title: Single-cell and spatial transcriptomics reveals memory CD8+ T cell differentiation fostered by monocytes through TGF-β signaling

city: xian

first: Cangang

state: State...

address: Yanta district, xi 'an city, shaanxi province

institute: xian jiaotong university

summary: Upon antigen recognition, CD8+ T cells undergo a robust expansion and differentiation to give rise to effector and memory CD8+ T cells. The spatial determinants for effector and memory CD8+ T cell fate decision during acute infection is poorly understood. By integrating single-cell RNA sequencing (scRNA-seq) and spatial resolved transcriptomics, we investigated the anatomical locations, cell-cell interactions and molecular mechanisms that contribute to memory CD8+ T cell differentiation. We found naïve CD8+ T cells adopted a divergent differentiation trajectory where they rapidly differentiated into memory precursors (MP) and IFN-responsive cells which represent the entry point of effector T cell lineage.In the spleen, monocytes were largely colocalized with CD8+ MP cells in an antigen stimulation-dependent manner. CD8+ T cells primed with monocytes, compared to DC populations, had significantly increased memory T cell differentiation. Mechanistically, monocytes promoted memory CD8+ T cell differentiation by providing TGF-β signaling. Our study reveals a novel spatial mechanism for memory CD8+ T cell fate decision, shedding lights on the importance of monocytes and TGF-β in fostering memory CD8+ T cell development.

overall_design: The spleen tissues were dissociated into single-cell suspensions from the mice 5 days post-infection (Recipient sample). In the in vivo T cell adoptive transfer model, donor CD8+ 581 T cells (CD45.2+CD8+7AAD-) 582 were sorted and subjected to scRNA-seq (Donor sample).

first: Baojun

first: Lina

last: Sun


title: Induction of secondary cell wall biosynthesis genes and their regulators by melatonin in Glycine max

status: Public on Jun 05 2025

city: Taipei

first: Ying-Chung Jimmy

last: Lin

address: No.1, Sec. 4, Roosevelt Rd., Da'an Dist.

department: Department of Life Sciences

country: Taiwan

institute: National Taiwan University

type: Expression profiling by high throughput sequencing

summary: Plant lodging severely reduced crop yield and quality. Different plant growth regulators (PGRs) have been applied to improve lodging resistance through the regulation of physiological changes, especially on the increase of stem thickness and strength. Melatonin is a pleiotropic PGR for the regulation of plant growth and development. In this study, we demonstrated that the exogenous treatment of melatonin to Glycine max significantly enhanced plant lateral growth by increasing stem diameter. In addition to the stem thickness, secondary cell wall (SCW) deposition acts as another critical factor for stem rigidity for lodging resistance. To understand whether exogenous treatment of melatonin would regulate SCW biosynthesis genes, we performed transcriptomic analyses on the stems of Glycine max with or without melatonin treatment. Through the differentially-expressed-genes (DEGs) analyses, many SCW biosynthesis genes were found to be regulated by melatonin, including the cellulose, hemicellulose and lignin biosynthesis enzymes. We also found that the two known master regulators, NAC and MYB, of SCW biosynthesis genes were induced under melatonin treatment, which further supported our observation on the differential expression of SCW biosynthesis genes. Our study highlighted the improvement of lodging resistance by the exogenous treatment of melatonin through the increase of plant stem thickness and the regulation of SCW biosynthesis genes and their upstream TFs in Glycine max.

overall_design: To reveal the potential mechanism of melatonin on the effect of stem thickness increase in Glycine max, we performed time-course transcriptomic analyses to find the differentially expressed genes (DEGs) after melatonin treatment.

first: Chung-Ting

last: Kao

first: Ya‑Wen

last: Huang

first: Jia‑Cheng

first: Tsai‑Pin

last: Chou

first: Chang‑Hung

last: Chen

first: Shang‑Che

last: Kuo

first: Cho‑Chun

first: Gui‑Jun

last: Li

first: Jhong‑He

last: Yu

first: Ying‑Lan

first: Kai

last: Xia

first: Ying‑Chung Jimmy


title: Inhibition of methylthioadenosine phosphorylase provides protection from experimental acute kidney injury

status: Public on Jun 04 2025

city: San Antonio

first: Yidong

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

country: USA

institute: UT Health Science Center at San Antonio

summary: This dataset presents transcriptomic profiling of mouse kidney cortex in a model of ischemia-reperfusion injury (IRI) with and without methylthioadenosine phosphorylase (MTAP) inhibition (MTAPi). Male C57BL/6J mice were assigned to sham (n=6), IRI with vehicle (n=6), or IRI with MTAP inhibitor (MTAPi; n=5) groups. MTAPi (MT-DADMe-ImmA, AdooQ Bioscience, Cat# A13651) was administered intraperitoneally 4.5 hours before ischemia. Total RNA was extracted from kidney cortices, depleted of rRNA, and processed using a directional library prep kit. Libraries were sequenced on the Illumina NovaSeq 6000 platform (100 bp paired-end). Transcriptomic analysis revealed that MTAP inhibition reduced injury-associated gene expression, suppressed inflammatory and stress pathways, and preserved epithelial and metabolic gene programs. These data provide insight into the protective role of MTAP inhibition in acute kidney injury.

overall_design: Male C57BL/6J mice (10–12 weeks old) were assigned to three groups: sham surgery (n=6), ischemia-reperfusion injury (IRI) with vehicle (n=6), and IRI with MTAP inhibitor (MTAPi) treatment (n=5). IRI was induced by clamping both renal pedicles for 30 minutes, followed by 24 hours of reperfusion. MTAPi (MT-DADMe-ImmA, AdooQ Bioscience, Cat# A13651, 20 mg/kg) or vehicle (10% DMSO in PBS) was administered intraperitoneally 4.5 hours before ischemia. Kidney cortex tissue was collected for RNA extraction (QIAGEN RNeasy Mini Kit, Cat# 74104), rRNA depletion, and directional RNA library preparation. Libraries were sequenced using the Illumina NovaSeq 6000 with 100 bp paired-end reads.

first: Afaf

last: Saliba

first: Kumar

last: Sharma

first: Zhao

last: Lai


title: Creb5 controls its own expression and directly induces the joint-interzone regulatory program [ATACseq_Sox9_GFP]

city: Boston

first: Andrew

middle: Bruce

last: Lassar

state: MA

address: 240 Longwood Ave.

department: Biological Chemistry and Molecular Pharmacology

institute: Harvard Medical School

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Our prior work indicated that the transcription factor Creb5 plays a crucial role in the genesis of synovial joints.  Here, we perform an integrative analysis of the transcriptome, chromatin accessibility, and Creb5-chromatin occupancy in Creb5-expressing synovial joint progenitor cells. This analysis revealed that Creb5 directly binds to both its own two promoters and to the regulatory regions of the early joint markers, Gdf5 and Sfrp2; each of whose expression in the joint-interzone is Creb5-dependent.  Functional enhancer analysis indicated that Creb5 binding sites in its own two promoters are necessary for these sequences to drive transgene expression in a pattern that mimics endogenous Creb5 expression.  While Creb5 directly regulates both Gdf5 and Sfrp2 expression in the inner joint-interzone by binding to either CREs or TREs in their regulatory elements,  Creb5 activates Barx1 expression specifically in the outer joint-interzone; and thus initiates both the formation of the joint-interzone and regionalization of cell fates within this tissue.

overall_design: Guided by fluorescence of both tdTomato and EGFP, we dissected the developing knee joints of E14.5 Creb53XHA-P2A-tdTomato/+; Sox9iresEGFP/+ embryos.  Following collagenase treatment and filtration to isolate single cells, we sorted the cells from the developing knees into three populations: those that express either Creb5HA-tdTomato, Sox9-ires-EGFP, or both Creb5HA-tdTomato and Sox9-ires-EGFP.  To identify Creb5-dependent regulatory regions in knee joint progenitor cells we compared chromatin accessibility in Fluorescence-Activated Cell (FAC) sorted Creb5+Sox9+, Creb5+Sox9-, and Creb5-Sox9+ cells, isolated from the developing knees of E14.5 Creb53XHA-P2A-tdTomato/+; Sox9 iresEGFP/+ embryos from two independent litters by ATAC-Seq.

first: Cheng-Hai

last: Zhang

first: Aref

last: Shahini

first: Laura

last: Cook

first: Meeta

last: Mistry

first: Jongkil

last: Kim

first: Heather

last: Wick

first: Len

last: Pennacchio

middle: B


title: APOE Protects Against Severe Infection with Mycobacterium tuberculosis by Restraining Production of Neutrophil Extracellular Traps (scRNA-Seq)

city: Seattle

first: Aderem

last: Lab

state: WA

address: 307 Westlake Avenue North, Suite 500

institute: Seattle Children's Research Institute

summary: Mice lacking apolipoprotein E (APOE, Apoe-/- mice) are highly susceptible to infection with Mycobacterium tuberculosis (Mtb) but the underlying immune dysregulation has been unclear. We found that Mtb infected Apoe-/- mice have significantly elevated levels of neutrophils in their lungs compared to controls and demonstrated that depleting neutrophils, depleting pDCs, blocking type1 interferon signaling, and blocking LTB4 receptor signaling all improved the outcome of Mtb-infected Apoe-/- mice. Neutrophils can undergo a specialized form of cell death termed NETosis in which activation of the enzyme PAD4 leads to extrusion of neutrophil extracellular traps (NETs). NETs have been associated with severe tuberculosis in other mouse models and in humans but a role of NETs in the pathology has not been directly established. We demonstrate that blocking PAD4 activation leads to a decrease in NETs in the lungs and, strikingly, completely reverses the hypersusceptiblity of Apoe-/- mice.

overall_design:
To examine the role of Apoe in the responses of pulmonary immune cells to infection with Mtb in vivo, we fed B6, Apoe-/-, and Ldlr-/- mice a normal or high cholesterol diet, infected them with ~50 CFU of Mtb H37Rv via aerosol, and isolated CD45+, IV- cells from the lung prior to infection (day 0) or at 14 days following infection.
This population was analyzed by single-cell RNA-seq using the 10X Genomics platform.

first: Alan

middle: H

last: Diercks

first: Elizabeth

middle: S

last: Gold

first: Dong

last: Liu

first: Dat

last: Mai


title: Impact of molecular androgen and estrogen response scores on outcomes following neoadjuvant pembrolizumab in muscle-invasive bladder cancer

city: San Diego

first: Elai

last: Davicioni

state: California

address: 9725 Lusk Blvd

institute: Veracyte, Inc

summary: Gene expression study of the Phase 2 PURE-01 clinical trial

overall_design: Retrospective analysis of tumor transcriptomes from PURE-01 clinical trial

first: V

last: Tateo

first: J

middle: J

last: De Jong

first: A

last: Cigliola

first: B

middle: A

last: Maiorano

first: C

last: Mercinelli

last: Proudfoot

first: E

last: Briganti

first: F

last: Montorsi

last: Necchi


title: RNA sequencing of High Grade Serous Ovarian cancer

city: Oxford

first: Lena

last: Rai

address: Headley Way

department: Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

summary: In this study we have validated the prognostic strength of the Oxford Classic-defined EMT in an independent cohort of HGSOC patients- 'Brescia Ovarian Tumour'. Additionally, we carried out whole transcriptome sequencing of FLOW-sorted tumour epithelial cells from five HGSOC patients at diagnosis.

