title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [KRABscreen]

overall_design:
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging
iPSCs were maintained in StemCell mTeSR or mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging
Each screen was performed in triplicate with independent transductions. For each replicate, 24 x 106  SNRPN-2A-GFP VP64dCas9VP64 (maternal) or dCas9KRAB (paternal) iPSCs were dissociated using Accutase (Stemcell Tech, 7920) and transduced in suspension across five matrigel-coated 15-cm dishes in mTesR (Stemcell Tech 85850) supplemented with 10 μM Rock Inhibitor (Y-27632, Stemcell Tech, 72304). Cells were transduced at a MOI of 0.2 to obtain one gRNA per cell and ~430-fold coverage of the gRNA library. The medium was changed to fresh mTesR without Rock Inhibitor 18-20 h after transduction. Antibiotic selection was started 30 h after transduction by adding 1 μg/mL puromycin (Sigma, P8833) directly to the plates without changing the medium. The cells were fed daily and passaged as necessary maintaining library coverage until harvest. Cells were harvested for sorting 9 d after transduction of the gRNA library for all screens.
Cells were washed once with 1x PBS, dissociated using Accutase, filtered through a 30 μm CellTrics filter (Sysmex, 04-004-2326) and resuspended in FACS Buffer (0.5% BSA (Sigma, A7906), 2 mM EDTA (Sigma, E7889) in PBS). Before sorting, an aliquot of 4.8 x 106 cells were taken to represent a bulk unsorted population. The highest and lowest 10% of cells were sorted based on GFP expression and 4.8 x 106 cells were sorted into each bin. Sorting was done with a SH800 FACS Cell Sorter (Sony Biotechnology).

FASTQ files were aligned to custom indexes (generated from the bowtie2-build function) using Bowtie 2. Counts for each gRNA were extracted and used for further analysis. All enrichment analysis was done with R. Individual gRNA enrichment was determined using the DESeq2 package to compare between high and low conditions for each screen. Only forward read (read 1) of paired-end sequenced sequencing files was used for data processing and enrichment analysis.
Differentially-enriched gRNAs were gRNAs were identified via DESeq2 using an adjusted p-value cutoff of 0.05.

processed output file: DESeq2 results table and sequencing counts for each condition


title: Suboptimal prophylactic treatments with BDGR-49 in a lethal VEEV TC-83 mouse model

status: Public on Jan 06 2025

city: Memphis

first: Evan

middle: Peter

last: Williams

state: TN

address: 858 Madison Ave

department: Dept. of Microbiology, Immunology, and Biochemistry

country: USA

institute: University of Tennessee Health Science Center

type: Expression profiling by high throughput sequencing

summary: To study emergence of resistance to BDGR-49 during infection with Venezuelan equine encephalitis virus (VEEV), mice were treated suboptimal treatment regimen. C3H/HeN mice were prophylactically treated with BDGR-49 2 hours prior to infection for a total of 5 days. Mice were infected with a lethal challenge dose of VEEV TC-83. Following infection mice brains were euthanized on 3-, 5-, 7,9-, and 10-days post-infection and their brains collected and RNA-Seq performed on. RNA-Seq results were used to analyze for viral resistant mutants. Analysis revealed that only one of the mice contained a mutant that is potentially resistant to BDGR-49.

overall_design: C3H/HeN mice were intranasally infected Venezuelan equine encephalitis virus  (VEEV) TC-83,  prophylactically treated with BDGR-49  for a period of 5 days. At 3, 5, 7,9, and 10 days post-infection mice were euthanized and brains collected and RNA-Seq performed on.

first: Colleen

last: Jonsson


title: BDGR-49 protects BALB/c mice from lethal, intranasal challenge of Venezuelan Equine Encephalitis Virus

summary: Venezuelan, Western and Eastern Equine Encephalitis Virus (VEEV, WEEV and EEEV), genus Alphavirus, causes a febrile illness that may result in fatal neurological disease which has no FDA-approved antivirals for the prevention or treatment. To address this gap, we developed a novel brain-penetrant, small molecule, BDGR-49, which when administered subcutaneously at 6 mg/kg twice per day for 6 days conferred 100% protection against a lethal intranasal challenge of VEEV Trinidad donkey (TrD) in BALB/c mouse model. By eight days post-infection (dpi), viral load in the brain of BDGR-49-treated mice was significantly reduced whereas TrD-infected, sham-treated mice succumbed to disease on 5 dpi. Analysis of the host responses in the brains of VEEV TrD-infected, BDGR-49-treated, mice resulted in a significant reduction in expression of genes in pathways associated with inflammation and cell death.

overall_design: BALB/c mice were intranasally infected Venezuelan equine encephalitis virus  (VEEV),  prophylactically treated with BDGR-49 or sham-treated. At 2, 4, 5 (Sham-treated), and 8 (BDGR-49-treated) days post-infection mice were euthanized and brains collected and RNA-Seq performed on.

middle: P

first: Xue

last: Yi

middle: B


title: Expression analysis of WT, efk-1, cep-1, and zip-2 mutant C. elegans strains in fed and starved condition

city: Vancouver

first: Stefan

last: Taubert

state: BC

address: 950 W 28th Ave, CMMT Room 2024

department: Medical Genetics

country: Canada

institute: UBC

summary: Cells and organisms frequently experience starvation. To adapt and survive, they mount an evolutionarily conserved stress response. A vital component in the mammalian starvation response is eukaryotic elongation factor 2 (eEF2) kinase (eEF2K), which responds to starvation by phosphorylating and inactivating the translation elongation driver eEF2, thus shutting down translation and facilitating survival. C. elegans efk-1/eEF2K phosphorylates EEF-2/eEF2 on a conserved residue and is required for starvation survival, but how it promotes survival remains unclear. Surprisingly, we found that eEF2 phosphorylation is unchanged in starved C. elegans, suggesting that efk-1 promotes survival via a noncanonical pathway. We show that efk-1 upregulates transcription of the DNA repair pathways, nucleotide excision repair (NER) and base excision repair (BER), to promote starvation survival. Furthermore, efk-1 suppresses oxygen consumption and ROS production in starvation to prevent oxidative stress. Thus, efk-1 enables starvation survival by protecting animals from starvation-induced oxidative damage through a translation-independent pathway.

overall_design: Comparative gene expression profiling (RNA-seq) of N2, efk-1, zip-2 and cep-1 mutant L4 C. elegans hermpahrodites in fed and 8-hour starved conditions

first: Forum

last: Bhanshali

first: Junran

last: Yan

first: Xuanjin

last: Cheng


title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cance

city: Seoul

first: Jaemoon

last: Koh

state: Seoul

address: Jongnogu 101 Seoul National University hospital, B1, Department of Pathology

country: South Korea

institute: Seoul National University Hospital

summary: Background:  The tumor cell-intrinsic function of programmed cell death-ligand 1 (PD-L1) is poorly understood. The roles of the intrinsic function of PD-L1 in interleukin (IL)-6-mediated immunosuppression and the response to immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) were investigated.  Methods:  Cohorts of NSCLC patients treated with ICI and public datasets were analyzed. PD-L1-overexpressing and PD-L1-knockdown NSCLC cells were submitted to RNA-seq, in vitro analyses, ChIP-qPCR, CUT&Tag, and biochemical assays. Human myeloid-derived suppressor cells (MDSCs) sorted from peripheral blood mononuclear cells were co-cultured with NSCLC cells, and then assessed for their immunosuppressive activity on T-cells. Mouse Lewis lung carcinoma (LLC) cells with PD-L1 overexpression/or knockdown were subcutaneously injected into wild-type or PD-1-knockout C57BL/6 mice in the presence of IL-6 and/or PD-1 blockade.  Results:  In the ICI cohort with RNA-seq data, the IL-6/Jak/Stat3 pathway was enriched and IL-6 expression was higher in patients with no response to ICI in PD-L1-high NSCLCs. IL-6 expression in PD-L1-high NSCLCs correlated positively with MDSCs and Tregs, but negatively with cytotoxic lymphocytes. In another ICI cohort, a higher baseline serum IL-6 level was associated with poor clinical outcome after ICI therapy. PD-L1 activated Jak2/Stat3 signaling by binding to and inhibiting tyrosine phosphatase PTP1B. PD-L1 also bound to p-Stat3 in the nucleus, thus promoting the activity of p-Stat3 on the transcription of several cytokines (IL-6, TGFβ, TNFα, IL-1β) and chemokines (CXCL1, CXCL3, CXCL8). PD-L1-overexpressing NSCLC cells enhanced the migration and immunosuppressive activity of human MDSC in vitro, mediated by IL-6 and CXCL1. In both wild-type and PD-1-knockout mice, PD-L1-overexpressing mouse lung tumors were heavily infiltrated by MDSCs with high immunosuppressive function. Treg numbers were increased while the numbers of granzyme B+ or IFNγ+ CD8 T-cells decreased. These responses were shown to be mediated by IL-6 secreted from PD-L1-overexpressing tumor cells. Combined blockade of PD-1 and IL-6 was effective in tumor control and decreased MDSCs while increasing granzyme B+ or IFNγ+ CD8 T-cells.  Conclusions:  The tumor-cell-intrinsic function of PD-L1 contributes to immunosuppression and tumor progression by MDSCs through the IL-6/Jak/Stat3 pathway, which may serve as therapeutic target to improve ICI efficacy in patients with PD-L1-high NSCLC.

overall_design:
RNA sequencing of lung tissue from patients who received ICI therapy (PD-1 or PD-L1 blockade) using Illumina TruSeq Stranded mRNA Library prep and sequenced on Illumina HiSeq 2500.

