title: Minimizing far-extending chromatin perturbation in genome editing  preserves stem cell identity [PEM-seq]

first: Jiawei

last: Yu

overall_design: To investigate possible misinterpretation of editing-introduced effects, alongside long-read sequencing after editing, we conducted PEM-seq experiments to provide an independent view of possible large deletions or structural variations. In this study, PEM-seq was used to profile the spectrum of repair outcomes around the cut site, including deletion-size distribution and low-frequency rearrangement-like events.

last: Ming

last: Junsong


title: Destabilization of the noncanonical PRC1.1 complex via USP7 inhibition induces neuronal differentiation in neuroblastoma [ChIP-Seq NGP]

status: Public on Feb 26 2026

city: West Lafayette

first: Nathaniel

middle: Wesley

last: Mabe

state: Indiana

address: 201 S. University St.

department: Medicinal Chemistry and Molecular Pharmacology

country: USA

institute: Purdue University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Pediatric cancers are frequently driven by genomic alterations that result in impaired differentiation during tissue development.  To identify protein complex-level dependencies required for differentiation in neuroblastoma, a pediatric cancer of the developing peripheral nervous system, we curated a list of protein complexes using the CORUM database and mined the Dependency Map (DepMap) using single sample gene set enrichment analysis. This analysis identified the non-canonical PRC1.1 complex, which represses transcriptional activity through ubiquitination of histone 2A, lysine 119 (H2AK119Ub), as a selectively enriched dependency in neuroblastoma. Knockout of several PRC1.1 subunits reduced neuroblastoma growth, arrested the cell cycle, and induced a neuronal differentiation program. While no known direct inhibitors of PRC1.1 exist, co-dependency analysis of PRC1.1 subunits against all other genes in DepMap identified that the deubiquitinase USP7 strongly correlated with PRC1.1 dependency. Treatment with XL177A, a small molecule inhibitor of USP7, significantly reduced neuroblastoma growth in both cellular and animal models. Integrated RNA- and ChIP-sequencing showed that both PRC1.1 knockout and USP7 inhibition resulted in highly correlated transcriptional alterations and reduced H2AK119Ub deposition on chromatin, suggesting that USP7 inhibition reduced neuroblastoma growth through a PRC1.1-dependent mechanism. Mechanistically, global proteomics and ubiquitinomics revealed that USP7 inhibition disrupted non-canonical PRC1 complex assembly, resulting in destabilization of PRC1.1 and subsequent proteolysis. Our findings expand our understanding of the chromatin complexes required to maintain a de-differentiated state in neuroblastoma and suggest the therapeutic potential for USP7 inhibitors in the treatment of this disease.

overall_design: ChIPsequencing for H2AK119Ub at day 10 with 1 µM XL177A treatment in NGP cels.

middle: W


title: Dosage compensation defects due to roX RNA deletion are rescued by recalibration of X/autosome stoichiometry

city: Planegg-Martinsried

first: Fotios

last: Gkountromichos

address: Großhaderner Strasse 9

department: Molecular Biology

country: Germany

institute: Biomedical Center, Ludwig-Maximilians-Universität München

summary: Metazoa evolved regulatory networks to balance the expression of their sex chromosomes. In Drosophila, males have a single gene-rich X chromosome, whereas females have two. Balanced X/autosome expression is essential for viability, and in male flies is achieved by activation of genes on the X through the male-specific-lethal (MSL) dosage compensation complex (DCC). This ribonucleoprotein assembly contains long, non-coding roX RNAs. To dissect the functional requirements of roX in a cell-based system, we deleted the roX2 gene in male S2 cells and selected two independent lines lacking detectable roX RNA. In the absence of roX, the remaining MSL protein complex was unable to associate with known or newly identified binding sites and thus failed to activate transcription. Surprisingly, the X/autosome expression ratio appeared nevertheless compensated. Cytogenetic and genomic analyses revealed that both roX-deficient cell populations had acquired additional X chromosomes. Apparently, chromosome gains due to mis-segregation made up for the loss of DCC-mediated dosage compensation. Interestingly, ectopic expression of full-length roX2, but not of shortened derivatives, fully restored DCC binding and normalized the karyotype. These findings illustrate that X chromosome dosage compensation is critical for viability even in cultured cells and provide a striking example of rapid evolution under stringent selection.

overall_design: CUT&RUN of MSL2 and H4K16ac in wild-type and roX2-KO Drosophila Schneider S2 cells.

first: George

last: Yankson

first: Muhunden

last: Jayakrishnan

first: Aline

last: Campos Sparr

first: Marisa

last: Muller

first: Patrick

last: Heun

first: Peter

middle: B

last: Becker


title: NKG2A inhibition promotes NK cell-CD8+ T cell interactions in support of improved anticancer immunity in ovarian carcinoma

city: Prague

first: Michal

last: Hensler

state: Prague 7

address: Jankovcova 1518/2

country: Czech Republic

institute: Sotio Biotech a.s.

summary: Natural killer (NK) cells contribute to tumor immunosurveillance, yet their phenotypic and functional heterogeneity and impact on anti-tumor immunity remain incompletely understood. Here, using an integrated set of transcriptomic, spatial, and functional assays, we demonstrate that advanced non-small cell lung carcinoma (NSCLC), which is generally considered an immunologically “hot” tumor – contains elevated levels of intratumoral cytotoxic NK1 (CD56dim) and NK3 subsets, which exhibit robust clinically relevant effector functions, localze preferentially within tumor cores, and spatially interact with CD8+ T cells in mature tertiary lymphoid structures (mTLSs). In contrast, the NK cell compartment of immunological “cold” tumors such as high-grade serous ovarian carcinoma (HGSOC) – which generally contains immature TLSs and ICI-resistant TCF1-TIM3+CD8+ CTLs – is enriched in functionally exhausted NK2 (CD56bright) NK cells expressing high levels of TIM3 and NKG2A. Functional studies in HGSOC patient samples and syngeneic mouse models reveal that bidirectional crosstalk between NK cells and CD8⁺ T cells is essential for anti-tumor immunity, and that disrupting this interaction—via CD8⁺ T cell or NK cell depletion—promotes NK cell exhaustion and CD8⁺ T cell dysfunction. Importantly, blockade of NKG2A restores NK cytotoxicity and promotes CD8⁺ T cell effector responses, and when combined with PD1 inhibition, significantly enhances survival in murine HGSOC models. Our findings highlight the NKG2A–HLA-E axis as a clinically actionable checkpoint in tumors with impaired NK cell–mediated immunity, and delineate the spatial and functional interplay between NK cells and CD8⁺ T cells as a determinant of anti-tumor immune efficacy.

overall_design: A total of 4 samples were analyzed, including 2 HGSOC and 2 NSCLC samples.

first: Teraza

last: Lanickova

first: Artemis

last: Angelidou

first: Veronika

last: Niederlova

first: Jitka

last: Fucikova


title: H3K79me2 Profiling Reveals DOT1L Regulated Splicing in MLL-rearranged AML [RNA-Seq]

city: San Antonio

first: Bowen

last: Yang

state: TEXAS

address: 7703 Floyd Curl Drive, Mail code 7971

institute: University of Texas Health Science Center at San Antonio

summary: Aberrant H3K79 dimethylation by DOT1L is a defining feature of MLL-rearranged (MLLr) acute myeloid leukemia (AML), but whether this modification influences alternative splicing remains unclear. Here, we performed H3K79me2 ChIP-seq and RNA-seq on primary samples from 24 MLLr AML patients, 4 wild-type MLL AML patients, and 4 healthy bone marrow donors. We found that a subset of exon skipping (SE) events was enriched at H3K79me2-occupied loci in MLLr samples. DOT1L inhibition with EPZ5676 led to broad changes in SE patterns, and many switched events showed concurrent reduction in local H3K79me2 signal. Pathway analysis of genes harboring these events revealed enrichment for RNA processing and splicing-related functions, with additional involvement of DNA repair and apoptosis-associated pathways. To identify potential mediators, we performed rapid immunoprecipitation mass spectrometry (RIME) in MV4-11 and MOLM-14 cells and detected SRSF2 and hnRNP family proteins in complex with DOT1L; these interactions were confirmed by co-immunoprecipitation. Combined inhibition of DOT1L and selected splicing factors reduced cell proliferation more effectively than single-agent treatment, both in MLLr cell lines and in xenograft models. These results indicate that H3K79me2 contributes to alternative splicing regulation through splicing factor recruitment in MLLr AML and provide a rationale for coordinated epigenetic–splicing therapeutic strategies.

overall_design: RNA-seq and H3K79me2 ChIP-seq were performed to define DOT1L/H3K79me2–associated alternative splicing in normal bone marrow (n=4), wtMLL AML (n=4), and MLLr AML (n=24). In the MLLr cohort, matched samples were profiled before and after DOT1L inhibition (EPZ5676) to identify treatment-responsive exon-skipping events and corresponding H3K79me2 peak gain/loss at regulated exon regions.

first: Tianbao

last: Li

first: Qi

last: Liu

first: Victor

last: Jin


title: H3K79me2 Profiling Reveals DOT1L Regulated Splicing in MLL-rearranged AML [ChIP-Seq]


title: Single-nucleus multiome profiling of ventral hippocampus across female reproductive states

city: Bronx

first: Marija

last: Kundakovic

state: NY

address: 441 E. Fordham Road

department: Biological Sciences

institute: Fordham University

type: Expression profiling by high throughput sequencing

summary: This study used single-nucleus multiome (RNA + ATAC) sequencing to profile transcriptional and chromatin accessibility changes in the mouse ventral hippocampus across the estrous cycle, sex differences, and peripartum states. C57BL/6J mice were used, and nuclei were isolated from ventral hippocampal tissue and processed using the 10x Genomics Chromium Single Cell Multiome ATAC + Gene Expression platform.

overall_design: Ventral hippocampal tissue from C57BL/6J mice was collected across distinct physiological conditions (proestrus, diestrus, male, pregnancy, postpartum). Single-nucleus multiome sequencing was performed to jointly measure gene expression and chromatin accessibility.

first: Luisa

last: Demarchi

first: Maria

last: Tickerhoof

first: Ayyappa

last: Kumar Sista Kameshwar

first: Devin

last: Rocks

first: Laila

last: Ouldibbat

first: Ana

last: Milosevic

first: Masako

last: Suzuki


title: Single-nucleus transcriptomics of Parp7-/- lung

city: DALLAS

last: YAN

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center

summary: To explore the cell type-specific effects of PARP7 deficiency in the lung, we performed single-nucleus RNA sequencing (snRNA-seq) of lung from WT and Parp7-/- lung.

overall_design: 10-month-old wild-type and Parp7-/- mice (2 mice/genotype) were euthanized and lung were collected and snap frozen in liquid nitrogen. Nuclei were isolated from frozen lung and droplet-based snRNA-sequencing was performed using 10x Genomics.

last: Yan

first: Kun

first: Devon

last: Jeltema


title: B cell immunity to the Lassa virus glycoprotein is a correlate of vaccination-induced viral load control