overall_design: RNA sequencing of 139 fresh-frozen bulk tumours (Brescia samples) was carried out using NEB Next® Ultra II RNA Library Prep Kit for Illumina. Whole-transcriptome sequencing using SMARTer Stranded total RNA-Seq kit v2 Pico input was carried out on FLOW-sorted tumour epithelial cells from five High Grade Serous Ovarian cancer patients. Paired-end sequencing of the libraries was carried out on Illumina NovaSeq 6000 S4 flowcell with PE150.

first: Antonella

last: Ravaggi

first: Eliana

last: Bignotti

first: Franco

last: Odicino

first: Ahmed

last: Ahmed


title: Validation of single-cell transcriptomic profiles from Illumina and MGI Tech sequencing platforms.

city: Anderlecht

first: Valerie

last: Vandenbempt

state: Vlaams-Brabant

address: Lenniksebaan 808

country: Belgium

institute: Université Libre de Bruxelles

summary: Different companies have developed sequencing platforms with a wide variety of strengths and drawbacks. Illumina is currently used for high-throughput and highly accurate short-read sequencing, however, other companies, such as MGI Tech, have developed a similar sequencing method at a lower cost. The accuracy, sensitivity, and reproducibility of Illumina and MGI Tech have been compared, showing high similarity. While many comparisons exist, the performance of MGI on gene expression and cell clustering for single-cell applications is unknown. In this study, we performed single-cell RNA sequencing on three human breast cancer samples to compare the cell clustering or average gene expressions for the reads generated by Illumina and MGI sequencing. We observed unique platform-specific cells detected and show that these low-quality cells appear randomly and could only affect minor cell populations.

overall_design: Single cell RNA-sequencing by illumina or MGI sequencer

first: Monika

last: Mohenska

first: Kirk

last: Jensen

first: Kellie

last: Wise

first: Michael

last: Roach

first: Jose

last: Polo

first: Esteban

middle: N

last: Gurzov

first: Luciano

middle: G

last: Martelotto


title: Gene Regulatory Programs that Specify Age-Related Differences during Thymocyte Development (RNA-Seq)

city: Ithaca

first: Andrew

last: Grimson

state: NY

address: 445 Biotechnology Bldg

department: Molecular Biology and Genetics

institute: Cornell University

summary: T cell development is fundamental to immune system establishment, yet how this development changes with age remains poorly understood. Here, we construct a transcriptional and epigenetic atlas of T cell developmental programs in neonatal and adult mice, revealing the ontogeny of divergent gene regulatory programs. We identify a gene module that diverges with age from the earliest stages of genesis and includes programs that govern effector response and cell cycle regulation. Interestingly, the epigenetic atlas reveals that neonates possess more accessible chromatin during early thymocyte development, likely establishing poised gene expression programs that manifest later in thymocyte development. Finally, we leverage this atlas, employing a CRISPR-based perturbation approach coupled with single-cell RNA sequencing as a readout to uncover a conserved transcriptional regulator, Zbtb20, that contributes to age-dependent differences in T cell development. Taken together, our study defines transcriptional and epigenetic programs that regulate age-specific differences in T-cell development.

overall_design: We set out to determine how transcriptomic profiles change between adults and neonates during thymocyte development. To this end, we isolated cells from thymi and spleens of neonatal (5-7 days post-birth) and adult (12 weeks) mice and subjected these cells to RNA-seq. Antibodies to CD4, CD8, CD44 and CD25 were utilized for flow cytometry to isolate thymocytes at different stages of development. We obtained cells from four stages of thymocyte development: pooled Double Negative (DN1-3 cells; referred to as DN here), double-positive and single-positive CD8 cells (DP and SP8, respectively) and mature splenic naïve CD8+ T cells. We generated four biological replicates of RNA-seq profiles from both adult and neonatal mice for all four developmental stages (DN, DP, SP8 and CD8).

first: Divya

last: Ganapathi Sankaran

first: Viviana

middle: I

last: Maymi

first: Brian

middle: D

last: Rudd

middle: W


title: Generation of cellular diversity through temporal patterning of radial glia during mouse corticogenesis

city: Shanghai

first: XIAOSU

last: LI

state: Shanghai

address: 131 Dongan Road

country: China

institute: Fudan University

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

overall_design: Refer to individual Series


title: Gene Regulatory Programs that Specify Age-Related Differences during Thymocyte Development

summary: T cell development is fundamental to immune system establishment, yet how this development changes with age remains poorly understood.  Here, we construct a transcriptional and epigenetic atlas of T cell developmental programs in neonatal and adult mice, revealing the ontogeny of divergent gene regulatory programs. We identify a gene module that diverges with age from the earliest stages of genesis and includes programs that govern effector response and cell cycle regulation. Interestingly, the epigenetic atlas reveals that neonates possess more accessible chromatin during early thymocyte development, likely establishing poised gene expression programs that manifest later in thymocyte development. Finally, we leverage this atlas, employing a CRISPR-based perturbation approach coupled with single-cell RNA sequencing as a readout to uncover a conserved transcriptional regulator, Zbtb20, that contributes to age-dependent differences in T cell development. Taken together, our study defines transcriptional and epigenetic programs that regulate age-specific differences in T-cell development.

overall_design: We identify transcription factors whose activities likely underlie alternative gene expression programs and phenotypes in adults and neonatal thymocytes. In particular, we identify and focus on a conserved transcriptional regulator, Zbtb20, which is preferentially expressed in adult HSCs and during thymocyte development. We employ a CRISPR-based perturbation approach coupled with single-cell RNA sequencing as a readout to validate the role of Zbtb20 in thymocyte development. To compare T cell development in control and Zbtb20 knockdown ATOs, we generated ATOs from EGFP-Cas9 HSCs (Lineage−, Sca1(hi), c-Kit(hi) HSCs) that either received no gRNA, gRNAs against EGFP (sgGFP) or against Zbtb20 (sgZbtb20-1 or 2), and harvested thymocytes at different timepoints.

first: HongYa

last: Zhu


title: NOTCH1 reverses immune suppression in small cell lung cancer through reactivation of STING

city: Bethesda

first: Nitin

last: Roper

state: MD

address: 37 Convent Drive

department: Developmental Therapeutics Branch

institute: National Cancer Institute

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Transcriptomic profiling of vitiligo patients shows polar immune dysregulation in involved and uninvolved skin

status: Public on Jun 03 2025

city: New York City

first: Agata

last: Kurowski

address: 1180 5th Avenue

department: Department of Dermatology

institute: Icahn School of Medicine at Mount Sinai

summary:
Background: Vitiligo is a chronic autoimmune skin depigmenting disorder, with a major impact on quality of life. Therapeutic options are still limited, with only one topical JAK inhibitor being FDA-approved. Although vitiligo is primarily regarded as a Th1/IFN-driven disease, emerging evidence suggests the involvement of additional immune axes, but their relevance to disease pathogenesis remains unclear.
Objective: To obtain a global cutaneous transcriptomic profile of lesional and nonlesional vitiligo.
Results: Robust inflammatory dysregulation was captured not only in lesional, but also nonlesional vitiligo skin relative to healthy controls. Lesional samples demonstrated upregulation of Th1 (OASL, CXCL9, CXCL10), Th2 (IL4, IL4R, CCL13, CCL17, CCL22, CCL26), and Th17/22 (IL20, S100A7, S100A8, S100A9, PI3) related markers. Similarly, nonlesional samples demonstrated activation of Th1 (CXCL9, OASL), Th2 (IL4R, IL10, CCL13, CCL17, CCL22), and Th17/22 (PI3, DEFB4A) associated markers. Clinical severity scores (VASI and/or VIDA) significantly and positively correlated with multiple inflammatory mediators (i.e., CXCL14, IL25, IL17RC) in lesional and/or nonlesional vitiligo skin. On a single-cell level, IL13 and IFNG expression were primarily found in nonlesional helper T cells and in lesional proliferating T cells, respectively.
Conclusions: Our findings show that immune dysregulation in vitiligo involves immune axes beyond Th1/Tc1, with upregulation of particularly type 2 markers already in nonlesional skin, suggesting a role during early lesion formation.
Capsule Summary: We suggest that the inflammatory dysregulation of vitiligo is more complex than previously appreciated, involving not only Th1, but also Th2 and some components of Th17/22 immune axes, even in clinically normal appearing skin.
Clinical implications: The dysregulation of multiple inflammatory mediators in nonlesional vitiligo skin might have major implications for future targeted treatment approaches.

overall_design:
Methods: We performed bulk RNA-sequencing combined with real-time PCR and immunohistochemistry of skin biopsies from 15 lesional and nonlesional vitiligo samples and compared them to 14 matched healthy controls. Results were corroborated by single-cell RNA sequencing.

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

first: Patrick

middle: M

last: Brunner

first: Eden

last: David

first: Ester

last: Del Duca

first: Meredith

last: Manson

first: Malini

middle: P

last: Naidu

first: Lauren

middle: R

last: Port

first: Jesus

last: Gay-Mimbrera

first: Pedro

last: Gómez-Arias

first: Natalia

last: Alkon

first: Jessica

last: Beaziz Tordjman

first: Yeriel

last: Estrada

first: Yael

last: Renert-Yuval

first: Juan

last: Ruano

first: Emma

last: Guttman-Yassky


title: DNA methylomes undergo distinct waves of remodeling during thymic T cell development and do not acquire a proliferation-induced imprint.

city: Berlin

first: Julia

middle: K

last: Polansky

address: Augustenburger Platz 1

country: Germany

institute: Berlin Institute of Health @ Charité Universitätsmedizin Berlin

type: Methylation profiling by genome tiling array

summary: Dynamic changes of the DNA methylation (DNAmeth) landscape drive differentiation of mature T lymphocytes. However, the role of DNAmeth-mediated regulation in thymocyte development remains unclear. Thus, we generated genome-wide DNAmeth profiles of eight defined human thymocyte subsets, revealing two waves of DNAmeth remodeling: first before TCR rearrangement, then during final maturation. Transcriptomic changes occurred also during phases of DNAmeth stability, indicating decoupled dynamics of epigenetic and transcriptional regulation. In contrast to mature T cells, thymocytes were protected from proliferation-induced changes in the DNA methylome. Finally, the DNAmeth profiles were useful as biomarkers to assess the quality of an in vivo mouse model of human T cell development. This study revealed unexpected dynamics of DNAmeth-mediated control of human thymocyte development and displayed the capacity of DNAmeth profiles to serve as biomarkers for successful T cell generation in model systems and, as a possible future application, during manufacturing of therapeutic cell products.

overall_design: To assess the possible influence of patient's DNA-methylation status on human thymocyte development, we performed comparative analysis of eight thymocyte subsets (n=5). Additionally, DNA methylation profiles were compared to thymocytes from a humanized mouse model (NSGW41hlL7) after umbilical cord blood hematopoietic stem cell graft (n=4).

