title: Cytokine exposure of human macrophages induces disease-associated microglia transcriptional signatures

status: Public on Sep 01 2024

first: Anna

last: Podlesny-Drabiniok

state: NY

address: 1425 Madison Avenue

institute: Icahn School of Medicine at Mount Sinai

summary: Myeloid cells including brain-resident macrophages (microglia) and peripheral macrophages play a crucial role in various pathological conditions, including neurodegenerative diseases such as Alzheimer's disease (AD). They adapt to extracellular stimuli by acquiring various transcriptional states. Understanding of how these states are regulated is limited due to a lack of tools that enable robust reprogramming toward disease-associated phenotypes. In this study, we investigated the effects of a cytokine mix, including interleukin-4 (IL-4), colony stimulating factor 1 (CSF1/M-CSF), interleukin 34 (IL-34) and transforming growth factor beta (TGFβ), on human THP-1 macrophages to elucidate their potential reprogramming towards disease-associated phenotypes and subsequential functional changes. Our results indicate that treatment with the cytokine mix led to significant transcriptomic changes, driving human THP-1 macrophages towards a transcriptional state reminiscent of disease-associated microglia (DAM) and lipid-associated macrophages (LAM) collectively referred to as DLAM. Transcriptome profiling revealed gene expression changes related to oxidative phosphorylation, lysosome function, and lipid metabolism. Single-cell RNA sequencing revealed the activation of DLAM-like transcriptional responses across multiple cell clusters. Functional assays demonstrated alterations in macrophage motility, phagocytosis, lysosomal activity, and metabolic and energetic profiles. Our findings provide insights into the cytokine-mediated reprogramming of macrophages towards disease-relevant states, highlighting their role in neurodegenerative diseases and potential for therapeutic development.

overall_design: Human THP-1 monocytic leukemia line was differentiated to macrophages and treated with 1) three factors (3F): IL34, MCSF, TGFb; 2) IL4; 3) mix of 3F and IL4 (MIX): IL34, MCSF, TGFb, IL4. Control THP-1 macrophages were not treated (NT). Concentration of each cytokine in every treatment IL34 100ng/ml, TGFb 50ng/ml, MCSF 25ng/ml, IL4 20ng/ml

first: Tulsi

last: Patel

first: Yiyuan

last: Liu

first: Alison

last: Goate

first: Edoardo

last: Marcora


title: Synonymous mitochondrial DNA variation impairs CD8 T cell fate via codon affinity [Tcell Culture scATAC]

status: Public on Oct 31 2024

city: New York

first: Caleb

last: Lareau

state: New York

address: 417 E 68th St, Zuckerman - ZRC 1132

country: USA

institute: Memorial Sloan Kettering

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Somatic variation contributes to biological heterogeneity by modulating the cellular proclivity to differentiate, expand, adapt, or die. While large-scale sequencing efforts have revealed the foundational role of somatic variants to drive human tumor evolution, our understanding of the contribution of somatic variation to modulate cellular fitness in non-malignant contexts remains understudied. Here, we identify a somatic synonymous variant (m.7076A>G) in the mitochondrial DNA (mtDNA) encoded cytochrome c-oxidase subunit 1 gene (COX1), which was present at homoplasmy in 47% of immune cells from a healthy donor. Using single-cell multi-omics, we discover highly specific selection against the m.7076G mutant allele in the CD8+ effector memory T cell compartment in vivo, reminiscent of selection observed for pathogenic mtDNA alleles and indicative of lineage-specific metabolic vulnerabilities. Due to the limited transfer RNA (tRNA) pool in mitochondria, the m.7076G mutant allele requires wobble-dependent translation whereas the wildtype m.7076A allele can translate via Watson-Crick-Franklin (WCF) base-pairing. Mitochondrial ribosome profiling revealed that the synonymous m.7076G mutant allele altered codon-anticodon affinity by 33% at the wobble position, stalling translation at the glycine residue within COX1 encoded at m.7076. Leveraging population-based sequencing analyses, we identify evidence of synonymous somatic variants in tumor genomes and dozens of germline variants associated with complex diseases, suggesting broad functional effects of synonymous variation altering codon affinity across the mitochondrial genome. Together, these results provide a new ontogeny for mitochondrial genetic variation and elucidate a framework whereby somatic variation can impact cell state transitions in a lineage-specific manner.

overall_design: T cells from PBMCs were isolated and then stimulated and cultured to assess changes in chromatin accessibility and surface protein at a single-cell level


title: A multitargeting approach rewires cell identity in fusion-negative rhabdomyosarcoma

status: Public on Sep 03 2024

city: Leuven

first: Maurilio

last: Sampaolesi

address: Herestraat 49, ON4 - box 804

department: Department of Development and Regeneration

country: Belgium

institute: KU Leuven

type: Expression 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: Single-cell level gene expression profiles of immune cells within B16F10-OVA tumours treated with PBS or UDSP-Hep

status: Public on Aug 28 2024

city: Beijing

first: Yuanhanyu

last: Luo

state: Beijing

address: No.7, Science Park Road, Zhongguancun Life Science Park, Beijing

country: China

institute: National Institute of Biological Sciences

summary: Activation of the Alpha kinase 1 (ALPK1) is a promising strategy to be developed as a next-generation cancer immunotherapy. We used single cell RNA sequencing (scRNA-seq) to analyze the ALPK1 activation-triggered antitumour immune responses within the tumour microenvironment of B16F10-OVA tumours treated with PBS or UDSP-Hep.

overall_design: Immune cells within B16F10-OVA tumours treated with PBS or UDSP-Hep on days 10, 12, 14, and 16 were isolated by Fluorescence-activated cell sorting (FACS) according to the presence and absence of PE signal for further scRNA-seq analysis.

first: Jiaqi

first: Xiaoying

last: Tian

first: Yupeng

last: Wang


title: NR4a knock-down in HER2 CAR-T cells improves T cell longevity and promotes  solid tumor eradication

status: Public on Aug 30 2024

city: Shinjuku-ku

first: Taeko

last: Hayakawa

state: Tokyo

address: 35 Shinanomachi

department: Division of Tumor Immunology, Institute for Advanced Medical Research

country: Japan

institute: Keio University School of Medicine


title: The antiviral nuclear body protein SP140 represses Ifnb1 transcript stabilization by the novel regulator RESIST [WT and SP140 KO RNA-seq]

city: Berkeley

first: Russell

last: Vance

state: CA

address: Life Sciences Addition, University of California, Berkeley

institute: University of California, Berkeley

summary: Negative regulation of the antiviral cytokine IFN-I is critical to prevent host pathology, but is not fully understood. The highly conserved but understudied epigenetic reader and nuclear body protein SP140 represses IFN-I by an unknown mechanism. We describe the mechanism by which SP140 negatively regulates IFN-I, and include RNA-seq data of WT (B6) and SP140 KO BMMs untreated or treated with 100 ug/mL DMXAA for 8 hours in this dataset.  We found that SP140 represses the expression of a novel positive regulator of IFN-I previously annotated as an annexin II receptor, which we rename RESIST (REgulated Stimulator of IFN-I via Stabilization of Transcript). RESIST promotes IFN-I mRNA stability by binding the CCR4-NOT mRNA deadenylase complex and blocking recruitment of the TTP family of RNA-binding proteins, which negatively regulate IFN-I mRNA stability, to CCR4-NOT. Mechanistically, RESIST uses N and C-terminal alpha-helices respectively to either dock to the CCR4-NOT complex or block TTP family binding. Our work reveals a new regulator of IFN-I and a new mechanism regulating IFN-I mRNA stability.

overall_design: Primary bone-marrow derived macrophages from WT (B6) or SP140 KO mice were treated with 100 ug/mL DMXAA or left untreated for 8 hours. Cells were then harvested for RNA for RNA-seq.

first: Kristen

last: Witt

first: Adam

last: Dziulko

first: Edward

middle: B

last: Chuong

middle: E


title: The antiviral nuclear body protein SP140 represses Ifnb1 transcript stabilization by the novel regulator RESIST [RNA-seq]

summary: Negative regulation of the antiviral cytokine IFN-I is critical to prevent host pathology, but is not fully understood. The highly conserved but understudied epigenetic reader and nuclear body protein SP140 represses IFN-I by an unknown mechanism. We describe the mechanism by which SP140 negatively regulates IFN-I, and include RNA-seq of IFNAR KO and SP140 KO IFNAR KO BMMs untreated or treated with 10 ug/mL DMXAA for 4 hours in this dataset.  We found that SP140 represses the expression of a novel positive regulator of IFN-I previously annotated as an annexin II receptor, which we rename RESIST (REgulated Stimulator of IFN-I via Stabilization of Transcript). RESIST promotes IFN-I mRNA stability by binding the CCR4-NOT mRNA deadenylase complex and blocking recruitment of the TTP family of RNA-binding proteins, which negatively regulate IFN-I mRNA stability, to CCR4-NOT. Mechanistically, RESIST uses N and C-terminal alpha-helices respectively to either dock to the CCR4-NOT complex or block TTP family binding. Our work reveals a new regulator of IFN-I and a new mechanism regulating IFN-I mRNA stability.

overall_design: We generated RNA-seq data for bone-marrow derived macrophages from Ifnar KO and Sp140 Ifnar KO mice. Cells were untreated or treated with 10 ug/mL DMXAA for 4 hours.


title: Circadian Clock Regulation in Soybean Senescence: A Transcriptome Analysis of Early and Late Senescence Types

status: Public on Aug 23 2024

city: Chuncheon

first: Prakash

last: Basnet

state: Gangwon-do

address: Department of Agriculture and Life Industry, Kangwon National University, Bldg. Asangwan Rm.424, 1 K

department: Agriculture and Life Industries

country: South Korea

institute: Kangwon National University

summary: We obtained early-senescence (ES)- and late-senescence (LS)-type F7 plants from a cross between a hybrid (Glycine max × Glycine soja) and the Glycine max cultivar. The ES-type plants presented the reproductive (R2) growth stage at 50 days after sowing (DAS) and the R7 growth stage at 95 DAS, whereas the LS-type plants presented the beginning of the R1 and R6 growth stages at 50 and 95 DAS, respectively. To understand the molecular mechanisms underlying this senescence, we performed transcriptome analysis of leaves from 50 and 95 DAS of ES- and LS-type plants. A total of 2,414 and 2,471 genes at 50 and 95 DAS, respectively, were differentially expressed between ES-type and LS-type plants. Twenty-three candidate genes associated with the circadian clock, chlorophyll biosynthesis, phytohormones, and protein kinases were identified, and their expression levels were analyzed. In addition, we analyzed the expression patterns of circadian clock-related genes such as CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), CONSTANS-LIKE 9 (COL9), EARLY FLOWERING 3 (ELF3) and pseudo response regulator 5 (PRR5) in ES- and LS-type plants under light and dark conditions. The expression patterns of circadian clock-related genes were similar in the ES- and LS-type plants. However, the transcription levels of these genes were compared between ES- and LS-type plants, and the expression of these genes was greater than that in LS-type plants during the period when expression increased. Therefore, each set of candidate genes regulated senescence in each plant by regulating their expression level.

