title: Lung NR3C1+ and CXCR6 High CD4 T cells distinguish immunopathogenesis of human emphysema

city: Houston

first: Francesca

last: POlVERINO

state: Texas

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: There is a significant knowledge gap in how T cells promote emphysema in smokers with chronic obstructive pulmonary disease (COPD). Single-cell RNA sequencing (scRNA seq) analysis of human samples and relevant clinical data could provide new mechanistic insights into disease pathogenesis. We generated an in-house human lung scRNA seq dataset with extensive disease characteristic annotation and analyzed a second independent scRNA seq dataset to examine the pathophysiological role of T cells in emphysema. Comparisons of pulmonary immune landscapes in emphysematous (E)-COPD, non-emphysematous (NE)-COPD, and controls showed positive enrichment of T cells in E-COPD. Pathway analyses identified upregulated inflammatory states in CD4 T cells a distinguishing feature of E-COPD. Compared to controls, glucocorticoid receptor NR3C1 expressing CD4 T cells were enriched in NE-COPD but were reduced in E-COPD. Interactions between macrophages and NR3C1+ CD4 T cell subset via CXCL signaling were strongly predicted in E-COPD but were absent in NE-COPD and controls. The relative abundance of CD4 CXCR6high effector memory T cells positively correlated with preserved lung function in E-COPD but not in NE-COPD. Overall, these findings suggest that NR3C1+ and CXCR6high effector memory subsets of CD4 T cells distinguish the immune-pathophysiological features of emphysema in human lungs. Molecular targeting of relevant T cell subsets in emphysema could provide new therapeutic opportunities.

overall_design:
Single cell RNA sequencing on human lung samples collected from 3 groups of patients: patients with no history of COPD, patients with nonemphysematous COPD and patients with emphysematous COPD. The primary goal of this study is to define the differences in tissue immune landscapes between emphysematous and nonemphysematous COPD.
NOTE: CellRanger 8 was not able to generate      count matrices for  wo  samples S18      and S59,  thus no cellranger processed files are provided for those samples.  Their raw data      fastq files were uploaded in case future studies may  be able to generate count matrices with these files.

first: Yun

last: ZHANG

first: Maor

last: SAULER

first: David

last: CORRY

first: Scott

middle: A

last: OCHSNER

first: Sarah

last: PERUSICH

first: Li-Zhen

last: SONG

first: Joshua

last: MALO

first: Raul

last: SAN JOSE ESTEPAR

last: POLVERINO

first: Farrah

last: KHERADMAND


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - thirdBatch1

status: Public on Nov 13 2025

city: Rehovot

first: Miguel

middle: A.

last: Garcia-Campos

state: Center District

address: Herzl 234, Meyer Bldg., Room 1410

department: Molecular Genetics

country: Israel

institute: Weizmann Insitute of Science

type: Expression profiling by high throughput sequencing

summary: Over 170 modifications are deposited on RNA, across the tree of life. A major bottleneck to systematically dissecting RNA modifications has been the lack of methods allowing to acquire systematic, high-confidence maps of many RNA modifications and at scale. Here we have developed Pan-Mod-seq, which permits de-novo, sensitive and specific identification of 16 distinct modifications in dozens of samples in parallel. We applied Pan-Mod-seq to RNA from 14 different species from all 3 domains of life, most of which sampled under highly variable chemical/physical/biological gradients, aiming to systematically explore the plasticity of rRNA modifications. While dynamically modified sites are relatively rare in mesophiles, we find that these are widespread in hyperthermophiles, where ~50% of identified modifications were induced with temperature. We focused on dissection of three key dynamically induced modifications: m5C, ac4C and Ψ. We uncover an m5C program encompassing dozens of targets across both rRNA subunits, all of which are dramatically induced with growth temperature in two of the sampled hyperthermophiles. In both cases m5C is introduced at a single consensus motif, via a single enzyme which is essential for growth at higher temperatures. ac4C in turn is dynamically induced among all three sampled hyperthermophiles at hundreds of sites, also at a single consensus motif. Remarkably, the methylation and acetylation overlap, and together form a tandemly modified temperature induced G-m5C-ac4C-G sequence motif.  Temperature-dependent induction of both enzymes can be recapitulated in vitro, establishing that it is an intrinsic property of the two enzymes rather than externally regulated. We obtain high-resolution cryoEM structures of ribosomes from WT strains at low and high temperatures and ones deleted of the methylating enzyme, and identify numerous mechanisms via which m5C confers structural stability. Finally, we also uncover a systematic induction in Ψ levels in T. kodakarensis, and reveal that it is largely coordinated by a single temperature-induced sRNA guiding pseudouridine formation at an unprecedented number of targets, whose loss results in growth deficiency at higher temperatures. Our findings offer a wide and systematic view of rRNA modification plasticity across representative species sampled from the tree of life, dissect the ability of different modifications to contribute to the stability of ribosomes at higher temperatures, and present a methodology allowing an unprecedented view on the the rRNA epitranscriptome in health and in disease.

overall_design: In-vitro treatment of WT and TkNat10 KO T. kodakarensis strains with Pfu-RsmB at at 85°C, followed by bisulfite treatment, in triplicates

first: Schraga

last: Schwartz

first: Joe

last: Georgeson

first: Ronit

last: Nir

first: Vinithra

last: Iyer

first: Anatoly

last: Kustanovich


title: Transcription factor ZNF263 primes human embryonic stem cells for pluripotency dissolution and early differentiation [RNA-Seq 2]

city: shanghai

first: shao

last: zhen

address: 320 Yueyang Road

country: China

institute: Shanghai Institute of Nutrition and Health

summary: Conventional human embryonic stem cells (hESCs) are capable of self-renewal and simultaneously poised for differentiation, and thus have been considered to resemble a primed pluripotent state. But the underlying regulatory mechanisms remain largely underexplored. Especially, little was known about the pivotal transcription factors (TFs) driving hESCs through the naïve-to-primed transition to prepare for differentiation. Here, we conduct a large-scale in-silico screening and identify ZNF263 as a new key TF in primed hESCs, as evidenced by its extensive binding capacity at the active and poised genes. Genetic and functional assays reveal that ZNF263 directly initiates the incipient expression of early differentiation genes and concurrently dampens the core pluripotency circuitry in hESCs, greatly tilting the balance from pluripotency maintenance to lineage priming. Importantly, ZNF263 deficiency in hESCs further led to clear defects in pluripotency dissolution and lineage commitment, suggesting it’s indispensably required for the whole process. Together, our results demonstrate a pivotal role of ZNF263 in coordinating the establishment of primed pluripotency state in hESCs and facilitating the differentiation into primary germ layer lineages, which opens a new gate for understanding the central regulatory network governing human early development.

overall_design: To investigate the role of ZNF263 in the process of pluripotency regulation, we conducted ZNF263 overexpression and cofactor(ZIC2) overexpression experiments to demonstrate corresponding molecular mechanism in hESCs, and then performed gene expression profiling using RNA-seq.

first: Zhen

last: Shao


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata4

overall_design: Total RNA from Thermococcus kodakarensis, WT, Tko105 KO, or Tko35 KO was subjected to BID-seq treatment. WT RNA was also left untreated (input). The WT strain was grown at 65 °C, 75 °C, 85 °C and 95 °C, while the Tko105 KO strain and Tko35 KO strain were grown at  75°C and 95°C. three replicates


title: RNAseq in melanoma

city: Nice

first: Corine

last: Bertolotto

address: 151 route Saint-Antoine de Ginestière

country: France

institute: C3M - U1065 INSERM - CENTRE MEDITERRANEEN DE MEDECINE MOLECULAIRE

summary: Compare control vs knockdown cells

overall_design: Compare control vs knockdown cells for SETDB1 in uveal melanoma

first: Thomas

last: Strub

first: Imene

last: Krossa


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata1

overall_design: Total RNA from Pyrococcus furiosus strain VC1, either WT or knockout (ΔRsmB), grown at 95 °C, was subjected to bisulfite sequencing, three replicates


title: The skeletal muscle of aged male mice exhibits a sustained growth regulatory transcriptional profile following prolonged glucocorticoid exposure compared to young males

city: Tallahassee

first: Cynthia

last: Vied

state: Florida

address: 1115 W. Call St

department: Translational Science Laboratory

country: USA

institute: Florida State University

summary: Excess glucocorticoids induce a skeletal muscle myopathy by changing gene expression. Advanced age augments glucocorticoid-mediated muscle phenotypes, although transcriptional responses underlying those augmented phenotypes are unclear. The purpose of this study was to determine glucocorticoid-induced transcriptional landscapes in young and aged muscle following acute exposure to the hormone and following longer glucocorticoid treatment. Young (4-month-old) or aged (24-month-old) male mice were administered an acute injection of dexamethasone (DEX) or vehicle or treated with DEX or vehicle for 7 days. Muscles were harvested 6.5 h after the only/last injection(s). The tibialis anterior (TA) was analyzed because mass was lower following DEX treatment only in aged males. RNA from the TA was subjected to RNA sequencing. In silico analyses determined pathways associated with glucocorticoid-sensitive genes and predicted transcription factors regulating transcriptional changes. Acute DEX altered similar numbers of genes in young (950) vs. aged males (913) although aged males had greater magnitudes of fold change. The transcriptional response in aged males following 7 days of DEX treatment was greater than acute exposure (1,196 vs 913) whereas the transcriptional response in young males was less pronounced after 7 days (599 vs 950). The glucocorticoid-sensitive genes in aged males were related to growth regulation regardless of treatment duration whereas that predominant enrichment was not evident in young males. Despite transcriptional differences, the transcription factors predicted to regulate the glucocorticoid-sensitive genes were similar in young and aged males. These data expand our understanding of transcriptional changes elicited by glucocorticoids in response to advanced age

overall_design: Young (4months) and aged (24months) mice were each randomized into 2 groups of equal body weight. One group from each age was given daily subcutaneous injections of saline while the other group from each age was given daily subcutaneous injections of dexamethasone (1mg/kg/day). Injections were administered for 7 days. On the final day, mice were given a final injection and the tibialis anterior muscle was harvested 6.5 hr later. RNA was extracted from the TA muscle and subjected to RNA sequencing analysis.

first: Grant

middle: R

last: Laskin

first: David

middle: S

last: Waddell

first: Bradley

last: Gordon


title: c-JUN Enhances CRISPR Knock-In anti-B7-H3 CAR T-cell Function for Treatment of Small Cell Lung Cancer and thoracic SMARCA4-deficient undifferentiated tumors

city: Stanford

first: Yiyun

last: Chen

state: California

address: 265 Campus Dr Room G2105

department: Stanford Cancer Institute

institute: Stanford University

summary: Small Cell Lung Cancer (SCLC), a highly lethal disease, limits T-cell responses by downregulating Major Histocompatibility (MHC) class I molecules. Because chimeric antigen receptor (CAR) T-cells are not MHC-restricted, they may provide a powerful strategy against SCLC. However, few CAR targets for SCLC have been identified. Here we show that B7-H3/CD276 is expressed on the surface of SCLC and thoracic SMARCA4-deficient undifferentiated tumor (UT) cells that can clinicopathologically mimic SCLC. Thoracic SMARCA4-deficient UT, despite high B7-H3 expression, limit killing by B7-H3 CAR-T cells via secretion of transforming growth factor beta 1 (TGFB1). To overcome tumor-driven CAR-T cell suppression, we knocked-in c-JUN alongside a B7-H3 CAR into the TRAC locus of primary human T-cells utilizing CRISPR/Cas9. Non-viral c-JUN+B7-H3 CAR-T cells show enhanced killing of both SCLC cells with low antigen density and thoracic SMARCA4-deficient UT, providing a platform to address these highly aggressive entities. We also provide evidence that GMP clinical scale manufacturing is feasible for c-JUN+B7-H3 CAR-T cells.

