title: 10x single-cell RNAseq profiling of influenza Virus infected Human Lung Tissue Explants (HLTE)

status: Public on May 29 2024

city: Hannover

first: Frank

last: Pessler

state: Lower Saxony

address: Feodor-Lynen-Str. 7

department: Experimental Infection Research

institute: TWINCORE Centre for Experimental and Clinical Infection Reearch

summary: This study investigates the interactions between Influenza A virus (IAV) and lung tissue at the single-cell level using the 10x Genomics single-cell RNA sequencing (scRNAseq) platform. Lung tissue samples from a donor with COPD were infected with IAV, and both infected and uninfected samples were analyzed 24 hours post-infection. Single-cell suspensions from multiple infected and uninfected tissue pieces were pooled and analyzed to identify cell types and their responses. A total of 10 cell types were identified, including various airway epithelial cells and immune cells. Apoptotic T cells, characterized by high mitochondrial DNA content, were excluded from gene expression analyses. The presence of viral mRNA was used to indicate viral transcription activity, although distinguishing between intracellular synthesis and extracellular uptake of viral mRNA remains challenging.

overall_design: Human Lung tissue explant  from COPD patients were cultrured in in vitro . These tissue pieces were infected with influenz A virus for 24 hours . After infection, tissue was digested into single cell suspension.

first: Robert

last: Geffers

first: Aaqib

last: Sohail


title: Optimizing RNA Extraction Methods for High Throughput Transcriptome Sequencing of  Fresh Frozen and Formalin-Fixed Paraffin-Embedded Cardiac Tissues Specimens

status: Public on May 23 2024

city: Rochester

first: Jin Sung

last: Jang

address: 200 1st st sw

country: USA

institute: Mayo Clinic

type: Expression profiling by high throughput sequencing

summary: In this study, the efficiency of four RNA extraction methods was compared on 23 FFPE cardiac tissue specimens. The Qiagen AllPrep DNA/RNA FFPE kit (Method QP), Qiagen AllPrep DNA/RNA FFPE kit, with protocol modification on the ethanol wash step after deparaffinization (Method QE), CELLDATA RNAstorm 2.0 FFPE RNA Extraction kit (Method BP) and CELLDATA RNAstorm 2.0 FFPE RNA Extraction Kit with protocol modifications on the lysis step (Method BL). In comparing RNA quality metrics across FFPE RNA extract, nucleic acids extracted with Method QE and QP had the highest RNA yield. However, Method QE outperformed Method QP as more extract from Method QE had DV 200 values above 30%. Both method BL and BP produced a similar range of RNA purity and yield but more extract from Method BL had DV 200 values above 30% compared to Method BP. When accessing distribution value, Method BL outperformed Methods BP, QE, and QP as more extracts from Method BL had DV 200 values above 30% (16/23 samples) compared to other methods (PDV200<0.001; Kruskal-Wallis). However, method QE outperformed other methods in terms of RNA yield. The sequencing performance of RNA extracts from Method QE and Method BL was further tested on 8 matching samples with high RNA yield and high DV200 value respectively. RNA extracts from Method QE which yielded the highest RNA quantity among all methods exhibited comparable sequencing performance to extract obtained through Method BL, which yielded extract with high DV200 value. This study suggests that the DV200 and RNA yield are both reliable pre-analytic metrics in determining a suitable method for successful transcriptome sequencing of FFPE samples and have important implications for future studies exploring transcriptome sequencing of FFPE cardiac specimens.

overall_design: To generate quality transcriptome sequences for the identification of biomarkers predicting mortality in cardiac disease, we sought to generate a reliable and reproducible RNA extraction method. Four nucleic acid extraction methods (four methods) were compared on 23 FFPE cardiac tissue specimens. The RNA yield, RNA integrity, as reflected by the distribution value 200 (DV 200); and RNA purity, as reflected by the 260/280 and the 260/230 nm absorbance ratios, were determined. RNA sequencing was performed by deploying the  Illumina Stranded Total RNA Ligation protocol on FFPE RNA extract. Bioinformatic analysis was performed to access transcriptome at gene and transcript level.

first: Nkechi

middle: M

last: Odogwu

first: Jin

middle: S

first: Timothy

middle: J

last: Nelson


title: Transcriptome analysis of VapC13 and VapC26 overexpression in Mycobacterium tuberculosis

status: Public on May 17 2024

city: Bangalore

first: Munmun

last: Bhasin

state: Karnataka

address: CV Raman Rd

department: Molecular Biophysics Unit

country: India

institute: Indian Institute of Science

summary: Using Total RNA sequencing, We report the effects of VapC13 and VapC26 overexpression on the transcriptome of M. tuberculosis. For RNA-seq experiments, early-log phase cultures (OD600nm ~ 0.2-0.3) of M. tuberculosis strains harboring either pTetR or pTetR-vapC13 or pTetR-vapC26 were induced with the addition of 50 ng/ml Atc for 24 h. For total RNA isolation, induced cultures were harvested by centrifugation, washed twice with 1x PBS and lysed by bead beating in Trizol. Total RNA was extracted by phenol-chloroform, precipitated with isopropanol and eluted using Qiagen RNA isolation kit as per manufacturer’s instructions. We report that VapC13 or VapC26 resulted in increased transcripts levels of stress responsive genes of M. tuberculosis.

overall_design: Cells harbouring pTetR (Vector only) or pTetR VapC13 or pTetR VapC26 were used for RNA-Seq analysis. Experiments were performed thrice for each strain.

first: Arun

last: Sharma

first: Neelam

last: Singh

first: Prabhakar

last: Tiwari

first: Pankaj

last: Chopra

first: Raghavan

last: Vardarajan

first: Ramandeep


title: Banxia Xiexin decoction promotes gastric lymphatic pumping by regulating lymphatic smooth muscle cell contraction and energy metabolism in a stress-induced gastric ulceration rat model

status: Public on Jun 01 2024

city: Beijing

first: Shutao

last: Pan

address: Liangxiang University Town, Fangshan District, Beijing, 102488, China

country: China

institute: Beijing University of Chinese Medicine

summary: The roles of lymphatic pumping disorder in gastrointestinal diseases have drawn increasing research attentions. A Traditional Chinese medicine (TCM) formula Banxia Xiexin decoction (BXD) has definite therapeutic effect in treating stress-induced gastric ulceration (SIGU) and many other gastrointestinal diseases, but its effect on gastric lymphatic pumping (GLP) remains unclear. This study used tracer-aided in vivo imaging to illustrate BXD’s GLP recovery effect in a SIGU rat model, and explored the underlying mechanisms via RNA sequencing (RNA-Seq) and transcriptomic analysis of gastric lymphatic vessel (GLV). The result indicated the possible mechanisms included smooth muscle contraction signaling pathway activation, energy supply restoration, energy metabolic program modulation and fatty acid degradation reduction. The most probable target of these mechanisms was the lymphatic smooth muscle cells (LSMCs) which drove the GLP. The transcriptomic results were further validated by the assessments for expression level of key genes and proteins. By GLP recovery and lymphostasis clearance, BXD effectively ameliorates the accumulation of inflammatory cytokines and metabolic wastes in the stomach, which contributes a lot to its efficacy in treating SIGU and other gastrointestinal diseases.

overall_design: In vivo GLP imaging were performed on SIGU rat model, and the lymphatic dynamic parameters were evaluated. Gastric antrum tissues and serum were collected for macroscopic, histopathological and ulcerative parameters analysis. Gastric lymphatic vessel (GLV) tissues were collected for RNA-Seq assays. Differentially expressed genes (DEGs) were screened from RNA-Seq result and submitted for transcriptomic analysis. Key DEGs and their derivative proteins were measured by qRT-PCR and WB.

first: Xue

last: Yu

first: Mingyu

last: Liu

first: Jiaqi

first: Chunguo

last: Wang

first: Yao

last: Zhang

first: Fei

last: Ge

first: Yushan

last: Gao

first: Meng

last: Chen

first: Dongmei


title: Multiple direct and indirect roles of the Paf1 Complex in elongation, splicing, and histone post-translational modifications [RNA-seq]

status: Public on May 15 2024

city: Pittsburgh

first: Karen

last: Arndt

state: PA

address: 4249 Fifth Ave

department: Biological Sciences

institute: University of Pittsburgh

summary: Eukaryotes employ a set of conserved transcription elongation factors to modulate the behavior of RNA polymerase II (RNAPII). Disruptions of one such factor, the Paf1 complex (Paf1C), generate subunit-specific phenotypes, including distinct changes to co-transcriptional histone modifications. How individual Paf1C subunits impact transcription and coupled processes remains ambiguous. By comparing conditional depletion and steady-state deletion of Paf1C subunits, we determine direct and indirect contributions of Paf1C to gene expression in Saccharomyces cerevisiae. Through nascent transcript sequencing, RNAPII profiling, and mechanistic modeling of transcription elongation dynamics, we find evidence for unique roles of Paf1C subunits in regulating RNAPII processivity, elongation rate, mRNA stability, transcript splicing, and repression of antisense transcripts. Through genetic suppression, we attribute increased antisense transcription, but not other defects, of a PAF1 mutant to loss of H3 K36 methylation. This work comprehensively analyzes both the immediate and extended roles of Paf1C subunits in transcription elongation and transcript regulation.

sample_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae

overall_design: 4-thiouracil sequencing of Paf1C mutants, Paf1C-rapid depletion alleles, and genetic interrogation of H3K36me contributions to Paf1C-dependent phenotypes.

first: Alex

last: Francette


title: Multiple direct and indirect roles of the Paf1 Complex in elongation, splicing, and histone post-translational modifications [4tU-seq]

platform_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae


title: M6A reduction relieves FUS-associated ALS granules [CLIP-seq]

status: Public on May 06 2024

city: Rome

first: Irene

last: Bozzoni

address: Piazzale Aldo Moro 5

department: Biology and Biotechnologies "Charles Darwin"

country: Italy

institute: Sapienza University of Rome

summary: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motorneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by mutant protein aggregation, among which the RNA binding protein FUS. In this work we show that such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished as a consequence of the m6A writer METTL3 knock-down. These effects were obtained observed both in neuronal cell lines and in iPSC-derived human motor neurons expressing mutant FUS.  Importantly, stress granules formed in mutant conditionswhen mutant FUS is expressed/ALS condition showed a distinctive transcriptome with respect to control cells; interestingly, after METTL3 downregulation, it reverted to similar to control. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, a well characterized inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

overall_design: In order to unveil the RNAs bound to FUS(P525L), we sequenced RNA from input (INP) and immunoprecipitated (IP) RNA fraction isolated  from SK-N-BE cells carrying the doxycycline inducible overexpression of flag-tagged  FUSP525L, and constitutively expressing the SG marker G3BP1 tagged with GFP using an anti-flag antibody.