first: Frederik

last: Hamm

first: Emilie

last: Coppin

first: Mingxing

last: Yang

first: Alisier

last: Malard

first: Anne

last: Schulze

first: Marcel

last: Finke

first: Dania

last: Hamo

first: Abdulrahman

last: Salhab

first: Gilles

last: Gasparoni

first: Jörn

last: Walter

first: Mir-Farzin

last: Mashreghi

first: Joachim

last: Photiadis

first: Claudia

last: Waskow


title: Microvascular preservation & cardiomyocyte hyperplasia underlie adaptive right ventricle development in congenital heart disease-pulmonary arterial hypertension

city: San Francisco

first: Michael

last: Smith

state: CA

address: 550 16th St, 5th Floor

department: Pediatrics

institute: UC San Francisco

summary: Right ventricular (RV) failure is the primary cause of death among patients with pulmonary arterial hypertension (PAH). Patients with congenital heart disease-associated PAH (CHD-PAH) demonstrate improved outcomes compared to patients with other forms of PAH, which is related to the maintenance of an adaptively hypertrophied RV. In an ovine model of CHD-PAH, we aimed to elucidate the cellular, microvascular, and transcriptional adaptations to congenital pressure overload that support RV function. Fetal surgery was performed on late gestation lambs to insert an aortopulmonary graft, leading to a persistent congenital left-right shunt and RV pressure load. At 3 days and 4-6 weeks of life, shunt RV myocardial structure, growth mechanisms, and transcriptomes were compared to age-matched control and unoperated fetal RV. At 4-6 weeks of age, shunt lambs demonstrate significant RV enlargement (shunt 37.1±2.9g vs control 15.9±1.0g, p<0.001) but maintain stable microvascular density (fetal 3.0±0.6 vs shunt 2.9±0.3 vs control 3.1±0.6 capillaries per 1000 µm3, p>0.05). Shunt RV cardiomyocytes are significantly more numerous and smaller by cross-sectional area than age-matched controls (shunt 73.3±5.5µm2 vs control 99.2±4.9µm2, p=0.013). At 3 days, shunt RV cardiomyocytes show evidence of increased proliferative capacity and ongoing hyperplasia compared to controls. RNA sequencing analyses reveal a distinct transcriptomic profile in shunt RV consistent with a delay in terminal differentiation and metabolic adaptations to support adaptive function. This study provides novel insights into the roles of microvascular preservation and cardiomyocyte hyperplasia in the development of adaptive RV hypertrophy in CHD-PAH.

overall_design: RNA sequencing was performed on snap frozen tissue obtained from the right ventricle free wall of fetal lambs, 4-6 week old control lambs, and 4-6 week old shunt lambs. Shunt animals had previously undergone a fetal surgery to insert an aortopulmonary graft that introduced a congenital left-to-right shunt and pressure load on the right ventricle.

first: Eleana

last: Guardado

first: Jason

last: Boehme

first: Sanjeev

last: Datar

first: Emin

last: Maltepe

first: Naveen

last: Swami

first: Gary

last: Raff

first: Aaron

last: Bodansky

first: Prakash

last: Chelladurai

first: Joseph

last: Moreno

first: Annelise

last: Prince

first: Nevan

last: Powers

first: Guo

last: Huang

first: Vinicio

last: De Jesus Perez

first: Jeffrey

last: Fineman


title: PACT suppresses PKR activation through dsRNA binding and dimerization, and is a therapeutic target for triple-negative breast cancer

city: West Lafayette

first: Kyle

last: Cottrell

state: IN

address: 201 S University Street

department: Medicine

institute: Purdue University

summary: Triple-negative breast cancer (TNBC), the deadliest breast cancer subtype, lacks broadly applicable targeted therapies. Induction of ‘viral mimicry’ through activation of viral double-stranded RNA (dsRNA) sensors has potential therapeutic applications for TNBC and other cancers. Suppressors of dsRNA sensing prevent sensing of endogenous dsRNAs and resulting autoimmunity. Depletion of the suppressor of dsRNA sensing ADAR1 causes activation of dsRNA sensors and cell death in many cancer cell lines. These ADAR1-dependent cells are generally also dependent on the dsRNA-binding protein PACT, which is highly expressed and essential in many TNBC cell lines. While PACT is known as an activator of the dsRNA sensor PKR, overexpression of PACT had no effect on activation of PKR in multiple breast cancer cell lines. Conversely, depletion of PACT in PACT-dependent cell lines caused robust activation of the dsRNA sensor PKR and cell death, in addition to induction of integrated stress response genes and NF-κB targets. These phenotypes were entirely dependent on PKR. Rescue experiments revealed that PACT dimerization and dsRNA binding is required to suppress PKR activation. While depletion of PACT alone in ADAR1/ PACT-independent cell lines had no effect on PKR activation, combined depletion of both PACT and ADAR1 in those cell lines caused robust PKR activation and cell death, supporting a partially redundant role for ADAR1 and PACT in suppression of dsRNA sensing. Taken together, these findings support a vital role for PACT in suppression of PKR activation, and highlight the therapeutic potential of targeting PACT to treat TNBC.

overall_design: Gene expression analysis using RNAseq for two triple-negatvie breast cancer cell line with (sgPACT-2) or without (sgC1 or sgNTA)  depletion of PACT by CRISPR-Cas9. Three replicates for each condition.

first: Addison

last: Young


title: Activin A prevents hyper-responsiveness to VEGF in pathological blood vessels by perturbing trafficking of activated VEGFR2

city: Chicago

first: Andrius

last: Kazlauskas

state: IL

address: 1855 W Taylor Street

department: Ophthalmology & Visual Sciences

institute: University of Illinois Chicago

summary: While quality of life compromising afflictions such as diabetic retinopathy (DR) are driven by vascular endothelial growth factor (VEGF), there is a growing appreciation that the concentration of VEGF is not the only influencer of vascular dysfunction within the retina. Activin A (activin), a ligand of the transforming growth factor β superfamily (TGF-β), attenuated VEGF-induced VE-cadherin disorganization, pore formation and permeability of primary human retinal endothelial cells (HRECs). Efforts to further investigate the mechanism of this phenomenon revealed that activin reduced expression of Rab11, which was required for the activin effect. The activin effect was not observed in cells with suppressed expression of the endosomally-localized protein tyrosine phosphatase, (PTP1b), whereas PTPs present on the plasma membrane were dispensable. Activin attenuated VEGF-mediated phosphorylation of VEGF receptor 2 (VEGFR2) at time points 30 min and longer post stimulation. Together these data support the concept that activin suppressed VEGF-induced barrier relaxation by perturbing trafficking of activated VEGFR2. This activin-based suppression of responsiveness to VEGF was compromised in the endothelium of pathological blood vessels from patients who developed end-stage proliferative diabetic retinopathy (PDR). This defect rendered HRECs hyper-responsive to VEGF and was not observed in retinal vessels of diabetic mice, which do not develop the angiogenic forms of DR. These data provide novel insights regarding the pathogenesis of PDR in patients.

overall_design: RNA-Seq analysis of human retinal endothelial cells (HRECs) treated with activin (50ng/mL) or vehicle for 24h.

first: Basma

last: Baccouche

first: Maximilian

last: McCann

first: Janani

last: Rajasekar

first: Nguyễn Thị Thanh

last: Nhàn

first: Kaori

last: Yamada


title: Uncovering the novel signaling components in warm temperature mediated regulation of transcriptome in Arabidopsis thaliana

city: New Delhi

first: Mukesh

last: Jain

state: New Delhi

address: New Mehrauli Road

country: India

institute: Jawaharlal Nehru University

summary: Transcriptome analysis suggests that lrb123 and pif3 mutants have similar gene expression profiles with pif4 mutants, suggesting that LRBs and PIF3 have overlapping functions with PIF4. Further analysis revealed that most of the genes induced by PIF4 in a temperature-dependent manner are strongly downregulated. These genes belong to the Auxin/BR growth hormone pathways, wherein genes involved in auxin/BR biosynthesis and signalling are severely perturbed in response to 27°C compared to 22°C. These results indicate that LRBs and PIF3-mediated induction of PIF4 target genes in response to warm temperature is likely through PIF4. Moreover, our data also demonstrate that both LRBs and PIF3 inhibit the expression of light-responsive genes in response to warmth, further highlighting the role of LRBs and PIF3 in promoting thermomorphogenesis, is by directly promoting growth-responsive genes, and second by inhibiting light signalling genes. Together, we conclude that LRBs and PIF3 are novel components of the thermosensory pathway and are essential to promote themomorhogensis, likely by enhancing the PIF4 activity and down gene expression.

overall_design: To understand the mechanism of regulation of thermomorphogenesis by LRB E3 ubiquitin ligases and PIF3 transcription factor, whole transcriptome (RNA-seq) analysis of lrb123 and pif3 were performed in comparison to the wild-type (Col-0) under normal and warm temperature conditions. Also, we compared their gene expression profiles with the pif4-101 mutant to know the extent of overlapping function in regulating temperature-mediated transcriptome.

first: Gangappa

last: Sreeramaiah

first: Singhal

last: Chirag

first: Das

last: Sreya

first: Rajkumar

last: Mohan

first: Jain

last: Mukesh


title: The catalytic efficiency of METTL16 affects cellular methyltransferase activities by governing the intracellular S-adenosylmethionine setpoint

city: Dallas

first: Nicholas

last: Conrad

state: TX

address: 6000 Harry Hines Blvd

department: Microbiology

institute: UT Southwestern Medical Center

summary: S-adenosylmethionine (SAM) regulates multiple cell processes as the methyl donor for cellular methyltransferases. The N6-methyladenosine (m6A) methyltransferase METTL16 regulates the expression of the SAM synthetase MAT2A, but it has been unclear if this regulation supports SAM homeostasis in a physiologically relevant way. Here, we used a degron and complementation strategy in HCT116 cells to demonstrate that disruption of MAT2A regulation by METTL16 has consequences for a wide variety of SAM-dependent processes including histone methylation, translation, m6A and m6Am RNA methylation. We also identified several U6 snRNA psuedogenes, including those with non-canonical consensus motifs, that are methylated by METTL16. Complementation by a catalytically hyperactive METTL16 abrogates MAT2A regulation and decreases intracellular SAM concentrations. Moreover, these complemented cells are hypersensitive to treatment with a MAT2A inhibitor and to deletion of the MTAP gene, which is lost in nearly 15% of all cancers. These findings support the conclusion that the catalytic efficiency of METTL16 helps establish the SAM setpoint in cells and that this function could be exploited as a potential treatment for MTAP-deficient cancers.

overall_design: 3 biological replicates of GLORI-Seq +/- 72 hr of auxin treatment in EV, K163A, and WT complemented METTL16-AID cells. Riboerased RNA.

