title: Decoding the Multiple Functions of ZBP1 in the Mechanism of Sepsis-Induced Acute Lung Injury

city: Pittsburgh

first: Jie

last: Fan

address: 200 Lothrop Street

department: Department of Surgery

institute: University of Pittsburgh School of Medicine

summary: Sepsis-induced acute lung injury (ALI), characterized by severe hypoxemia and pulmonary leakage, remains a leading cause of mortality in intensive care units. The exacerbation of ALI during sepsis is largely attributed to uncontrolled inflammatory responses and endothelial dysfunction. Emerging evidences suggest an important role of Z-DNA binding protein 1 (ZBP1) as a sensor in innate immune to drive inflammatory signaling and cell death during infections. However, the role of ZBP1 in sepsis-induced ALI has yet to be defined. We utilized ZBP1 knockout mice and combined single-cell RNA sequencing with experimental validation to investigate ZBP1's roles in the regulation of macrophages and lung endothelial cells during sepsis. We demonstrate that in sepsis, ZBP1 deficiency in macrophages reduces mitochondrial damage and inhibits glycolysis, thereby altering the metabolic status of macrophages. Consequently, this metabolic shift leads to a reduction in the differentiation of macrophages into pro-inflammatory states and decreases macrophage pyroptosis triggered by activation of the NLRP3 inflammasome. These changes significantly weaken the inflammatory signaling pathways between macrophages and endothelial cells, and alleviate endothelial dysfunction and cellular damage. These findings reveal important roles for ZBP1 in mediating multiple pathological processes involved in sepsis-induced ALI by modulating the functional states of macrophages and endothelial cells, thereby highlighting its potential as a promising therapeutic target.

overall_design: These cells were isolated from the lungs of WT and Zbp1-/- mice at 24 hours post-CLP and sham procedures.

first: Ting

last: Gong

first: Yu

last: Fu

first: Qingde

last: Wang

first: Patricia A

last: Loughran

first: Yuehua

last: Li

first: Timothy R

last: Billiar

first: Zongmei

last: Wen

first: Youtan

last: Liu


title: Endogenous self-peptides guard CNS immune privilege [TCR_Dura-dCLN]

status: Public on Oct 11 2024

city: St. Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

country: USA

institute: Washington University in St. Louis

type: Expression profiling by high throughput sequencing

summary: The central nervous system (CNS), despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system1,2. In fact, it remains physically connected to and can be influenced by the peripheral immune system. How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding conundrum. In searching for molecular cues that derive from the CNS and allow its direct communication with the immune system, we discovered a repertoire of CNS-derived endogenous regulatory self-peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders. During homeostasis, a preponderance of these regulatory self-peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of regulatory self-peptides diminished. Upon boosting the presentation of these regulatory self-peptides, a population of suppressor CD4+ T cells could be expanded, controlling CNS autoimmunity in a CTLA-4 and TGF dependent manner. This unexpected discovery of CNS-derived autoimmune self-peptides may be the molecular key adapting the CNS to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.

overall_design: Empty extracellular vesicles, MOG35-55, or MBP160-175-containing extracellular vesicles were introduced into C57BL/6J male mice via intracisterna magna injection (n=10 per group). 36 hours later cells from the dura as well as the CNS-draining dCLNs were harvested as described in “single cell isolation.” Single cell suspensions were then sorted to capture cells negative for DAPI (live cells) and positive for Thy1.2 and TCRß.

first: Min Woo

middle: W

last: Kim

first: Wenqing

last: Gao

first: Cheryl

middle: F

last: Lichti

first: Xingxing

last: Gu

first: Taitea

last: Dykstra

first: Jay

last: Cao

first: Igor

last: Smirnov

first: Pavle

last: Boskovic

first: Denis

last: Kleverov

first: Andrea

last: Salvador

first: Antoine

last: Drieu

first: Kyungdeok

first: Susan

last: Blackburn

first: Claire

last: Crewe

first: Maxim

middle: N

last: Artyomov

first: Emil

middle: R

last: Unanue


title: mRNA sequencing of MDA-MB-231 engineered for doxycycline-inducible overexpression of wild-type or mutant myocardin related transcription factor A

status: Public on Oct 10 2024

first: Ian

last: Eder

state: Pennsylvania

address: 300 Technology Drive

department: Bioengineering

institute: University of Pittsburgh

summary: Myocardin Related Transcription Factor A (MRTF-A), a co-factor or serum response factor (SRF), has been implicated in breast cancer cell migration and metastatic potential. The goal of this sequencing effort was to determine which mRNA were regulated by MRTF-A overexpression and which mRNA were differentially regulated when function-inhibited (SAP-domain deleted or SRF-binding deficient) mutant MRTF-A were overexpressed. GFP and luciferase expressing MDA-MB-231 cells engineered with doxycycline inducible overexpression of wild-type or mutant MRTF-A were treated with doxycycline for 72 hours prior to RNA collection. Two mutant versions of MRTF-A were used 1) a SAP-domain deleted mutant (deleted base-pairs 343-378) and 2) an SRF binding deficient mutant (mutated K237A, Y238A, H239A, Y241A). The cells were plated in a tissue-treated culture dish or on top of matrigel during doxycycline induction. RNA was extracted using the Qiagen RNeasy Plus Mini Kit according the manufacturer's instructions. Libraries were obtained starting from total RNA following the Illumina Stranded Total RNA preparation Ligation with Ribo-Zero Plus protocol. mRNA was then sequenced on the Illumina NextSeq2000.

overall_design: To investigate how MRTF-A overexpression impacts the RNA transcriptomic profile, we triggered doxycline overexpression of either wildtype or mutant MRTF-A (described in the abstract). We then performed gene expression profiling analysis using data obtained from the RNA-seq across 4 different cell lines in 2 different culture conditions (2D or 3D).

first: Eder

last: Ian

first: Gau

last: David

first: Boone

first: Roy

last: Partha


title: Dual inhibition of oxidative stress and CREB transcription activity by melanin-like nanoparticles slows cyst growth in autosomal dominant polycystic kidney disease

city: Tianjin

first: Zhang

last: Lirong

state: Tianjin

address: No.22 Qixiangtai Road

country: China

institute: Tianjin Medical University

summary: Melanin-like nanoparticles (MNPs) have recently emerged as valuable agents in antioxidant therapy due to their excellent biocompatibility and potent capacity to scavenge various reactive oxygen species (ROS).  However, previous studies have mainly focused on acute ROS-related diseases, leaving a knowledge gap regarding their potential in chronic conditions. Furthermore, apart from their well-established antioxidant effects, it remains unclear whether MNPs target other intracellular molecular pathways. In this study, we synthesized ultra-small polyethylene glycol-incorporated Mn2+-chelated MNP (MMPP). We discovered that MMPP traversed the glomerular filtration barrier and specifically accumulated in renal tubules. Autosomal dominant polycystic kidney disease (ADPKD) is a chronic genetic disorder closely associated with increased oxidative stress and featured by the progressive enlargement of cysts originating from various segments of the renal tubules. Treatment with MMPP markedly attenuated oxidative stress levels, inhibited cyst growth, thereby improving renal function. Interestingly, we found that MMPP effectively inhibits a cyst-promoting gene program downstream of the cAMP-CREB pathway, a crucial signaling pathway implicated in ADPKD progression. Mechanistically, we observed that MMPP directly binds to the bZIP DNA-binding domain of CREB, leading to competitive inhibition of CREB's DNA binding ability and subsequent reduction in CREB target gene expression. Furthermore, by engineering an orally administrable formulation, we demonstrated that orally delivered-MMPP also effectively inhibits cyst growth. In summary, our findings highlight the potential of MMPP in delaying the progression of ADPKD by simultaneously targeting oxidative stress and CREB transcriptional activity.

overall_design: RNA-seq were performed in freshly isolated Dolichos biflorus lectin (DBA) positive cells from WT, ADPKD or mmpp treated ADPKD mice kidneys.

first: Yupeng

last: Chen

first: Xudan

last: Dou


title: Bidirectional histone monoaminylation dynamics regulate neural rhythmicity

city: New York

first: Benjamin

middle: H

last: Weekley

state: NY

address: 1470 Madison Ave

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

type: Genome binding/occupancy profiling by high throughput sequencing

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Trabectedin Promotes Oncolytic Virus Antitumor Efficacy, Viral Gene Expression, and Immune Effector Function in Models of Bone Sarcoma

city: Columbus

first: Emily

middle: M.

last: Ringwalt

state: OH

address: 575 Children's Xrd

department: Center for Childhood Cancer

institute: Nationwide Children's Hospital

summary: We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular antiviral response which increases viral transcript presence in the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing infiltrating immunosuppressive CD4 T and myeloid cells and stimulating granzyme expression in infiltrating T and natural killer cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

overall_design: We demonstrated the efficacy of oncolytic herpes simplex virotherapy (oHSV) and trabectedin in numerous preclinical human xenograft and mouse models of Ewing sarcoma and osteosarcoma. To characterize the mechanisms behind the synergy we observed, we analyzed oHSV presence, enriched pathways, and the cellular infiltrates in Ewing sarcoma and osteosarcoma tumors from treated mice using single-cell RNA transcriptomics. We tested the reliance of the combinatorial synergy in the Ewing sarcoma and osteosarcoma models on the infiltrating immune effector cells through natural killer cell and T cell depletions. Our work was funded by the NIH/NCI through U54 CA232561-01A1 for the Pediatric Ohio-New York Cancer (Peds-ONC) Immunotherapy Center as well as F31 CA278353 and T32 CA269052.

middle: M

first: Mark

middle: A

last: Currier

last: Glaspell

first: Chun-Yu

first: Matthew

middle: V

last: Cannon

first: Maren

last: Cam

first: Amy

middle: C

last: Gross

last: Gust

first: Pin-Yi

last: Wang

first: Louis

last: Boon

first: Laura

middle: E

last: Biederman

last: Schwarz

first: Prajwal

last: Rajappa

first: Dean

last: Lee

first: Elaine

last: Mardis

first: William

last: Carson 3rd

first: Ryan

middle: D

last: Roberts

first: Timothy

middle: P

last: Cripe


title: Expansion of peripheral T cell clonotypes predict response in patients with small cell neuroendocrine bladder and prostate cancers treated with chemo-immunotherapy in a phase I clinical trial

city: Los Angeles

first: Yiqian

last: Gu

state: CA

address: 5000D Terasaki Life Sciences Building

institute: University of California Los Angeles

summary: To explore the impact of immunotherapy, we report a single institution, dual cohort phase Ib study (NCT 03582475) of platinum-based chemotherapy in combination with PD-1 antagonist pembrolizumab to evaluate safety and preliminary efficacy in patients with small cell bladder and small cell/neuroendocrine prostate cancers. To assess the immunologic basis for responses, single cell gene expression and T cell receptor (TCR) sequencing of serial peripheral blood samples revealed a diverse T cell repertoire with expansion of large clonotypes correlating with response.

overall_design: Serial peripheral blood samples from patients with small cell bladder and small cell/neuroendocrine prostate cancers were collected. We utilized scRNA-seq and single cell immune profiling to investigate T cell repertoire related with response.