overall_design: CAR T cells were derived from the plate-bound stimulation assay described above and collected after ~20 hours of incubation. Cells were counted to ensure a maximum number of 500000 per sample.

first: Hyatt

last: Balke-Want

first: Elena

last: Sotillo

first: Crystal

middle: L

last: Mackall

first: Steven

last: Feldman


title: Genome-wide profile of Mpp8- and Tasor-DNA binding in mouse embryonic stem cells (mESCs) [CUT&Tag]

city: Nanjing

first: Bowen

last: Wang

address: 12 Xue-fu Road, Pukou District

institute: Nanjing University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Purpose: To determine the genome-wide binding sites of Mpp8 and Tasor in mESCs

overall_design: CUT&Tag of Mpp8 and Tasor in mESCs


title: Hypoxia increases susceptibility of human intestinal epithelial cells to rotavirus infection through repression of interferon induction

city: Gainesville

first: Sorin

middle: Ophelie

last: Jacobs

address: 1200 Newell Dr. ARB R2-273

department: Molecular Genetics and Microbiology

institute: University of Florida

summary: Intestinal epithelial cells (IECs) serve as both a physical barrier and a source of robust antiviral interferon (IFN) response. As such, they constitute the primary barrier that enteric viruses, such as rotavirus, need to overcome to initiate infection. The gut is characterized by very low oxygen levels (hypoxia) within the lumen, resulting in a unique hypoxic physiological environment in which rotavirus infection occurs. Depending on the tissues or viruses, conflicting results have been described for the role of hypoxia in regulating viral infections, where hypoxia could have either a proviral or antiviral function. Since intestinal epithelial cells naturally exist in a hypoxic environment, it is essential to investigate how these conditions affect rotavirus infection. We found that hypoxia promotes rotavirus infection, resulting in increased virus replication and production of infectious virus particles. We showed that this increased production of rotavirus particles under hypoxia is due to a decreased induction of both type I and III IFNs leading to a decreased expression of IFN stimulated genes (ISGs) and antiviral protection. RNA sequencing showed a robust decrease in ISG production in hypoxia for both rotavirus infection and poly I:C transfection, suggesting a conserved inhibition of IFN responses in hypoxia for IECs. Functional analyses revealed that hypoxia impairs signal transduction leading to IFN expression by negatively regulating the activation of the master signaling molecule TBK1. Mechanistically, we determined that hypoxia induces the expression of the protein phosphatase PP2A which is responsible for the hypoxia-induced impairment of TBK1 activation. Importantly, we observed that this hypoxia-mediated dampening of immune response was not restricted to rotavirus infection but dampened the IFN induction of a broad range of viruses and immune stimuli. Together, we propose that hypoxia creates an immune-suppressive environment through downregulation of IFN, representing a novel proviral mechanism for hypoxia in the human gastro-intestinal tract.

overall_design: Bulk RNA seq on intestinal epitheial T84 cells cultured in normoxia (20% O2) or hypoxia (1% O2), and either treated with poly I:C (8hr) or infected with rotavirus (8hr and 16 hr). For each condition, cells were seeded in 24-well plates with four replicates. Sequencing was conducted using the Illumina NovaSeq 6000 platform, with paired-end 150 bp reads.

middle: O

first: Stephanie

last: Muenchau

first: Zina

middle: M

last: Uckeley

first: Gianna

middle: V

last: Passarelli

first: Asher

last: David

first: Megan

last: Stanifer

first: Steeve

last: Boulant


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata2

first: Miguel

last: Garcia-Campos

sample_organism:
Pyrococcus furiosus
Saccharomyces cerevisiae

overall_design: In-vitro methylation followed by bisulfite sequencing. A mixture of total RNA of Saccharomyces cerevisiae (WT) and Pyrococcus furiosus (ΔRsmB) were in-vitro methylated with either Pfu-RsmB or TK1935under different temperatures (37 °C, 55 °C, 75 °C, 85 °C, 95 °C, and 105 °C), and a no enzyme control kept at 4 °C. Samples were subjected to bisulfite sequencing. One replicate per condition.

first: Schraga

last: Schwartz

first: Joe

last: Georgeson

first: Ronit

last: Nir

first: Vinithra

last: Iyer

first: Anatoly

last: Kustanovich


title: FOXP3 Expression Depends on Cell-Type Specific Cis-Regulatory Element and Transcription Factor Circuitry [mouse_Tconv_ChIP-seq]

city: San Francisco

first: Alexander

last: Marson

state: CA

address: 1650 Owens St.

institute: UCSF Gladstone Institutes

summary: FOXP3 is a lineage-defining transcription factor (TF) for immune-suppressive regulatory T cells (Tregs). While mice exclusively express FOXP3 in Tregs, humans also transiently express FOXP3 in stimulated conventional CD4+ T cells (Tconvs). Mechanisms governing these distinct expression patterns remain unknown. Here, we performed CRISPR screens tiling the FOXP3 locus and targeting TFs in human Tregs and Tconvs to discover cis-regulatory elements (CREs) and trans-regulators of FOXP3. Tconv FOXP3 expression depended on a subset of Treg CREs and Tconv-selective positive (NS+) and negative (NS-) CREs. Combinatorial silencing of Tconv CREs revealed their epistatic logic. The CREs are occupied and regulated by TFs we identified as critical regulators of FOXP3. Finally, mutagenesis of murine NS- revealed that it is critical for restriction of FOXP3 expression to Tregs. We discover CRE and TF circuitry controlling FOXP3 expression and reveal cell and species-specific regulation of a gene indispensable to immune homeostasis.

overall_design: ChIP-seq in mouse T cells. ChIP-seq was conducted in using DSG/formaldehyde fixation.

first: Jennifer

last: Umhoefer

first: Maya

last: Arce

first: Sivakanthan

last: Kasinathan

first: Sean

last: Whalen

first: Rama

last: Dajani

first: Sanjana

last: Subramanya

first: Laine

last: Goudy

first: Julia

last: Belk

first: Royce

last: Zhou

first: Minh

middle: T

last: N Pham

first: Wenxi

last: Zhang

first: Rosmely

last: Hernandez

first: Carinna

last: Tran

first: Nikhita

last: Kirthivasan

first: Jacob

middle: W

last: Freimer

first: Cody

last: Mowery

first: Vinh

last: Nguyen

first: Mineto

last: Ota

first: Benjamin

middle: G

last: Gowen

first: Dimitre

last: Simeonov

first: Gemma

last: Curie

first: Zhongmei

last: Li

first: Andy

middle: Y

last: Chen

middle: E

last: Corn

first: Howard

last: Chang

first: Qizhi

last: Tang

first: Luke

last: Gilbert

first: Ansuman

last: Satpathy

first: Katherine

last: Pollard


title: Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukemia [ChIP-Seq]

city: Melbourne

first: Emily

last: Gruber

state: VIC

address: 305 Grattan St

country: Australia

institute: Peter MacCallum Cancer Centre

summary: The ubiquitous metabolite heme has diverse enzymatic and signaling functions in most mammalian cells. Through integrated analyses of mouse models, human cell lines and primary patient samples, we identify de novo heme biosynthesis as a selective dependency in acute myeloid leukemia (AML). The dependency is underpinned by a propensity of AML cells, and especially leukemic stem cells (LSCs), to downregulate heme biosynthesis enzymes (HBEs) which promotes their self-renewal. Inhibition of HBEs causes collapse of mitochondrial Complex IV (CIV) and dysregulates the copper-chaperone system inducing cuproptosis, a form of programmed cell death brought about by the oligomerization of lipoylated proteins by copper. Moreover, we identify pathways that are synthetic lethal with heme biosynthesis, including glycolysis, which can be leveraged for combination strategies. Altogether, our work uncovers a heme rheostat that controls gene expression and drug sensitivity in AML and implicates HBE inhibition as a novel cuproptosis trigger.

overall_design: Hoxb8 cells expressing empty vector (EV), DNMT3AR882H, or DNMT3AR882H and treated with Hemin were isolated for ChIPseq.

first: Lev

last: Kats


title: Microbiota-derived bile acid ameliorates lung inflammation through metabolic rewiring of alveolar macrophages

city: Warsaw

first: Bartosz

last: Wojtas

address: Pasteura 3

country: Poland

institute: Nencki Institute of Experimental Biology

summary: Using mouse models of airway inflammation, we identified a causal link between the microbiota-dependent bile acid isolithocholic acid and protection against lung immunopathology. We further found that this protection involves Sphingosine-1-phosphate receptor 2–dependent metabolic rewiring of alveolar macrophages as a key mechanism. Collectively, our findings demonstrate that isolithocholic acid, a microbiota-derived bile acid, can ameliorate lung inflammation through previously unrecognized pathways, highlighting its therapeutic potential—and that of its derivatives—in lung disease.

overall_design: Acute lung injury was induced in six wild-type C57BL/6J mice by intranasal instillation of LPS. Following induction, half of the mice were treated with isolithocholic acid for three days, while the other half received vehicle. A control group of mice that did not receive LPS underwent the same treatment regimen. After four days, lungs were collected, dissociated into single-cell suspensions, and analyzed using scRNA-seq.


title: Age and cell type specific specific effects of histone variant H2BE in the brain [E2F_CUT&Tag]

city: Philadelphia

first: Sean

last: Louzon

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: Transcription is regulated in part through histone proteins that complex with DNA and control access to genes. Histones can be replaced with variant forms that are particularly critical in the brain and accumulate throughout lifespan. Recently, we defined the first broadly expressed H2B variant, H2BE, and demonstrated that it regulates chromatin structure, neuronal transcription, and mouse behavior. However, the role of H2BE in other cell types and its role throughout lifespan remain unknown. Here, we discovered that H2BE is highly expressed in astrocytes as well as neurons and accumulates with age in both cell types. Using single-nucleus RNA-sequencing, we demonstrate that loss of H2BE robustly affects gene expression in both astrocytes and neurons, with divergent effects in young and aging brains. Interestingly, loss of H2BE in young brains causes similar gene expression changes as aging and some effects of aging are reversed with H2BE loss. Lastly, behavioral testing demonstrates that H2BE loss disrupts long-term memory but improves working memory in aging mice. Together, these data provide novel links between histone variants, aging-related gene expression changes in both astrocytes and neurons, and memory.