***************************************************************
Due to our institution's security policy, we are unable to provide raw data.
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first: Se-Hoon

last: Lee

first: Yoon Kyung

last: Jeon


title: Broad spectrum efficacy of BDGR-164 against neurotropic alphaviruses and correlates of protection

summary: Infections by the New World alphaviruses, Eastern Equine encephalitis virus  (EEEV), Venezuelan Equine encephalitis virus  (VEEV), and Western Equine encephalitis virus  (WEEV), cause febrile illness that can progress to fatal disease in humans and equids. Currently there are no FDA-approved antivirals for prophylactic or therapeutic treatment of human infection by these viruses. To combat these infections, we have developed a novel small molecule, BDGR-164, which has subnanomolar potency against VEEV, EEEV, and WEEV.  Using an intranasal route of virus infection in a lethal BALB/c model, prophylactic subcutaneous administration of BDGR-164 conferred 100% (VEEV), 88% (EEEV), and 63% (WEEV) survival. To evaluate the ability of BDGR-164 to reduce viral RNA/antigen, inflammation, and pathogenesis, we used RNASeq and histopathology of whole brain at 4 days post-infection (dpi). Viral RNA levels and antigen were reduced significantly in virus-infected and BDGR-164-treated versus virus-infected, sham-treated mice. Moreover, there was a significant reduction in host immune responses associated with inflammatory signaling, immune cell recruitment, and programmed cell death in virus-infected, BDGR-164 treated mice. Cytokine analyses of sera corroborated the reduction in upregulation of the immune response in virus-infected, BDGR-164 treated mice. Limited antiviral resistance to BDGR-164 was detected in one mouse on 4 dpi at NSP2:Y102C. In conclusion, our studies suggest that BDGR-164 has broad and potent prophylactic efficacy against the neurotropic alphaviruses.

overall_design: BALB/c mice were either intranasally mock-infected or infected with Eastern equine encephalitis virus  (EEEV), Venezuelan equine encephalitis virus  (VEEV), or Western equine encephalitis virus (WEEV) prophylactically treated with BDGR-164 or sham-treated. At 4 days post-infection mice were euthanized and brains collected and RNA-Seq performed on.


title: Glutathione triggers leaf-to-leaf, calcium-based plant defense signaling

city: Beijing

first: daoxin

last: Xie

address: Tsinghua University Haidian District

country: China

institute: Tsinghua University

summary: To get deeper insights into the GSH-triggered systemic defense signaling events in plants, we performed transcriptome profiling on leaf 6 collected after one hour application of GSH to leaf 1 of WT plants. To further investigate the relations between GSH and Glu in plant wound response, we generated transcriptome data sets from leaf 6 one hour after wounding leaf 1 or application of Glu to leaf 1 of WT plants.

overall_design: Arabidopsis WT plants growing in MS medium at 22℃ with a 10 h/14 h light/dark photoperiod for 22-24 days were used for GSH, Glu, and wounding treatment. Transcriptome profiling was performed on leaf 6 respectively collected one hour after wounding leaf 1 or application of 100 mM GSH or 100 mM Glu to leaf 1 of WT plants.

first: Rui

last: Li

first: Yongfang

last: Yang

first: Jianbin

first: Xiaoyi

last: Shan

first: Haidong

last: Chen

first: Guo-Liang

last: Wang

first: Daoxin


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing [RNA-seq]

city: Durham

first: Charles

last: Gersbach

state: North Carolina

address: 101 Science Dr.

department: Biomedical Engineering

institute: Duke University

summary: Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

overall_design:
iPSCs were maintained in StemCell mTeSR Plus, with ROCK inhibitor (Y-27632) after seeding or passaging.
Stable polyclonal Tet1-dCas9 or VP64-dCas9-VP64 cell lines were established by transducing with lentivirus and selecting for transgene-expressing cassette with 1.5 ug/mL blasticidin for 5 days. Then, cells were transduced with gRNA lentivirus and selected for gRNA-expressing cassette with puromycin (1ug/mL) for 3 days.
14 days after gRNA virus transduction, cells were harvested with Accutase, pelleted, and total RNA was extracted using Norgen Total RNA Plus Micro kit. Total RNA was submitted to Genewiz (Azenta) for library prep and total RNA sequencing.
RNA libraries were prepared by Genewiz/Azenta Life Sciences. Total RNA library was constructed with rRNA depletion method.

Raw reads were trimmed using Trimmomatic toe remove adapters.
Trimmed reads were aligned to hg19 genome using STAR aligner.
Gene counts were obtained with featureCounts from the subread package (v1.4.6-p4) using the comprehensive gene annotation in Gencode v22.
Differential expression analysis was determined with DESeq2 where gene counts are fitted into negative binomial generalized linear models (GLMs) and Wald statistics to determine significantly differentially expressed genes.

Genome: hg19

first: Dahlia

last: Rohm

middle: A


title: Quantification of subcellular RNA localization through direct detection of RNA oxidation

status: Public on Jan 03 2025

city: Aurora

first: Matthew

last: Taliaferro

state: CO

department: Biochemistry and Molecular Genetics

institute: University of Colorado Anschutz Medical Campus

summary: Across cell types and organisms, thousands of RNAs display asymmetric subcellular distributions. The study of this process often requires quantifying abundances of specific RNAs at precise subcellular locations. To analyze subcellular transcriptomes, multiple proximity-based techniques have been developed in which RNAs near a localized bait protein are specifically labeled, facilitating their biotinylation and purification. However, these complex methods are often laborious and require expensive enrichment reagents. To streamline the analysis of localized RNA populations, we developed Oxidation-Induced Nucleotide Conversion sequencing (OINC-seq). In OINC-seq, RNAs near a genetically encoded, localized bait protein are specifically oxidized in a photo-controllable manner. These oxidation events are then directly detected and quantified using high-throughput sequencing and our software package, PIGPEN, without the need for biotin-mediated enrichment. We demonstrate that OINC-seq can induce and quantify RNA oxidation with high specificity in a dose- and light-dependent manner. We further show the spatial specificity of OINC-seq by using it to quantify subcellular transcriptomes associated with the cytoplasm, ER, and the inner and outer membranes of mitochondria. Finally, using transgenic zebrafish, we demonstrate that OINC-seq allows proximity-mediated RNA labeling in live animals. In sum, OINC-seq together with PIGPEN provide an accessible workflow for the analysis of localized RNAs across different biological systems.

sample_organism:
Danio rerio
Homo sapiens

platform_organism:
Danio rerio
Homo sapiens

overall_design: Cells and tissues were treated with and without conditions that drive localized RNA oxidation at a variety of subcellular locations, including treatment with the singlet oxygen producer Halo-DBF and treatment with green light to activate Halo-DBF. The extent of oxidation was then calculated at the gene level using PIGPEN software.


title: ZBTB16/PLZF regulates self-renewal and differentiation of juvenile spermatogonial stem cells through an extensive transcription factor-chromatin poising network

city: Salt Lake City

first: Bradley

middle: R.

last: Carins

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: University of Utah

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions feature broad H3K4me3 and H3K27ac, DNA hypomethylation, RNAPol2 and often CTCF. Hi-C analyses reveal robust 3D physical interactions among these co-bound promoters, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation, even when active. Overall, ZBTB16 promotes uSPG cell cycle progression and colocalizes with SALL4, SOX3, CTCF and RNAPol2 to establish a novel and extensive chromatin poising network.

overall_design: To investigate the function of ZBTB16 in vivo, we isolated undifferentiated spermatogonia (uSPG) from the testis and conducted RNA-seq and histone modification ChIP-seq. We also performed ChIP-seq for ZBTB16, SALL4, SOX3, TBP, and RNAPol2 (Pan, CTD Ser5P, and Ser2P) using whole testis samples. Given that ZBTB16 expression is restricted to uSPG at postnatal day 7 (P7), these experiments aimed to identify genes directly regulated in postnatal mouse spermatogonia.

first: Chongil

last: Yi

middle: R

last: Cairns


title: ZBTB16/PLZF regulates self-renewal and differentiation of spermatogonial stem cells through an extensive transcription factor-chromatin poising network

summary: Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression (Ccnd1) to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network.

overall_design: To better understand ZBTB16 function in vivo, we isolated spermatogonia and then performed RNA-seq and histone modification ChIP-seq. We also performed ChIP-seq for the transcription factors ZBTB16, SALL4 and SOX3 using whole testis to reveal directly regulated genes in postnatal mouse spermatogonia.


title: PARP inhibitors differentially regulate immune responses in distinct genetic backgrounds of high-grade serous tubo-ovarian carcinoma

city: New York City

first: Alireza

last: Khodadadi-Jamayran

state: NY

address: 550 1st Ave, MSB 304

department: Division of Advanced Research Technologies (DART)

institute: New York University, NYU Langone Medical Center

summary: Immune checkpoint inhibitors (ICIs) have revolutionized treatment for several tumor indications without demonstrated benefit for ovarian cancer patients. To improve the therapeutic ratio of ICIs in ovarian cancer patients, several different clinical trials are testing combinations with poly (ADP-ribose) polymerase (PARP) inhibitors. Comparing the immunomodulatory effects of clinically advanced PARP inhibitors may help to identify the best partner to combine with ICIs. We examined the treatment effect of talazoparib (a PARP trapper) and veliparib (a solely PARP enzymatic inhibitor) in homologous recombination deficient (HRD) and homologous recombination proficient (HRP) high-grade serous tubo-ovarian carcinoma (HGSC) cell lines on immune-related gene expression. We discovered and validated that CXCL8, IL-6, and TNF gene expression were upregulated after talazoparib treatment in both OVCAR3 (HRD) and CAOV3 (HRP) HGSC cell lines. In contrast, veliparib treatment slightly elevated similar genes exclusively in a HRD HGSC cell line model. We expanded these studies to include olaparib, a PARP trapper less potent than talazoparib, and found effects specific to COV361 (BRCA1 mutant) and OVCAR8 (BRCA1 methylated) HGSC cells but not all HRD HGSC cell lines. Our studies also identified differences among PARP trappers versus veliparib on augmenting CXCL10 expression. Finally, we show that talazoparib modulates the CXCL10 response in cGAS-defective cell lines, independent of the cGAS-STING pathway.  These mechanistic studies advance our understanding of how different PARP inhibitors affect the immune system in various genetic backgrounds.