first: Ann

last: Ly

first: Arnold

middle: I

last: Chin


title: Loss of NUMB drives aggressive bladder cancer via a RHOA/ROCK/YAP signaling axis.

city: Milan

first: Salvatore

last: Pece

address: Via Adamello, 16

department: Experimental Oncology

country: Italy

institute: European Institute of Oncology IRCCS,

summary: We have discovered a previously uncharacterized tumor suppressor function of Numb in the homeostasis of the normal bladder mucosa. Targeted ablation of Numb in the basal layer of the urothelium drives spontaneous bladder tumorigenesis, driving progression from preneoplastic to preinvasive and overtly invasive tumors, and accelerates tumorigenesis in the presence of oncogenic insults. Using 3D-Matrigel organoid cultures to recapitulate bladder tumorigenesis in vitro, we found that Numb deficiency enhances the proliferative and invasive potential of both mouse and human bladder cancer cells. Integrative transcriptomic and functional analyses revealed that downregulation of the canonical Hippo pathway, resulting in enhanced YAP transcriptional activity, underlies the biological aggressiveness of Numb-deficient bladder cancer. These molecular events are dependent on the activation of RhoA/ROCK signaling subsequent to Numb loss. Thus, a dysfunctional Numb–RhoA/ROCK–Hippo/YAP regulatory network is at play in aggressive Numb-deficient bladder cancers, opening avenues for the development of targeted therapies. Furthermore, retrospective cohort studies revealed that a Numb-deficient status correlates with worse overall survival in post-cystectomy muscle-invasive bladder cancer (MIBC) patients and increased risk of progression to MIBC in non-muscle-invasive bladder cancer (NMIBC) patients. The Numb-deficient status was associated with a 27-gene prognostic signature that has the potential to identify high-risk NMIBC patients who are eligible for targeted RhoA/ROCK/YAP therapies.

overall_design: Gene expression profiling of NUMB-Low (HT1376, CLS439, 5637) and NUMB-High cell lines (KK47, RT4, RT112); NUMB-High cell lines (KK47, RT4, RT112) silenced for NUMB or scramble; NUMB-High cell lines (KK47, RT4, RT112) silenced for NUMB and treated with verterporfin, ROCKi or vehicle; RT4 cell line silenced for NUMB and then silenced for CYR61 and CTGF or scramble; Mouse Bladder Organoids (MBO) derived from WT and NUMB-KO mice; MBO NUMB-KO organoids treated with verteporfin, ROCKi or vehicle; tumor cells derived from WT and NUMB-KO mice treated with BBN; BBN NUMB-KO tumor cells treated with verteporfin, ROCKi or vehicle; BBN NUMB-KO tumor cells expressing shCYR61+shCTGF or shLuc.

first: Francesco

last: Tucci

first: Rosa

last: Pennisi

first: Damiano

last: Rigiracciolo

first: Maria

middle: G

last: Filippone

first: Roberta

last: Bonfanti

last: Romeo

first: Stefano

last: Freddi

first: Elisa

last: Guerrera

first: Chiara

last: Soriani

first: Simona

last: Rodighiero

first: Rosalind

last: Gunby

first: Giovanna

last: Jodice

first: Francesca

last: Sanguedolce

first: Giuseppe

last: Renne

first: Nicola

last: Fusco

first: Pier

last: Di Fiore

first: Giancarlo

last: Pruneri

first: Giovanni

last: Bertalot

first: Gennaro

last: Musi

first: Gianluca

last: Vago

first: Daniela

last: Tosoni


title: Pervasive off-target activity in CRISPR-interference (CRISPRi) system [RNA-seq]

city: South San Francisco

first: Rohit

last: Reja

address: 1 DNA Way

institute: Genentech

summary: CRISPR/Cas system and its dCas derivatives have been widely and effectively used to alter the intended genomic target. However, the presence of off-targets due to unintended binding of sgRNAs to sequences that closely resemble the target sequence, is still a major challenge. Here, we utilized a genome-wide sgRNA library for the dCas9-KRAB CRISPRi system to investigate the presence of off-target activity and its effects on gene expression. Our study provides strong evidence that CRISPRi off-targets affect the transcriptome of a cell extensively. We also highlight that mis-matches to the target DNA are tolerated while sgRNA binding and the length of the contiguous matches in the PAM-proximal region is one of the most predictive features of binding, along with open chromatin and the DNA-RNA hybrid energy. Additionally, we provide a curated random forest model that can be used to predict the off-target activity in the dCas systems.

overall_design: RNAseq across two different cell lines, Jurkat and EA.hy926, infected with single guides for ADAT1, HMGB1, GRK4, and LRRC34 respectively, with controls

first: R

first: N

last: Rohatgi


title: Hepatoprotective effects of late-stage clinical drug candidates and dietary intervention in the non-obese CDAA-HFD mouse model of advanced MASH with progressive fibrosis

city: Hørsholm

first: Henrik

last: Hansen

address: Hørsholm Kongevej 11

department: Histology & NGS

country: Denmark

institute: Gubra

summary: We applied RNA sequencing (RNA-seq) to study the gene expression profile in the liver of mice fed the CDAA-HFD for 3 weeks (n=9), 6 weeks (n=10), 12 weeks (n=8) and 20 weeks (n=8) and lean chow controls (n=8). At all timepoints, CDAA-HFD mice displayed a widespread increase in the expression of MASH candidate genes related to inflammation, hepatocellular injury and ECM organization as compared to chow-fed controls. Gene expression was further studied in response to chow reversal (n=9), semaglutide (n=9) and lanifibranor (n=10) as compared to Vehicle (n=10). Chow reversal and lanifibranor, induced wide-spread transcriptional changes in MASH candidate genes while semaglutide showed only discrete effects on candidate genes.

overall_design: RNA-seq of RNA isolated from mice fed CDAA-HFD for 3-20 weeks (n=8-10/group) and healthy controls (n=8), and CDAA-HFD fed mice treated with Vehicle, Semaglutide, Lanifibranor and Chow-reversal (n=9-10/group).

first: Maja

last: Andersen

first: Malte

last: Nielsen

first: Michael

last: Feigh


title: The activity of tertiary lymphoid structures in high grade serous ovarian cancer is governed by site, stroma, and cellular interactions

first: Ian

middle: Philip

last: MacFawn

state: PA

address: 5115 Centre Ave

department: Immunology

summary: Most high grade serous ovarian cancers (HGSOC) originate in the fallopian tube but spread to the ovary and peritoneal cavity, highlighting the need to understand antitumor immunity across HGSOC sites. Using spatial analyses, we discover that tertiary lymphoid structures (TLS) within ovarian tumors are less developed compared with TLS in fallopian tube or omental tumors. We reveal transcriptional differences across a spectrum of lymphoid structures, demonstrating that immune cell activity increases when residing in more developed TLS and produce a prognostic, spatially derived TLS signature from HGSOC tumors. We interrogate TLS-adjacent stroma and assess how normal mesenchymal stem cells MSCs (nMSCs) may support B cell function and TLS, contrary to cancer-educated MSCs (CA-MSCs) which negate the prognostic benefit of our TLS signature, suggesting that pro-tumorigenic stroma could limit TLS formation.

overall_design: FFPE embedded HGSOC specimens were selected from the following anatomic sites: Fallopian Tube, Omentum and Ovary. Non-malignant lymph node was selected as a control. Samples were stained with morphology markers: CD20, CD45, PanCk and processed through the Nanostring GeoMx Digital Spatial Profiler. Using morphology stains, ROI categories were called according to staining combinations. The (v1.0) Human NGS Whole Transcriptome Atlas RNA was used for all samples.

first: MacFawn

last: Ian

first: Bruno

last: Tullia

first: Coffman

last: Lan

first: Bao

last: Riyue


title: ARF alters PAF1 complex integrity to selectively repress oncogenic transcription programs upon p53 loss

city: Dallas

first: Ivan

last: D'Orso

state: TX

address: 5323 Harry Hines

department: Microbiology

institute: UT Southwestern Medical Center

summary:
In this study, we found that the ARF tumor suppressor directly binds the PAF1 subunit to block the assembly of PAF1 complex (PAF1c) and its interaction with RNAPII, thereby dampening PAF1c-dependent transcription in vitro and in cells. To investigate whether ARF regulates RNAPII and PAF1c occupancy at putative target genes identified through RNA-seq, we performed ChIP-seq (RNAPII, PAF1, and CTR9) in ARF  (shARF) vs. control  (shNT) knockdown MEFs. Our data show that loss of ARF increases RNAPII, PAF1, and CTR9 occupancy on upregulated genes, consistent with ARF-mediated gene-specific repression.
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for RNA polymerase subunit RPB3 and PAF1 complex subunits PAF1 and CTR9 in mouse embryonic fibroblasts.

overall_design: Comparative analysis of ChIP-seq data for ARF (shARF) and negative control (shNT) knockdown  MEFs.

first: Jinli

last: Wang

first: Ashutosh

last: Shukla

first: Ashwini

last: Challa

first: Iván

last: D’Orso


summary: In this study, we found that the ARF tumor suppressor directly binds the PAF1 subunit to block the assembly of PAF1 complex (PAF1c) and its interaction with RNAPII, thereby dampening PAF1c-dependent transcription in vitro and in cells. Using RNA-seq of control (shNT) and ARF knockdown (shARF) mouse embryonic fibroblasts (MEFs) we first define transcriptome changes upon ARF loss. Through downstream mechanistic analysis we then validate direct ARF-mediated inactivation of PAF1c-dependent RNAPII transcription and their cellular consequences.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for ARF (shARF) and control knockdown (shNT) MEFs.

first: Jeon

last: Lee


title: Gastric adenocarcinoma of fundic gland type vs matched normal mucosa

city: Tokyo

first: Kazushi

last: Fukagawa

address: 7-3-1 Hongo, Bunkyo-ku

department: Department of Gastroenterology, Graduate School of Medicine

country: Japan

institute: The University of Tokyo

summary:
Background: Gastric adenocarcinoma of fundic gland type (GA-FG) is rare, and the mechanisms of carcinogenesis and growth progression have not to be elucidated. Results: Transcriptional profiles of 3 fresh-frozen GA-FG and surrounding normal mucosa were compared. Key features of gene expression analysis suggested that TTF-1/NKX2-1 acts as an important transcription factor in GA-FG. This study will provide insight into the mechanism of development.
All samples were obtained prior to resection of target lesions in order to minimize effects of cauterization.

overall_design: Biopsy samples from GA-FG and matched normal mucosa were endoscopically obtained from each patients using endoscopic biopsy forceps. The tissues were collected prospectively with informed consent and the approval of the institutional review board.

