo latent effector differentiation following thymic selection, but this process is subject to PD-1 regulation. We further demonstrate that PD-1 limits the contribution of thymic output of CD8⁺ T cells in shaping the TCR repertoire within the tumor tissues for tumor immunosurveillance. Although PD-1 inhibition facilitates the expansion of effector CD8⁺ T cells in the periphery, these cells gradually lose antitumor activity within tumors due to accelerated exhaustion in the absence of PD-1. Thus, while latent effector differentiation of thymic CD8⁺ T cells enable a rapid response to malignant cells in the periphery, PD-1 restrains this process to prevent overt or terminal effector differentiation, which could compromise balanced and durable peripheral immunity.

overall_design: Selected tissue sections from four patients with muscle-invasive bladder cancer were evaluated to define regions of interest (ROIs) enriched for NKG7⁺ and NKG7⁻ CD8⁺ T cells. ROIs containing NKG7⁺ CD8⁺ T cells and ROIs containing NKG7⁻ CD8⁺ T cells were subsequently profiled using spatial transcriptomics.

first: Zhiming

last: Mao

first: Ying

last: Li


title: NuRD-Enabled CTCF-TET Crosstalk Orchestrates Epigenome Reprogramming and Genome Architecture

first: Jian

last: Yan

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

sample_organism:
Escherichia coli
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: PD-1 regulates latent effector differentiation of thymic cytotoxic CD8+ T cells [scRNA-Seq]

overall_design: Isolated thymic CD8+ T cells and splenic CD8+ T cells at baseline from Pdcd1fl/fl and Pdcd1 cKO mice were performed single cell RNA sequencing. CD8+ tumor infiltrating lymphocytes were isolated from B16-OVA at day 12 post tumor injection in Pdcd1fl/fl and Pdcd1 cKO mice and analysed by single cell RNA sequencing.


title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell type-specific transcription [scRNA-seq]

city: New York

first: Elizabeth

last: LaMarca

state: NY

address: 1470 Madison Avenue

institute: Icahn School of Medicine at Mount Sinai

summary: Here, we construct genome-scale maps for R-loops, three-stranded nucleic acid structures comprised of a DNA/RNA hybrid and a displaced single strand of DNA, in the proliferative and differentiated zones of the human prenatal brain. We show that R-loops are abundant in the progenitor-rich germinal matrix, with preferential formation at promoters slated for upregulated expression at later stages of differentiation, including numerous neurodevelopmental risk genes. RNase H1-mediated contraction of the genomic R-loop space in neural progenitors shifted differentiation toward the neuronal lineage and was associated with transcriptomic alterations and defective functional and structural neuronal connectivity in vivo and in vitro. Therefore, we conclude that R-loops are important for fine-tuning differentiation-sensitive gene expression programs of neural progenitor cells.

overall_design: scRNA-seq files from hiPSC-derived neurons transduced with an RNase H1-overexpression construct or a blue fluorescent protein (BFP) lentiviral backbone control. Data were collected from independent cultures from 2 donors. Samples were multiplexed using hashtag oligonucleotides (HTOs) and sequenced as a single pooled GEX and HTO library. Raw FASTQs, HTO hastags, and processed files (barcodes, features, matrix) are provided, along with sample-level metadata.

first: Atsushi

last: Saito

first: Amara

last: Plaza-Jennings

first: Sergio

last: Espeso-Gil

first: Allyse

last: Hellmich

first: Michael

last: Fernando

first: Benham

last: Javidfar

first: Will

last: Liao

first: Molly

last: Estill

first: Kayla

last: Townsley

first: Anna

last: Florio

first: James

last: Ethridge

first: Catherine

last: Do

first: Benjamin

last: Tycko

first: Li

last: Shen

last: Kamiya

first: Nadejda

last: Tsankova

first: Kristen

middle: J

last: Brennand

first: Schahram

last: Akbarian


title: The HSF2-HSP110 Axis Promotes Genome Stability by Supporting RNA Polymerase II Dependent Transcription and DNA Repair Gene Expression [CUT&Run]