overall_design: Cortical astrocytes were isolated from WT P1 mouse pups. They were cultured for 7 days before collection for control samples. Passaged samples were split after 7 days in culture and then grew for an additional 12 days before collection for Cut&Tag-sequencing. (n = 3 biological replicates)


title: Spatial Transcriptomic Analysis of Mouse Parathyroid Gland Cells Expressing an Activating Variant of Gcm2

city: Bethesda

first: James

last: Iben

state: MD

address: 10 CENTER DR

department: NICHD

institute: NIH

summary: Spatial transcriptomics can capture RNA from even a small number of cells in tissue sections. Using this technology, we analyzed the transcriptome of parathyroid gland cells in FFPE tracheal tissue sections of mice with a Gcm2 variant p.Y392S (Gcm2+/Y392S) compared to WT mice. The mouse Gcm2 variant p.Y392S corresponds to the human GCM2 variant p.Y394S that is seen in a subset of patients with primary hyperparathyroidism. Spatially resolved transcriptome analysis detected increased Lgals3 (Galectin-3) in Gcm2+/Y392S parathyroid gland cells, a marker of parathyroid carcinoma, indicating the growth and malignant potential of parathyroid glands expressing an activating Gcm2 variant.

overall_design: FFPE fixed mouse parathyroid tissue from WT or Gcm2 variant p.Y392S isolated from male and female mice in 4 replicates each, processed with the 10x Visium probe-based spatial transcriptome capture profiling

first: Priyanka

last: Bolel

first: Jeremie

last: Piña

first: Fabio

last: Faucz

first: Wafa

last: Abbas

first: Smita

last: Jha

first: William

middle: F

last: Simonds

first: Lee

last: Weinstein

first: Sunita

middle: K

last: Agarwal


title: Gene expression profile at single cell level of immune cells from ileum of ArpC5 mutant and control mice

city: London

first: Probir

last: Chakravarty

address: 1 Midland Road

country: United Kingdom

institute: Francis Crick Institute

summary: ArpC5 mutant mice develop spontaneous enteritis. We sought to investigate the gene expression profile in the single cell level to have insight into the pathology observed comparing to control conuterparts.

overall_design: immune cells from small intestine lamina propria (ileum) were isolated by Fluorescence-activated cell sorting (FACS) and analyzed using scRNAseq.

first: Luiz

last: Ricardo C. Vasconcellos

first: Michael

last: Way


title: Th1 effector cells and macrophages facilitate respiratory IgA after mucosal vaccination

city: Charlottesville

first: Arka

last: Sen Chaudhuri

state: VA

address: 345 Crispell Drive

department: Carter Immunology Center

institute: University of Virginia

summary: Mucosal immunization and mucosal IgA offer significant promise in protecting against airborne pathogens, including SARS-CoV-2. However, the conditions and mechanisms that lead to the robust induction of mucosal IgA responses following mucosal vaccination remain poorly understood. It is also currently debatable whether mucosal vaccination is still warranted given that most individuals in developed countries have established a hybrid immunity from vaccination and infection. Here we characterized respiratory mucosal immune responses after SARS-CoV-2 infection, vaccination or both in humans. We found that hybrid immunity resulted in moderately increased mucosal IgA and neutralizing antibody responses compared to infection or vaccination alone. However, a direct comparison of hybrid immunity and a mucosal adenovirus-based booster vaccination in animal models revealed that respiratory booster immunization elicited markedly stronger and more durable mucosal IgA, T cell response, and protective immunity against SARS-CoV-2, supporting the promise of respiratory mucosal vaccination. Mechanistically, we found that mucosal booster immunization induced local IgA-secreting cells in the respiratory mucosa, aided by pulmonary CD4+ T cells in situ. Strikingly, local IL-21-producing Blimp-1+ Th1 effector cells were critical in mediating the CD4+ T cell help for IgA production. Furthermore, lung macrophages were important for this mucosal IgA response via the production of TGF-β. Consequently, Spike mRNA coated with pulmonary surfactant (PS) containing liposomes, rationally designed to target lung macrophages, elicits a stronger mucosal IgA response. Collectively, our results uncover a local cellular network supporting enhanced mucosal IgA responses, with implications for the development of optimal mucosal immunization strategies against SARS-CoV-2 and other respiratory pathogens.

overall_design: After mRNA-S plus Ad5-S immunization, CD4+ T cells in the BAL, lung parenchyma, and draining lymph nodes in IL-21-VFP reporter mice were enriched with negative selection by using biotin-labeled CD8a, TER-119, and B220 antibody and streptavidin magnetic beads (Miltenyi Biotec) before surface markers staining. Live CD4+CD44+VFP+ and CD4+CD44+VFP- cells were sorted by Sony MA900 Cell Sorter and then resuspended in Trizol for RNA extraction. Two replicates of each cell subset were obtained. RNA quality was assessed and sequenced using Illumina 2 x 150 platform provided by Admera Health. cDNA libraries construction, sequencing alignment to the mouse reference genome through services were provided by Admera Health. Differentially expressed genes between each group were identified using DESeq2. For functional analysis, Gene Set Enrichment Analysis was performed to identify enriched gene sets from MSigDB.

first: Jinyi

last: Tang

first: Jie

last: Sun


title: The chromatin remodeling factor BAP18 promotes non-small cell lung cancer progression via the recruitment of β-Catenin with the transcriptional coactivator complex ACTL6A-PAF1

city: Shenyang

first: Qilin

last: Hu

state: Liaoning

address: No. 155 Nanjing North Street

institute: The First Hospital of China Medical University

summary: Non-small cell lung cancer (NSCLC) is a prevalent and deadly form of lung cancer, with treatment challenges including drug resistance and limited therapeutic targets, despite advances such as immune checkpoint inhibitors (ICIs). This study investigated the role of BAP18, a chromatin-associated protein, in NSCLC progression and its potential as a therapeutic target.

overall_design: RNA sequencing and enrichment analyses identified the pathways involved in BAP18-regulated NSCLC progression. A549 cells were collected 48 hours after siRNA transfection for RNA extraction and transcriptome sequencing. The experimental group was transfected with siRNA targeting BAP18, while the control group was transfected with non-specific siRNA. Each group had three biological replicates.


overall_design: For library preparation, BAL cells or sorted BAL CD4+ T cells from immunized mice in each group were pooled together and each group was hashtagged using BioLegend TotalSeq™-A antibodies according to the provided protocol. The library of hashtagged pooled groups was then constructed using the 10x Genomics Chromium Next GEM Single Cell 3’ Reagent Kit with the v3.1 (Dual Index) protocol. Sequencing was performed using the NovaSeq X Plus platform, a service provided by Novogene. The sequenced data were aligned using Cell Ranger. The curated data then underwent demultiplexing, quality assurance, filtering, differential gene expression analysis, and cluster identification, all conducted using the standard Seurat protocol with Seurat (v 5.1.0) in R (R-4.3.3).


title: Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukemia [RNA-Seq OCIAML3_ALAS1_KO_and_SA]

first: Emily

last: Gruber

overall_design: OCI-AML3 cells containing sgRNAs targeting ALAS1 (or scrambled) were treated with doxycyline to induce sgRNA expression and knockout ALAS1 for 7 days. Additionally, OCI-AML3 cells were treated with SA (500uM) or vehicle for 6 days and RNA isolated for RNAseq.

first: Lev

last: Kats


title: Age and cell type specific specific effects of histone variant H2BE in the brain [Cortex_CUT&Tag]

first: Sean

last: Louzon

overall_design: Cortex tissue was isolated from male mice at 3, 12, and 18 months of age and then underwent Cut&Tag-sequencing with H2BE (Millipore ABE1384) or IgG antibody (Sino Biological CR1). 2 H2BE biological replicates per age, 1 IgG biological replicate per age.


title: Lewy body dementia promotion by air pollutants

city: Baltimore

first: Xiaodi

last: Zhang

address: 733 N. Broadway, MRB 710

institute: Johns Hopkins School of Medicine

summary: Evidence links air pollution to dementia, yet its role in Lewy body dementia (LBD) remains unclear. Here we show in a cohort of 56.5 million individuals across the U.S. that PM2.5 exposure significantly raises LBD risk. Mechanistically, we find PM2.5 exposure leads to brain atrophy in wild-type mice, an effect not seen in α-synuclein (αSyn)-deficient mice. PM2.5 exposure generates a highly pathogenic αSyn strain, PM-PFF, with enhanced proteinase K-resistance and neurotoxicity, resembling αSyn LBD strains. PM2.5 samples from China, the U.S., and Europe consistently induce proteinase-resistant αSyn strains and in vivo pathology. Transcriptomic analyses reveal shared responses between PM2.5-exposed mice and LBD patients, underscoring PM2.5’s role in LBD and the stresses the need for interventions to reduce air pollution and its associated neurological disease burden.

overall_design: 2-month-old humanized WT αSyn mice (FVB; 129S6-Sncatm1Nbm Tg (SNCA)1Nbm/J, Strain: 010710), obtained from The Jackson Laboratory (Bar Harbor, ME, USA), were used for RNA sequencing experiments. The mice were divided into five groups: the PBS group (intranasal administration of PBS), the PM2.5 group (intranasal administration of PM2.5), the PFF group (PFF injection), the PM-PFF group (PM-PFF injection), and the PM-PFF with PM2.5 exposure group (PM-PFF injection followed by intranasal PM2.5 administration one week later). All treatments were administered for a duration of 2 months.

first: Haiqing

last: Liu

first: Xiao

last: Wu

first: Longgang

last: Jia

first: Kundlik

last: Gadhave

first: Lena

last: Wang

first: Kevin

first: Hanyu

last: Li

first: Rong

last: Chen

first: Ramhari

last: Kumbhar

first: Ning

first: Chantelle E.

last: Terrillion

first: Bong Gu

last: Kang

first: Bin

last: Bai

first: Minhan

last: Park

first: Ma. Cristine Faye

last: Denna

first: Shu

first: Wenqiang

last: Zheng

first: Denghui

last: Ye

first: Xiaoli

last: Rong

first: Yang

first: Lili

last: Niu

first: Han Seok

last: Ko

first: Weiyi

last: Peng

first: Lingtao

last: Jin

first: Mingyao

last: Ying

first: Liana S

last: Rosenthal

first: David W

last: Nauen

first: Alex

last: Pantelyat

first: Mahima

last: Kaur

first: Kezia

last: Irene

first: Liuhua

last: Shi

first: Rahel

last: Feleke

first: Sonia

last: García-Ruiz

first: Mina

last: Ryten

first: Valina

last: Dawson

first: Francesca

last: Dominici

first: Rodney

middle: J

last: Weber

first: Xuan

first: Pengfei

first: Ted

first: Shizhong

last: Han

first: Xiaobo

last: Mao


title: Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukemia [RNA-Seq Hoxb8_R882H_v_EV]

first: Emily

last: Gruber

overall_design: RNA from Hoxb8 cells expressing empty vector (EV) or DNMT3AR882H was isolated for RNAseq.

