title: PlexinD1 drives the lineage plasticity in advanced prostate cancer

city: Spokane

first: Boyang

last: Wu

state: Washington

address: 412 E. Spokane Falls Blvd

institute: Washington State University

summary: PlexinD1 is an axon guidance molecule which is upregulated in neuroendocrine prostate cancer (PC). In order to better understand its role in mediating the development and disease progression in PC, we performed the RNA-seq in PC cells in lack of or with enforced PlexinD1 expression comparing to the control cells. We compared the gene expression profile and conducted downstream pathway analysis. Our data dememstrated that PlexinD1 regulates the cell lineage in castration-resistant PC by activating Gli1 signaling.

overall_design: To investigate the function of PlexinD1, we knocked down PlexinD1 in C4-2B ENZR and 22Rv1 cells and overexpress PlexinD1 in LNCaP cells using lentivirues. Two parental prostate cancer cell lines, C4-2B ENZR and LNCaP, were also included to characterize the gene signature of enzalutamide-resistant prostate cancer cells. Differentially exrepressed genes and pathway regulated by PlexinD1 were further analyzed using R, IPA and GSEA software.

first: Jing

last: Wang


title: Multiplex generation and single cell analysis of structural variants in mammalian genomes

status: Public on Dec 01 2024

city: Seattle

first: Sudarshan

last: Pinglay

state: WA

address: 3720 15th Ave NE

country: USA

institute: University of Washington

type: Expression profiling by high throughput sequencing

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: RNA-Targeting CRISPR/CasRx System Relieves Disease Symptoms in Huntington's Disease Models

city: guangzhou

first: yizhi

last: chen

state: guangdong

address: No.601, West Huangpu Avenue

department: Guangdong-Hong Kong-Macau Institute of CNS Regeneration

country: China

institute: Jinan university

summary: Huntington's disease (HD) is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (HTT) gene. Recent advances in gene editing technologies, such as CRISPR/CasRx, have opened new avenues for therapeutic interventions. In this study, we explored the efficacy of CRISPR/CasRx, which can specifically and accurately digest single-stranded RNA and down-regulate the expression of related genes, in targeting the HTT mRNA and its potential as a treatment strategy for HD. Our results showed that CRISPR/CasRx could significantly down-regulate HTT mRNA in different models, including human embryonic kidney (HEK) 293T cells, HD140Q-knockin (HD 140Q-KI) mice at various disease stages, and Huntingtin knockin (HD-KI) pigs, and lead to a subsequent decrease in the expression of mutant Huntingtin (mHTT) protein. Moreover, this intervention could significantly ameliorate the neurological symptoms in HD 140Q-KI mice and HD-KI pigs. These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.

sample_organism:
Sus scrofa
Mus musculus

platform_organism:
Sus scrofa
Mus musculus

overall_design: Huntington's disease (HD) is a well-known transcription disorder related to neurodegenerative diseases. To further confirm the effects of our CasRx treatment, we performed RNA-seq on both treated pigs and mice,  with three replicates for each group.

first: Yingqi

last: Lin

first: Caijuan

last: Li

first: Yizhi

last: Chen

first: Sen

last: Yan


title: Optimized Method to Generate Well-Characterized Macrophages from Induced Pluripotent Stem Cells

city: Cleveland

first: Jonathan

middle: D

last: Smith

state: OH

address: Cleveland Clinic Box ND50, 9500 Euclid Avenue

department: Cardiovascular & Metabolic Sciences

institute: Cleveland Clinic

summary: Background: Macrophages play a pivotal role in various pathogenic processes, necessitating the development of efficient differentiation techniques to meet the high demand for these cells in research and therapy. Human macrophages can be obtained via culturing of peripheral blood monocytes; however, this source has limited yields and requires patient contact for each proposed use. In addition, it would be difficult to perform gene editing on peripheral blood monocytes.  The differentiation of induced pluripotent stem cells (iPSCs) into macrophages can meet these needs for recurrent studies with high yields with the possibility of gene editing.  Methods: We refined the traditional embryoid body-based differentiation strategy to create a novel three-phase method that optimizes yield, consistent quality, and reproducibility. This approach incorporates the use of microwell plates and cell filtration to standardize the production of embryoid bodies and subsequent macrophage progenitors. Using up to five independent iPSC donors, we performed several assays for macrophage functions and polarization.  such as marker protein staining by flow cytometry, lipoprotein uptake, phagocytosis, cytokine release, inflammasome activation, and the effects of M1-like and M2-like polarization. RNA sequencing was performed to determine segregation of cells at different stages of differentiation and by iPSC donor, as well as to identify marker genes for each stage of differentiation. Results: The iPSC-derived macrophages generated through this method exhibit characteristic features and cell marker proteins, as well as classical macrophage activities including lipoprotein uptake, bacterial phagocytosis, cytokine release, and inflammasome activation. We demonstrate the effects of M1-like and M2-like polarization on cytokine release.  The first three principal components of the RNA sequencing data showed clear clustering by differentiation stage, while the fourth and fifth principal components clustered the differentiated macrophages by their respective iPSC donor.  Marker genes were identified for each stage of differentiation and polarization. Conclusions: The methods provide an optimized and simplified procedure to produce iPSC-derived macrophages. Our results demonstrate the reproducibility of this method in generating high-quality macrophages suitable for a variety of biomedical applications.

overall_design:
Several iPS cell lines were subjected to differentiation into macrophage progenitors and then to M0 macrophages, followed by polarization into M1-like and M2-like macrophages. We preformed paired end bulk RNAseq on these samples in order to determine clustering by cell type and iPS donor as well as to determine marker genes for each cell state.  Oligo-dT cDNA libraries were prepared and paired end 100 bp sequencing was performed on a NovaSEQ-X instrument at the University of Chicago Genomics Core.  RNAseq quality control was performed using the FastQC program and alignments were performed using STAR. Normalized counts per million (CPM), differential gene expression, and principal component analysis (PCA) was determined using DESeq2.