overall_design: To investigare the differentially expressed genes between early and late senscence type soybean plants in 50 and 95 days

first: Ik-Young

last: Choi

first: Taeyoung

last: Um

first: MOON

middle: K

last: Hee

first: Sevin

last: Lee


title: Activation of polo-like kinase 1 correlates with selective motor neuron vulnerability in familial ALS

status: Public on Aug 27 2024

city: Rostock

first: Andreas

last: Hermann

state: Mecklenburg-Western Pomerania

address: Gehlsheimer Str.20

department: Neurology

country: Germany

institute: University Medical Center Rostock

summary: Mutations in the Fused in Sarcoma (FUS) gene cause familial amyotrophic lateral sclerosis (ALS), characterized by selective degeneration of spinal motor neurons (sMNs) with relative sparing of cortical neurons (CNs). The mechanisms underlying this cell-type vulnerability remain unclear. Here, we compare CNs and sMNs derived from FUS-ALS models to assess differential responses to FUS mutations. We find that CNs are less affected than sMNs in DNA damage repair, axonal organelle trafficking, and stress granule dynamics. RNA sequencing (RNA-seq) reveals distinct transcriptomic signatures, with sMNs uniquely activating DNA damage responses involving cell cycle regulators, particularly polo-like kinase 1 (PLK1). PLK1 is highly expressed in sMNs but not CNs, correlating with greater nuclear FUS loss and splicing defects in sMNs. Cross-comparison with other familial ALS RNA-seq datasets highlights PLK1 upregulation as a shared molecular feature. These findings identify intrinsic differences between CNs and sMNs in FUS-ALS and suggest PLK1 as a potential driver of sMN vulnerability.

overall_design: Patient-derived isogenic spinal motoneurons (MN) or cortical neurons (CN) expressing either normal (FUS WT) or mutant P525L FUS-eGFP (FUS P525L) were matured for 21 DIV. The main cell model used in this study were neurons derived from FUS-WT-EGFP and FUS-P525L-EGFP that were generated as a part of previous study (Naumann, Pal et al. 2018). Briefly, fibroblasts carrying R521C FUS mutation obtained from a 58-year-old female were reprogrammed into iPSC using cDNA of OCT4, SOX2, KLF4 and cMYC delivered via retroviral vectors. Next, CRISPR/Cas9 genome editing was used to i) correct the R521C mutation into WT FUS and to tag it with EGFP to generate the FUS WT EGFP line or ii) replace the R521C mutation into P525L FUS mutation and to tag it with EGFP to generate FUS P525L EGFP line. Differentiation of hiPSC into spinal motor neurons (MNs) was carried out based on the protocol from Reinhardt et al. 2013. Differentiation of hiPSC into cortical neurons (CN) was based on the protocol published by Burkhardt et al.(Burkhardt, Martinez et al. 2013, Japtok, Lojewski et al. 2015).

first: Barbara

last: Szewczyk

first: Julia

last: Japtok

first: Banaja

middle: P

last: Dash

last: Petzold

first: Mathias

last: Lesche


title: The antiviral nuclear body protein SP140 represses Ifnb1 transcript stabilization by the novel regulator RESIST [ATAC-seq]

summary: Negative regulation of the antiviral cytokine IFN-I is critical to prevent host pathology, but is not fully understood. The highly conserved but understudied epigenetic reader and nuclear body protein SP140 represses IFN-I by an unknown mechanism. We describe the mechanism by which SP140 negatively regulates IFN-I, and include ATAC-seq data of WT (B6) and SP140 KO BMMs untreated or treated with 100 ug/mL DMXAA for 8 hours in this dataset.  We found that SP140 represses the expression of a novel positive regulator of IFN-I previously annotated as an annexin II receptor, which we rename RESIST (REgulated Stimulator of IFN-I via Stabilization of Transcript). RESIST promotes IFN-I mRNA stability by binding the CCR4-NOT mRNA deadenylase complex and blocking recruitment of the TTP family of RNA-binding proteins, which negatively regulate IFN-I mRNA stability, to CCR4-NOT. Mechanistically, RESIST uses N and C-terminal alpha-helices respectively to either dock to the CCR4-NOT complex or block TTP family binding. Our work reveals a new regulator of IFN-I and a new mechanism regulating IFN-I mRNA stability.

overall_design: Primary bone-marrow derived macrophages from WT (B6) or SP140 KO mice were treated with 100 ug/mL DMXAA or left untreated for 8 hours. Cells were then harvested  for ATAC-seq (Buenrostro, 2015).


title: The antiviral nuclear body protein SP140 represses Ifnb1 transcript stabilization by the novel regulator RESIST

summary: Negative regulation of the antiviral cytokine IFN-I is critical to prevent host pathology, but is not fully understood. The highly conserved but understudied epigenetic reader and nuclear body protein SP140 represses IFN-I by an unknown mechanism. We describe the mechanism by which SP140 negatively regulates IFN-I, and include CUT&RUN data in this dataset. We performed anti-HA CUT&RUN on SP140KO macrophages transduced with HA-tagged or untagged SP140. We found that SP140 represses the expression of a novel positive regulator of IFN-I previously annotated as an annexin II receptor, which we rename RESIST (REgulated Stimulator of IFN-I via Stabilization of Transcript). RESIST promotes IFN-I mRNA stability by binding the CCR4-NOT mRNA deadenylase complex and blocking recruitment of the TTP family of RNA-binding proteins, which negatively regulate IFN-I mRNA stability, to CCR4-NOT. Mechanistically, RESIST uses N and C-terminal alpha-helices respectively to either dock to the CCR4-NOT complex or block TTP family binding. We also find SP140 does not bind the Ifnb1 gene itself. Consistent with previous results, SP140 binds genes involved in embryonic development.  Our work reveals a new regulator of IFN-I and a new mechanism regulating IFN-I mRNA stability.

overall_design: We performed anti-HA CUT&RUN on 0.5e6 SP140 KO bone marrow derived macrophages (BMMs) transduced with untagged or HA-tagged SP140, treated with the STING agonist DMXAA at 100 ug/ml for 8 hours. We used triplicates (separate CUT&RUN reactions) for these conditions. We also performed CUT&RUN using an isotype control (rabbit) on 0.5e6 WT BMMs.


title: mRNA Sequencing of human monocyte cell lines exposed to Brugia malayi filarial helminth-derived extracellular vesicles.

status: Public on Aug 20 2024

city: Bethesda

first: Sasisekhar

last: Bennuru

state: MD

address: 10 Center Dr, Bldg 10, Rm 11N208

department: LPD

institute: NIH/NIAID

summary: Live microfilariae (Mf) and mf-derived extracellular vesicles (EVs) have been shown to modulate human antigen presenting cell (APC) function, most notably by suppressing the induction of IL-12 (and other pro-inflammatory cytokines) following activation with LPS and interferon-gamma.  To explore further how EVs alter human APC function, we studied the effect of mf and EVs on monocyte celllines isolated from human donors, following the exposure to Mf, Mf-derived excretrory/secretory (E/S) products, E/S depleted of EVs through ultracentrifugation and purified EVs using RNAseq analysis.

overall_design: We performed comparative RNAseq analysis of human monocytes following exposure to live Brugia malayi filarial helminth, EVs, E/S, or EV-depleted E/S.

first: Thomas

last: Nutman

first: Pedro

middle: H

last: Guimaeres-Gazzinelli

first: Alessandra

last: Ricciardi

first: Gayatri

last: Sanku


title: Brugia malayi filarial helminth-derived extracellular vesicles suppress antigen presenting cell functions and antigen-specific CD4+ T cell responses

summary: Live microfilariae (Mf) and mf-derived extracellular vesicles (EVs) have been shown to modulate human antigen presenting cell (APC) function, most notably by suppressing the induction of IL-12 (and other pro-inflammatory cytokines) following activation with LPS and interferon-gamma.  To explore further how EVs alter human APC function, we studied the effect of mf and EVs on human elutriated monocyte-derived DCs following exposure to Mf, Mf-derived excretrory/secretory (E/S) products, E/S depleted of EVs through ultracentrifugation and purified EVs using RNAseq analysis. In our analyses of the data for the DC, using a false discovery rate (FDR)<0.05,  EV-exposed DCs had induced the expression of 212  differentially expressed genes (DEGs) when compared to unexposed DC and 157 when compared to ES-depleted EVs.  These genes were enriched in GO biological processes associated with neutrophil degranulation and 15 DEGs associated with KEGG Lysosome pathways. IPA analysis point to immune dysregulation.

overall_design: We performed comparative RNAseq analysis of human DC following exposure to live Brugia malayi filarial helminth, EVs, E/S, or EV-depleted E/S.


title: Intratumoural microbiome-derived lipopolysaccharides contributes to non-small-cell lung cancer progression via activating TLR4-mTOR-NF-κB-IL-6 axis

status: Public on Aug 19 2024

city: Jinan

first: guomeng

last: Sha

address: Beiyuan Street

institute: Second Hospital of Shandong University

summary: Background: As emerging component of the tumor microenvironment, intratumoural microbiota imperceptibly influences the progression of various human malignancies. However, the critical intratumoural microbiota and its role in non-small-cell lung cancer (NSCLC) progression has not been fully elucidated. Results: Herein, we reveal significant abnormalities in the intratumoural microbiota of NSCLC by analyzing the microbiota profiles of tumor and non-malignant tissues from NSCLC patients. Specifically, Gram-negative bacteria were significantly increased in intratumoural bacteria, and network analysis revealed that Escherichia-Shigella and unclassified_f__Enterobacteriaceae, which had strong ability to synthesize bacterial toxin lipopolysaccharides (LPS), could significantly promote tumor proliferation. Mechanistically, we found that Escherichia-Shigella and unclassified_f__Enterobacteriaceae-derived LPS activated the TLR4-mTOR-NF-κB-IL-6 axis to facilitate the proliferation of NSCLC, while rapamycin could effectively postpone LPS-induced tumor proliferation through in vitro cell culture and in vivo murine model. In addition, the receiver operating characteristic (ROC) curves showed that the combined diagnosis of intratumoural bacterial concentration, Escherichia-Shigella, and unclassified_f__Enterobacteriaceae had higher diagnostic validity than the individual diagnosis in predicting the probability of NSCLC, and their detection in blood has the potential to be combined with imaging for non-invasive diagnosis of NSCLC. Conclusions: Collectively, this research discovered increased Gram-negative bacteria Escherichia-Shigella, and unclassified_f__Enterobacteriaceae in NSCLC tumors, and clarified the mechanism of these bacteria-derived LPS in promoting tumor development, providing new strategies for the treatment and diagnosis of NSCLC from a microbial perspective.

overall_design: Patient recruitment and sample collection，Cell culture and reagents，DNA extraction and high-throughput sequencing，Animal models，RNA sequencing and data analysis

first: Guomeng

first: Zhengwen

last: Wu

first: Weiquan

last: Zhang

first: Xiaogang

last: Zhao


title: Dissection of tumor-intrinsic and tumor-extrinsic features of MIT/TFE translocation renal cell carcinoma via single-nucleus RNA sequencing

status: Public on Aug 11 2024

city: Boston

first: Srinivas

middle: Raghavan

last: Viswanathan

state: Massachusetts

address: 450 Brookline Avenue

institute: Dana-Farber Cancer Institute

summary: We used single-nucleus profiling to study tumor heterogeneity and tumor microenvironment in tRCC sample.