overall_design:
We examined transcriptome changes among ovarian cancer cell lines treated with various PARP inhibitors through RNA-seq and GSEA
We validated findings through RT-PCR and immunoblotting in select ovarian cancer cells
We confirmed findings for interferon related genes and proteins using RNA interference

first: Douglas

last: Levine


title: Pterostilbene targets hallmarks of aging in the gene expression landscape in blood of healthy rats

status: Public on Jan 02 2025

first: Barbara

last: Stefanska

address: 2205 East Mall, FNH 248

department: Faculty of Land and Food Systems

institute: The University of British Columbia

summary: Scope: Polyphenols from the phytoestrogen group, including pterostilbene (PTS), are known for their antioxidant, anti-inflammatory and anti-cancer effects. In recent studies, phytoestrogens attenuate age-related diseases, however their pro-longevity effects in healthy models in mammals remain unknown. As longevity studies demonstrate age-related transcriptomic signatures in human blood, we propose that phytoestrogen-supplemented diet may induce changes in gene expression that ultimately confer pro-longevity benefits. Methods and Results: In the present study, we conducted RNA sequencing to determine transcriptome-wide changes in gene expression in whole blood of healthy rats consuming diets supplemented with phytoestrogens. Ortholog cell deconvolution was applied to analyze the omics data. We discovered that PTS leads to changes in the gene expression landscape and PTS-target genes are associated with functions counteracting hallmarks of aging, including genomic instability, epigenetic alterations, compromised autophagy, mitochondrial dysfunction, deregulated nutrient sensing, altered intercellular interaction, and loss of proteostasis. These functions bridge together under anti-inflammatory effects through multiple pathways, including immunometabolism, where changes in cellular metabolism (e.g., ribosome biogenesis) impact the immune system. Conclusion: Our findings provide a rationale for pre-clinical and clinical longevity studies and encourage investigations on PTS in maintaining cellular homeostasis, decelerating the process of aging, and improving conditions with chronic inflammation.

overall_design: A total of 24 male Fischer 344 rats at 4 weeks of age were obtained from Charles River (Indianapolis, IN, USA) and housed 2 rats per cage in a temperature-controlled (24°C) room with a 12-h light/dark cycle, and ad libitum access to water and a chow diet [i.e., choline-suﬃcient amino acid-defined (CSAA)]. After a week of acclimation, rats were randomly divided into 4 groups, each containing 6 animals. Using the biological and analytical variability from previous data, it could be determined that 6 rats per group (n = 6) were required to detect a 50% difference with the variation of the endpoint 25% (i.e., gene expression measured by qRT-PCR) and with 80% power at a nominal significance of P < 0.05. The 4 groups were as follows: Group 1 - fed the CSAA diet; Group 2: fed a CSAA diet supplemented with resveratrol (CSAA+RSV, 1.2 g/kg of diet); Group 3 – fed a CSAA diet supplemented with pterostilbene (CSAA+PTS, 1.34 g/kg of diet); Group 4 – fed a CSAA diet supplemented with chlorogenic acid (CSAA+CGA, 1.9 g/kg of diet). All diets were pelleted at Dyets Inc. (Bethlehem, PA, USA). The compounds (BIOTANG Inc., Lexington, MA, USA) were used at equal molar concentrations. Following a 20-day feeding period, blood samples were collected by retro-orbital plexus sampling from anesthetized rats, where the retro-orbital plexus was penetrated with a capillary tube to promote blood flow from the capillaries behind the eye. The amount of collected blood did not exceed 10% of circulating blood volume based on body weight. Blood samples were collected from each rat into spray coated K2EDTA tubes (BD & Co,. Franklin Lakes, NJ, USA) and stored in -80°C until RNA was extracted. Rats were treated strictly following the animal use protocol #1112000342 approved by the Institutional Animal Care and Use Committee at the involved institutions. Mouse RiboPure™-Blood RNA Isolation Kit (Invitrogen) was used to extract RNA from rat whole blood samples as recommended by the manufacturer.

first: Marco

last: Tello-Palencia

first: Tony

first: Olga

last: Sularz

first: Louis

middle: E

last: Demers

first: Cayla

last: Boycott

first: Huiying

last: Zhang

first: Katarzyna

last: Lubecka-Gajewska

first: Sadhri

last: Kumar

first: Benjamin

middle: S

last: Ramsey

first: Sandra

last: Torregrosa-Allen

first: Bennett

middle: D

last: Elzey

first: Nadia

last: Atallah Lanman

first: Keegan

last: Korthauer


title: Contribution of Stroma to Inflammatory Bowel Disease Progression Revealed in Patient-Derived Human Colon Chips

city: Boston

first: Alican

last: Ozkan

state: MA

address: 201 Brookline Ave, Suite 401

department: Wyss Institute

institute: Harvard University

summary: Inflammatory bowel disease (IBD) patients exhibit compromised intestinal barrier function, enhanced inflammation, and increased cancer risk, with symptoms sometimes being exacerbated in women during pregnancy. To investigate the IBD phenotype and disease progression in human intestine, we leveraged organ-on-a-chip (Organ Chip) technology to engineer human Colon Chips lined by homotypic or heterotypic recombinants of intestinal epithelium interfaced with stromal fibroblasts isolated from Healthy or IBD patient-derived colon resections. Chips created with both epithelial and stromal cells from the same ulcerative colitis or Crohn's disease patient showed increased IBD-associated gene pathway activation, elevated pro-inflammatory cytokines, reduced mucus layer thickness, increased intestinal barrier permeability, compared to homotypic Healthy Colon Chips. When heterotypic tissue recombinants were created, the IBD stromal fibroblasts induced the IBD phenotype in healthy epithelium, and healthy colonic fibroblasts partially reduced inflammation in the IBD epithelium. Using this system, we discovered that peristalsis-like mechanical deformations directly impact colon tissue physiology by increasing mucin gene expression, mucus thickness, and barrier permeability in both Healthy and IBD Chips, and that they only stimulate a fibrotic response in IBD Chips.  We also found that both inflammation and fibrosis were accentuated in IBD Chips created with cells from a female patient that were exposed to pregnancy-associated hormones. Interestingly, exposure to a carcinogen (N-ethyl-N-nitrosourea) for 3 weeks in vitro induced histological changes and gene duplication in the IBD Chips, but not in the Healthy Chips. These data suggest that the stroma plays a key role in driving inflammation and disease progression in IBD patients and that human Organ Chip technology may represent a new preclinical tool to gain insight into these mechanisms as well as to identify novel diagnostic biomarkers and therapeutics.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for IBD pathology (Crohn's disease and Ulcerative Colitis) modeled in Human Colon Chips.

first: Megan

last: Sperry

first: Viktor

last: Horvath

first: Donald

last: Ingber


title: Neuronal Hyperactivity in Neurons Derived from Individuals with Grey Matter Heterotopia

city: Martinsried

first: Tobias

last: Straub

address: Großhadener Str. 9

department: Biomedical Center, Bioinformatics

country: Germany

institute: LMU Munich

summary: Periventricular heterotopia (PH), the most common form of grey matter heterotopia, represents a cortical malformation that is often associated with developmental delay and drug-resistant seizures1,2. The detailed neurophysiological underpinnings of PH symptoms in humans remain, however, elusive. Human cerebral organoids (hCOs) derived from patients with causative mutations in FAT4 or DCHS1 exhibit key features of PH3, but neuronal activity in these 3D models has not yet been investigated. Here, using silicon probe recordings, we detected exaggerated spontaneous spike activity in FAT4 and DCHS1 hCOs, suggesting functional changes in neuronal networks. Patch-clamp recordings revealed a decreased spike threshold exclusively for DCHS1 neurons, which presumably results from an enhanced density of somatic voltage-gated sodium channels. Furthermore, single-cell morphological reconstructions and immunostainings demonstrated greater morphological complexity of neurons and synaptic alterations, rather than an imbalance of excitatory-inhibitory neuron number, as contributing to the hyperactivity observed in FAT4 and DCHS1 hCOs. The morphological phenotype was rescued by an expression of wild-type DCHS1 in DCHS1 neurons. In addition, transcriptome and proteome analyses uncovered changes in GO terms associated with neuronal morphology and synaptic function. Overall, we provide detailed new insights into cellular alterations likely contributing to the emergence of symptoms in grey matter heterotopia.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for neuronal nuclei sorted from FAT4, DCHS1 and controls human cerebral organoids (hCOs)

first: Rossella

last: Di Giaimo

first: Ane

last: Martin

first: Francesco

last: Di Matteo


title: ZBTB16/PLZF regulates self-renewal and differentiation of spermatogonial stem cells through an extensive transcription factor-chromatin poising network [RNA-Seq]

first: Bradley

last: Carins

summary: Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression (Ccnd1) to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network.Spermatogonial stem cells (SSCs) balance self-renewal and differentiation to ensure continuous sperm production in the testis. The transcription factor Zbtb16 (PLZF) supports undifferentiated SSC maintenance through partly unknown mechanisms. We combined genomics (RNA-seq and ChIP-seq) and genetic approaches to reveal multiple functions of Zbtb16 in juvenile mouse SSCs. Zbtb16-bound loci show a striking correlation with active promoters bearing H3K4me3 and the activator Sall4. Zbtb16 activates genes that support SSC self-renewal and cell cycle progression (e.g., Ccnd1) that help maintain undifferentiated SSC pools, including both self-renewing SSCs and transit-amplifying progenitors. Zbtb16 also attenuates certain genes, including meiotic genes and specific retrotransposons that confer genome instability. Notably, Zbtb16 genome localization and its impact on the transcriptome are dynamic, displaying mesenchymal gene targets in vivo, which are not maintained in cultured SSCs. Our data reveal dynamic roles for Zbtb16 in ensuring SSC identity, amplification, and maintenance in vivo.

overall_design: To better understand Zbtb16 function in vivo, we isolated SSCs and then performed RNA-seq and histone modification ChIP-seq. We also performed ChIP-seq for the transcription factors Zbtb16 and Sall4 using whole testis to reveal directly regulated genes in postnatal mouse spermatogonia.

first: Chongil

last: Yi

last: Cairns


title: ZBTB16/PLZF regulates juvenile spermatogonial stem cell development via an extensive transcription factor poising network

summary: Spermatogonial stem cells balance self-renewal and differentiation/spermatogenesis to ensure continuous sperm production. Here, we identify roles for  the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG) in promoting self-renewal and cell cycle progression to maintain uSPG and transit-amplifying states. Notably, ZBTB16, SALL4, SOX3 co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions largely share broad H3K4me3 and H3K27ac, DNA hypomethylation, RNAPol2 and often CTCF. Hi-C analyses show robust 3D physical interactions among these co-bound promoters, suggesting the existence of a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not notably occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation, even when active. Overall, ZBTB16 promotes uSPG cell cycle progression and colocalizes with SALL4, SOX3, CTCF and RNAPol2 to help establish an  extensive and interactive chromatin poising network.