first: Lev

last: Kats


title: HTGTS-TCR-seq for profiling of mouse and human T-cell receptor α and β gene rearrangements and diversity [HTGTS-TCRseq]

city: Shanghai

first: Haiqiang

last: Dai

address: 320 Yue Yang Road

institute: Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell  Science

summary: T-cell receptor (TCR) diversity is generated through V(D)J recombination, which are essential for adaptive immunity. Although many methods have been applied, a simple method to detect TCR rearrangement at the DNA level remains lacking. Here we introduce HTGTS-TCR-seq, a novel approach derived from high-throughput genome-wide translocation sequencing (HTGTS), to comprehensively and unbiasedly analyze TCR recombination. Unlike multiplex PCR requiring extensive V-J segment primers, HTGTS-TCR-seq enables unbiased TCR profiling through targeted enrichment and deep sequencing by a single primer. This method identifies biased V(D)J segment usage in developing T cells through V/J primer-based amplification and provides unique complementarity determining region 3 (CDR3) profiles in mature T cells via J primers. HTGTS-TCR-seq detects rearrangement at both alleles, enabling quantification of DJβ and VβDJβ rearrangement products with productive and nonproductive profile. Applied to human peripheral blood mononuclear cells (PBMCs), HTGTS-TCR-seq reveals a shared CDR3 motif between humans and mice. HTGTS-TCR-seq is a robust tool for T cell immunity research, with potential application in immune responses analysis, cancer research, and autoimmune diseases investigation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We employed HTGTS-TCR-seq to asess rearrangement events and TCR repertoire changes across developmental stages and T cell subsets at DNA level. DN3 thymocytes, preselection DP thymocytes, splenic CD4+ and CD8+ T cells from mice and CD4+ and CD8+ T cells from human PBMCs were FACS sorted. Total thymocytes from young and aged mice were collected. Genome DNA were extracted, sonicated and utilized to conduct HTGTS-TCR-seq.

first: Rui

last: Luo

first: Yawei

last: Song

first: Meichen

last: Wang

first: Longhao

last: Zou

first: Tiange

last: Yang

first: Guangchuan

first: Wei

last: Wu

first: Hai-Qiang


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata3

city: Rehovot

first: Miguel

last: Garcia-Campos

address: Herzl 234, Meyer Bldg., Room 1410

institute: Weizmann Insitute of Science

overall_design: total RNA extracted from a ΔTkNat10 Thermococcus kodakarensis strain was subjected to in-vitro acetylation using purified TkNat10, grown at 37 °C, 55 °C, 65 °C, 75 °C, 85 °C, or 95 °C; RNA was subjected to ac4C-seq using NACNBH3 or was left untreated (control), one replicate

first: Schraga

last: Schwartz

middle: A

first: Joe

last: Georgeson

first: Ronit

last: Nir

first: Vinithra

last: Iyer

first: Anatoly

last: Kustanovich


title: Senataxin Promotes RNA Polymerase II Elongation [Hap1_TTseq]

city: Copenhagen

first: Zhong

last: Han

address: Blegdamsvej 3B

department: ICMM, Institut for Cellulær og Molekylær Medicin

country: Denmark

institute: University of Copenhagen

summary: Senataxin (SETX) is recognized as a critical regulator of RNA Polymerase II (RNAPII) transcription and plays a pivotal role in maintaining genomic stability. Despite its importance, the direct in vivo functions of SETX remain inadequately characterized. Employing the degradation tag (dTAG) system for acute protein depletion, we demonstrate that SETX does not influence transcription termination but significantly enhances RNAPII elongation. Through in vitro reconstitution assays, we show that SETX actively drives RNAPII forward across complex transcriptional landscapes, akin to the way bacterial ribosomes mitigate transcriptional pauses. In vivo analyses further reveal an accumulation of pausing RNAPII at early transcription stages and a corresponding increase in R-loop formation at these sites following SETX depletion. These insights not only redefine our understanding of SETX's role in transcriptional regulation but also highlight its potential as a therapeutic target in disorders linked to transcriptional dysregulation.

overall_design: Determination of nascent RNA synthesis by TT-seq under DMSO vs dTAGV1 treatment (4h)

first: Jesper

middle: Q

last: Svejstrup


title: Molecular and neural control of social hierarchy by forebrain-thalamocortical circuit

city: Cambridge

first: Catherine

last: Dulac

address: 52 Oxford St

institute: Harvard University

summary: Many animal groups are organized hierarchically, which generates behavioral states that facilitate social interactions. Although generally stable, social status can change, underscoring the plasticity of underlying neural circuits. We examined competition among unfamiliar male mice and uncovered how the molecular and biophysical characteristics of a forebrain-thalamocortical circuit affect hierarchy. We identify the mediodorsal thalamus (MDT) as a hub receiving inputs from orbitofrontal cortex and basal forebrain and projecting to caudal anterior cingulate cortex (cACC) to regulate competitive performance. This circuit becomes potentiated or depressed in high- and low-rank males, respectively, in part through altered expression of the voltage-gated ion channel Trpm3 and synaptic plasticity. In high-rank mice, MDT projections drive inhibition of cACC pyramidal cells, promoting winning, in a pattern strikingly opposite to dorsomedial prefrontal cortex, where winners display increased pyramidal cell activity. Our data suggests a model in which hierarchy modulation relies on coordinated remodeling of multiple forebrain-thalamocortical circuits.

overall_design: Single cell RNA-seq of thalamic nuclei from male mice from within established social hierarchies (dominant and subordiate), as well as non-hierarchy control animals.

first: Vikrant

last: Kapoor

first: Adam

last: Nelson


title: Multiomics profiling of molecular and cellular dynamics of spinal cord injury in a rat [mRNA-seq]

city: Vestec

first: Ruslan

last: Klassen

address: Průmyslová 595

department: Institute of Biotechnology

country: Czech Republic

institute: Czech Academy of Science

summary: Spinal cord injury (SCI) is a debilitating condition with no effective treatment. The injury triggers a complex cascade of molecular and cellular events that drive both damage and repair processes. To explore these mechanisms, we performed a comprehensive multiomics analysis in a rat compression model of SCI, focusing on the acute phase. Transcriptomic profiling revealed extensive gene dysregulation, highlighting early inflammation, neuronal death, and synaptic dysfunction, followed by the initiation of reparative processes. Cell type composition analysis showed a rapid infiltration of peripheral immune cells, activation of microglia, and loss of neurons, astrocytes, and oligodendrocytes. miRNA profiling uncovered a highly dysregulated miRNA landscape, with the miR-17~92 cluster emerging as a key regulator of neurogenesis, synaptic activity, and cell survival. Integrative miRNA-mRNA-protein analysis identified potential therapeutic targets, including miR-20a, whose inhibition in vitro supported neurogenesis and reduced apoptosis under oxidative stress. Our findings provide new insights into the molecular mechanisms of SCI and highlight miRNAs as potential targets for therapeutic intervention.

overall_design: Adult male Wistar rats (n=64) were used as an experimental model. The rats were obtained from the facility's breeding center (Physiological Institute of the Academy of Sciences of the Czech Republic, Prague, Czech Republic). Animals undergoing surgery weighed 300 ± 30 g and were 10 weeks old. Animals were maintained on a 12-h light/dark cycle with ad libitum access to food and water. Rats were randomly divided into experimental groups: SCI 3 hpi (n=4), 12 hpi (n=4), 24 hpi (n=4), 3 dpi (n=4), 7 dpi (n=4), time- corresponding shame-operated groups (n=4 for each group) and an intact control group (n=4), also 8 animals were used for behavioral tests and 12 animals were used for immunohistochemical analysis.

first: Sarka

last: Chytilova

first: Ivan

last: Arzhanov

first: Daniel

last: Zucha

first: Eva

last: Rohlova

first: Peter

last: Androvic

first: Pavel

last: Abaffy

first: Lucia

last: Urdzikova-Machova

first: Mikael

last: Kubista

first: Nataliya

last: Romanyuk

first: Lukas

last: Valihrach


title: Senataxin Promotes RNA Polymerase II Elongation [Hap1_DRBrelease_TTseq]

overall_design: Determination of RNAPII elongation speed by DRBrelease_TTseq under DMSO vs dTAGV1


title: Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

city: New York

first: Andrea

last: Terceros

address: 1230 York ave

institute: The Rockefeller University

summary: The molecular mechanisms that enable memories to persist over long time-scales from days to weeks and months are still poorly understood. To develop insights we created a behavioral task where, by varying the frequency of learned associations, mice formed multiple memories but only consolidated some, while forgetting others, over the span of weeks. We then monitored circuit-specific molecular programs that diverge between consolidated and forgotten memories. We identified multiple distinct waves of transcription, i.e., cellular macrostates, specifically in the thalamo-cortical circuit, that defined memory persistence. Notably, a small set of transcriptional regulators orchestrated broad molecular programs that enabled entry into these macrostates. Targeted CRISPR-knockout studies revealed that while these transcriptional regulators had no effects on memory formation, they had prominent, causal, and strikingly time-dependent roles in memory stabilization. In particular, the calmodulin-dependent transcription factor Camta1 was required for initial memory maintenance over days, while Tcf4 and the histone methyl-transferase Ash1l were required later to maintain memory over weeks. These results identify a critical Camta1-Tcf4-Ash1l thalamo-cortical transcriptional cascade required for memory stabilization, and puts forth a model where the sequential, multi-step, recruitment of circuit-specific transcriptional programs enable memory maintenance over progressively longer time-scales.

overall_design: Cells from rodent brain were isolated and processed using scRNA-seq, at different time points during training and retrieval, for both HR and LR conditions. At least 3 replicates were included per time point and condition.

first: Celine

last: Chen

first: Yujin

last: Harada

first: Tim

last: Eilers

first: Millennium

last: Gebremedhin

first: Richard

last: Koche

first: Pierre-Jacques

last: Hamard

first: Roshan

last: Sharma

first: Priya

last: Rajasethupathy


title: The Function of Tetrameric MADS-box Transcription Factors in Floral Fate Determination [Mnase-seq]

city: Berlin

first: Manuel

last: Neumann

address: Philippstr. 13

country: Germany

institute: HU Berlin

summary: MADS-domain transcription factors (TF) are critical in determining floral cell fate and floral organ identities. However, their function molecular mechanisms are still poorly understood. Here, we show that effective DNA-binding in closed chromatin and target gene regulation of APETALA1 (AP1), a  key floral MADS-box TF requires tetramerisation, indicating the fundamental role of tetrameric MADS-box TFs in floral cell fate programming.

overall_design: MNase-seq were generated with seedling tissues induced by dexamethasone for 24 hours for AP1 expression. In addition,same amount of mock-treated seedlings were collected as a control. Three biological replicates were generated.