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

country: Switzerland

institute: University of Basel

summary: Lassa fever, a viral hemorrhagic fever caused by the arenavirus Lassa virus (LASV), affects thousands of individuals annually, highlighting the need for a vaccine. Yet, immunological correlates of viral load control remain poorly defined. Studying vaccination-induced immunity in a surrogate LASV challenge model we found that LASV-cross-reactive B cell immunity induced by the glycoprotein of distantly related arenaviruses, such as lymphocytic choriomeningitis virus (LCMV), provided significant viral load control in mice. Counter to common concepts, suppression of viremia was observed in the absence of CD8 T cells or neutralizing antibodies but correlated with non-neutralizing glycoprotein-specific antibody responses. Adoptive cell transfer experiments with monoclonal LCMV-specific B cells demonstrated that these cells suppressed viral loads when previously activated by a heterologous cross-reactive glycoprotein and diversified by somatic hypermutation. These findings establish vaccination-induced B cell immunity to the LASV glycoprotein as a correlate of viral load control, independently of virus-neutralizing antibody titers at the time of challenge.

overall_design: The immunizing GPC determines the extent of HkiL cell hypermutation and clonal diversification. At week -1, TgL mice were depleted of CD8 T cells. At wk0 they were given 104 HkiL cells and were immunized with either rLCMV/LASVLIIA, rLCMVA or rLCMVA/LASVLIVC. At wk5 HkiL cell progeny in spleen were analyzed.

first: Tiago

last: Abreu-Mota

first: Anna-Friederike

last: Marx

first: Dorothee

last: Winterberg

first: Karen

last: Tintignac

first: Jonas

last: Fixemer

first: Florian

last: Geier

first: Nicole

last: Brodmann

first: Cemre

last: Seven

first: Claudia

last: Reichmuth

first: Anna Lena

last: Kastner

first: Min

last: Lu

first: Weldy

middle: V

last: Bonilla

first: Mirela

last: Dimitrova

first: Mehmet

last: Sahin

first: Gert

last: Zimmer

first: Matthias

last: Peipp

first: Daniel

middle: D

last: Pinschewer


title: Enhanced mitophagy promotes lung repair and regeneration by restoring epithelial metabolic fitness [AT2s RNA-Seq]

city: Shanghai

first: pei

last: wu

address: 393 Middle Huaxia Road

country: China

institute: ShanghaiTech University

summary: Alveolar Type II cells (AT2s) are the stem cells in the alveoli, responsible for both alveolar homeostasis and regeneration. Mitochondrial dysfunction has been reported in the AT2 cells from both chronic and acute injury induced alveolar diseases, including idiopathic pulmonary fibrosis (IPF) and viral pneumonia. Here, we demonstrate that genetic depletion of Ubiquitin Specific Peptidase 30 (USP30), a negative regulator of mitophagy, boosts mitophagy and restores mitochondrial function, leading to protection from injury induced AT2 apoptosis and increased AT2 stem cell activity. Both global Usp30 knockout (KO) and AT2-specific Usp30 KO protects the mice from bleomycin induced lung fibrosis and influenza puneumonia. Moreover, pharmaceutical inhibition of USP30 using a small molecule inhibitor effectively alleviates lung fibrosis and influenza pneumonia. Our study underscores a therapeutic potiential of USP30 inhibition for the treatment of injury induced alveolar diseases, offering a promising strategy for treating injury induced diseases

overall_design: RFP+ AT2 cells were flow-sorted from tamoxifen-treated WT-sftpc-creert2  and Usp30-Sftpc-creert2 mice at day 14 after influenza infection

first: Ying

last: Xi

first: Pei

last: Wu

first: Jiawei

last: Chen


title: CRYPTID-exon: streamlined detection of cryptic exons from RNA-seq data

city: Worcester

first: Athma

middle: A

last: pai

state: MA

address: 55 Lake Avenue North

department: RNA Therapeutics Institute

institute: UMass Chan Medical School

summary: Cryptic splicing has emerged as a pervasive feature of mammlian gene expression with recent studies discovering thousands of previously unannotated splice sites. Despite its prevalence, the functional consequences of this hidden layer of splicing remain largely unknown due to challenges in identifying the exact exonic regions introduced into mRNA transcripts. Here, we introduce a novel computational approach, CRYPTID-exon, that accurately predicts exon boundaries by modeling RNA-seq read coverage anchored on empirically derived splice sites. We use CRYPTID-exon to identify and characterize thousands of cryptic exons in nascent and mature RNA from human cells. Additionally, we demonstrate that CRYPTID-exon is well powered to identify exons that are sensitive to translation-mediated degradation processes. Finally, given the growing interest in leveraging cryptic exons to modulate gene expression levels, we use our approach to identify cryptic exons in disease-relevant genes. We see that targeting these exons with splice-switching antisense oligonucleotides (ASOs) can alter gene expression and splicing patterns of the parent genes. Our study provides a framework to systematically identify and characterize cryptic exons, which will enable downstream insights into their impact on mRNA stability and translation.

overall_design:
Targeted RNA sequencing: RNA-seq following combined transcript pulldown against 13 genes from nuclear RNA isolated from KNS60 cells. (Methods: Targeted sequencing of nuclear RNA from specific genes)
Amplicon sequencing: PCR amplification using primer pairs covering: (a) upstream - exon region, (b) exon- downstream region, and (c) upstream - downstream of exon region, using cDNA generated from total RNA isolated from KNS60 cells. (Methods: Amplicon sequencing to validate cryptic exons)
RNA-seq following ASO treatment:  ASO treatment of KNS60 cells against target gene, using scrambled ASO (non-targeting control) and unstransfected (untreated) cells as controls, followed by transcript pulldown for target gene, and RNA-seq. (Methods:Antisense oligonucleotide (ASO) treatments)

first: Eraj

middle: S

last: Khokhar

first: Kaitlyn

last: Brokaw

first: Zachary

middle: J

last: Kartje

first: Marina

last: Krykbaeva

first: Ezequiel

last: Calvo-Roitberg

first: Adam

last: Hedger

first: Jonathan

last: Lee

first: Atish

last: Wagh

middle: K

last: Watts

last: Pai


title: A dietary pan-amino acid dropout screen in vivo reveals a critical role for histidine in T-ALL

city: Zürich

first: Puneet

last: Sharma

state: Zürich

address: Otto-Stern-Weg 3

department: Institute of Biochemistry

institute: ETH

summary: Dietary interventions show therapeutic potential in cancer, but systematic comparisons are lacking. We performed a dietary pan-amino acid dropout screen in an orthotopic model of NOTCH1-driven T-cell acute lymphoblastic leukemia and identified histidine depletion as uniquely antileukemic. Histidine-restricted diets extended survival in a dose-dependent manner, while remaining well-tolerated. Mechanistically, multi-omic profiling revealed that histidine deprivation strongly suppresses SREBF2 and cholesterol biosynthesis pathways critical for leukemic proliferation, imposing a therapeutic vulnerability. Dietary cholesterol supplementation partially rescued the antileukemic effects of histidine restriction in vivo. Overall, these findings couple translational control in T-ALL to cholesterol metabolism, suggesting that defined dietary amino acid restrictions may expose broader therapeutic vulnerabilities in cancer.

overall_design: Bulk RNA-seq and ribosome profiling of Jurkat and DND41 cells grown in media supplemented with different histidine concentrations.

first: Komal

last: Mandleywala

first: Simona

last: Ulrich

first: Victoria

last: da Silva-Diz

first: Christopher

last: Thai

first: Oekyung

last: Kim

first: Maya

last: Aleksandrova

first: Gwendolyn

last: Chung

first: Cristian

last: Eggers

first: Dieter

last: Lütjohann

first: Tanaya

last: Kulkarni

first: Amartya

last: Singh

first: M Elena

last: Díaz-Rubio

first: Michael

last: Wierer

first: Sebastian

last: Leidel

first: Xiaoyang

last: Su

first: Eileen

middle: P

last: White

first: Joshua

last: Rabinowitz

first: Raphael

last: Morscher

last: Herranz


title: Minimizing far-extending chromatin perturbation in genome editing  preserves stem cell identity [RNA-Seq]

city: Haidian District

first: Jiawei

last: Yu

state: Beijing

address: Tsinghua  University

department: School of Pharmaceutical Sciences

institute: Tsinghua University

summary: While CRISPR/Cas9 holds therapeutic promise, broader application demands understanding complications in vast non-coding regions. We found that CRISPR/Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated ATAC/RNA approach (AR-seq) and identified editing-induced chromatin accessibility change, with its scale varying by cell types. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and ultimately loss of stemness identity. To minimize chromatin perturbations and preserve cell identity we refined gene editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for safer and broader application of genome editing technologies.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: To define the transcriptional consequences of genome editing at regulatory-adjacent non-coding loci, we designed an RNA-centered framework that prioritizes spatially resolved, mechanism-informative readouts rather than endpoint differential expression alone. Specifically, we used the RNA arm of AR-seq to quantify locus-proximal and distal expression perturbation after editing, and formalized these effects as a distance-aware metric (ΔRNA/ΔRNA span) to test whether transcriptional disruption propagates outward from the cut site. This design was coupled to matched chromatin measurements and perturbation controls (including p53 inhibition and repair-pathway modulation) to separate generic stress responses from editing-linked regulatory rewiring. We further integrated 3D genome context to evaluate whether genes proximal in chromatin contact space are preferentially affected relative to genes closer only in linear genomic distance. In aggregate, this study design enables a causal interpretation in which editing-induced transcriptional changes are modeled as structured, distance- and architecture-dependent regulatory perturbations, rather than as nonspecific global expression drift.

first: Zhu

last: Ming

first: Yuan

last: Junsong

first: Meng

last: Qiuchen

first: Yu

last: Jiawei


title: Deconvolution of regulatory T cell heterogeneity identifies cycling precursors as conductors of immune escape during breast carcinoma progression

city: Boston

first: Kornelia

last: Polyak

address: 450 Brookline Ave. S1070

institute: Dana-Farber Cancer Institute

summary: Tumor-immune microenvironment is instrumental in shaping breast cancer tumor evolution. We previously identified an evolutionary bottleneck demarcating ductal carcinoma in situ (DCIS) from invasive ductal carcinoma (IDC). How the microenvironment evolves during such transition remains elusive. We dissected, in parallel, single-cell composition of DCIS and IDC patients and rat tumors that faithfully recapitulate human breast cancer. We discovered T cell diversity distinguishing DCIS and IDC and a novel cycling Treg state occupying the pseudotime bifurcation between DCIS and IDC, preceding more activated Treg subtypes. Multiscale analysis of patient cohorts as part of HTAN Consortium established that cycling Treg predicts recurrence in low-grade DCIS. We proved cycling Treg as Treg progenitors critical for immune escape and demonstrated that OX40 agonism restored immunotherapeutic sensitivity, where responders displayed increased inflammatory CAF subtype heterogeneity. Our study thus established cycling Treg’s importance in preserving immune escape and underscored its prognostic value to stratify future recurrence.

overall_design: Dataset 5: Outbred Sprague Dawley rats were injected with NMU carcinogen (50 mg/kg, twice, 7 days apart, starting at 40 days of age rat) to induce rat mammary tumors. Tissues from mammary glands with microlesions (MG) and and progressing DCIS tumors from NMU-treated rats collected at different timepoints since NMU injection were dissociated by collagenase 4/hyaluronidase, and subjected to droplet based 10X scRNAseq (5'HTv3) pipeline and Illumina NovaSeq6000. Downstream analyses of Datasets 1-3 include basic filtering and quality control, removal of cell doublets, batch-effect control/integration, cell cluster identification, and module scoring.