middle: R

first: Prathyusha

last: Konda


title: Mogat1 drives metabolic adaptations to evade immune surveillance

status: Public on Aug 16 2024

city: Hangzhou

first: Huan

last: Wei

address: sandun street

institute: westlake university

summary: Despite immune checkpoint blockades have achieved remarkable success, most  patients with solid tumors do not respond or develop resistance, suggesting additional  treatment strategies are needed. Concentrated efforts are directed toward revitalizing  the IFNγ pathway or amplifying antigen presentation capabilities, alternative  mechanisms underlying immune evasion remain poorly understood. Using an unbiased  whole-genome in vivo natural selection screen, we identified an uncharacterized role of  Monoacylglycerol O-Acyltransferase 1 (Mogat1) as a critical modulator of tumor  immune evasion. As tumors progress and expand, they undergo systemic metabolic  adaptations to evade immune surveillance. Tumor cells exploit Mogat1 to sequester  excess fatty acids into triglycerides, orchestrating metabolic reprogramming that  simultaneously fuels tumor growth and creates an immunosuppressive  microenvironment. Mogat1 inhibition suppresses tumor growth by promoting T-cell  infiltration and cytotoxicity. Remarkably, Mogat1 loss facilitates the circumvention  of PD-1 checkpoint blockade resistance. This heightened inflammatory response,  characterized by increased interferon sensitivity, circumvents the need for conventional  antigen presentation. Our findings reveal a novel lipid metabolism-centered mechanism  of immune evasion and offer a potential strategy to enhance cancer immunotherapy  efficacy.

overall_design: To unveil the molecular underpinnings of tumor immune evasion and growth, we performed RNA sequencing on tumor cells or CD8T cells isolated at early (2 weeks), middle (3 weeks), and late (4 weeks) stages of tumor development in our MMTV-PyMT model.What's more,to elucidate the molecular mechanisms underlying Mogat1's effects, we performed RNA-seq on MMTV-PyMT cells or CD8T cells isolated from shCTRL and shMogat1 xenografts.

first: Congyi

last: Niu


title: Metabolic reprogramming driven by TFE3 fusions unveils novel vulnerabilities in translocation renal cell carcinoma [RNA-Seq]

status: Public on Aug 10 2024

first: Dipanjan

last: Chowdhury

state: MA

address: 4 Blackfan Circle

summary: Translocation renal cell carcinoma (tRCC) is an aggressive subtype of kidney cancer driven by TFE3 gene fusions, which act via poorly characterized downstream mechanisms. Here we report that TFE3 fusions transcriptionally rewire tRCCs toward oxidative phosphorylation (OXPHOS), contrasting with the highly glycolytic metabolism of most other RCCs. The transcriptional program driven by TFE3 fusions sustains high NRF2 signaling and glutathione production, which offsets reactive oxygen species generated by OXPHOS but renders tRCC cells sensitive to reductive stress. Genome-scale CRISPR screening identifies tRCC-selective vulnerabilities linked to maintaining this metabolic balance, including EGLN1, which hydroxylates HIF-1α and targets it for proteolysis. Inhibition of EGLN1compromises tRCC cell growth by stabilizing HIF-1a and promoting glycolytic reprogramming. Our study defines a distinctive tRCC-essential metabolic program driven by TFE3 fusions and nominates EGLN1inhibition as a therapeutic strategy to counteract fusion-induced metabolic rewiring.

overall_design: To inverstigate the regulatory role of TFE3 fusion in tRCC, we performed RNA-Seq upon TFE3 fusion knockout/knockdown in tRCC.

first: Jiao

last: Li


title: Recruitment to the nuclear periphery enhances repression of H3K9me2-marked genes and transposons to shape cell fate [CUT&RUN]

status: Public on Aug 09 2024

city: San Francisco

first: Harold

last: Marin

address: 555 Mission Bay Boulevard South

department: Smith Cardiovascular Institute

institute: University of California San Francisco

summary: Heterochromatic loci marked by histone H3 lysine 9 dimethylation (H3K9me2) are enriched at the nuclear periphery in metazoans, but the functional importance of this organization is unknown. Both the nuclear lamins and the nuclear membrane protein lamin B receptor (LBR) have been linked to heterochromatin positioning in mammals, yet embryonic stem cells (mESCs) lacking all lamins retain both LBR and H3K9me2 at the nuclear periphery. We ablated LBR in this context and show that heterochromatin detaches from the nuclear periphery in lamin + LBR quadruple knockout (QKO) cells. QKO mESCs sustain naïve pluripotency and maintain H3K9me2 at normal levels across the genome but cannot repress H3K9me2-marked genes or transposons. Further, their capacity to differentiate into epiblast-like cells (EpiLCs) is impaired. While normal EpiLCs expand H3K9me2 across the genome, QKO EpiLCs exhibit abnormal patterning of this mark. QKO cells can exit naïve pluripotency and activate epiblast-stage genes but also express markers of alternative fates including the primitive endoderm. These results establish that recruitment of H3K9me2 to the nuclear periphery controls the spatial position, dynamic remodeling, and repressive capacity of this mark to shape cell fate decisions.

overall_design: Cleavage Under Target and Release Under Nuclease (CUT & RUN) for the histone modification di-methylated lysine 9 on histone H3 (H3K9me2) in naive mouse embryonic stem cells (mESCs) and epiblast-like cells (EpiLC).

first: Eric

last: Simental

first: Charlie

last: Allen

last: Martin

first: Barbara

last: Panning

first: Bassem

last: Al-Sady

first: Abigail

last: Buchwalter


title: HMGA2 overexpression induces plasticity in myometrial cells and a transcriptomic profile more similar to that of uterine fibroids

status: Public on Aug 14 2024

city: Grand Rapids

first: Jose

middle: M

last: Teixeira

state: MI

address: 400 Monroe Ave NW

department: Ob/Gyn

institute: Michigan State University

summary:
Objective: To study the possible role for HMGA2 overexpression in differentiated myometrial cells and its potential to induce a stem cell-like or dedifferentiating phenotype and drive fibroid development.
Design: Myometrial cells were immortalized and transduced with an HMGA2 lentivirus to produce HMGA2hi cells. In vitro stem cell assays were conducted, and ribonucleic acid from HMGA2hi and control cells as well as fibroid-free myometrial and HMGA2 fibroid (HMGA2F) tissues were submitted for ribonucleic acid sequencing.
Setting: University research laboratory.
Patient(s): Women who underwent hysterectomy for symptomatic uterine fibroids or other gynecological conditions. Intervention(s): Not applicable.
Main Outcome Measure(s): In vitro stem cell-like properties from myometrial cell lines. Ribonucleic acid sequencing and collagen production of HMGA2-overexpressing primary leiomyoma tissue and cell lines.
Result(s): HMGA2hicellshadenhancedself-renewalcapacity,decreasedproliferation,andagreaterabilitytodifferentiateintoother mesenchymal cell types. HMGA2hi cells exhibited a stem cell-like signature and shared transcriptomic similarities with HMGA2F. Moreover, dysregulated extracellular matrix pathways were observed in both HMGA2hi cells and HMGA2F.
Conclusion(s): Our findings show that HMGA2 overexpression may drive myometrial cells to dedifferentiate into a more plastic phenotype and provide evidence for an alternative mechanism for fibroid etiology, suggesting that fibroids arise not only from a mutated stem cell but also from a mutated differentiated myometrial cell.

overall_design:
TERT immortalized myometrium cells depleted for CRIP1 transduced with an HMGA2 lentivirus (HMGA2hi) and control were send for RNA-sequencing. Fibroid-free myometrium (MyoN) and HMGA2 fibroid (HMGA2F) tissues were also sequenced.
The raw files for two samples in this Series were already in SRA under BioProject PRJNA498292 (GSM8297308: SRX4936739; GSM8297309: SRX4936736).

first: Emmanuel

middle: N

last: Paul

first: Tyler

middle: J

last: Carpenter

first: Laura

middle: A

last: Pavliscak

middle: Z

last: Bennett

first: Maria

last: Ochoa-Bernal

first: Asgerally

middle: T

last: Fazleabas


title: Sex-Specific modulation of Anxiety-Like behavior by Forebrain Neuronal SMC3 in Mice

city: Safed

first: Evan

last: Elliott

address: Henrietta Szold Street, P.O. Box 1589

department: The Azrieli Faculty of Medicine

country: Israel

institute: Bar-Ilan University

summary: SMC3 is a chromatin binding factor that plays central roles in genome organization and in proper neurodevelopment. Mutations in SMC3 gene (SMC3) induce neurodevelopmental and behavioral phenotypes in humans, including changes in anxiety behavior and self-injury. However, it is not clear what are the exact roles of SMC3 in behavior in adulthood or if its effects are only developmental. Using an adulthood forebrain excitatory neuron specific Smc3 knockout mouse model, the current study determined specific sex-dependent effects of SMC3 ablation during the adulthood. Behavioral tests identified anxiolytic effects of Smc3 knockout in females and anxiogenic effects in males four weeks after initiation of adulthood knockout. The prefrontal cortex, a regulator of anxiety behavior, also displayed sex-dependent effects in dendritic complexity. Transcriptional analysis revealed differential effects of Smc3 knockout in males and females, including changes in anxiety-related genes and relevant transcriptional pathways. While anxiety behavior was sex-specific, both males and females developed self-injury behavior at approximately ten weeks after induction of knockout. The current study demonstrates that neuronal SMC3 regulates anxiety during the adulthood in a sex-specific manner.

overall_design: To understand the function of SMC3 in the adulthood brain, we established a neuron-specific knockout of Smc3 specifically in adulthood in mice.  Mice with floxed Smc3 and Camk2a-cre/ERT2+ (Cre+) or Camk2a-cre/ERT2- (Cre-). Mice with Cre+ named as cKO due the lack of Smc3 gene after tamoxifen treatment; mice with Cre- served as WT.

first: Natalia

last: Saleev

first: Dmitriy

last: Getselter


title: Recruitment to the nuclear periphery enhances repression of H3K9me2-marked genes and transposons to shape cell fate [RNAseq_shLBR]

last: Marin

overall_design: Total RNAseq in naive mouse embryonic stem cells (mESCs) and epiblast-like cells (EpiLC).


title: Engraftable multilineage hematopoietic cells differentiated from induced human pluripotent stem cells

status: Public on Aug 03 2024

city: Melbourne

first: Jacky

middle: Yisheng

last: Li

state: Victoria

address: 50 Flemington Rd

department: Cellular Biology

country: Australia

institute: MCRI

summary: The production of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) would have significant implications for medicine and biotechnology. We differentiated iPSCs to blood cells resembling the first repopulating HSCs of the human embryo. In immune deficient mice transplanted with iPSC-derived CD34+ cells, long term, low level bone marrow engraftment was seen in most animals. Although many animals were engrafted by myeloid, or myeloid and B lymphoid restricted progeny, 18% of mice receiving cells exposed to retinoids at day 3 of differentiation developed multilineage erythroid, myeloid and lymphoid cell engraftment (MLE), reflecting in vitro generation of HSCs or MPPs (termed iPSC-derived (i)HSCs). In a second cohort of mice, we observed in 36% of animals transplanted with cells receiving retinoids throughout differentiation. These studies identify conditions that yield engrafting iHSCs differentiated from human iPSCs and provide a foundation for the generation of cells for clinical translation.