first: K

first: Y

last: Takahashi

last: Sakaguchi


title: LSD1 Conditional Knockout Mice Have an Increased Bone Mass Due to Increased IFN-B Gene Expression

city: Minneapolis

first: Kim

last: Mansky

state: MN

address: 515 Delaware St SE

institute: University of Minnesota

summary: Osteoclasts are large multinucleated cells that degrade bone mineral and extracellular matrix. Investigating the mechanisms by which osteoclast differentiation is epigenetically regulated is a key to understanding the pathogenesis of skeletal related diseases such as periodontitis and osteoporosis. Lysine specific demethylase 1 (LSD1/KDM1A) is a member of the histone demethylase family that regulates gene expression via the removal of mono- and dimethyl groups from H3K4 and H3K9. Prior to our study, little was known about the effect of LSD1 on skeletal development and osteoclast differentiation. Here we show conditional deletion of LSD1 in the myeloid lineage or macrophage/osteoclast precursors results in a decrease in osteoclast differentiation and activity. Our bulk RNA-SEQ data suggests that osteoclast differentiation is inhibited in LSD1cKO mice due to an increase in expression of IFN-b target genes. Lastly, we demonstrate that LSD1 can form a complex with CoREST, HDAC1 and HDAC2 suggesting a mechanism by which the combination of methylation and acetylation of histone residues regulates osteoclast gene expression.

overall_design: Bone marrow macrophages were isolated from LSD1 WT mice (LSD1 floxed mice) and LSD1cKO mice (LSD1;LysM-Cre) female mice.  Bone marrow macrophages were cultured in M-CSF and RANKL for 2 days. We performed gene expression analysis of RNA-SEQ of three mice of each genotype at one time point.

first: Juan

last: Abrahante Llorens

first: Kristina

last: Astleford-Hopper

first: Elizabeth

last: Bradley


title: Spermidine Metabolism Regulates Leukemia Stem and Progenitor Cell Function through KAT7 Expression

status: Public on Oct 09 2024

city: Toronto

first: Soheil

last: Jahangiri

state: Ontario

address: 610 University Ave

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

summary: To invesetigate the transcriptional changes as a result of polyamine depletion in AML leukemic stem cells and normal hematopoietic stem cells

overall_design: Leukemic stem cells enriched from patient samples and hematopoietic sten cells enriched from normal bone marrow samples were treated with polyamine analog DENSpm or control and collected for RNA sequencing.

first: Vincent

last: Rondeau

first: Jacob

last: Berman

first: Tianyi

last: Ling

first: Cristiana

last: O'Brien

first: Rachel

last: Culp-Hill

first: Julie

last: Reisz

first: Mark

last: Wunderlich

first: Yun

last: Chueh

first: Karina

last: Jiménez-Camacho

first: Christina

last: Sexton

first: Katharine

last: Carter

first: Cody

last: Stillwell

first: Jonathan

last: St-Germain

first: Duhan

last: Yendi

first: Aarushi

last: Gupta

first: Mary

last: Shi

first: Aleksandra

last: Bourdine

first: Vikram

last: Paralkar

first: Kristin

middle: J

last: Hope

first: Anastasia

last: Tikhonova

first: Andrea

last: Arruda

last: Minden

first: Brian

last: Raught

first: Angelo

last: D'Alessandro

first: Courtney

middle: L

last: Jones


title: Understanding the dosage-dependent role of Dicer1 in thyroid tumorigenesis [2]

city: brussels

first: MARIA

last: ROJO PARDILLO

address: Rte de Lennik 808, 1070 Anderlecht

country: Belgium

institute: IRIBHM J.E. DUMONT

summary: Multiple studies suggest Dicer1 as a haploinsufficient tumor suppressor gene: while the loss of one allele promotes tumorigenesis, the complete loss of Dicer1 prevents tumor formation. To study the impact of Dicer1 partial or total loss in papillary thyroid carcinoma cells in vitro, we generated stable Dicer1 (+/-) cell lines by CRISPR-Cas9 from TPC1 cell line. No Dicer1 (-/-) cell lines could be generated. Therefore, siRNA against Dicer1 was transfected into Dicer1 (+/-) cell lines to further decrease its expression. RNA sequencing and transcriptomic analysis revealed alterations in proliferation, cell cycle and cell locomotion. BrdU staining revealed a slow-down of the cell cycle, with lower percentages of cells in S-phase and higher percentages of cells in G0-G1-phase. Furthermore, transwell invasion and migration assays showed a decrease of invasive and migrating cells following transfection of Dicer1 siRNA. These results were confirmed in two supplementary thyroid cell lines, BCPAP and H-Tori3. Additionally, to achieve high protein levels, we performed Dicer1 plasmid transfection and we observed reduced proliferation and invasion as well as increased apoptosis. Globally, our results allow us to better understand the function of Dicer1 in thyroid cancer tumorigenesis and suggest Dicer1 as an attractive target for novel therapeutic strategies.

overall_design: To study the impact of partial loss of Dicer1 we generated stable Dicer1 (+/-) cell lines by Crispr-cas9 from TPC1 cell line.

first: Maria

last: Rojo-Pardillo

first: Ludivine

last: Godefroid

first: Genevieve

last: Dom

first: Frederick

last: Libert

first: Anne

last: Lefort

first: Bernard

last: Robaye

first: Carine

last: Maenhaut


title: Role of Forkhead Box P3 in Interferon g-mediated PD-L1 Expression and Bladder Cancer Differentiation

first: Yiqian

last: Gu

summary: Antagonism of the PD-1/PD-L1 axis is a critical therapeutic strategy for advanced bladder cancer patients. IFNg functions as a key regulator of PD-L1 in both immune as well as cancer cells. Forkhead Box P3 (FOXP3) is a transcription factor synonymous in T regulatory cell function, but with increasingly described functions in cancer cells. Here we investigated the relationship between FOXP3 and PD-L1 in bladder cancer. We showed that FOXP3 is critical in the ability for IFNg to activate PD-L1 in bladder cancer cells. FOXP3 can bind to the PD-L1 promoter and induces a gene program that leads to regulation of multiple immune-related genes and genes involved in epithelial-to-mesenchymal transition. Using in vitro and in vivo human and murine models, we showed that FOXP3 can regulate bladder cancer differentiation as well as promote metastases. Furthermore, FOXP3 may be a convergent factor for multiple activators of PD-L1 including by cisplatin.

overall_design: Total RNA was isolated from HT1376 WT and HT1376 FOXP3 KO cells treated with IFNg. To investigate the relastionship between FOXP3 and PD-L1 in bladder cancer, RNA-seq data was obtained to perform gene expression analysis between four groups: WT, WT IFNg, KO, and KO IFNg.

first: Hanwei

last: Zhang

first: Ann

last: Ly

first: Arnold

last: Chin


title: Asymmetric dynamics of GABAergic system and paradoxical responses of GABAergic neurons in piriform seizures

city: Shang Hai

first: Yan

last: Tao

address: Dong An Road

institute: Fudan University

summary: The piriform cortex (PC) plays a critical role in epileptogenesis, where an excitation/inhibition imbalance contributes to epilepsy etiology. However, the epileptic dynamics of the GABA system and the precise role of GABAergic neurons within the PC in epilepsy remain unclear. Here, we combined Ca2+ and GABA sensors to investigate the dynamics of Gad2-expressing neurons and GABA levels, and selectively manipulated GABAergic neurons in the PC through chemogenetic inhibition and caspase3-mediated apoptosis targeting Gad2 interneurons. The chronic deficiency of PC Gad2 neurons triggered spontaneous recurrent seizures. These findings uncover the dynamic interplay within PC inhibitory components and elaborate counteractive mechanisms in seizure regulation.

overall_design: To delve into the epileptic pathological features underlying this knock-down model, we conducted bulk RNA-seq on PC samples with Gad2 neuron loss in the transcriptome. We employed a knock-down strategy using genetically engineered caspase3, which triggers apoptosis upon activation. Cre-dependent caspase3 was injected to specifically target and eliminate Gad2-positive neurons in the PC.

first: Yu

middle: X

last: Zhao


title: Understanding the dosage-dependent role of Dicer1 in thyroid tumorigenesis

overall_design: To study the role of Dicer1 we knocked down Dicer1 expression by Dicer1 siRNA transfection. Therefore, cells were non transfected (NT), transfected with the negative control (NC) or transfected with Dicer1 siRNA(siDicer)


first: Benjamin

last: Weekley

summary: H3 monoaminylation dynamics are bidirectionally controlled by TG2 and facilitate neural rhythmicity.

overall_design:
RNA-seq experiment, sequencing mRNA from the TMN across the ZT (6 timepoints) from wild type animals or animals transduced with an AAV expressing a GFP only control, H3.3 WT overexpression, or H3.3Q5A mutant overexpression
Reanalyzed samples were submitted to GEO previously in GSE209834.

first: David

last: Vinson

first: Ryan

last: Bastille

first: Qingfei

last: Zeng

first: Yael

last: David

first: Ian

last: Maze

first: Tom

last: Muir


title: Therapeutic benefits of maintaining CDK4/6 inhibitors and incorporating CDK2 inhibitors beyond progression in breast cancer

first: Jessica

middle: Sarah

last: Armand

department: Pathology and Cell Biology

institute: Columbia University Irving Medical Center

summary: The combination of CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) has significantly improved outcomes for patients with hormone receptor-positive (HR+) metastatic breast cancer. However, most patients eventually develop resistance, leading to treatment discontinuation. The therapeutic benefits of maintaining CDK4/6i or transitioning to CDK2 inhibitors (CDK2i) beyond disease progression remain unclear. Here, we show that maintaining CDK4/6i and ET or combining them with CDK2i effectively suppresses the growth of drug-resistant HR+ breast cancer cells by prolonging G1-phase progression. CDK4/6i maintenance triggers a non-canonical pathway for Rb inactivation through post-translational degradation, resulting in attenuated E2F activity and slowed G1 progression. ET further augments this effect by inhibiting c-Myc-mediated E2F amplification. Although the maintenance of CDK4/6i and ET outperforms CDK2i with ET, the triple combination of CDK4/6i, CDK2i, and ET most effectively suppresses both E2F activity and tumor growth. Moreover, while overexpression of both cyclin E and A can promote resistance to the CDK4/6i and CDK2i combination, cyclin E plays a more pivotal role in developing resistance. These findings highlight the potential of sustained CDK4/6i therapy and the incorporation of CDK2i to mitigate resistance in HR+ breast cancer.

overall_design: To gain insight into differences in gene expression upon introduction of CDK2i after dual resistance, palbociclib (CDK4/6i) +fulvestrant (ET) resistant MCF-7 cells were either discontinued from treatment, maintained in palbociclib + fulvestrant, switched to INX-315 (CDK2i) + fulvestrant, or switched to palbociclib + INX-315 + fulvestrant for 20 days then submitted to bulk RNA sequencing.