overall_design: Refer to individual Series


title: Transcriptome analyses of Leishmania donovani promastigotes depleted of SET7 and SET29 proteins, under normal and oxidative growth environment [exp 2]

status: Public on Jan 01 2025

city: New Delhi

first: Swati

last: Saha

state: Delhi

address: Benito Juarez Road

department: Department of Microbiology

country: India

institute: University of Delhi South Campus

summary: This study was performed with the goal of understanding the roles of Leishmania donovani SET7 and SET29 proteins in gene regulation. Using homologous recombination to knock out set7 and set29 genes, set7 and set29 mutant parasites were created. This was folllowed by transcriptome analyses of wild type parasites, set7 mutant parasites and set29 mutant parasites, that were carried out using RNA-seq.  In the first experimental set, transcriptomes of wild type parasites cultured under normal conditions as well as under hydrogen peroxide (H2O2) -induced oxidizing conditions were analysed, along with transcriptomes of set7 mutant parasites that were similarly grown under normal growth conditions as well as under H2O2-induced oxidizing conditions. Using the data obtained, the transcriptome of wild type parasites exposed to H2O2 has been compared with the transcriptome of wild type parasites grown under normal conditions. The transcriptome of set7 mutant parasites grown under H2O2-induced oxidizing conditions versus normal conditions have been similarly compared. In addition, the transcriptome of set7 mutant parasites grown under normal conditions has been compared to that of similarly grown wild type parasites. In the second experimental set, transcriptomes of wild type parasites cultured under normal growth conditions, set29 mutant parasites grown under normal conditions, as well as set29 mutant parasites grown under H2O2-induced oxidizing conditions, were analysed. Using the data obtained, the transcriptome of set29 mutant parasites grown under normal conditions has been compared to that of similarly grown wild type parasites. The transcriptome of set29 mutant parasites grown under H2O2-induced oxidizing conditions versus normal conditions have been similarly compared. Our data reveals, that knock out of set7 modulates transcription of specific clusters of genes on particular chromosomes, with almost 10 % of the total genes being regulated. On the other hand, depletion of SET29 modulates transcription of a very small set of genes distributed primarily in two clusters. This datasheet is for second experimental set.

overall_design: Two experimental sets were performed, each with two biological replicates of all samples. For transcriptome analysis, we first depleted the cells of LdSET7 and LdSET29 expression by sequentially knocking out two alleles of both the genes, which led to creation of set7-/- and set29-/-/+ cell lines. In the first experimental set, RNA was isolated  from logarithmically growing wild type and set7-/- promastigotes (two biologicals). RNA was also isolated  from logarithmically growing wild type and set7-/- promastigotes (two biologicals) that had been exposed to H2O2 (100 µM) for 5h followed by transfer of cells into fresh medium for 3 hours. In the second set of experiments, RNA was isolated  from logarithmically growing wild type and set29-/-/+ promastigotes (two biologicals). RNA was also isolated  from logarithmically growing set29-/-/+ promastigotes (two biologicals) that had been exposed to H2O2 (100 µM) for 5h followed by transfer of cells into fresh medium for 3 hours.This was followed by RNA sequencing. We then performed comparative gene expression profile analysis, among different samples. This datasheet is for second experimental set

first: Varshni

last: Sharma

first: Jyoti

last: Pal

first: Somdeb

last: Chattopadhyay

first: G

last: Arimbasseri


title: Identification of L1TD1-associated transcripts in DNMT1 KO HAP1 cells

city: Vienna

first: Christian

last: Seiser

address: Schwarzspanierstrasse 17

department: Center for Anatomy and Cell Biology

country: Austria

institute: Medical University of Vienna

summary: In this study, we aimed at determining RNA interactors of L1TD1 RNPs. To achieve this,  DNMT1 KO and DNMT1 L1TD1 DKO HAP1 cells were generated by Crispr/Cas9 gene editing tool and L1TD1 RIP was performed by using these cell lines. Our study reveals a comprehensive analysis of differentially enriched transcripts in L1TD1 containing RNPs.

overall_design: We performed RIP-seq by L1TD1 immunoprecipitation followed by RNA isolation from immunoprecipitated complexes using DNMT1 KO and DNMT1 L1TD1 DKO HAP1 cells in biological triplicates. Differential enrichment analysis was done using DESeq2. 10% input used as a reference to for the enrichment of the transcripts in relate to their expression. DNMT1 L1TD1 DKO cells were used as a negative control as they lack L1TD1 expression.


title: Nup107 contributes to the maternal to zygotic transition by regulating the nuclear exit of pri-miRNA 427

city: Exeter

first: Nick

last: Owens

state: Exeter

address: Barrack Road

country: United Kingdom

institute: University of Exeter Medical School

summary: High temporal resolution profiling of Xenopus tropicalis transcriptome during early development in control and nup107 MO conditions.

overall_design: Synchronously developing embryos were collected, harvesting 10 embryos every 30 mins from stage 6 (2.5 hours post fertilization) until stage 12 (12 hours post fertilization) in both control and nup107 morpholino conditions.

first: Valentyna

last: Kostiuk

first: Mustafa

last: Khokha


title: Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer II

first: Steven

last: Balk

address: 3 Blackfan Circle

department: Hematology/Oncology

institute: Harvard Medical School

summary: Androgen receptor (AR) splice variants, of which ARv7 is most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which they drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that was resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full length AR (ARfl), despite its continued high level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in these cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and X knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for ARfl and ARv7 in ENZ-resistant VCaP16 cells in response to NFIB and NFIX knokdown with siRNA (siNFIBX) vs siRNA non-targeted control (siNTC).

first: Larysa

last: Poluben


title: Expression profile of DNMT1 KO and DNMT1 L1TD1 HAP1 cells by high throughput sequencing

summary: In this study, we aimed at analyzing gene expression profile of DNMT1 KO and DNMT1 L1TD1 DKO HAP1 cells using high throughput sequencing. Our study reveals a comprehensive analysis of differentially expressed transcripts in the absence of L1D1.

overall_design: To delineate the role of of L1TD1 in HAP1 cells, we deleted L1TD1 in DNMT1 KO background as WT HAP1 cells do not express L1TD1. We analyzed differentially expressed transcripts in DNMT1 KO with respect to the negative control  DNMT1 L1TD1 DKO HAP1. the experiment included biological triplicates for each genotype.


title: Gliomagenesis mimics an injury response orchestrated by neural crest-like cells

city: Toronto

first: Akram

last: Hamed

department: Molecular Genetics

institute: University of Toronto

summary:
To explore the early steps in glioma formation, we utilized conditional gene deletion and lineage tracing in tumour mouse models, coupled with serial magnetic resonance imaging to initiate and then closely track tumour formation. We isolated labeled and unlabeled cells at multiple stages -- before the first visible abnormality, at the time of the first visible lesion, and then through the stages of tumour growth -- and subjected each stage to single-cell profiling. We identify a malignant cell state with a neural crest-like gene expression signature that is highly abundant in the early stages, but relatively diminished in the late stage of tumour growth. Genomic analysis based on the presence of copy number alterations suggests that these neural crest-like states exist as part of a heterogenous clonal hierarchy that evolves with tumour growth. By exploring the injury response in the wounded normal mouse brain, we identify cells with a similar signature that emerge following injury and then disappear over time, suggesting that activation of an injury response programme occurs during tumourigenesis. Indeed, our experiments reveal a non-malignant injury-like microenvironment that is initiated in the brain following oncogene activation in cerebral precursor cells. Collectively, our findings provide insight into the early stages of gliomagenesis, identifying a unique stem cell-like state and an injury response programme tied to early tumour formation. This will have implications on glioblastoma therapies and raises exciting new possibilities for early disease diagnosis and prevention.
Some of the data in this series can also be explored through the following App link: gbm.stem-cells.ca.

overall_design:
1) Tumourigenesis atlas: we explored the early stages in tumour formation by combining glioma mouse modelling with serial magnetic resonance imaging (MRI) and single-cell profiling. We used genetically engineered mouse models to trace the fate of subpopulations of cells, with labelling initiated in either the pre- or postnatal brain. Brain tissue was harvested from mutant mice at four MRI-defined stages of tumourigenesis and samples were characterized by scRNA-seq.
2) Brain injury: to create an injury phenotype, we implanted an intracranial cannula in the right hemisphere of 4-6 week old non-mutant Sox2eGFP mice at stereotactic coordinates that place the cannula near the subventricular zone. The cannula was connected to an osmotic pump to slowly infuse the brain with saline over a period of 5 days. This resulted in structural tissue damage on the ipsilateral hemisphere.


title: Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing

status: Public on Jan 05 2025

first: Charles

last: Gersbach

type: Methylation profiling by high throughput sequencing

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


title: scRNAseq of the TCRs and BCRs of aqueous and blood immune cells in human uveitis

last: Concepcion

state: Colorado

institute: University of Colorado Denver Anschutz Medical

summary: We profiled using single cell RNA sequencing the immune cells of the aqueous humor from patients with uveitis as well as their peripheral blood. Then processed their BCR repertoire and TCR repertoire

overall_design: We sampled 100-200 ul of the inflamed anterior chamber fluid using a 25- to 30-gauge needle. We pelleted cells by centrifugation (400 x g, 5 min) to remove the aqueous fluid and cryopreserved cells in fetal bovine serum containing 10% DMSO. To profile cells by single cell RNA sequencing, we used the 10x Genomics 5'v2 gene expression kit.

first: Lynn

middle: M

last: Hassman


title: Distinct molecular features in early-onset lung adenocarcinoma

city: Shenyang

first: Rui

last: Ma

address: 4 Chongshan East Road

institute: The Fourth Affiliated Hospital of China Medical University

summary: To investigate the difference in gene expression between early-onset and late-onset lung adenocarcinoma, we performed gene expression profiling analysis using data obtained from RNA-seq of 14 fresh tumor specimens

overall_design: Comparative gene expression profiling analysis of RNA-seq data for early-onset and late-onset lung adenocarcinoma.