first: Gaia

middle: D

last: Timoteo

first: Andrea

last: Giuliani

first: Adriano

last: Setti


title: M6A reduction relieves FUS-associated ALS granules [RNA-Seq]

summary: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motorneurons (MN) degeneration1. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by mutant protein aggregation, among which the RNA binding protein FUS2. In this work we show that such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished as a consequence of the m6A writer METTL3 knock-down. These effects were obtained observed both in neuronal cell lines and in iPSC-derived human motor neurons expressing mutant FUS.  Importantly, stress granules formed in mutant conditionswhen mutant FUS is expressed/ALS condition showed a distinctive transcriptome with respect to control cells; interestingly, after METTL3 downregulation, it reverted to similar to control. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, a well characterized inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

overall_design: In order to unveil the impact of m6A on SG RNA composition in physiological and ALS-linked conditions, we sequenced RNA from input (INP) and immunoprecipitated  granules (SGenr) isolated from stresses (ARS+) SK-N-BE cells carrying the doxycycline inducible overexpression of either wild type FUS (FUSWT) or mutant FUS (FUSP525L), and constitutively expressing the SG marker G3BP1 tagged with GFP.   We treated FUSWT or FUSP525L cells with the combination of two shRNAs against METTL3 (shMETTL3), treated (DOXY+) or not (DOXY-) with doxycycline.

last: Di Timoteo


title: M6A reduction relieves FUS-associated ALS granules [m6A-IP]

summary: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motorneurons (MN) degeneration1. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by mutant protein aggregation, among which the RNA binding protein FUS. In this work we show that such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished as a consequence of the m6A writer METTL3 knock-down. These effects were obtained observed both in neuronal cell lines and in iPSC-derived human motor neurons expressing mutant FUS.  Importantly, stress granules formed in mutant conditionswhen mutant FUS is expressed/ALS condition showed a distinctive transcriptome with respect to control cells; interestingly, after METTL3 downregulation, it reverted to similar to control. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, a well characterized inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

overall_design: In order to correlate the methylation status of the transcripts with their localization, we performed MeRIP-Seq in FUSP525L cells


title: Tuft cells act as regenerative stem cells in the human intestine

status: Public on May 01 2024

city: Utrecht

first: Amir

last: Giladi

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: In mice, intestinal tuft cells have been described as a long-lived, post-mitotic cell type of which two distinct subsets have been identified, named tuft-1 and tuft-2.By combining analysis of primary human intestinal resection material with organoid technology, we identify four distinct states of human tuft cells, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that its numbers rapidly increase upon interleukin (IL)-4 and IL-13 exposure, as reported previously in mouse. This, however, is induced through proliferation of pre-existing tuft cells, rather than through increasedde novo generation from stem cells. Indeed, proliferative tuft cells occurin vivoboth in adult and in fetal human intestine. Single mature proliferating Tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem- and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

overall_design: Human ntestinal organoids were grown in a  medium that promotes diferentiation to Tuft cells with or without the stimulants IL-4 and IL-13. Single cells suspension was processed by 10x platform or with SORT-seq, using KIT or AVIL-CLOVER expression in order to enrich for tuft cells.

first: Lulu

last: Huang

first: Jochem

middle: H

last: Bernink

first: Daniel

last: Krueger

first: Gijs

last: van Son

first: Maarten

last: Geurts

first: Georg

last: Busslinger

first: Lin

last: Lin

first: Maurice

last: Zandvliet

first: Peter

last: Peters

first: Carmen

last: Iglesias

first: Christianne

last: Buskens

first: Willem

last: Bemelman

first: Harry

last: Begthel

first: Hans

last: Clevers


title: MSA: Scalable DNA methylation BeadChip for human trait screening

status: Public on May 11 2024

city: Philadelphia

first: Wanding

last: Zhou

address: 3501 Civic Center Blvd

department: Pathology and Lab Medicine

institute: Children's Hospital of Philadelphia

summary: We designed and introduced a new methylation array concentrating on human trait screening and discovery. The new MSA (Methylation Screening Array) leveraged the massive Infinium platform-based data from epigenome-wide association studies, combined with updated knowledge from the latest single cell and cell type-resolution whole genome methylome profiles, to achieve scalable screening of epigenetics-trait association in an ultra-high sample-throughput. Our design encompassed diverse human trait associations, including those with genetic, biological, and demographical variables, environmental exposures, and common human diseases such as neurodegenerative, genetic, cardiovascular, infectious, and immune diseases. We comprehensively evaluated this array's reproducibility, accuracy, and capacity in supporting 5-hydroxymethylation profiling and comprehensive cell-type deconvolution in diverse human tissues. Our first data using this platform uncovered dynamic chromatin and tissue contexts of DNA modification variations and genetic variants with human trait associations.

overall_design: Cell line (GM12878, K562, LNCaP, HeLa, Raji, MCF7, Jurkat, HCT116 and other Coriell cell lines), and 25 human tissue (Adrenal, Bladder, Cerebellum, Colon, Esophagus, Eye, Heart, Kidney, Larynx, Liver, Lung, Lymph Node, Motor cortex, Ovary, PBMC, Pancreas, Placenta, Prostate, Skin, Spinal Cord, Spleen, Stomach, Testis, Thymus, Thyroid, Trachea) and purified cell DNA (CD4+ T cells, CD8+ T cells, total T cell, NK cells, monocytes) and methylation titration control DNA is run on the MSA BeadChip to evaluate MSA reproducibility, accuracy, performance at low input ranges, and capability at capturing biological methylation variation. Tissues including cerebellum, motor cortex, colon, pancreas, skin, heart are additionally processed with bACE conversion to obtain 5hmC profiles.

first: David

last: Goldberg

first: Nicole

last: Renke


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity (mouse_vivo_cgas_RT_rnaseq)

status: Public on Apr 30 2024

city: Cambridge

first: Robert

middle: T

last: Manguso

state: MA

address: 75 Ames Street, Rm 7053

institute: Broad Institute of MIT & Harvard

summary: Isocitrate Dehydrogenase 1 (IDH1) is the most commonly mutated metabolic gene across human cancers. Mutant IDH1 (mIDH1) generates the oncometabolite (R)-2-hydroxyglutarate, disrupting enzymes involved in epigenetics and other processes. A hallmark of IDH1-mutant solid tumors is T cell exclusion, whereas mIDH1 inhibition in preclinical models restores anti-tumor immunity. Here, we define a cell-autonomous mechanism of mIDH1-driven immune evasion. IDH1-mutant solid tumors show striking, selective hypermethylation and silencing of the cytoplasmic dsDNA sensor, CGAS, compromising innate immune signaling. mIDH1 inhibition restores DNA demethylation, derepressing CGAS and transposable element (TE) subclasses. dsDNA produced by TE-reverse transcriptase activates cGAS, triggering viral mimicry and stimulating anti-tumor immunity. Thus, we demonstrate that mIDH1 epigenetically suppresses innate immunity and link endogenous reverse transcriptase activity to the mechanism of action of an FDA-approved oncology drug.

overall_design: Immunocompetent mice harboring subcutaneous allograft tumors generated with murine CKIR132C ICC cells (2205-sgCtrl, -sgCgas, -sgRT) were treated (~100 mm^3 starting volume) with vehicle or AG120 (150 mg/kg; twice daily), and tumors were harvested at serial time points (Vehcle, AG120-Day3 and AG120-Day 6). sgCtrl, sgCgas and sgRT CKIR132C ICC cells were isolated by magnetic bead–mediated depletion of stromal populations from tumors treated with vehicle or AG120 for 3 or 6 days.

first: Meng

last: Wu

first: Ashwin

middle: V

last: Kammula

first: Nabeel

last: Bardeesy


title: Brd4::Nutm1 fusion gene initiates NUT carcinoma in vivo [WGS]

status: Public on May 07 2024

city: East Lansing

first: Bin

last: Gu

state: MI

address: 755 Woodlot Drive

department: OBGYN

institute: Michigan State University

type: Genome variation profiling by high throughput sequencing

summary: NUT carcinoma (NC) is an aggressive cancer with no effective treatment.  About 70% of NUT carcinoma is associated with chromosome translocation events that lead to the formation of a BRD4::NUTM1 fusion gene. Because the BRD4::NUTM1 gene is unequivocally cytotoxic when ectopically expressed in cell lines, questions remain on whether the fusion gene can initiate NC. Here, we report the first genetically engineered mouse model (GEMM) for NUT carcinoma that recapitulates the t(15;19) chromosome translocation in mice. We demonstrated that the mouse t(2;17) syntenic chromosome translocation, forming the Brd4::Nutm1 fusion gene, could induce aggressive carcinomas in mice. The tumors present histopathological and molecular features similar to human NC, with an enrichment of undifferentiated cells. Similar to the reports of human NC incidence, Brd4::Nutm1 can induce NC from a broad range of tissues with a strong phenotypical variability. The consistent induction of poorly differentiated carcinoma demonstrated a strong reprogramming activity of BRD4::NUTM1.  The new mouse model provided a critical preclinical model for NC that will lead to better understanding and therapy development for NC.

overall_design: whole genome sequencing (WGS) of two mNC tumors and matched liver tissues as control

first: Dejin

last: Zheng

first: Chenxiang

last: Luo


title: Single-cell Analysis of Adipose Tissues Revealed the Potential Cellular and Molecular Mechanism of Cold and Disease Resistance in Mongolian Cattle

status: Public on Jun 02 2024

city: Taigu

first: Ruiwen

last: Fan

address: Mingxian South Road 1

institute: Shanxi Agricultural University

summary: Adipose tissue plays essential roles in adapting to harsh environmental conditions in  Mongolian cattle. To dig out the cellular and molecular mechanism of adipose tissue taking part in the tolerance of rough feeding, cold temperature and disease of Mongolian cattle, here, we applied single-nucleus RNA-seq to map the landscape of cell types and profiles of transcriptome of adipose tissues at different anatomical depots at single-nucleus resolution. The entire adipogenic trajectory from preadipocyte commitment to mature adipocytes was analyzed. The results showed that the existence of different adipocyte subpopulations at subcutaneous, perirenal and greater omentum for which acting as a storage depot for body energy to protect vital organs and to insulate the body against heat loss.The interaction between macrophages and adipocytes  and as an endocrine and immunologically active organ to regulate systemic energy and metabolism homeostasis and to modulate T cell activity and subsequent adipose tissue inflammation through antigen presentation of adipocytes to T cells.The data provide a powerful resource for future hypothesis-driven investigations of the mechanisms of adipocyte differentiation and adipose tissue plasticity.

overall_design: Adipose tissues at greaer omental, subcutaneous, perirenal depots of Mongolian cattle were collected. The cells and cell subtypes are identified busing snRNA-seq.