city: Augusta

first: Nahid

middle: F

last: Mivechi

state: GA

department: Georgia Cancer Center

institute: Augusta University

summary: The precise regulation of RNA polymerase II (RNAPII) is essential for transcriptional fidelity and genome stability. Here, we identify a previously unrecognized genotoxic stressresponsive transcriptional axis composed of Heat Shock Factor 2 (HSF2) and its client chaperone HSP110, which is activated by ionizing radiation (IR). Loss of HSF2 or HSP110 leads to increased DNA damage and heightened IR sensitivity. Mechanistically, the HSF2-HSP110 axis safeguards genome stability by sustaining RNAPII processivity and its C-terminal domain (CTD) phosphorylation, particularly at serine 7 (S7). Disruption of this axis leads to transcriptional dysregulation, altered pre-mRNA splicing, increased transcription-replication conflict, and downregulation of DNA damage response genes, resulting in DNA damage. In vivo, loss of HSF2 accelerates IR-induced lymphomagenesis by impairing transcriptional regulation, specifically inhibiting DNA repair gene expression. These findings define the HSF2-HSP110 axis as an essential transcriptional mechanism in the genotoxic stress response and reveal a therapeutic vulnerability that could be exploited to sensitize tumors to genotoxic therapies.

overall_design: CUT&RUN analyses using an RNAPII antibody in parental and sgHSF2 K562 cells, either untreated or exposed to IR, followed by 2- or 4-hour recovery periods

first: Xiongjie

last: Jin

first: Caixia

last: Xi

first: Bhaumik

last: Pandya

first: Junfeng

last: Pang

first: Binnur

last: Eroglu

first: Demetrius

last: Moskophidis


title: The HSF2-HSP110 Axis Promotes Genome Stability by Supporting RNA Polymerase II Dependent Transcription and DNA Repair Gene Expression [PRO-Seq]

overall_design: Parental and sgHSF2 K562 cells (in duplicates) were rinsed with ice-cold PBS and werepermeabilized using buffer containing 10mM Tris-HCl pH 8.0, 10% glycerol, 250mM sucrose,5mM MgCl2, 1mM EGTA, 10mM KCl 0.1 % (v/v) Igepal CA-630, 0.05 % (v/v) Tween-20.0.5mM DTT and SUPERase IN (0.2ml/ml). Subsequent steps were performed in the NascentTranscriptomics Core at Harvard University (Garrido-Martin et al., 2018).


title: PD-1 regulates latent effector differentiation of thymic cytotoxic CD8+ T cells [TCR-Seq]

first: Haidong

last: Dong

overall_design: In this cohort of TCR sequencing, thymic and splenic CD8+ T cells at baseline as well as thymic and tumor-infiltrating CD8+ T cells isolated from B16-OVA-bearing, and B16-F10-bearing Pdcd1fl/fl and Pdcd1 cKO mice were isolated and analysed for clonotype information.

first: Zhiming

last: Mao

first: Ying

last: Li


title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell type-specific transcription [prenatal brain DRIP-seq]

first: Elizabeth

last: LaMarca

overall_design:
DNA/RNA-Immunoprecipiation Sequencing (DRIP-seq) from hiPSC-derived neurons transduced with an RNase H1-overexpression construct or a catalytically-inactive RNase H1-D145N mutant construct or a blue fluorescent protein (BFP) lentiviral backbone control. Data were collected from independent cultures from 2 donors.  DRIP-seq bigwigs, and DRIP-seq peak data are provided. Additionally included are DRIP-seq bigwigs and peak files from human second trimester prenatal brain (17-21 gestational weeks. Germinal matrix n = 3 and cortical plate n = 3).
Submitters state that raw FASTQs are not provided due to patient privacy concerns (as the samples are prenatal autopsy samples).