middle: S

first: Heewon

last: Yang


title: The transcriptional program of Staphylococcus aureus phage K is affected by a host rpoC mutation that confers phage K resistance

city: Silver Spring

first: Ramchander Rohit

last: Kongari

state: Maryland

address: 10903 New Hampshire Ave

department: Department of Bacterial, Parasitic and Allergenic products

institute: United States Food and Drug Administration

summary: To better understand host/phage interactions and the genetic bases of phage resistance in a model system relevant to potential phage therapy, we isolated several spontaneous mutants of the USA300 S. aureus clinical isolate NRS384 that were resistant to phage K.  Six of these had a single missense mutation in the host rpoC gene, which encodes the RNA polymerase beta prime subunit. To examine the hypothesis that the mutations in the host RNA polymerase affect the transcription of phage genes, we performed RNA-seq analysis on total RNA samples collected from NRS384 wild-type (WT) and rpoC G17D mutant cultures infected with phage K, at different time points after infection. Infection of the WT host led to a steady increase of phage transcription relative to the host.  Our analysis allowed us to define different early, middle, and late phage genes based on their temporal expression patterns and group them into transcriptional units. Predicted promoter sequences defined by conserved -35, -10, and in some cases extended -10 elements were found upstream of early and middle genes. However, sequences upstream of late genes did not contain clear, complete, canonical promoter sequences, suggesting that factors in addition to host RNA polymerase are required for their regulated expression. Infection of the rpoC G17D mutant host led to a transcriptional pattern that was similar to the WT at early time points. However, beginning at 20 minutes after infection, transcription of late genes (such as phage structural genes and host lysis genes) was severely reduced. Our data indicate that the rpoCG17D mutation prevents the expression of phage late genes, resulting in a failed infection cycle for phage K. In addition to illuminating the global transcriptional landscape of phage K throughout the infection cycle, these studies can inform our investigations into the bases of phage K’s control of its transcriptional program as well as mechanisms of phage resistance.

overall_design: For transcriptomic analysis, four replicate 25 mL cultures of NRS384WT and rpoC G17D were grown to an OD600 of ~0.3, and phage K was added at an MOI of 5. 1 mL samples were collected at 2, 5, 10, 20, 30, 40 min after addition of phage, and total RNA was extracted from these samples using the QIAGEN RNEasy Mini Kit. The samples were further treated with Turbo DNAse to degrade phage/bacterial DNA, and the RNA quality was measured using an Agilent 2100 Bioanalyzer. Ribosomal RNA was depleted using the Illumina Ribozero plus kit, and Illumina RNA-seq libraries were prepared without any enrichment. A strand-specific RNA seq Novaseq S2 100-bp paired end run was performed on the samples using an Illumina Novaseq 6000 instrument

first: Ramchander

last: Rohit Kongari

first: Melissa

last: Ray

first: Susan

last: Lehman

first: Roger

last: Plaut

first: Debbie

last: Hinton

first: Earle

last: Stibitz


title: Identification of genes bound by NFAT5-SMADs complex

city: Newark

first: Pingping

last: Hou

state: New Jersey

address: 205 South Orange Ave

department: Microbiology, Biochemistry and Molecular Genetics

institute: Rutgers University

summary: Oncogenic KRAS is now considered a druggable target; however, multiple mechanisms contribute to the development of resistance to KRAS-targeted therapy. A significant factor in therapy resistance is the alteration in cell state or cellular plasticity, exemplified by the epithelial-to-mesenchymal transition (EMT) phenotype. In pancreatic ductal adenocarcinoma (PDAC), the negative correlation between addiction to oncogenic KRAS signaling and EMT has been observed, yet the role of cell plasticity and its underlying mechanisms in governing resistance remain unclear. Our findings reveal that the pivotal EMT driver, TGFβ, facilitates KRAS bypass in PDAC through the nuclear factor NFAT5. NFAT5 interacts with canonical TGFβ factors SMAD3 and SMAD4, inducing EMT and therapy resistance. To identify DNA bound by the NFAT5-SMADs complex, we conducted Chromatin IP followed by next-generation sequencing (ChIP-seq) using antibodies binding to NFAT5, SMAD2, SMAD3, and SMAD4.

overall_design: We conducted ChIP-seq using antibodies binding to endogenous NFAT5, SMAD2, SMAD3, and SMAD4 in mouse PDAC cells.

first: Daiyong

last: Deng


title: Demethylzeylasteral inhibits oxidative phosphorylation complex biogenesis by targeting LRPPRC in lung cancer

city: Beijing

first: Lina

last: Wang

address: No. 2, Bei Yi Street, Zhongguancun, Haidian District, Beijing

institute: Institute of Chemistry, Chinese Academy of Sciences

summary: Targeted inhibition of mitochondrial oxidative phosphorylation (OXPHOS) complex generation is an emerging and promising cancer treatment strategy, but limited targets and specific inhibitors have been reported. Leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is an atypical RNA-binding protein that regulates the stability of all 13 mitochondrial DNA-encoded mRNA (mt-mRNA) and thus participates in the synthesis of the OXPHOS complex. LRPPRC is also a prospective therapeutic target for lung adenocarcinoma, serving as a promising target for OXPHOS inhibition. In this study, we identified Demethylzeylasteral (T-96), a small molecule extracted from the Chinese herb Tripterygium wilfordii Hook. f., as a novel inhibitor of LRPPRC. T-96 directly bound to the RNA-binding domain of LRPPRC, inhibiting its interaction with mt-mRNA. This led to instability in both mt-mRNA and LRPPRC protein. Treatment with T-96 significantly reduced the mRNA and protein levels of the OXPHOS complex. As a consequence of LRPPRC inhibition, T-96 treatment induced a defect in the synthesis of the OXPHOS complex, inhibiting mitochondrial aerobic respiration and ATP synthesis. Moreover, T-96 exhibited potent antitumor activity for lung adenocarcinoma in vitro and in vivo, and the antitumor effect of T-96 was dependent on LRPPRC expression. In conclusion, this study not only identified the first traditional Chinese medicine monomer inhibitor against OXPHOS complex biosynthesis as well as a novel target of Demethylzeylasteral, but also shed light on the unique antitumor mechanism of bioactive compounds derived from traditional Chinese medicine.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for A549 cells and T-96 treated A549 cells.

first: Wei

last: Zhou

first: Wenxi

first: Yuxin

last: Liang

first: Qiqi

last: Xue

first: Zhen

last: Zhang

first: Jinghe

last: Yuan

first: Xiaohong

last: Fang


title: Determination of the tumor microenvironment remodeling by KRAS targeted therapy in pancreatic cancer

summary: Oncogenic KRAS is now recognized as a viable target for drug intervention; nevertheless, the efficacy of KRAS-targeted therapy is impeded by various resistance mechanisms. The intricate interplay between cancer cells and the cells within the tumor microenvironment (TME) actively contributes to the mutual promotion of resistance to KRAS-targeted therapies. To discern specific cell populations orchestrating tumor responses in pancreatic ductal adenocarcinoma (PDAC), we conducted single-cell RNA sequencing (scRNA-seq) on spontaneous tumors obtained from genetically engineered mouse models. This analysis was performed both before and after the inhibition or genetic ablation of KRAS, shedding light on the dynamic changes occurring at the single-cell level.

overall_design: Spontaneous tumors from KPC or iKPC mice were dissected into single cells for library preparation.


title: Nuclear Factor NFAT5 Governs Cellular Plasticity-Induced KRAS-Targeted Therapy Resistance in Pancreatic Cancer

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

overall_design: Refer to individual Series


title: Bidirectional histone monoaminylation dynamics regulate neural rhythmicity

first: Benjamin

middle: H

last: Weekley

overall_design: CUT&RUN experiments across the ZT in the mouse TMN include H3K4me2, H3K4me3, H3K4me3Q5ser, H3K4me3Q5his, and WDR5. CUT&RUN in HeLa cells including H3K4me3Q5his and H3Q5his.

first: David

last: Vinson

first: Ryan

last: Bastille

first: Qingfei

last: Zeng

first: Yael

last: David

first: Ian

last: Maze

first: Tom

last: Muir


title: Gene expression profiles of each subset of human intestinal mononuclear phagocytes (MNPs)

status: Public on Oct 08 2024

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: MNPs play an important role in intestinal homeostasis. The interaction of intestinal metabolites with G protein-coupled receptors (GPCRs) expressed on human MNPs has not been well characterized. We report here that GPR31 is specifically expressed in cDC1 in human ileal  MNPs by bulk RNA sequencing.

overall_design: Macrophages, type1 conventional dendritic cells (cDC1),  type2 conventional dendritic cells (cDC2) and  plasmacytoid dendritic cells (pDC) from unaffected small intestinal mucosa were isolated by Fluorescence-activated cell sorting (FACS) and analyzed using RNAseq.

first: Eri

last: Oguro-Igashira

first: Mari

last: Murakami

last: Okuzaki

first: Kiyoshi

last: Takeda


title: Intracellular zinc protects tumors from T cell-mediated cytotoxicity [MC38 RNAseq]

city: Melbourne

first: Jane

last: Oliaro

state: Victoria

address: 305 Grattan St

country: Australia

institute: Peter MacCallum Cancer Centre

summary: We performed RNA sequencing on parental (WT) and Cbfb-deficient (CbfbKO) MC38 cells. Cells were generated by electroporation with Cas9-sgRNA complexes using a non-targeting sgRNA (WT) or Cbfb-targeting sgRNA (CbfbKO)

overall_design: At least 1 week after electroporation with Cas9-sgRNA (non-targeting or Cbfb-targeting), KO was confirmed by western blot and approximately 1 million WT and CbfbKO cells were collected for RNA extraction and sequencing. 3 replicates per group were collected. 2 replicates from each group were used for downstream library preparation, and the 3rd replicate from each group was not used due to poor quality RNA.

first: Emily

last: Lelliott


title: Combined transcriptome and proteome profiling reveal cell-type-specific functions of Drosophila garland and pericardial nephrocytes

city: Kiel

first: Judith

last: Bossen

address: Am Botanischen Garten 3-9

department: Molecular Physiology

country: Germany

institute: CAU Kiel, Zoological Institute

summary: Drosophila nephrocytes are specialised cells that share critical functional, morphological, and molecular features with podocytes of the mammalian kidney, including the slit diaphragm, which is a functional filtration barrier. Accordingly, nephrocytes are considered highly suitable invertebrate models for glomerular disease in humans. In this study, we established a method to individually isolate the garland and pericardial nephrocytes from Drosophila of different developmental stages. This allowed us to collect nephrocyte samples that were devoid of any contamination by other cell types. We used a combination of mass spectrometry-based proteomics and RNA-Seq-based transcriptomics to characterise the proteome and transcriptome of the respective cells in an integrated manner. In addition to identifying a set of factors that were expressed exclusively in nephrocytes, we observed characteristic changes in the cellular proteome and transcriptome as a consequence of ageing. Furthermore, functional enrichment analyses suggested that larval and adult nephrocytes fulfil distinct physiological functions, which was also indicated for garland and pericardial nephrocytes in adult animals. In addition, the latter cells were characterised by transcriptomic and proteomic profiles that were suggestive of a cell-type-specific energy metabolism. Finally, both types of nephrocytes displayed transcriptomic signatures that indicated elevated immune signalling, implying that nephrocytes are immune competent. The data generated in this study may represent a valuable basis for a more specific application of the Drosophila model system in the analysis of renal cell function in health and disease.

overall_design: Dissected nephrocytes (pericardial and garland cells) were used for proteomic and transcriptomic analysis. This included nephrocytes from larvae and adults of different age.