first: Muren

last: Herrid

first: Zhiduan

last: Chi

first: Hongrui

last: Ren


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [ChIP-Seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Murine ICC (2205) cells were treated for 10 days with either DMSO or 1 μM mIDH inhibitor (AG120). Where indicated, 20ng/mL IFNγ was added for the final two days of culturing. 20 million cells per replicate for each condition (DMSO, AG120, IFNγ, AG120 + IFNγ) were cross-linked using 1% formaldehyde for 10 minutes at room temperature and quenched using glycine for 5 minutes. ChIP experiments were performed using SimpleChIP Enzymatic Chromatin IP Kit based on manufacturer's protocol. Sequencing libraries were prepared using the NEBNext Ultra II DNA library preparation kit (New England Biolabs) and were sequenced using NovaSeq 6000 (Illumina) with paired-end 150 bp reads.

first: Hiroyuki

last: Kato


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [mouse_vivo_tet2_rnaseq]

overall_design: Immunocompetent mice harboring subcutaneous allograft tumors generated with murine CKIR132C ICC cells (2205-sgCtrl, -sgTet2) were treated (~100 mm^3 starting volume) with vehicle or AG120 (150 mg/kg; twice daily), and tumors were harvested at serial time points (Vehcle, AG120-Day3 and AG120-Day 6). sgCtrl and sgTet2 CKIR132C ICC cells were isolated by magnetic bead–mediated depletion of stromal populations from tumors treated with vehicle or AG120 for 3 or 6 days.


title: Impact of Pclaf/Paf knock-out on murine lung regeneration (scRNA-seq)

status: Public on May 31 2024

city: Houston

first: Bongjun

last: Kim

state: Texas

address: 6565 MD Anderson BLVD

department: Experimental Radiation Oncology

institute: MD Anderson Cancer Center

summary: The spatiotemporal orchestration of lung stem/progenitor cells is essential for lung regeneration, of which failure leads to lung disease, including fibrosis. We sought to identify mechanisms controlling lung stem/progenitor cells during lung regeneration. We previously found that PCLAF/PAF/KIAA0101 remodels the DREAM complex for cell quiescence exit and cell proliferation. PCLAF is expressed explicitly in the pulmonary proliferative cells (PPC), along with the DREAM target genes. Pclaf expression and Pclaf-expressing cells were acutely increased upon lung injury. Intriguingly, Pclaf knock-out mice exhibited lung fibrosis with impaired alveolar regeneration, resulting from the loss of alveolar type I cells. The single-cell transcriptome and organoid analyses showed that Pclaf transactivated the DREAM target gene expression for alveolar type I cells repopulation from alveolar type 2 cells. Additionally, pharmacological mimicking of the Pclaf-mediated transcriptome markedly increased alveolar regeneration in vitro and in vivo, without neoplasia. Together, our study unveiled the pivotal roles of the PCLAF-DREAM axis in controlling stem/progenitor cell activation and cell lineage, further proposing the potentially viable option for lung regenerative medicine.

overall_design: After 1 week of bleomycin (1.35 U/kg) insitllation into Pclaf WT and Pclaf KO mice, lung epithelial cells (Cd31-/Cd45-/Epcam+) were isolated by FACS and subjected to generate single-cell RNA library.

first: Yuanjian

last: Hwang

first: Kyung-Pil

last: Ko

first: Jae-Il

last: Park


title: Affymetrix SNP array data for T-cell acute lymphoblastic leukemia samples

status: Public on May 26 2024

city: Lublin

first: Borys

last: Styka

state: Lubelskie

address: ul. Prof. Antoniego Gębali 6

department: Genetic diagnostics department

country: Poland

institute: University Children's Hospital in Lublin

summary: Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved diagnostic bone marrow samples.

overall_design: Copy number analysis of Affymetrix CytoScanHD SNP arrays was performed for 172 pediatric T-ALL samples.

first: Monika

last: Lejman

first: Agata

last: Pastorczak


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [human_cgas_AZT_rnaseq]

status: Public on Apr 28 2024

first: Robert

last: Manguso

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Human ICC cells (SNU1079 sgControl or sgcGAS) were treated for five days with either DMSO or 1 μM mIDH inhibitor (AG120) with or without Zidovudine/AZT. Where indicated, 10ng/mL IFNγ was added for the final two days of culturing. Cells were collected for RNAseq on the fifth day.

first: Meng

last: Wu

first: Omar

middle: I

last: Avila

first: Ashwin

last: Kammula


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [mouse WGBS]

type: Methylation profiling by high throughput sequencing

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Murine ICC (2205) cells were treated for five days with either DMSO or 1 μM mIDH inhibitor (AG120). Where indicated, 10ng/mL IFNγ was added for the final two days of culturing. Cells were collected for WGBS on the fifth day. The normal livers were from vehicle- or AG120-treated mice for 6 days.

first: Nabeel

last: Bardeesy


title: Multiple direct and indirect roles of the Paf1 Complex in elongation, splicing, and histone post-translational modifications [ChIP-seq]

first: Karen

last: Arndt

sample_organism:
Saccharomyces cerevisiae
Kluyveromyces lactis

overall_design: ChIP sequencing of RNAPII localization upon long and short term inactivaiton of the Paf1 complex

first: Alex

last: Francette


title: Single-cell RNA sequencing of healthy mouse colon and mouse colon with acute or chronic colitis induced by DSS

status: Public on May 08 2024

city: Yongin-si

first: Seokhyun

last: Yoon

state: Gyeonggi-do

address: Dept of Electronics and Electrical Eng.,, 152 Jukjeon-ro, Suji-gu

department: Electronics and Electrical Eng.

country: South Korea

institute: Dankook University

summary: In this study, we attempted to dissect the dynamic changes during inflammation using well-prepared scRNA-seq dataset. Specifically, we tried to characterize the pathology and the biological mechanism underlying the ulcerative colitis separately for the acute and chronic colitis. We observed a significant reduction in epithelial populations during acute colitis, indicating tissue damage, with a partial recovery observed in chronic inflammation. Analyses of cell-cell interactions demonstrated shifts in networking patterns among different cell types during disease progression. Notably, macrophage phenotypes exhibited diversity, with a pronounced polarization towards the pro-inflammatory M1 phenotype in chronic condition, suggesting the role of macrophage heterogeneity in disease progression. Analysis of the intestinal microbiome revealed significant alterations in composition and metabolism pathways, particularly the nicotinamide pathway. Additionally, dysbiosis was linked to dysregulation of NAD homeostasis through NAMPT, providing insights into potential therapeutic strategies. The study also highlighted the role of T cell differentiation in the context of dysbiosis and its implications in colitis progression, emphasizing the need for targeted interventions to modulate the inflammatory response and immune balance in colitis.

overall_design: We conducted single-cell RNA sequencing (scRNA-Seq) using Chromium (10X Genomics) on samples from 3 healthy mouse colons, 3 mouse colons with acute colitis induced by DSS, and 4 mouse colons with chronic colitis induced by DSS.

first: Dawon

last: Hong

first: Hyo Keun

first: Wonhee

last: Yang

first: Chanjin

first: Minsoo

first: CHul-Su


title: miRNA seq mouse liver HFD, PCB126, Aroclor1260 exposure

status: Public on Apr 23 2024

city: Louisville

first: Eric

middle: Christian

last: Rouchka

state: Kentucky

address: 522 East Gray Street

department: Biochemistry and Molecular Genetics

institute: University of Louisville

type: Non-coding RNA profiling by high throughput sequencing

summary: Exposure to high fat diet (HFD) and persistent organic pollutants including polychlorinated biphenyls (PCBs) is associated with liver injury in human populations and with non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in animal models. Exposure of HFD-fed male mice to the non-dioxin-like (NDL) PCB mixture Aroclor1260 or to dioxin-like (DL) PCB126 or to the combination caused steatohepatitis and differentially altered the liver proteome with pathways involving epigenetic regulation of gene expression. Here unbiased RNA sequencing of miRNA (miRNA-seq) and subsequent network analysis to characterize the biological pathways altered by HFD and PCB exposure compared to HFD alone. Distinct miRNA expression patterns reveald a potential role of miRNAs in the pathogenesis of NAFLD. These results demonstrate miRNA and transcriptome pathways in PCB-related hepatic inflammation and fibrosis in a mouse model of NAFLD.

overall_design: Male C57Bl/6J mice were fed a high fat diet (HFD, 42% kCal from fat) for 12 weeks and exposed to a single oral gavage of vehicle control (corn oil), Aroclor1260 (20 mg/kg), PCB126 (20 µg/kg), or the combination of the doses of Aroclor1260 + PCB126. miRNA was isolated from five mouse livers/experimental exposure using Qiagen miRNA kits. Libraries were prepared from 1 µg of mouse liver RNA the QIAseq miRNA Library Kit. Two sequencing runs were performed on the Illumina NextSeq 500 using the NextSeq 500/550 High Output Kit v2.5 (75 cycles).

first: Belinda

last: Petri

first: Kellianne

last: Piell

first: Bandrida

last: Wahlang

first: Kimberly

middle: Z

last: Head

first: Kalina

last: Andreeva

middle: C

first: Jianmin

last: Pan

first: Shesh

middle: N

last: Rai

first: Matthew

last: Cave

first: Carolyn

last: Klinge


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [mouse_ICC_rnaseq]

first: Robert

last: Manguso

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Murine ICC (2205) cells were treated for five days with either DMSO or 1 μM mIDH inhibitor (AG120). Where indicated, 10ng/mL IFNγ was added for the final two days of culturing. Cells were collected for RNAseq on the fifth day.

first: Meng

last: Wu

first: Ashwin

last: Kammula

first: Nabeel


title: RYBP coordinates with Trithorax complex and selectively recruits Polycomb complex for cell fate control

status: Public on Apr 26 2024

city: Guangzhou

first: Chao

last: Wei

address: No.74 Zhongshan Rd.2

institute: Sun Yat-Sen University

summary: The selective expression is pivotal in orchestrating human development, with the Trithorax Group (TrxG) and Polycomb Group (PcG) complexes playing crucial roles in transcriptional activation and repression, respectively. However, mechanism underlying selective regulation of transcription by TrxG and PcG remains poorly understood. In this study, RYBP was observed to interact with TrxG and PcG components. RYBP and TrxG co-localized loci selectively enriches RING1B. STAT3 enriches at RYBP-TrxG co-localized loci devoid of RING1B, while displaying minimal enrichment at RING1B-enrichred loci. Introduction of STAT3 at RYBP loci disrupted RING1B aggregation and chromatin binding. RYBP-deficiency impairs TrxG deposition at DNA repair genes in RYBP-TrxG loci, consequently diminishing their expression and inducing DNA damage. These results facilitate the transition of embryonic stem cells to 2-cell-like cells. Additionally, RYBP-deficiency attenuates PcG deposition at lineage-specific genes within RYBP-TrxG-RING1B loci, thereby promoting ESC differentiation. Collectively, these results provide novel insights into the selective regulation of gene expression.