overall_design: Single-cell RNA sequencing (scRNA seq) of differentiated iPSCs was performed to find transcriptional differences that might account for functional differences between cells differentiated with and without retinoid and to allow comparisons with published data sets of human AGM. Two iPSC lines were profiled, the RM TOM line and an independent line in which the mtagBFP2 fluorescent protein was expressed from the GAPDH locus of the PB1.1 iPSCs (denoted PB1.1 BFP). Mesoderm was induced with 4µM CHIR and either 3ng/ml BMP4 and 5ng/ml ACTIVIN A (3B5A) or 30ng/ml ACTIVIN A (30A), and differentiated for 14 days. In each case some Swirling EBs were treated with retinyl acetate (RETA) at d3, or for a more prolonged period (RETA added every 2 days from d3 until d11, d13 or d14). Blood cells in suspension and disaggregated Swirling EBs were harvested after 14 days and subjected to scRNA seq using the 10X Genomics platform. In total, 252,607 cells, comprising 12 RM TOM and 16 PB1.1 BFP samples were analysed.

first: Elizabeth

last: Ng

middle: Y

first: Andrew

middle: G

last: Elefanty


title: Translation efficiency covariation across cell types is a conserved organizing principle of mammalian transcriptomes

status: Public on Aug 01 2024

city: Austin

first: Can

last: Cenik

state: TX

address: Moffett Molecular Biology Building 2.122 2500 Speedway

department: Molecular Biosciences

institute: The University of Texas at Austin

summary: Characterization of shared patterns of RNA expression between genes across conditions has led to the discovery of regulatory networks and novel biological functions. However, it is unclear if such coordination extends to translation, a critical step in gene expression. Here, we uniformly analyzed 3,819 ribosome profiling datasets from 117 human and 94 mouse tissues and cell lines. We introduce the concept of Translation Efficiency Covariation (TEC), identifying coordinated translation patterns across cell types. We nominate potential mechanisms driving shared patterns of translation regulation. TEC is conserved across human and mouse cells and uncover novel gene functions. Moreover, our observations indicate that proteins that physically interact are highly enriched for positive covariation at both translational and transcriptional levels. Our findings establish translational covariation as a conserved organizing principle of mammalian transcriptomes.

overall_design: Matched ribosome profiling and RNA-seq of PARK7, VIM, and USP42 knockout cell lines

first: Yue

last: Liu

first: Ian

last: Hoskins

first: Michael

last: Geng

first: Qiuxia

last: Zhao

first: Jonathan

last: Chacko

first: Logan

last: Persyn

first: Jun

last: Wang

first: Dinghai

last: Zheng

first: Yochen

last: Zhong

first: Shilpa

last: Rao

first: Dayea

last: Park

first: Elif

last: Sarinay-Cenik

first: Vikram

last: Agarwal

first: Hakan

last: Ozadam


title: Transcriptomic analysis of response of Vibrio parahaemolyticus to excess copper.

city: 河南

first: 翟

last: 怡梦

address: 河南省濮阳市华龙区

institute: 翟怡梦

summary: We studied the transcriptome changes of Vibrio parahaemolyticus under normal culture condition and copper stress by RNA sequencing. The data showed that genes in copA and cusFABC operon were significantly upregulated by copper.

overall_design: The transcription profiles of Vibrio parahaemolyticus after copper treatment and water treatment were compared. Each condition contains three biological copies.

first: Chengkun

first: Yimeng

last: Zhai

first: Mengxian


title: The short-chain dehydrogenases/reductase DHRS13 suppresses differentiation and mitophagy in glioma cells by modulating retinoic acid signaling and mitochondrial ROS

status: Public on Aug 07 2024

city: Seoul

first: Min Ji

institute: Korea University

summary: To better understand the complex interplay of undifferentiated cancer cells in tumor malignancy, we have focused on the crucial mechanisms that maintain the undifferentiated state of cancer stem-like cells, which drive tumor growth and therapy resistance. Here, we have identified a protein called dehydrogenase/reductase 13 (DHRS13) abundant in undifferentiated glioblastoma cells. DHRS13 is primarily located in the mitochondria and functions as a retinaldehyde reductase, converting all-trans retinaldehyde to all-trans retinol with a high affinity for NADPH. Remarkably when we deplete DHRS13 from these cells, we observe a significant decrease in tumor initiation and progression. Mechanistically, DHRS13 prevents these cells from differentiating by blocking retinoic acid signaling, thereby maintaining their undifferentiated state. Additionally, depletion of DHRS13 results in mitochondrial reactive oxygen species-driven mitophagy and cell death. These findings hold promise for the development of novel strategies to target undifferentiated cancer cells and potentially leading to improved treatment outcomes.

overall_design: RNA sequencing was performed at the Beijing Genomics Institute (BGI, Shenzhen, China). Raw fragments per kilobase million (FPKM) and probability values (PA) were calculated using the performer’s algorithm and used for further analysis. Genes with PA > 0.8, whose expression levels increased or decreased by more than 2-fold after the knockdown of DHRS13, were grouped and designated as DEGs.

first: Sunyoung

last: Seo

first: Min Gi

first: Hyunggee

last: Kim


title: RNA-seq analysis of the transcriptomic profiles of ATO+DAC-treated THP-1 cell lines expressing p53-R282W, vector control, or p53-R282W with IRF7 knockdown

status: Public on Jul 31 2024

city: Shanghai

first: Huaxin

last: Song

address: Shanghai Jiao Tong University School of Medicine

summary: To investigate the transcriptomic effects of ATO and DAC co-treatment in THP-1 cells expressing different p53 contexts, RNA-seq analysis was conducted on cell lines expressing p53-R282W, vector control, or p53-R282W with IRF7 knockdown. Cells were treated with arsenic trioxide (1 μg/mL) and decitabine (5 μM) prior to total RNA extraction. Purified mRNA was subjected to high-throughput sequencing using the Illumina HiSeq platform. Sequencing reads that passed quality control were trimmed using Cutadapt and used for downstream transcriptomic analysis.

overall_design: RNA-seq was performed to evaluate transcriptional activity of mutant p53 in response to the indicated treatments.

first: Min

last: Lu

first: Yuting

last: Dai


title: Histone acetylation readers Bdf1 and Yaf9 guide targeting of SWR1 remodeler  to +1 nucleosome

status: Public on Jul 29 2024

city: Ithaca

first: Justin

middle: Sungsoo

last: Cha

address: 526 Campus Rd

department: Computational Biology

institute: Cornell University

summary: The histone variant H2A.Z marking permissive chromatin is preferentially deposited at promoter-proximal +1 nucleosomes by the multicomponent SWR1 chromatin remodeler. Although SWR1 targeting to nucleosome-depleted regions (NDRs) is directed by its free DNA length sensing module, how the remodeler is guided preferentially to flanking +1 nucleosomes has been enigmatic. Here, we show by live-cell, single-molecule tracking (SMT) that SWR1 subunits Bdf1 and Yaf9 harboring bromo and YEATS acetyl-histone reader domains are required for quantitative chromatin binding via distinct kinetic mechanisms: Bdf1 increases SWR1 association, Yaf9-YEATS reduces disassociation.  Notably, SMT and genome-wide ChIP-exo reveals Bdf1 and Yaf9 contributions to SWR1 targeting globally and histone exchange at +1 nucleosomes. Our findings highlight the native biochemistry of histone readers and suggest a broadly applicable, two-stage mechanism wherein acetyl-histone interactions initially constrain 3D diffusion of SWR1 to increase local concentration, followed by stochastic 1D diffusion at NDRs with directional capture by acetylated +1 nucleosomes.

overall_design: Chromatin Immunoprecipitation DNA-sequencing with exonuclease treatment (ChIP-exo) for histone variant H2A.Z and chromatin remodeler Swr1 in S. Cerevisiae with genetic modifications in the SWR1 complex

first: Anand

last: Ranjan

first: Ejlal

last: Elalaoui

first: Xiaona

last: Tang

first: Robert

last: Louder

first: Kevin

last: Nguyen

first: Joseph

last: Ye

first: Muhammad

last: Bennani

first: Anne

last: Gardner

first: B

middle: F

last: Pugh

first: Carl

last: Wu


title: Kinome reprogramming of G2/M kinases and repression of MYCN contribute to superior efficacy of lorlatinib in ALK-driven neuroblastoma

city: Philadelphia

first: Steven

last: Pastor

state: PA

address: 3401 Civic Center Blvd.

institute: Children's Hospital of Philadelphia

summary: Mutations in the tyrosine kinase domain of the Anaplastic Lymphoma Kinase (ALK) oncogene in neuroblastoma occur most frequently at one of three hotspot amino acid residues, with the F1174* and F1245* variants conferring de novo resistance to first and second generation ALK inhibitors including crizotinib and ceritinib. Lorlatinib, a third generation ALK/ROS inhibitor, overcomes de novo resistance and induces complete tumor regressions in patient-derived xenograft (PDX) models unresponsive to crizotinib. Lorlatinib has now completed Phase 1 testing in children and adults with relapsed/refractory ALK-driven neuroblastoma, and entered pivotal Phase 3 testing within the Children’s Oncology Group. To define mechanisms underlying the superior activity of lorlatinib, we utilized a chemical proteomics approach to quantitatively measure functional kinome dynamics in response to the ALK inhibitors lorlatinib and crizotinib, in clinically relevant ALK-driven neuroblastoma PDX models. Lorlatinib was a markedly more potent inhibitor of ALK and preferentially downregulated several kinases implicated in G2/M cell cycle transition compared to crizotinib. Lorlatinib treatment also led to the repression of MYCN expression and its occupancy at promoters of the same G2/M kinases. These data providing mechanistic insight in neuroblastoma into the far improved efficacy of lorlatinib over crizotinib for the treatment of ALK-driven neuroblastoma.

overall_design: To investigate transcriptomic changes upon treatment with ALK inhibitors in neuroblastoma models, we treated mice bearing NB1643 cell derived xenograft (CDX) or COG-N-453 patient derived xenograft (PDX) with vehicle, crizotinib or lorlatinib for 2 days. We performed comparative gene expression analysis on the RNA seq data obtained from the two models.

first: Smita

last: Matkar


title: Single cell gene expression profile of bronchoalveolar lavage fluid (BALF) cells from Apoa1-/- and Apoa1+/+ mice challenged with LPS and house dust mite

first: Stewart

last: Levine

state: Maryland

address: Bldg 10, Room 6D03, MSC 1590

department: Cardiovascular & Pulmonary Branch

institute: NHLBI, NIH

summary: Neutrophils play important roles in inflammatory airway diseases.  Here, we assessed whether apolipoprotein A-I (apoA-I) modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation.  Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 days. Single cell RNA sequencing was performed on cells recovered in bronchoalveolar lavage fluid (BALF) on day 4.  Unsupervised profiling identified 10 clusters of neutrophils in BALF from Apoa1-/- and Apoa1+/+ mice.  LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8, and had high maturation, aggregation, and TLR4 binding scores.  There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing interferon-stimulated genes were decreased.  An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice.  LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages.  Increased expression of a common set of pro-inflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice.  Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of pro-inflammatory genes.  This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apoA-I attenuates acute airway inflammation.

overall_design: Apoa1-/- and Apoa1+/+ mice (n = 3) were challenged daily with LPS (1 ug) plus house dust mite (HDM) (25 ug) in a volume of 20 ul for 3 days, and bronchoalveolar lavage was performed on day 4. The single cell gene expression experiment was performed on BALF cells that were pooled from three Apoa1-/- and three Apoa1+/+ mice using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (catalogue number: PN-1000121) (10x Genomics, Pleasanton, CA).

first: Xianglan

last: Yao

first: Neelam

last: Redekar


title: RNA splicing controls organ-wide maturation of postnatal heart in mice

city: Beijing

first: Wang

last: Li

address: 167 Beilishi Rd

department: State Key Laboratory of Cardiovascular Disease

institute: Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences

summary: Postnatal cardiac development requires the orchestrated maturation of diverse cellular components, for which unifying control mechanisms are still lacking. Using full-length sequencing, we examined the transcriptomic landscape of the maturating mouse heart (E18.5-P28) at single-cell and transcript isoform resolution. We identified dynamically changing intercellular networks as a molecular basis of the maturing heart, and alternative splicing (AS) as a common mechanism that distinguished developmental age. Manipulation of RNA-binding proteins (RBPs) remodeled the AS landscape, cardiac cell maturation, and intercellular communication through direct binding of splice targets, which were enriched for functions related to general, as well as cell type-specific, maturation. Overexpression of an RBP NCBP2 in neonatal hearts repressed cardiac maturation. Together, our data suggest AS regulation by RBPs as an organ-level control mechanism in mammalian postnatal cardiac development, and provide insight into the possibility of manipulating RBPs for therapeutic purposes.

overall_design:
We designed a time course covering all critical postnatal (P) stages up until early adulthood (P1-7, P10, P14, and P28) ans isolate the cardiomyocytes and non-cardiomyocytes for single-cell sequencing.