first: Heiko

last: Meyer

first: Maren

last: Janz

first: Xenia

last: Müller

first: Thomas

last: Roeder

first: Achim

last: Paululat


title: Differing Epithelial and Immunologic Activation Patterns Following Food Reintroduction Reveal Differential Transcriptional Profiles in Active Eosinophilic Esophagitis

city: Salt Lake City

first: Kathryn

middle: Anne

last: Peterson

state: Utah

address: 30 N Mario Capecchi Dr

department: Internal Medicine

institute: University of Utah

summary: During food trigger reintroductions, eosinophilia can recur in a patchy manner both endoscopically and histologically. We postulated that areas containing low eosinophils represented the early stage of EoE recurrence, while areas with >15 eos/HPF represented established active EoE. We identified ten patients with prior pan-esophageal EoE who experienced patchy eosinophilia during trigger food reintroduction. The progression of recurrent EoE is illustrated by the transcriptional changes occurring from baseline remission through recurrent low and high eosinophil tissues in those with paired time points. We identified novel genes of cytokine regulation distinct to the low eosinophil tissue, indicating involvement of such cytokines before the onset of diagnostic eosinophilic infiltration. The advancement to robust eosinophil is associated with more severe epithelial injury in conjunction with further inflammation. Utilization of a food antigen re-exposure model elucidates immunological and epithelial changes that instigate and perpetuate the development of EoE.

overall_design: To investigate transcriptional shifts associated with the elimination and reintroduction of an antigen. Formalin-fixed, paraffin-embedded esophageal biopsies from patients with EoE at paired time points: (1) “baseline remission” with trigger foods eliminated (n=5, <10 eos/HPF); (2) trigger food reintroduction with patchy eosinophilia consisting of (2a) “recurring low eosinophil” (n=10, <13 eos/HPF) and (2b) “recurring high eosinophil” (n=10, >18 eos/HPF). Non-EoE control esophageal samples (n=22) were included from patients  with normal histology.

first: Ashley

last: Pyne

first: Amiko

last: Uchida

first: Mark

middle: W

last: Hazel

first: Maria

last: Pletneva

first: Kristina

last: Allen-Brady


title: Molecular diversity and migration of GABAergic neurons in the developing ventral midbrain

city: Utrecht

first: Özge

last: Düdükcü

address: Universiteitsweg 100

country: Netherlands

institute: University Medical Center Utrecht

summary: single-cell RNA sequencing data (plate-based CelSeq2) of developing mouse ventral midbrain GABAergic neurons. vmbGABA series contains E16 and P0 VGAT mouse ventral midbrain and E16 and P0 VGAT/PITX3 co-expressing mouse ventral midbrain.

overall_design: These cells were isolated from VGAT-Cre:Ai14:Pitx3-GFP mice using FACS sorting for td-tomato signal to isolate GABAergic neurons and td-tomato + GFP  signal to isolate the co-expressing population. single-cell RNA sequencing data (plate-based CelSeq2) of isolated GABAergic neurons in developing mouse ventral midbrain.

first: Divya

last: Raj

first: Lieke

last: van de Haar

first: Laurens

middle: M

last: Grossouw

first: Louisa

last: Linders

first: Oxana

last: Garritsen

first: Youri

last: Adolfs

first: Nicky

last: van Kronenburg

last: Broekhoven

first: Troy

last: Kapteijns

first: Frank

last: Meye

first: R

last: Pasterkamp


title: FNDC4 alleviates cardiac ischemia/reperfusion injury through facilitating HIF1α-dependent cardiomyocyte survival and angiogenesis in male mice

city: Wuhan

first: Xin

last: Zhang

state: Hubei

address: 238 Jiefang Road

department: Geriatrics

institute: Renmin Hospital of Wuhan University

summary: Fibronectin type III domain-containing (FNDC) proteins play critical roles in cellular homeostasis and cardiac injury, and our recent findings define FNDC5 as a promising cardioprotectant against doxorubicin- and aging-related cardiac injury. FNDC4 displays a high homology with FNDC5; however, its role and mechanism in cardiac ischemia/reperfusion (I/R) injury remain elusive. Here, we show that cardiac and plasma FNDC4 levels are elevated during I/R injury in a hypoxia-inducible factor 1α (HIF1α)-dependent manner. Cardiac-specific FNDC4 overexpression facilitates, while cardiac-specific FNDC4 knockdown inhibits cardiomyocyte survival and angiogenesis in I/R-stressed hearts of male mice through regulating the proteasomal degradation of HIF1α. Interestingly, FNDC4 does not directly stimulate angiogenesis of endothelial cells, but increases the expression and secretion of fibroblast growth factor 1 from cardiomyocytes to enhance angiogenesis in a paracrine manner. Moreover, therapeutic administration of recombinant FNDC4 protein is sufficient to alleviate cardiac I/R injury in male mice, without resulting in significant side effects. In this work, we reveal that FNDC4 alleviates cardiac I/R injury through facilitating HIF1α-dependent cardiomyocyte survival and angiogenesis, and define FNDC4 as a promising predictive and therapeutic target of cardiac I/R injury.

overall_design: To investigated the role of FNDC4 in cardiac I/R injury, mice were overexpressed with FNDC4 by AAV9 vectors or control AAV9, and then subjected to I/R surgery, with the hearts collected for transcriptome analysis 24 h postI/R injury


title: Gene expression profile of human intestinal mononuclear phagocytes (MNPs) at the single-cell level II

first: Daisuke

last: Motooka

summary: MNPs play an important role in intestinal homeostasis. The interaction of intestinal metabolites with G protein-coupled receptors (GPCRs) expressed on human MNPs has not been well characterized. We used single-cell RNA sequencing (scRNA-seq) to characterize human intestinal NMPs and identify cell type-specific GPCRs.

overall_design: MNPs from unaffected intestinal mucosa were isolated by Fluorescence-activated cell sorting (FACS) and analyzed using scRNA-seq.

first: Eri

last: Oguro-Igashira

first: Mari

last: Murakami

first: Kiyoshi

last: Takeda


title: Immunotherapies that repolarize macrophages and regulatory T cells enhance the effect of chemotherapy in high-grade serous ovarian cancer (Human)

city: London

first: Samar

last: Elorbany

address: Barbican

department: Tumour Microenvironment

country: United Kingdom

institute: Queen Mary University of London

summary:
We conducted single-cell RNA sequencing (scRNAseq) of tumor-infiltrating immune cells in high-grade serous ovarian cancer (HGSOC) omental biopsies to identify potential targets that could enhance response to neo-adjuvant chemotherapy (NACT). Analysis of 64,097 cells identified NACT-induced overexpression of stabilin-1 (clever-1) on macrophages and FOXP3 in Tregs and these were validated at the protein level. STAB1 inhibition in vitro induced anti-tumor macrophages. FOXP3 anti-sense oligonucleotide (FOXP3-ASO), repolarised Tregs to an effector T cell phenotype.
ScRNAseq on 69,781 cells from an HGSOC syngeneic mouse model recapitulated the patients’ data. Combining chemotherapy with anti-stabilin1 antibody and/or Foxp3-ASO significantly increased survival of mice with established peritoneal disease in two HGSOC syngeneic models and progression-free survival in a third model. Long-term survivors (300 days+) were resistant to tumor rechallenge. Anti-stabilin1 antibody enriched the tumors with CXCL9+ macrophages and Foxp3-ASO increased TBET cell infiltration. Our results suggest targeting these molecules may improve chemotherapy response in patients.

overall_design: Tumours were taken either during primary debulking surgery pre-NACT (n=3) or three week after chemotherapy in the post-NACT(n=4).

first: Chiara

last: Berlato

first: Larissa

last: Carnevalli

first: Eleni

last: Maniati

first: Jun

last: Wang

first: Simon

middle: T

last: Barry

first: Ranjit

last: Manchanda

first: Julia

last: Kzhyshkowska

first: Frances

last: Balkwill


title: Chemotherapy induces myeloid-driven spatial T-cell exhaustion in ovarian cancer

city: Helsinki

first: Matías

last: M. Falco

address: Biomedicum Helsinki 1, Haartmaninkatu 8

department: Faculty of Medicine

country: Finland

institute: University of Helsinki

summary: To characterize the spatial tumor microenvironment (TME) of high-grade serous ovarian cancer (HGSC) at the single cell level, we performed single-cell RNA sequencing (scRNA-seq) on 48 tumor or ascites samples from 29 HGSC patients. This data set includes paired scRNA-seq data from chemo-naive and post-neoadjuvant chemotherapy (IDS) samples of 22 HGSC patients.

overall_design:
Gene expression profiles of single cells from chemo-naïve and IDS tumor tissue and ascites samples from 29 HGSC patients. Raw data will be deposited in EGA due to patient privacy concerns.

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Submitter states that missing raw data are being made available for controlled access in EGA.
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first: Inga-Maria

last: Launonen

first: Erdogan

last: Pekcan Erkan

first: Matias

last: Marin Falco

first: Anna

last: Vähärautio

first: Anniina

last: Färkkilä


title: ZBTB18 hinders cytokine expression to impair microglia and macrophage recruitment and the establishment of a tumor-supportive microenvironment in glioblastoma

city: Freiburg

first: Eyleen

last: Corrales

address: Breisacherstr. 153

department: Medizinische Fakultät

institute: Institut für Medizinische Bioinformatik und Systemmedizin

summary:
Background. Glioma associated macrophages/microglia (GAMs) are massively recruited to the tumor site where they commit to a tumor promoting phenotype, driving glioblastoma progression. GAMs secrete several factors that facilitate tumor proliferation and invasion, and prevent an effective immune response against glioblastoma. Here, we investigate how the tumor suppressor ZBTB18 affects GAMs and ultimately shapes the tumor microenvironment.
Methods. The regulation of cytokine expression and secretion was assessed by gene expression and cytokine arrays. The effect of ZBTB18 expression on microglia properties was investigated in vivo in mouse xenografts and by RNAseq of microglia cells conditioned with the medium of ZBTB18-expressing patient-derived GBM cells. The analysis was further extended to TAM scRNAseq data (GBMap).
Results. Here, we demonstrate that ZBTB18, a transcriptional repressor with tumor suppressive function in glioblastoma, impairs the production of key chemokines responsible for GAM recruitment. Consistently, we observe a reduced migration of GAMs towards ZBTB18-expressing glioblastoma cells, both in cell culture and in vivo experiments. Moreover, RNA sequencing analysis shows that the presence of ZBTB18 in glioblastoma cells alters the commitment of conditioned microglia, suggesting the loss of the immunosuppressive phenotype.
Conclusions. Our data indicate that, by blocking the release of key cytokines, ZBTB18 modifies microglia behavior, counteracting their tumor-promoting function. Thus, therapeutic approaches to increase ZBTB18 expression in glioblastoma cells could represent an effective adjuvant to immunotherapy in the treatment of this kind of tumor.

overall_design: To investigate how ZBTB18 shape glioma associated macrophages/microglia (GAMs), patient-derived glioblastoma stem cell-like with no endogenous expression of ZBTB18 were transduced to express full length ZBTB18 (ZBTB18-FL). Transcriptional changes were addressed by RNA-seq in comparison to empty vector control cells (EV).