overall_design: RYBP KO for ChIP seq and RNA-seq

first: Jun

last: Sun

first: Mulan

last: Wang

first: Zhuoyan

last: Liu

first: Jin

last: Tan

first: Xiaona

last: Huang

first: Ranran

last: Dai

first: Junjun

last: Ding


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [RRBS]

overall_design: We used reduced-representation bisulfite sequencing (RRBS) of a set of human ICC cell lines to investigate DNA methylation status on the promoter region of interested genes in mIDH1 model tested (N=3 cell lines), as compared with all IDH1 WT models (N=12 cell lines)

first: Jonathan

last: Sussman

last: Bardeesy


title: A glial circadian gene expression atlas reveals cell type and disease-specific reprogramming in response to amyloid pathology or aging

status: Public on Jun 26 2024

city: Worcester

first: Patrick

last: Sheehan

address: 364 Plantation Ave.

department: Neurobiology

institute: University of Massachusetts Medical School

summary: While circadian rhythm disruption may promote neurodegenerative disease, how aging and neurodegenerative pathology impact circadian gene expression patterns in different brain cell types is unknown. Here, we used translating ribosome affinity purification methods to define the circadian translatomes of astrocytes, microglia, and bulk cerebral cortex, in healthy mouse brain and in the settings of amyloid-beta plaque pathology or aging. Our data reveal that glial circadian translatomes are highly cell type-specific and exhibit profound, context-dependent reprogramming of rhythmic transcripts in response to amyloid pathology or aging. Transcripts involved in glial activation, immunometabolism, and proteostasis, as well as nearly half of all Alzheimer Disease (AD)-associated risk genes, displayed circadian oscillations, many of which were altered by pathology. Amyloid-related differential gene expression was also dependent on time of day. Thus, circadian rhythms in gene expression are cell- and context dependent and provide important insights into glial gene regulation in health, AD, and aging.

overall_design: Mice were sacrificed at specified time points and perfused with cycloheximide.  Cortex was immediatley isolated and tissue frozen. After tissue processing, astrocyte- or microglia-specific RNA were isolated by incubating tissue with anti-GFP or anti-HA coated beads, respectively.  Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 10ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.RNA-seq reads were then aligned and quantitated to the Ensembl release 101 primary assembly with an Illumina DRAGEN Bio-IT on-premise server running version 3.9.3-8 software.

middle: W

first: Erik

last: Musiek


title: M6A reduction relieves FUS-associated ALS granules

first: Irene

last: Bozzoni

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

overall_design: Refer to individual Series

first: Gaia

last: Di Timoteo

first: Andrea

last: Giuliani

first: Adriano

last: Setti


title: KMT2A-driven cyclin D2 upregulation and chronic inflammation as potential drivers of sporadic parathyroid adenoma pathogenesis

city: Callaghan

first: Ting

last: La

state: NSW

address: University Drive

country: Australia

institute: UON

summary: Sporadic parathyroid adenoma (PA) is the most common cause of hyperparathyroidism, but the mechanisms involved in its pathogenesis remain incompletely understood. Here we present a single-cell transcriptomic atlas detailing the cellular differences between human PA and normal parathyroid gland (PG) tissues and delineating the transcriptome of individual cell types. We show that there is a pervasive increase in gene transcription in PA cells (PACs) compared with PG cells (PGCs), with transcriptional upregulation of cyclin D2 driven by the transcriptional coactivator, histone-lysine N-methyltransferase 2A (KMT2A) through the transcription factors signal transducer and activator of transcription 3 (STAT3) and GATA binding protein 3 (GATA3) potentially involved in promoting PAC proliferation. Moreover, we demonstrate that PA tissues are heavily infiltrated with myeloid cells, and that fibroblasts, endothelial cells (ECs), as well as macrophages in the PA microenvironment are commonly enriched with proinflammatory gene signatures relative to their counterparts in PG tissues. Collectively, these results provide new insights into the etiology of PA, where the pathogenesis likely involves the net contribution of the dysregulated KMT2A-STAT3/GATA3-cyclin D2 axis in PACs and the chronic inflammation of the microenvironment. These findings provide practical implications for the treatment of PA through KMT2A targeting and anti-inflammation therapies.

overall_design:
Five parathyroid glands of patients with sporadic parathyroid adenoma and three parathyroid glands of patients with Thyroid cancer

NOTE: Processed data file GSM8016804_ctrl1-matrix.mtx.gz is corrupt. The submitter has been contacted.

first: Qin

last: Xu

first: Li

first: Xu

last: Zhang

first: Feng-Min

last: Shao

first: HuiXia

last: Cao


title: High CD44 expression identifies a quiescent/chemoresistant leukemic population with enhanced E-Selectin binding in T-ALL

city: Fontenay-aux-Roses

first: Julien

last: Calvo

address: 18, route du panorama

department: Institut de Radiobiologie Cellulaire et Moléculaire

country: France

institute: Commissariat à l'Energie Atomique

summary: We used single cell RNA sequencing (scRNA-seq) to decipher cell heterogeneity of  human T-cell Acute Lymphoblastic Leukemia (M18) recovered from Bone Marrow Adipose Tissue (BMAT)-poor (Femur and Thoracic Vertebrae) and -rich (Tail Vertebrae) sites of xenografted immunodeficient mice (NSG,nonobese diabetic/severe combined immunodeficiency / interleukin-2Rγ null). We wanted to know whether some human T-ALL cells recovered in BMAT-poor sites resemble to those from BMAT-rich site.

overall_design: hT-ALL cells recovered from Femur, Thoracic and Tail Vertebrae of xenograft immunodeficient mice were purified according to human CD45 expression through immuno magnetic selection and analyzed using scRNAseq.

first: Françoise

last: Pflumio


title: CDK12 Loss Promotes Prostate Cancer Development While Exposing Vulnerabilities to Paralog-Based Synthetic Lethality [scRNA-Seq]

status: Public on Apr 17 2024

city: Ann Arbor

first: Arul

middle: M.

last: Chinnaiyan

state: MICHIGAN

address: 1500 E. Medical Center Dr

institute: University of Michigan

summary: Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a unique molecular subtype of metastatic castration resistant prostate cancer (mCRPC).  It remains unclear, however, whether CDK12 loss per se is sufficient to drive prostate cancer development—either alone, or in the context of other genetic alterations—and whether CDK12-mutant tumors exhibit sensitivity to specific pharmacotherapies. Here, we demonstrate that tissue-specific Cdk12 ablation is sufficient to induce preneoplastic lesions in the mouse prostate. Allograft-based CRISPR screening demonstrated that Cdk12 loss is positively associated with p53 inactivation, but negatively associated with Pten inactivation—similar to what is seen in human mCRPC. Consistent with this, ablation of Cdk12 in prostate organoids with concurrent p53 loss promotes their proliferation and ability to form tumors in mice, while Cdk12 knockout in the Pten-null prostate cancer mouse model abrogates tumor growth. Bigenic CDK12 and p53 loss allografts represent a new syngeneic model for the study of prostate cancer.  Cdk12-null organoids (either with or without p53 co-ablation) and patient-derived xenografts from tumors with CDK12 inactivation are highly sensitive to inhibition or degradation of its paralog kinase, CDK13. Together, these data identify CDK12 as a bona fide tumor suppressor gene with impact on tumor progression and paralog-based synthetic lethality is a promising strategy for treating CDK12 mutant mCRPC.

overall_design:
To investigate role of CDK12 in prostate cancer development, we established Cdk12 KO vs WT mouse prostate organoids from GEMM model and conducted scRNA on the organoids.
We also performed scRNA sequencing  on mouse prostate tissues on Cdk12 KO mice and compared gene profiling to WT mice

first: Jean

last: Tien

first: Yuping

first: Yu

last: Chang

first: Yunhui

last: Cheng

first: Rahul

last: Mannan

first: Palak

last: Shan

first: Xiaoming

last: Wang

first: Sanjana

last: Eyunni

first: Caleb

first: Brian

last: Magnuson

first: Lanbo

last: Xiao

first: Marcin

last: Cieslik

first: Abhijit

last: Parolia

first: Yi

last: Bao

first: Shuqin

last: Li

first: Alice

first: Fengyun

last: Su

first: Xuhong

first: Yi-Mi

last: Wu

first: Dan

last: Robinson

first: George

first: Ke


title: RNA sequencing analysis of gene expresssion profiles in Cic-null MZB cells

status: Public on Apr 29 2024

city: Pohang-Si

first: Jongseok

last: Park

address: Cheongam-Ro, 77

department: Department of Life Science

institute: POSTECH

summary: Purpose: The goal of this study is to compare and examine the transcriptional profiles in Cd19-Cre (control) versus Cicf/f;Cd19-Cre (Cic-null) MZB cells,by mRNA sequencing. Methods: MZB cells (CD19+CD93-CD21+CD23-) were sorted by a MoFlo-Astrios (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. . A library for mRNA sequencing was prepared using the Truseq Stranded mRNA/Total Library Prep Kit (Illumina) according to the manufacturer’s instructions. Sequencing was performed with Novaseq 6000 (Illumina). Results: Tophat (v2.0.13) was used to map reads for each sample to the mm10 RefSeq reference genome. The aligned results were then added to Cuffdiff (v2.2.0) to identify the differentially expressed genes (DEGs).  In total, 775 genes were differentially expressed in Cic-null MZB cells (505 upregulated and 270 downregulated) compared to control cells (Log2 fold-change > 0.5 and P < 0.01). Conclusions: Our study presents the first comparative gene expression analysis of MZB cells from control and B cell-specific Cic-deficient mice. We concluded that CIC deficiency attenuates MZB cell development through downregulation of the NOTCH signaling pathway. The data reported here also provide references for the gene expression profiles of murine MZB cells through TPM.

overall_design: mRNA profiles of MZB cells from the spleen of Cd19-Cre (control) and Cicf/f;Cd19-Cre (Cic-null) mice. Two replicates for each genotypes were anaylzed.

first: Yoontae

last: Lee


title: Response to mRNA vaccine BNT162b2 in hemodialysis patients

status: Public on Apr 22 2024

city: Chicago

first: Kai

last: Huang

state: IL

address: 909 S. Wolcott Ave COMRB Room 3060

department: Medicine

institute: University of Illinois at Chicago

summary: End-stage renal disease patients experience uremia-driven immune compromise  characterized by complex alterations of both innate and adaptive immunity, and results in higher  susceptibility to infection and lower response to vaccination. This immune compromise, coupled with  greater risk of exposure to infectious disease at hemodialysis (HD) centers, motivates an examination of  immune response to the COVID-19 mRNA-based BTN162b2 vaccine. We performed gene expression profiling by RNA-seq across 6 time points to assess vaccine response in healthy controls and hemodialysis patients over time.

overall_design: mRNA extracted from peripheral blood mononuclear cells of healthy controls and hemodialysis patients receiving the BTN162b2 vaccine were sequenced and compared. We assessed differences in gene expression across time points between and within the two groups.