first: Ye

last: Tian


title: Loss of ARID1A leads to a cold tumor phenotype via suppression of IFNγ signaling

first: Pamela

middle: Morgan

last: Watson

state: Tennessee

address: 71 South Manassas

department: Genetics

institute: University of Tennessee Health Science Center

summary: The collapse of inflammatory signaling that recruits cytotoxic immune cells to the tumor microenvironment contributes to the immunologically cold tumor phenotype in neuroblastoma (NB) and is a barrier to NB immunotherapy. Multiple studies have reported that MYCN amplification, a trait of high-risk NB, correlates with a loss of inflammatory signaling; but MYCN also correlates with 1p36 deletions in NB where the SWI/SNF chromatin remodeling complex subunit ARID1A (1p36.11) is located. ARID1A is known to support inflammatory signaling in adult cancers but its role in NB inflammatory signaling is unexplored.  We find MYCN overexpression causes a stronger and ARID1A knockdown causes a weaker inflammatory response to interferon-gamma (IFNγ). ARID1A silencing reduces IFNγ induced gene signatures for the transcription factor interferon response factor 1 (IRF1). We found ARID1A is a functional interactor of IRF1 by co-immunoprecipitation studies, and ARID1A silencing causes loss of activating chromatin marks at the IRF1 target gene CXCL10. We model that IRF1 uses ARID1A containing SWI/SNF to promote IRF1’s target genes in response to IFNγ. This work finds that ARID1A is a critical regulator of inflammatory signaling in NB and provides rationale for testing immune therapies in MYCN amplified NB that are effective in adult ARID1A mutated cancers.

overall_design: SK-N-DZ cells were transfected with doxycycline inducible shRNA to ARID1A. Samples were then treated with interferon gamma.

first: Chelsea

last: DeVaux

first: Kevin

last: Freeman


title: Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer [scATAC-Seq]

overall_design: To study adaptational mechanisms of ENZ-resistance in prostate cancer,  VCaP cells were cultured in medium with increasing concentrations of ENZ (up to 16 uM) over ~2 months to select for resistance. A subline resistant to ENZ was generated (VCaP16). We studied chromatin architecture changes on a single-cell level by single-cell ATAC-seq (scATAC) in responce to short 10uM ENZ treatment for 96hrs in VCaP and in ENZ-resistant VCaP16 cells compared to parental VCaP in DMSO.

first: Joshua

last: Russo

first: Mannan

last: Nouri


title: Stem cell niche ageing involves coordinated changes in transcription and alternative splicing

status: Public on Dec 31 2024

city: Barcelona

first: Manuel

last: Irimia

state: Barcelona

address: 88 Dr. Aiguader

department: MELIS

country: Spain

institute: Universitat Pompeu Fabra - Centre for Genomic Regulation

summary: Gene expression (GE) determines the functionality and properties of cell types. Alternative splicing (AS) enhances the complexity of the transcriptome, expanding the protein interactome. As GE and AS jointly contribute to the formation of new interaction networks with potentially significant cellular functions, they could serve as the foundation for fundamental biological processes like ageing. Here, we investigate ageing in the Drosophila female germline stem cell (GSC) niche and describe functional changes in both GE and AS in aged flies. The GSC niche comprises escort cells, terminal filament cells and cap cells. Comparison of their transcriptomes reveals that, while all these cell types share genes exhibiting differential GE and AS variations related to cell adhesion, cytoskeleton and neural signalling during ageing, each population also experiences distinctive changes. This demonstrates unique ageing signatures within niche cell types. In addition and depending on the niche cell population, sets of genes display changes in both GE and AS, revealing a coordinated effort in the regulation of transcription and splicing during niche ageing. One such gene is Fasciclin 2, a neural adhesion molecule which we report to be essential for niche functioning. Furthermore, some genes involved in AS undergo changes in GE and/or AS themselves, providing a mechanistic explanation for the coordination of these two processes in the regulation of stem cell niche ageing. This is the case of the splicing factor Smu1, described here as a key element necessary for ovarian niche homeostasis.

overall_design:
To investigate the changes in gene expression and alternative splicing during ageing in different populations of support cells in the ovarian niche of the Drosophila female. We compared niche cells isolated from 1 week (1w)-old and 4w-old ovaries. We generated 2 replicates of each age per cell type group (group 1: terminal filament cells + cup cells; group 2: escort cells)
We also studied the differentially expressed genes and alternative splicing in niche cells with reduced levels of the splicing factor Smu1. Generated 2 replicates of control cells and 2 of experimental cells.

first: Dilamm

last: Even-Ros

first: Judit

last: Huertas-Romero

first: Miriam

last: Marín-Menguiano

first: Gretel

last: Nusspaumer

first: Miguel

last: Borge

first: Federico

last: Zurita

first: Acaimo

last: González-Reyes


title: Neuroinflammation results in dysfunctional mitral cells and olfactory impairment

city: Hamburg

first: Charlotte

last: Schubert

address: Falkenried 94

department: Department of Neurology, University Hospital Hamburg - Eppendorf

institute: Institute of Neuroimmunology and Multiple Sclerosis Hamburg

summary: Olfactory dysfunction is an underestimated symptom in multiple sclerosis (MS). Here, we examined the pathogenic mechanisms underlying inflammation-induced dysfunction of the olfactory bulb using the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Reduced olfactory function in EAE was associated with the degeneration of short-axon neurons, immature neurons, and mitral cells, together with their synaptic interactions and axonal repertoire. To dissect the mechanisms underlying the susceptibility of mitral cells, the main projection neurons of the olfactory bulb, we profiled their responses to neuroinflammation by single-nucleus RNA sequencing. Neuroinflammation resulted in the induction of potassium channel transcripts in mitral cells, which was reflected in reduced halothane-induced outward currents of these cells, likely contributing to the impaired olfaction in EAE animals.

overall_design: HuC/D+ nuclei were isolated by fluorescence activated cell sorting (FACS) representing mainly neuronal nuclei of olfactory bulb of 10-week old female C57Bl/6J mice at the acute phase of the MS model experimental autoimmune encephalomyelitis (EAE) together with their healthy control littermates.

first: Marcel

last: Woo

first: Alexandros

last: Hadjilaou

first: Max

last: Kaufmann


title: Sex-specific and multiomic integration enhance accuracy of peripheral blood biomarkers of major depressive disorder [EPIC]

city: Strasbourg

first: Pierre-Eric

last: Lutz

address: 8 allée du général Rouvillois

country: France

institute: Institut des Neurosciences Cellulaires et Intégratives, INCI CNRS UPR3212

type: Methylation profiling by genome tiling array

summary: Major depressive disorder (MDD) is a leading cause of disability and reduced life expectancy, with a two-fold increase in prevalence in women compared to men. Over the last few years, identifying reliable molecular biomarkers of MDD has proved challenging, likely reflecting the fact that, in addition to sex-differences, a variety of environmental and genetic risk factors are implicated. Recently, epigenetic processes have been proposed as mediators of the impact of life experiences on functional regulation of the genome, with the potential to contribute to MDD biomarker development. In this context, here we characterized and integrated transcriptomic gene expression data with two upstream mechanisms for epigenomic regulation, DNA methylation and micro-RNAs. The 3 molecular layers were analyzed in peripheral blood samples from a well-characterized cohort of individuals with MDD (n=80) and healthy controls (n=89), and processed using 3 complementary bioinformatic strategies. First, we conducted case-control comparisons for each single omic layer, and contrasted sex-specific adaptations. Second, we leveraged network theory to define gene co-expression modules, followed by step-by-step annotations across omic layers. Finally, we implemented a genome-wide and multiomic integration strategy that included cross-validation and bootstrapping. The approach was used to systematically compare the accuracy of MDD prediction across 6 methods for dimensionality reduction and, importantly, for every combination of 1, 2 or 3 types of molecular data. Results showed that accuracy was higher when female and male cohorts were analyzed separately, rather than combined, and also progressively increased with the number of molecular datasets considered. While multiomic informational gain has already been illustrated in other medical fields, our results pave the way towards similar advances in molecular psychiatry, and have practical implications towards developing clinically useful biomarkers of MDD.

overall_design: Examination of DNA methylation in human peripheral blood samples from depressed inviduals and healthy controls

first: Amazigh

last: Mokhtari

first: El

last: Cherif Ibrahim

first: Arnaud

last: Gloaguen

first: Claire-Cécile

last: Barrot

first: Raoul

last: Belzeaux

first: Andrée

last: Delahaye-Duriez


title: Sex-specific and multiomic integration enhance accuracy of peripheral blood biomarkers of major depressive disorder [RNA-Seq]

overall_design: Examination of gene expression and micro-RNAs in human peripheral blood samples from depressed inviduals and healthy controls


title: Epithelial regeneration is modulated by chitin and chitinase activity

city: St Louis

first: Steven

last: Van Dyken

state: MO

address: 425 S. Euclid Ave

institute: Washington University in St. Louis

summary: Epithelial injury underlies fibrotic and inflammatory lung diseases during which regenerative responses become dysregulated or recurrently engaged.  Although environmental exposures are risk factors for severe lung disease, specific drivers of persistent epithelial and immune dysfunction are poorly understood.  Here we identify a feedback circuit triggered by chitin, a common component of airborne particulate matter, that impacts lung health and regeneration after epithelial injury.  In mice, damage to epithelial cells results in loss of homeostatic lung chitinase activity and accumulation of environmental chitin substrates; these disturbances impair epithelial renewal and drive activation of group 2 innate lymphoid cells (ILC2s).  ILC2s, in turn, restore chitinase activity by inducing acidic mammalian chitinase (AMCase) in regenerating epithelial cells, thereby promoting chitin degradation, epithelial differentiation, and inflammatory resolution.  Mice lacking AMCase or ILC2s fail to clear airway chitin and exhibit exacerbated inflammation, impaired epithelial regeneration, and increased mortality following epithelial injury.  These effects are ameliorated by chitinase replacement therapy or AMCase overexpression, demonstrating that chitin degradation is crucial for restoring lung homeostasis after perturbation.  The ILC2-chitinase response circuit thus comprises a tissue adaptation to a widespread environmental constituent and may be a target for alleviating persistent post-injury lung epithelial and immune dysfunction.

overall_design: To investigatae whether chitin treatment in WT and AMCase deficient epithelial cells alters differentiation  proliferation program, mice were intransally challenged with chitin particles prior to sacrifice after 12 hours for isolation and analysis of  mature lung or progenitor-like epithelial cells through RNA sequencing.