**Raw data are not provided due to donor privacy concerns**

first: Qimin

last: Hai

first: Peter

last: Bazeley


title: Stress response factor ATF4 drives liver fibrosis by governing a stress-independent enhancer program to activate hepatic stellate cells

status: Public on Nov 30 2024

city: HOUSTON

first: chuangye

last: Qi

state: Texas

address: One plaza

department: Huffington Center

institute: Baylor College of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ATF4 promotes HSC activation and drives liver fibrosis independent of its canonical functions in stress signaling.

overall_design: We used HSC human cell lines and HSC-specific conditional knockout mice to investigate the function of ATF4 in liver fibrosis. RNA sequencing and ChIP sequencing technologies were applied to investigate ATF4-targeted transcriptome and its chromatin binding.

first: Yuxiong

last: Feng


title: Transcriptomic predictors of rapid progression from mild cognitive impairment to Alzheimer's Disease

city: Taipei

first: Yi-Long

last: Huang

state: Not US or Canada

address: No.155, Sec.2, Linong Street, Peitou

department: Center for Healthy Longevity and Aging Sciences

country: Taiwan

institute: National Yang Ming Chiao Tung University

summary: Background: Effective treatment for Alzheimer’s disease (AD) remains an unmet need. Thus, identifying patients with mild cognitive impairment (MCI) who are at high-risk of progressing to AD is crucial for early intervention. Methods: Blood-based transcriptomics analyses were performed using a longitudinal study cohort to compare progressive MCI (P-MCI, n=28), stable MCI (S-MCI, n=39), and AD patients (n=49). Statistical DESeq2 analysis and machine learning methods were employed to identify differentially expressed genes (DEGs) and develop prediction models. Results: We discovered a remarkable gender-specific difference in DEGs that distinguish P-MCI from S-MCI. Machine learning models achieved high accuracy in distinguishing P-MCI from S-MCI (AUC 0.93), AD from S-MCI (AUC 0.94), and AD from P-MCI (AUC 0.92). An 8-gene signature was identified for distinguishing P-MCI from S-MCI.

overall_design:
RNA-seq profiling of white blood cells from progressive MCI (P-MCI, n=28), stable MCI (S-MCI, n=39), and AD patients (n=49)

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

first: Zhao-Qing

last: Shen

first: Pei-Ning

first: Ting-Fen

last: Tsai


title: Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION 1 knockout for biofuel production

status: Public on Nov 29 2024

city: Denton

first: Amira

last: Rasoul

address: 1504 W Mulberry St

department: Biology

institute: University of North Texas

summary: The aviation industry’s growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays)  and soybean (Glycine max)  to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION 1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs)  from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multi-omics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to three days earlier at 10 ºC. However, these alterations led to a trade-off between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.

overall_design: RNA-seq profiling of the wildtype (WT) and FAE1 knockout mutant embryos at 14, 17, and 20 days after pollination (DAP)

first: Christopher

last: Johnston

first: Jordan

last: LaChance

first: John

middle: C

last: Sedbrook

first: Ana

middle: P

last: Alonso


title: Piwi regulates the usage of alternative transcription start sites in the Drosophila ovary

city: New Haven

first: Jiaying

state: CT

address: 10 Amistad St Rm 220

department: Genetics

institute: Yale University

summary: Transcription from alternative transcription start sites (TSSs) is prevalent among mammalian genes and has important biological functions. However, how TSSs are selected remains elusive. We identified 87 genes with altered TSS usage (ATU) in Piwi-deficient Drosophila ovaries using the cap-analysis gene expression sequencing (CAGE-seq) method. Piwi regulates TSS usage of ATU genes in germ cells and somatic cells in ovaries as well as in cultured ovarian somatic cells (OSCs). Correspondingly, RNA Pol II initiation and elongation at TSSs of ATU genes were affected in germline-piwi-knockdown ovaries and piwi-knockdown OSCs. We identified a FACT complex component, Ssrp, as a novel interactor of Piwi in the nucleus. Temporally controlled knockdown of ssrp affected TSS usage of ATU genes whereas overexpression of ssrp partially rescued the TSS usage of ATU genes in piwi mutant ovaries. Thus, Piwi may interact with Ssrp to regulate TSS usage in Drosophila ovaries by affecting Pol II initiation and elongation.

overall_design: To investigate how Piwi affected Ssrp in germ cells, chromatin immunoprecipitation DNA-sequencing (ChIP-seq) was conducted to profile the occupancy of Ssrp in young germline-egfp-knockdown and germline-piwi-knockdown ovaries.

last: Liu

first: Haifan

last: Lin

first: Yuedong

first: Nils

last: Neuenkirchen

first: Hongying


title: Gene Expression is Co-regulated by Non-nucleolar RNA Polymerase I [Hi-C]

city: Evanston

first: vadim

last: backman

state: IL

address: 2170 Campus Drive, Silverman Hall 3627

department: Bioengineering

institute: Northwestern University

summary: Due to its role in mRNA synthesis, many studies of the relationship between transcription and chromatin organization have focused on the regulation of RNA polymerase II (Pol-II) function by supranucleosomal structure and vice-versa. In contrast, there is little work on the function of RNA polymerase I (Pol-I) in non-nucleolar chromatin. Prior work has shown that Pol-I engages with components of Pol-II on rDNA, but it’s role in global transcription and chromatin structure beyond the nucleolus has largely been ignored. By pairing auxin-inducible degron technology with nanoscopic imaging, RNA-Seq, and Hi-C, we found that Pol-I and Pol-II co-regulate conformationally defined chromatin domains and mRNA synthesis. Mechanistically, Pol-I maintains the positioning of intronic and intergenic chromatin within domains for the proper expression of exon elements. Consequently, Pol-I loss disrupts genome connectivity, in situ chromatin domains, and the expression of mRNA, genome-wide.