first: Triet

last: Bui

first: Ernesto

last: Rojas

first: Julieann

last: Puleo

first: Simona

last: Cristea

first: Franziska

last: Michor


title: Destabilization of the noncanonical PRC1.1 complex via USP7 inhibition induces neuronal differentiation in neuroblastoma

first: Nathaniel

last: Mabe

overall_design: CUT&RUN for PCGF1 RING1B, EZH2 and ChIP-seq for H2AK119Ub and H3K27me3 in parental NBSD cells


title: Telomere damage enhances immunogenicity of neuroblastoma and accelerates response to anti-PD-L1 treatment

city: Cologne

first: Matthias

last: Fischer

address: Kerpener Str. 62

department: Department of Experimental Pediatric Oncology

institute: University Children’s Hospital of Cologne

summary: In human neuroblastoma cell lines, absence of baseline cGAS expression and lack of cGAS induction upon stimulation by cytosolic DNA has been reported, whereas the functionality of the cGAS-STING pathway in primary neuroblastoma has remained uncertain. To assess whether cGAS-STING pathway may be active in human neuroblastoma, we analyzed CGAS expression in primary tumor samples and compared it with that in human neuroblastoma cell lines.

overall_design: RNA-seq and gene expression analysis of 14 human neuroblastoma cell lines. The RNA-seq of primary tumor samples is part of GSE49711.

first: Stefanie

last: Höppner


title: Autism-like phenotypes and increased NMDAR2D expression in mice with KDM5B histone lysine demethylase deficiency

city: Exeter

first: Michiel

middle: Albert

last: Basson

address: Hatherly Labs, Prince of Wales Road

country: United Kingdom

institute: University of Exeter

summary: Loss-of-function mutations in genes encoding lysine methyltransferases (KMTs) and demethylases (KDMs) specific for trimethylated of lysine 4 of histone 3 (H3K4me3) are associated with neurodevelopmental conditions, including autism spectrum disorder and intellectual disability. To study the role of KDM5B (Lysine DeMethylase-5B)-mediated H3K4me3 demethylation, we investigated neurodevelopmental phenotypes in mice without KDM5B demethylase activity. These mice exhibited autism-like behaviours and increased brain size. H3K4me3 levels and the expression of neurodevelopmental genes were increased in the developing Kdm5b mutant neocortex. These included elevated expression of Grin2d. The Grin2d gene product NMDAR2D was increased in synaptosomes isolated from the Kdm5b-deficient neocortex and treating mice with the NMDAR antagonist memantine rescued deficits in ultrasonic vocalisations and reduced repetitive digging behaviours. These findings suggest that increased H3K4me3 levels and associated Grin2d gene upregulation disrupt brain development and function, leading to socio-communication deficits and repetitive behaviours, and identify a potential therapeutic target for neurodevelopmental disorders associated with KDM5B deficiency.

overall_design: Single nuclei RNA sequencing was performed on neocortices from wild-type and Kdm5b deltaARID/deltaARID mice at postnatal day 5. Nuclei were isolated and samples were processed with Parse Biosciences Evercode WT kit (chemical v3).

first: Leticia

last: Pérez-Sisqués

first: Shail

middle: U

last: Bhatt

first: Angela

last: Caruso

first: Josephine

middle: L

last: Robb

first: Alex

last: Donovan

first: Rosemary

last: Bamford

first: Alejo

last: Torres-Cano

first: Shoshana

last: Spring

first: Eleanor

last: Hendy

first: Talia

middle: E

last: Gileadi

first: Martyna

last: Panasiuk

first: Jed

last: Trengove

first: Neeru

last: Jindal

first: Mohi

last: Ahmed

first: Joyce

last: Taylor-Papadimitriou

first: Diana

last: Cash

first: Nicholas

last: Clifton

first: Jacob

last: Ellegood

first: Laura

middle: C

last: Andreae

first: Jason

last: Lerch

first: Maria Luisa

last: Scattoni

first: K

last: Giese

first: Cathy

last: Fernandes

first: M


title: A long non-coding RNA, afu-182, regulates in vitro fungal azole response and virulence in Aspergillus fumigatus

city: Riverside

last: Stajich

state: CA

address: 900 University Ave

department: Microbiology and Plant Pathology

institute: University of California-Riverside

summary: Azole-resistant Aspergillus infections are a source of serious concern, as only three classes of drugs are available for treatment. However, as most infections are still caused by azole-sensitive AF strains, there is a need to better understand fungal response to azole drugs. Here, we have identified a long non-coding RNA, afu-182, as a major regulator of erg11-independent fungal azole recalcitrant colony growth (ARC), without an apparent change in azole’s minimum inhibitory concentration. As strains are currently classified in a binary system of resistant or susceptible to drugs, we show that afu-182 is a negative regulator of azole drug response, leading to azole recalcitrant biofilms and poor disease outcomes in an animal model upon azole treatment. Furthermore, overexpression of afu-182 significantly reduced fungal burden in animals treated with azole drugs, showing a genetic basis of action. Whole transcriptome analysis revealed that azole drugs lead to upregulation of 7-transmembrane domain proteins of the RTA1 family, and these proteins are negatively regulated by afu-182. Here, we characterized two RTA1 family genes downstream of afu-182 and showed their individual and additive effects in regulating fungal ARC. Taken together, our data show a trans-acting role of long non-coding RNA afu-182 in regulating fungal response to azole drugs both in vitro and in vivo. This provides a promising novel approach to determine the treatment outcomes, given that MIC alone is a poor predictor for treatment outcomes in infections caused by ASAF strains.

overall_design: Whole transcriptomic profiling of WT CEA10 Δafu-182, comp-182 and OE-182 strains in the absence and presence of 0.15µg/ml voriconazole, grown as stationary cultures for 48 hours.

first: Nava

middle: R

last: Poudyal

first: Ryan

middle: T

last: Mehlem

first: Priyanka

last: Doneparthi

first: Tabitha

last: Cady

first: Anne

last: Strong

first: Sven

last: Willger

first: Maximiliano

last: Ortiz

first: Rooksana

last: Noorai

first: Robert

last: Cramer

first: Sourabh

last: Dhingra


title: Sensitive CAR T cells redefine targetable CD70 expression in solid tumors [ATAC-seq]

city: New York

first: Sophie

last: Hanina

address: 630 West 168th Street

institute: Columbia University

summary: CD70 expression in solid tumors is heterogeneous. We demonstrate that cells appearing CD70-negative retain ultra-low levels of CD70 expression. These cells that appear negative by conventional detection methods. Bulk ATAC-seq analysis of FACS-sorted CD70⁻ and CD70⁺ tumor populations revealed differential chromatin accessibility, indicating epigenetic regulation of CD70 expression in both ccRCC and HGSOC models.

overall_design: NSG mice (n = 3 per model) were implanted with ccRCC patient-derived xenograft (PDX) lines (K5 and K7) under the renal capsule. Fifteen days post-implantation, tumors were isolated and dissociated for FACS sorting into CD70⁻ and CD70⁺ tumor cell populations. Protein, RNA, and chromatin accessibility (ATAC-seq) were profiled at isolation (day 0 ex vivo) and during culture at day 2 and day 8 ex vivo. An analogous procedure was performed for an ovarian carcinoma (HGSOC) PDX line 32 days post tumor implantation.


title: Macrophage phagocytosis of Coccidioides promotes its differentiation into the parasitic form

city: San Francisco

first: Mark

last: Voorhies

address: 513 Parnassus, S472

department: Microbiology and Immunology

institute: University of California

summary: Coccidioides is an endemic fungus that is increasing in prevalence and can cause life threatening disease in otherwise immunocompetent people. In the environment the spores (arthroconidia) develop into hyphae, yet when they are inhaled by a mammalian host, they develop into a unique form called the spherule. The transition to spherule can be triggered in vitro with elevated temperatures and high CO2 levels, but the signals and host cells that might trigger Coccidioides spherulation in vivo are not known. We used live imaging to investigate how macrophages affect the fate of Coccidioides arthroconidia. Under tissue culture conditions, arthroconidia quickly developed into hyphae. The addition of macrophages promoted spherule development and delayed hyphal formation, indicating that innate immune cells can influence Coccidioides development into the pathogenic form. Exposure of arthroconidia to macrophage supernatants was not sufficient to stimulate spherulation, which was dependent on phagocytosis of arthroconidia by macrophages. Transcriptomics analysis of Coccidioides co-cultured with macrophages revealed a signature concordant with spherules grown in vitro and allowed the identification of a core set of spherule-specific transcripts. In addition, we identified Coccidioides transcripts with significantly higher abundance in the presence of macrophages compared to in vitro spherules, suggesting these factors may be needed to survive and thrive in the presence of innate immune cells. This work lays a foundation for uncovering host-pathogen signaling as well as Coccidioides factors that are critical for pathogenesis.

overall_design: Wild type (WT) or ryp1 mutant Coccidioides was germinated from arthroconidia either alone or in the presence of arthroconidia or in the presence of bone marrow derived macrophages (BMDMs) at two different multiplicities of infection (MOI) with three biological replicates.  RNA was collected at 48h for Coccidioides alone, 24h for hi MOI, and 24h and 48h for lo MOI for a total of 24 samples.

first: Jane

last: Symington

first: Bevin

last: English

first: Apoorva

last: Dabholkar

first: Anita

last: Sil


title: PRMT5 Inhibition Triggers Functional ATM Deficiency and Sensitizes Pancreatic Cancer to CHK1 Blockade

city: Milwaukee

first: Angela

last: Mathison

state: WI

address: 8701 Watertown Plank Rd, HM034, HRC5125

department: Department of Surgery

institute: Medical College of Wisconsin

summary: PRMT5 inhibitors are under clinical investigation for pancreatic ductal adenocarcinoma (PDAC), but strategies to maximize their therapeutic efficacy remain undefined. Here, we report that pharmacologic inhibition of PRMT5 markedly reduces levels of the DNA damage response kinase ATM across multiple PDAC cell lines and different PRMT5 inhibitors, leading to a state of functional ATM haploinsufficiency. We find that this functional deficiency rewires checkpoint signaling, rendering PDAC cells reliant on the ATR-CHK1 pathway for survival. Consequently, we tested combined PRMT5 and CHK1 inhibition and found synergistic suppression of PDAC growth, accompanied by enhanced Caspase 3/7 activation, Annexin V staining, and DNA damage accumulation. Congruent with these results, RNA-seq demonstrated downregulation of cell-cycle and DNA repair genes along with the upregulation of cell death pathways, providing mechanistic insight into the cooperative effect. Moreover, in subcutaneous xenografts, the combination substantially reduced tumor volume, prolonged median survival, and did not affect overall health or body weight. Treated tumors showed reduced ATM and Ki67 with elevated γH2A.X. Together, these findings identify PRMT5 inhibition as a trigger of functional ATM haploinsufficiency that exposes a therapeutically actionable vulnerability to CHK1 inhibition, offering a rational, mechanistically-based combination strategy for this dismal disease.

overall_design: Pancreatic mRNA profiles of L3.6pl cells that were treated with 200 nM EPZ015938 and 1.95-2.34 nM prexasertib, alone or in combination, for 5 days. Three biological replicates per condition.  DMSO controls after 5 and 7 days of treatment were treated as a single control.

first: Madeline

last: Dzikowski

first: Gareth

last: Pollin

first: Shawna

last: Butler

first: Byambasuren

last: Enkhtuul

first: Jennifer

last: Gavina Chavez

first: Michael

last: Zimmermann

first: Raul

last: Urrutia

first: Gwen

last: Lomberk


title: Wnt-materials sustain H3K14-acetylation in human skeletal stem cells for tissue engineering and bone repair

city: Hangzhou

first: Dong

last: Fang

state: Zhejiang

address: 866 Yuhangtang Road

department: Life Sciences Institute

institute: Zhejiang University

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

overall_design: Refer to individual Series


title: CUT&RUN profiling of EZH2, H3K27me3, and H3K4me3 enrichment in FACS-sorted CD70⁻ and CD70⁺ tumor cells from untreated ex vivo ccRCC and HGSOC patient-derived xenografts.