first: Haerin

last: Jung


title: Transcriptomics Unveil Canonical and Non-Canonical Heat Shock-Induced Pathways in Human Cell Lines

city: Fullerton

first: Nikolas

last: Nikolaidis

state: CA

address: 800 N. State College Blvd

department: Biological Science

institute: California State Univeristy Fullerton

summary: The cellular stress response (CSR) is a conserved mechanism that protects cells from environmental and physiological stressors. The heat shock response (HSR), a critical component of the CSR, utilizes molecular chaperones to mitigate proteotoxic stress caused by elevated temperatures. We hypothesized that while the canonical HSR pathways are conserved across cell types, specific cell lines may exhibit unique transcriptional responses to heat shock. To test this, we compared the transcriptomic responses of HEK293, HepG2, and HeLa cells under control conditions immediately following heat shock and after an 8-hour recovery period. RNA sequencing revealed conserved activation of canonical HSR pathways, including the unfolded protein response, alongside enrichment of the non-canonical “Receptor Ligand Activity” pathway across all cell lines. Cell line-specific variations were also observed, with HepG2 cells displaying higher transcriptional activity under stress conditions. Validation by qPCR confirmed the activation of key genes within the “Receptor Ligand Activity” pathway across time points. These findings provide insights into conserved and context-specific aspects of the HSR, contributing to a more comprehensive understanding of stress response mechanisms a cross mammalian cells.

overall_design: A comprehensive RNA sequencing (RNA-seq) analysis was conducted comparing cancerous (HeLa, HepG2) and non-cancerous (HEK293) cell lines exposed to heat stress.

first: Andrew

last: Reinschmidt

first: Luis

last: Solano

first: Yonny

last: Chavez

first: William

last: Hulsy


title: Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer

last: Balk

overall_design: Refer to individual Series


title: Sex-specific and multiomic integration enhance accuracy of peripheral blood biomarkers of major depressive disorder

type: Non-coding RNA profiling by high throughput sequencing


title: Bulk RNA seq on cerebral cortices derived from WT and Mecp2 null mice (P60) tretaed or not with rhNGF

city: Verona

first: Marzia

last: Rossato

state: Italy

address: Strada Le Grazie,15

department: Department of Biotechnology

country: Italy

institute: University of Verona

summary: MECP2 deficiency causes a broad spectrum of neuropsychiatric disorders that can affect both genders, among which Rett syndrome is the most frequent. Main feature of Rett syndrome is an apparent normal development followed by a regression phase in which patients lose most of the previously acquired skills. After this dramatic period, several symptoms progressively get in, including severe intellectual disability, epilepsy, apraxia, breathing abnormalities and motor deterioration. MECP2 codes for an epigenetic transcriptional factor particularly abundant in brain; accordingly, several transcriptional defects characterize the Rett syndrome brain. The well recognized deficiency in different neurotrophins and growth factors in Rett patients and mouse models of Mecp2 deficiency led us to investigate the therapeutic potential of nerve growth factor. The study was performed administrating a GMP-grade recombinant human NGF (rhNGF). To identify  molecular mechanisms triggering the observed benefits an unbiased RNAseq study was performed.

overall_design: 6 WT and 6 Mecp2 null mouse male brain cortices (P60) treated or not (control) with rhNGF from P30 to P60. Untreated mice were treated with vehicle (PBS).

first: Pozzer

last: Diego

first: Breccia

last: Martina

first: Florio

last: Elena

first: Antonio

last: Fadda

first: Nicoletta

last: Landsberger


title: Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer [ATAC-Seq]

overall_design: Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) in VCaP, VCaP in DMSO, VCaP treated with ENZ for 96hrs and enzalutamide-resistant VCaP16 cells.

first: Larysa

last: Poluben

first: Joshua

last: Russo

first: Mannan

last: Nouri


title: Hypoxia induced ATF3 escalates breast cancer invasion by increasing collagen deposition

city: Bhopal

first: Sanjeev

last: Shukla

state: Madhya Prades

address: Bhopal Bypass Road, Bhauri Bhopal 462 066 Madhya Pradesh, INDIA

department: Biological Sciences

institute: Indian Institute of Science Education and Research Bhopal

summary: Activating transcription factors (ATFs), members of the adaptive-response gene family, participate in cellular processes to aid adaptations in response to extra- and/or intracellular changes. In this study, we observed that one of the ATFs Activating transcription factor 3 (ATF3) is upregulated under hypoxia via alterations in the epigenetic landscape of its promoter, followed by transcriptional upregulation. Under hypoxic conditions, Hypoxia-inducible factor 1-alpha (HIF1ɑ) alleviates methylation at the ATF3 promoter by recruiting TET1 and  induces ATF3 transcription. Additionally, our RNA-seq analysis showed that ATF3 globally affects transcription under hypoxia and controls the processes of EMT and cancer invasion by stimulating the transcription of Prolyl 4-Hydroxylase Subunit Alpha 1 (P4HA1), an enzyme which enhances invasion conducive extracellular matrix (ECM) under hypoxic condition. Prolyl hydroxylases play a critical role in the hydroxylation and deposition of collagen in the extracellular matrix (ECM) during the evolution of cancer, which is necessary for metastasis. Importantly, P4HA1 undergoes alternative splicing under hypoxia, where the inclusion of exon 9a is increased. It is interesting to note that ATF3's involvement in P4HA1 splicing was also evident, as binding of ATF3 at intron 9a led to demethylation of this DNA region via recruitment of TET1. Further, we also show that the demethylated DNA region of intron 9a then becomes accessible to CTCF. Thus, a cascade of demethylation via ATF3 recruited TET1, followed by increased RNA PolII pause via CTCF at the intron 9a leads to inclusion of exon 9a. The P4HA1 9a isoform leads to enhanced invasion under hypoxic conditions by increasing deposition of collagen in the ECM. These results provide a novel  hypoxia-induced HIF1ɑ-ATF3-P4HA1 axis which can be potentially exploited as a therapeutic target to impede EMT and ultimately breast cancer invasion.

overall_design: shControl and shATF3 MCF7 cells were treated under hypoxic conditions for 24 hours

first: Shruti

middle: G

last: Dhamdhere


title: Radiation-Induced Cellular Plasticity Primes Glioblastoma for Forskolin-Mediated Differentiation [bulk RNA-seq]

status: Public on Dec 30 2024

city: Los Angeles

first: Frank

last: Pajonk

address: 10833 Le Conte Ave

institute: UC Los Angeles

summary: Glioblastoma is the deadliest brain cancer in adults and all patients succumb to the tumor. While surgery followed by chemo-radiotherapy delays disease progression, these treatments do not lead to tumor control and targeted therapies or biologics have failed to further improve survival. Utilizing a transient radiation-induced state of multipotency, we used the adenylcyclase activator forskolin to alter the fate of irradiated glioma cells. The effects of the combined treatment on neuronal marker expression, cell cycle distribution and proliferation were studied. Gene expression profiling was conducted using bulk RNA-seq. Changes in cell populations were investigated using single cell RNA-seq. Effects on glioma stem cells were studied in extreme limiting dilution assays and the effects on median survival were studied in both syngeneic and PDOX mouse models of glioblastoma. The combined treatment induced the expression of neuronal markers in glioma cells, reduced proliferation and led to a distinct gene expression profile. scRNA-seq revealed that the combined treatment forced glioma cells into a microglia- and neuron-like phenotype. In vivo, this treatment led to a loss of glioma stem cells and prolonged median survival. Collectively, our data suggest that revisiting a differentiation therapy with forskolin in combination with radiation could lead to clinical benefit.

overall_design: HK-374 cells were seeded into 6-well plates as monolayer cultures at 40,000 cells/well. 4 days after seeding the cells were irradiated with 4 Gy. Controls were sham irradiated. 48 hours later cells were treated with either forskolin or DMSO for 5 consecutive days.


title: CUT&Tag analysis of H3K4me3, H3K27Ac,H2A119Ub, H3K27me3 and H3K36me3 in KPC1199 murine PDAC cell line

city: Shanghai

first: Ningning

last: Niu

address: 160 Pu Jian Road, Shanghai

department: Clinical Stem Cell Research Center

institute: Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital

summary: To gain mechanistic insight into how Epigenetic factors reprogramming metabolism in response to glutamine starvation. we performed CUT&Tag sequencing in murine PDAC cells.  KPC1199 cells were treated in Cont group or Low Gln group , and harvested after 72 hours. CUT&Tag assay was performed following the manual of hyperactive pG-Tn5/pA-Tn5 transposase for CUT&Tag kit (TD901, Vazyme). DNA library were prepared according to manufacturer’s instructions of Trueprep index kit v2 (TD202, Vazyme).

overall_design: For library preparation, murine PDAC cell lines with Cont, Low Gln were first established. CUT&Tag and DNA library were prepared according to manufacturer’s instructions.

first: Xuqing

last: Shen

first: Jing

last: Xue


title: Radiation-Induced Cellular Plasticity Primes Glioblastoma for Forskolin-Mediated Differentiation [Day21]

overall_design: HK-374 gliomaspheres were plated onto the Poly-D-Lysine/Laminin 6-well plate and irradiated with 4 Gy the next day. 48 hours after the irradiation, the cells were treated with forskolin (10 µM) for 21 consecutive days.

first: Ling

last: He


first: Steven

last: Van Dyken

overall_design: To investigate whether influenza virus infection in WT and AMCase deficient epithelial cells alters differentiation and proliferation program, mice were intransally inoculated with PFU 250 of H1N1/PR8 strain of influenza virus intranasally under isoflurane anesthesia prior to sacrifice 14 days post infection for isolation and analysis of progenitor-like epithelial cells through RNA sequencing.

first: Haerin

last: Jung


title: Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer [WES]

last: Balk

overall_design: Whole Exome Sequencing (WES) in VCaP and enzalutamide-resistant VCaP16 cells.