overall_design: To investigate the structure-function relationship between Pol-I and Pol-II transcription and genome organization,  we took advantage of two auxin inducible degron lines targeting either POLR1A or POLR2A. After treating 2 technical replicates (different plates, but treated on the same day) with auxin for 6 hours, DMSO for 6 hours, or 1 hour of ActD (5 µg/mL), we generated Hi-C for each set of samples using the Qiagen Epitect Hi-C Kit (Qiagen, #59971). We processed each replicate using the Juicer pipeline and generated .hic files for each replicate. Replicates were merged to maximize resolution

first: Carter

last: Lucas

first: Backman

last: Vadim

first: Almassalha

last: Luay


title: Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [scRNA-Seq]

city: Utrecht

first: Gijs

middle: Jacob Fransiscus

last: van Son

address: Heidelberglaan 25

country: Netherlands

institute: Prinses maxima centrum

summary: Matrigel®/BME®, a solubilized basement membrane-like preparation, is especially rich in the Extra-Cellular Matrix (ECM) protein laminin-111. This hydrogel supports the formation and long-term growth of epithelial 3D organoids from adult stem cells (ASC)1,2. Laminin111 activates integrin β1 complexes, thus preventing anoikis and driving epithelial polarization. Here, we address if a bacterial integrin-activating protein can mimic Matrigel/BME. Enteropathogenic Yersinia bacteria invade human gut epithelial cells using their outer membrane protein Invasin. This protein binds and activates a diversity of integrin β1 complexes, including the laminin111-specific α6β1 receptor. A recombinant 25 kDa C-terminal integrin-binding fragment of Invasin allowed adhesion of gut epithelial cells, when coated on culture plates. Upon addition of organoid growth factor medium, the epithelial cells grew out in 2D and could be expanded and passaged long-term. Polarization, junction formation and generation of various intestinal cell types (i.e. enterocytes, goblet cells, Paneth cells, and enteroendocrine cells) was stable over time. Sustained expansion of multiple other human-, mouse-, and even snake epithelia was accomplished under comparable conditions. The 2D culturing format holds advantages over the 3D ‘in gel’ format in terms of imaging, accessibility of basal and apical domains and automation for high throughput screening approaches. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel/BME.

overall_design: Ileum N39 triple reporter line (Goblet cells; MUC2-GFP, enteroendocrine (EEC) cells; CHGA-iRFP and Paneth cells: DEFA5-dsRED) obtained from44 were passaged on the integrin-binding domain of Invasin (5 µg/ml) in transwell system with 3 µm pore size (Greiner Bio-one, 662630) using expansion medium. Differentiation was induced using a 2-step differentiation protocol. The first step is to add 500 µl of patterning medium to the lower compartment of the transwell system for 7 days (refresh medium every 2 days with patterning medium), the upper compartment is just air. The second step is to induce Paneth cells,  500 µl maturation medium (as described in44) was added to the lower compartment of the transwell system, with just air in the upper compartment. This culture condition was maintained for 7 days with refreshing maturation medium in the lower compartment every day.

first: Joost

last: Wijnakker

first: Daniel

last: Kreuger

first: Willine

last: van de Wetering

first: Carmen

last: Lopez-Iglesias

first: Robin

last: Schreurs

first: Fenna

last: van Rijt

first: Sangho

last: Lim

first: Lin

first: Peter

last: Peters

first: Ralph

last: isberg

first: Claudia

last: Yanda

first: Wim

last: de Lau

first: Hans

last: Clevers


first: Jiaying

last: Chen

summary: Alternative transcription initiation, which refers to the transcription of a gene from different transcription start sites (TSSs), is prevalent in mammalian systems and has important biological functions. Although transcriptional regulation has been extensively studied, the mechanism that selects one TSS over others in a gene remains elusive. We discovered Piwi, an RNA-binding protein, regulated TSS usage of genes and identified 87 genes with significantly altered TSS usage (ATU) in piwi-deficient Drosophila ovaries using the cap-analysis gene expression sequencing (CAGE-seq) method. This regulation occurs in both germline and somatic cells in ovaries as well as in cultured ovarian somatic cells (OSCs). Correspondingly, RNA Pol II initiation and elongation at TSSs of ATU genes were affected in germline-piwi-knockdown ovaries and piwi-knockdown OSCs. We identified a FACT complex component, Ssrp, which is essential for mRNA elongation, as a novel interactor of Piwi in the nucleus. Temporally controlled knockdown of ssrp affected TSS usage of ATU genes whereas overexpression of ssrp partially rescued the TSS usage of ATU genes in piwi mutant ovaries. Thus, Piwi may interact with Ssrp to regulate TSS usage in Drosophila ovaries by affecting Pol II initiation and elongation.

overall_design: To investigate the role of Piwi on transcription in germ cells, we conducted RNA-seq experiments using total RNA extracted from mature ovaries of germline-gfp-knockdown and germline-piwi-knockdown flies.

last: Liu

first: Haifan

last: Lin

first: Yuedong

last: Huang

first: Nils

last: Neuenkirchen

first: Hongying

last: Qi


overall_design: To investigate the role of Piwi in transcription start site usage, we conducted CAGE-seq experiments using total RNA extracted from young ovaries of wildtype flies and piwi mutant flies.


title: Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [RNA-Seq]

first: Gijs

last: van Son

overall_design: Fresh colon tissue was cultured on BME and INV for 10 days before RNA extraction and mRNA sequencing

first: Joost

last: Wijnakker

first: Daniel

last: Kreuger


overall_design: To investigate how Piwi affected RNA Polymerase II initiation and elongation in germ cells, chromatin immunoprecipitation DNA-sequencing (ChIP-seq) was conducted to profile the occupancy of Ser5-phosphorylated and Ser2-phosphorylated forms of Pol II in germline-egfp-knockdown and germline-piwi-knockdown ovaries.


overall_design: To investigate the role of Piwi on RNA Polymerase II initiation and elongation in ovarian somatic cells (OSC), Cleavage Under Targets and Release using Nuclease (CUT&RUN) was conducted to profile the occupancy of Ser5-phosphorylated and Ser2-phosphorylated forms of Pol II in OSCs nucleofected with siRNA targeting egfp or piwi.


title: Uncovering the Role of PIM2 in Modulating MCL1 Dependency and Activation of ISR-Mediated NOXA Expression

city: Rennes

first: Simon

last: Leonard

address: 2 avenue du Prof Léon Bernard

country: France

institute: Université Rennes

summary: Our study delves into the intricate dynamics of the integrated stress response (ISR) axis, focusing on the pivotal role of PIM2 kinase and its interaction with the BCL2 family of proteins, revealing crucial mechanisms governing cell survival and tumor progression. Elevated PIM2 expression is a hallmark of various cancers, often correlating with disease aggressiveness. Using a model of normal and malignant plasma cells, we unveil that inhibiting PIM2 kinase triggers not only the production of phosphorylated BAD but also activates ISR-mediated NOXA expression. This shift towards heightened dependence on MCL1 underscores the synergy achieved through combined PIM/MCL1 inhibition, largely propelled by ISR-mediated NOXA expression. In mouse xenograft models, dual targeting of PIM2 and MCL1 effectively controls tumor growth, a response reversed by ISR-specific inhibition, concomitant with the upregulation of genes associated with tumor cell dissemination. These findings illuminate the molecular intricacies of PIM2 inhibition and its implications for cancer therapy, particularly in tumors marked by elevated PIM2 expression.