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

first: Atsushi

last: Saito

first: Amara

last: Plaza-Jennings

first: Sergio

last: Espeso-Gil

first: Allyse

last: Hellmich

first: Michael

last: Fernando

first: Benham

last: Javidfar

first: Will

last: Liao

first: Molly

last: Estill

first: Kayla

last: Townsley

first: Anna

last: Florio

first: James

last: Ethridge

last: Do


title: A ceramide synthase is important for filamentous fungal biofilm morphology and antifungal drug susceptibility

city: Hanover

first: Charles

last: Puerner

state: NH

address: 1 Rope Ferry Rd

department: Microbiology and Immunology

institute: Geisel School of Medicine at Dartmouth College

summary: The complex structure of the biofilm generates microenvironments which impact the fitness of the biofilm community as a whole. Contributions to fitness include the development of emergent properties resulting in the recalcitrance to external stressors such as environmental stress or drug stress in the context of a pathogen. The biofilm developed by the filamentous fungal pathogen Aspergillus fumigatus develops zones of low oxygen which contribute to a reduction in antifungal drug susceptibility, however the mechanisms driving this have remained elusive. Here we have utilized a transcriptomic approach to probe the biofilm structure in comparison to a planktonic lifestyle to identify unique biofilm transcriptional patterns. Importantly we utilized two phenotypically diverse strains. From this analysis we have identified biofilm specific gene co-expression networks as well as a ceramide synthase, designated barA, with a striking increase in transcript abundance specifically in the biofilm. Null mutants for barA in two strain backgrounds display altered biofilm morphology with some strain specific differences but interestingly do not impact virulence. Importantly we find the BarA ceramide synthase has a role in regulating susceptibility to classes of ergosterol targeting antifungal drugs. In the absence of barA, treatment with voriconazole leads to significant cellular damage in the form of lysis. Through this we have identified a novel mechanism driving antifungal drug resistance in the biofilm of filamentous fungal pathogen.

overall_design: RNAseq profiling of A. fumigatus strains AF293 and CEA10 submerged biofilm and planktonic tissue harvested at 18--hours of development in four biological replicates. Planktonic tissue was with and without exposure to 0.2% oxygen for 30-minutes.

first: Owen

last: Wilkins

first: Robert

last: Cramer


title: RNA-seq profiling of iPSC-derived microglia (iMicroglia) with wild-type or GPNMB knockout genotype

status: Public on May 06 2026

city: Philadelphia

first: Alice

last: Chen-Plotkin

address: 165 Johnson Pavilion

institute: University of Pennsylvania

summary: Glycoprotein nonmetastatic melanoma B (GPNMB) is critical to cellular uptake of pathological forms of alpha-synuclein (aSyn), the hallmark disease protein in Parkinson's Disease (PD). Here, we demonstrate that the non-membrane-anchored, extracellular domain of GPNMB can function in a non-cell-autonomous manner. In human brain, GPNMB is widely expressed in neurons and microglia. In induced pluripotent stem cell-derived microglia (iMicroglia), GPNMB expression and secretion increase with exposure to apoptotic neurons. In the aSyn fibril-seeded model of PD, iMicroglia-derived GPNMB enhances neuronal aSyn uptake and development of aSyn pathology, including in GPNMB knockout neurons. This dataset contains bulk RNA-seq data from iPSC-derived microglia (iMicroglia) differentiated using the Brownjohn protocol from isogenic GPNMB wild-type (WT) and GPNMB knockout (KO) iPSC lines (3 biological replicates each), treated with PBS as vehicle control.

overall_design: Bulk RNA-seq profiling of iPSC-derived microglia (iMicroglia) differentiated via the Brownjohn protocol from isogenic GPNMB wild-type (WT) and GPNMB knockout (KO) iPSC lines. Three biological replicates per genotype were sequenced under basal (PBS vehicle control) conditions to characterize transcriptomic differences resulting from GPNMB loss-of-function in iMicroglia.

first: Marc

last: Carceles-Cordon

middle: D

last: Gallagher

first: Travis

middle: L

last: Unger

first: Cooper

middle: K

last: Penner

middle: S


title: Effect of BHLHE40 silencing using short-hair pin RNA on CD34+ cells isolated from human cord blood, during TGF-B1 depedent Langerhans cell differentiation

city: Graz

first: Sally

last: Connolly

state: Steiermark

address: Otto Loewi Forschungszentrum - Immunologie und Pathophysiologie Medizinische Universität Graz Heinrichstraße 31a A-8010 Graz

country: Austria

summary: Langerhans cells (LCs) are resident antigen presenting cells of the epidermis whose differentiation, immature state and tissue residency critically depend on transforming growth factor–β1 (TGF-β1) signaling. Although several downstream transcriptional regulators of TGF-β1 have been identified, how LC epithelial identity and homeostasis are transcriptionally stabilized remains incompletely understood.Human CD34⁺ hematopoietic progenitors were differentiated into LCs or monocyte-derived cells in vitro using cytokine-defined conditions, combined with lentiviral knockdown or ectopic expression of BHLHE40. Bulk-RNA seq of shBHLHE40 cells reveals blunted induction of TGF-β1 responsive genes.