first: Yishin

last: Chang

first: Jessica

first: Christen

middle: L

last: Vagts

first: Chrstian

last: Ascoli

first: Md-Ruhul

last: Amin

first: Mahmood

last: Ghassemi

first: Claudia

last: Lora

first: Russell

last: Edafetanure-Ibeh

first: Yue

first: Ruth

middle: A

last: Cherian

first: Nandini

last: Sarup

first: Samantha

middle: R

last: Warpecha

first: Sunghyun

last: Hwang

first: Rhea

last: Goel

first: Benjamin

last: Turturice

first: Cody

last: Schott

first: Montserrat

last: Hernandez

first: Yang

last: Chen

first: Julianne

last: Jorgensen

first: Wangfei

first: Mladen

last: Rasic

first: Richard

last: Novak

first: Patricia

last: Finn

first: David

last: Perkins


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [human_glioma_rnaseq]

first: Robert

last: Manguso

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Human glioma (MGG152) cells were treated for five days with either DMSO or 1 μM mIDH inhibitor (AG120). Where indicated, 10ng/mL IFNγ was added for the final two days of culturing. Cells were collected for RNAseq on the fifth day.

first: Meng

last: Wu

first: Ashwin

last: Kammula

first: Nabeel

last: Bardeesy


title: CDK12 Loss Promotes Prostate Cancer Development While Exposing Vulnerabilities to Paralog-Based Synthetic Lethality [RNA-Seq]

first: Arul

last: Chinnaiyan

overall_design:
To investigate role of CDK12 in prostate cancer development, we established Cdk12 KO vs WT mouse prostate organoids from GEMM model
We then performed gene expression analysis using RNA-seq of Cdk12 KO vs WT organoid line
We also knockout sgP53 using CRISPR technology in Cdk12 KO vs WT organoids and compaired gene expression profiling from 4 different organoid lines (WT-sgNT, WT-sgP53, CDK12 KO-sgNT, CDK12 KO-sgP53)

first: Jean

last: Tien

first: Yuping

last: Zhang

first: Yu

last: Chang

first: Yunhui

last: Cheng

first: Rahul

last: Mannan

first: Palak

last: Shan

first: Xiaoming

last: Wang

first: Sanjana

last: Eyunni

first: Caleb

first: Brian

last: Magnuson

first: Lanbo

last: Xiao

first: Marcin

last: Cieslik

first: Abhijit

last: Parolia

first: Yi

last: Bao

first: Shuqin

last: Li

first: Alice

last: Xu

first: Fengyun

last: Su

first: Xuhong

last: Cao

first: Yi-Mi

first: Dan

last: Robinson

first: George

first: Ke

last: Ding

middle: M


title: PSAT1 mediated changes in gene expression in EGF-stimulated lung cancer

first: Eric

last: Rouchka

summary: In addition to supporting increased cellular metabolism for untethered neoplastic growth, recent reports have indicated that certain metabolic enzymes may exhibit alternative functions in human cancers. Despite the ability to import serine from the microenvironment, lung tumors significantly elevate expression of biosynthetic enzymes responsible for the de novo production of serine, notably phosphoserine aminotransferase 1 (PSAT1). Highlighting the relevance of PSAT1 in lung cancer, patients with increased expression demonstrated poorer overall survival, so characterizing its role in lung tumor progression may ultimately identify different therapeutic strategies targeting this disease. Our preliminary studies now demonstrate a potential alternative function, beyond serine synthesis, of PSAT1 in lung tumor cells. Specifically, we found that PSAT1 localizes to the nucleus upon epidermal growth factor stimulation [via activating EGFR mutation (PC9, inhibited by the EGFR-mutant specific inhibitor, Erlotinib) or EGF stimulation (A549, EGFR-WT)] and regulates the nuclear import of pyruvate kinase M2 (PKM2). Importantly, nuclear PKM2 has been demonstrated to directly phosphorylate histones and modify gene transcription. In further support of this functional interaction, within yeast, PKM2 association with serine synthetic enzymes resulted in pervasive chromatin remodeling and alteration in global gene expression. Therefore, our objective in this proposal is to define gene signatures promoted by PSAT1-mediated nuclear PKM2 translocation in response to EGF-signaling in human lung cancer cells.

overall_design: In pursuit of this objective, we have stably-silenced PSAT1 expression in PC9 human lung cancer lines for comparison to control cells.  In total, we have two distinct sample groups with 3 replicates each (PC9: Control and PSAT1 shRNA).

last: Clem

first: Rumeysa

last: Biyik-Sit

first: Kalina

last: Andreeva


title: Genome-wide profiling of DNA repair proteins in single cells

first: Kim

last: de Luca

summary:
Accurate repair of DNA damage is critical for maintenance of genomic integrity and cellular viability. Because damage occurs non-uniformly across the genome, single-cell resolution is required for proper interrogation, but sensitive detection has remained challenging. Here, we present a comprehensive analysis of repair protein localization in single cells using DamID and ChIC sequencing techniques. This study reports genome-wide binding profiles in response to DNA double-strand breaks induced by AsiSI, and explores variability in genomic damage locations and associated repair features in the context of spatial genome organization.
By unbiasedly detecting repair factor localization, we find that repair proteins often occupy entire topologically associating domains, mimicking variability in chromatin loop anchoring. Moreover, we demonstrate the formation of multi-way chromatin hubs in response to DNA damage. Notably, larger hubs show increased coordination of repair protein binding, suggesting a preference for cooperative repair mechanisms. Together, our work offers new insights into the heterogeneous processes underlying genome stability in single cells.

overall_design:
Genome-wide profiling of DNA repair protein binding/occupancy by high-throughput sequencing (using DamID and ChIC), characterizing the response to sequence-specific double-strand break induction in U2OS cells.
Single-molecule chromatin conformation profiling by Tri-C and SPRITE.


title: Effect of piR-1254 on the differential expression in Neuroblastomacell lines

status: Public on Apr 07 2024

city: Regensburg

first: Tilman

last: Heise

address: Franz Josef Strauss Allee 11

country: Germany

institute: Uniklinik Regensburg

summary: Small non-coding RNAs (sncRNAs), including microRNAs and piRNAs, play crucial roles in regulating gene expression in cancer, influencing key processes such as cell proliferation, apoptosis, and metastasis. This also includes piR-1254, a 32 nucleotides long non-coding RNA. Here it is shown that piR-1254 has a broad influence on the expression of more than 2000 different genes. The expression of half of the genes is reduced and that of the other half is increased.

overall_design: To investigate the influence of piR-1254 on differential expression in the Neuroblastoma cell line Kelly, the cells were reverse transient transfected with piR-1254 or piR-Ctrl as control. 48 h after transfection, the RNA was harvested and a total RNA-seq was performed.

first: Marina

last: Sauer

first: Phillipp

last: Torkler

first: Gunhild

last: Sommer


title: Defining the Inflammation and Immunity Transcriptome in Severe Malarial Anemia for Immunotherapeutic Discovery

status: Public on Apr 16 2024

city: Albuquerque

first: Douglas

middle: Jay

last: Perkins

state: NM

address: MSC10 5550, 1 University of New Mexico

department: Center for Global Health

institute: University of New Mexico

summary: Malaria continues to pose a significant public health threat, with millions of cases and hundreds of thousands of deaths reported annually, primarily in sub-Saharan Africa. The disease disproportionally affects children under five years of age residing in holoendemic Plasmodium falciparum transmission regions, who account for 94% of the cases and 80% of the mortality. Young children are highly vulnerable to developing life-threatening severe malarial anemia [SMA, hemoglobin (Hb)<5.0 g/dL]. The overall goal of the project was to identify critical gene pathways within the transcriptome that mediate disease severity and then target these specific genes with compounds that elicit expression profiles witnessed in children with milder forms of disease. To achieve this goal, we are investing the following specific aims: 1) Determine how changing temporal dynamics of gene pathways in the Malarial Immunity Transcriptome promote SMA during acute disease; 2) Determine how changes in gene pathways in the Malarial Immunity Transcriptome mediate malarial severity throughout the development of naturally acquired immunity; and 3) Identify immunotherapeutic targets in the Malarial Immunity Transcriptome that can be used to reduce malaria disease severity and improve clinical outcomes in future trials. To successfully complete these aims, we are determining how host profiles impact on acute disease over 14 days.  We will utilize Ex vivo samples from the cohort to test the effect of immunotherapeutic compounds on host expression profiles. Accomplishing these goals will have broad reaching translational implications for: (1) identifying at-risk groups, and (2) prioritizing compounds that can be used to improve clinical outcomes in future immunotherapy trials.

overall_design:
To compare transcriptomic profiles and immune cell responses between children with SMA and non-SMA, we analyzed the entire expressed peripheral blood transcriptome of children aged, 0-59 months.
Children with P. falciparum infections (any density parasitemia) were stratified into two groups based on Hb concentrations: Hb≥6.0 g/dL (non-SMA, n=41), and Hb<6.0 g/dL (SMA, n=25).
Children with any detectable co-infections , namely HIV-1, and laboratory confirmed blood-borne bacterial cultures were omitted from the analysis. Children with hemoglobinopathies (Glucose-6-Phosphate Dehyrogrenase deficiency [G6PD], Sickle cell trait [HbAS] and Sickle cell disease [HbSS]) were included in the analysis.

first: Qiuying

last: Cheng

first: Ivy

last: Hurwitz

first: Christophe

middle: G

last: Lambert

first: Philip

last: Seidenberg

first: Kristan

last: Schneider

first: Benjamin

last: McMahon

first: Nick

last: Hengartner

first: Harshini

last: Mukundan

first: Collins

last: Ouma

first: Evans

last: Raballah

first: Samuel

middle: B

last: Anyona

first: Clinton

middle: O

last: Onyango

first: Anne

last: Ong'ondo

first: Nicholas

last: Ondiek

first: Everlyne

last: Modi

first: Joan

last: Ochieng

first: Vincent

last: Omanje

first: Rodney

last: Mongare

first: Sharley

last: Wasena

first: Shamim

last: Osata

first: Ananias

last: Escalante

first: Maria

last: Pacheco


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [human WGBS]

first: Robert

middle: T

last: Manguso

summary: Isocitrate Dehydrogenase 1 (IDH1) is the most commonly mutated metabolic gene across human cancers. Mutant IDH1 (mIDH1) generates the oncometabolite (R)-2-hydroxyglutarate, disrupting enzymes involved in epigenetics and other processes. A hallmark of IDH1-mutant solid tumors is T cell exclusion, whereas mIDH1 inhibition in preclinical models restores anti-tumor immunity. Here, we define a cell-autonomous mechanism of mIDH1-driven immune evasion. IDH1-mutant solid tumors show striking, selective hypermethylation and silencing of the cytoplasmic dsDNA sensor, CGAS, compromising innate immune signaling. mIDH1 inhibition restores DNA demethylation, derepressing CGAS and transposable element (TE) subclasses. dsDNA produced by TE-reverse transcriptase activates cGAS, triggering viral mimicry and stimulating anti-tumor immunity. Thus, we demonstrate that mIDH1 epigenetically suppresses innate immunity and link endogenous reverse transcriptase activity to the mechanism of action of an FDA-approved oncology drug.