title: Sex-specific and multiomic integration enhance accuracy of peripheral blood biomarkers of major depressive disorder [miRNA-Seq]

first: Pierre-Eric

last: Lutz

first: Amazigh

last: Mokhtari

first: El

last: Cherif Ibrahim

first: Arnaud

last: Gloaguen

first: Claire-Cécile

last: Barrot

first: Raoul

last: Belzeaux

first: Andrée

last: Delahaye-Duriez


title: Condensation of ZFP207 and U1 snRNP promotes spliceosome assembly [RAP-seq]

city: Hangzhou, Zhejiang, China

first: yuenan

last: zhou

address: Yuhangtang Road, Hangzhou, Zhejiang, 310058, China

department: School of Medicine

institute: Zhejiang University

summary: U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 largely recapitulates the molecular phenotypes observed with the depletion of SNRNP70, a core component of U1 snRNP. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to U1 snRNA, while its C-terminus undergoes phase separation via intrinsically disordered regions (IDRs) to forms biomolecular condensates with U1 snRNP. These condensates create a crowded molecular environment that increases the local concentration of splicing snRNPs and regulators, thereby accelerating the speed of spliceosome assembly by facilitating interactions between U1 snRNP and other snRNPs. Collectively, our study demonstrates the critical role of phase separation in ensuring proper U1 snRNP function and efficient spliceosome assembly.

overall_design: Briefly, harvest the ZFP207 AID mESCs with or without IAA treatment and incubate them with 0.5 mg/ml AMT solution on ice for 15 minutes. Transfer the cell suspension to a 6 cm dish and irradiate with 365 nm UV for 15 minutes. Collect the cells, wash with PBS, and spin down. Then add nuclear lysis buffer, and sonicate the samples at 30% amplitude for 5 minutes. Biotinylated U1 probes were added and hybridize at 39°C for 1 hour, then add streptavidin beads and incubate at 39°C for another 1 hour. After thorough wash with 0.2×SSC washing buffer (0.2×SSC, 1% SDS), elute the RNA with RNase H digestion. The eluent RNAs were further reverse-crosslinked by irradiating with 254 nm UV in the presence of acridine orange for 15 minutes, and then used for subsequent library construction.

first: Yuenan

last: Zhou

first: Chong

last: Tong

first: Xiaoyu

last: Li

first: Yafei

last: Yin


title: Radiation-Induced Cellular Plasticity Primes Glioblastoma for Forskolin-Mediated Differentiation [Day 5]

first: Frank

last: Pajonk

overall_design: HK-374 gliomaspheres were plated onto the Poly-D-Lysine/Laminin 6-well plate and irradiated with 4 Gy the next day. 48 hours after the irradiation, the cells were treated with forskolin (10 µM) for 5 consecutive days.


title: Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer [ChIP-Seq]

city: Boston

summary: Androgen receptor (AR) splice variants, of which ARv7 is most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which they drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that was resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full length AR (ARfl), despite its continued high level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in these cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and X knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for ARfl and ARv7 in VCaP and ENZ-resistant VCaP16 cells in response to 10uM ENZ 4hrs or 10nM DHT 4hrs treatment. ChIP-seq for H3K27Ac and H3K4me1 in VCaP and ENZ-resistant VCaP16 cells. ChIP-seq for ARv7  in LNCaP95 cells in responce to 10nM DHT, 100nM dexamethasone (DEX) or DMSO for 2hrs. ChIP-seq for H3K27Ac, BRD4 and p300  in VCaP16 cells in responce to 500nM ARCC-32 or DMSO for 24hrs. ChIP-seq for ARv7  in VCaP16 cells in responce to 500nM ARCC-23 or DMSO for 36hrs.

first: Larysa

last: Poluben

first: Joshua

last: Russo

first: Mannan

last: Nouri


title: TGFβ links EBV to multisystem inflammatory syndrome in children

city: Berlin

first: Frederik

middle: Richard

last: Heinrich

state: Berlin

address: Chariteplatz 1

department: Bioinformatics

summary: In a subset of children and adolescents, SARS-CoV-2 infection leads to multisystem inflammatory syndrome in children (MIS-C), a severe hyperinflammatory shock occurring 4-8 weeks post-infection. MIS-C is characterized by a specific expansion of TCRVβ21.3+ T-cells and systemic hyperinflammation, with an unclear pathogenesis. Here, we show that acute MIS-C exhibits impaired reactivation of virus-specific memory T cells due to elevated TGF-β levels, mirroring severe COVID-19 in adults. Functionally, the impaired T-cell cytotoxicity is accompanied by TGF-β-response signatures and reduced antigen-presentation capabilities of monocytes, reversible by TGF-β blockade. MIS-C TCRVβ21.3+ T cells resemble Epstein-Barr Virus (EBV)-reactive T cells, displaying enhanced capability in eliminating EBV-infected B cells. Clinically, active MIS-C correlates with TGF-β-induced defects in T-cell cytotoxicity, elevated EBV seroprevalence, and EBV-reactivation.  Our findings establish a connection between SARS-CoV-2 infection and COVID-19 sequelae in children, where reduced T-cell cytotoxicity induced by SARS-CoV-2-triggered TGF-β overproduction leads to EBV-reactivation and contributes to hyperinflammation.

overall_design: This experiment was designed for an in-depth analysis of activated human T cells, memory B cells and monocytes from patients with MIS-C. To do so, single cell transcriptomes and TCR sequencing of sorted memory B cells, activated T cells and monocytes from 11 patients hospitalized with MIS-C plasmablasts sorted from the blood of 3 patients with MIS-C and 4 children 6 weeks after infection with SARS-CoV-2 were prepared. Additionally sorted paired samples from 4 patients before and after treatment with methylprednisolone (20mg/kg/day for 3 days) were prepared for single cell transcriptomics. Furthermore cells from healthy donors were incubated with viral peptides (derived from SARS-CoV-2, EBV, CMV, AdV and measles) and subsequently T cells were sorted for peptide specific reactivation. In addition, cells were also incubated with DNA barcoded antibodies for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). Sequencing was performed using the 10X Genomics platform with the Single Cell 5’ Library & Gel Bead Kit v2 (dual index) and Illumina NextSeq2000 or NextSeq500.

first: Carl Christoph

last: Goetzke

first: Mona

last: Massoud

first: Gabriela Maria

last: Guerra

first: Jan

last: Licha

first: Katrin

last: Lehmann

first: Pawel

last: Durek

first: Tilmann

last: Kalliinich

first: Mir-Farzin

last: Mashreghi


title: Condensation of ZFP207 and U1 snRNP promotes spliceosome assembly

first: yuenan

last: zhou

summary: U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 results in a series of molecular phenotypes indicative of U1 snRNP dysregulation. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to the stem-loop 3 (SL3) of U1 snRNA, while its C-terminal intrinsically disordered regions (IDRs) undergo phase separation to form biomolecular condensates with U1 snRNP. These condensates create a crowded molecular environment that increases the local concentration of splicing snRNPs and regulators, thereby accelerating the speed of spliceosome assembly by facilitating interactions between U1 snRNP and other snRNPs. Collectively, our study demonstrates the critical role of phase separation in ensuring effective U1 snRNP function and promoting efficient spliceosome assembly.

first: Zhou

last: Yuenan

first: Tong

last: Chong

first: Shi

last: Zuokun

first: Zhang

last: Yan

first: Xiong

last: Xushen

first: Shen

last: Xiaohua

first: Li

last: Xiaoyu

first: Yin

last: Yafei


title: ZFP207 interacts with U1 snRNP to promote spliceosome assembly via phase separation  [ZFP207_AID_TT-seq]

summary: U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 recapitulates the molecular phenotypes observed with the depletion of SNRNP70, a core component of U1 snRNP. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to U1 snRNA, while its C-terminus undergoes phase separation via intrinsically disordered regions (IDRs). The coordination between the N-terminus and C-terminus of ZFP207 drives the formation of biomolecular condensate with U1 snRNP, which creates a molecular environment to promote spliceosome assembly by facilitating the interactions between U1 snRNP and other splicing regulators. Collectively, our study demonstrates the critical role of ZFP207-mediated phase separation in ensuring proper U1 snRNP function and spliceosome assembly.

overall_design:
To elucidate the hypothesis of the depletion of ZFP207 impairs the efficiency of spliceosome assembly, we employed transient transcriptome sequencing (TT-seq) to assess splicing efficiency.
This was achieved by determining the ratio of spliced reads to newly synthesized reads during a 10-min window of 4sU labeling.
To minimize potential confounding effects from other RNA synthesis and processing processes, our analysis specifically focused on reads that spanned the boundary between the last nucleotide of the intron and the initial nucleotide of its adjacent downstream exon, and calculated the spliced-to-unspliced reads ratio to serve as a representative indicator of the splicing efficiency of the corresponding intron.

first: Yuenan

last: Zhou

first: Chong

last: Tong

first: Xiaoyu

last: Li

first: Yafei

last: Yin


title: The role of compensatory metabolic reprogramming in ensuring PDAC cell survival under the limitation of glutamine

first: Ningning

last: Niu

summary: KPC1199 cells were treated in Cont group(2 mmol/L glutamine and 2.25 g/L glucose) or Low Gln group (0.4 mmol/L glutamine and 2.25 g/L glucose) , and harvested after 72 hours for RNA preparation.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for KPC1199 cells with or without glutamine starvation

first: Xuqing

last: Shen

first: Jing

last: Xue


title: Condensation of ZFP207 and U1 snRNP promotes spliceosome assembly [RIP-Seq]

overall_design: Firstly, we cultured ZFP207AID mESCs in medium containing 200 μM 4sU for 24 hours, then harvested the cells and crosslinked them with UV365 (200 mJ). We then performed ZFP207 bioRIP without RNase I digestion to ensure the integrity of U1 snRNA. Two sequential streptavidin beads pulldowns were performed to increase the signal-to-noise ratio. The eluted RNA was extracted, and reverse transcription was performed using a specific primer targeting the 3’ end of U1 snRNA. The full-length U1 snRNA was then amplified, and a library was constructed for deep sequencing.

first: Zuokun

last: Shi


title: ZFP207 interacts with U1 snRNP to promote spliceosome assembly via phase separation  [ZFP207_rescue_4sU]

first: yuenan

last: zhou

overall_design:
To systematically assess the effectiveness of various truncations and mutations in restoring the immediate function of ZFP207, we employed 4sU-seq to scrutinize the expression and processing of newly synthesized transcripts within ZFP207AID mESC rescued with different truncations and mutation.
We then performed gene expression profiling analysis using data obtained from RNA-seq of two independent replicates in different trunction/mutation-rescued ZFP207AID groups.
Comparative gene expression profiling analysis of RNA-seq data of different trunction/mutation-rescued ZFP207AID groups.