summary: CD70 expression in solid tumors is heterogeneous. We demonstrate that cells appearing CD70-negative retain ultra-low levels of CD70 expression. These cells that appear negative by conventional detection methods. Bulk ATAC-seq analysis of FACS-sorted CD70⁻ and CD70⁺ tumor populations revealed differential chromatin accessibility, indicating epigenetic regulation of CD70 expression in both ccRCC and HGSOC models. CUT&RUN profiling of EZH2, H3K27me3, and H3K4me3 in FACS-sorted CD70⁻ and CD70⁺ tumor cells derived from ccRCC and HGSOC PDXs revealed enrichment of differential repressive and activating histone modifications.

overall_design: NSG mice (n = 3 per model) were implanted with ccRCC patient-derived xenograft (PDX) lines (K5 and K7) under the renal capsule. Fifteen days post-implantation, tumors were isolated and dissociated for FACS sorting into CD70⁻ and CD70⁺ tumor cell populations. CUT&RUN were profiled at isolation (day 0 ex vivo) and during culture at day 2 and day 8 ex vivo. An analogous procedure was performed for an ovarian carcinoma (HGSOC) PDX line 32 days post tumor implantation.


title: Role of the SAF-A ATPase and RGG domains in X inactivation, transcription, splicing, and cell proliferation.

last: Blower

address: 72 E. Concord St. K123C

department: Biochemistry and Cell Biology

institute: Boston University

summary: SAF-A/HNRNPU is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including a central ATPase domain and a C-terminal RGG domain. In this study we tested for the role of the ATP binding, ATP hydrolysis and RNA binding in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the ATPase and RGG domains are required to maintain XIST RNA localization and XIST-dependent histone modifications on the inactive X chromosome, to execute normal protein dynamics, and to maintain normal cell proliferation. We found that the ATPase and RGG domains are not required to maintain gene expression but play a major role in mRNA splicing. We propose a model whereby RNA binding, ATP binding and ATP hydrolysis promote SAF-A interaction with nascent RNA to promote normal mRNA splicing and XIST localization and function.

overall_design: RPEa cells were engineered with a inducible SAF-A degron. These cell lines were then transduced with dox-inducible lentiviruses to express SAF-A WT, SAF-A Walker A, SAF-A Walker B, or SAF-A Delta RGG. SAF-A was depleted and rescue constructs were induced for 24 hours prior to harvesting total RNA.

first: Blower

last: Michael D.


title: Single cell and Spatial RNA-seq in Oral Squamous Cell Carcinoma with Different Stage

city: Luzhou

first: Fengtian

last: Li

address: Zhongshan Road

institute: Southwest Medical University

summary: To investigate the dynamic changes in the tumor microenvironment during the progression of oral squamous cell carcinoma (OSCC), we collected 16 treatment-naïve OSCC samples for single-cell RNA sequencing (scRNA-seq). Among these, 6 cases were additionally subjected to spatial transcriptomic profiling to capture the tissue architecture and spatial organization of the tumor microenvironment.

overall_design: 16 oral squamous cell carcinoma (OSCC) samples from 16 individuals with different stage were used to single cell RNA-seq by Mobinova-100 system. 6 samples from the 16 samples were also used to perform 10X visium spatial RNA-seq.

first: Hu

last: Chen


title: Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes moonlighting transporter functions

city: Kongens Lyngby

first: Oscar

last: Puiggene

address: Kemitorvet 220

department: DTU Biosustain

country: Denmark

institute: Novo Nordisk Foundation Center for Biosustainability

summary: Fluoride (F–) is the anionic form of fluorine, the 13th most abundant element in Earth crust, and it is toxic to many organisms above a threshold concentration. Environmental bacteria can withstand relatively high F– concentrations, but the only mechanism described so far is the CrcB dependent efflux. CrcB mediated export is the primary mechanism of F– tolerance in the model environmental bacterium Pseudomonas putida, yet spontaneous NaF-tolerant mutants arise in the absence of the CrcB transporter, showing that this is not the sole pathway of tolerance. We performed a genome wide search for factors affecting F– tolerance in P. putida. Screening >141,000 transposon mutants, we identified PP_3125, a Cro/cI type transcription regulator, as a determinant of high F– tolerance in a strain lacking the CrcB transporter. Proteomic and transcriptomic analyses revealed genes controlled by PP_3125, among which the benzoate transporter BenE-I was linked to the tolerance phenotype. These results show that transporters can adopt moonlighting functions, an aspect unexplored in bacteria. More broadly, the findings reveal alternative fluoride tolerance mechanisms in P. putida and define targets for engineering stress resilient microbial strains for industrial applications.

overall_design: P. putida KT2440 (WT) or mutants derived (∆crcB = DC; ∆crcB ∆PP_3125 = DD; ∆PP_3125 = D3) grown in the presence (extra "F") or absence of NaF.

first: Lea

last: Ets

first: Heili

last: Ilves

first: Tanel

last: Ilmjärv

first: Òscar

last: Puiggené

first: Pablo

middle: I

last: Nikel

first: Maia

last: Kivisaar


title: Destabilization of the noncanonical PRC1.1 complex via USP7 inhibition induces neuronal differentiation in neuroblastoma [ChIP-Seq]

city: West Lafayette

first: Nathaniel

middle: Wesley

last: Mabe

address: 201 S. University St.

institute: Purdue University

summary: Pediatric cancers are frequently driven by genomic alterations that result in impaired differentiation during tissue development.  To identify protein complex-level dependencies required for differentiation in neuroblastoma, a pediatric cancer of the developing peripheral nervous system, we curated a list of protein complexes using the CORUM database and mined the Dependency Map (DepMap) using single sample gene set enrichment analysis. This analysis identified the non-canonical PRC1.1 complex, which represses transcriptional activity through ubiquitination of histone 2A, lysine 119 (H2AK119Ub), as a selectively enriched dependency in neuroblastoma. Knockout of several PRC1.1 subunits reduced neuroblastoma growth, arrested the cell cycle, and induced a neuronal differentiation program. While no known direct inhibitors of PRC1.1 exist, co-dependency analysis of PRC1.1 subunits against all other genes in DepMap identified that the deubiquitinase USP7 strongly correlated with PRC1.1 dependency. Treatment with XL177A, a small molecule inhibitor of USP7, significantly reduced neuroblastoma growth in both cellular and animal models. Integrated RNA- and ChIP-sequencing showed that both PRC1.1 knockout and USP7 inhibition resulted in highly correlated transcriptional alterations and reduced H2AK119Ub deposition on chromatin, suggesting that USP7 inhibition reduced neuroblastoma growth through a PRC1.1-dependent mechanism. Mechanistically, global proteomics and ubiquitinomics revealed that USP7 inhibition disrupted non-canonical PRC1 complex assembly, resulting in destabilization of PRC1.1 and subsequent proteolysis. Our findings expand our understanding of the chromatin complexes required to maintain a de-differentiated state in neuroblastoma and suggest the therapeutic potential for USP7 inhibitors in the treatment of this disease.

overall_design: ChIP-seq of NBSD cell line treated with vehicle or XL177A

middle: W


title: AID Orchestrates MYC/E2F Target Gene Activation and Cell-of-Origin Transcriptional Programs in Diffuse Large B-cell Lymphoma

city: Amsterdam

first: Linda

last: Gijsbers

address: Meibergdreef 9

department: Pathology

country: Netherlands

institute: Amsterdam UMC - location AMC


title: CATCH-seq analysis of 2PN, P1PN, Parthenogenetic, and Androgenetic zygotes.

city: Kobe

first: Hirohisa

last: Kyogoku

state: Hyogo

address: 1-1, Rokkodai-chou

department: Agricultural Science

country: Japan

institute: Kobe university

summary: Embryogenesis begins with a zygote, a single cell with two pronuclei that separately enclose maternal and paternal chromosomes. The functional significance of the separation of parental chromosomes into distinct pronuclei remains unexplored, despite the fact that one-pronuclear biparental zygotes are used clinically. Here, using a combination of mouse zygote manipulation, quantitative imaging and theoretical approaches, we show a cytoplasm-mediated competition mechanism between separate parental pronuclei that ensures developmental potential. This mechanism limits pronuclear volume and prevents epigenetic mark dysregulation, including loss of maternal trimethylated histones. One-pronuclear biparental zygotes lack this mechanism, resulting in a reduced rate of development to term. This low developmental potential can be rescued by competition-based or drug-based restoration of epigenetic marks. This study provides a spatial mechanism linking fertilization to the establishment of the full developmental potential for the next generation, highlighting caveats in clinical use of one-pronuclear biparental zygotes.

overall_design: 2PN, P1PN, Parthenogenetic, and Androgenetic zygotes were analyzed by CATCH-seq for H3K4me3, H3K9me3 and H3K27me3.

first: Tappei

last: Mishina

first: Tomoya

last: Kitajima

first: Masahiro

last: Matsuwaka

first: Mami

last: Kumon

first: Azusa

last: Inoue


title: Human myeloid ontogeny drives differentiation potential in the brain environment.

city: Austin

first: Anna

last: Warden

state: TX

address: 2500 Speedway

institute: University of Texas at Austin


title: ZNF200 interacts with DDX17 in the RBP-J promoter region to regulate the transcriptional activity of RBP-J.[ATAC-Seq]

city: hefei

first: xiaozheng

last: wei

address: tanghelu

institute: Anhui Medical University

summary: We respectively knocked down ZNF200 and DDX17 in the H520 and A549 cell lines. Since H3K4me3 is an epigenetic modification that marks the active state of gene promoter regions and is typically associated with the activation of gene expression, we subsequently performed ATAC-seq experiments using antibodies against histone H3K4me3.

overall_design: Knockdown of ZNF200 and DDX17 in H520 and A549 cell lines, followed by ATAC-seq using an H3K4me3 antibody compared to the control group.

first: Weixiao

last: Zheng

first: Qian

last: Dai

first: Yanke

last: Zhang


title: ZNF200 interacts with DDX17 to regulate the transcriptional activity of RBP-J. [ChIP-Seq]

summary: In order to explore the molecular mechanism of ZNF200 in the development of NSCLC, we conducted CUT&Tag experiments in the H1299 cell line using IgG and ZNF200 antibodies. Based on previous exploratory experiments, we found an interaction between DDX17 and ZNF200. Therefore, we stably transfected DDX17-HA into H1299 cells and performed CUT&Tag experiments using IgG and ZNF200 antibodies. We then discovered that both ZNF200 and DDX17 act on the promoter region of RBP-J. Consequently, in the DDX17-HA stable transfectants of H1299,H520 and A549 cells, we knocked down ZNF200 and performed CUT&Tag experiments using HA antibodies. At the same time, we knocked down ZNF200 and DDX17 in the H520and A549 cell lines, respectively. Since H3K4me3, as an epigenetic modification, is a hallmark of the active status of gene promoter regions and is usually associated with the activation of gene expression, we subsequently used antibodies against histone H3K4me3 for the CUT&Tag experiments.

overall_design: CUT&Tag of IgG and ZNF200 in H1299 cells, CUT&Tag of IgG and ZNF200 after stable transfection of DDX17-HA in H1299 cells, and after knocking down ZNF200 in H1299,H520 and A549 cells stably transfected with DDX17-HA, as well as CUT&Tag of HA, and also knocking down ZNF200 and DDX17 in H520 and A549 cell lines compared to the control group for H3K4me3 CUT&Tag.