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Human ICC (SNU1079) cells were treated for five days with either DMSO or 1 μM mIDH inhibitor (AG120). Where indicated, 10ng/mL IFNγ was added for the final two days of culturing. Cells were collected for WGBS on the fifth day.

first: Meng-Ju

last: Wu

first: Ashwin

last: Kammula

first: Nabeel

last: Bardeesy


title: CDK12 Loss Promotes Prostate Cancer Development While Exposing Vulnerabilities to Paralog-Based Synthetic Lethality

first: Arul

last: Chinnaiyan


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [mouse_glioma_rnaseq]

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Murine glioma (PIC496-3) spheroid cells were treated for five days with either DMSO or 1 μM mIDH inhibitor (AG120). Where indicated, 10ng/mL IFNγ was added for the final two days of culturing. Cells were collected for RNAseq on the fifth day.

first: Meng

middle: J

middle: V


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Single cell RNA sequencing immune cells (macrophages, CD4 T and B cells) from male and female mice, at 0,1,2,3 hours after IFNa injection.

status: Public on Apr 01 2024

city: Boston

first: CBDM

last: Lab

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: Single cell RNA sequencing dataset collected in two independent experiments, including ~24,061 cells (reffered as Dataset A in the paper), and ~13,995 cells (reffered as Dataset B in the paper). Datasets include male and female mice, in 4 consecutive time points - 0h (no IFN, PBS), and 1,2,3 h after IFNa administration. Single cell RNA-sequencing was performed on the 10X platform.

overall_design: Data was collected in two independent experiments (Datasets A and B) for a total of 8 male and 7 female mice. In each experiment, C57BL/6J five-week-old male and female mice injected i.v. with IFNα or 100 μL PBS. After 1 h, two mice of each sex (one from the IFN group and one from the PBS group) were sacrificed. After 2 and 3 h, one mouse of each sex in the IFN group was sacrificed. *Mice in Dataset A were littermates; Dataset B did not include a female mouse at 2 h. Hashtag oligos (HTOs) were used to distinguish between mice of different sexes, injection types and time points. From each mouse, CD4 T and B cells from the spleen and macrophages from the peritoneal cavity were double sorted before sequencing. Single cell RNA-sequencing was performed on the 10X platform.

first: Shani

last: Gal Oz

first: Alev

last: Baysoy

first: Brinda

last: Vijaykumar

first: Sara

last: Mostafavi

first: Christophe

last: Benoist

first: Tal

last: Shay


title: Identification and removal of unexpected proliferative off-target cells emerging after implantation of iPSC-derived pancreatic islet cells

city: Fujisawa

first: Hideyuki

last: Hiyoshi

state: Kanagawa

address: 26-1, Muraoka-Higashi 2-Chome

country: Japan

institute: Takeda Pharmaceutical Company Limited

summary: The differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSC) has been thoroughly investigated for their application in cell therapy against diabetes. However, there is little information available on the long-term safety of these differentiated cells. Here we show that implantation of induced PSC-derived pancreatic islet cells (iPIC) results in the appearance of unexpected proliferative off-target cells via in vivo maturation. Since the unexpected cells have characteristics of both mesenchymal stem cells (MSC) and uterine tissue, we named these cells proliferative MSC and uterus like cells (PMUC). To detect PMUC without implantation, we developed four-week-extended culture system and found that PMUC can be reduced by compound treatment, especially a taxane Docetaxel. Indeed, when we implanted Docetaxel-treated iPIC, the appearance of PMUC disappeared. The present study provides useful insights into identifying and resolving safety issues which is particularly important in the field of cell-based medicine using PSC.

overall_design:
Single-cell mRNA sequencing of cadaveric human islet, and pluripotent stem cells differentiating in vitro towards pancreatic islet like cells.

***Raw data was returned to privacy due to patient privacy concerns***

first: Kensuke

last: Sakuma

first: Shinya

last: Asano

first: Hirokazu

last: Matsumoto

first: Ryo

last: Ito

first: Taro

last: Toyoda


title: Tissue-specific immune transcriptional signatures in the bordering tissues of the mouse brain and retina

city: Bentley

first: Paul

middle: James

last: Whatmore

state: Western Australia

address: Kent St

department: eResearch

institute: Curtin University

summary: Bordering the central nervous system (CNS) parenchyma are the pia mater enveloping the brain and the choroid underlying the retina. Although in close proximity to the neural parenchyma, the pia mater and choroid are external to the immune privileged environment of the brain and retina and are distinct immune compartments. This study generated a single cell transcriptomic atlas of immune cells within the healthy adult mouse pia and choroid. Comparative analysis of immune populations within these tissues revealed heterogeneous subtypes of T cells, B cells, monocytes/macrophages and dendritic cells, which displayed tissue-specific transcriptomic signatures and potential functional specialisations. Single cell RNA-sequencing of choroidal immune cells from human eye tissue identified populations with similar transcriptional profiles to mouse choroidal immune cells.  This study provides a detailed understanding of the molecular signatures of immune cells within the bordering tissues of the brain and retina, and their potential roles in CNS immune surveillance.

overall_design: Immune cells (CD45+ cells) were isolated from (i) pooled mouse choroid (n=20 C57Bl/6J healthy female mice, 7 weeks of age); (ii) pooled mouse pia mater (n=20 C57Bl/6J healthy female mice, 7 weeks of age); and (iii) human choroid (n=1 tissue donor), by FACS.  Sorted cells were partitioned into single cell droplets using the Chromium 10X Controller. Subsequently, scRNA-seq libraries were prepared using a Chromium single cell reaction 3’ v3.1 kit. Single cell libraries were sequenced on an Illumina NovaSeq S1 instrument, producing paired-end reads.

first: Fazeleh

last: Etebar

first: Damien

last: Harkin

first: Samantha

last: Dando


title: A "living biobank" of Crohn's Disease

status: Public on Mar 31 2024

city: La Jolla

first: Debashis

last: Sahoo

state: California

address: 9500 Gillman Drive

department: Pediatrics

institute: UCSD

summary: Patient-derived organoids (PDOs) from the colons of patients with Crohn's Disease (or healthy controls) were subjected to in-depth genotype, transcriptome and phenotype assessment. These studies revealed that CD may be classified broadly into two molecular subtypes, each with its unique profile of dysregulated celluar processes. We further show that these phenotypes can be rectified with matched therapeutics, hence making such intervention personalized. Findings show that CD-PDOs could serve as pre-clinical platforms for drug discovery and personalized medicine.

overall_design:
Patients representing various subtypes of Crohn's disease (CD) [Montreal classification-B1/2/3, perianal] were enrolled into this study to obtain tissue biopsies which would serve as source of adult stem cells for the generation of patient-derived organoids (PDOs). Standard cell biological, biochemical, functional and phenotypic assays were used to assess the PDOs for: 1) barrier integrity, 2) apoptosis, 3) proliferation, 4) ROS induced DNA/RNA damage; 5) senescence and DNA damage; 6) bacterial clearance, 7) induction of inflammatory cytokines, and morphology/growth characteristics.
Please note that the cell biological, biochemical, and functional assays are not done on these GEO samples prior to RNA analysis, but characterization that is independent of the samples sequenced. The current records are the content of the associated manuscript to validate RNA seq findings.

first: Soumita

last: Das

first: Pradipta

last: Ghosh


title: Epigenome analysis of WHS patients

city: Setagaya

first: Tomoko

last: Kawai

state: Tokyo

department: Department of Maternal-Fetal Biology

institute: National Research Institute for Child Health and Development

type: Methylation profiling by genome tiling array

summary: Genome wide DNA methylation profiling of peripheral blood cells from WHS and several disease patients. The Illumina Infinium EPIC Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 850,000 CpGs in peripheral blood samples. Samples included, 20 WHS, 1 patient with heterozygous deletion in 4p16.2p15.31, 2 Sotos syndrome patients, 2 Kabuki syndrome patients, 1 CHARGE syndrome patient, and  3 patients who harbor NSD2 de novo variants.

overall_design: Bisulphite converted DNA from normal controls and several disease patients were hybridized to the Illumina Infinium EPIC Human DNA methylation Beadchip

first: Kenihiro

last: Hata


title: Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [human_ICC_rnaseq]

city: Cambridge

first: Robert

last: Manguso

address: 75 Ames Street, Rm 7053

institute: Broad Institute of MIT & Harvard

overall_design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Human ICC cells (SNU1079) were treated for five days with either DMSO or 1 μM mIDH inhibitor (AG120). Where indicated, 10ng/mL IFNγ was added for the final two days of culturing. Cells were collected for RNAseq on the fifth day.

first: Meng-Ju

last: Wu

first: Ashwin

last: Kammula

first: Nabeel

last: Bardeesy


title: Impact of Folate Status on the Sperm DNA Methylome of Mice with the 677C>T Variant in 5,10-Methylenetetrahydrofolate Reductase

city: Montreal

first: Jacquetta

last: Trasler

state: Quebec

address: 1001 Decarie Boul, Block E, ES1.4380

department: Pharmacology & Therapeutics; Human Genetics

country: Canada

institute: McGill University

overall_design: Refer to individual Series


title: Chronic viral mimicry induction following p53 loss promotes immune evasion

status: Public on Mar 26 2024

city: Toronto

first: Sajid

last: Marhon

state: ON

address: 101 College Street

institute: Princess Margaret Cancer Centre


title: Mechanisms Underlying Dilated Cardiomyopathy Associated with FKBP12 Deficiency

status: Public on Mar 21 2024

city: Houston

first: Susan

last: Hamilton

state: TX

address: One Baylor Plaza

department: Integrative Physiology

institute: Baylor College of Medicine

summary: To define the role of the immunophilin FKBP12 in cardiac function, two conditional models of FKBP12 deficiency were created using Fkbp1a floxed (FL) mice expressing Cre+ recombinase under the control of different promoters (α-myosin heavy chain, αMHC, and muscle creatine kinase, MCK) that differ in both developmental stage of expression (E9 versus E 13) and Cre+ expression levels.  In adult mice FKBP12 was reduced by 96% and 86% in αMHC-Cre+- and MCK-Cre+-FKBP12 deficient mice, respectively, compared to FL mice. FL mice and mice hemizygous for the αMHC Cre+ recombinase only (to assess potential cytotoxic effects of the Cre+ recombinase) were used as controls. Of these four strains of mice, only the αMHC-Cre+-FKBP12 deficient mice developed an early-onset dilated cardiomyopathy (DCM) and displayed both increased cardiomyocyte sarcoplasmic reticulum Ca2+ leak and large elevations (assessed with RNAseq and western blots) in Ankyrin repeat domain 1 protein (ANKRD1), a negative regulator of cardiac gene expression in response to stress. These data suggest that an FKBP12 deficiency that  begins during cardiac development and reduces FKBP12 below a critical level causes RyR2 mediated Ca2+ leak and ANKRD1 elevation, leading to cardiac structural remodeling and the development of DCM. This Ca2+ leak/ANKRD1 pathway may represent a common and targetable mechanism for DCM.