overall_design: To investigate the contribution of the ISR pathway for the synergy of the combinaison of AZD1208 and AZD5991 in a xenograft model of XG7 human MM cell line, we treated mice (receiving the combinaison) with the molecule ISRIB. At mice sacrifice, human tumoral cells were sorted and we performed gene analysis profiling using data obtained from RNAseq of 12 tumoral samples from 12 differents mice receiving the combinaison AZD1208 and AZD5991 and grouped in 2 groups whose one (6 mice/samples) were treated with ISRIB (tested group) and the other (6 mice/samples) are not treated with ISRIB (control group). Comparative gene analysis profiling of the RNAseq data was performed in human tumor cells sample control group and the tested group.

first: Thierry

last: Fest

first: Haas

last: Marion

first: Sabrina

last: Cherfa


title: Intestinal Akkermansia muciniphila complements the therapeutic efficacy of PD1 therapy by reshaping the immunosuppressive microenvironment in nonalcoholic fatty liver disease (NAFLD)-induced hepatocellular carcinoma [16S]

city: Shatin

first: Karl Kam Hei

last: So

state: New Territories

address: 608A, Lo Kwee-Seong Integrated Biomedical Sciences Building, The Chinese University of Hong Kong

department: School of Biomedical Sciences

country: Hong Kong

institute: The Chinese University of Hong Kong

summary: 16S rRNA sequencing showed that Akkermansia muciniphila (Akk) decreased during the course of HCC tumor development, and daily administration of Akk not only ameliorated liver steatosis and cholesterol biosynthesis but also effectively attenuated the development of NAFLD-induced HCC.

overall_design: NAFLD-induced HCC mouse model was established upon injection of diethylnitrosamine (DEN) and a high-fast diet (HFD) using C57BL/6 mice. The mice developed hepatocellular carcinoma in 35 weeks. During the course of HCC tumor development, feces were collected at five different time points, and corresponding fecal DNA was extracted for 16S rRNA sequencing.

first: Fan

last: Ying

first: Xue

last: Wu

first: Katherine

last: Chung

first: Carmen

last: Leung

first: Karl

first: Terence

last: Lee


title: Intestinal Akkermansia muciniphila complements the therapeutic efficacy of PD1 therapy by reshaping the immunosuppressive microenvironment in nonalcoholic fatty liver disease (NAFLD)-induced hepatocellular carcinoma [scRNA-Seq]

summary: Using scRNA-seq coupled with immunoprofiling analyses, we found that Akk decreased the populations of immunosuppressive cells, including monocytic myeloid-derived suppressor cells (m-MDSCs) and M2 macrophages, by suppressing the differentiation of these cells from monocytes, which led to T-cell proliferation and activation.

overall_design: C57BL/6 mice were fed orally with 5x10^9 CFU of Akk or control PBS for 2 weeks prior to the start of the MCD diet for 2 weeks followed by orthotopic implantation of luciferase labelled RIL-175 cells for establishment NAFLD-induced HCC mouse model. The treatment continued in the period of MCD diet. After orthotopic transplantation, feeding of Akk was paused for 6 days to allow mice to recover from surgery, and the treatment was resumed until the end of the experiment. CD45+ cells from tumors treated with either Akk or PBS were extracted for studying the effect of Akk on the regulation of innate immunity in the tumor microenvironment using single RNA sequencing.


title: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

status: Public on Nov 28 2024

city: Urbana

first: Thomas

last: Gaj

address: 1406 W. Green St.

institute: University of Illinois Urbana-Champaign

summary: An abnormal expansion of a G4C2 hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

overall_design: Analyzed differentially expressed genes in a transgenic C9-BACexp mouse model and wild-type C57BL/6J littermates for a high-fidelity RfxCas13d variant, RfxCas13d-N2V8, programmed to target human C9ORF72. RNA-seq was conducted in EGFP-KASH+ nuclei enriched by FACS. Call caro delivered by AAV. N = >3. Each replicate is an independent biological replicate.

first: Tristan

middle: X

last: McCallister

first: Colin

middle: K

last: Lim

first: Mayuri

last: Singh

first: Sijia

last: Zhang

first: Najah

middle: S

last: Ahsan

first: William

middle: M

last: Terpstra

first: Alisha

middle: Y

last: Xiong

first: Maria

last: Zeballos

first: Jackson

middle: E

last: Powell

first: Jenny

last: Drnevich

first: Yifei

last: Kang


title: A decision-point between programmed cell death and transdifferentiation in  pancreatic cancer

city: Strasbourg

first: Frank

last: Jühling

address: 3 Rue Koeberlé

institute: Université de Strasbourg, Inserm, Institut de Recherche sur Les Maladies Virales et Hépatiques UMR_S1110

summary: Transdifferentiation and changes of cellular identity are hallmarks of malignant transformation1-3. As such, the process of acinar-to-ductal metaplasia (ADM) represents a fundamental step in pancreatic ductal adenocarcinoma (PDAC) development, but regulatory pathways controlling ADM are not fully understood4,5. Here, we show that murine pancreatic acinar cells upregulate expression of CASPASE3, CASPASE8, RIPK3 and MLKL - key molecules driving both apoptosis and necroptosis6,7 - during transdifferentiation towards a duct-like phenotype. Spontaneous activation of these respective programmed cell death (PCD) pathways during KRAS-driven transdifferentiation is counterbalanced by transforming growth factor (TGF)-β-activated kinase 1 (TAK1), a kinase transmitting inhibitory phosphorylation signals towards the apoptosis- and necroptosis activator RIPK18. Genetic deletion or pharmacological inhibition of TAK1 simultaneously triggered apoptosis and necroptosis and thereby prevented KRAS-driven ADM in vitro as well as KRAS-driven PDAC in vivo. In line, TAK1 expression is upregulated in human PDAC cells, and pharmacological inhibition of TAK1 triggered PCD in organoid and spheroid cells derived from PDAC patients. Collectively, these findings suggest that TAK1 defines a decision point between ADM and PCD in pancreas cells and represents a promising pharmacological target for the prevention and treatment of PDAC patients.