title: Human myeloid ontogeny drives differentiation potential in the brain environment [ATAC-seq]

summary: Microglia, the brain-resident macrophages, play key roles in neurodevelopment and aging, including neurogenesis, synaptic pruning, and myelination. Dysregulated microglia are implicated in various central nervous system (CNS) disorders. Developing safe microglia replacements and eliminating toxic microglia are promising but challenging approaches for treating CNS diseases. Microglia uniquely originate from primitive yolk-sac hematopoiesis, unlike other tissue-resident macrophages that arise from the postnatal bone marrow. Previous studies show that peripheral macrophages fail to adopt microglial characteristics after transplantation. We aim to understand the transcriptional networks underlying the ontological and environmental differences between microglia and peripheral macrophages, which may guide therapeutic strategies. Using human stem cell-derived hematopoietic progenitors (iHPCs) in a humanized mouse model deficient in resident microglia, we demonstrate that iHPCs can replicate the yolk-sac-derived fate of human microglia. We transplanted iHPCs, CD34+ hematopoietic progenitor cells, and umbilical cord blood (UCB)-derived monocytes into murine brains and livers, comparing their differentiation into microglia/macrophages. We found that iHPCs generated more microglia-like cells in the brain compared to CD34+ cells and UCB-derived monocytes, with significant differences in migration and differentiation based on the tissue environment. These findings reveal transcriptional network differences influenced by ontogeny and environment, providing insights for developing microglial-based therapies for CNS disorders.

overall_design:
To investigate  the transcriptional networks underlying ontological and evironmental differences between transplanted microglia and peripheral macrophages, we transplanted iHPCs, CD34+ hematopoietic progenitor cells and umbilical cord blood monocytes into murine brains and livers.
We then performed gene expression profiling of these celltypes across enrgraftment sites to determine the effect of both ontology and transplantation site. We leveraged comparitive gene expression methods (DESeq2) to determine differential expression, gene patterns (DEGreport) and upstream regulators (IPA).

first: Jing

last: Wang

first: Bing

last: Xia

first: Katie

last: Mostoller

first: Benjamin

last: Brandon

first: Michelle

last: Du

first: Nathan

last: Spann

first: Rick

middle: Z

last: Li

first: Karen

last: Mestan

first: Fatir

last: Qureshi

first: Olivia

last: Corradin

first: Christopher

last: Glass

first: Nicole

middle: G

last: Coufal


title: ZNF200 RNA-seq in H1299 cell line

summary: We aimed to detect the mRNA expression levels in stable-transfected H1299 cells after transfection with ZNF200 knockdown or control virus.

overall_design: Extract total RNA from H1299 cell line after transfection with ZNF200 knockdown or control virus, each sample use one well in 6 wells plate H1299 cells.


title: Destabilization of the noncanonical PRC1.1 complex via USP7 inhibition induces neuronal differentiation in neuroblastoma [RNA-Seq]

first: Nathaniel

last: Mabe

overall_design: RNAsequencing at day 10 after infection with sgChr2.2, sgPCGF1.1, or sgPCGF1.2 in KELLY, NB1, and NBSD cell lines.


title: Human myeloid ontogeny drives differentiation potential in the brain environment [ChIP-seq]

first: Anna

last: Warden

overall_design:
To investigate  the transcriptional networks underlying ontological and evironmental differences between transplanted microglia and peripheral macrophages, we transplanted iHPCs, CD34+ hematopoietic progenitor cells and umbilical cord blood monocytes into murine brains and livers.
We then performed ChIP-seq for all celltypes across enrgraftment sites to determine the effect of both ontology and transplantation site.


title: Transcriptional profiling of fasted H3K27ac-deficient Caenorhabditis elegans nematodes.

first: Fasih

middle: Mubtasim

last: Ahsan

address: 185 Cambridge Street, CPZN 6500

department: Center for Genomic Medicine

institute: Massachusetts General Hospital

summary: Exposure to low levels of environmental challenges, known as hormetic stress, such as nutrient deprivation and heat shock, fosters subsequent stress resistance and promotes healthy aging in later life. However, specific mechanisms governing transcriptional reprogramming upon hormetic nutrient stress remain elusive. In this study, we identified histone H3 lysine 27 acetylation (H3K27ac) as a crucial driver of transcriptomic adaptation to hormetic fasting. Beyond its immediate function of enhancing lipid catabolism for alternative energy sources, stress-induced H3K27ac activates lifelong antioxidant defenses, thereby reducing reactive oxygen species (ROS) produced by stress-induced fatty acid oxidation and their accumulation during aging. The increase in H3K27ac, mediated by pioneer factor PHA-4/FOXA and cooperating transcription factor NHR-49/HNF4, is crucial for lifespan extension under hermetic nutrient stress in Caenorhabditis elegans. Our findings establish H3K27ac as a key transcriptional switch that bridges nutrient status with transcriptomic reprogramming, underpinning the pro-longevity effects of hormetic fasting through orchestrating lipid catabolism and antioxidative defenses.

overall_design: Wild-type (N2) animals and H3K27ac-deficient animals, bearing jmjd-3.1(gk384) null mutation and cbp-1 RNAi knockdown, were synchronized to adult day 1 (D1), transferred to ad libitum E. coli or fasted NGM RNAi plates for 24 hours, and subsequently collected for total RNA extraction in biologically independent quadruplicates (16 total samples).

middle: M

first: Yifei

last: Zhou

first: Sainan

first: Alexander

middle: A

last: Soukas


title: mitfa-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

city: Iowa City

first: Colin

last: Kenny

state: Iowa

address: CBRB, 5269, 285 Newton Road

department: Surgery

institute: University of Iowa

summary: Melanocytes reside in diverse microenvironments that influence their susceptibility to oncogenic transformation, however, studying rare melanoma subsets has been hindered by the lack of suitable animal models. We developed a primary, immune-competent zebrafish model to study uveal melanoma (UM), utilizing choroidal-targeted injection and electroporation of plasmids containing human GNAQQ209L and CRISPR/Cas9 cassettes for tumor suppressor gene deletion. Single-cell transcriptional profiling of genetically identical eye- and skin-derived tumors revealed distinct oncogenic pathways, highlighting the importance of studying melanoma subtypes in their correct anatomical context. Additionally, we identified a population of tfec- and pax3a-expressing melanocyte progenitor cells in mitfa-deficient embryos and adult zebrafish eyes, which were highly susceptible to GNAQ-driven transformation. While previous studies have linked mitfa deficiency to accelerated UM onset, our findings suggest that an expanded progenitor population in mitfa-deficient animals drives this susceptibility. Our study establishes a critical role for MITF-independent melanocyte progenitors in UM pathogenesis.

overall_design: Eye and skin tumors were harvested with surrounding tissue from 4-6 month old AB wildtype and casper (mitfa -/-, mpv17 -/-) zebrafish. Uninjected eyes and uninjected regions of skin were also included as controls. Tumors were derived by injecting mitfa:GNAQQ209L-mitfa:GFP, mitfa:Cas9, u6:sgRNA-tp53, u6:sgRNA-ptena, u6:sgRNA-ptenb, PCS2FA Tol2 into either eye or skin of adult zebrafish and electroporated to induce uptake.

first: Julius

last: Yevdash


title: Sensitive CAR T cells redefine targetable CD70 expression in solid tumors [RNA-seq RCC]

first: Sophie

last: Hanina

summary: Overcoming tumor antigen heterogeneity in solid tumors is a major challenge for cancer immunotherapies including chimeric antigen receptor (CAR) T cells. Unlike CD19 for B-cell malignancies, no target with pan-cellular expression in solid tumors and absence in normal vital cells has been identified. CD70 is a potential candidate, confined to immune cell subsets and aberrantly expressed in many solid tumors, albeit heterogeneously. We find that CD70 expression in heterogeneous tumors is not binary but ranges from high to very low, appearing negative. We show that CD70-heterogeneous tumors are efficiently eliminated by highly sensitive CD70 receptors where prototypic CAR T cells fail. We further identify an epigenetic signature that predicts targetable expression. These findings provide a potential strategy to treat a broad range of solid tumors.

overall_design: Mice bearing ccRCC tumor at both the kidney and lung site within the same mouse were used for bulk ATAC and RNA sequencing. For the ccRCC kidney model, 5×10E5 FFLuc-Tdtomato K5 or K7 ccRCC cells were injected into the renal subcapsule of male NOD.Cg-Prkdc scid IL2rg tm1Wjl/SzJ (NSG, Jackson Laboratory) mice, aged 6-8 weeks. For the ccRCC lung model, 7.5×105 FFLuc-Tdtomato K5 or K7 ccRCC cells were administered via tail vein injection into the same NSG mouse bearing tumor at the kidney site. K5 PDX tumor cells or K7 PDX tumor cells at kidney site and lung site were digested into single cell suspensions using the Tumor Dissociation Kit, human (Miltenyi Biotec) according to the manufacturer’s instructions and isolated by fluorescence-activated cell sorting 15 days post orthotopic kidney tumor implantation. Live cells were sorted for TdtomatoPOS. CD70LO and CD70HI tumor cells from kidney site or lung site were isolated by FACs, denoted as day 0 of isolation, d0. Isolated CD70LO and CD70HI tumor cells from kidney site, and CD70HI tumor cells from lung site were then cultured for 8 days. CD70 transcript expression and chromatin accessibility was assessed for each biological replicate in CD70LO and CD70HI tumor cells at d0 of isolation, d2 and d8 of culture.


title: Systematic evaluation of the impact of promoter proximal short tandem repeats on gene expression

city: La Jolla

first: Alon

last: Goren

address: 9500 Gilman Drive

institute: University of California San Diego

summary: This study uses a massively parallel reporter assay (MPRA) to investigate how short tandem repeats (STRs) within promoter regions influence gene regulation. A custom DNA library encoding promoter variants with different STR configurations was linked to a GFP reporter and unique barcodes for expression tracking. We transfected this MPRA to HEK293T cells in triplicate which showed highly reproducible reporter expression across replicates (R=0.98). We identified 1,366 out of 19,818 loci with significant associations between repeat copy number and expression, with a strong bias toward positive effect sizes (p<1.04e-110, binomial distribution testing). We designed a second array to perform more detailed study of top regulatory STRs by modifying the repeat unit, orientation, and length, for a total of 200-300 perturbations per locus. This second array identified repeat unit sequence as the key driver of differences in expression associations across loci, whereas strand and sequence context had far weaker effects. Overall, in this work we identified optimal conditions for studying STRs using MPRAs, allowing us to perform the first in depth interrogation of the impact of STR characteristics on transcriptional regulation and study the critical roles STRs play in genome function.

overall_design: This MPRA study was designed to assess the regulatory effects of short tandem repeats (STRs) located within promoter regions on gene expression. A synthetic oligonucleotide library containing promoter sequences with variable STR lengths and motifs was cloned upstream of a GFP reporter gene, with unique barcodes in the 3′ UTR to track expression. The plasmid library was transfected into human cells, and barcode abundance was quantified from both RNA and input DNA to determine the transcriptional activity of each STR-containing promoter variant.

first: Xuan

last: Zhang

first: Lingzhi

first: Ellice

last: Wang

first: Susan

last: Benton

first: Eduardo

last: Modolo


title: The Colon Contains an Immature/Intermediate iNKT Cell Population That is Expandable by CD1d Expressing Goblet Cells and Can be Protective in Colitis

city: St. Louis

first: Rodney

middle: D

last: Newberry

address: 660 S. Euclid Ave

department: Medicine

institute: Washington University School of Medicine

summary: scRNA-seq from sorted murine and human colonic iNKT populations to determine subsets compared to published human thymic data. For murine samples mice were treated with EGFRi or vehicle for 4 days prior to sort.