first: Md Abul Hassan

last: Samee

first: Kevin

middle: S

last: Ho


title: NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis [ChIP-seq]

status: Public on Mar 20 2024

first: Arul

last: Chinnaiyan

address: 1500 East Medical Center Dr

department: MCTP

summary: Androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 is ectopically expressed in prostate cancer cells and catalytic inhibition of NSD2 impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites or greater than 65% of its cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and contain the canonical palindromic motifs instead. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Therapeutically exploiting these insights, we developed a dual NSD1/2 PROTAC degrader, called LLC0150, which was preferentially cytotoxic in AR-dependent prostate cancer and synergized with enzalutamide. In a pan-cancer screen, comprising over 120 cell lines from 22 distinct lineages, NSD1/2 co-degradation triggered apoptotic cell death in AR-addicted prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the AR neo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 paralog co-targeting as a novel and potent therapeutic strategy.

overall_design: Comparative RNA profiles of prostate cancer cells upon CRISPR (stable) or siRNA-mediated (transient) inactivation of genes belonging to the NSD family of histone methylatransferases or treatment with the EZH2 inhibitor EPZ6438 (72h at 2uM).

first: Abhijit

last: Parolia

first: Eleanor

last: Young

first: Irfan

last: Asangani


title: NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis [RNA-seq]

overall_design: Comparative RNA profiles of prostate cancer cells upon CRISPR or siRNA-mediated inactivation of NSD1, NSD2, and/or NSD3 genes, treatment with the EZH2 inhibitor EPZ6438 (72h at 2uM), or treatment with NSD1/2 PROTAC degrader LLC0150 (2um for 24h).


title: Transcriptional analysis of placenta samples exposed to maternal smoking during pregnancy

status: Public on Mar 07 2024

city: Beaverton

first: Lyndsey

middle: Elizabeth

last: Shorey-Kendrick

state: Oregon

address: 505 NW 185th Ave

institute: Oregon Health and Science University

summary: RNAseq analysis of placental gene expression in samples obtained from 59 smokers (enrolled in the VCSIP RCT: NCT03203603) and 20 never-smokers for reference.

overall_design: Comparative gene expression profiling analysis of RNA-seq data in placentas from pregnant smokers randomized to supplemental vitamin C versus placebo

middle: E

first: Cindy

last: McEvoy

last: Tepper

first: Eliot

last: Spindel


title: Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function.

first: Domenic

last: Narducci

state: MA

address: Building 56 Room 787B, 32 Vassar Street

department: Biological Engineering

institute: Massachusetts Institute of Technology

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


title: Transcription factor genomic occupancy is determined by multiple, overlapping DNA binding sites

status: Public on Mar 06 2024

first: Shubham

last: Khetan

institute: Brigham and Women's Hospital

summary: Transcription factors (TFs) regulate gene expression by interacting with DNA in a sequence-specific manner. High-throughput in vitro technologies, such as PBMs and HT-SELEX, have revealed the DNA binding specificities of hundreds of TFs. However, they have limited ability to reliably identify lower affinity binding sites, which are increasingly recognized as important for precise spatial and temporal control of gene expression. To address this limitation, we developed a novel technology to measure protein affinity to DNA by in vitro transcription and RNA sequencing (PADIT-seq). Using PADIT-seq, we comprehensively assayed the binding preferences of four human and two yeast TFs to all possible 10-bp DNA sequences, detecting hundreds of novel lower affinity binding sites. The expanded repertoire of lower affinity binding sites unexpectedly revealed that nucleotides flanking high affinity DNA binding sites create overlapping lower affinity sites that together modulate TF genomic occupancy in vivo. We propose a model where TF binding is not determined by individual binding sites, but rather by the sum   of multiple, overlapping binding sites. Formation of such extended recognition sequences stems from an inherent property of TF binding sites to interweave each other. Consequently, competition between paralogous TFs for shared high-affinity binding sites is controlled by flanking nucleotides that create differential numbers of overlapping lower affinity binding sites. Noncoding variants alter multiple, overlapping binding sites to influence gene expression and human traits, including diseases.

overall_design: Binding preferences of 6 TFs to all possible 10-mers using PADIT-seq, a novel technology to detect TF-DNA interactions across the entire spectrum of affinities.

first: Brent

last: Carroll

first: Martha

last: Bulyk


title: PD-1 regulates ILC3-driven intestinal immunity and homeostasis

status: Public on Feb 29 2024

city: Melbourne

first: Cyril

last: Seillet

address: 1G Royal Parade

institute: Walter and Eliza Hall Institute

summary: PD-1 is constitutively expressed on a fraction of ILC3 in the gut. To identify the transcriptional differences between PD1+ and PD1- ILC3, we have sorted these subsets and analysed their transcriptional regulation by RNA-Seq.

overall_design: PD1 expressing and PD1 negative ILC3 from the small intestine of adult female rorgt-reporter mice. ILC3 NCR+ were excluding, ILC3 were sorted as CD45+Lineage- (CD3, CD11b, B220, TER119, TCRb, CD19, NK1.1, F4/80) CD90.2+RORγt+ NCR-

first: Jacquelot

last: Nicolas

first: Seillet

last: Cyril

first: Yu

last: Huiyang

first: Le

last: Xiong

first: Azin

last: Sayad


title: Manganese homeostasis modulates fungal virulence and stress tolerance in Candida albicans

status: Public on Feb 13 2024

first: Adnane

last: Sellam

address: 5000, rue Bélanger

institute: Universite de Montreal

summary: As transition metal availability is very limited inside the human host, fungal pathogens have evolved sophisticated mechanisms to uptake and utilize these micronutrients at the infection interface. While considerable attention was turned into iron, copper and zinc acquisition mechanisms and their importance in fungal fitness, less was done regarding either the role of Mn in infectious processes or the cellular mechanism by which fungal cells achieve their Mn-homeostasis. Here, we undertook a transcriptional profiling of the pathogenic fungus Candida albicans experiencing both Mn starvation and excess to comprehensibly capture biological processes that are modulated by Mn. We uncovered that Mn scarcity influence diverse biological processes associated with fungal fitness including morphogenetic switch, invasion of host cells and antifungal sensitivity. We also uncovered that Mn levels influence the abundance of iron and zinc emphasizing the complex crosstalk between ions metals. Deletion of SMF12, a member of Mn Nramp transporters confirmed its contribution to Mn uptake. In accordance with the RNA-seq data, smf12 was unable to form hyphae and damage host cells, and exhibited sensitivity to azole antifungals. We also found that unfolded protein response (UPR), likely activated by decreased glycosylation under Mn limitation, was essential to promote C. albicans growth. RNA-seq profiling of cells exposed to Mn excess uncovered that the UPR signaling was also essential to bypass Mn toxicity. Collectively, this study underscores the importance of Mn homeostasis in fungal virulence, and comprehensively provides a transcriptional portrait of biological functions that are modulated by Mn in a fungal pathogen.

overall_design: In this study, we identified transcripts modulated under manganese (Mn) starvation by comparing the transcriptome of cells growing in Mn-depleted medium to those growing in Mn-replete medium. We also profiled cells growing under manganese excess by comparing the transcriptome of cells treated with 7.5 mM MnCl2 to that of the control condition.

first: Manon

last: Henry

first: Inès

last: Khemiri

first: Faiza

last: Tebbji

first: Rasmi

last: Abu-Helu

first: Antony

last: Vincent


title: Bojungikki-Tang Enhances the Efficacy of Pembrolizumab Treatment in Human PBMC-Injected H460 Tumor-Bearing Mice

status: Public on Mar 05 2024

city: Daejeon

first: Heerim

last: Yeo

address: 99, Daehak-ro, Yuseong-gu

institute: Chungnam National University

summary: Immune checkpoint inhibitors (ICIs) have become a major treatment option for non-small cell lung cancer, but their efficacy is still limited because only a subset of patients responded to ICIs. Bojungikki-Tang (BJIKT), a widely used herbal medicine extracted from 10 medicinal plants, has been largely investigated as a combined treatment with anticancer agents. In this study, we examined the potential anti-tumor effect of co-administration of BJIKT and pembrolizumab in human peripheral blood mononuclear cell (PBMC)-injected H460 tumor-bearing MHC Ⅰ/Ⅱ double knockout NSG mice. We found that combination treatment with BJIKT and pembrolizumab significantly suppressed tumor growth by regulating the activation of immune cells. Immunohistochemistry analysis showed that the combination therapy decreased the exhausted proportion of CD8(+) and CD4(+) T cells expressing PD-1 and LAG-3 markers in the tumor tissues. Our transcriptome analysis indicated that BJIKT strengthened T cell function and TNF signaling and inhibited TGF-β signaling to induce cell cycle arrest and apoptosis. Additionally, BJIKT synergistically exhibited anti-tumor effects along with immune-related pathways when combined with pembrolizumab. Taken together, combination therapy altered immune cells and regulated anti-tumor immune response, paving the way for a more effective understanding of the role of BJIKT in the tumor microenvironment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for H460 tumor tissues by pembrolizumab and/or bojungikki-tang treatment

first: SeWon

first: Jin-Mu

last: Yi

first: Sang-Min

last: Park

first: Jaemoo

last: Chun

first: Mi-Kyung

last: Jeong


title: CRISPR screening identifies nucleolar RPL22 as a heterochromatin destabilizer and senescence driver

status: Public on Feb 14 2024

city: Beijing

first: jiang

middle: xiao

last: yu

address: 15 Datun Road Chaoyang District

institute: institute of zoology, CAS

summary: Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, that RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1γ (HP1γ) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process.

overall_design: For our CRISPR screen, we first constructed a RAG library containing single guide RNAs (sgRNAs) targeting ribosome-related genes and based on the lentiCRISPRv2 plasmid (Addgene # 52961) as previously described. Then, we obtained a lentivirus library and infected senescent hMPCs at a low multiplicity of infection (MOI) (0.3) to ensure that one cell receives no more than one sgRNA. At the same time, we set up a control in which hMPCs were transduced with non-targeting control (NTC) at the same MOI. After two days, the cells transduced with sgRNA were put under puromycin selection (1 μg/ml) (InvivoGen) for six days, after which the cells switched to fresh medium without puromycin and passaged until the control cells underwent growth arrest. We harvested cells at different time points and extracted the genomic DNA using TIANamp Genomic DNA Kit (TIANGEN), according to the manufacturer’s protocol.