first: Kerstin

last: Kaufmann

first: Xiaocai

last: Xu


title: Multiomics profiling of molecular and cellular dynamics of spinal cord injury in a rat [miRNA-seq]

first: Ruslan

last: Klassen

type: Non-coding RNA profiling by high throughput sequencing

first: Sarka

last: Chytilova

first: Ivan

last: Arzhanov

first: Daniel

last: Zucha

first: Eva

last: Rohlova

first: Peter

last: Androvic

first: Pavel

last: Abaffy


title: Use of DNA methylation profiling as a molecular classification tool: a pediatric population-based study in tumors from central nervous system

city: Campinas

first: Felipe

last: Silva

address: R. Márcia Mendes, 506

country: Brazil

institute: Boldrini

type: Methylation profiling by genome tiling array

summary: Pediatric central nervous system (CNS) tumors are the second most common type of cancer in children and the leading cause of cancer-related death in this age group. Diagnosing these tumors based solely on histopathology is challenging and can lead to misdiagnosis, even among experienced pathologists. DNA methylation profiling classifies CNS tumors into molecular classes providing a more reliable diagnosis with the potential to impact patient’s management. This approach allows for faster and more accurate diagnoses combined with histopathology and other biomarkers. In this study, we performed DNA methylation analysis using the Infinium MethylationEPIC BeadChip (Illumina®) on a cohort of 182 pediatric CNS tumor samples from a reference center for pediatric cancer treatment in the countryside of São Paulo, Brazil. The data were analyzed using the DKFZ/Heidelberg CNS tumor platform (v12.8). Of the 182 tumors, excluding cases classified as control tissue, 82.8% (135 of 163) achieved a satisfactory calibrated score of ≥ 0.9 for a methylation family. Methylation profiling refined the initial diagnosis in 88 cases (65.7%) and provided a new diagnosis for 28 (20.9%) Several rare tumors only identified by methylation profile disclose the importance of analyzing full and consecutive samples, which allows the heterogeneity assessment and provide a more comprehensive insight into CNS tumors. Our work highlights the value of DNA methylation profiling as a molecular classification tool in a pediatric cohort from a middle-income country, which is often underrepresented in general databases.

overall_design: The study used fresh frozen tissues without a pathological review of the frozen sample but rather from a mirror sample embedded in paraffin for diagnostic purposes, enabling us to exclude normal, necrotic, and inflammatory tissues for most cases. All patients received the standard-of-care histopathologic diagnosis (tumor histology and IHC), which was used for patient management.   DNA was extracted with the GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Co. LLC, St Louis, MO, USA; cat. ID: G1N70-1KT) and measured with Qubit® dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA; cat. ID: Q322853), reporting an average recovery of 100 ng/uL. Samples with 260/280 >1.7, as measured by BioDrop Duo (Biochrom Ltd, Waterbeach, Cambridge, UK) was bisulfite converted with Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA, USA; cat. ID: D5002) and then hybridized on the 850K Infinium HD EPIC Methylation Array Bead Chip version 1 (Illumina, San Diego, CA, USA). Beadchip arrays were scanned on the NextSeq550 (Illumina, San Diego, CA, USA), which generated IDAT files.

first: Mariana

last: Maschietto

first: Mayara

last: Euzebio


title: A Split Luciferase Biosensing Platform for Detection and Imaging of Chromatin Loops in Individual Live Cells

city: Davis

first: Henriette

last: O'Geen

address: 451 Health Sciences Drive, GBSF 4617A

department: Genome Center

institute: UC Davis

summary: Eukaryotic cells regulate higher-order chromatin architecture, gene expression, and gene recombination via compaction of the genome into chromatin loops and topologically associating domains (TADs). While chromatin architecture has been thoroughly characterized for many eukaryotic genomes using cell-destructive techniques such as 3C-based methods, live-cell biosensing tools that can probe three-dimensional chromatin contacts in real-time are lacking. Using a dual dCas9 DNA biosensor based on a split NanoLuc luciferase reporter, we directly detected chromatin loops in live cells using luminescence quantification in a luminometer. We were able to see signal above background ratios of up to 10-fold. In addition, we directly visualized chromatin looping at the MYC TAD in live cells using high-resolution, low light live-cell imaging. Our biosensing platform therefore provides a useful methodology for live-cell, real-time detection of known or novel loops and for monitoring looping dynamics upon alterations in cell state.

overall_design: 4C-seq of cell-type specific chromatin loops in WT HCT116 cells, WT K562 cells, HCT116 E7 enhancer deletion cells, and HCT116 RAD21-mAC cells treated with either 1 uM DMSO or 1 uM auxin for 120 min using a non-blind viewpoint centered on the conserved CTCF binding site upstream of the MYC promoter with three replicates per cell type.

first: Nicholas

last: Heath

first: Antonio

last: Gomez

last: O’Geen

first: David

last: Segal


title: Dermal adipocytes activate hair follicle stem cells through metabolic communication [SP]

city: Taipei

first: Kang-Yu

last: Tai

address: Yonglin Biomedical Engineering Hall, No.49, Fanglan Rd.

department: Genome and Systems Biology Degree Program

country: Taiwan

institute: National Taiwan University

summary: The ability of tissue stem cells to sense and respond to their surroundings enables them to adapt their activity to meet the needs of the organism. External irritation has long been observed in humans to cause acquired hypertrichosis, but the mechanism is unknown. We discovered that external irritation activates hair follicle stem cells (HFSCs), leading to substantial hair regeneration in mice. The external irritation is sensed by niche adipocytes, which undergo lipolysis to activate HFSCs, rather than by the HFSCs themselves. Fatty acids released by adipocytes are taken up by HFSCs and activate them by priming them for fatty acid oxidation and increasing the mitochondrial oxidative phosphorylation capacity for ATP production. Inhibiting lipolysis, fatty acid uptake, or fatty acid oxidation restricts irritation-induced hair regeneration. As a result, adipocytes create an injury-sensing niche, allowing HFSCs to respond to external irritation by initiating a new round of hair growth to protect the skin.

overall_design: To determine the mechanism of the influence of irritant contact dermatitis induced by SDS on the whole skin, four groups of comparisons were performed. The control group is day 0, and the experimental group is treated with SDS on days 1, 2, 3, 4 and 5, respectively. Mice were sacrificed, and whole skin samples were collected for histopathologicand spatial transcriptomical analyses. Ten micrometer-thick FFPE sections were sliced and mounted on a Visium HD glass slide before undergoing HE staining.

first: Sung-Jan

last: Lin


title: Dermal adipocytes activate hair follicle stem cells through metabolic communication [SDS II]

overall_design: To determine the mechanism of the influence of unsaturated fatty acids versus saturated fatty acids on hair follicle stem cells, three groups of comparisons were performed. The control group is ETOH alone, the unsaturated fatty acid treatment group is C18:1, and the saturated fatty acid treatment group is C18:0. Hair follicle stem cells were extracted for examination three days after the mouse dorsal skin was treated using Cd34+/Lgr5GFP+/Sca1- fluorescence-activated cell sorting


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [RNAseq_ZNF398_OE]

city: Turin

first: Andrea

last: Lauria

address: Via Accademia Albertina, 13

department: Life Sciences and Systems Biology

country: Italy

institute: University of Turin

summary: Pluripotency is the ability to give rise to all cell types of the body and is first observed in a mass of disorganised cells of the embryo. Upon implantation, pluripotent cells form a columnar epithelium and undergo lumenogenesis. At gastrulation, a portion of the pluripotent epiblast will undergo epithelial to mesenchymal transition (EMT), forming the primitive streak (PS). It still remains unclear what molecular mechanism supports the epithelial identity of the pluripotent epiblast before gastrulation. Here we developed an optimised, chemically defined 3D model of human pluripotent epiblast formation in which conventional pluripotent stem cells (PSCs) self-organise into a columnar epithelium with a lumen in 48 hours. From 72 hours we observed spontaneous symmetry breaking and specification of PS-like cells, as confirmed by single-cell RNA sequencing. We found that Insulin and FGF signalling are both required for the proliferation and survival of the pluripotent epiblast model. Conversely, TGF-beta signalling maintains epithelial identity. Epithelial identity appears uncoupled from the expression of canonical pluripotency markers OCT4, NANOG and PRDM14, but under the control of ZNF398. Once the pluripotent epithelium is established, TGF-beta inhibition is inconsequential, and stimulation with Activin A leads to highly efficient PS induction. We conclude that TGF-beta dynamically orchestrates epithelial identity of human pluripotent cells.

overall_design: Bulk RNAseq profiling of KiPS stably expressing the empty vector control without or with SB43 (TGF-beta inhibition), or expressing ZNF398 without or with SB43, in 2D and 3D after 4 days of self-organisation. 12 samples, 2 replicates per condition.

first: Irene

last: Zorzan

first: Elena

last: Carbognin

first: Valentina

last: Proserpio

first: Davide

last: Benvegnù

first: Susana

last: De Juambelz Urias

first: Caterina

last: Dalrio

first: Giorgia

last: Panebianco

first: Rebecca

last: Scarfò

first: Eleonora

last: Pensabene

first: Mattia

last: Arboit

last: Paolucci

last: Drusin

first: Monika

last: Sledziowska

first: Paola

last: Braghetta

last: Ditadi

first: Gianluca

last: Amadei

first: Salvatore

last: Oliviero

first: Graziano

last: Martello


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [scRNAseq_HES3-MIXL1-GFP_hESC]

last: Lauria

overall_design: scRNA-seq profiling of 3D-hE-gastruloids generated from MIXL1-GFP HES3 reporter hESC line, collected at 0, 2 and 5 days of gastrluoid generation. 654 samples.

last: Zorzan

last: Carbognin


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [scRNAseq]

overall_design: scRNA-seq profiling of 3D-hE-gastruloids generated from H9 hESC line, collected at 0, 2 and 6 days of gastrluoid generation. 394 samples.

last: Proserpio

last: Benvegnù

last: De Juambelz Urias


title: Sphingolipids in extracellular vesicles released from the skeletal muscle plasma membrane control muscle stem cell fate during muscle regeneration

city: Pierre Bénite

first: emmanuelle

last: meugnier

address: Cens Eli Bat 2 D- Hopital Lyon Sud

summary: Extracellular vesicles (EVs) represents a cytokine-independent pathway by which skeletal muscle (SkM) cells modulate the fate of neighbouring cells to control SkM homeostasis. Here we show that SkM release a bulk of small (sEVs) and large (lEVs), both generated from plasma membrane (PM). Conversely to lEVs, sEVs are synthesized from membrane folds enriched in Alix and TSG101 proteins and lipid-rafts, which have never been described for other cell types. During muscle regeneration, sEVs promoted M1 macrophage polarization and migration and MuSC differentiation, contributing to accelerated muscle regeneration, whereas lEVs influenced the shift from a pro-inflammatory to an anti-inflammatory response, promoting MuSC proliferation. The lipid composition of sEVs and lEVs, particularly the ratios of sphingosine 1-phosphate (d18:1 and d16:1), explained their differential effects, with TNF-α altering these ratios and modifying SkM-EV actions. Our work highlight the role of sub-populations of SkM-EVs in muscle regeneration, and their potential for therapeutic applications.

overall_design: Biopsies from vastus lateralis were obtained from 5 healthy donors (Geneva Hospital and Danish Muscle Center approved the study protocols #NAC 06–093 and #jr. nr. H-A-2007–0016). MuSC were isolated and differentiated as in Bouzakri K et al.Diabetes 2003; 52:1319-1325.  Myotubes were treated for 24h with serum-free DMEM (4.5g/L glucose) containing BSA 2% + 20ng/mL TNF‐α.