overall_design: iNKT were isolated from colonic lamina propria and stained for CD45, TCRb, CD3, and CD1d-loaded tetramer

first: Vini

last: John

first: Bibiana

last: Barrios

first: Jackie

first: Sreeram

last: Udayan

first: Alexandria

middle: N

last: Floyd

first: Elisabeth

last: Joyce

first: Ellen

last: Schill

first: Sushma

last: Sundas

first: Keely

last: McDonald

first: Richard

middle: S

last: Blumberg

first: Brigida

last: Rusconi


title: Sensitive CAR T cells redefine targetable CD70 expression in solid tumors [CITE-seq]

first: Sophie

last: Hanina

overall_design: 5×10E5 FFLuc-Tdtomato K5 or K7 ccRCC cells were injected into the renal subcapsule of male NOD.Cg-Prkdc scid IL2rg tm1Wjl/SzJ (NSG, Jackson Laboratory) mice, aged 6-8 weeks. K5 PDX tumor cells or K7 PDX tumor cells at kidney site were digested into single cell suspensions using the Tumor Dissociation Kit, human (Miltenyi Biotec) according to the manufacturer’s instructions and isolated by fluorescence-activated cell sorting 15 days post tumor administration.


title: Sensitive CAR T cells redefine targetable CD70 expression in solid tumors [RNA-seq OV]

overall_design: For the orthotopic ovarian model, 5×10E5 FFLuc-Tdtomato OV4 were injected into the ovary of female NSG mice aged 6-8 weeks. 32 days later, tumor cells from the ovary were digested into single cell suspensions using the Tumor Dissociation Kit, human (Miltenyi Biotec) and CD70LO and CD70HI tumor cells were isolated by FACs for bulk RNA and ATAC sequencing.


title: Sensitive CAR T cells redefine targetable CD70 expression in solid tumors [Multiomics]

overall_design: Kidney tumors collected from patients undergoing surgery, were digested into single cell suspensions using the Tumor Dissociation Kit, human (Miltenyi Biotec) according to the manufacturer’s instructions. Red cell blood lysis was performed using ACK buffer (Lonza). Prior to FACS sorting, cell suspensions were blocked with Human FcR blocking reagent for 10 min at room temperature (Miltenyi Biotec) and then stained with human CD45 antibody (HI30, Biolegend) for 30 min on ice. DAPI and calcein (Invitrogen) were used to sort live cells and CD45NEG cells were excluded except for patient 3


title: CHD1 is a synthetic lethal vulnerability in MYC-driven breast cancer [RNA-Seq]

city: Toronto

first: Brandon

middle: Alexander

last: Cho

state: ON

address: 25 Orde Street, Room 6-1001 MRS

country: Canada

institute: Lunenfeld-Tanenbaum Research Institute

summary: c-MYC (MYC) is a transcription factor that regulates gene expression during growth stages under developmental and physiological conditions, and its dysregulation often results in cancer. Understanding how over-activation of MYC leads to transformation and tumour growth may provide new therapeutic avenues in a variety of cancers. The function of Chromodomain-Helicase DNA-binding 1 (CHD1), a chromatin remodeler that plays essential roles in stem/progenitor cells during development, has not been explored in MYC-driven cancer. By intersecting two independent genome-wide screens, we identified loss of CHD1 as a potential synthetic lethal relationship with MYC activation in breast cancer. Using a xenograft model of MYC-driven breast cancer, we found that knockdown of CHD1 suppresses tumour growth. This finding is further supported in vitro, where knockdown of CHD1 in breast epithelial cells results in significant decreases in cell proliferation and transformation, and in increased cell death. Importantly, this CHD1 dependency is observed only when MYC is over-expressed. Knockdown of CHD1 leads to a prominent reduction in chromatin accessibility, in particular at genomic regions associated with tumour progression and invasion. Transcriptional analyses and follow-up experiments reveal that knockdown of CHD1 induces defects in nucleolar architecture and activation of p53. Taken together, these findings shed light on the chromatin-level regulation of MYC-driven breast cancer and uncover CHD1 as a novel therapeutic target.

overall_design: RNA-seq profiling of non-transformed (MCF10A PE) and MYC-transformed (MCF10A PM) breast cancer cells investigating the effect of CHD1 knockdown (shCHD1) relative to control conditions (shGFP)

first: Giacomo

last: Furlan

first: Peter

last: Lin

first: Mélissa

last: Shen

first: Kirti

last: Mittal

first: Andrew

last: Mazzanti

first: Linda

last: Penn

first: Miguel

last: Ramalho-Santos


title: UHRF1 drives subtype-independent aggressiveness and immune evasion in small cell lung cancer through GATA2-ST6GALNAC5 and PRC2 interactions

city: Irvine

first: Claudia

last: Benavente

address: 839 Health Sciences Road, Sprague Hall B200

institute: University of California, Irvine

summary: Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma characterized by early metastasis, immune evasion, and limited therapeutic progress. While its genomic landscape is well defined, the epigenetic programs sustaining its lineage fidelity, metastatic potential, and immune evasion remain incompletely understood. We used inducible CRISPR-Cas9 knockout models, transcriptomic and epigenetic profiling, immune-competent allografts, and genetically engineered mouse models (GEMMs) to investigate the role of the chromatin regulator UHRF1 in SCLC progression. We identify the chromatin remodeler UHRF1 as a subtype-independent oncogenic driver in SCLC, with high expression correlating with poor survival. UHRF1 loss impaired proliferation, invasion, and metastasis across ASCL1-, NEUROD1-, and POU2F3-driven subtypes. Mechanistically, UHRF1 maintained neuroendocrine lineage identity and suppressed immune gene expression via DNA methylation and interaction with PRC2. UHRF1-deficient tumors displayed increased infiltration of CD8⁺ T cells, monocytes, neutrophils, and dendritic cells, along with a higher CD8⁺/Treg ratio. Secretome profiling revealed upregulation of chemokines, linking UHRF1 loss to active recruitment of immune effector cells. In parallel, UHRF1 regulated the pro-metastatic sialyltransferase ST6GALNAC5 via the transcription factor GATA2. UHRF1 loss downregulated GATA2 and ST6GALNAC5, impairing tumor dissemination. Lastly, UHRF1 depletion increased expression of cancer/testis antigens such as MAGE-A4, without altering DLL3 levels, suggesting compatibility with both antigen-specific and DLL3-targeted immunotherapies. In conclusion, UHRF1 is a master regulator of tumor progression in SCLC, promoting lineage stability, immune evasion, and metastatic spread. Its inhibition reprograms tumors toward an inflamed, less metastatic, and more immunogenic state. These findings establish UHRF1 as a compelling therapeutic target for overcoming the epigenetic and immune resistance barriers in small cell lung cancer.

overall_design: Gene expression profiling analysis of RNA-seq data from 1) NCI-H526 VC and UHRF1 KO   5-week orthotopic xenograft tumors in NSG mice, 2) NCI-H446 VC and UHRF1 KO 5-week orthotopic xenograft tumors in NSG mice, 3) mouse SCLC tumors from genetically engineered mouse models Tp53/Rb1 DKO (TR DKO) and Tp53/Rb1/Uhrf1 TKO (TRU TKO).

first: Yijun

last: Gu

first: Yasamin

last: Fazeli


title: Effects of Histidine restriction in DND-41 cells in vitro

city: New Brunswick

first: Daniel

last: Herranz

state: New Jersey

address: 195 Little Albany St

institute: Rutgers Cancer Institute of New Jersey

summary: Here we report the gene expression profile of T-ALL cell line - DND-41, cultured in different levels of Histidine restricted media and collected at two time points -  Day 6  and Day 9

overall_design: RNAseq analysis of DND-41 cells cultured in different levels of Histidine restricted media and collected at two time points - Day 3 and Day 6

first: Thai

last: Christopher

first: Mandleywala

last: Komal

first: Herranz

last: Daniel


title: CHD1 is a synthetic lethal vulnerability in MYC-driven breast cancer [ATAC-seq]

overall_design: ATAC-seq of MYC-transformed (MCF10A PM) breast cancer cells to investigate the effect of CHD1 knockdown (shCHD1) relative to control conditions (shGFP)


title: Discovery of a paralog-selective p300 protein degrader with potent anti-cancer activity in hematological malignancies

city: South San Francisco

first: Anlu

last: Chen

address: 1000 Gateway Blvd

department: Quantitative Medicine & Genomics

institute: AbbVie Inc

summary: The E1A-associated protein p300 (EP300) is a key regulator of oncogenic transcription factors, making it a promising target for cancer therapy. However, high sequence similarity to its paralog, CREB-binding protein (CBP), has hindered the development of selective inhibitors, leading to toxic side effects. Here, we describe a highly potent and selective p300 degrader. Unlike dual p300/CBP degraders, this compound forms a stronger, more stable ternary complex with p300, induces higher level of p300 ubiquitination and recruitment to the proteosome, and ubiquitinates a unique lysine site on p300. Hematological cancers, including multiple myeloma, non-Hodgkin’s lymphoma and acute myeloid leukemia, showed the greatest sensitivity to the p300-selective degrader, which induced a lethal phenotype in cells and demonstrated antitumor efficacy in xenograft models. These findings highlight the therapeutic potential of selective p300 degradation as a novel strategy for the treatment of hematological malignancies and the inhibition of cancer progression.

overall_design: RNAseq profiling in NCI-H929 cells 1) treated with p300 degrader 27 at 3 nM for 6 hours when compared with DMSO as vehicle and 2) p300 knockout when compared with NTC

first: Asem

last: Marwa


title: Hematological Consequences of Environmental Change During Dewilding of Rhesus Macaques

city: Bethesda

first: P'ng

last: Loke

state: MD

address: 9000 Rockville Pike

department: NIAID

institute: NIH

summary: The environment shapes immune system development and the ability to regulate inflammatory responses, which may affect the prevalence of immune mediated inflammatory diseases. However, the hematological consequences of a major environmental change, such as those experienced during migration, remain poorly understood. Here, we comprehensively assessed the immunological consequences in rhesus macaques as they transitioned from a natural outdoor provisioned environment to a quarantined laboratory facility in a process we term ‘dewilding.’ Dewilding led to a sharp drop in neutrophils and increased lymphocytes in the peripheral blood, a skewing toward a proinflammatory TH1 response, and increased T cell activation. In the bone marrow, we observed increased granulopoiesis, reduced lymphocytes, and reduced hematopoietic stem cells and progenitors, with their shift toward less committed progenitor states. Single-cell nuclear RNA sequencing revealed increases in erythrocytes and their progenitors in the bone marrow, along with upregulation of genes involved in hemoglobin control and erythropoiesis. Notably, we also found that vaccination response was dampened when vaccination occurred earlier in the dewilding process. Together, our findings demonstrate how dewilding alters immune homeostasis, with implications for understanding immune adaptation in migrants from rural to urban environments and for optimizing immunization strategies during environmental change.

overall_design: Following the environmental transition of rhesus macaques from provisioned outdoor environment to indoor research facilities in a process called dewilding, nuclei were isolated from bone marrow samples and analyzed using snRNA-seq.