title: Development of a 6-gene mesothelioma-specific prognostic signature using whole transcriptomic analysis of mouse and human tumors

status: Public on Feb 15 2024

city: Nedlands

first: Scott

last: Fisher

address: 35 Stirling Highway, Nedlands

department: Institute for Respiratory Health

institute: The University of Western Australia

summary:
Background: Mesothelioma is an aggressive, fatal cancer that is inextricably linked to asbestos exposure. Recent trials using a combination of the immune checkpoint inhibitors ipilimumab and nivolumab has significantly improved treatment outcomes, however durable treatment responses remain restricted to a subset of patients (15-20%), highlighting the need to identify strategies that better predict treatment response.
Method: Here, we performed RNAseq on a large tumor biobank from genetically diverse mouse model, CC-MexTAg model to compare gene expression profiles of tumors from mice with different overall survival to develop a prognostic gene signature.
Results: while the variation in gene expression data of tumors did not associate with 3-fold variation in overall survival of CC-MexTAg mice, we identified two distinct tumor clusters characterized with immune and non-immune phenotypes, in which immune cluster tumours showed the better potential of response to cancer therapies. We used 20 hub genes associated with these tumor phenotypes to develop a 6-gene signature that could predict survival in four independent mesothelioma datasets and showed a potential to respond to cancer immunotherapy.

overall_design: 167 asbestos induced tumours harvested from 119 mice, representing 35 CC-MexTAg strains.

first: Kiarash

last: Behrouzfar


title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

status: Public on Mar 08 2024

city: Amsterdam

first: Mikhail

last: Magnitov

address: Plesmanlaan 121

institute: The Netherlands Cancer Institute

summary: Gene expression is orchestrated by transcription factors (TFs), which bind to DNA in a sequence-specific manner, and by spatial genome structure, which constrains and shapes TF activity. Zinc finger protein 143 (ZNF143/ZFP143) is a TF that has been implicated in both gene activation and 3D genome organisation. We have generated an acute ZNF143/ZFP143 depletion system to study its direct consequences on chromatin looping and gene transcription. The effects of ZNF143/ZFP143 depletion are inconsistent with it being a looping factor, which we further confirmed by systematic analysis of previous studies. However, degradation of ZNF143/ZFP143 led to the down-regulation of hundreds of its targets, which were found to be enriched in nuclear-encoded mitochondrial genes. By studying the consequences of ZNF143/ZFP143 loss in monoculture and in an in vitro embryonic development model, we establish ZNF143/ZFP143 as a conserved transcriptional regulator of cell proliferation and differentiation by modulating mitochondrial functions.

overall_design: Analysis of chromatin occupancy (ZFP143 and CTCF), nascent RNA, 3D genome structure in ZFP143 transcription factor depletion using the dTAG degron system in mESCs. Analysis of open chromatin and stable RNA in ZFP143 depletion in stem cell-derived differentiation models.

middle: D

first: Michela

last: Maresca

first: Noemí

last: Alonso Saiz

first: Hans

last: Teunissen

first: Elzo

last: de Wit


title: Single-Cell Identification of Melanoma Biomarkers in Circulating Tumor Cells

status: Public on Feb 07 2024

city: Portland

first: Rosalyn

last: Fey

state: OR

address: 3181 S.W. Sam Jackson Park Road

institute: Oregon Health & Science University

summary: The current standard for investigating tumors is surgical biopsy, which is costly, invasive, and difficult to perform serially. As an adjunct, circulating tumor cells (CTCs)—cells that have broken away from the primary tumor or metastatic sites—can be obtained from a blood draw and offer the potential for obtaining serial genetic information and serving as biomarkers. Here, we detail the potential for melanoma CTCs to serve as biomarkers and discuss a clinically viable methodology for single-cell CTC isolation and analysis that overcomes previous limitations. We explore the use of melanoma CTC biomarkers by isolating and performing single-cell RNA sequencing on CTCs from melanoma patients. We then compared transcriptional profiles of single melanoma CTCs against A375 cells and peripheral blood mononuclear cells to identify unique genes differentially regulated in circulating melanoma tumor cells. The information that can be obtained via analysis of these CTCs has significant potential in disease tracking.

overall_design: Isolation and single-cell RNA sequencing of circulating tumor cells from melanoma patients, healthy control peripheral blood mononuclear cells, and A375 melanoma cells.

first: Reilly

last: Fankhauser

first: Matthew

last: Chang

first: Zachary

last: Garrison

first: Rachel

last: Berryman

first: Olivia

last: Lucero

first: Allison

last: Fuiten

first: Nicholas

last: DePatie

first: Hilary

last: Seifert

first: Rajan

middle: P

last: Kulkarni


title: Altered miRNA expression in liver and serum of NASH mice

status: Public on Jan 31 2024

city: Hirosaki

first: Mitsuru

last: Chiba

state: Aomori

address: 66-1 Hon-cho

department: Graduate School of Health Sciences

institute: Hirosaki University

summary: Non-alcoholic steatohepatitis (NASH) is a fatty liver disease that does not involve alcohol consumption and is characterized by fatty degeneration, inflammation, and hepatocellular damage. Therefore, predicting future fibrosis is necessary in the early stages of NASH to prevent developing diseases. This study examined histological changes in the liver as well as microRNA expression changes in the liver and serum of NASH mice model to search for potential biomarker candidates that may predict early fibrosis. This study used 6-week-old C57BL/6NJcl male mice and fed the control and NASH groups with a food-breeding solid diet (CE-2) and a high-fat diet (choline-deficient high-fat and 0.1% [w/v] methionine supplemented diet), respectively. We used Agilent Technologies miRNA microarray to examine microRNA expression in the liver and serum.

overall_design: We purchased 5-week-old C57BL/6NJcl male mice from Clea Japan. Treatment was initiated after a 1-week acclimatization. The NASH model group was fed a choline-deficient high-fat (CDAHFD) and 0.1% (w/v) methionine-supplemented diet (Research Diets Inc., A06071302). The Control group was fed a solid diet CE-2 (Clea Japan). Both groups received feed and water ad libitum, which were replaced once a week. Blood and livers were removed from mice at 1, 2, and 6 weeks after ingestion of the high-fat diet or food-breeding solid diet, and miRNA expression was examined by miRNA microarray.

first: Kana

last: Matsumoto


title: A Panoramic View of Cell Population Dynamics in Mammalian Aging

city: New York

first: Zehao

last: Zhang

state: NY

address: 1230 York Ave, The Rockefeller University

institute: The Rockefeller University

summary: To elucidate the aging-associated cellular population dynamics throughout the body, here we present PanSci, a single-cell transcriptome atlas profiling over 20 million cells from 623 mouse tissue samples, encompassing a range of organs across different developmental stages, genders, and genotypes. This comprehensive dataset allowed us to identify more than 3,000 unique cellular states and catalog over 200 distinct aging-associated cell populations experiencing significant depletion or expansion. Our panoramic analysis uncovered four distinct, temporally structured aging-related cell population dynamic waves that are organ- and lineage-specific. Moreover, we investigated aging-associated alterations in immune cell populations, revealing both widespread shifts and organ-specific changes. We further explored the regulatory roles of the immune system on aging and pinpointed specific age-related cell population expansions that are lymphocyte-dependent. The breadth and depth of our 'cell-omics' methodology not only enhance our comprehension of cellular aging but also lay the groundwork for exploring the complex regulatory networks among varied cell types in the context of aging and aging-associated diseases.

overall_design:
Single nucleus RNA-seq profiling for C57BL/6 mouse cells from lung, heart, liver, kidney, muscle, stomach, duodenum, jejunum, ileum, colon, brown adipose tissue, inguinal adipose tissue, gonadal adipose tissue cells at 3,6,12,16,23 months. Single nucleus RNA-seq profiling for B6.129S7-Rag1tm1Mom/J and B6.Cg-Prkdcscid/SzJ mouse cells from lung, heart, liver, kidney, muscle, stomach, duodenum, jejunum, brown adipose tissue, inguinal adipose tissue, gonadal adipose tissue cells at 3 and 16 months.
** Please note that the library for each organ is made out of pooled samples. For example, only one library (splitted into 9 sample/part records) was constructed and sequenced for kidney. After demultiplexing, there were over 2000 FASTQ files, which were submitted as multiple SRA experiments due to the maximum file number (256) for each library.  All FASTQ files (generated from all corresponding organ *part sample records) are from the same pooled sample and each processed data (linked to the corresponding *part1 sample records) was generated from all pooled samples.
** 20240213_PanSci_all_cells_adata.h5ad:  The h5ad file is generated with Scanpy where we pooled all the count matrix and metadata from GSE247719 and GSE212606, containing cells from brain, lung, heart, liver, kidney, muscle, stomach, duodenum, jejunum, ileum, colon, brown adipose tissue, inguinal adipose tissue, gonadal adipose tissue.

first: Chloe

last: Schaefer

first: Weirong

last: Jiang

first: Ziyu

last: Lu

first: Jasper

last: Lee

first: Andras

last: Sziraki

first: Abdulraouf

last: Abdulraouf

first: Wei

last: Zhou

first: Junyue

last: Cao


title: Clr4SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing via heterochromatic phase transitions

status: Public on Feb 01 2024

city: Cold Spring Harbor

first: Hyun Soo

last: Kim

state: New York

address: 1 Bungtown Rd

department: Plant

institute: Cold Spring Harbor Laboratory

summary: Transcriptional silencing by RNAi paradoxically relies on transcription, but how the transition from transcription to silencing is achieved has remained unclear. The Cryptic Loci Regulator complex (CLRC) in Schizosaccharomyces pombe is a cullin-ring E3 ligase required for silencing that is recruited by RNAi. We found that the E2 ubiquitin conjugating enzyme Ubc4 interacts with CLRC and mono-ubiquitinates the histone H3K9 methyltransferase Clr4SUV39H1, promoting the transition from co-transcriptional gene silencing (H3K9me2) to transcriptional gene silencing (H3K9me3). Ubiquitination of Clr4 occurs in an intrinsically disordered region (IDR), which undergoes robust liquid-liquid phase separation (LLPS), along with Swi6HP1 the effector of transcriptional gene silencing. Phase separation of Clr4 and Swi6 is exquisitely sensitive to non-coding RNA (ncRNA), which promotes dimerization, chromatin association, and di-, but not tri- methylation instead. Ubc4-CLRC also targets the transcriptional co-activator Bdf2BRD4, down-regulating centromeric transcription and small RNA production. The deubiquitinase Ubp3 counteracts both activities.

overall_design: Enrichment of H3K9me2, H3K9me3, RNA Pol II, Flag-Clr4, Epe1-Flag and Bdf2-Flag by ChIP-seq. Enrichment of sRNA by sRNA-seq.

first: Hyun

first: Benjamin

last: Roche

first: Robert

middle: A

last: Martienssen


title: NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis

city: Ann Arbor

first: Arul

last: Chinnaiyan

state: MI

institute: University of Michigan