CLIP-seq data of G3BP1, NCBP2 and PTBP1 of cardiomyocytes, endothelial and fibroblast from nenatal heart.

Different Lentivirus or adenovirus were concentrated, and the efficiencies were tested in P1 CMs, ECs, and FBs in vitro. For transduction, 200 ?l lentivirus and 8 µg/ml polybrene or 4 ?l  adenovirus were added to cultured P1 CMs, ECs, FBs and adult CMs . Twenty-four hours later, cell medium was changed to DMEM supplemented with 10% FBS (Gibco, 11965092), 1% penicillin-streptomycin (Gibco, 15140122), and 5 µg/ml puromycin (Thermo, A1113802) for CMs and FBs or DMEM/F12 (Gibco, 11330032) supplemented with 10% FBS (Gibco, 11965092), 1% penicillin-streptomycin (Gibco, 15140122), 1 × ECGS (ScienceCell, 1052), and 5 µg/ml puromycin (Thermo, A1113802) for ECs. Forty-eight hours post transduction, cells were collected for subsequent experiments.

AAV expressing GFP (control), Ncbp2, G3bp1, or Ptbp1 were directly injected into the anterior myocardial wall of P1 mice. Validation of overexpression by qPCR and immunofluorescence (IF) staining were performed at age P14 (2 weeks, 2W) and assessment of cardiac function by echocardiography was performed at 2W and P35 (5 weeks, 5W) for all RBPs, respectively. At endpoint (5W), scRNA-seq was performed for GFP OE and Ncbp2 OE hearts.

first: Zheng

first: Quanyi

last: Zhao

first: Dandan

first: Mingzhi

last: Zhang

first: Lin

last: Mao

first: Bingying

last: Zhou

first: Li

last: Wang


title: Recruitment to the nuclear periphery enhances repression of H3K9me2-marked genes and transposons to shape cell fate [RNAseq_epilc]

first: Harold

last: Marin

first: Eric

last: Simental

first: Charlie

last: Allen

last: Martin

first: Barbara

last: Panning

first: Bassem

last: Al-Sady

first: Abigail

last: Buchwalter


title: Single-cell and spatial transcriptomics uncovers the role of senescent vascular cells in pathological arterial remodeling during murine atherosclerosis

status: Public on Jul 28 2024

first: Desiree

last: Tillo

address: 41 Center Dr, Room D310

institute: NIH/NCI

summary: Studying cardiovascular senescence is crucial in understanding the diverse manifestations of disease-related changes in the cardiovascular system and their implications for overall health and disease.  To systematically investigate the heterogeneity of senescent vascular cells in atherosclerosis, we employed the senescence reporter mouse p16TdTomato+/-, injected with an adeno-associated virus expressing PCSK9 to induce atherosclerosis when fed a high-fat diet (HFD).  We then treated this mouse with the senolytic drug ABT-737 or vehicle.  Whole-aorta single-cell RNA-sequencing (scRNA-seq) and Gene Set Enrichment Analysis (GSEA) using the SenMayo panel, uncovered 10 cell clusters displaying increased senescent features that were reduced by treatment with ABT-737.  Interestingly, these clusters do not express ‘classical’ senescence markers such as p16 and p21, but were senescent based on SenMayo GSEA, indicating that the senescence transcriptomic signature in this niche requires deeper characterization.  Using unbiased subclustering of the top four potentially senescent clusters, we identified subsets of cells increasing by HFD and reduced by ABT-737 treatment.  Accordingly, unique transcripts associated with each subpopulation, such as Spp1, Ctsb, and Tnfrsf11b, implicated these cells in senescence-related signaling pathways.  We then validated these findings by spatial transcriptomic profiling, locating senescent cells in the cap and core of brachiocephalic arteries from atherosclerotic mice.  Our results uncover a new vascular-specific transcriptomic signature that may be exploited for therapeutic targeting in age-related vascular diseases.

overall_design: We investigated senescence by feeding high-fat diet (HFD, 42% calories from fat from Harlan-Teklad, TD.88137) to Ldlr–/– and Ldlr–/–;3MR mice for 190 days, where treating (PBS vehicle or 5 mg/kg GCV) during the last 100 days on a 5 days on, 14 days off cycle.

first: Krystyna

last: Mazan-Mamczarz

first: Dimitrios

last: Tsitsipatis

first: Angelica

middle: E

last: Carr

first: Bennett

last: G Childs

first: Carla

last: Rocha Dos Santos

first: Carlos

last: Anerillas

first: Brigette

last: Romero

first: Jordan

last: M Gregg

first: Marc

last: Michel

first: Rachel

last: Munk

first: Jennifer

last: L Martindale

first: Yulan

last: Piao

first: Jinshui

last: Fan

first: Maria

last: O Hernandez

first: Noemi

last: Kedei

first: Madeline

last: M.F. Wong

first: Olga

last: Fedorova

first: Mona

last: Batish

first: Supriyo

last: De

first: Darren

last: J Baker

first: Myriam

last: Gorospe

first: Allison

last: B Herman


title: iSB09 and iCB elicit ABA-responsive gene expression in Solanum lycopersicum seedlings

status: Public on Jul 22 2024

city: Valencia

first: Pedro

middle: L.

last: Rodriguez

state: Valencia

address: C/ Ingeniero Fausto Elio, s/n

country: Spain

institute: IBMCP

summary: To compare the genome-wide transcriptional effect of ABA and iSB09 in tomato plants, we performed RNA-seq analysis of mock-, 10 uM ABA- or 20 uM iSB09-treated plants. Differential gene expression analysis between mock- and ABA-treated or iSB09-treated seedlings was done with DESeq2 and genes with an absolute value of log2 fold change (log2FC) > 1 or (log2FC) < -1 and p-adjusted value (padj) < 0.05 were selected. iSB09 upregulated and downregulated genes represent a subset of the ABA-responsive genes, which reflects the activation of PYL1-like and PYL4-like ABA receptors in tomato seedlings. Additionally, to compare the genome-wide transcriptional effect of ABA and iCB in tomato plants, we performed RNA-seq analysis of mock-, 10 uM ABA- or 10 uM iCB-treated plants. Differential gene expression analysis between mock- and ABA-treated or iCB-treated seedlings was done with DESeq2 and genes with an absolute value of log2 fold change (log2FC) > 1 or (log2FC) < -1 and p-adjusted value (padj) < 0.05 were selected. iCB mimics ABA transcriptional response through activation of the three subfamilies of ABA receptors.

overall_design: S. lycopersicum seedlings (3 d.a.g.) were transferred from plates to flashes with 3 ml of liquid Murashige and Skoog (MS medium) for 8 days in a growth chamber at 22ºC under long-day conditions (16 h light/8 h dark). Before treatment, the medium was refreshed and supplemented with either 0.1% DMSO (mock) or 10 μM ABA or 20 µM iSB09 or 10 µM iCB. Samples were collected after 3 hours of incubation, and the experiment was conducted with three independent biological replicates.  We performed genome-wide expression profiling analysis using data obtained from RNA-seq of the three biological replicates. Comparative analysis of transcriptional profiles using RNA-seq data for mock, iSB09, iCB or ABA treatments (WT genotype).

first: Jonatan

last: Illescas-Miranda

first: Javier

last: Forment

middle: L


title: Gene expression profile of Baf3 cells overexpressing TPR-FGFR1 fusion gene

status: Public on Jul 21 2024

city: Nanjing

first: Mengyao

last: Lv

address: 305 East Zhongshan Road, Nanjing

institute: Department of Hematology, Jinling Clinical Medical College, Nanjing University of Chinese Medicine

summary: Nowadays, fibroblast growth factor receptor 1 (FGFR1) inhibitors are considered as an effective kinase inhibitor for 8p11 myeloproliferative syndrome (EMS) treatment. However, targeting FGFR1 alone may not be sufficient in EMS patients with TPR-FGFR1 rearrangement. In this study, we established TPR FGFR1 expressing Baf3 cells and performed RNA-Seq analysis. RNA-Seq analysis revealed that genes associated with TPR-FGFR1 expression mainly participated in the epidermal growth factor receptor (EGFR) pathway (|FC| > 1.80, P < 0.05). Gene set enrichment analysis (GSEA) identified significant enrichment in the PI3K/AKT pathways. We found that EGFR is another activation pathway for AKT in TPR-FGFR1-expressing Baf3 cells. Moreover, we found that treatment with FGFR1 inhibitors alone could completely inhibit FGFR1 phosphorylation, but could not completely inhibit AKT, which is the downstream molecule of FGFR1. Compared to FGFR1 inhibitors alone, co-treatment with PD-166866 (FGFR1 inhibitor) and MK-2206 (AKT inhibitor) could simultaneously inhibit the phosphorylation of FGFR1 and AKT. In addition, FACS results indicated that the combination therapy of PD-168866 and MK-2206 significantly increased the rate of cells apoptosis than monotherapy of PD-166866 (P < 0.0001). Collectively, the data obtained in the present study provides strong evidence for dual targeting therapy of FGFR1 and AKT in EMS patients with TPR-FGFR1 rearrangement, offering a new direction for EMS treatment.

overall_design: Baf3 cells were infected with lentivirus and selected for 6 days in 5 μg/ml puromycin to produce Baf3 cell with stably expression of TPR-FGFR1.