overall_design: To investigate the functional effects of TAK1 inhibitor on immune cells in pancreatic tumor, we generated pancreatic tumorspheroids, treated them with TAK1 inhibitor or DMSO (vehicle CTRL) and isolated CD45+ immue cell populations by FACS sorting. We then analyzed the effect of the compound by scRNA-Seq

first: Emilie

last: Crouchet

first: Sarah

last: Durand

first: Clara

last: Ponsolles

middle: F

last: Baumert


title: Intestinal Akkermansia muciniphila complements the therapeutic efficacy of PD1 therapy by reshaping the immunosuppressive microenvironment in nonalcoholic fatty liver disease (NAFLD)-induced hepatocellular carcinoma [bulk RNA-Seq]

first: Karl Kam Hei

last: So

summary: The mechanism underlying negative regulatory role of Akk in hepatic steatosis was examined using bulk RNA sequencing.

overall_design: C57BL/6 mice were fed orally with 5x10^9 CFU of Akk or control PBS for 2 weeks prior to the start of the MCD diet for 2 weeks followed by orthotopic implantation of luciferase labelled RIL-175 cells for establishment NAFLD-induced HCC mouse model. The treatment continued in the period of MCD diet. After orthotopic transplantation, feeding of Akk was paused for 6 days to allow mice to recover from surgery, and the treatment was resumed until the end of the experiment. Liver tumors were harvested for RNA extraction.

first: Fan

last: Ying

first: Xue

last: Wu

first: Katherine

last: Chung

first: Carmen

last: Leung

first: Karl

first: Terence

last: Lee


title: SULFORAPHANE ACUTELY ACTIVATES MULTIPLE STARVATION RESPONSE PATHWAYS

city: Oklahoma City

first: Jonathan

last: Wren

state: OK

address: 825 N.E. 13th Street

department: Genes & Human Disease Research Program

institute: OMRF

summary: Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables that ameliorates various disease models in rodents (e.g., cancer, diabetes, seizures) that are likewise mitigated by dietary restriction leading us to test the hypothesis that this compound elicits cellular responses consistent with being a fasting mimetic. Using untransformed human retinal pigment epithelial (RPE-1) cells, we report that SFN impacted multiple nutrient-sensing pathways consistent with a fasting state.  SFN treatment (i) increased mitochondrial resistance to oxidative stress, (ii) acutely suppressed markers of mTORC1/2 activity via inhibition of insulin signaling, (iii) upregulated autophagy and further amplified autophagic flux induced by rapamycin or nutrient deprivation while concomitantly promoting lysosomal and mitochondrial biogenesis, (iv) acutely decreased glucose uptake and lactate secretion followed by an adaptive rebound that coincided with suppressed protein levels of thioredoxin-interacting protein (TXNIP) due to early transcriptional down-regulation.  This early suppression of TXNIP mRNA expression could be overcome with exogenous glucosamine consistent with SFN inhibiting glutamine F6P amidotransferase (GFAT), the rate limiting enzyme of the hexosamine biosynthetic pathway.  SFN also altered multiple glycolytic and tricarboxylic acid (TCA) cycle intermediates while reducing the inhibitory phosphorylation on pyruvate dehydrogenase, indicative of an adaptive cellular starvation response directing pyruvate into acetyl coenzyme A for uptake by the TCA cycle. RNA-seq of cells treated for 4hrs with SFN confirmed the activation of signature starvation-responsive transcriptional programs. Concomitant with stabilizing and activating the transcription factor NF-E2-related factor 2, we posit that the fasting-mimetic properties of SFN underlie both the therapeutic efficacy and potential toxicity of this phytochemical.

overall_design: mRNA profiling of tissue culture cells (human telomerase-immortalized RPE-1 cells) that were treated with either sulforaphane or vehicle (DMSO) for 4 hours (N=5 replicates for each treatment)

first: Kendra

last: Plafker

first: Scott

first: Constantin

last: Georgescu

first: Atul

last: Pranay

first: Nathan

last: Pezant


first: Julien

last: Moehlin

institute: Institute for Translational Medicine and Liver Disease (ITM)

summary: Mecanism of action of TAK inhibitor on immune cells and cancer cells extracted from pancreatic tumor (PDAC)

overall_design: PDAC tumor tissue was processed and immune cells were isolated from other cell types that were grown as 3D tumorspheroids. Tumorspheroids were then treated with TAK inhibitor or DMSO as control. Conditionned media were harvested and used to stimulate immune cells. Immune cells were then analyzed by single cell RNA-Seq (SORT-Seq)

first: Frank

first: Charlotte

last: Bach

first: Thomas


title: HKDC1 Deletion in the Liver Mitigates Western Diet-Induced MASH

status: Public on Nov 27 2024

city: Chicago

first: MD Wasim

last: Khan

address: 835 S Wolcott

department: Division of Endocrinology, Diabetes & Metabolism, Department of Medicine,

institute: University of Illinois at Chicago

summary: Metabolic-associated steatohepatitis (MASH), the inflammatory stage of MASLD, will soon be one of the primary causes of liver-related complications, including advanced fibrosis and hepatocellular carcinoma. Its steady increase in both the US and the global population is devastating. Between the two genders, more women are suffering from MASH compared to the man due to estrogen deficiency caused by polycystic ovary syndrome or menopause. Our published data shows a clear positive association between the progression of liver disease and the expression of novel hexokinase HKDC1 in the liver. Targeting HKDC1, which is highly expressed in pathological hepatocytes (in MASH) but negligible in normal hepatocytes, represents a highly selective approach. Therefore, we hypothesized that liver-specific deletion of HKDC1 will protect against diet-induced obesity and the progression of MASH to fibrosis. We used an early-onset liver knockout (LKO) of HKDC1 by crossing HKDC1 floxed mice with Albumin Cre mice to test our hypothesis. We fed the Western Diet to HKDC1LKO female mice along with HKDC1 floxed mice (HKDC1fl/fl; as Controls) for 28 weeks. Our data shows that HKDC1 deletion significantly decreases body weight and fat mass compared to HKDC1fl/fl mice with no changes in food intake and energy expenditure. Knockout mice have better glucose tolerance and lower fasting glucose levels than HKDC1fl/fl mice. Our data also shows that the knockout group has smaller livers, healthier liver parameters, less steatosis, and lower NAS and fibrosis scores. Furthermore, HKDC1 knockout alters hepatic gene expression, and we found significantly low proinflammatory and profibrogenic gene expression in the LKO mice. Although we did not see any changes in triglyceride levels, there was a significant reduction of serum cholesterol concomitant with enhanced expression of cholesterol metabolism genes in the knockout group. Our data suggest a promising protective role of HKDC1 deletion against diet-induced obesity and MASH mice.