overall_design: RNA-seq profiling of shCTRL and shBHLHE40 transduced CD34+ cells  at 0 hours and 24 hours post-culturee with TGF-β1 to promote Langerhans cell differentiation

first: Hebert

last: Strobl


title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell type-specific transcription [RNA-seq]

first: Elizabeth

last: LaMarca

overall_design: RNA-seq files from hiPSC-derived neurons transduced with an RNase H1-overexpression construct or a catalytically-inactive RNase H1-D145N mutant construct or a blue fluorescent protein (BFP) lentiviral backbone control. Data were collected from independent cultures from 2 donors. Raw FASTQs and an RNA count matrix are provided.

first: Atsushi

last: Saito

first: Amara

last: Plaza-Jennings

first: Sergio

last: Espeso-Gil

first: Allyse

last: Hellmich

last: Fernando

last: Javidfar

first: Catherine

first: Benjamin

last: Tycko

first: Li

last: Shen

last: Kamiya

first: Nadejda

middle: M

last: Tsankova

first: Kristen

last: Brennand

first: Schahram

last: Akbarian


title: Brain FGF2 and NCAM1 contribute to FGFR1-dependent progression of ER+ breast cancer brain metastases in young and aged hosts [NanoString_DSP]

city: Aurora

first: Diana

last: Cittelly

state: CO

address: 12801 East 17th Avenue, Research Complex 1 South, L18-5402G

institute: University of Colorado

summary: Estrogen receptor positive (ER+) breast cancer (BC) represents a significant proportion of BC brain metastasis (BCBM) but remains understudied. Here, we report that FGFR1-amplification, a well-established driver of ER+ BC endocrine resistance, promotes ER+ BCBM colonization in young and aged mice, through brain-dependent mechanisms. FGFR1-dependent brain colonization in young and aged mice occurs via canonical FGF2/FGFR1 signaling and non-canonical NCAM1/FGFR1  interactions. Astrocytic FGF2-mediated paracrine activation of FGFR1 promoted BCBMs in estrogen36 treated young mice, but FGF2 signaling decreased in the brain with aging and estrogen-depletion. Neuronal and glial NCAM1, which remain unchanged in young and aged brains, promoted adhesion to neurons and migration of ER+ BC cells, suggesting that interactions with astrocytes and neurons facilitate early ER+ BCBM colonization through FGFR1. Importantly, FDA-approved FGFR inhibitors effectively blocked early but not late metastatic progression only in young mice, suggesting limited efficacy of FGFR inhibitors to block non-kinase-dependent FGFR1 functions in vivo.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: NanoString digital spatial profiling of UCD65 human breast cancer brain metastases and mouse glial cells in brain metastases within aged OVX, aged sham, young OVX, and young sham NSG mice.

first: Morgan

last: Fox

first: Jenny

last: Jaramillo-Gómez

first: R. Alejandro

last: Marquez-Ortiz

first: Karen

last: Alvarez-Eraso

first: Maria

last: Contreras-Zárate

first: Stella

last: Koliavas

first: Peter

last: Kabos

first: Natalie

last: Serkova

first: Carol

last: Sartorius

last: Wellberg


title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell type-specific transcription [hiPSC-derived neuron DRIP-seq]

first: Elizabeth

last: LaMarca

overall_design: DNA/RNA-Immunoprecipiation Sequencing (DRIP-seq) from hiPSC-derived neurons transduced with an RNase H1-overexpression construct or a catalytically-inactive RNase H1-D145N mutant construct or a blue fluorescent protein (BFP) lentiviral backbone control. Data were collected from independent cultures from 2 donors. Raw FASTQs, DRIP-seq bigwigs, and DRIP-seq peak data are provided. Additionally included are DRIP-seq bigwigs and peak files from human second trimester prenatal brain (17-21 gestational weeks. Germinal matrix n = 3 and cortical plate n = 3).