first: Smith

last: Annabelle

first: Kiwanuka

last: Kasalina

first: Pessenda

last: Gabriela

first: Rahmberg

last: Andrew

first: Flynn

last: Jacob

first: Markowitz

last: Tovah

first: Herbert

last: Richard

first: Brenchley

last: Jason

first: Loke

last: P'ng


title: Effects of 7 EAA restriction (Histidine, Methionine, Valine, Leucine, Isoleucine, Phenylalanine and Tryptophan) in Jurkat cells in vitro

summary: Here we report the gene expression profile of T-ALL cell line - Jurkat, cultured in control vs 25% restricted media of 7 Essential amino acids (Histidine, Methionine, Valine, Leucine, Isoleucine, Phenylalanine and Tryptophan) and collected at Day 3

overall_design: RNAseq analysis of Jurkat cells cultured in 7 different essential amino restricted media and collected at Day 3

last: Chris


title: Splicing fidelity infuences the fate of mRNA molecules

city: Worcester

first: Athma

last: pai

address: 55 Lake Avenue North

department: RNA Therapeutics Institute

institute: UMass Chan Medical School

summary: RNA splicing has historically been thought to be highly efficient and accurate, with little opportunity for deviation from regulated alternative splicing decisions. This dogma has been challenged by recent observations that suggest that biological noise may contribute substantially to transcriptome diversity. However, quantitative understanding of stochastic variations in splicing is challenging because these transcripts are likely subject to rapid degradation. Here, we use ultra-deep sequencing across RNA compartments to track splicing intermediates in human cells and see abundant cryptic splicing associated with genomic features that promote splicing noise. We observe pervasive usage of low-fidelity splice sites, likely due to stochasticity in recruitment or binding of the spliceosome. These sites are most likely degraded in the nucleus rather than targeted by translation-dependent degradation processes, suggesting widespread surveillance and quality control of non-productive RNA transcripts. Our findings provide unprecedented insights into the propensity for error in RNA processing mechanisms to buffer mechanistic noise.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
RNAseq from nascent, nuclear and polyA enriched fractions from K562, KNS60, AM38, SY5Y and HepG2 cell lines.
K562 cells were treated with cycloheximide, NMDi14, uridine, 4SU and DMSO, followed by polyA enriched RNAseq.
K562 cells were treated with cycloheximide and NMDi14 followed by polyA enriched RNAseq.
K562 cells were treated with IdU, with or without 4sU labelling, followed by nuclear isolation, without or without 4sU pulldown and RNAseq

first: Eraj

last: Khokhar

first: Kaitlyn

last: Brokaw

first: Zachary

middle: J

last: Kartje

first: Valeria

last: Sanabria

first: Nida

last: Javeed

first: Ayush

last: Kumar

first: Jonathan

middle: K

last: Watts

last: Pai


title: Variation of Human Transfer RNA Demand and Supply [ncRNA-seq]

city: Frankfurt am Main

first: Marius

last: Külp

address: Theodor-Stern-Kai 7

department: Department of Medicine II, Hematology/Oncology

institute: Goethe University Hospital Frankfurt

type: Non-coding RNA profiling by high throughput sequencing

summary:
Codons function as translation units in open-reading-frames (ORF) of genes to encode for proteins. Transfer RNAs (tRNAs) mediate the connection of every codon to its cognate amino acid. Despite the cooperation between messenger and transfer RNA during translation, approaches to integrate codon usage and tRNA quantities remain to be established.
Using matched mRNA- and tRNA-sequencing of peripheral blood cells, we apply a precision-biology approach quantitatively integrating transcriptomic codon- and corresponding tRNA-abundance. Thereby, we classify codons as highly or lowly supplied and compare optimality of synonymous codons. Additionally, we describe substantial differences regarding the conservation of a codon’s tRNA-supply among healthy donors.
A meta-ORF-analysis demonstrates depletion of lowly supplied codons at translation start sites. Discrepancy between codon- and tRNA-abundance, and codon-preference depending on the distance to the translation start site, seem to be non-random and could affect translational speed and thus provide a novel level of regulation of protein abundance.

overall_design: tRNA-seq profiling of peripheral blood cells derived from 10 healthy human individuals

first: Halvard

last: Bonig

first: Michael

last: Rieger


title: Variation of Human Transfer RNA Demand and Supply [MACE-seq]

overall_design: mRNA-seq profiling (MACE-seq) of peripheral blood cells derived from 10 healthy human individuals


title: Enhanced mitophagy promotes lung repair and regeneration by restoring epithelial metabolic fitness

city: Shanghai

first: pei

last: wu

address: 393 Middle Huaxia Road

institute: ShanghaiTech University

summary: Alveolar Type II cells (AT2s) are the stem cells in the alveoli, responsible for both alveolar homeostasis and regeneration. Mitochondrial dysfunction has been reported in the AT2 cells from both chronic and acute injury induced alveolar diseases, including idiopathic pulmonary fibrosis (IPF) and viral pneumonia. Here, we demonstrate that genetic depletion of Ubiquitin Specific Peptidase 30 (USP30), a negative regulator of mitophagy, boosts mitophagy and restores mitochondrial function, leading to protection from injury induced AT2 apoptosis and increased AT2 stem cell activity. Both global Usp30 knockout (KO) and AT2-specific Usp30 KO protects the mice from bleomycin induced lung fibrosis and influenza puneumonia. Moreover, pharmaceutical inhibition of USP30 using a small molecule inhibitor effectively alleviates lung fibrosis and influenza pneumonia. Our study underscores a therapeutic potiential of USP30 inhibition for the treatment of injury induced alveolar diseases, offering a promising strategy for treating injury induced diseases

overall_design: On day 18, CD45+ and CD45- cells were flow-sorted from bleomycin-treated wild-type (WT) and USP30-/- mice for single-cell RNA sequencing (scRNA-seq) analysis.

first: Ying

last: Xi

first: Pei

last: Wu

first: Jiawei

last: Chen


title: Human BRD2 impedes pluripotency reprogramming mainly by suppressing lipogenesis

city: Fort Worth

first: Kejin

last: Hu

address: 3400 Camp Bowie Boulevard

department: Microbiology, Immunology and Genetics

institute: UNT Health Science Center

summary: Induction of pluripotent stem cells (iPSCs) from human somatic cells encounters extensive and significant molecular barriers. Our understanding of the reprogramming barriers remains limited. Lipids are the fundamental cellular building blocks, energy sources, nutrition, signaling molecules, and components for protein lipidation. The roles of lipids and lipogenesis in reprogramming are unknown.  Here, we proved that BRD2 is a major molecular barrier to human iPSC (HiPSC) reprogramming. Human pluripotent stem cells (hPSCs) displayed a much weaker transcriptional program in matrisome compared to that of the reprogramming starting cells. BRD2 legitimately reprogrammed a set of matrisome and the related genes from the higher somatic to the weaker pluripotent states. BRD2 also suppressed expression of genes in lipogenesis and lipid metabolism in the reprogramming cells. The two rate-limiting enzymes of lipogeneses, SCD and HMGCR, enhanced iPSC reprogramming while chemical inhibition of HMGCR abrogates reprogramming. Critically, the supplementation of lipids rescues BRD2 suppression of reprogramming. ET tail of BRD2 suppresses reprogramming and the transcriptional programs of lipogenesis and lipid metabolism, but positively regulates gene expression of matrisome and the related genes. These discoveries advance our molecular understanding of HiPSC reprogramming, matrisome, and lipogenesis, and improve HiPSC technology.

overall_design:
We conducted RNA-seq on human fibroblast cells under reprogramming in different conditions. This research focused on different BRD2 conditions including overexpression, shRNA knockdown, CRISPR KO, ET tail deletion, and luciferase shRNA controls. The starting cells for reprogramming is humana BJ fibroblasts. RNA was extracted at early stage of reprogramming (48 hours and 72 hours post transduction).

ChIP-seq of BJ fibroblasts transduced with lentiviral vector preparations of shRNA/sgRNA targeting BRD2 gene. Scrambled  vector was used as control for shRNA knockdown. For gene disruption, BJ fibroblasts with a dox-inducible Cas9 construct was used.

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The table below lists GEO accessions reused/reanalyzed for this study.
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last: HU

first: Ricardo

last: Cevallos


title: Effects of Histidine restriction in Jurkat cells in vitro

first: Daniel

last: Herranz

summary: Here we report the gene expression profile of T-ALL cell line - Jurkat, cultured in different levels of Histidine restricted media and collected at two time points - Day 3 and Day 6

overall_design: RNAseq analysis of Jurkat cells cultured in different levels of Histidine restricted media and collected at two time points - Day 3 and Day 6

first: Thai

last: Christopher

first: Mandleywala

last: Komal

first: Herranz

last: Daniel


title: Mitochondrial RNA degradation is increased in AML and controls differentiation, stem cell function and sensitivity to immune-mediated killing

first: Veronique

last: Voisin

address: 101 College St, room 8-353

institute: University Health Network

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

overall_design: Refer to individual Series


title: Mitochondrial RNA degradation is increased in AML and controls differentiation, stem cell function and sensitivity to immune-mediated killing [RNA-seq]

summary: Eukaryotic cells have two separate genomes that are replicated independently; nuclear DNA organized in chromosomes and circular DNA within mitochondria. Mitochondrial DNA is transcribed bi-directionally to generate mitochondrial RNA (mtRNA). As a by-product of the bi-directional transcription, dsRNA is also generated. By gene expression analysis and functional studies, we demonstrated that mtRNA transcription and degradation were increased in AML (Acute Myeloid Leukemia) cells and stem cells resulting in higher rates of mtRNA turnover. We discovered that the mitochondrial degradosome, SUV3 and PNPase, was upregulated in AML cells and stem cells and functionally important for the degradation of mtRNA and mitochondrial dsRNA in AML. Depleting SUV3 or PNPase impaired mtRNA degradation and promoted the accumulation of dsRNA. dsRNA that accumulated after depleting SUV3 or PNPase, stimulated IFN-I signaling that induced AML differentiation, decreased stem cell function and increased sensitivity of AML cells to immune-mediating killing. Thus, this work highlights new mitochondrial biology in AML and a novel mechanism by which the turnover of mtRNA regulates AML differentiation and stem cell function

overall_design: Double-strand RNA (dsRNA) was extracted from AML cell line OCI AML-2 using immunoproecipation. Samples were extracted from 3 biological replicates after SUV3 knock-down and from 3 biological replicated after PNPT1 knock-down

first: Geethu

middle: E

last: Thomas

first: Aaron

last: Schimmer


title: Human myeloid ontogeny drives differentiation potential in the brain environment [RNA-seq]

first: Anna

last: Warden

summary: Microglia, the brain-resident macrophages, play key roles in neurodevelopment and aging, including neurogenesis, synaptic pruning, and myelination. Dysregulated microglia are implicated in various central nervous system (CNS) disorders. Developing safe microglia replacements and eliminating toxic microglia are promising but challenging approaches for treating CNS diseases. Microglia uniquely originate from primitive yolk-sac hematopoiesis, unlike other tissue-resident macrophages that arise from the postnatal bone marrow. Previous studies show that peripheral macrophages fail to adopt microglial characteristics after transplantation. We aim to understand the transcriptional networks underlying the ontological and environmental differences between microglia and peripheral macrophages, which may guide therapeutic strategies. Using human stem cell-derived hematopoietic progenitors (iHPCs) in a humanized mouse model deficient in resident microglia, we demonstrate that iHPCs can replicate the yolk-sac-derived fate of human microglia. We transplanted iHPCs, CD34+ hematopoietic progenitor cells, and umbilical cord blood (UCB)-derived monocytes into murine brains and livers, comparing their differentiation into microglia/macrophages. We found that iHPCs generated more microglia-like cells in the brain compared to CD34+ cells and UCB-derived monocytes, with significant differences in migration and differentiation based on the tissue environment. These findings reveal transcriptional network differences influenced by ontogeny and environment, providing insights for developing microglial-based therapies for CNS disorders.