overall_design: To understand hepatic HKDC1 role in MASH development, liver of HKDC1 hepatic speicific knockout  (HKDC1LKO) and HKDC1 floxed(HKDC fl/fl) female mice were harvested after 20 weeks western diet feeding from age of 8 weeks. RNA were extrected from liver of harvested mice and send for transcriptomic sequencing.

first: Kai

last: Xu

first: Irene

last: Covila-Corona

first: Dhruvi

last: Makhanasa

first: Pratham

last: Shah

first: Medha

last: Priyadarshini

first: Md. Wasim


title: Genome-wide identification of circular RNAs in eIF4A3 IP

city: Daejeon

first: Sung Ho

last: Boo

address: 291, Daehak-ro

country: South Korea

institute: Korea Advanced Institute of Science and Technology

summary: One-color microarray experiment to identify eIF4A3-associated circular RNAs.

overall_design: Relative enrichement of each circular RNAs in eIF4A3 IP analyzed and normalized.

first: SungHo

first: Min-Kyung

last: Shin

first: HyunJung

last: Hwang

first: YoonKi

last: Kim


title: Intestinal Akkermansia muciniphila complements the efficacy of PD1 therapy in MAFLD-related hepatocellular carcinoma

first: Karl Kam Hei

last: So

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

overall_design: Refer to individual Series


title: Circular RNAs affect mRNA stability

type:
Expression profiling by array
Non-coding RNA profiling by array

first: Hyun Jung

first: Yoon Ki


title: Enrichment of CBP80-associated and eIF4E-associated linear RNAs in SMG6 depleted cells

summary: One-color microarray experiment comparing the enrichment of CBP80-associated and eIF4E-associated linear RNAs.

overall_design: Relative enrichement of each linear RNAs in CBP80/eIF4E IP analyzed and normalized. Relative ratio (SMG6 siRNA/Control siRNA) was calculated.


title: Effective Inhibitor Removal from Wastewater Samples Increases Sensitivity of RT-dPCR and Sequencing Analyses and Enhances the Stability of Wastewater-Based Surveillance

city: Berlin

first: Emanuel

last: Wyler

address: Robert Roessle Str 10

department: Berlin Institute for Medical Systems Biology

country: Germany

institute: Max Delbrück Center for Molecular Medicine

summary: Wastewater-based surveillance (WBS) is a proven tool for monitoring population-level infection events. Wastewater contains high concentrations of inhibitors, which contaminate total nucleic acids (TNA) extracted from these samples. We found that TNA extracts from raw influent of Berlin wastewater treatment plants contained highly variable amounts of inhibitors that impaired molecular analyses like dPCR and next-generation sequencing (NGS). By using dilutions, we were able to detect inhibitory effects. To enhance WBS sensitivity and stability, we applied a combination of PCR inhibitor removal and TNA dilution (PIR+D). This approach led to a 26-fold increase in measured SARS-CoV-2 concentrations, practically reducing the detection limit. Additionally, we observed a substantial increase in stability of the time series. We define suitable stability as a mean absolute error (MAE) below 0.1 log10 copies/l and a geometric mean relative absolute error (GMRAE) below 26%. Using PIR+D, the MAE could be reduced from 0.219 to 0.097 and the GMRAE from 65.5% to 26.0% and even further in real-world WBS. Furthermore, PIR+D improved SARS-CoV-2 genome alignment and coverage in amplicon-based NGS for low to medium concentrations. In conclusion, we strongly recommend both the monitoring and removal of inhibitors from samples for WBS.

overall_design: Communal wastewater of three wastewater treatment plants (EU UWW Codes: Ruhleben DETP_BE01, Schönerlinde DETP_BE02, Waßmannsdorf DETP_BE03) in Berlin and the airport BER was collected from raw influent. Samples were collected during low SARS-CoV-2 levels (February/March 2024) and high levels (September 2024). Total TNA was extracted and then, with or without inhibitor removal, subjected to high-throughput SARS-CoV-2 sequencing.

first: Nico

last: Linzner

first: Alexander

last: Bartel

first: Vera

last: Schumacher

first: José

middle: H

last: Grau

first: Henrike

last: Preuß

first: Sonja

last: Garske

first: Julia

last: Bitzegeio

first: Elisabeth

middle: B

last: Kirst

first: Karsten

last: Liere

first: Sebastian

last: Hoppe

first: Tatiana

middle: A

last: Borodina

first: Janine

last: Altmüller

first: Markus

last: Landthaler

first: Martin

last: Meixner

first: Daniel

last: Sagebiel

first: Uta

last: Böckelmann


title: Enrichment of CBP80-associated and eIF4E-associated circular RNAs in SMG6 depleted cells

first: Sung Ho

last: Boo

summary: One-color microarray experiment comparing the enrichment of CBP80-associated and eIF4E-associated circular RNAs.

overall_design: Relative enrichement of each circular RNAs in CBP80/eIF4E IP analyzed and normalized. Relative ratio (SMG6 siRNA/Control siRNA) was calculated.