first: Wenbing

last: Shangguan

first: Qian

last: Zhao

first: Yan

last: Li

first: Quan

first: Feng


title: Effect of LINC01315 depletion and overexpression on proliferation of OMM2.3 uveal melanoma cells

status: Public on Jul 16 2024

city: Shanghai, China

first: Zhu

last: Xie

state: China

address: Rm A601, Building for School of Life Sciences Fudan University Jiangwan Campus 2005 Songhu Rd, Shanghai 200433 China

institute: Fudan university

summary: Cell proliferation disorders contribute to several diseases including cardiac developmental defects and tumours,and identifying pivotal factors that modulate the cell proliferation process is important. In this study, we identified a short peptide, YAPer-ORF, encoded by the long-stranded non-coding RNA gene LINC01315. we clarified the biological function of YAPer-ORF in promoting cell proliferation using gene manipulation and examined various cell lines including OMM2.3. In terms of the molecular mechanism, we found that YAPer-ORF regulates YAP intranuclear kinase PRP4K, while coercing GNAQ/11 mutants to undergo nuclear translocation and inhibit transcription of critical factors in the Hippo signalling pathway to synergistically activate YAP activity.We finally validated our findings in transgenic mice with cardiac-specific expression and nude mouse models of tumour formation. Together, the present study elucidates a novel regulatory mechanism of the Hippo signalling pathway and provides a new intervention target for disorders arising from dysregulation of the Hippo signalling pathway.

overall_design: To investigate regulated action of YAPer-ORF on cell proliferation, we applied lentiviral infection to knockdown and overexpress target genes in uveal melanoma cell line OMM2.3.

first: Chao

first: Zhe

last: Zhang

first: Hongyan

last: Wang


title: RNAseq of Brassica napus seed

status: Public on Jul 15 2024

city: Winnipeg

first: Mark

last: Belmonte

state: Manitoba

address: 50 Sifton Rd.

department: Biological Sciences

country: Canada

institute: University of Manitoba

summary: mRNA expression profiling of the embryo, endosperm (micropylar, peripheral, chalazal), and seed coat (outer, inner, chalazal, chalazal proliferating tissue) of the developing Brassica napus seed. Tissues were isolated using laser microdissection (LMD) from Brassica napus seeds at the globular, heart, and mature green stages of seed development.

overall_design:
To investigate the transcriptome of the B. napus seed throughout space and time, we sampled seeds across four developmental stages (ovule, globular, heart, and mature green)
We fixed seeds from each stage and embedded them in parrafin wax, then dissected out four fililal subregions (embryo proper, micropylar endosperm, peripheral endosperm, chalazal endosperm) and four maternal subregions (inner seed coat, outer seed coat, chalazal seed coat, and chalazal proliferating tissue) using laser microdissection (LMD)
We then performed RNA-seq on the samples from each seed stage and each available subregion.

first: Dylan

last: Ziegler

first: Deirdre

last: Khan

first: Jenna

last: Kalichuk

first: Michael

last: Becker


title: Spinal motor neuron development and metabolism are transcriptionally regulated by Nuclear Factor IA [ChIP-Seq]

city: La Jolla

first: Stacey

middle: Marie

last: Glasgow

state: California

address: 9500 Gilman Dr, 3216 Pacific Hall

institute: University of California San Diego

summary: Neural circuits governing complex motor behaviors in vertebrates rely on the proper development of motor neurons and their precise targeting of limb muscles. Transcription factors are essential for motor neuron development, regulating their specification, migration, and axonal targeting. While transcriptional regulation of the early stages of motor neuron specification is well-established, much less is known about the role of transcription factors in the later stages of maturation and terminal arborization. Defining the molecular mechanisms of these later stages is critical for elucidating how motor circuits are constructed. Here, we demonstrate that the transcription factor Nuclear Factor-IA (NFIA) is required for motor neuron positioning, axonal branching, and neuromuscular junction formation. Moreover, we find that NFIA is required for proper mitochondrial function and ATP production, providing a new and important link between transcription factors and metabolism during motor neuron development. Together, these findings underscore the critical role of NFIA in instructing the assembly of spinal circuits for movement.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for transcription factor NFIA in E13.5 whole spinal cords

first: Julia

last: Gauberg

first: Kevin

middle: B

last: Moreno

first: Karthik

last: Jayaraman

first: Sara

last: Abumeri

first: Sarah

last: Jenkins

first: Alisa

last: Salazar

first: Hiruy

middle: S

last: Meharena


title: Spinal motor neuron development and metabolism are transcriptionally regulated by Nuclear Factor IA [RNA-Seq]

overall_design: To investigate the function of NFIA in mouse motor neuron development, we knocked down NFIA from the CNS. We performed gene expression profiling from 3 control and 3 conditional knockout E13.5 embryos.

middle: M


title: A developmental mechanism to Regulate Alternative Polyadenylation in an Adult Stem Cell Lineage [3'seq_KDs]

city: Stanford

first: Lorenzo

last: Gallicchio

state: california

address: Campus Drive W

department: developmental Biology

institute: Stanford University

summary: Alternative Cleavage and Polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3’UTRs from the same genetic locus, potentially impacting mRNA translation, localization and stability. Developmentally regulated APA can thus make major contributions to cell-type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, approximately 500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3’ UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of Cleavage Factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knock down of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell-type-specific APA at selected genes.

overall_design: 3'seq was performed on control testes (bamGal4) and testes in which PCF11, cbc, CstF64, CstF50, su(f), CPSF6 or PABP2 were knocked down in spermatocytes using bamGa4. Libraries were performed in duplictaes. Per each library roughly 150 pairs of testes were used.

first: Neruza

middle: R

last: Matias


title: Unraveling the elongating bovine conceptus microenvironment: Identification of gene transcripts and proteins along the conceptus-maternal interface in cattle

status: Public on Jun 30 2024

city: Knoxville

first: Daniel

last: Mathew

state: TN

address: 2506 River Drive

department: Animal Science

institute: University of Tennessee

summary: The bovine conceptus elongates near Day 16 of development and releases interferon-tau (IFNT), disrupting the endometrial luteolytic mechanism to sustain luteal P4 and pregnancy. Conceptus factors other than IFNT modify local endometrial activities in support of pregnancy; however, the microenvironment is largely uncharacterized. We utilized a bovine conceptus-endometrial culture system to elucidate the microenvironment in the form of RNA and protein. Estrus synchronized heifers remained cyclic (13) or were inseminated (9) to produce Day 16 cyclic endometrium and elongating conceptuses, respectively. Conceptus sections and cyclic endometrium were then used to generate tissue cultures in 1 mL of medium: (1) no tissue (Control Med; n=7), (2) mono-cultured conceptus (Conceptus; n=9), (3) mono-cultured endometrium (Endo; n=13), or (4) Endo-Conceptus co-culture (n=15). After 12 h, tissue RNA was sequenced (RNA-Seq) and media underwent proteomic analysis (LC-MS/MS). Compared to Conceptus and Endo, co-cultured conceptus and endometrial tissue contained 3,400 and 4,575 differentially expressed genes (DEG), respectively (P ≤ 0.01). Upregulated endometrial DEG, independent of IFNT, were associated with transcription whereas upregulated conceptus DEG were associated with protein homeostasis and metabolism (P ≤ 0.001).

overall_design: All procedures involving animals were approved by the University of Tennessee, Knoxville, Institutional Animal Care and Use Committee (IACUC 2020-67015-31617). Estrus synchronized Angus-Holstein heifers were used to generate cyclic Day 16 endometrial explants or elongated Day 16 bovine conceptuses. Endometrial explants (8 mm) and conceptus sections (5 X 2 mm) were cultured in medium separately (monoculture) or together in a co-culture system. After a 12 h incubation, the endometrial and conceptus tissue RNA was also isolated for RNA sequencing (RNA-Seq). Collectively, 13 cyclic uteri and and 9 conceptuses were used to produce endometrial explants and conceptus sections for mono- and co-cultures.

first: Katheryn

middle: D

last: Peterson

first: Trevor

last: Freeman

first: Shankar

last: Poudel

first: Hongyu

last: Liu

first: Tulio

last: Prado

first: Lew

last: Strickland

first: Jonathan

last: Beever

first: Susanta

middle: K

last: Behura

first: Thomas

last: Spencer

first: David

last: Kakhniashvili

last: Johnson

middle: J


title: TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

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

overall_design: Refer to individual Series


title: Impact of Retinoic Acid on directed differentiation of human iPSC-derived thyroid follicular cell progenitors

city: Boston

first: Pushpinder

last: Bawa

address: 670 Albany St

department: Boston University Medical Campus

institute: Center for Regenerative Medicine

summary: The goal of this experiment was to compare the transcriptome of human iPSC-derived thyroid follicular cell progenitors cultured in the presence or absence of Retinoic Acid in culture medium.

overall_design: Transcriptomic profiling at single cell resolution of human iPSC-derived thyroid follicular cell progenitors at 16 days of differentiation, cultured in thyroid specification medium containing or lacking Retinoic Acid (RA). 1) d16_RA_negative cells cultured without RA from day 2 to 16, 2) d16_RA_positive cells cultured with RA from day 2 to 16.

first: Hendrik

last: Undeutsch

first: Alberto

last: Posabella

first: Darrell

middle: N

last: Kotton

first: Anthony

last: Hollenberg


title: human iPSC-derived thyroid follicular cells maturation depends on time and duration of BMP-signaling

summary: The goal of this experiment was to analyse transcriptomic changes of human iPSC-derived thyroid follicular cell progenitors and follicular cells in a time-dependend manner and understanding the impact of prolonged BMP-signaling on thyroid maturation. We compared human iPSC-derived thyrocyte progenitors at day 12 of differentiation with human iPSC-derived thycytes at day 29 of differentiation that were continiously cultured in presence of BMP4 or were cultured with BMP4 until day 14, followed by 15 days of culture with BMP4-withdrawal.

overall_design: Transcriptomic profiling at single cell resolution of human iPSC-derived thyroid follicular cell progenitors at 12 days of differentiation,  and thyroid follicular cells at 29 days of differentiation cultured in thyroid maturation medium containing or lacking BMP4. 1) d12 cells cultured in thyroid specification medium until day 12, 2) d29_BMP_sustained cells cultured in thyroid specification medium until day 14 and in thyroid maturation medium supplemented with BMP4 from day 15 to day 29, 3) d29_BMP_withdrawn cells cultured in thyroid specification medium until day 14 and in thyroid maturation medium not containing BMP4 from day 15 to day 29


title: DDR1 regulates RUNX1-CBFβ to control breast stem cell differentiation

first: Colin

last: Trepicchio

address: 136 Harrison Ave

department: GSBS

institute: Tufts University

summary: The human breast is complex and comprised of multi-lineage and multi-structural elements. Recent work has shown that epithelial stem and progenitor cells use the collagen receptor Discoidin Domain Receptor 1 (DDR1) for differentiation into both basal and luminal cell lineages, which together are necessary for complex ductal-lobular morphogenesis. We developed a next-generation single cell derived organoid model that generates miniaturized breast tissue, to study how single stem cells can give rise to multiple cell types and compound tissue structures. We show that DDR1 activation triggers stem cell differentiation via RUNX1, in turn driving multilineage differentiation as well as complex ductal-lobular development. Mechanistically, DDR1 affects the interaction and expression of RUNX1 and its cofactor CBFβ, thereby regulating its activity. Together, these findings contribute to the current understanding of how the extracellular matrix component within the stem cell niche drives organogenesis and tissue regeneration.

overall_design: To investigate similarities between gene networks regulated by DDR1 and RUNX1, in an attempt to prove that they may be in the same pathway, MCF10A cells were treated with Collagen or Collagen and either DDR1 or RUNX inhibitors. RNA-seq was performed on these samples, followed by gene expression analysis comparison of differential gene sets from RUNXi and DDR1i treated samples compared to the control.