first: Maria

last: Carro

first: Melanie

last: Boerries

first: Geoffroy

last: Andrieux


title: PDGF-BB overexpression in p53 null oligodendrocyte progenitors increases H3K27me3 and induces transcriptional changes which favor proliferation

city: New York

first: Patrizia

last: Casaccia

address: 85 St Nicholas Terrace

department: Neuroscience

institute: ASRC-CUNY

summary: Proneural gliomas are aggressive brain tumors characterized by enrichment of oligodendrocyte progenitor cell (OPC) transcripts and genetic alterations. In this study we sought to identify transcriptional and epigenetic differences between OPCs with Trp53 deletion and PDGF-BB overexpression (BB-p53n), which form aggressive gliomas when transplanted in mouse brains, and those carrying only p53 deletion (p53n), which do not. We used unbiased histone proteomics and RNA-seq analysis on these two OPC populations and detected higher levels of H3K27me3 in BB-p53n compared to p53n OPCs. The BB-p53n OPC were characterized by higher levels of transcripts related to proliferation and lower levels of those related to differentiation. Pharmacological inhibition of histone H3K27 trimethylation in BB-p53n OPC reduced cell cycle transcripts and increased the expression of differentiation markers. These data suggest that PDGF-BB overexpression in p53 null OPC results in histone post-translational modifications and consequent transcriptional changes favoring proliferation while halting differentiation, thereby promoting the early stages of transformation.

overall_design: To investigate the effects of PDGF-BB expression in p53 null primary OPCs. Separately to investigate the effects of EZH2 inhibition on p53 null PDGF-BB expressing primary OPCs

first: Huang

last: Dennis

first: Mela

last: Angeliki

first: Canoll

last: Peter

first: Casaccia

last: Patrizia


title: Differential usage of shared binding sites drives functional specialization of an essential family of transcription factors [ChIP-seq]

city: Philadelphia

first: Aman

last: Husbands

email: aman.husbands@gmail.com, ayh@upenn.edu

address: 103B Caroline Lynch Bldg, 433 S University Ave

institute: University of Pennsylvania

summary: Functional divergence of transcription factors (TFs) has driven cellular and organismal complexity throughout evolution, but its mechanistic drivers remain poorly understood. Here we test for new mechanisms using CORONA (CNA) and PHABULOSA (PHB), two functionally diverged paralogs in the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) family of TFs. We show that virtually all genes bound by PHB ( ~ 99%) are also bound by CNA, ruling out occupation of distinct sets of genes as a mechanism of functional divergence. Further, genes bound and regulated by both paralogs are almost always regulated in the same direction, ruling out opposite regulation of shared targets as a mechanistic driver. Functional divergence of CNA and PHB instead results from differential usage of shared binding sites, with hundreds of uniquely regulated genes emerging from a commonly bound genetic network. Regulation of a given gene by CNA or PHB is thus a function of whether a bound site is considered ‘responsive’ versus ‘non-responsive’ by each paralog. Discrimination between responsive and non-responsive sites is controlled, at least in part, by their lipid binding START domain. This suggests a model in which HD-ZIPIII TFs use information integrated by their START domain to generate paralog-specific transcriptional outcomes from a shared network architecture. Taken together, our study identifies a mechanism of HD-ZIPIII TF paralog divergence and proposes the ubiquitously distributed START evolutionary module as a driver of functional divergence.

overall_design: To investigate genes differentially regulated after indiction of CLASS III HOMEOEDOMAIN LEUCINE ZIPPER (HD-ZIPIII) transcription factors. Sequencing was performed on 3 biological replicates of induced and mock induced genotypes of CORONA (CNA) and PHABULOSA (PHB) as well as chimeric CNA constructs.

first: Ashton

last: Holub

first: Sarah

last: Choudury

first: Ekaterina

last: Andrianova

first: Courtney

last: Dresden

first: Igor

last: Zhulin


title: Identification of genes regulated by NFAT5-SMADs complex

first: Pingping

last: Hou

summary: Oncogenic KRAS is now considered a druggable target; however, multiple mechanisms contribute to the development of resistance to KRAS-targeted therapy. A significant factor in therapy resistance is the alteration in cell state or cellular plasticity, exemplified by the epithelial-to-mesenchymal transition (EMT) phenotype. In pancreatic ductal adenocarcinoma (PDAC), the negative correlation between addiction to oncogenic KRAS signaling and EMT has been observed, yet the role of cell plasticity and its underlying mechanisms in governing resistance remain unclear. Our findings reveal that the pivotal EMT driver, TGFβ, facilitates KRAS bypass in PDAC through the nuclear factor NFAT5. NFAT5 interacts with canonical TGFβ factors SMAD3 and SMAD4, inducing EMT and therapy resistance. To unravel the regulatory role of the NFAT5-SMADs complex in KRAS* bypass, we conducted transcriptomic analysis in TGFβ-treated, KRAS*-depleted iKPC spheroids.

overall_design: To investigate the cooperative function of NFAT5-SMADs complex in the regulation of KRAS* bypass, we knocked down or inhibited NFAT5 and SMAD3 in mouse PDAC cells.

first: Daiyong

last: Deng


title: A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells

first: Justin

last: English

state: UT

address: 15 N Medical Drive East, Rm 4100

department: Biochemistry

summary: Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this library’s utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications.

sample_organism:
Homo sapiens
Mesocricetus auratus
Mus musculus
synthetic construct

overall_design: Multiple mammalian cell lines were transfected with the TRE-MPRA library for 24 hours and were then treated with growth factors or specific compounds for six hours. For some experiments, cells were co-transfected with specific G protein-coupled receptors and treated with receptor agonsists for six hours. Some treatments contained multiple technical replicates, while others were performed once. RNA and DNA barcode abundance was determined via Illumina next-generation sequencing.

first: Adam

last: Zahm


title: Mutated b-catenin Gene Signature to Identify CTNNB1-mutated and Immune Checkpoint Resistant Hepatocellular Cancers

city: Pittsburgh

first: Shuchang

last: Liu

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: Background & Aims: Patients with beta-catenin (encoded by CTNNB1)-mutated hepatocellular carcinoma (HCC) have demonstrated limited clinical benefit to first-line immunotherapy (IO). Animal models of HCC expressing mutant-beta-catenin and additional aberrations via hydrodynamic tail vein injection with sleeping beauty transposon/transposase (SB-HDTVI) represent clinically relevant human HCC subsets. Here, we perform transcriptomic analysis on multiple beta-catenin-mutated and non-mutated HCC animal models to identify Mutated beta-catenin Gene Signature (MBGS) for HCC patient stratification for CTNNB1-mutations and IO response. Methods: We co-expressed in mice mutant-NFE2L2 and hMET +- mutant-CTNNB1 via SB-HDTVI and monitored for HCC development. Bulk RNA-sequencing was assessed for transcriptional differences between various beta-catenin-mutated and non-mutated models. MBGS was generated for predictive ability of CTNNB1 mutations and IO resistance in multiple HCC patient cohorts. Results: Co-expression of S45Y-beta-catenin + G31A-NFE2L2 + hMet (beta-N-M) resulted in HCC development by 4.5 weeks while co-expression of G31A-NFE2L2 + hMet (N-M) led to HCC by 14 weeks with tumors being positive for expected targets by immunohistochemistry (IHC). Bulk RNA-sequencing comparing beta-catenin-driven versus non-beta-catenin driven models yielded 95 common upregulated genes. Differential gene expression analysis of the common genes comparing CTNNB1-mutated vs non-mutated TCGA patients narrowed the gene panel to 13-(or 10-) genes. This MBGS predicted CTNNB1-mutation status in TCGA (n=374) and French (n=398) patient cohorts (with ROC AUC of 0.90 and 0.94). High MBGS expression was also associated with no overall and progression-free survival benefit when comparing atezolizumab + bevacizumab versus sorafenib arms in IMbrave150 cohort implying fewer treatment effects. Conclusions: In an era of patient molecular stratification for HCC, MBGS may aid in optimally selecting patients for IO through diagnosis of a molecular subset which lacks optimal response.

overall_design: RNA-seq on mouse liver samples with five condition groups. G1: Wild-type FVB liver; G2: S45Y-CTNNB1 + G31A-NFE2L2 + hMET; G3: S45Y-CTNNB1 + hMET; G4: S45Y-CTNNB1 + G31A-NFE2L2; G5: G31A-NFE2L2 + hMET

first: Brandon

last: Lehrich

first: Tao

last: Junyan

first: Silvia

first: Catherine

last: Cao

first: Theo

middle: Z

last: Hirsch

first: Evan

middle: R

last: Delgado

first: Tyler

last: Yasaka

first: Xiangnan

last: Guan

first: Hartmut

last: Koeppen

first: Long

last: Pan

first: Yuqing

first: Sucha

last: Singh

first: Minakshi

last: Poddar

first: Aaron

last: Bell

first: Aatur

last: Singhi

first: Jessica

last: Zucman-Rossi

first: Yulei

last: Wang

first: Satdarshan

last: Monga


title: Interleukin-22 improves ovulation disorder via STAT3 signalling in polycystic ovary syndrome

first: Yanli

last: Pang

state: Beijing

address: No.49 Huayuan-Bei Road , Haidian District

institute: Reproductive Medicial Center , Peking University Third Hospital

summary: Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease which leads to serious impairment of reproductive health in women of child-bearing age. Anovulation or oligo-ovulation is a common clinical manifestation of PCOS patients. The disturbance of ovarian immune microenvironment contributes to the disorders of follicle development and ovulation, and the mechanism remains unclear. Here we demonstrated the protective effect of immune factor interleukin-22 (IL-22) on PCOS follicle development and ovulation. Follicular IL-22 levels were significantly lower in PCOS patients than in the control group and were positively correlated with the oocyte fertilization rate and the high-quality embryo rate. Additionally, IL-22 evidently improved follicle development in vitro and promoted ovulation-related gene expression, which was disrupted by the depletion of interleukin-22 receptor 1 (IL-22R1) or inhibition of STAT3 in granulosa cells, indicating that IL-22 acted through IL-22R1 and STAT3 signalling pathway to promote follicle development and ovulation in PCOS. In summary, this study elucidated the vital role of ovarian immune microenvironment in follicle development and ovulation, the application of IL-22 may provide new insights into the treatment of PCOS patients.

overall_design: Granulosa cells from Il22r+/+ or Il22r-/- mice were collected for RNA-sequencing, and there was three samples in  Il22r+/+ and Il22r-/- group

first: Baoying

last: Liao

first: Weixuan

last: Chen

first: Xinyu

last: Qi

first: Chuyu

last: Yun


title: Effect of pyruvate (PA), lactate (LA), or 12-hydroxyeicosatetraenoic acid (12-HETE) stimulation on type1 conventional dendritic cells (cDC1) from the human ileum

first: Daisuke

last: Motooka

summary: We here report that GPR31 is specifically expressed in cDC1 in human intestinal  mononuclear phagocytes. Since GPR31 has been previously reported to be activated by PA, LA, and 12-HETE, RNA-seq was performed to analyze the transcriptional changes in human ileal cDC1 under the stimulation of PA, LA, or 12-HETE.

overall_design: cDC1 were isolated from the human ileum of 4 individuals by Fluorescence-activated cell sorting (FACS), followed by the stimulation with PA,  LA, or 12-HETE and subjected to RNA-seq analysis for gene expression profiling.