first: SungHo

first: Min-Kyung

last: Shin

first: HyunJung

last: Hwang

first: YoonKi

last: Kim


title: Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer [scRNA-Seq]

city: Boston

first: Seong Won

state: MA

address: 77 Ave Louis Pasteur

institute: Harvard Medical School

summary: Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte TTN expression we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, while heterozygous mice demonstrated allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive the expression of a reporter gene in hiPSC-CMs. Deletion of E1 downregulated TTN expression, impaired sarcomerogenesis, and decreased contractility in hiPSC-CMs. Site-directed mutagenesis of predicted NKX2-5- and MEF2-binding sites within E1 abolished its transcriptional activity. Embryonic mice expressing E1 reporter gene constructs validated in vivo cardiac-specific activity of E1 and the requirement for NKX2-5 and MEF2 binding sequences. Moreover, isogenic hiPSC-CMs containing a rare E1 variant in the predicted MEF2 binding motif that was identified in a patient with unexplained DCM showed reduced TTN expression. Together, these discoveries define an essential, functional enhancer that regulates TTN expression. Manipulation of this element may advance therapeutic strategies to treat DCM caused by TTN haploinsufficiency.

overall_design: WT and mutant hiPSCs with TTN enhancer mutations were differentiated to cardiomyocytes. Cardiomyocytes from multiple independent differentiation were frozen. CellPlex kit was used to barcode each replicate and labeled cells were pooled together. Cells were sequenced using the standard single nuclei RNAseq protocol. TTN expression was compared between the WT hiPSC-CMs anad mutant hiPSC-CMs

first: Seongwon

first: Yuri

first: Joshua

last: M Gorham


title: Bulk RNA-seq of pericytes isolated from mouse brain and heart

city: New York

first: William

last: Coetzee

state: United States

address: 550 First Ave

department: Pathology

institute: NYU School of Medicine

summary: The goal of the experiment was to characterize the transcriptome of Pericytes from mouse brain and heart. Data were analyzed to examine the ion channels, exchangers and pumps that are enriched in pericytes

overall_design: Pdgfr-β-CreERT2 transgenic mice (Jackson: 029684) were crossed with Ai14 reporter mice (Jackson #007914) that express tdTomato fluorescence following Cre-mediated recombination. Offspring were treated with tamoxifen, which led to robust tdTomato expression in pericytes. Pericytes were isolated by enzymatic digestion from the brains and hearts of 5 mice, subjected to cell sorting and bulk RNA-sequencing.


title: Cell competition drives bronchiolization and pulmonary fibrosis

city: Rochester

first: Stijn

last: De Langhe

state: Minnesota

address: 200 First street SW

department: Medicine

institute: Mayo Clinic Rochester Minnesota

summary: Idiopathic pulmonary fibrosis (IPF) is a progressive respiratory scarring disease arising from the maladaptive differentiation of lung stem cells into bronchial epithelial cells rather than into alveolar type 1 (AT1) cells, which are responsible for gas exchange. Here, we report that healthy lungs maintain their stem cells through tonic Hippo and β-catenin signaling, which promote Yap/Taz degradation and allow for low level expression of the Wnt target gene Myc. Inactivation of upstream activators of the Hippo pathway in lung stem cells inhibits this tonic β-catenin signaling and Myc expression and promotes their Taz-mediated differentiation into AT1 cells. Vice versa, increased Myc in collaboration with Yap promotes the differentiation of lung stem cells along the basal and myoepithelial like lineages allowing them to invade and bronchiolize the lung parenchyma in a process reminiscent of submucosal gland development. Our findings indicate that stem cells exhibiting the highest Myc levels become supercompetitors that drive remodeling, whereas loser cells with lower Myc levels terminally differentiate into AT1 cells

overall_design: Lungs of mice were injured with intratracheal administration of bleomycin in which we inactivated Myc, Yap1/Wwtr1, Stk3/4 or overexpressed Myc. At 6 weeks after injury lungs were analyzed using sptaial transcritpomics.

first: Rachel

last: Warren


title: Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer [RNA-seq]

overall_design: RNAseq of WT or mice carrying heterozygous E0 deletion. We generated E0del/+ mice in a hybrid genetic background by crossing C57BL/6 E0del/+ mice and 129SvEv WT mice. We performed RNAseq of transcripts from left ventricle (LV) and skeletal muscle where Ttn is highly expressed.

first: Hiroko

last: Wakimoto

last: Gorham


title: Raising NAD+ Level Stimulates Short-Chain Dehydrogenase/Reductase Proteins to Alleviate Heart Failure Independent of Mitochondrial Protein Deacetylation

city: Seattle

first: Matthew

last: Walker

address: 850 Republican

institute: University of Washington

summary: BACKGROUND: Strategies to increase cellular NAD+ (oxidized nicotinamide adenine dinucleotide) level have prevented cardiac dysfunction in multiple models of heart failure, but molecular mechanisms remain unclear. Little is known about the benefits of NAD+-based therapies in failing hearts after the symptoms of heart failure have appeared. Most pretreatment regimens suggested mechanisms involving activation of sirtuin, especially Sirt3 (sirtuin 3), and mitochondrial protein acetylation. METHODS: We induced cardiac dysfunction by pressure overload in SIRT3-deficient (knockout) mice and compared their response with nicotinamide riboside chloride treatment with wild-type mice. To model a therapeutic approach, we initiated the treatment in mice with established cardiac dysfunction. RESULTS: We found nicotinamide riboside chloride improved mitochondrial function and blunted heart failure progression. Similar benefits were observed in wild-type and knockout mice. Boosting NAD+ level improved the function of NAD(H) redox sensitive SDR (short-chain dehydrogenase/reductase) family proteins. Upregulation of Mrpp2 (mitochondrial ribonuclease P protein 2), a multifunctional SDR protein and a subunit of mitochondrial ribonuclease P, improves mitochondrial DNA transcripts processing and electron transport chain function. Activation of SDRs in the retinol metabolism pathway stimulates RXRα (retinoid X receptor α)/PPARα (proliferator-activated receptor α) signaling and restores mitochondrial oxidative metabolism. Downregulation of Mrpp2 and impaired mitochondrial ribonuclease P were found in human failing hearts, suggesting a shared mechanism of defective mitochondrial biogenesis in mouse and human heart failure. CONCLUSIONS: These findings identify SDR proteins as important regulators of mitochondrial function and molecular targets of NAD+-based therapy. Furthermore, the benefit is observed regardless of Sirt3-mediated mitochondrial protein deacetylation, a widely held mechanism for NAD+-based therapy for heart failure. The data also show that NAD+-based therapy can be useful in pre-existing heart failure.