first: Sophie

last: Rome

first: Rhyma

last: Hakkar

first: Caroline

last: Brun

first: Emmanuelle

last: Meugnier


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [RNAseq_CRISPRi_ZNF398]

first: Andrea

overall_design: Bulk RNA-seq profiling of gRNA-ZNF398 PSCs in 2D and 3D-hE-gastruloids, with (ZNF398 Knock-Down) or without (Control) Dox+TMP induction. 8 samples, 2 replicates per condition.

first: Irene

first: Elena

first: Valentina

first: Davide

last: Dalrio

last: Panebianco

last: Scarfò

last: Pensabene

last: Arboit

last: Paolucci

last: Drusin

last: Sledziowska


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [RNAseq_H9_KiPS]

last: Lauria

overall_design: Bulk RNA-seq profiling of 3D-hE-gastruloids generated from H9 and KiPS cell lines, collected at 0, 4 and 8 days of gastruloid formation. 12 samples, 2 replicates per condition.

last: Zorzan

last: Carbognin

first: Susana

first: Caterina

first: Giorgia

first: Rebecca

first: Eleonora

last: Braghetta

last: Ditadi

last: Amadei

last: Oliviero

last: Martello


title: Enhanced phase separation of BRD4S derived from CDK12 inactivation-induced IPA drives cancer metastasis

city: Rochester

first: LIGUO

last: WANG

state: MN

address: 200 1st St SW

department: Division of Computational Biology

institute: Mayo Clinic

summary: CDK12 gene is frequently deleted/mutated in human cancers and CDK12 loss associates with intronic polyadenylation (IPA). Here, we show that CDK12 deficiency induces IPA usage in BRD4 gene locus and expression of the BRD4 short form (BRD4S). Different from the full-length BRD4 (BRD4L) that possesses a C-terminal intrinsically disordered region (IDR), surprisingly, BRD4S has a greater capacity to form phase separation in a manner dependent on oligomerization mediated by a base-interacting structural domain (BID). Through CUT&Tag profiling, we identify BRD4S specifically binds to the promoters of and transcriptionally upregulates TGF-β signaling genes including TGFB2 and LTBP1. Our study identifies IPA usage at the BRD4 gene locus as a previously unrecognized mechanism responsible for BRD4S genesis. Our findings also reveal an essential role of BRD4S condensation formation and a unique ability of BRD4S to turn on TGF-β signaling gene expression.

overall_design: Genome-wide binding profiling of BRD4L and BRD4S by CUT&Tag in OVCAR8 cells ectopically expressing FLAG-tagged BRD4L or BRD4S individually.

first: Rui

last: Sun

first: Liguo

last: Wang

first: Haojie

last: Huang


title: Genome structure in single nucleoli by sequencing of laser capture nucleolar microdissection (scRNA-Seq)

city: Zurich

first: Raffaella

last: Santoro

address: Winterthurerstrasse 190

department: Dep. of Molecular Mechanisms  of Disease

country: Switzerland

institute: University of Zurich

summary: The three-dimensional architecture of the eukaryotic genome is a critical determinant in the regulation of gene expression programs. Repressive chromatin regions are localized either at the nuclear lamina or the nucleolus. While the role of the nuclear lamina in genome organization has been widely studied, the role of the nucleolus has only recently come under investigation. Here, we present a new method called nucleolus architecture mapping (NAM) for identifying nucleolus-associated genomic domains (NADs) at a single nucleolus resolution. NAM combines laser capture microdissection and DNA sequencing. We applied NAM to embryonic stem cells (ESCs) and neural progenitor cells and observed a distinct pattern of nucleolus-to-nucleolus heterogeneity for NADs. NAM not only identified characteristic features of NADs, such as high levels of H3K9me2, depletion of H3K27me3, low active histone marks, and repressed gene expression but also revealed that the genomic domains seen in cell population studies to contact both nucleoli and nuclear lamina (NAD/LAD regions) can contact the nucleolus in one cell and the NL in another cell, but rarely both nucleolus and NL in the same cell. Additionally, we found that ribosomal protein (RP) genes are frequently associated with the nucleolus, suggesting a potential direct crosstalk between the nucleolus and the regulation of RP genes, and hence ribosome biogenesis. We also performed NAM upon perturbation of the nucleolus structure by inhibiting PolI transcription with Actinomycin D. This showed a loss of mostly NADs from the nucleolus other than the chromosomes containing rRNA genes, demonstrating that nucleolar integrity is required for genomic contacts with the nucleoli. Additionally, NAM applied to hybrid ESCs demonstrates a predominantly monoallelic distribution of NADs in single cells. We identify parental-specific NADs exhibiting a non-uniform distribution across different chromosomes. Our findings underscore NAM as an invaluable tool for investigating nucleolar organization in individual cells and, potentially, genome organization in other large phase-separated organelles in the future.

overall_design: scRNAseq F123 ESCs

first: Kaivalya

last: Walavalkar


title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

city: Daejon

first: Semi

last: Kim

address: 125 Gwahak-ro

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology

summary: Epithelial-mesenchymal transition (EMT) is implicated in tumor progression, and EMT-inducing transcription factors play multifaceted roles; however, the molecular mechanisms underlying these processes are not well understood. Previously, we showed that ZEB2 acts cooperatively with the transcription factor SP1 to function as a transcriptional activator that promotes cancer cell invasion and survival, as well as angiogenesis. Here, we investigated whether cooperation between ZEB2 and SP1 promotes immunosuppressive activity of tumor cells, and characterized the underlying mechanisms. The effects of ZEB2 on expression of cytokines and PD-L1 were investigated using mRNA-seq analysis of ZEB2-overexpressing tumor cells.

overall_design: RNA-seq profiling of wildtype SW480 cells and ZEB2-overexpressing SW480 cells

first: Dongjoon

last: Ko


title: Transcriptional profiles of castration resistant prostate cancer cells treated with ITRI-148 PROTAC targeting AR and AR-V7

city: Taoyuan

first: Ling-Yu

address: No.259, Wenhua 1st Rd., Guishan Dist.,

department: Biochemistry and Molecular Biology

institute: Chang Gung University

summary: Androgen receptor (AR) signaling remains a key driver of castration-resistant prostate cancer, with AR splice variants like AR-V7 mediating resistance to second-generation antiandrogens. Targeting the AR N-terminal domain (NTD) offers a strategy to bypass ligand-binding domain-mediated resistance. We developed a VHL-based (ITRI-90) and a CRBN-based (ITRI-148) PROTAC degrader capable of targeting AR and AR-V7 via NTD. These compounds demonstrate potent antitumor activity towards castration-resistant and enzalutamide resistant xenograft models. Transcriptomic analyses confirm robust suppression of AR and AR-V7 target genes by ITRI-148 in VCaP cells. The transcriptomic effects of these AR-NTD degraders in enzalutamide-resistant C4-2B/MDVR cells closely mirror that of AR knockdown.

overall_design: RNA-seq profiling of VCaP cells treated with DMSO or 1 μM ITRI-148 for 48 hours; and the profiling of enzalutamide-resistant C4-2B/MDVR cells treated with DMSO, 5 μM ITRI-90, 5 μM ITRI-148 or infected with Lentivirus carrying pLKO1 control vector or AR-knockdown shRNA.

first: Chiu-Lien

last: Hung


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [ChIP-seq]

city: Philadelphia

first: YEQIAO

last: ZHOU

address: 421 Curie Boulevard

department: Pathology

institute: University of Pennsylvania

summary: Multiple enhancers, often located across vast genomic distances, regulate key genes in cancer. However, how organization of chromatin topology at individual allele enables cancer type-restricted multi-enhancer gene regulation remains unclear. Using acute protein degradation and time-course population-average chromatin conformation capture in lymphoma, we found that the B-cell lineage-determining transcription factor EBF1 preferentially positions multiple enhancers at loci containing sparsely distributed genes essential for B-cell identity and oncogenesis. Our time-resolved sub-diffraction optical tracing of chromatin architecture of over 100,000 alleles further revealed diverse topological conformations that facilitate multi-enhancer interactions in individual lymphoma cells. Mechanistically, we found that positioning of enhancers at the topological center of the allele is required for their interactions with the target promoter, with EBF1 serving as a barrier to the loop extruding cohesin on the enhancers. These findings, which we demonstrate their generalizability to the T-cell lineage-determining transcription factor TCF1 in T-cell leukemia, suggest that lineage-determining transcription factors establish local radial positioning of enhancers and promoters to enable multi-enhancer regulation of key genes in cancer.

overall_design: ChIPseq was performed to assess protein binding in Granta519 cells with or without EBF1

first: Robert

middle: B

last: Faryabi

first: Yeqiao

last: Zhou


title: Single cell resolution of an open chromatin signature in persister tumor cells

first: Alexandre

last: Gaspar-Maia

address: 200 First street SW

summary: To characterize transcriptional and chromatin accessibility changes that promote survival of residual cancer following chemotherapy, we performed single-cell multiomic profiling (snRNA+snATAC) on a cohort of non-malignant fallopian tube, treatment-naive, and neoadjuvant chemotherapy (NACT)-treated high-grade serous ovarian cancer (HGSOC) samples. From this analysis, we identified an epigenetic signature that precedes expression and defines the drug-tolerant persister state.

overall_design: Nuclei from ovarian cancer tissue were isolated and processed for single cell Multiome sequencing following 10x Genomics protocol.

first: Mihai

last: Dumbrava

first: Wazim

last: Mohammed Ismail

first: Leticia

last: Sandoval

first: Amelia

last: Mazzone

first: Xiaonan

last: Hou

first: Shariska

last: Harrington

first: Scott

middle: H

last: Kauffmann

first: S

last: Weroha


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [HiChIP]

overall_design: HiChIP was performed in Granta519 cells to assess the EBF1 and SMC1 mediated chromatin folding


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [Hi-C]

overall_design: Hi-C was performed to assess genome folding in Granta519 Cas9 cells with or without EBF1


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [Micro-C]

overall_design: Micro-C was performed in Granta519-EBF1-FKBP-KI cells to assess the impact of EBF1 on chromatin folding


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [RNA-seq]

overall_design: RNA was performed to assess gene expression in Granta519 with or without EBF1


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [ATAC-seq]

overall_design: ATAC-seq was performed to assess chromatin accessibility in Granta519 Cas9 cells with or without EBF1


title: Enhancer rewiring orchestrates inflammation and loss of cell identity during muscle stem cell aging [ATAC-seq]

city: Baltimore

first: Supriyo

last: De

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

institute: NIA-IRP, NIH

summary: Loss of regeneration is a key feature of aging organs, often linked to stem cell exhaustion. Skeletal muscle stem cells (MuSCs) undergo age-related numerical and functional decline, contributing to reduced regenerative potential. Using low-input multi-omics, we systematically profiled the epigenome, transcriptome, and 3D genome of MuSCs from individual mice across 3 age groups (young, old, and geriatric) and both sexes. At baseline, young male MuSCs showed reduced expression of cell cycle-related mRNAs. In aged mice, particularly males, MuSCs exhibited early alterations (emerging during the transition from young to old age), including enhanced proinflammatory signaling, and loss of cell identity. Late alterations (emerging during the transition from old to geriatric age) included heightened inflammation, widespread enhancer activation, and extensive 3D genome rewiring. Proinflammatory pathways were enriched for interferon signaling and correlated with endogenous retroviral expression and NFκB activity. Late-stage epigenome and 3D genome rewiring reflected downstream degenerative changes in muscle organization, response to cytokines, and loss of myogenic identity. Thus, progressive molecular shifts may explain the aggravated proliferative deficit and functional impairment observed in MuSCs during aging.