first: Juliana

last: Flaherty

first: Enakshi

last: Sivasudhan

first: Matthew

last: Tegowski

first: Zheng

last: Xing

first: Madeline

last: McGinnis

first: Olga

middle: V

last: Hunter

first: Kyah

last: Featherston

first: Komal

last: Sethia

first: Benjamin

last: Tu

first: Kate

last: Meyer


title: Transcriptional analysis of developing Aspergillus fumigatus biofilms reveals metabolic shifts required for biofilm maintenance

city: Hanover

first: Charles

last: Puerner

state: NH

address: 1 Rope Ferry Rd

department: Microbiology and Immunology

institute: Geisel School of Medicine at Dartmouth College

summary: Aspergillus fumigatus is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in a broad spectrum of individuals. Diagnosis and treatment of aspergillosis often occur during stages of infection when A. fumigatus has formed dense networks of hyphae within the lung. These dense hyphal networks are multicellular, encased in a layer of extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, characteristics which are defining features of a microbial biofilm. A mode of growth similar to these dense hyphal networks observed in vivo can be recapitulated in vitro using a static, submerged biofilm culture model. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized an RNA sequencing approach to evaluate changes in transcript levels during A. fumigatus biofilm development. These analyses revealed an increase in transcripts associated with fermentation and a concomitant decrease in oxidative phosphorylation related transcripts. Further investigation revealed that ethanol and butanediol fermentation is important for mature biofilm biomass maintenance. Correspondingly, a gene (silG), a predicted transcription factor, was observed to also be required for mature biofilm biomass maintenance. Taken together, these data suggest temporal changes in A. fumigatus metabolism during biofilm development are required to maintain a fully mature biofilm.

overall_design: RNAseq profiling of A. fumigatus strain CEA10 submerged biofilm tissue harvested at 12-, 18-, 24-, and 30-hours of development in three biological replicates.

first: Kaesi

last: Morelli

first: Joshua

last: Kerkaert

first: Jane

middle: T

last: Jones

first: Katherine

last: Quinn

first: Sandeep

last: Vellanki

first: Robert

last: Cramer


title: A distinct mechanism of epigenetic reprogramming silences PAX2 and initiates endometrial carcinogenesis

first: Subhransu

last: Sahoo

address: 5323 Harry Hines Blvd

department: Pathology


title: NEXN protects against vascular calcification by promoting SERCA2 SUMOy-lation and stabilization

city: Guangzhou

first: Canzhao

last: Liu

address: Gongye Street

institute: Southern Medical University

summary: Vascular calcification, a key risk factor for cardiovascular diseases, is driven by the phenotypic transition of vascular smooth muscle cells (VSMCs) from a contractile to an osteogenic phenotype. NEXN, a protein highly associated with heart function, has also been implicated as a potential susceptibility factor in the development of coronary artery disease, but its role in the progression of vascular calcification remains unclear. In this study, multi-transcriptomics analysis and various animal models were used to explore the cell-specific roles and molecular mechanisms of NEXN in vascular calcification. VSMC-specific NEXN knockout exacerbated calcification, while NEXN overexpression alleviated it. NEXN was shown to interact with SERCA2, enhancing its SUMOylation, stability, and function, thereby protecting against calcification. These findings suggest that NEXN modulates vascular calcification through SERCA2 stabilization, offering potential therapeutic strategies by targeting NEXN-SERCA2 interactions or enhancing SERCA2 SUMOylation to prevent vascular calcification and its complications.

overall_design: RNA-seq profiling of HASMCs pre-transfected with NC or Ad-NEXN followed by high-Pi stimulation.

first: Wenjie

last: Guo

first: Wenjing

first: Boliang

last: Chen

first: Zexuan

last: Lin

first: Zhuohua

last: Wen

first: Jiamin

last: Ye

first: Wei

last: Feng

first: Xin

first: Jianyun

last: Yan

first: Pingzhen

last: Yang

first: Kunfu

last: Ouyang

first: Yifei

last: Li

first: Hanyan

first: Caiwen

last: Ou


title: Peptidylarginine deiminase 4 deficiency improves elastase-induced formation of neutrophil extracellular traps and emphysema in mouse lung

city: Chiba

first: Jun

last: Ikari

state: Chiba

address: 1-8-1 Inohana Chuo-ku Chiba, Chiba, Japan

country: Japan

institute: Chiba University

summary: Neutrophil extracellular traps (NETs) are associated with the extracellular release of nuclear chromatin decorated with cytoplasmic proteins. Excessive release of NETs has been reported in chronic lung diseases, including chronic obstructive pulmonary disease (COPD). However, the role of NETs in the pathogenesis of COPD remains unclear. Peptidylarginine deaminase 4 (PAD4) contributes to NET formation. Therefore, in an elastase (ELS)-induced emphysema mouse model, we examined the role of PAD4 using the Padi4 gene knockout (KO) mice. First, we confirmed that ELS induced NET formation in the lungs. Although PAD4 deficiency did not affect the cellular profile of bronchoalveolar lavage fluid (BALF), PAD4 deficiency suppressed ELS-induced neutrophil migration and NET expression in the parenchyma of the lung. Additionally, PAD4 deficiency ameliorated emphysema and apoptosis in lung cells. Finally, we examined the effects of PAD4 on comprehensive gene expression signatures using RNA sequencing. Enrichment analysis of the transcriptomic data revealed that the expression of several genes associated with COPD pathogenesis was altered in the KO mice. Overall, the results suggest that PAD4 deficiency improves NET formation and emphysema in the lungs; this pathway can be a potential therapeutic target for the treatment of COPD.

overall_design: To examine the inhibitory effect of PAD4 on the formation of NETs and emphysema, we used PAD4-knockout (PAD4-KO) mice. ELS was administered intratracheally to wild-type (Wt) and PAD4-KO mice to create a model of emphysema. The control group was administered PBS. On days 1 and 14, bronchoalveolar lavage fluid (BALF) and lung tissue were examined histopathologically, and whole lung RNA sequencing was performed.

first: Megumi

last: Katsumata

first: Akira

last: Urano

first: Eiko

last: Suzuki

first: Kazuto

last: Kugou

first: Yoshinori

last: Hasegawa

first: Koichiro

last: Tatsumi

first: Takuji


title: Early innate immune response and evolution of a SARS-CoV-2 furin cleavage site inactive variant in bat cells

city: Stanford

first: Daniel

last: Richard

address: 290 Jane Stanford Way

department: Genetics

institute: Stanford University

summary: SARS-CoV-2 has caused the largest known coronavirus pandemic and is believed to have emerged from insectivorous bats. Little is known about the evolution of these viruses in their reservoir bat species. In this study, we investigated SARS-CoV-2-host interaction using human and bat cells. Bat cells mount a robust and early antiviral response but elicit a dampened pro-inflammatory response upon SARS-CoV-2 infection compared to human cells. Furthermore, an inactivating R685P mutation within the furin cleavage site (FCS) of the SARS-CoV-2 spike protein was naturally selected for in infected bat cells. Taken together, our data demonstrate that insectivorous bat cells have evolved a differential antiviral immune response against SARS-CoV-2 infection, likely to mitigate immunopathology that is observed in humans. Our study sheds light on the evolution of sarbecoviruses in bats and extends molecular evidence to data from field studies that have demonstrated that SARS-CoV-2-related viruses in wild-caught bats lack an intact FCS.

sample_organism:
Homo sapiens
Eptesicus fuscus

platform_organism:
Homo sapiens
Eptesicus fuscus

overall_design: Clinical isolate of ancestral SARS-CoV-2 (SARS-CoV-2/SB2) was used for infections. Cells at ~80% confluence were infected with SARS-CoV-2 at indicated multiplicity of infection (MOI). Control cells were treated with serum-free DMEM alone (mock infected). Infected and mock infected cells were incubated at 37 °C for 1 h with gentle rocking every 15 min. After 1 h, the virus inoculum was aspirated, cells were washed 1x with phosphate-buffered saline (PBS), and complete growth medium was added. All work with infectious SARS-CoV-2 was done in BSL3 laboratories at the Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Canada and McMaster University, Canada following approved protocols. Cells were infected with SARS-CoV-2 at an MOI of 0.1 for 12 and 24 h. Time-matched, sham infected cells served as controls. RNA was isolated from whole cell lysates using RNeasy Mini kit (Qiagen, 74106). Sequencing was conducted at the McMaster Genomics Facility, Farncombe Institute at McMaster University.

first: Kaushal

last: Baid

first: Andrew

last: Doxey

first: Arinjay

last: Banerjee


first: Subhransu

last: Sahoo

summary: DNA-level alterations (mutations) conferring selective cell growth advantages are well-established in cancer initiation and progression. Changes in cell states not due to alterations in DNA sequence (epimutations) are also proposed to contribute to cancer, but concrete examples involving specific genes are elusive. Endometrial cancer is a common, lethal malignancy of women, understood to arise through the stepwise selection of heritable changes that result first in the formation of a preinvasive precursor known as endometrioid intraepithelial neoplasia, followed by progression to cancer. PAX2 is required for the development of endometrium in embryos, and PAX2 endometrial expression persists throughout life. Per clinical studies, loss of PAX2 protein occurs in 80% of endometrial cancers. However, the molecular basis of PAX2 loss—and its functional significance—remain unknown. Here, we show that loss of PAX2 protein occurs through a specific transcriptional gene silencing event specifically targeting PAX2. This RNA-level silencing event occurs in early microscopic neoplastic clones preceding endometrioid intraepithelial neoplasia, linking PAX2 silencing with the initiation of most endometrial cancers. Integrated genomic, transcriptomic, epigenomic, 3D genomic, and machine learning approaches showed that loss of PAX2 expression is associated with replacement of open/active chromatin features (H3K27ac and H3K4me3) with inaccessible chromatin features (H3K27me3). The spread of the H3K27me3 signal resembles a pearl necklace, with its length modulated by cohesin loops, preventing transcriptional dysregulation of neighboring genes, thereby limiting PAX2 silencing as a focal epigenetic event. PAX2 broadly regulates enhancer activity and transcription in endometrial cancer cells including PGR, rationalizing its activity as an oncodevelopmental tumor suppressor. Functional studies, including of a novel mouse model and tumor-derived organoids based on endometrial-specific Pax2 inactivation, proved that Pax2 is a bona fide tumor suppressor in vivo that potently cooperates with Pten, the second most frequently inactivated tumor suppressor in endometrial cancer. PAX2 silencing was reversible in human cell lines, suggesting that PAX2 may be a druggable target. Taken together, our findings establish a new paradigm for cancer-driving epimutations and open new lines of investigation into the origins of endometrial cancer, with diverse implications for its diagnosis and treatment.

overall_design: Transcriptomic profiling of PAX2+ PDX and PAX2- PDX tumor samples.

first: Diego

last: Castrillon

first: Chao

last: Xing

first: Ashwani

last: Kumar


summary: DNA-level alterations conferring selective cell growth advantages (mutations) are a well-established source of heritable variation underpinning cancer initiation and progression. Heritable changes in cell states not due to alterations in DNA sequence (epimutations) are also proposed to contribute to cancer, but concrete examples involving specific genes are elusive. Endometrial cancer is a common, lethal malignancy of women, understood to arise through the stepwise selection of heritable changes that result first in the formation of a preinvasive precursor known as endometrioid intraepithelial neoplasia, followed by progression to cancer. PAX2 is required for the embryological specification of endometrium, and PAX2 endometrial expression persists throughout life. Per clinical studies, loss of PAX2 protein occurs in 80% of endometrial cancers. However, the molecular basis of PAX2 loss—and its functional significance—remain unknown. Here, we show that loss of PAX2 protein occurs through a specific transcriptional gene silencing event (an epimutation) specifically targeting PAX2. This RNA-level silencing event occurs in early microscopic neoplastic clones preceding endometrioid intraepithelial neoplasia, linking PAX2 silencing with the initiation of most endometrial cancers. PAX2 silencing is associated with loss of a promoter-proximal active enhancer, and 3D genome organization studies of cell lines and patient-derived tumors showed that repressive H3K27me3 marks span an insulated gene neighborhood formed via a looping mechanism, restraining spread of the H3K27me3 domain. PAX2 broadly regulates enhancer activity and transcription in endometrial cancer cells including PGR, rationalizing its activity as an oncodevelopmental tumor suppressor. Functional studies, including of a novel mouse model and tumor-derived organoids based on endometrial-specific Pax2 inactivation, proved that Pax2 is a bona fide tumor suppressor in vivo that potently cooperates with Pten, the second most frequently inactivated tumor suppressor in endometrial cancer. PAX2 silencing was reversible in human cell lines, suggesting that PAX2 may be a druggable target. Taken together, our findings establish a new paradigm for cancer-driving epimutations and open new lines of investigation into the mechanistic basis of endometrial cancer, with diverse implications for its diagnosis and treatment.