overall_design:
To investigate  the transcriptional networks underlying ontological and evironmental differences between transplanted microglia and peripheral macrophages, we transplanted iHPCs, CD34+ hematopoietic progenitor cells and umbilical cord blood monocytes into murine brains and livers.
We then performed gene expression profiling of these celltypes across enrgraftment sites to determine the effect of both ontology and transplantation site. We leveraged comparitive gene expression methods (DESeq2) to determine differential expression, gene patterns (DEGreport) and upstream regulators (IPA).

first: Jing

last: Wang

first: Bing

last: Xia

first: Katie

last: Mostoller

first: Benjamin

last: Brandon

first: Michelle

last: Du

first: Nathan

last: Spann

first: Rick

last: Li

first: Karen

last: Mestan

first: Fatir

last: Qureshi

first: Olivia

last: Corradin

first: Christopher

last: Glass

first: Nicole

last: Coufal


title: TopBP1 controls conventional dendritic cell development and Flt3L-driven cDC1 differentiation via PU.1 and IRF8

city: Suwon-si

first: Minsuk

last: Cha

address: 2066, Seobu-ro, Jangan-gu

country: South Korea

institute: Sungkyunkwan University

summary: DNA topoisomerase II-binding protein 1 (TopBP1) plays a vital role in V(D)J recombination during B and T cell development. However, its role in the development of conventional dendritic cells (cDCs) remains unexplored. Mice with DC-specific depletion of TopBP1 (TopBP1cKO) showed accelerated tumor progression due to impaired anti-tumor immunity, characterized by cDC deficiency and pre-DC accumulation. Notably, Flt3 ligand (Flt3L)-mediated tumor immunotherapy was ineffective in TopBP1cKO tumor-bearing mice. Our study demonstrates that TopBP1 is required not only for the steady-state differentiation of total cDCs, including both cDC1 and cDC2, but also for the terminal differentiation of XCR1⁻CD24⁺ emergency progenitors (EPs; CD11c⁺cKit⁺) into XCR1⁺CD24⁺ cDC1s in response to Flt3L. Furthermore, we revealed that TopBP1 directly interacts with PU.1 and IRF8, key transcription factors (TFs) required for cDC development, triggering the expression of their downstream target genes. These findings identify TopBP1 as a crucial factor for cDC development and Flt3L-driven EP differentiation into cDC1s, revealing that the function of key TFs for cDC development is mediated via interaction with TopBP1. Our work underscores the importance of TopBP1 in promoting cDC development and the therapeutic efficacy of Flt3L-mediated tumor immunotherapy.

overall_design: RNA-seq profiling of CD24⁺ EP(Emergency progenitor; CD11c+CD117+)s from the spleen of TopBP1^fl/fl (littermate) and TopBP1 fl/fl, CD11c-cre (cKO) mice on day 9 after Flt3L-Ig injection

first: Min-Suk

first: Yong-Soo

last: Bae


title: Time-Course RNA Sequencing of Alveolar Type 2 Cells Following Influenza Infection

first: pei

last: wu

summary: The influenza-infected mouse model serves as an acute lung injury model, leading to extensive collapse of the alveolar regions. In this project, we aimed to investigate the effects of influenza infection on the transcriptome of type II alveolar epithelial cells. We isolated type II alveolar epithelial cells at various time points post-infection for transcriptome sequencing.

overall_design: Alveolar type 2 cells (CD45- EPCAM+ CD31- Integrin β4-) were isolated from the lungs of influenza-infected mice at 0, 2, 6, and 11 days post-infection (dpi) using fluorescence-activated cell sorting (FACS). Subsequently, RNA was extracted from these cells for RNA sequencing.

first: Ying

last: Xi

first: Pei

last: Wu

first: Shenxi

last: Shen


title: An InDel Genomic Variant within a Bifunctional Super-Enhancer for LINC00636 and CD47 Regulation in Breast Cancer  [ATAC-seq]

city: San Francisco

first: Carolina

last: Di Benedetto

address: 2340 Sutter Street, S371

institute: University of California, San Francisco

summary: Highly accessible genomic super-enhancers often upregulate tumor-promoting genes in cancer, yet the role of genomic variation within them remains unclear. We identified a bifunctional super-enhancer that regulates the expression of cancer-promoting genes LINC00636 and CD47 in breast cancer. Our study revealed that a common germline insertion variant within the super-enhancer is associated with better progression-free survival in breast cancer patients and reduced chromatin accessibility at the super-enhancer locus. By deleting the insertion allele in breast cancer cells, we observed increased chromatin accessibility, leading to upregulation of LINC00636 and CD47, delayed cell death, and reduced infiltration of CD80+ pro-inflammatory macrophages—events that promote tumor growth. The absence of the insertion also activated a protumoral transcriptional program through LINC00636 overexpression. Our findings highlight an insertion/deletion variant that fine-tunes the regulatory function of a bifunctional super-enhancer and suggest a protective role for the insertion in breast cancer.

overall_design: ATAC-seq was performed in parental MCF7 cells or in the clones M11 (MCF7Δ#1) and M46 (MCF7Δ#2), in which the germline insertion variant rs58296163 was deleted by CRISPR. This insertion variant is located within a core element of a bifuntional super-enhancer for the regulation of LINC00636 and CD47 gene expression. Samples were processed in duplicates.

first: Paola

last: Betancur


title: Aberrant NOTUM+ Program Induced in LGR5+ Crypt Base Columnar Cells Maintains an Immunosuppressive Niche in Colorectal Cancer

city: Calgary

first: Arshad

last: Ayyaz

state: Alberta

address: BI238D, 507 Campus Dr nw

institute: University of Calgary

summary: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with treatment failure largely driven by cancer stem-like cells that resist conventional chemoradiation and subsequently initiate tumor recurrence. While immune checkpoint blockade is effective in microsatellite instability-high (MSI-H) CRCs, the majority of CRCs are microsatellite stable (MSS) and exhibit immune exclusion, rendering them refractory to immunotherapy. Here, we identify a previously uncharacterized cancer cell subtype, which we term cancerous Crypt Base Columnar (canCBC) cells. These cells transcriptionally resemble normal LGR5+ CBC cells but activate an aberrant WNT/β-catenin signalling inhibitory program, marked by NOTUM expression. We show that canCBC cells are specifically enriched in MSS tumors, where their presence correlates with reduced CD8⁺ T cell infiltration, broader immune exclusion, and a propensity for regional lymphatic dissemination. Consistently, targeted ablation of canCBCs enhances the tumor-clearing potential of CD8⁺ T cells. This study identifies a novel therapeutic target for overcoming immune exclusion and improving immunotherapy responses in MSS CRCs.

overall_design: Local colorectal cancer biopsies were extracted and converted into patient derived colorectal cancer speroids organoids. Organoids were then singled and scRNA sequencing was conducted.

first: Julian

last: Chua


title: Mitochondrial RNA degradation is increased in AML and controls differentiation, stem cell function and sensitivity to immune-mediated killing [NS_Immunology_v2_C2328]

first: Veronique

last: Voisin

summary: Eukaryotic cells have two separate genomes that are replicated independently; nuclear DNA organized in chromosomes and circular DNA within mitochondria. Mitochondrial DNA is transcribed bi-directionally to generate mitochondrial RNA (mtRNA).   As a by-product of the bi-directional transcription, dsRNA is also generated. By gene expression analysis and functional studies, we demonstrated that mtRNA transcription and degradation were increased in AML (Acute Myeloid Leukemia) cells and stem cells resulting in higher rates of mtRNA turnover. We discovered that the mitochondrial degradosome, SUV3 and PNPase, was upregulated in AML cells and stem cells and functionally important for the degradation of mtRNA and mitochondrial dsRNA in AML. Depleting SUV3 or PNPase impaired mtRNA degradation and promoted the accumulation of dsRNA. dsRNA that accumulated after depleting SUV3 or PNPase, stimulated IFN-I signaling that induced AML differentiation, decreased stem cell function and increased sensitivity of AML cells to immune-mediating killing. Thus, this work highlights new mitochondrial biology in AML and a novel mechanism by which the turnover of mtRNA regulates AML differentiation and stem cell function

overall_design: Expression of immune pathway genes after SUV3 and PNPase knockdown was measured using the NanoString NS_Immunology_v2_C2328, chip that contains probes for 579 genes involved in immune related processes. OCI-AML2 cell line was used and 2 biological replicates were used for both the SUV3 and PNPase knockdown experiments. Samples were taken at day3, day4, day 5 and day 6 for eah replicate.

first: Geethu

last: Thomas

first: Aaron

last: Schimmer


title: Pulmonary Organoid Models Demonstrate Compositionally Driven Epithelial Plasticity and Immune Polarization

city: New Haven

first: Micha Sam

middle: Brickman

last: Raredon

state: Connecticut

address: 10th Amistad Street

department: Department of Anesthesiology

institute: Yale University

summary: Aberrant epithelial regeneration and immune remodeling are hallmarks of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF), COPD, and post-viral syndromes. Yet how cellular context shapes these trajectories remains unresolved. We present a tunable, primary rat-derived lung organoid model that systematically varies immune, epithelial, and mesenchymal inputs to reveal how composition alone dictates epithelial plasticity and macrophage polarization. Across conditions, we observed the spontaneous emergence of disease-relevant transitional cell states, including Sox9⁺ stressed progenitors, RAS/AT0-like intermediates, and hillock-like cells, alongside distinct macrophage activation profiles. In mesenchyme-rich contexts, epithelial, immune, and mesenchymal crosstalk appeared to reinforce inflammatory signaling and stabilize transitional persistence, while immune-dominant inputs favored ATI-like repair and squamous remodeling. Notably, hillock-like cells displayed context-specific polarization and expressed immune-regulatory genes, suggesting a role as epithelial orchestrators that help calibrate inflammatory response during regeneration. Connectomic analysis revealed that regenerative outcomes were associated with dynamic multicellular signaling networks that integrate stress sensing, immune coordination, and epithelial fate. This platform provides a tractable system for modeling context-specific repair and regenerative mechanisms and could inform therapeutic strategies aimed at redirecting epithelial fate in chronic lung disease.

overall_design: Whole native rat lungs were enzymatically dissociated to generate heterogeneous starting populations containing epithelial, immune, and mesenchymal cells (PD_1, PD_2, PD_3) and sequenced. Additionally, primary immune and epithelial cells isolated via bronchoalveolar lavage (BAL) from native rat lungs were sequenced (BAL_1, BAL_2, BAL_3). Both PD and BAL suspensions were seeded into organoid culture and expanded for 10 days. Three organoid conditions were generated: PD_3D (100% PD), Mixed_3D (50:50 mix of PD and BAL), and BAL_3D (100% BAL). This design was intended to explore how varying the initial immune, epithelial, and mesenchymal composition shapes downstream cell fate, regenerative plasticity, and multicellular signaling.

first: Sophie E.

last: Edelstein

first: Satoshi

last: Mizoguchi

first: Maria

last: Tomàs Gracia

first: Nuoya

last: Wang

first: Vi

last: Lee

first: Hahram

last: Kim

first: Connor

last: Haynes

first: Colten

last: Danelski

first: Tomoshi

last: Tscuchiya

first: Maor

last: Sauler

first: Micha Sam Brickman