overall_design: Sorted MuSCs (young (n=8), old (n=8), and geriatric (n=7)) were pelleted by centrifugation, lysed, and transposed. The transposed DNA fragments were PCR-amplified, purified, and sequenced on the Illumina platform.

first: Changyou

last: Shi

first: Payel

last: Sen


title: Enhancer rewiring orchestrates inflammation and loss of cell identity during muscle stem cell aging [CUT&RUN]

overall_design: CUT&RUN was performed to profile enhancer histone modifications H3K4me1 and H3K27ac from sorted MuSCs (young (n=5-6), old (n=7-9), and geriatric (n=4-5)) following guidelines in the CUTANA CUT&RUN Protocol (EpiCypher).


title: Enhancer rewiring orchestrates inflammation and loss of cell identity during muscle stem cell aging [RNA-seq]

overall_design: RNA from sorted MuSCs (young (n=7), old (n=8), and geriatric (n=8)) was converted to Illumina libraries using SMART-Seq v4 Ultra Low Input RNA Kit (Takara) according to manufacturer’s instructions.


title: Genome-wide discovery of multiple sclerosis genetic risk variant allelic regulatory activity

city: Cincinnati

first: Matthew

last: Weirauch

state: Ohio

address: 3333 Burnet Avenue

department: Center for Autoimmune Genomics and Etiology (CAGE)

institute: Cincinnati Children's Hospital Medical Center

summary: Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) with a complex etiology involving environmental and genetic factors. Numerous genetic risk loci for MS have been nominated through genome-wide association studies, with most associated variants residing in non-coding regions. However, further work is needed to understand how genetic variation contributes to disease-related alterations to gene expression. Here, we use massively parallel reporter assays (MPRAs) to identify genetic risk variants with genotype-dependent enhancing or silencing activity within a set of 14,276 variants distributed among 233 independent multiple sclerosis risk loci that have reached genome-wide or suggestive significance. We applied our MPRA library to a commercially available B cell line (GM12878) as well as B cell lines derived from two patients with MS. In total, our approach discovered 150 allelic enhancer variants and 286 allelic silencing variants, representing 83 independent MS risk loci. Collectively, our systematic, genome-scale approach implicates causal genotype-dependent gene regulatory mechanisms for 38% of the known or suggestive MS risk loci, providing a unique resource for the discovery of the genetic mechanisms underlying this chronic inflammatory disease.

overall_design: Massively parallel reporter assays to study MS-associated variants for allele-dependent transcriptional enhancer activity, 5 replicates, 3 cell types

first: Leah

last: Kottyan


title: Analysis of nascent transcription by PRO-seq in HUDEP-2 cells following CRISPR editing of BCL11A enhancer

city: Memphis

first: Jian

last: Xu

state: Tennessee

address: 262 Danny Thomas Place, MS 345

institute: St. Jude Children's Research Hospital

summary: CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as a gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. Ehancer RNAs transcribes from enhancer play a role in gene regulation. Here, we apply PRO-seq in wild-type and sg1617-edited HUDEP-2 cells to determine the effects of sg1617 editing on enhancer RNA transcription.

overall_design: PRO-seq was performed in wild-type HUDEP-2 cells and sg1617-edited single-cell clones with 1 bp insertion, 13 bp deletion, or 15 bp deletion.

first: Kaili

last: Wang


title: Analysis of chromatin occupancy in erythroid cells by CUT&RUN following CRISPR editing of BCL11A enhancer

summary: The mechanisms underlying the remarkable efficacy of CRISPR-mediated BCL11A enhancer ablation remain poorly understood. Here, we employ CUT&RUN to investigate active and repressive histone modifications, as well as cohesin and CTCF binding, to uncover epigenetic changes under various editing conditions.

overall_design: The CUT&RUN assay was performed following enhancer knockout or sg1617 editing to assess chromatin alterations. The inactive chromatin mark H3K27me3, as well as the occupancy of cohesin, and CTCF, were examined in wild-type cells, single-cell clones with different INDELs, and bulk-edited CD34+ HSPCs.


title: Analysis of chromatin structure of the BCL11A locus in HUDEP-2 cells by Capture-Pore-C (CPC)

summary: CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as a gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. We describe an evolutionarily conserved, enhancer-dependent chromatin architecture, the enhancer-CTCF rosette, essential for chromatin insulation and the developmental expression of BCL11A in hematopoiesis. CRISPR-mediated disruption of lineage-specific BCL11A enhancers impairs enhancer RNA transcription and NIPBL-cohesin loading, leading to destabilized chromatin configuration, impaired chromatin insulation, and epigenetic silencing of BCL11A. Antisense oligonucleotide-mediated depletion of enhancer RNA silences BCL11A by disrupting chromatin insulation in adult erythroid cells.

overall_design: Capture-Pore-C data were generated from wild-type HUDEP-2 cells, sg1617-edited cells, and enhancer knockout single-cell clones enriched using biotin-labeled probes targeting CTCF binding sites or erythroid enhancers E+55, E+58, and E+62.

first: Juan


title: Enhancer rewiring orchestrates inflammation and loss of cell identity during muscle stem cell aging [Hi-C]

summary: Loss of regeneration is a key feature of aging organs, often linked to stem cell exhaustion. Skeletal muscle stem cells (MuSCs) undergo age-related numerical and functional decline, contributing to reduced regenerative potential. Using low-input multi-omics, we systematically profiled the epigenome, transcriptome, and 3D genome of MuSCs from individual mice across 3 age groups (young, old, and geriatric) and both sexes. MuSCs from aged mice, particularly males, exhibited early alterations (evident in old), including enhanced proinflammatory signaling with loss of cell identity. Late alterations (evident in geriatric) included heightened inflammation, widespread enhancer activation, and 3D genome rewiring. Proinflammatory pathways were enriched for interferon signaling and correlated with endogenous retroviral expression and NFκB activity. Late-stage epigenome and 3D genome rewiring reflected downstream degenerative changes in muscle organization, response to cytokines, and loss of myogenic identity. These progressive molecular shifts may explain the aggravated proliferative deficit observed in MuSCs during aging.

overall_design: Hi-C of sorted MuSCs was performed with a modified low cell number protocol using the Arima-HiC kit (Arima Genomics). ~800,000 MuSCs from 3-4 male mice was used per replicate ((young (n=2), old (n=2), and geriatric (n=4) replicates).


title: Analysis of chromatin occupancy in erythroid cells by CUT&Tag following CRISPR editing of BCL11A enhancer

summary: The mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. Here, we employ CUT&Tag to investigate histone modifications following BCL11A enhancer editing or CTCF disruption.

overall_design: CUT&Tag analysis of H3K27ac was performed in wild-type HUDEP-2 cells, enhancer knockout cells, sg1617-edited cells, and double CBS knockout single-cell clones, each with two independent biological replicates. CUT&Tag analysis of NIPBL was perfromed in wild-type HUDEP-2 cells, sg1617-edited cells, and ASO treated cells. Additionally, CUT&Tag of H3K27ac was conducted in control AAVS-edited and sg1617-edited CD34+ HSPCs following erythroid differentiation.


title: Analysis of CTCF-mediated chromatin interactions at the BCL11A locus in HUDEP-2 cells by Micro-Capture-C (MCC)

summary: Three-dimensional chromatin structures, including promoter-enhancer interactions and CTCF looping, are essential for gene regulation. In this study, we apply Micro-Capture-C (MCC) to analyze chromatin structures at the BCL11A locus.

overall_design: Micro-Capture-C was performed at the BCL11A locus using probes targeting seven CTCF binding sites (CBS) as baits, with experiments conducted in two biological replicates.

first: YoonJung

last: Kim


title: p53 drives epigenetic programs associated with luminal cell identity in breast cancer [RNA-seq]

city: Rehovot

first: Efrat

last: Shema

state: Israel

address: 234 Hertzel

department: Department of Immunology & Regenerative Biology

institute: Weizmann Institute of Science

summary: Breast cancer is one of the most commonly diagnosed cancers among women and the leading cause of death in women under 50. The majority of breast cancers are estrogen receptor α-positive (ER+). Historically, these cancers are treated with therapies, such as tamoxifen, that inhibit ER activity. The TP53 tumor suppressor gene, encoding the p53 protein, is the most frequently mutated gene in breast cancer, and TP53 mutations are associated with diminished tamoxifen response and worse prognosis. Here, we report that in breast cancer cells p53 and ER cooperate to regulate the expression of a set of genes encoding chromatin modifiers. The net result is a global increase in H3K4me3 and decrease in H3K9me3 chromatin marks. The resultant “open” chromatin is associated with increased transcription of luminal cell identity genes and augmented tamoxifen sensitivity. Conversely, diminished p53 control of these chromatin modulators is associated with the evolution of tamoxifen resistance and cancer stem cell properties.

overall_design: MARseq analysis of WT or p53 KO MCF7 cells treated with Tamoxifen or Nutlin

first: Yael

last: Aylon

first: Noa

last: Furth

first: Moshe

last: Oren


title: Analysis of nascent transcription by PRO-cap in HUDEP-2 cells

summary: CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, offering a potential gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. Enhancer RNAs, transcribed from enhancers, play a role in gene regulation. We apply PRO-cap to map the location of active polymerases and transcription start sites.

overall_design: PRO-cap was performed on wild-type HUDEP-2 cells with two biological replicates.


title: Trimethylamine-N-oxide affects cell type-specific pathways and networks in mouse aorta to promote atherosclerotic plaque vulnerability

city: Los Angeles

first: Jenny

last: Cheng

address: 675 Charles E. Young Dr. S

institute: University of California, Los Angeles

summary: We performed single cell RNA-sequencing (scRNAseq) identify which aortic cell types, differentially expressed genes, and biological pathways are affected by trimethylamine-N-oxide (TMAO). We also modeled cell-cell communications and intracellular gene regulatory networks to identify gene networks perturbed by TMAO feeding. The objective of this study was to characterize the cell type-dependent and independent effects of TMAO on key vascular cell types involved in atherosclerosis progression in vivo.

overall_design: We performed single cell RNA-sequencing (scRNAseq) on aortic athero-prone regions, including the acsending aorta, aortic arch, and thoracic aorta, of female Ldlr-/- mice fed control Chow, high-cholesterol (HC), or HC+TMAO diets for three months

first: Xia

last: Yang

first: Diana

last: Shih


title: Loss of function of chromatin remodeler OsCLSY4 led to RdDM-mediated mis-expression of endosperm-specific genes effecting grain qualities

city: Bangalore

first: Shivaprasad

last: Padubidri V.

address: UAS-GKVK Campus

country: India

institute: National Centre for Biological Sciences - TIFR

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Analysis of chromatin accessbility in erythroid cells following CRISPR editing or ASO perturbation of BCL11A enhancer

overall_design: ATAC-seq analysis was performed on wild-type and sg1617-edited HUDEP-2 cells, as well as control AAVS-edited CD34+ HSPCs and Cas9-sg1617 RNP-edited CD34+ HSPCs following erythroid differentiation. Additionally, ATAC-seq was conducted on HUDEP-2 cells treated with either control ASO or E+58 eRNA ASO.