first: Charlotte

last: Kuperwasser


title: Expression pattern of circRNA in primary and recurrent glioblastoma.

status: Public on Jun 29 2024

city: Poznań

first: Katarzyna

last: Rolle

address: Noskowskiego 12/14

department: Department of Molecular Neurooncology

country: Poland

institute: Institute of Bioorganic Chemistry Polish Academy of Sciences

type: Non-coding RNA profiling by high throughput sequencing

summary: Circular RNAs are single-stranded covalently closed non-coding RNAs  expressed in a variety of tissues and cells. Glioblastoma (GBM) is the most aggressive and lethal tumor in the central nervous system, characterized by high recurrence and mortality rates. Here we explore circRNA expression profiles in 26 primary and 3 recurrent GBM samples and compare them to healthy brain tissue. Additionally we compare expression of circRNA in GBM patients blood in comparison to healthy donor blood.

overall_design: CircRNA expression profiles in 26 primary and 3 recurrent GBM samples in comparison to healthy brain tissue RNA libraries. Additionally expression profiles of circRNAs in GBM patients blood (n=3) in comparison to healthy donors blood (n=4)

first: J

last: Latowska

first: Z

last: Zarebska

first: M

middle: P

last: Sajek

first: K

last: Kuczynski

last: Kosinski

last: Zywicki

last: Misiorek

first: R

last: Piestrzeniewicz

first: S

last: Smol

first: A

last: Barciszewska


title: TSPO deficiency promotes the progression of malignant peripheral sheath tumors by regulating the G2/M phase of the cell cycle via CDK1

status: Public on Jun 26 2024

first: Chenhao

last: Hu

address: No.119 South Fourth Ring West Road, Fengtai District, Beijing, China

institute: Beijing Neurosurgical Institute

summary: Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann cell-derived sarcomas that are sporadic or associated with NF1 gene mutations. Traditional therapies are `usually ineffective for treating MPNSTs, so new targets need to be identified for the treatment of MPNSTs. In the present study, the role of the mitochondrial translocator protein (TSPO) in the regulation of cell proliferation and the cell cycle in MPNSTs was investigated. TSPO expression was lower in MPNSTs than in NFs. Loss-of-function experiments revealed that TSPO deficiency promoted MPNST cell growth, migration, and invasion and influenced the cell cycle in vitro and in vivo. In addition, TSPO depletion suppressed cell apoptosis by downregulating the expression of caspase-3, caspase-8, HSP60, p27, p53, and BCL-2 and suppressed the cell cycle by upregulating CDK1, CDK2, CCNB1 and CCNA2. Furthermore, CDK1 was determined to be an upstream target of TSPO-mediated regulation via RNA-seq, qPCR, and Western blotting. Specifically, depletion of CDK1 weakened the effect of TSPO deficiency on cell proliferation and migration. More importantly, CDK1 knockdown induced significant cell cycle arrest in the G2/M phase. In summary, TSPO deficiency regulates the cell cycle in MPNSTs by targeting CDK1, which may be an effective molecular target for prognosis evaluation and treatment.

overall_design:
Genomic DNA from 3 MPNST tumor samples and 3 NF tumor samples was subjected to RNA-seq.
Three cell lines were subjected to RNA-seq(ipNF05.5,MPNST8814 and sNF96.2).
Two conditions: siControl and TSPO knockdown

first: Xingnan

last: Zhang

first: Shengqiao

last: Sun

first: Chao

last: Guo

first: Yakun

last: Bu

first: Zicong

last: Wang

first: Song

last: Liu


title: TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase (HiC HiChIP)

first: Martin

last: Eilers

summary:
MYC proteins bind to virtually all active promoters transcribed by RNA polymerase II (RNAPII), but whether they interact with the three-dimensional chromatin architecture is unknown. Here we used HiChIP sequencing of the MYCN oncoprotein and found that MYCN localizes to three-dimensional hubs formed by active promoters and enhancers. In these hubs, MYCN interacts with TFIIIC, an architectural protein complex. MYCN recruits TFIIIC to promoters when transcription elongation is inhibited, and the complex of both proteins induces premature transcription termination.
Termination correlates closely with the TFIIIC-dependent removal of MYCN from promoter hubs and with corresponding alterations in the three-dimensional interactions of cohesin complexes. This limits DNA damage by removing RNAPII that stalls proximal to double-strand breaks. Binding of TFIIIC to MYCN is limited by competition with Aurora-A and this protects genes involved in mRNA processing from termination, arguing that MYCN contributes to the unusual proliferative capacity of neuroblastoma cells via removing stalled RNAPII from promoter hubs and via increasing the capacity for RNA processing.

overall_design: pLHiC, pLHiChIP and spLHiChIP sequencing was done with SH-EP NMYC-ER cells where the NMYC expression can be induced with 4-OHT. For some experiments, SH-EP NMYC-ER cells were used that carry a doxycycline inducible shRNA against TF3C5. For some other experiments, SH-EP NMYC-ER cells were treated with either DRB or MLN inhibitor. The immunoprecipitation in these experiments were done for either NMYC, TF3C5 or RAD21.

first: Raphael

last: Vidal

first: Eoin

last: Leen

first: Mareike

last: Müller

first: Christina

last: Schülein-Völk

first: Ursula

first: Dimitrios

last: Papadopoulos

first: Isabelle

last: Röschert

first: Steffi

last: Herold

first: Carsten

last: Ade

first: Peter

last: Gallant

first: Richard

last: Bayliss

first: Gabriele

last: Büchel


title: MUC1-C auto-regulatory complex with EBNA1 is responsible for latent Epstein-Barr virus-associated gastric cancer progression

status: Public on Jun 28 2024

city: Brookline

first: Hiroki

last: Ozawa

address: 450 Brookline

institute: Dana-Farber Cancer Institute

summary: Latent Epstein-Barr Virus (EBV) infection promotes cancers derived from B-lymphocytes and epithelial cells. EBV-encoded nuclear antigen 1 (EBNA1) is uniformly expressed in EBV-associated cancers. The MUC1 gene evolved in mammals to protect barrier tissues from viral infections. We report that MUC1 and the encoded oncogenic MUC1-C protein are upregulated in EBV-associated gastric cancers (EBVaGCs). We demonstrate that EBNA1 forms a complex with MUC1-C that regulates EBNA1 and MUC1-C expression. EBNA1 appropriates MUC1-C for (i) inducing DNA methyltransferases and DNA methylation, (ii) suppressing p21 and driving cell cycle progression, (iii) increasing survivin in promoting survival, and (iv) regulating MYC as an effector of EBV latency. MUC1-C thereby functions in maintaining EBV episomes and regulating EBV latency and lytic genes. In regard to EBVaGC progression, MUC1-C regulates expression of the SOX2 and NOTCH1-3 stemness genes, self-renewal capacity, and tumorigenicity. These findings indicate that EBNA1 co-opts MUC1-C functions that promote EBV latency and that MUC1-C is necessary for EBVaGC pathogenesis.

overall_design: RNA Seq in EBVaGC Cell line with MUC1-C shRNA silencing. We conpared DMSO and DOX treatment cells for differential gene expression analysis. MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma, St. Louis, MO, USA)  was inserted into the pLKO-tet-puro vector (Plasmid #21915; Addgene, Cambridge, MA, USA) . Vector-transduced cells were selected for growth in 1–2 μg/ml puromycin. Cells were treated with 0.1% DMSO as the vehicle control or 500 ng/ml doxycycline (DOX; Millipore Sigma).


title: Porcine ovary tissues : Minipig ovary vs. Pig ovary

status: Public on Jun 15 2024

city: Ansung

first: SangHwan

last: Kim

state: Gyeonggi-do

address: 327, Jungang-ro

department: School of Animal Life Convergence Science

institute: Hankyong National University

summary: Successful production of offspring in mammals is determined by the growth and apoptosis pathway, which is responsible for maintaining the balance between the estrous cycle. It is also believed that the development of the porcine ovary is regulated similarly; however, the molecular mechanism underlying differences in follicle development in the minipig and pig has yet to be elucidated. The present study aimed to identify developmental-associated genes differentially expressed in the minipig vs. pig.

overall_design: Experiment, Minipig ovary vs. Pig ovary

first: Sang-Hwan


title: FBL catalyzes internal 2′-O-Methylation in mRNA to promote RNA stability

status: Public on Jun 20 2024

first: Yanqiang

last: Li

address: 300 Longwood Ave.

department: Cardiology Department

institute: Boston Children's Hospital

summary: 2′-O-Methylation is prominent in ribosome RNA catalyzed by Fibrillarin (FBL). However, the stoichiometry of 2′-O-methylation at base resolution in the internal mRNA and its functions remain to be explored. Here, we firstly investigate the effect of the 2′-O-methylation on the mRNA stability and expression at a genome-wide scale. Combined with Nanopore sequencing and a machine learning strategy, we identified thousands of RNA methylation sites in mRNA and rRNA at base resolution. The mRNA half-life profiling of FBL deficient cells established a direct effect of RNA methylation and FBL binding on the mRNA stability. We further determined the RNA methylation on the expression levels and tested the post-transcriptional effect of elevated FBL mediated 2′-O-Methylation in mRNA within the prostate cancer model. Besides the role of the translational regulation of FBL in rRNA, elevated FBL promotes cancer progress by stabilizing the mRNA through RNA binding and 2′-O-Methylation. Our results reveal a novel mechanism that FBL regulates the 2′-O-methylation and turnover of mRNA at whole transcriptome levels.

overall_design:
Examination of mRNA half-life, in replicative analysis with NGS RNA-seq, with 2 replicates at siCTRL, siFBL and shNOP56 at time point 0h, 6h, 12h, 24h for HEK293T cells. Examination of mRNA half-life, in replicative analysis with NGS RNA-seq, with 2 replicates at siCTRL, siFBL in C4-2 cell line at 0h, 3h, 6h, 12h. Nanopore directive RNA-seq for the RNA modification detection for siCTRL and siFBL were performed with two replicates.
Examination of mRNA half-life, in replicative analysis with SLAM-seq RNA-seq, with 2 replicates at siCTRL, siFBL in C4-2 cell line at  0h, 6h,12h,24h.

first: Yang

last: Yi

first: Qi

last: Cao

first: Kaifu

last: Chen


title: ChREBPα protects against diet-induced non-alcoholic fatty liver disease

status: Public on Jun 12 2024

city: Ann Arbor

first: Sujuan

last: Wang

address: 2800 Plymouth Road

department: Molecular & Integrative Physiology

institute: University of Michigan

summary: To investigate the role of ChREBPα in the development and progression of non-alcholic fatty liver disease, we investigated how Chrebpα deficiency or overexpression affect the development and progression of NAFLD.

overall_design: We compared the gene expression profiling of RNA-seq data from the livers of Chrebpα-flox/flox mice injected with AAV-TBG-GFP (Chrebpα-WT) and AAV-TBG-Cre (Chrebpα-LKO) and fed NASH diet (40 kcal% Fat (Either Palm Oil), 20 kcal% Fructose and 2% Cholesterol) for 12 weeks, and compared the gene expression profile of the livers of wildtype mice injected with Adenovirus overexpressing GFP (Ad-GFP) and Chrebpα (Ad-Chrebpα) and fed HFLMCD diet (45 kcal% Fat, Without Choline and With 0.1% Added Methionine).