first: Eri

last: Oguro-Igashira

first: Mari

last: Murakami

last: Okuzaki

first: Kiyoshi

last: Takeda


title: Gene expression profile of human intestinal mononuclear phagocytes (MNPs) at the single-cell level


title: PU.1 Eviction at Lymphocyte-Specific Chromatin Domains Mediates Glucocorticoid Response in Acute Lymphoblastic Leukemia

city: Sydney

first: Dominik

last: Beck

state: NSW

address: Level 10, UTS Building 11 (CB11 81-113, Broadway, Ultimo)

department: School of Biomedical Engineering

institute: University of Technology Sydney

summary: Glucocorticoids are an essential part of many multi-agent treatment regimens for lymphoid malignancies where the emergence of glucocorticoid resistance is a significant barrier to a cure. We have previously identified that aberrant chromatin accessibility, at lymphocyte-specific glucocorticoid-response elements (GREs), is associated with drug resistance in acute lymphoblastic leukaemia (ALL). To gain a deeper understanding of regulatory mechanisms leading to these epigenetic alterations, we conducted a multi-omics study, to examine changes in chromatin conformation, following the in-vivo treatment of patient-derived xenografts (PDXs) with glucocorticoid i.e., dexamethasone (DEX). We found that glucocorticoid treatment led to distinct patterns of topological associated domains (TADs) in DEX sensitive and resistant ALLs. Pre-treatment, these TADs were primed by a development-related pioneer transcription factor PU.1, which also occupied GREs in glucocorticoid sensitive but not resistant ALLs. PU.1 binding was associated with lymphocyte-specific activation of GREs and GRE-interacting super-enhancers within the TADs. Furthermore, the PU.1-associated TADs modulated epigenetics and promoted expression of multiple genes, including BIM, ZBTB16, RASA1 and other genes mediating glucocorticoid-induced apoptosis in ALL. The inhibition of PU.1 enhanced sensitivity of ALL cells to glucocorticoid treatment. Overall, this study illustrated a link between the lineage-specific efficiency of glucocorticoids and the developmental epigenetic modulators and provides insights into the role of fundamental epigenetics in clinical practice.

overall_design: Hi-C and ChIP-Seq libraries from ALL xenograft pre and post treatment with Dexamethasone.  ALL xenograft cells were inoculated by tail-vein injection into NSG mice, and engraftment was monitored weekly as previously described (PMID: 30537513). Mice were randomized and treated with either dexamethasone (15 mg/kg) or vehicle control by intraperitoneal injection when the %huCD45+ cells in the peripheral blood reached 70% for in vitro molecular biology experiments. Briefly, mice were treated when > 70% %huCD45+ cells in the peripheral blood, and sacrificed 8 hours thereafter. Cell suspensions of spleens were prepared, and mononuclear cells enriched to 97% human by density gradient centrifugation. After harvesting, cells were immediately resuspended in fetal calf serum (FCS) containing 10% DMSO, frozen and stored at liquid nitrogen for further use.   Hi-C was performed using a modified version of a previously described protocol (PMID: 28435001). Briefly, 10 million cells were collected and cross-linked in 10 ml of PBS containing 1% formaldehyde for 10 minutes at room temperature. The reaction was stopped by 0.125M glycine solution. Cross-linked cell pellet was used to prepare nuclei, which were subjected to digestion with restriction enzyme followed by end-filling to create biotin-labeled blunt ends. Blunt ends were ligated by T4 DNA ligase at room temperature for 4 hours with rotation. Nuclei were digested with proteinase K and 10% sodium dodecyl sulfate (SDS) at 55°C for 30 minutes. Following sodium acetate and pure ethanol precipitation, DNA was isolated and dissolved in Tris buffer. DNA was sonicated using Bioruptor sonicator (Diagenode SA, Belgium), to obtain fragments ranging in size from 150 bp to 300 bp. Sonicated material was then size selected using AMPure XP beads with the 0.55X and 0.3X for HiC. HiC material was then biotinylated, washed, and purified using Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602). While on beads, DNA ends were blunt ended and dA tailed, followed by ligation of the Illumina sequencing adapter with the T4 DNA ligase. PCR reactions were performed on beads involving 8 cycles of amplification (95°C for 2 min, followed by 94°C for 80 s, 65°C for 30 s, 72°C for 30 s). DNA library was size selected using 1.5% agarose gel to obtain fragments ranging in size from 400bp to 700bp. Sequencing was performed on the Illumina HiSeq 2500 to obtain 150bp paired-end reads. Samples were sequenced to depth of 100 million reads.   ChIP-seq were carried out as previously described (PMID: 30537513). Briefly, frozen PDX cells that were harvested from mice after dexamethasone or vehicle control treatment in vivo were revived from cryostorage, and fixed with 1% formaldehyde for 10 min at room temperature. Nuclei were extracted from fixed cells by 10 min incubation in lysis buffer (0.2% NP40 in 10 mM Tris buffer, pH 8.0) followed by centrifugation at 1250 × g for 5 min at 4 °C. Chromatin was fragmented using a Bioruptor sonicator (Diagenode SA, Belgium) on high power at 4 °C with 30 s on/off for 10 min. Separate immunoprecipitates were produced using immunoglobulin raised against the GR (Cell Signaling, Danvers, MA, USA), CTCF (Cell Signaling), Histone (N-terminal acetylated histone H3 in general, Millipore, Billerica, MA, USA; H3K4Me3, H3K27Ac and H3K27Me3, Cell Signaling) and processed according to the manufacturer’s instructions. DNA from protein-associated complexes and corresponding input samples was recovered using phenol/chloroform/isoamyl alcohol with phase lock gel tubes (5 Prime, Hilden, Germany). The ChIP DNA samples were dissolved in 20 µl water and sequenced using Hiseq2500 (50SE) platform with 20 M reads per sample.

first: Richard

middle: B

last: Lock

first: Charles

last: de Bock

first: Duohui

last: Jing


title: Role of ZBTB18 in cytokine production and microglia phenotype in GBM

first: Maria

middle: Stella

last: Carro

state: Deutschland

address: Breisacherstrasse 64

institute: Uniklinik Freiburg

summary: Glioma associated macrophages (GAMs) are known to play an important role in glioblastoma progression, due to their massive recruitment in the tumor microenvironment and polarization to a tumor promoting phenotype. GAMs secrete a variety of cytokines, which promotes tumor cell growth and invasion while inhibiting an immune tumor response by other immune cells. Here, we demonstrate that ZBTB18, a transcriptional repressor with tumor suppressive function in glioblastoma, impairs the production of key cytokines, which functions as chemoattractant for GAMs. Consequently, we observe a reduced migration of GAMs when ZBTB18 is expressed by GBM cells, both in cell culture and in vivo experiments. Moreover, the presence of ZBTB18 halts GBMs cells-mediated GAMs polarization to a tumor suppressive phenotype characterized by a reduced engage in the oxidative phosphorylation pathway for energy supply. Thus, our findings suggest that therapeutic approaches to increase ZBTB18 expression in GBM cells could have a broad impact on both tumor cells and the surrounding immune microenvironment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from human immortalized microglia conditioned with medium from GBM cells transduced with empty vector (EV), ZBTB18 full lenght (FL) or ZBTB18 Nte-Short form (SF)

first: Roberto

last: Ferrarese

first: Dieter

last: Heiland


title: Clearance of p21-highly-expressing senescent cells accelerates wound healing

status: Public on Oct 07 2024

city: Farmington

first: William

middle: F

last: Flynn

state: CT

address: 10 Discovery Drive

institute: The Jackson Laboratory

summary: Targeting senescent cells for therapeutic purposes is gaining momentum across various organ systems. However, concerns about potential off-target effects have been raised. Previous studies have shown that removing senescent cells expressing high levels of p16 (p16high) can hinder processes like wound healing. Here, we identify a distinct senescent cell population during dermal wound healing characterized by high expression level of p21 (p21high) using the p21-Cre mouse model. Using a standard cutaneous injury model, we find that eliminating p21high cells can expedite wound closure, in contrast to the effects of removing p16high cells. Through Xenium, a single cell spatial imaging platform, we show that p21high cells are distinct from p16high cells, with p21high cells mainly comprising fibroblasts, immune cells, keratinocytes, and endothelial cells with a pro-inflammatory profile. Moreover, inhibition of NF-kB signaling specifically from p21high cells partially contributes to the accelerated wound healing rates. These findings highlight the heterogeneity of senescent cells during wound healing responses within the skin and likely in other conditions.

overall_design: 15 cores from mouse unwounded skin and wounds 6 days post injury (both treated with 6 doses of tamoxifen) collected from p21-Cre/+tdTomato/+ animals profiled by 10x Genomics Xenium In Situ Analyzer

first: Nathan

last: Gasek

first: Junyu

last: Zhu

first: Ming

last: Xu


title: Whole blood RNAseq of SIVmac239-infected Mamu-B*08+/- indian rhesus macaques with and without heterologous adenovirus-vector SIV vaccination

city: Minneapolis

first: Michael

last: Gale

state: Minnesota

address: 689 23rd Ave SE

department: Microbiology and Immunology

institute: University of Minnesota

summary: Investigation of the transcriptional response in whole blood following SIVmac239 infection in Macaca mulatta vaccinated with heterologous prime-boost adenovirus-vector SIVmac239 Vif and Nef.

overall_design: Comparative gene expression analysis in vaccinated Macaca mulatta whole blood of each time point following production SIVmac239 infection in animals with binary allelic background (Mamu-B*08pos or neg) and unvaccinated Mamu-B*08neg control. Pre-infection vaccination was heterologous prime-boost with recombinant vectors expressing SIVmac239 Vif and Nef (rAd5-Vif and rAD5-Nef; rVSV-Nef-Tat-Vif; rRRV-Rev-Tat-Nef, rRRV-Nef, rRRV-Vif). Animals were repeatedly challenged with a maginal infectious dose of SIVmac239 until infection was established. Only the challenge establishing infection and the respective pre-challenge baseline were sequenced.

first: Kaitlin

last: Sawatzki

first: Brandon

middle: C

last: Rosen

first: Inah

last: Golez

first: Elise

last: Smith

first: Jennifer

last: Tisoncik-Go

first: Leanne

last: Whitmore

first: David

middle: I

last: Watkins

last: Gale Jr


title: Transcriptomic analysis of cell-of-origin CNS neuroblastoma, FOXR2 activated

city: Montréal

first: Claudia

last: Kleinman

state: Québec

address: 3999 Côte Ste-Catherine Road

department: Human Genetics

institute: Lady Davis Institute for Medical Research

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

overall_design: Refer to individual Series


title: Transcriptomic analysis of cell-of-origin CNS neuroblastoma, FOXR2 activated [RNA-seq]

summary: The transcription factor FOXR2 is the universal driver of childhood central nervous system neuroblastoma, FOXR2 activated (NB-FOXR2). NB-FOXR2 tumors arise exclusively in the brain hemispheres, and despite morphological similarities to other pediatric brain tumors, they are a molecularly distinct entity based on DNA methylation profiling. The cell-of-origin is unknown. Here, we profiled a cohort of rare NB-FOXR2 tumors by bulk and single-cell transcriptomics. Through systematic comparative analyses, we delineate tumor transcriptional states and candidate cell-of-origin. More broadly, we demonstrate systematic molecular profiling of childhood cancers to orient oncogenic targeting for in vivo modeling, a critical resource for the study of rare tumors and development of therapeutics.

overall_design: 3 replicates of a cell line originating from genetically-engineered In utero electroporation mouse model with brain tumor (FOXR2_p53LoF) was sequenced for bulk RNA-seq.