title: Analysis of nascent transcription by EU-seq in HUDEP-2 cells following CTCF degredation

summary: CTCF plays a crucial role in looping and insulation, yet its role in BCL11A regulation remains unclear. Here, we utilize EU-seq to measure nascent transcription in HUDEP-2 cells with CTCF-AID.

overall_design: EU-seq of CTCF-AID HUDEP-2 cells with 5-ph-IAA treatment for 0h, 2h, 4h and 7h.


title: ZNF469 promotes extracellular matrix production in normal and keloid dermal fibroblasts

city: Bangkok

first: Thammachanok

last: Boonto

address: 254 Phaya Thai Rd, Khwaeng Wang Mai, Pathum Wan

department: Biochemistry

country: Thailand

institute: Faculty of Medicine, Chulalongkorn University

summary: Aberrant production of extracellular matrix (ECM) from dermal fibroblasts drives fibrotic skin diseases, which significantly affect the quality of patients’ lives. Current treatment options for skin fibrosis remain limited or ineffective; therefore, new antifibrotic strategies are urgently needed. Gaining insights into molecular control of ECM production could lead to the identification of new therapeutic targets. Recent studies suggest Zinc Finger 469 (ZNF469) as a positive regulator for ECM production; however, its role in skin fibrosis has not been investigated. In this study, we found that knockdown of ZNF469 impaired proliferation, migration, and contraction in human dermal fibroblasts. More importantly, depletion of ZNF469 significantly caused a reduction in collagen production. RNA-sequence analysis of ZNF469-knockdown cells revealed significant suppression of several ECM genes and genes involved in the process of collagen biosynthesis, suggesting ZNF469 as a key regulator of ECM production. To find the clinical relevance, we examined expression of ZNF469 by reanalyzing public bulk and single-cell RNA-sequence data sets of hypertrophic scars and keloids as a representation of skin fibrosis. The RNA sequence of bulk tissues showed that hypertrophic scar and keloid tissues had higher levels of ZNF469 and that ZNF469 and ECM genes were positively correlated. Single-cell RNA sequence of keloid scars revealed restricted expression of ZNF469 in the fibroblast population. Further analysis of fibroblast subpopulations showed specific expression of ZNF469 in mesenchymal fibroblasts, a subpopulation that has been demonstrated to be crucial for collagen overexpression in keloids. Psuedotime analysis indicated ZNF469 as a pioneering factor to establish the mesenchymal fibroblast subpopulation. Supporting this finding, we observed downregulation of various mesenchymal fibroblast markers from our RNA sequence results of ZNF469-depleted dermal fibroblasts, suggesting ZNF469 as a crucial factor for maintaining mesenchymal phenotypes. Finally, to explore the therapeutic potential of ZNF469, we silenced it in human keloid fibroblasts and demonstrated that its depletion reduced proliferation, migration, contraction, and collagen production. Moreover, expression of key mesenchymal fibroblast markers was significantly downregulated in ZNF469-knockdown keloid fibroblasts. Together, we illustrated that ZNF469 promoted the ECM production in dermal fibroblasts and could serve as a novel target for treating skin fibrosis.

overall_design: To investigate the function and impact of ZNF469 on ECM production, ZNF469-knockdown human dermal fibroblasts cell lines were established with short-hairpin RNAs.  Three technical replicated were performed for each group (Control and sh_ZNF469) to obtaine the expression profile followed by trascriptomic analysis.

first: Chaiyaboot

last: Ariyachet

first: Denita

last: Charoenthanakitkul

first: Archittapon

last: Nokkeaw


title: ChIP-seq analysis of control and DOT1L HET cortical nuclei.

first: Marissa

last: Maroni

address: 3400 Civic Center Blvd

summary: We aimed to understand the changes in H3K79me2 deposition upon monoallelic DOT1L loss in cortical nuclei from 10 week old mice.

overall_design: Control and DOT1L HET cortical nuclei were isolated from 10-week-old mice. DOT1L HET mice were heterozygous for floxed DOT1L and both Controls and DOT1L HET were heterozygous for CMV-cre (Strain #:006054). Cortical nuclei were isolated and prepared using the TruSeq ChIP Library Preparation Kit. (n = 4 biological replicates per genotype, 2 male mice and 2 female mice)


title: p53 drives epigenetic programs associated with luminal cell identity in breast cancer [ATAC-seq]

country: Israel

overall_design: ATACseq analysis of WT or p53 KO MCF7 cells treated with Tamoxifen


title: Identification of the bacterial actors of the anaerobic plant biomass delignification

city: MARSEILLE

first: Nicolas

last: VITA

address: 31 ch Joseph Aiguier

institute: CNRS

summary: The anaerobic plant biomass delignification was questioned till recently despite being a key process for plant cell wall degradation in anaerobic biotopes where plant debris accumulate, the carbon mineralization being the limiting step of the Carbon cycle. Besides no actors were identified till now and no data were available on this processus performed by anaerobic cellulolytic bacteria or even on its existence  Using RNAseq, we here report the idenfication of secreted (including cellulosomal) enzymes, especially SGNH hydrolase family members known to be esterases but never described to act on lignin.  We also propose based on our genetic, biochemical and bioinformatic approaches, a new role for the SGNHs in the remodeling of lignin to favor the access to polysaccharides by plant polysaccharides degrading proteins, function that could be useful for the design of biotechnological applications.

overall_design: RNA-seq profiling of R. cellulolyticum grown on more or less lignified substrates at the mid and end of the exponential growth phases

last: Vita

first: Séverine

last: Gagnot

first: Felipe

last: Meija

first: Marion

last: Holmière


title: DOT1L activity limits transcription elongation velocity and favors RNAPII pausing to facilitate mutagenesis by AID. [ChIP-seq]

city: Montréal

first: Javier

last: Di Noia

state: QC

address: 110 Pine Avenue West

country: Canada

institute: Institut de recherches cliniques de Montréal (IRCM)

summary: Activation-induced deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation of immunoglobulin genes, which are required for efficient antibody responses. Collaterally, AID can also mutate a set of off-target genes with oncogenic consequences. The mechanisms that make such loci susceptible to AID are not fully understood. We find that the H3K79 histone methyltransferase DOT1L is located close to AID in the nucleus. Not only does DOT1L activity facilitate CSR, but it also facilitates AID off-target activity, including Igh-cMyc translocations. Genes mutated by AID display high DOT1L activity. DOT1L deficiency causes mostly increased nascent transcription, accompanied by a modest reduction in RNAPII occupancy. By integrating different genomic approaches, we discovered that DOT1L restricts the velocity of transcription elongation proportionally to the level of higher order H3K79 methylation (H3K79me2/3) and favors RNAPII pausing. These transcriptional conditions enhance AID occupancy and, consequently, its activity. Our data indicate a role for DOT1L and H3K79me2/3 in limiting transcription and highlight stronger RNAPII pausing and attenuated RNAPII elongation velocity as critical steps for productive AID association and mutagenesis at target loci. Furthermore, increased transcription elongation velocity could explain both upregulation and downregulation of genes in DOT1L-deficient cells.

overall_design: Quantitative ChIP-seq for RNAPII, S2P-RNAPII, S5P-RNAPII, Rad21 and Nipbl in WT and Dot1l-/- CH12F3-G cells; and ChIP-seq for H3K79me2 and H3K79me3 in WT CH12F3-G cells.

first: Poorani Ganesh

last: Subramani

first: Noé

last: Seija

first: Jana

last: Ridani

first: Jonathan

last: Boulais

first: Paul

last: Donnelly

first: Mélanie

last: Provencher

first: Halil

last: Bagci

first: Christian

last: Poitras

first: Jean-François

last: Côté

first: François

last: Robert


title: DOT1L activity limits transcription elongation velocity and favors RNAPII pausing to facilitate mutagenesis by AID. [TT(chem)-seq]

overall_design:
To investigate changes in nascent transcription upon Dot1l depletion, CH12F3-G Dot1l knockout cells were generated by CRIPSR.
We then perform paired-end TT(chem)-seq of unstimulated WT and Dot1l-/- CH12F3 cells, using S. cerevisiae (strain BY4741) cells as spike-in controls.


title: p53 drives epigenetic programs associated with luminal cell identity in breast cancer [CUT&RUN]

first: Efrat

last: Shema

overall_design: H3K4me3 Cut&Run analysis of WT or p53 KO MCF7 cells

first: Yael

last: Aylon

first: Noa

last: Furth

first: Moshe

last: Oren


title: Heat Shock Transcription Factors (HSFs) Mediate Multilayer Regulations of Heat-Induced Chromatin Dynamics in Plants [DAP-seq]

city: Taipei

first: Lavakau

last: Thalimaraw

address: 128 Sec. 2, Academia Rd, Nankang

institute: Institute of Plants and Microbial biology, Academia Sinica

summary: Eukaryotic organisms remodel chromatin landscapes to regulate gene expression in response to environmental stress. In plants, heat stress (HS) induces widespread chromatin changes, yet the role of early responsive Heat Shock Transcription Factors (HSFs) in chromatin remodeling and their evolutionary conservation remain unclear. Here, we use chromatin accessibility profiling and transcriptomics in Marchantia polymorpha hsf mutants to reveal HSFA1 as a key determinant in positioning cis-regulatory elements (CREs) for stress-induced gene activation. Comparative analysis suggests that this HSFA1-dependent chromatin remodeling mechanism is evolutionarily conserved across land plants. Multi-layered gene regulatory network (GRN) analysis further identifies MpWRKY10 and MpABI5B as HSF-independent regulators of HS responses, providing insights into parallel transcriptional subnetworks. We develop a cross-species and cross-condition machine learning framework that accurately predicts chromatin accessibility and gene expression, demonstrating a conserved regulatory logic of stress-responsive transcription. Our findings establish a scalable computational approach for decoding gene regulatory networks and provide a conceptual framework for how TFs coordinate chromatin architecture to drive stress adaptation in plants and other eukaryotes.

overall_design:
DAP-seq of Marchantia polymorpha HSFA1 in Tak-1 genomic DNA at Day14 of germmalings.
We investigated the binding sites and locations of MpHSFA1 in regulating chromatin dynamics and gene expression under heat stress (HS) using DNA Affinity Purification Sequencing (DAP-seq). The HSFA1_DAP sample consists of in vitro translated MpHSFA1 proteins incubated with Tak-1 genomic DNA. The sequencing results represent the MpHSFA1-bound DNA fragments. For peak calling, we used the Golden Gate expression vector pICH47732 (which was used to express the MpHSFA1 CDS) as a negative control to remove non-specific binding sites. The Empty_DAP sample represents this control.

first: Ming-Jen

last: Yen

first: Kuan-Hung

last: Lin

first: Hsiu-Ru

last: Yang

first: Kulaporn

last: Boonyaves

first: Chia-Yi

last: Cheng

first: Ting-Ying

last: Wu