overall_design: Single cell RNA-Seq of uterine cells isolated from Pax2 cKO mouse.


title: Gene Regulatory Programs that Specify Age-Related Differences during Thymocyte Development  (ATAC-Seq)

last: Grimson

overall_design: We set out to determine how chromatin accessibilty profiles change between adults and neonates during thymocyte development. To this end, we isolated cells from thymi and spleens of neonatal (5-7 days post-birth) and adult (12 weeks) mice and subjected these cells to ATAC-seq. Antibodies to CD4, CD8, CD44 and CD25 were utilized for flow cytometry to isolate thymocytes at different stages of development. We obtained cells from four stages of thymocyte development: pooled Double Negative (DN1-3 cells; referred to as DN here), double-positive and single-positive CD8 cells (DP and SP8, respectively) and mature splenic naïve CD8+ T cells. We generated four biological replicates of ATAC-seq profiles from both adult and neonatal mice for all four developmental stages (DN, DP, SP8 and CD8).

first: Divya

last: Ganapathi Sankaran

first: Viviana

last: Maymi

first: Brian

last: Rudd


type: Methylation profiling by high throughput sequencing

summary: DNA-level alterations (mutations) conferring selective cell growth advantages are well-established in cancer initiation and progression. Heritable changes in cell states not due to alterations in DNA sequence (epimutations) are also proposed to contribute to cancer, but concrete examples involving specific genes are elusive. Endometrial cancer is a common, lethal malignancy of women, understood to arise through the stepwise selection of heritable changes that result first in the formation of a preinvasive precursor known as endometrioid intraepithelial neoplasia, followed by progression to cancer. PAX2 is required for the embryological specification of endometrium, and PAX2 endometrial expression persists throughout life. Per clinical studies, loss of PAX2 protein occurs in 80% of endometrial cancers. However, the molecular basis of PAX2 loss—and its functional significance—remain unknown. Here, we show that loss of PAX2 protein occurs through a specific transcriptional gene silencing event specifically targeting PAX2. This RNA-level silencing event occurs in early microscopic neoplastic clones preceding endometrioid intraepithelial neoplasia, linking PAX2 silencing with the initiation of most endometrial cancers. Integrated genomic, transcriptomic, epigenomic, 3D genomic, and machine learning approaches showed that loss of PAX2 expression is associated with replacement of open/active chromatin features (H3K27ac and H3K4me3) with inaccessible chromatin features (H3K27me3). The spread of the H3K27me3 signal resembles a pearl necklace, with its length modulated by cohesin loops, preventing transcriptional dysregulation of neighboring genes, thereby limiting PAX2 silencing as a focal epigenetic event. PAX2 broadly regulates enhancer activity and transcription in endometrial cancer cells including PGR, rationalizing its activity as an oncodevelopmental tumor suppressor. Functional studies, including of a novel mouse model and tumor-derived organoids based on endometrial-specific Pax2 inactivation, proved that Pax2 is a bona fide tumor suppressor in vivo that potently cooperates with Pten, the second most frequently inactivated tumor suppressor in endometrial cancer. PAX2 silencing was reversible in human cell lines, suggesting that PAX2 may be a druggable target. Taken together, our findings establish a new paradigm for cancer-driving epimutations and open new lines of investigation into the origins of endometrial cancer, with diverse implications for its diagnosis and treatment.

overall_design: Targeted methylation sequencing (Methyl-Seq) of 8 human endometrial cancer cell lines.

first: Xunzhi

last: Zhang


title: Single-cell RNA-seq analysis of wildtype and ETV2-overexpressing embryoid body-derived progenitor cells

city: Minneapolis

last: Garry

state: Minnesota

address: 2231 6th Street SE, Suite 4-156

department: Lillehei Heart Institute

institute: University of Minnesota

summary: Wild-type (WT) and Etv2-overexpressing (OE) mouse embryonic stem cells (mESC) were cultured and differentated to embryoid bodies (EBs) in mesodermal conditions. Induction of Etv2 in OE EBs was achieved by the addition of doxycycline. Cells were collected at different time points (i.e., D2, D3, D4 for WT and D3, D4 for OE EBs) following FACS based on the presence or absence of two surface markers: Flk1 (F+/-) and Pdgfrα (P+/-). We performed single-cell RNA sequencing (scRNA-seq) on the FACS sorted cells at different time points. The transcriptomic profiles revealed that ETV2 overexpression promotes HE lineage specification while repressing cardiac and skeletal muscle programs.

overall_design: A total of 13 samples were used for the capture and analysis. The following list shows the sample with the annoation format as Day_Surface-Marker_Condition:  For WT EBs the samples are D2_P-F-_WT,  D3_P-F-_WT, D3_P+F-_WT, D3_P+F+_WT, D3_P-F+_WT, D4_P-F-_WT, D4_P+F-_WT, D4_P+F+_WT and D4_P-F+_WT. For OE EBs the samples are D3_P-F-_OE, D3_P-F+_OE, D4_P-F-_OE, and D4_P-F+_OE.

first: Xiao

last: Ma

first: Satyabrata

last: Das

first: Kidus

last: Hailemariam


title: High-resolution spatial mapping of cell state and lineage dynamics in vivo with PEtracer

city: Cambridge

first: Kathryn

last: Yost

state: Massachusetts

address: 455 Main St

department: Biology

institute: Whitehead Institute

summary: Charting the spatiotemporal dynamics of cell fate determination in development and disease is a long-standing objective in biology. Here we present the design, development, and extensive validation of PEtracer, a prime editing-based, evolving lineage tracing technology compatible with both single-cell sequencing and multimodal imaging methodologies to jointly profile cell state and lineage in dissociated cells or while preserving cellular context in tissues with high spatial resolution. Using PEtracer coupled with MERFISH spatial transcriptomic profiling in a syngeneic mouse model of tumor metastasis, we reconstruct the growth of individually-seeded tumors in vivo and uncover distinct modules of cell-intrinsic and cell-extrinsic factors that coordinate tumor growth. More generally, PEtracer enables systematic characterization of cell state and lineage relationships in intact tissues over biologically-relevant temporal and spatial scales.

overall_design: 10X Genomics scRNA-seq (3' v3.1) libraries were generated from cell lines engineered with PEtracer lineage tracing components. Multiple libraries can be associated with a single sample and library type is indicated in the sample name, including: 1) transcriptome data, _GEX; 2) lineage tracing cassette data, _LTC; 3) integration barcode data, _intBC; 4) Puromycin or blasticidin static barcode data, _PURO or _BLAST; 5) pegRNA mismatch data from CROP-seq vector, _pegRNA

first: Luke

last: Koblan

middle: E

first: Pu

last: Zheng

first: William

last: Colgan

first: Matthew

last: Jones

first: Dian

last: Yang

first: Arhan

first: Jaspreet

last: Sandhu

first: Alexandra

last: Schnell

first: Dawei

last: Sun

first: Can

last: Ergen

first: Reuben

last: Saunders

first: Xiaowei

last: Zhuang

last: Allen

first: Nir

last: Yosef

first: Jonathan

last: Weissman


first: Subhransu

last: Sahoo

state: Texas

overall_design: Transcriptomic profiling of AN3CA shEV and AN3CA shPAX2 cells.

first: Diego

last: Castrillon

first: Chao

last: Xing

first: Ashwani

last: Kumar


title: Control of Inflammatory Response by Tissue Microenvironment [ChIP-seq]

city: Boston

first: Zhongyang

last: Wu

address: 300 Longwood Ave,61 Binney Street, Enders building, EN622

institute: Boston Children's Hospital

summary: Inflammation is an essential defense response but operates at the cost of normal functions. Whether and how the negative impact of inflammation is monitored remains largely unknown. Acidification of the tissue microenvironment is associated with inflammation. Here we investigated whether macrophages sense tissue acidification to adjust inflammatory responses. We found that acidic pH restructured the inflammatory response of macrophages in a gene-specific manner. We identified mammalian BRD4 as a novel intracellular pH sensor. Acidic pH disrupts the transcription condensates containing BRD4 and MED1, via histidine-enriched intrinsically disordered regions. Crucially, decrease in macrophage intracellular pH is necessary and sufficient to regulate transcriptional condensates in vitro and in vivo, acting as negative feedback to regulate the inflammatory response. Collectively, these findings uncovered a pH-dependent switch in transcriptional condensates that enables environmental sensing to directly control inflammation, with a broader implication for calibrating the magnitude and quality of inflammation by the inflammatory cost.

overall_design: to investigate the effect of acidic pH on the inflammatory response in macrophages, we pretreated the cells using medium at pH 7.4 and pH 6.5 for 4 h. Subsequently, we added LPS stimulation for another 4 h. The antibodies H3K4me3, H3K27Ac, p65, IRF3, BRD4 and BRD9 were applied to perform ChIP-seq.

first: Nasiha

last: Ahmed

first: Scott

last: Pope


title: A pilot trial of perioperative IDH Inhibition in treatment-naïve IDH-mutant glioma

status: Public on Jun 02 2025

city: Melbourne

first: Saskia

last: Freytag

address: 1G Royal Parade, Parkville

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: Mutant-IDH (mIDH) inhibition significantly improves progression free survival in patients with mIDH WHO grade 2 glioma, however almost all patients will progress, and there is a lack of understanding on the mechanisms of adaptation to mIDH inhibition. In the first pre- and post-treatment perioperative trial in patients with mIDH1 low-grade glioma, we tested safusidenib (AB-218 / DS-1001b), an oral, blood brain barrier penetrant IDH1 R132X inhibitor, in a single arm, open label study. The primary endpoint demonstrated the feasibility and acceptability of conducting a two-stage perioperative trial and treatment was well tolerated, with ongoing follow-up for safety and efficacy. Tumor 2-hydroxyglutarate quantification revealed on-target activity, resulting in significant alterations in differentiation programs and neural excitability, functionally validated by patch clamp electrophysiology. Taken together, these results provide a detailed understanding on mechanisms of mIDH inhibition in glioma and the safety and feasibility of this perioperative approach, which can be applied broadly in clinical trial design, advancing drug development in glioma. Funded by AnHeart Therapeutics (a Nuvation Company) and the Victorian Government; ClinicalTrials.gov number NCT05577416.

overall_design: Single nuclei gene expression was profiled using the Chromium Single Cell Gene Expression Flex (10x Genomics) from tumor resections of patients with IDH-mutated low grade glioma (n = 8 astrocytoma, n = 3 oligodendroglioma).