first: Atsushi

last: Saito

first: Amara

last: Plaza-Jennings

first: Sergio

last: Espeso-Gil

first: Allyse

last: Hellmich

first: Michael

last: Fernando

first: Benham

last: Javidfar

first: Will

last: Liao

first: Molly

last: Estill

first: Kayla

last: Townsley

first: Anna

last: Florio

first: James

last: Ethridge

last: Do

last: Tycko


title: Single-cell V(D)J-seq of tumors from Eif3e cg1cre KO mice

city: xiamen

first: Lianghua

last: Lin

state: Fujian

address: Xiang An Nan Lu 4221

institute: 生命科学学院

summary: white nodules collected from tissues with evident tumor development in organs of cKO mice, such as liver, lung, and spleen were predominantly enriched with B cells or CD4+ T cells, and the lymphocytes within these tissues exhibited highly monoclonal enrichment (in one case, oligoclonal enrichment was observed), indicating that lymphocytes in the tumor tissues of cKO mice display characteristics of monoclonal expansion.

overall_design: White nodules were collected from tissues with evident tumor development in cKO mice, such as liver, lung, and spleen covered with white nodules. The nodules were preserved in tissue storage solution and sent to SeekGene for tissue dissociation. After dead cell removal, both single-cell RNA-seq and single-cell V(D)J-seq were performed simultaneously to assess clonal expansion of cells within the tissues.

first: Jiazhen

last: Wang

first: Yunjie

last: Zhang

first: Huilin

last: Song


title: Single-cell sequencing study of splenocytes and YFP+ B cells to investigate differences between Eif3e cg1cre knockout mice and control mice

summary: To gain insights into the cellular mechanisms underlying the lymphoproliferative disorder developed in cKO mice, we performed single cell RNA-seq (scRNA-seq) analysis of YFP+ and total splenocytes from cKO and control mice at the age of 2 months, when lymphocyte activation and proliferation had not yet become obvious.Through our study, we found that Eif3e-deficient B cells are impaired in their development into GCB and PC, becoming blocked at the pre-GCB stage. Additionally, all B cells exhibited high expression of MHC-II. Among CD4+ T cells, the TFH cell population was significantly expanded in cKO mice and showed high expression of IL4. Based on the early-stage phenotype of this mouse model, we hypothesize that Eif3e-deficient B cells promote IL4 expression in CD4+ T cells, which in turn stimulates upregulation of MHC-II on all B cells. The increased MHC-II further enhances CD4+ T cell activation, forming a positive feedback loop.

overall_design: For each batch, spleens from 2-3 cKO or control mice (2 months old) were pooled. Single-cell sequencing was then performed on sorted total splenocytes and YFP+ cells.

first: Chao

last: Huang


title: Bulk RNA sequencing study of iGCB day3 cells to investigate differences between Eif3e cg1cre knockout B cells and control  B cells

summary: To obtain a larger number of Eif3e-deficient B cells induced by cg1cre, we utilized an in vitro germinal center B cell (iGCB) culture system. By day 3 of culture, we harvested abundant YFP+ Eif32-deficient B cells and their corresponding control cells. RNA-seq comparison revealed that Eif3e-deficient B cells upregulated some co-stimulatory molecules, including Cd80 and Icam1. GO enrichment analysis showed that upregulated genes were primarily associated with ribosome-related pathways, while downregulated genes were mainly enriched in lipid metabolism. The upregulation of co-stimulatory molecules may confer Eif3e-deficient B cells with the capacity to promote CD4+ T cell activation.

overall_design: Three biological replicates each for cKO and control groups were included, with naïve B cells derived from individual mice. The cells were cultured for 3 days with 40LB cells irradiated at 120 Gy in 1640 complete medium supplemented with IL-4. YFP+ cells (indicating cg1cre expression) were sorted for RNA-seq analysis.

first: Pengda

last: Chen

first: Xinyong