first: Selin

last: Jessa

first: Antonella

last: De Cola

first: Bhavyaa

last: Chandarana

last: McNicholas

first: Steven

last: Hébert

first: Damien

last: Faury

first: Manav

last: Pathania

first: Nada

last: Jabado


title: Analysis of gliomas DNA methylation: Assessment of pre-analytical variables

city: Seattle

first: Karol

last: Bomsztyk

state: WA

address: 850 Republican St

department: Medicine

institute: University of Washington

type: Methylation profiling by high throughput sequencing

summary: MeDIP-Seq for assessment of the effect of pre-analytical variables on DNA methylation

overall_design: Human frozen and FFPE brain tissue were obtained and analyzed using MeDIP, qPCR, and Sequencing. Data was compared using correlation scatterplots, differential expression, IGV (sequencing read profile snapshots), PCA, PCR Crunch (5mC levels)

first: Bomsztyk

last: Karol

first: Mar

last: Daniel

first: Denisenko

last: Oleg

first: Powell

last: Suzanne

first: Vishnoi

last: Monika

first: Yin

last: Zheng

first: Delegard

last: Jennifer

first: Hadley

last: Caroline

first: Tandon

last: Nitin

first: Patel

last: Akash

last: Anoop

first: Ellenbogen

last: Richard

first: Ramakrishna

last: Rohan

first: Rostomily

last: Robert


title: Transcriptomic analysis of cell-of-origin CNS neuroblastoma, FOXR2 activated [CUT&RUN]

first: Claudia

last: Kleinman

overall_design: CUT&RUN chromatin immunocleavage sequencing with antibody against V5 tag was performed on cell lines originating from genetically-engineered In utero electroporation(IUE) mouse models with brain tumor. 2 replicates on cell lines with FOXR2 p53LoF IUE vector containing the V5 tag and 2 replicates from cell lines of a separate IUE mouse model lacking V5 tag (G34R) as control were used.

first: Selin

last: Jessa

first: Antonella

last: De Cola

first: Bhavyaa

last: Chandarana

first: Michael

last: McNicholas

first: Steven

last: Hébert

first: Damien

last: Faury

last: Pathania


title: G3BP2B stress granules regulate mRNA expression under ER stress [Riboseq_Exp2]

city: San Francisco

first: José

middle: M.

last: Liboy-Lugo

state: CA

address: 513 Parnassus Ave.

institute: UCSF

summary: Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the nucleation of proteins such as G3BPs. G3BPs are RNA-binding proteins that condense into SGs following translation shutoff during the integrated stress response (ISR). Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of the different G3BP paralogs to stress granule formation and stress-induced gene expression changes are incompletely understood. Here, we identify a molecular tool to study G3BP condensation into SGs by mutating residue V11 of G3BPs. This conserved amino acid potentiates the G3BP-Caprin-1 complex, hence promoting SG assembly. Ribosome profiling revealed that disruption of G3BP condensation impacts mRNA levels and ribosome engagement during the ISR. Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.

overall_design: To investigate differences in regulation of gene expression by G3BP paralogs, we performed Ribo-seq and total RNA-seq in U-2OS wild type cells, U-2OS G3BP1/2 KO cells (Cell line shared by Dr. Nancy Kedersha, which was developed via CRISPR Cas9 deletions), and G3BP1/2 KO cells stably expressing either transgenic G3BP1, G3BP2A, or G3BP2B N-terminally tagged to monomeric EGFP. All cell lines were treated with either DMSO or 1 µM thapsigargin (Tg) for 2 hours and harvested using two replicates per sample for Ribo-seq and total RNA-seq library preparation. For differential expression analysis, cell lines were analyzed relative to G3BP1/2 KO as the reference sample. We also performed differential expression analysis by comparing Tg treated samples against DMSO treated samples to validate the activation of the integrated stress response.

first: Stephen

middle: N

last: Floor


title: G3BP2B stress granules regulate mRNA expression under ER stress [2]


title: G3BP2B stress granules regulate mRNA expression under ER stress [3]


title: G3BP2B stress granules regulate mRNA expression under ER stress [Riboseq_Exp3]

overall_design: To investigate differences in regulation of gene expression by G3BP2B condensation, we performed Ribo-seq and total RNA-seq in U-2OS G3BP1/2 KO cells (Cell line shared by Dr. Nancy Kedersha, which was developed via CRISPR Cas9 deletions), and G3BP1/2 KO cells stably expressing either transgenic G3BP2B WT, or G3BP2B V11A N-terminally tagged to monomeric EGFP. All cell lines were treated with either DMSO or 1 µM thapsigargin (Tg) for 2 hours and harvested using two replicates per sample for Ribo-seq and total RNA-seq library preparation. For differential expression analysis, cell lines were analyzed relative to G3BP1/2 KO as the reference sample. We also performed differential expression analysis by comparing Tg treated samples against DMSO treated samples to validate the activation of the integrated stress response. Furthermore, G3BP2B V11A was analyzed relative to G3BP2B WT to determine gene expression changes sensitive to stress granule deficiency.


title: G3BP2B stress granules regulate mRNA expression under ER stress [RNAseq_Exp3]

summary: Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Their function is not completely understood. Formation of these organelles is driven by the nucleation of proteins such as G3BPs. G3BPs are RNA-binding proteins that condense into SGs following translation shutoff during the integrated stress response (ISR). Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. Here, we found a molecular tool to study G3BP condensation into SGs by mutating residue V11 of G3BPs. This conserved amino acid potentiates the G3BP-Caprin-1 complex, hence promoting SG assembly. Ribosome profiling revealed that disruption of G3BP condensation impacts mRNA expression during the ISR. Moreover, we found that G3BP2B robustly condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. This indicates that G3BP paralogs differentially regulate SG assembly and gene expression programs. Together, this work suggests that stress granule assembly promotes changes in gene expression to mediate stress-response during the ISR.


title: G3BP2B stress granules regulate mRNA expression under ER stress [RNAseq_Exp1]

overall_design: To investigate differences in regulation of gene expression by G3BP1 condensation, we performed Ribo-seq and total RNA-seq in U-2OS G3BP1/2 KO cells (Cell line shared by Dr. Nancy Kedersha, which was developed via CRISPR Cas9 deletions), and G3BP1/2 KO cells stably expressing either transgenic G3BP1 WT, or G3BP1 V11A N-terminally tagged to monomeric EGFP. All cell lines were treated with either water or 200 µM arsenite (NaAs) for 2 hours and harvested using two replicates per sample for Ribo-seq and total RNA-seq library preparation. For differential expression analysis, cell lines were analyzed relative to G3BP1/2 KO as the reference sample. We also performed differential expression analysis by comparing NaAs treated samples against water treated samples to validate the activation of the integrated stress response. Furthermore, G3BP1 V11A was analyzed relative to G3BP1 WT to determine gene expression changes sensitive to stress granule deficiency.


title: Intracellular zinc protects tumors from T cell-mediated cytotoxicity [E0771 RNAseq]

city: Melbourne

first: Jane

last: Oliaro

address: 305 Grattan St

institute: Peter MacCallum Cancer Centre

summary: We performed RNA sequencing on parental (WT) and Cbfb-deficient (CbfbKO) E0771 cells. Cells were generated by electroporation with Cas9-sgRNA complexes using a non-targeting sgRNA (WT) or Cbfb-targeting sgRNA (CbfbKO)

overall_design: At least 1 week after electroporation with Cas9-sgRNA (non-targeting or Cbfb-targeting), KO was confirmed by western blot and approximately 1 million WT and CbfbKO cells were collected for RNA extraction and sequencing. 3 replicates per group were collected.

first: Emily

last: Lelliott


title: G3BP2B stress granules regulate mRNA expression under ER stress [Riboseq_Exp1]

summary: Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the nucleation of proteins such as G3BPs. G3BPs are RNA-binding proteins that condense into SGs following translation shutoff during the integrated stress response (ISR). Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of the different G3BP paralogs to stress granule formation and stress-induced gene expression changes are incompletely understood. Here, we identify a molecular tool to study G3BP condensation into SGs by mutating residue V11 of G3BPs. This conserved amino acid potentiates the G3BP-Caprin-1 complex, hence promoting SG assembly. Ribosome profiling revealed that disruption of G3BP condensation impacts mRNA levels and ribosome engagement  during the ISR. Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.


title: Integration of genetic and epigenetic data pinpoints autoimmune specific remodelling of enhancer landscape in CD4+ T cells

city: Heidelberg

first: Nila

middle: Hendrika

last: Servaas

address: Meyerhofstrasse 1

department: Structural and Computational Biology

institute: EMBL

summary: CD4+ T cells play a crucial role in adaptive immune responses and have been implicated in the pathogenesis of autoimmune diseases (ADs). Despite numerous studies, the molecular mechanisms underlying T cell dysregulation in ADs remain incompletely understood. Here, we used transcriptomic and epigenomic data from CD4+ T cells of healthy donors and patients with systemic lupus erythematosus (SLE), psoriasis, juvenile idiopathic arthritis (JIA), and Graves' disease to investigate the role of enhancers in AD pathogenesis. By generating enhancer-based gene regulatory networks (eGRNs), we identified disease-specific dysregulated pathways and potential downstream target genes of enhancers harbouring AD-associated single-nucleotide polymorphisms, which we also validated using CRISPRi in primary CD4+ T cells. Our results suggest that alterations in the regulatory landscapes of CD4+ T cells, including enhancers, contribute to the development of ADs and provide a basis for developing new therapeutic approaches.

overall_design: Chromatin immunoprecipitation DNA-Sequencing (ChIP-seq) data for histone modification H3K27ac in CD4+ T cells obtained from healthy donors, psoriasis and systemic lupus erythematosus (SLE) patients

first: Neha

last: Daga

first: Kai

last: Kisand

first: Dewi

last: Moonen

first: Christian

last: Arnold

first: Armando

last: Reyes-Palomares

first: Epp

last: Kaleviste

first: Külli

last: Kingo

first: Reet

last: Kuuse

first: Katrin

last: Ulst

first: Lars

last: Steinmetz

first: Pärt

last: Peterson

first: Nikolina

last: Nakic

first: Judith

last: Zaugg


title: G3BP2B stress granules regulate mRNA expression under ER stress [RNAseq_Exp2]

first: José

last: Liboy-Lugo

first: Stephen

last: Floor


title: Transcriptomic analysis of cell-of-origin CNS neuroblastoma, FOXR2 activated [Multiome]

first: Claudia

middle: L

last: Kleinman

overall_design:
3 genetically-engineered In utero electroporation mouse model with brain tumor sequenced for single-cell multiome. All the samples have a Foxr2 induced overexpression and one has also a p53 loss of function.
The 3 scMultiome samples have been split into 2 parts (RNA-seq and ATAC-seq; 6 sample records). Each raw data fastq file is specific to one data type only. However, the processed files contain information for both RNA-seq and ATAC-seq in the same file (as indicated in the sample description field).

first: Selin

last: Jessa

first: Antonella

last: De Cola

first: Michael

last: McNicholas

first: Damien

last: Faury

first: Steven

last: Hébert

first: Nada

last: Jabado

first: Manav

last: Pathania