first: Katharine

last: Drummond

first: Montana

last: Spiteri

first: Sarah

last: Cain

first: Jordan

last: Jones

first: Sammy

last: Shaya

first: Monique

last: Topp

first: Robert

last: Tobler

first: Adam

middle: L

last: Valkovic

first: Zachery

last: Moore

first: Oluwaseun

last: Fatunla

first: Jurgen

last: Kriel

first: Joel

last: Moffet

first: Heidi

last: McAlpine

first: Marius

last: Rosier

first: Hefei

last: Guan

first: James

last: Dimou

first: Verena

last: Schadewaldt

first: Samuel

last: Robets-Thomson

first: David

last: McArdle

first: Elaine

last: Lui

first: Moritz

last: Voelker-Albert

first: Simone

last: di Sanzo

first: Brunda

last: Nijagal

first: Vinod

last: Narayana

first: Camilla

middle: B

last: Mitchell

first: Joseph

last: Vissers

first: Sean

last: Grimmond

first: Mark

last: Rosenthal

first: Lucy

last: Palmer

last: Best

last: Whittle


title: GD2TIF cells as a platform for single-dose and long-term delivery of biologics

city: Beijing

first: Guangyue

last: Zhang

address: Haidian  District

institute: Tsinghua University

summary: The efficacy of biologics, such as peptide and protein drugs, is often limited by their short half-lives in vivo. GD2TIF cells expanded in immunocompetent mice without the need for chemotherapeutic conditioning and persisted as a polyclonal population, safely and almost indefinitely. Thus, GD2TIF cells be leveraged as a versatile platform for long-term delivery of biologics. In multiple disease models, GD2TIF cells were proved can be taken as a general platform for long-term delivery of biologics, offering durable therapeutic efficacy with a single infusion.

overall_design: Splenic CD8+Thy1.1+ GD2TIF cells were sorted for scRNA-seq and TCR-seq at 2 months post transfer, for characterization of GD2TIF cells in a higher resolution.

last: Yin

first: Min

last: Peng


title: Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

city: St. Louis

first: Richard

middle: Keith

last: Slotkin

state: Missouri

address: 975 N Warson Rd

institute: Donald Danforth Plant Science Center

summary: Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of transcript cleavage by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein / RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation, rather than RNAi. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and editing rates. In addition, transgenes with the engineered protein / RNA-binding module had improved long-term durability of transgene expression, as after four generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.

overall_design: Methylation profiling by high throughput sequencing of 4 GmRPS5a:Cas9 with various 3'UTR transgenic Arabidopsis lines, with 2-6 biological replicates for each line.

first: Yu-Hung

last: Hung


title: The histone acetyltransferase MOZ is a molecular dependency and therapeutic target in NUP98-rearranged acute myeloid leukemia [WGS, ATACseq, CUT&RUN, scRNAseq]

city: MEMPHIS

first: QINGSONG

last: GAO

state: Tennessee

address: 262 DANNY THOMAS PLACE

institute: ST JUDE CHILDREN'S RESEARCH HOSPITAL

summary: NUP98 fusion oncoproteins (FOs) are a hallmark of childhood acute myeloid leukemia (AML), and drive leukemogenesis through liquid-liquid phase separation-mediated nuclear condensate formation. However, the composition and consequences of NUP98 FO-associated condensates are incompletely understood. Here we show that histone acetyltransferase (HAT) complex proteins including MOZ associate with NUP98 FOs, and that BRPF1, an epigenetic writer that associates with MOZ, is a molecular dependency in NUP98::KDM5A AML. Inactivation of Brpf1 as well as HAT complex member Moz, Hbo1, Brd1 or Meaf6 in Nup98::Kdm5a;Vav-Cre cells impaired fitness of NUP98-rearranged cells.  MOZ acetyltransferase inhibition decreased global H3K23ac levels, displaced FO from chromatin at the Meis1 locus, and led to myeloid cell differentiation. Additionally, MOZ inhibition decreased leukemic burden in multiple NUP98-rearranged leukemia xenograft mouse models, synergized with Menin inhibitor treatment, and was efficacious in Menin inhibitor-resistant cells. In summary, we show that MOZ is a potentially targetable dependency in NUP98-rearranged AMLs.

overall_design: IgG, NUP98, MOZ, Menin, and KMT2A chromatin binding sites were identified in CHRF-288-11 cells [CUT&RUN].  MSKG5191 (NUP98::NSD1) PDX-bearing mice were treated with vehicle, MOZ inhibitor PF9363, Menin inhibitor SNDX-5613, or combination, and spleen samples were analyzed to identify drug-induced changes in gene expression [scRNAseq]. For scRNAseq, spleens from three mice were pooled for each individual sample.

first: Nicole

last: Michmerhuizen

first: Charles

last: Mullighan


title: NOTCH1 reverses immune suppression in small cell lung cancer through reactivation of STING [CHIP-seq]

city: Bethesda

first: Nitin

last: Roper

address: 37 Convent Drive

institute: National Cancer Institute

summary: Downregulation of antigen presentation and lack of immune infiltration are defining features of small cell lung cancer (SCLC) limiting response to immune checkpoint blockade (ICB), While a high MHC Class I, immune-inflamed subset benefits from ICB, underlying mechanisms of immune response in SCLC have yet to be elucidated. Here we show that in the landmark IMpower133 clinical trial high, but not low, NOTCH1 expression is significantly associated with longer survival with the addition of ICB to chemotherapy among ~80% of SCLC patients with neuroendocrine-enriched tumors (ASCL1-enriched, HR 0.39, P=0.0012; NEUROD1-enriched, HR 0.44, P=0.024). Genetic or pharmacologic activation of NOTCH1 in ASCL1 and NEUROD1 SCLC cell lines dramatically up-regulates MHC Class I through epigenetic up-regulation of STING. In syngeneic mouse models, Notch1 activation reprograms SCLC tumors from immune-excluded to immune-inflamed, facilitating durable, complete responses with ICB combined with a STING agonist. STING1 expression is significantly enriched in high compared to low NOTCH1 expressing tumors in IMpower133 thereby validating our proposed mechanism. Our data reveal a previously undiscovered role for NOTCH1 as a critical driver of SCLC immunogenicity and a potential predictive biomarker for ICB in SCLC. NOTCH1 activation may be a therapeutic strategy to unleash anti-tumor immune responses in SCLC and other neuroendocrine cancers in which NOTCH1 is typically suppressed.

overall_design: We performed H3K27Ac ChIP-sequencing in human small cell lung cancer cells with either NOTCH1 overexpression or activation of Notch signaling by LSD1 inhibition.

first: Yoo

middle: S

last: Kim

first: Xinyu

last: Wen


title: Rapid depletion of the cohesin subunits and CTCF reveals their role in maintaining high-order genome architecture [Hi-C]

city: Rotterdam

first: Ning Qing

last: Liu

address: Wytemaweg 80

department: Hematology

country: Netherlands

institute: Erasmus University Medical Center Rotterdam

summary: Cohesin stalling at CTCF binding sites represents one of the main principles of interphase chromosome organization. In the current studies, we dissect the role of cohesin and CTCF, both alone and in combination, in 3D genome organization by depleting these proteins using acute protein degradation techniques. By systematic examination of interactomic, epigenomic and transcriptomic changes using various sequencing techniques, our studies reveal the functions of cohesin and CTCF in mediating the formation of chromatin loops, topologically associating domains, chromosome compartments and nuclear lamina associating domains. Our studies describe fundamental principles of how the architectural proteins contribute to genome folding at multiple genomic scales and transcriptional regulation.

overall_design: Acute depletion of WAPL, CTCF/WAPL and CTCF/WAPL/RAD21 using AID-mediated and dTAG13-mediated protein depletion, followed by a Hi-C experiment.

first: Ning

middle: Q

first: Elzo

last: de Wit


title: Expression data from the transplanted kidney and the native kidney of NEP25 mice with/without the treatment of LMB2

city: Toyama

first: Jun-Dal

state: Toyama

address: 2630 Sugitani

department: Department of Complex Biosystem Research (CBR), Institute of National Medicine (INM)

institute: University of Toyama

summary: A previous study revealed the appropriate amount of LMB2 (1.75 ng/g body wt dissolved in 0.1 ml of PBS containing 0.1% bovine serum albumin [BSA]) needed to induce reproducible a focal segmental glomerulosclerosis (FSGS) lesions in NEP25 mice. We performed kidney transplantation between NEP25 mice and their wild-type littermates (WTm) which have the same genetic background (C57BL/6N). In this study, the transcriptomic profiles of transplanted and native kidneys with/without LMB2 in nephrotic model mice were investigated using the next-generation RNA sequencing (RNA-seq). Reads were mapped on mm10 mouse reference genome and quantified using CLC Genomics Workbench version 10.1.1 (QIAGEN).

overall_design: mRNA profiles of murine kidney were generated by RNA sequencing using the NextSeq 500 (Illumina).

first: Kazuo

last: Sakamoto

first: Akiyoshi

last: Fukamizu

first: Michio

last: Nagata


title: A Common Mechanism of Singular Gene Choice is Revealed by Broadly Expressed Odorant Receptor Transgenes within the Olfactory Epithelium

status: Public on Jun 01 2025

city: NY

first: Paul

last: Feinstein

address: 695 Park Ave

institute: CUNY Hunter College

summary: In the mouse, more than 1,100 odorant receptors (ORs) are expressed in a monogenic and monoallelic fashion, referred to as singular gene expression. Here we utilize multimers of a naturally occurring 21bp enhancer (x21) and radically increase the number of olfactory sensory neurons (OSNs) choosing an OR transgene that is normally expressed by Class II cell types. Surprisingly, hundreds of ORs derived from Class I, Class II and TAAR cell types have reduced expression and are replaced by the OR transgene expression leading to novel glomeruli formed in three segregating domains of glomeruli on the olfactory bulb: DI (Class I), DII (Class II), and DIII (TAAR). The reduction of expression across distinct cell types that were thought to have distinct mechanisms of singular gene choice in favor of the expression of the x21-OR transgene argues that a common mechanism of singular gene choice is present for all OR-expressing OSNs.

overall_design: We have employed a single cell RNA sequencing approach using 10x Genomics scRNAseq to study odorant receptor gene expression profile in our transgenic mouse and wildtype mouse of olfactory sensory neurons and the precursor cells.

first: Mélanie

last: Makhlouf

first: Charlotte

last: D’Hulst

first: Masayo

last: Omura

first: Alessandro

last: Rosa

first: Raena

last: Mina

first: Sergio

last: Bernal-Garcia

first: Eugene

last: Lempert

first: Luis

last: Saraiva


title: RNA-seq profiling of C/EBPβ-overexpressing myoblasts [differentiating myoblasts]

city: Ottawa

first: Nadine

last: Wiper-Bergeron

state: Ontario

address: 451 Smyth Road

department: Cellular and Molecular Medicine

country: Canada

institute: University of Ottawa

summary: We previously identified C/EBPβ as an inhibitor of myogenic differentiation and its role in muscle satellite cell’s self-renewal and quiescence. C/EBPβ is regulated during the transition from proliferation to differentiation in myoblasts and that overexpression of C/EBPβ inhibits myocyte differentiation. To identify the molecular mechanism by which C/EBPβ inhibits myogenic differentiation, we performed RNA-seq on C/EBPβ-overexpressing differentiating myoblasts.