title: LUC7 proteins define two major classes of 5' splice sites in animals and plants

city: Cambridge

first: Connor

middle: J

last: Kenny

address: 31 Ames Street

department: Biology

institute: MIT

summary: Mutation or deletion of the U1 snRNP-associated factor LUC7L2 is associated with myeloid neoplasms, and knockout of LUC7L2 alters cellular metabolism. Here, we uncover that members of the LUC7 protein family differentially regulate two major classes of 5' splice sites (5'SS) and broadly regulate mRNA splicing in both human cell lines and leukemias with LUC7L2 copy number variation. We describe distinctive 5'SS features of exons impacted by the three human LUC7 paralogs: LUC7L2 and LUC7L enhance splicing of “right-handed” 5'SS with stronger consensus matching on the intron side of the near-invariant /GU, while LUC7L3 enhances splicing of “left-handed” 5'SS with stronger consensus matching upstream of the /GU. We validated our model of sequence-specific 5’SS regulation both by mutating splice sites and swapping domains between human LUC7 proteins. Evolutionary analysis indicates that the LUC7L2/LUC7L3 subfamilies evolved before the split of animals and plants. Analysis of Arabidopsis thaliana mutants confirmed that plant LUC7 orthologs possess similar specificity to their human counterparts, indicating that 5'SS regulation by LUC7 proteins is deeply conserved.

sample_organism:
Arabidopsis thaliana
Homo sapiens

platform_organism:
Arabidopsis thaliana
Homo sapiens

overall_design: Flag-tagged human LUC7L, LUC7L2 and LUC7L3 ORFs were transfected into HEK293 RMCE cells as described above. RNA was extracted 24 hours after transfection using the Qiagen RNeasy Mini kit (cat. no. 74104) according to the manufacturer’s instructions with the optional on-column DNase digestion (cat. No 79254). RNA was eluted in nuclease-free water and quantified using Nanodrop. Illumina-compatible libraries were prepared by MIT BioMicroCenter using NEB II Ultra Directional RNA with poly(A) selection and sequenced on NovaSeq 6000 with 2 x 150 bp reads. Seeds of Wildtype Col0, luc7 single mutants (luc7a-1, luc7a-2, luc7b-1 and luc7rl-1), luc7 double mutants (luc7a-1 luc7b-1, luc7a-2 luc7b-1, luc7a-1 luc7rl-1 and luc7b-1 luc7rl-1) and a luc7 triple mutant (luc7a-2 luc7b-1 luc7rl-1) were surface sterilized with chlorine-gas and then grown on half-strength Murashige Skoog (MS) plates containing 0.8% phytoagar in continuous light at 22°C for 10 days. Seedlings were collected and flash frozen in liquid nitrogen. Total RNA was isolated using RNeasy® Plant Mini Kit (Quiagen, cat. nos. 74904) according to the manufacturer’s instructions. mRNA stranded library preparation and sequencing (PE150) was done by Novogene (Cambridge, United Kingdom) using an Illumina Novaseq6000 system.

first: Kenny

last: Connor


title: ZFP207 interacts with U1 snRNP to promote spliceosome assembly via phase separation  [ZFP207_CLIP]

overall_design:
We did the CLIPFB-seq to identify the binding sites of ZFP207.
Peak calling using data obtained from CLIPFB-seq of each replicate in control and ZFP207AID group.
Comparative peak and motif analysis of CLIPFB-seq data of control and ZFP207AID  group.


title: ZFP207 interacts with U1 snRNP to promote spliceosome assembly via phase separation  [ZFP207_AID_4sU]

overall_design:
To identify the function of ZFP207 in the regulation of U1 snRNP function, we established ZFP207AID mESCs cell lines in which ZFP207 degradation.
We then performed gene expression profiling analysis using data obtained from RNA-seq of three independent replicates in control and ZFP207AID group.
Comparative gene expression profiling analysis of RNA-seq data of control and ZFP207AID  group.


title: ZFP207 interacts with U1 snRNP to promote spliceosome assembly via phase separation  [CRISPR screen]

overall_design:
To identify other regulators of U1 snRNP and U1-PAS-mediated gene repression, we sought to conduct a genome-wide CRISPR interference (CRISPRi) screen on U1-PAS reporter cells, selecting cells in which reporter genes become upregulated.
We performed two independent screens in parallel and amplified the genomic sequences encoding the sgRNA from surviving cells after 5 and 8 days of blasticidin selection, as well as non-selected control cells for deep-sequencing analysis.
Gene enrichment analysis for CRISPRi screen data (D5 vs D0, D8 vs D0) using MAGeCK (model-based analysis of genome-wide CRISPR–Cas9 knockout).


title: H19 Regulates Metabolic Reprogramming Through Encoding a Novel Protein GMRSP and Is Protective Against Aortic Dissection

city: Guangzhou

first: Jianfang

last: Luo

state: Please Select

address: # 106 Zhongshan Second Road, Yuexiu District

institute: Guangdong Provincial People's hospital

summary: To explore the potential regulators in the pathophysiology of aortic dissection

overall_design: Total RNA obtained from dissected ascending aorta compared to normal ascending aorta


title: ZFP207 interacts with U1 snRNP to promote spliceosome assembly via phase separation  [SNRNP70_AID_4sU]

overall_design:
To compare the function of ZFP207 with SNRNP70 in the regulation of U1 snRNP function, we established SNRNP70AID mESCs cell lines in which SNRNP70 degradation.
We then performed gene expression profiling analysis using data obtained from RNA-seq of two independent replicates in control and SNRNP70AID group.
Comparative gene expression profiling analysis of RNA-seq data of control and ZFP207AID group.


title: H19 Regulates Metabolic Reprogramming Through Encoding a Novel Protein GMRSP and Is Protective Against Aortic Dissection.

summary: To explore the mechanism by which GMRSP influence the aortic dissection, RNA-sequence was performed  after overexpressing the GMRSP in  PDGFBB-stimulated human-derived SMC.

overall_design: (1) HASMC stimulated with PDGFBB; (2) HASMC stimulated with PDGFBB, and overexpressed with GMRSP


title: Astrocytic EphA4 signaling is important for the elimination of excitatory synapses in Alzheimer’s disease

status: Public on Dec 29 2024

city: Hong Kong

first: Nancy

last: Ip

address: Room 6313, Academic Building The Hong Kong University of Science and Technology Clear Water Bay, Kowloon

summary: Cell surface receptors, including erythropoietin-producing hepatocellular A4 (EphA4), are important in regulating hippocampal synapse loss, which is the key driver of memory decline in Alzheimer’s disease (AD). However, the cellular-specific roles and mechanisms of EphA4 are unclear. Here, we show that EphA4 expression is elevated in hippocampal CA1 astrocytes in AD conditions. Specific knockout of astrocytic EphA4 ameliorates excitatory synapse loss in the hippocampus in AD transgenic mouse models. Single-nucleus RNA sequencing analysis revealed that EphA4 inhibition specifically decreases a reactive astrocyte subpopulation with enriched complement signaling, which are characteristics associated with synapse elimination by astrocytes in AD. Importantly, astrocytic EphA4 knockout in an AD transgenic mouse model decreases complement tagging on excitatory synapses and excitatory synapses within astrocytes. These findings suggest an important role of EphA4 in the astrocyte-mediated elimination of excitatory synapses in AD and highlight the crucial role of astrocytes in hippocampal synapse maintenance in AD.

overall_design: Hippocampus from aCSF-treated WT, aCSF-treated APP/PS1 mice and KYL-treated APP/PS1 mice

first: Nancy Y

first: Xin

last: YANG


title: NKG2D blockade impairs cytotoxic CD8+ tissue resident memory T cell accumulation and attenuates severity of chronic lung allograft dysfunction after transplantation

city: Pittsburgh

first: Kaveh

last: Moghbeli

state: PA

address: 3459 Fifth Avenue

institute: University of Pittsburgh

summary: Chronic lung allograft dysfunction (CLAD) significantly limits long-term survival following lung transplantation. To identify potential targets for CLAD prevention, T cells from explanted CLAD lungs and lung-draining lymph nodes, as well as diseased and non-diseased controls were isolated and single-cell RNA sequencing and TCR sequencing were performed. TCR sequencing revealed a clonally expanded population of tissue resident memory (TRM) CD8+ T cells with high cytotoxic potential including upregulation KLRK1, encoding the co-receptor NKG2D. These cytotoxic CD8+ TRM accumulated around the CLAD airways and had 100-fold increase in clonal overlap with lung draining lymph nodes when compared to non-CLAD lungs. Using a murine model of orthotopic lung transplant, we confirmed that cytotoxic CD8+ TRM accumulation was due to chronic rejection and not transplant alone. Furthermore, blocking NKG2D in-vivo attenuated the airway remodeling following transplantation and diminished airway accumulation of CD8+ T cells. Our findings support NKG2D as a potential therapeutic target for CLAD, affecting cytotoxic CD8+ TRM accumulation.

overall_design:
Surgically explanted lung and hilar lymph node (HLN) from patients were obtained (lung-transplant recipients with chronic lung allograft dysfunction undergoing re-transplantation, healthy donor controls without chronic lung disease who were deemed not to be candidates for organ transplantation, and patients with idiopathic pulmonary fibrosis undergoing lung transplantation). Individual samples were tagged with unique hashtag oligonucleotides (HTOs, Biolegend). Recipient-derived T cells from each of these samples were isolated via FACS-sorting for live singlets that were CD45+Lineage (CD20, CD19, CD56, CD14)-CD4 and/or CD8+pan-human leukocyte antigen (HLA)+recipient-specific HLA+ as previously described. Sorted cells were captured for single-cell RNA and T cell receptor (TCR) sequencing using the 10x Genomics 5’ assay. Libraries were sequenced using the Illumina platform. Alignment, filtering, barcode counting, and unique molecular identifier counting were performed using 10x Genomics CellRanger v5 and CellRanger VDJ pipelines.
Please note that processed data files generated from both HTO and GEX samples are linked to the corresponding GEX sample records.

first: Madeline

last: Lipp

first: Mark

last: Snyder