first: Deqiang

last: Zhang

first: Lei

last: Yin


title: RNASeq of MuSCs and single myofibers

status: Public on Jun 11 2024

city: Montreal

first: Vahab

middle: D.

last: Soleimani

state: Quebec

address: 3755 Chem. de la Côte-Sainte-Catherine

department: Lady Davis Institute for Medical Research

institute: Jewish General Hospital

summary: The maintenance of cell lineage and cell fate are essential for the function of adult stem cells. Despite this, the epigenetic mechanisms that regulate muscle stem cell (MuSC) identity are not well understood. In this study, we performed Cut&Tag of the activating histone marks H3K4me3 and H3K27ac on freshly isolated quiescent MuSCs and found that a large number of genes without transcription still maintained the permissive mark H3K4me3 but lacked the marker of active enhancers, H3K27ac. These genes included those that are essential for non-myogenic lineage determination. We found that many of the genes that were not transcribed but enriched for H3K4me3, were also enriched for the RE-1 binding motif, the motif recognized by the repressive transcription factor REST. Using a genetic mouse model where REST is conditionally knocked out of MuSCs, we further investigated the role of REST in the maintenance of quiescent MuSC cell identity. Investigation of the transcriptome and chromatin accessibility of WT and REST deficient MuSCs determined that the loss of REST results in the gain of expression of key genes of several non-myogenic tissues, particularly neuronal genes.  Additionally, the loss of REST led to the dramatic reduction of the MuSC pool and the induction of muscle atrophy.  The unstable cell identity caused by the genetic deletion of REST results in the MuSCs undergoing apoptosis and is the main driver of the observed loss of the MuSC pool. Together, the data presented in this study establishes a novel function of the transcription factor REST, where it safeguards the identity and myogenic lineage of MuSCs, through the repression of alternative lineages.

overall_design: 1000 Muscle stem cells (MuSCs) (ITGA7+/VCAM1+/CD11b-/CD45-/CD31-/Ly6a-) were isolated by Fluorescence Activated Cell Sorting (FACS) from 3 male WT and 3 male REST-cKO mice (4-6 weeks old) and sorted directly into SMART lysis bufer for RNA-Seq of quiescent MuSCs and the remaining isolated cells were cultured for 7 days. After 1 week in culture, myoblasts were counted and 1000 myoblasts were obtained for RNA-Seq of myoblasts. RNA-Seq libraries were created using SMART-Seq to make cDNA (TAKARA,cat  # 634456), followed by Nextera XT (Illumina, #cat FC-131-1024) for incorporation of Illumina sequencing adaptors. Single myofibers were isolated from 3 WT and 3 REST-cKO mice (4-6 weeks old, male) and lysed in SMART lysis buffer. Single myofiber RNA-Seq was performed as previously described  (Blackburn, D.M. et al. Journal of Biological Chemistry, 2019).


title: ATAC-Seq of single myofibers and muscle stem cells (MuSCs)

overall_design: 5000 MuSCs (ITGA7+/VCAM1+/CD11b-/CD45-/CD31-/Ly6a-) were isolated by Fluorescence Activated Cell Sorting (FACS) from 4 REST-cKO and 4 WT male mice (4-6 weeks old) and sorted directly into ATAC-Seq lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin). Samples were processed as previously described OMNI-ATAC-Seq protocol (Corces, M.R. et al. Nature Methods, 2017). Single myofiber ATAC-Seq (smfATAC-Seq) was performed on uninjured Extensor Digitorum Longus (EDL) myofibers isolated from 2 REST-cKO and 2 WT male mice (4-6 weeks old) (same mice used for smfRNA-Seq). Single myofibers were isolated and smfATAC-Seq was performed as previously described (Sahinyan K. et al. eLife, 2022 , Sahinyan K. et al. bioProtocol, 2022). All the ATAC-Seq libraries underwent 150 bp paired end sequencing on NovaSeq6000 Sprime Paired End (PE) 150 bp.

first: Marie-Michelle

last: Simon


title: Cut& Tag of quiescent muscle stem cells (MuSCs)

overall_design: 45000 MuSCs (ITGA7+/VCAM1+/CD11b-/CD45-/CD31-/Ly6a-) were isolated by Fluorescence Activated Cell Sorting (FACS) from 2 WT (C57BL/6) and 2 RESTc-KO male mice (4-6 weeks old). MuSCs were sorted directly into 2%FBS in PBS and Cu&Tag was performed by using CUT&Tag-IT Assay Kit (Active Motif, 53160). Cut&Tag of H3K4me3 was performed on WT MuSCs using the H3K4me3 antibody. Cut&Tag of H3K27ac was performed on WT and REST-cKO MuSCs using the H3K27ac antibody.


title: ITGB4 up-regulated by STAT3 reduces the sensitivity of bladder cancer to cisplatin by suppressing p53

status: Public on Jun 07 2024

city: changsha

first: ruijiang

last: Zeng

address: No.139 Renmin Road Central

institute: The Second Xiangya Hospital of Central South University

summary: Background: Cisplatin-based chemotherapy is the first-line treatment for patients with advanced bladder cancer (BC). However, the development of cisplatin resistance limits its antitumor effects. While, the mechanism of cisplatin resistance remains unclear. Methods: Bioinformatics techniques were used to analyze genes and pathways associated with cisplatin therapy resistance.  A variety of biological techniques were used to identify the role of ITGB4 in cisplatin sensitivity in BC and its potential molecular mechanism. Results: In this study, we demonstrated that ITGB4 plays a key role in regulating the sensitivity of p53 wild-type (WT) BC to cisplatin therapy. Our findings revealed that ITGB4 reduces the sensitivity of BC to cisplatin by inhibiting phosphorylation of the p53-S15 site and promoting MDM2 binding to p53, thereby reducing p53 expression. Additionally, we showed that ITGB4 influences the antitumor effects of MDM2 inhibitors when they are combined with cisplatin therapy. Furthermore, we found that ITGB4 levels were elevated in cisplatin-resistant BC cells, a process mediated by STAT3 activation. The combination of STAT3 inhibitors can enhance the antitumor effect of cisplatin in BC. Conclusions: ITGB4 is a key molecule influencing cisplatin sensitivity in p53 WT BC, and the combination of STAT3 inhibitors can enhance the antitumor effect of cisplatin.

overall_design:
Comparative gene expression profiling analysis of RNA-seq data for the RT4 cells. Flag_EV vs. Flag_ITGB4
Normal and cisplatin-resistant RT4 bladder cancer cell lines

first: Ruijing

first: Zhuo

last: Xing


title: Seclidemstat blocks the transcriptional function of multiple FET-fusion oncoproteins

city: Columbus

first: Cenny

last: Taslim

address: 700 Children's Drive

institute: Nationwide Children's Hospital

summary: Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in several rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting pediatric, adolescent, and young and middle-aged adults, with prognosis depending on the fusion and whether the disease is localized, metastatic, or relapsed. New therapies are needed for these patients. These translocations fuse the 5’ portion of the FET family gene with a 3’ partner gene encoding a transcription factor. The resulting fusion proteins have the intrinsically disordered low complexity domain (LCD) of the FET protein paired with a DNA binding domain and these chimeras function as oncogenic transcription factors. FET fusion proteins have proven stubbornly difficult to target directly and promising new therapeutic strategies target the critical regulators and co-regulators of these proteins. One such protein is the histone lysine specific demethylase 1 (LSD1). LSD1 physically interacts with and is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 gene regulation and prior studies showed that treatment with the noncompetitive inhibitor SP-2509 blocked the transcriptional activity of the fusion. A similar molecule, seclidemstat (SP-2577), was identified as a promising lead for clinical development and is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against EWSR1::FLI1 or any other FET fusion protein has yet to be demonstrated. In this study, we sought to evaluate the in vitro potency of seclidemstat against multiple cell lines derived from different FET-rearranged sarcomas, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also defined the transcriptomic effect of seclidemstat treatment in these diseases and evaluated the activity of seclidemstat against FET fusion transcriptional regulation in multiple cell lines. Seclidemstat showed potent activity in cell viability assays in all four FET-rearranged sarcomas and disrupted the transcriptional function of all the tested fusion proteins. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas

overall_design: To evaluate the pharmacologic effects of seclidemstat treatment on the transcriptomes of FET-rearranged sarcoma cell lines, we treated 9 cell lines expressing a variety of FET fusion oncogenes with either DMSO or the IC90 dose of seclidemstat for 48 hours, collected RNA, and performed RNA-seq. Additionally, to compare with another sarcoma without a FET-rearrangement, we treated 3 fusion positive rhabdomyosarcoma cell lines with 2 uM SP-2509. RNA from these samples was collected at 48 hours and analyzed by RNA-seq.

first: Galen

last: Rask

first: Ariunaa

last: Bayanjargal

first: Julia

last: Selich-Anderson

first: Emily

last: Theisen

first: Benjamin

last: Stanton


title: The 18S rRNA Methyltransferase DIMT-1 Regulates Lifespan in the Germline Later in Life

status: Public on Jun 06 2024

first: Wayne

last: Mitchell

address: 77 Avenue Louis Pasteur

institute: Brigham and Women's Hospital

summary: There is a marked dysregulation of the proteome as organisms age. Regulation of both protein production and degradation can become dysregulated as an organism ages. However, whether and how aging-responsive transcripts are selectively translated is unknown. Changes in the composition of the ribosome can lead to differential binding and translation of specific transcripts. Here we examined the role of ribosomal RNA (rRNA) methylation in maintaining appropriate translation of age-dependent transcripts. In a directed RNAi screen we identified the 18S rRNA N6’-dimethyl adenosine (m6,2A) methyltransferase, dimt-1, as a regulator of C. elegans lifespan and stress resistance. Lifespan extension induced by dimt-1 deficiency required a functional germline and the Rag GTPase, raga-1, which links amino acid sensing to mechanistic target of rapamycin (mTOR) complex (mTORC)1. Using an auxin-inducible degron tagged version of dimt-1 we demonstrate that DIMT-1 functions in the worms germline after mid-life to regulate lifespan. We further found that knock-down of dimt-1 leads to selective translation of transcripts important for stress resistance and lifespan regulation in the C. elegans germline in mid-life. Our findings highlight a new role for rRNA modifications such as m6,2A and ribosome heterogeneity more broadly, in maintaining appropriate translation later in life to promote healthy aging.

overall_design:
To investigate the role of germline dimt-1 in regulating C. elegans lifespan, we IP'd FLAG-tagged germline ribosomes from dimt-1 knock down and control worms. The purified ribosome-bound transcripts were then subject to mRNA-seq. As a control, we also performed mRNA-seq on dimt-1 knock down and control worms using total RNA from whole-worm lysates.
To further investigate the role of germline dimt-1 in regulating C. elegans lifespan, we IP'd FLAG-tagged muscle ribosomes from dimt-1 knock down and control worms as a separate control experiment. The purified ribosome-bound transcripts were then subject to mRNA-seq. As another control, we also performed mRNA-seq on dimt-1 knock down and control worms using total RNA from whole-worm lysates.

first: M. Hafiz

last: Rothi

first: Joseph

last: Al Haddad

first: Gautam

middle: C

last: Sarkar

first: Albert

last: Ying

first: Nancy

last: Pohl

first: Roberto

last: Sotomayor

last: Natale

first: Scarlett

last: Dellacono

first: Vadim

last: Gladyshev

first: Eric

last: Greer