overall_design: Differentiating C2C12s mRNA profiles of C/EBPβ-overexpressing and empty vector myoblasts.

first: Hamood

last: AlSudais


title: A distinct mechanism of epigenetic reprogramming silences PAX2 and initiates endometrial carcinogenesis

city: Dallas

first: Subhransu

last: Sahoo

institute: UT Southwestern Medical Center

overall_design: Transcriptomic profiling of Ishikawa (ISK) and ISK-PAX2 cells.

first: Diego

middle: H

last: Castrillon

first: Chao

last: Xing

first: Ashwani

last: Kumar


title: Reproducible single cell annotation of programs underlying T cell subsets, activation states, and functions

first: Soumya

last: Raychaudhuri

state: MA

address: 75 Francis St

institute: Brigham and Women's Hospital

summary: CITE-sequencing and TCR-sequencing of sorted activated, unactivated, and mock T cells derived from blood. T cells from five healthy human donors were stimulated with a peptide pool (CEFX, JPT) and anti-CD28/CD49d co-stimulation. Cells were then sorted based on activation (OX40 and PD-L1 for CD4s, CD137 for CD8s) and labeled with corresponding hashing antibodies prior to sequencing. In total, this dataset encompasses 42,370 cells (12,743 AIM-positive, 15,369 AIM-negative, 14,258 Mock).

first: Dylan

last: Kotliar

first: Michelle

last: Curtis


title: Pulmonary fibroblast-derived stem cell factor promotes neutrophilic asthma by augmenting IL-17A production from ILC3s

city: Taipei

first: Jheng-Syuan

last: Shao

address: 128 Sec. 2, Academia Rd.

institute: Academia Sinica

summary: Group 3 innate lymphoid cells (ILC3s) have emerged as an important player in the pathogenesis of neutrophilic asthma. However, the regulatory mechanism supporting ILC3 responses in lung remains largely unclear. Here, we demonstrated that stem cell factor (SCF) expression is significantly increased and positively correlated with IL-17A and MPO expression in asthmatic patients. Notably, we identified ILC3 as a major IL-17A-producing responder to SCF in lung. In mice, SCF synergized with IL-1β/IL-23 to enhance pulmonary ILC3 activation and neutrophilic inflammation. Mechanistically, SCF promoted ILC3 proliferation and cytokine production. Transcriptomic analysis revealed that SCF treatment upregulated the genes related to proliferation and Th17 differentiation, associated with increased AKT and STAT3 signaling. In contrast, deficiency of SCF receptor, c-Kit, reduced ILC3 proliferation and IL-17A production, resulting in the amelioration of airway hyperreactivity (AHR) and neutrophilic inflammation in mouse neutrophilic asthma model. Furthermore, genetic deletion of SCF in fibroblasts revealed fibroblasts as the primary source of SCF for ILC3 activation in lung. Moreover, administration of imatinib, a c-Kit inhibitor, alleviated LPS, air pollution or ovalbumin/LPS-induced AHR and neutrophilic inflammation. Our findings elucidated a positive modulatory role of SCF/c-Kit signaling in ILC3 responses during neutrophilic inflammation, offering a potential therapeutic target for neutrophilic asthma.

overall_design: mRNA profiles of sorted mouse small intestine ILC3s treated with either IL-1β/IL-23 or IL-1β/IL-23 plus SCF, in four replicates, using a Illumina NovaSeq 6000 system.


title: NOTCH1 reverses immune suppression in small cell lung cancer through reactivation of STING [RNA-seq]

first: Nitin

last: Roper

overall_design: We activated Notch signaling (through NOTCH1 and REST overexpression or LSD1 inhibition) in human and mouse small cell lung cancer cells and assessed for gene expression changes in innate immune response genes by bulk RNA-sequencing.

first: Yoo

last: Kim

first: Xinyu

last: Wen


title: The development of a high-plex spatial proteomic methodology for the characterisation of the head and neck tumour microenvironment  (GeoMx WTA)

city: Brisbane

first: Arutha

last: Kulasinghe

state: QLD

address: 37 kent st

institute: University of Queensland

summary: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer globally. Despite advancements in treatment modalities, patient prognoses remain unimproved. Insight into the tumour microenvironment (TME) using spatially resolved approaches, and its association with clinical endpoints, may provide useful prognostic tools and refine current treatment outcomes. We profiled 84 mucosal HNSCC tissue samples using next-generation ultra-high plex spatial protein and transcriptome mapping (580-proteins, 18,000 mRNA targets). We found patient survival outcomes (both disease-free and overall survival) were associated with anatomical subsite and tumour stage. We identified proteomic and transcriptomic functional T cell markers within discrete TME regions that were associated with survival. GSEA revealed elevated glycolysis and hypoxia pathways in patients with worse outcomes. Key proteomic findings (including CD34 and CD44) were independently validated using single-cell protein profiling. This study provides a systematic workflow for high-plex protein and transcriptomic profiling in mucosal HNSCC.

overall_design: GeoMx Human Whole Transcriptome Atlas (GeoMx Hu WTA, 18,000+ protein-coding genes, Nanostring, USA) was applied to tumour cores collected from 61 chemotherapy and/or radiotherapy treated patients with tissues acquired from a range of sites across the mucosal head and neck regions (i.e. tongue, oral cavity, pharynx and lip). Each core were segmented into tumour and stroma regions based on PanCK staining, resulting in 122 fields of view (FoVs) across 61 cores. After QC filtering, 117 FoVs across 61 cores were used for downstream analyses. The following cell surface markers were used for morphology visualization: SYTO-13, CD45,  PanCk and nuclei marker DAPI.

first: Chin Wee

last: Tan

first: James

last: Monkman


title: Ncoa4-mediated ferritinophagy as a dependence in Tet2-deficient hematopoiesis

first: Peter

middle: G

address: 360 Longwood Ave, LC8202

department: Medical Oncology

institute: Dana Farber Cancer Institute

summary: TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies, thus, the ability to identify selective dependencies in TET2 deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2 deficient HSPCs in a physiological setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared to wild-type (WT). Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron (Fe2+) in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial ATP production correlates with increased cellular iron requirements, and in turn, promotes Ncoa4-dependent ferritinophagy.  Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.

overall_design: Expression profiling by single cell high throughput sequencing of Tet2 KO HSPCs. Following transplantation of Cas9+ WT or Tet2 KO HSCs transduced with nontargeting (NTC) sgRNAs or Ncoa4 sgRNAs, Lin-Sca1+Kit+ (HSPCs) were sorted via flow cytometry and subjected to scRNAseq.

first: Justin

last: Loke

first: Thuy

last: Nguyen

first: Meaghan

last: Boileau

first: Marie

last: McConkey

first: Aidan

last: Miller

first: Wesley

last: Shin

first: Christopher

last: Hergott

first: Maria

last: Ericsson

first: Anja

last: Nordstrom

first: Paula

last: Llopis

first: Scott

last: Armstrong

first: Joseph

last: Mancias

first: Benjamin

last: Ebert


title: Control of Inflammatory Response by Tissue Microenvironment [RNA-seq]

first: Zhongyang

last: Wu

overall_design: To investigate the effect of acidic pH on the inflammatory response in macrophages, we pretreated the cells using medium at pH 7.4 and pH 6.5 for 4 h. Subsequently, we added LPS stimulation for another 4 h. Additionally,  macrophages were treated with medium at pH 6.5 were switched to pH 7.4 medium for 0 h, 2 h and 4 h before LPS stimulation.  We then performed gene expression profile analysis using data obtained from bulk RNA-seq.

first: Diana

last: Leung

first: Xu

last: Zhou


title: Human myometrial transcriptome and DNA methylome of testosterone treated patients appear more similar to myometrium from fibroid patients

city: Grand Rapids

first: Jose

last: Teixeira

state: MI

address: 400 Monroe Ave NW

department: Ob/Gyn

institute: Michigan State University

summary: Uterine fibroids, or leiomyomas, are noncancerous tumors of the myometrium and the most common reproductive tract tumors in women, with an incidence of up to 80% by age 50. Currently, hysterectomy is the only definitive cure, and effective non-hormonal therapeutics are lacking. Understanding the etiology of fibroids may lead to alternative, less invasive treatments. Several obstetric disorders, including polycystic ovary syndrome (PCOS), have been linked to uterine fibroids, and women with PCOS often exhibit hormonal imbalances, particularly elevated serum testosterone levels. However, the impact of testosterone on the myometrium remains poorly understood. We hypothesize that elevated testosterone may increase the risk of developing uterine fibroids. Using RNA sequencing and MethylationEPIC array analysis, we compared myometrial tissue from women without fibroids (MyoN, n=33), with fibroids (MyoF, n=66), and after testosterone therapy (MyoT, n=7). The transcriptomic and methylation profiles of MyoT clustered with MyoF and were distinct from MyoN. We identified 1,321 differentially expressed protein-coding genes between MyoT and MyoN, while only 494 were found between MyoT and MyoF. Disease ontology analysis of MyoT vs. MyoN revealed enrichment of the fibroid tumor gene set. Fibroid associated genes including TGFβ3, CCND1, SERPINE1, and FGFR1 were upregulated in MyoT and MyoF samples compared to MyoN samples. The DNA methylation profiles of MyoT were closer to those of MyoF, but no correlation was observed between methylation status and gene expression. Our preliminary data suggest that testosterone induces transcriptional and methylation changes in the myometrium consistent with those observed in MyoF tissues. These findings suggest that elevated testosterone may be associated with an increased risk of developing uterine fibroids.

overall_design:
Fibroid-free myometrium (MyoN, n=33), myometrium from fibroid patient (MyoF, n=66) and myometrium from patient treated with testosterone (MyoT, n=7) were sent for RNA-sequencing. C: Caucasian, AA: Black/African American, H: Hispanic, A: Asian, Multi: Multi-race

NOTE:
The 'Myometrium from fibroid patient (MyoF), patient NW2' raw data (which was submitted to SRA; PRJNA498292, SAMN10287966, SRR8110208/SRX4936752) was downloaded and re-analyzed in the current study.
The re-analyzed sample information and resulting processed data file is linked as Series supplementary file.

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

first: Emmanuel

middle: N

last: Paul

first: Tyler

middle: J

last: Carpenter

first: Andrew

last: Bossick

first: Ghassan

last: Allo

first: Ganesa

last: Wegienka


title: Time- and Dose-Dependent Effects of Irradiation on Endothelial and Tumor Endothelial Cells: Transcriptional, Molecular, and Functional Changes Driving Activation In Vitro and In Vivo

city: Ljubljana

first: Bostjan

last: Markelc

state: Ljubljana

address: Zaloska cesta 2

department: Department of Experimental Oncology

country: Slovenia

institute: Institute of Oncology Ljubljana

summary: Background: Irradiation (IR) targets cancer cells, but also the tumor microenvironment, including the tumor’s blood vessels. In addition to tumor endothelial cell (TEC) apoptosis, IR can lead to TEC activation, potentially increasing immune cell infiltration. However, the changes underlying the IR-induced activation of endothelial cells (ECs) are poorly understood. This study investigated dose- and time-dependent molecular and functional responses of murine and human EC lines to IR in vitro and TECs in vivo in murine tumor models of colorectal carcinoma. Methods: HUVEC, EA.hy926, and Hulec5a, as well as murine bEND.3, 2H11, and SVEC4-10 EC lines, were irradiated with single doses of 2–10 Gy. EC proliferation and survival after IR were assessed by staining all nuclei (Hoechst 33342) and dead cells (propidium iodide) every 24 h for 5 days using the Cytation 1 Cell Imaging Multi-Mode Reader. RNA sequencing analysis of HUVECs irradiated with 2 Gy and 5 Gy at 24 h and 72 h after IR was conducted, focusing on processes related to EC activation. To validate the RNA sequencing results, immunofluorescence staining for proteins related to EC activation, including Stimulator of Interferon Response cGAMP Interactor 1 (STING), Nuclear factor kappa B (NF-κβ), and Vascular cell adhesion molecule 1 (VCAM-1), was performed. To validate the in vitro results, the response of TEC in vivo was analyzed using publicly available RNA sequencing data of TECs isolated from MC38 colon carcinoma irradiated with a single dose of 15 Gy. Finally, murine CT26 colon carcinoma tumors were immunofluorescently stained for STING and NF-κβ 24 and 48 h after IR with a clinically relevant fractionated regimen of 5 × 5 Gy. Results: Doses of 2, 4, 6, 8, and 10 Gy led to a dose-dependent decrease in proliferation and increased death of ECs. RNA sequencing analysis showed that the effects on the transcriptome of HUVECs were most pronounced 72 h after IR with 5 Gy, with 1014 genes (661 down-regulated and 353 up-regulated) being significantly differentially expressed. Irradiation with 5 Gy resulted in HUVEC activation, with up-regulation of the immune system and extracellular matrix genes, such as STING1 (log2FC = 0.81) and SELE (log2FC = 1.09), respectively; and down-regulation of cell cycle markers. Furthermore, IR led to the up-regulation of immune response- and extracellular matrix (ECM)-associated signaling pathways, including NF-κβ signaling and ECM–receptor interaction, which was also observed in the transcriptome of irradiated murine TECs in vivo. This was confirmed at the protein level with higher expressions of the EC activation-associated proteins STING, NF-κβ, and VCAM-1 in irradiated HUVECs and irradiated TECs in vivo. Conclusions: IR induces changes in ECs and TECs, supporting their activation in dose- and time-dependent manners, potentially contributing to the anti-tumor immune response, which may potentially increase the infiltration of immune cells into the tumor and thus, improve the overall efficacy of RT, especially in combination with immune checkpoint inhibitors.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for irradiated and non-irradiated Human umbilical vein endothelial cells (HUVECs).

first: Iva

last: Santek

first: Gregor

last: Sersa


title: Catalytic-dependent and independent functions of the histone acetyltransferase CBP promote pioneer factor-mediated zygotic genome activation [CUT&Tag]

city: Madison

first: Melissa

last: Harrison

state: Wisconsin

address: 6204B Biochemical Sciences Building, 440 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin- Madison

summary: Immediately after fertilization the genome is transcriptionally quiescent. Maternally encoded pioneer transcription factors reprogram the chromatin state and facilitate the transcription of the zygotic genome. In Drosophila, transcription is initiated by the pioneer factor Zelda. While Zelda-occupied sites are enriched with histone acetylation, a post-translational mark associated with active cis-regulatory regions, the functional relationship between Zelda and histone acetylation in zygotic genome activation remained unclear. We show that Zelda-mediated recruitment of the histone acetyltransferase CBP is essential for zygotic transcription and embryonic development. CBP catalytic activity is necessary for release of RNA Polymerase II (Pol II) into transcription elongation. However, CBP largely activates zygotic transcription independent of acetylation through Pol II recruitment. Neither acetylation nor CBP are required for the pioneering function of Zelda. Our data suggest that pioneer factor-mediated recruitment of CBP is a conserved mechanism required to activate zygotic transcription but that this role is separable from the function of pioneer factors in restructuring chromatin accessibility.

overall_design: This data set contains CUT&Tag data from the bulk Drosophila melanogaster embryos 2-3 hours after egg laying (AEL) (nuclear cycle 14) during the maternal-to-zygotic transition. CUT&Tag was executed on GFP-CBP;his2AV-RFP (control) and GFP-CBP;nos-deGrad/+;his2AV-RFP embryos to measure the effect CBP knockdown has on level and distribution of chromatin marks.  CRY2-CBP;his2av-RFP were treated with blue light to inactivate CBP catalytic activity via the CRY2 optogenetic tag or left in the dark as a control to assay the function of CBP mediated acetylation during ZGA. To corroborate the assay of the catalytic function we used an orthogonal approach with a catalytic mutant CBPHAT (F2161A); the mutant protein is exclusively loaded in embryos with the help of Dominant Female Sterile technique to produce germline clones, compared to a control line (w1118).

first: Audrey

last: Marsh

first: Sergei

last: Pirogov

first: Abby

last: Ruffridge

first: Suresh

last: Sajwan

last: Gibson

first: George

last: Hunt

first: Mattias

last: Mannervik


title: Catalytic-dependent and independent functions of the histone acetyltransferase CBP promote pioneer factor-mediated zygotic genome activation [PRO-Seq]

overall_design: This data set contains qPRO-seq on embryos 2-3 hours AEL (nuclear cycle 14) during the maternal-to-zygotic transition. GFP-CBP;his2AV-RFP (control) and GFP-CBP;nos-deGrad/+;his2AV-RFP embryos were used to quantify the effect CBP knockdown has on transcription during zygotic genome activation.CRY2-CBP;his2av-RFP were treated with blue light to inactivate CBP catalytic activity via the CRY2 optogenetic tag or left in the dark as a control to assay the function of CBP mediated acetylation during ZGA.


title: Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [sRNA-Seq]

first: Richard

last: Slotkin

type: Non-coding RNA profiling by high throughput sequencing

overall_design: Examination of small RNA accumulation in 4 GmRPS5a:Cas9 with various 3'UTR transgenic Arabidopsis lines, with 6 biological replicates for each line.

first: Yu-Hung

last: Hung


title: Catalytic-dependent and independent functions of the histone acetyltransferase CBP promote pioneer factor-mediated zygotic genome activation [RNA-Seq]

overall_design: This data set contains bulk RNA-seq on CBPHAT embryos 2h15min -3 hours AEL (nuclear cycle 14). To assay the catalytic function of CBP we used a catalytic mutant CBPHAT (F2161A). The mutant protein is exclusively loaded in embryos with the help of Dominant Female Sterile technique to produce germline clones, compared to a control line (w1118), both genotypes were analyzed in three replicates.


title: Transcriptome profiling of human bladder cancer cell line RT112 infected with lentivirus expressing either an shRNA targeting ELOVL6 (shE6) or a scrambled shRNA (shCtrl)

city: Tsukuba

first: Takashi

last: Matsuzaka

state: Ibaraki

address: 1-1-1 Tennodai

department: Internal Medicine

institute: University of Tsukuba

summary: ELOVL6 is a member of mammalian fatty acid elongase family responsible for converting C16 saturated and monounsaturated fatty acids into C18 species. To investigate the role of ELOVL6 in human bladder cancer, we generated RT112 cells (a human bladder cancer cell line) infected with lentivirus expressing either an shRNA targeting ELOVL6 (shE6) or a scrambled shRNA (shCtrl). The aim of this experiment was to assess the effects of ELOVL6 knockdown on the transcriptome of human bladder cancer cells.

overall_design: Total RNA was extracted from RT112 cell lines stably expressing shControl (shCtrl, n=3), shELOVL6#1 (shE6#1, n=3), or shELOVL6#2 (shE6#1, n=3) that were cultured in E-MEM supplemented with 5% FBS for 48 hours. RNA amplification and RNA sequencing were outsourced to Takara Bio, Inc (Shiga, Japan). Mapped reads per gene (raw tag counts) were quantified as gene expression levels, and between-sample normalization of gene expression was performed on the raw count data using Genedata Profiler Genome software (Genedata).

first: Hitoshi

last: Shimano


title: Spatial constraints and cell surface molecule depletion structure a randomly connected learning circuit

city: Ann Arbor

first: Eleanor

middle: Josephine

last: Clowney

address: 1105 North University, 4218

department: MCDB

institute: University of Michigan

summary: The brain can represent almost limitless objects to “categorize an unlabeled world” (Edelman, 1989). This feat is supported by expansion layer circuit architectures, in which neurons carrying information about discrete sensory channels make combinatorial connections onto much larger postsynaptic populations. Combinatorial connections in expansion layers are modeled as randomized sets. The extent to which randomized wiring existsin vivois debated, and how combinatorial connectivity patterns are generated during development is not understood. Non- deterministic wiring algorithms could program such connectivity using minimal genomic information. Here, we investigate anatomic and transcriptional patterns and perturb partner availability to ask how Kenyon cells, the expansion layer neurons of the insect mushroom body, obtain combinatorial input from olfactory projection neurons. Olfactory projection neurons form their presynaptic outputs in an orderly, predictable, and biased fashion. We find that Kenyon cells accept spatially co-located but molecularly heterogeneous inputs from this orderly map, and ask how Kenyon cell surface molecule expression impacts partner choice. Cell surface immunoglobulins are broadly depleted in Kenyon cells, and we propose that this allows them to form connections with molecularly heterogeneous partners. This model can explain how developmentally identical neurons acquire diverse wiring identities.

overall_design: Kenyon cells (KC) genetically labeled with GFP, olfactory projection neurons (PN) labeled with tomato, and neurons negative for both (DN) were profiled at 45 hours after puparium formation and in adult.

first: E


title: Catalytic-dependent and independent functions of the histone acetyltransferase CBP promote pioneer factor-mediated zygotic genome activation

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

overall_design: Refer to individual Series


title: Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [Chromatin RNA-seq]

city: Berlin

first: Dubravka

last: Vucicevic

address: Hannoversche Straße 28

department: Berliner Institut for Medical Systems Biology

institute: Max Delbrück Center for Molecular Medicine in the Helmholtz Association

summary: Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the identification of CRISPRa- responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted- SingLe- cell- Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing, and identified functional CaRE-target gene pairs. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks active outside of a given experimental system.

overall_design: Cellular fractionation was performed in SH-SY5Y cell line according to (Conrad and Ørom, 2017). Two replicates of chromatin RNA were extracted using Trizol and Direct-zol RNA MiniPrep Kit (Zymo Research #R2052) according to the manufacturer’s instructions. The library was prepared using NEXTflex Rapid Directional qRNA-Seq Kit (BiooScientific #NOVA-5130-01D) according to the manufacturer’s instructions and paired-end sequencing (2x75nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 150 cycles.

last: Vučićević

first: Che-Wei

last: Hsu

first: Lorena

last: Lopez Zepeda

first: Martin

last: Burkert

first: Antje

last: Hirsekorn

first: Ilija

last: Bilić

first: Nicolai

last: Kastelić

first: Markus

last: Landthaler

first: Scott

middle: A

last: Lacadie

first: Uwe

last: Ohler