overall_design: We performed RNA sequencing of Sham and TAC hearts treated with NR or vehicle. We then examined the enrichment of genes that belong to known molecular pathways from the Reactome database using gene set enrichment analysis (GSEA). GSEA was performed on a Preranked gene list as described in Nat Protoc. 2019 February ; 14(2): 482–517. doi:10.1038/s41596-018-0103-9.

first: Wang

last: Yuliang


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [CUT&Run]

city: Frankfurt am Main

first: Timothy

last: Warwick

state: Hesse

address: Theodor-Stern-Kai 7

department: Institute for Cardiovascular Physiology

institute: Goethe Universität Frankfurt am Main

summary: The coordination of chromatin remodeling is essential for DNA accessibility and gene expression control. The highly conserved and ubiquitously expressed SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex plays a key role in regulating gene expression in a context-dependent manner. SWI/SNF actively maintains open chromatin states across the genome and responds dynamically to cellular signals. However, the precise mechanisms determining how SWI/SNF is targeted to specific genomic sites remain elusive. In this study we demonstrate that long non-coding RNAs (lncRNAs) are pivotal in the binding of the SWI/SNF complex to specific genomic targets. The interaction between SWI/SNF and lncRNAs is essential for the recruitment of the complex to gene regulatory elements, where it plays a critical role. We show that trans-acting lncRNAs direct the SWI/SNF complex to cell-specific enhancers, with lncRNA knockdowns leading to a genome-wide redistribution of SWI/SNF away from these enhancers. This redistribution impacts the expression of genes connected to these enhancers, underscoring the critical role of lncRNAs in the specific targeting of SWI/SNF to DNA. This insight into the targeting mechanisms of SWI/SNF by lncRNAs has broad implications, from understanding the processes of gene expression control to identifying therapeutic targets in diseases associated with SWI/SNF dysfunction, such as cancer.

overall_design: To investigate which gene regulatory elements depend on lncRNA-recruited BRG1 for their chromatin accessibility, BRG1 CUT&RUN was performed in HUVEC after lncRNA knockdown, which in some cases was followed by overexpression of the given lncRNA to rescue the effects.

first: James

last: Oo

first: Matthias

last: Leisegang

first: Ralf

last: Brandes


title: Transcriptional coupling of telomeric retrotransposons with the cell cycle [CUT&RUN]

city: New Orleans

first: Jun-yuan

last: Ji

state: LA

address: 1700 Tulane Avenue

department: Department of Biochemistry and Molecular Biology

institute: Tulane University

summary: Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in Drosophila, these are HeT-A, TART, and TAHRE. Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. Here we report the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in Drosophila. Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division.

overall_design: We performed the CUT&RUN assay follwing the same protocol outlined previously (Liu et al., 2023).


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [ATAC-Seq]

overall_design: To investigate which gene regulatory elements depend on lncRNA-recruited BRG1 for their chromatin accessibility, ATAC-seq was performed in HUVEC after knockdown of BRG1-binding lncRNAs.


title: Long-range Atoh1 enhancers maintain competency for hair cell regeneration in the inner ear

city: Los Angeles

first: Tuo

last: Shi

state: California

address: 1425 San Pablo St BCC 403

institute: University of Southern California

summary: During tissue regeneration, lineage-related cells can switch their fate to replace missing cells. This cell plasticity is particularly prominent in more regenerative vertebrates such as zebrafish, yet the molecular basis by which cells transdifferentiate into another cell type upon injury remains unclear. Here we investigate the epigenetic basis of regenerative transdifferentiation in the inner ear, where supporting cells (SCs) generate mechanosensory hair cells (HCs) upon damage. By comparing the chromatin landscapes in regenerative zebrafish and green anole lizards versus non-regenerative mice, we identified a class of enhancers that function in progenitors to generate HCs and then are selectively maintained in SCs of regenerative vertebrates to regenerate HCs. In particular, we uncovered a syntenic class of long-range enhancers for Atoh1, a master transcription factor for HC differentiation. In the absence of injury, these enhancers maintain accessibility in SCs through adulthood but are prevented from driving zebrafish atoh1a expression through Notch repression. Deletion of these enhancers not only impaired atoh1a expression and HC formation during development but also blocked the ability of SCs to transdifferentiate into HCs during regeneration. Moreover, defects were specific to the inner ear versus the lateral line, revealing distinct mechanisms of regeneration in these mechanosensory organs. These findings reveal a class of regenerative enhancer that maintains competency of inner ear SCs to upregulate atoh1a and transdifferentiate into HCs upon damage. We propose that the continued accessibility of developmental enhancers for one cell fate in lineage-related cells may be a common theme underlying adult cell plasticity in regenerative vertebrates.

sample_organism:
Danio rerio
Anolis carolinensis

platform_organism:
Danio rerio
Anolis carolinensis

overall_design: Inner ears were dissected from the Sox10Cre;UbiSwitch zebrafish at 14dpf (n=40) and 12mpf (n=6) zebrafish. Converted cells were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of mCherry signal and analyzed using 10x genomics based snATACseq or multimome ATAC+Gene Expression kits. scRNA/snRNA associated with these two timepoints were previous deposted at GSE211728. Reads were aligned to danRer11 using the Cellranger pipeline. Inner ear saccules were dissected from adult green anole lizards heads (n=7, heads were 2.5-3cm) and FACS sorted for live cells by DAPI exlucsion. 10x Genomics multiome ATAC+gene expression library was constructed and reads were aligned to AnoCar2.0v2 using the cellranger pipline

first: Yeeun

first: Juan

last: Llamas

first: Xizi

last: Wang

first: Peter

last: Fabian

first: Thomas

last: Lozito

first: Neil

last: Segil

first: Ksenia

last: Gnedeva

first: J Gage

last: Crump


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [RedChIP]

overall_design: To investigate RNA binding in proximity to both DNA and BRG1, RedChIP was performed in HUVECs.


title: Transcriptional coupling of telomeric retrotransposons with the cell cycle

first: Liu

last: Mengmeng

first: Xie

last: Xiao-Jun

first: Li

last: Xiao

first: Ren

last: Xingjie

first: Lin

last: Zhen

first: Ji

last: Jun-Yuan


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [iCLIP]

first: Timothy

last: Warwick

overall_design: To investigate RNA binding to BRG1, BRG1 iCLIP was performed.

first: James

last: Oo

first: Matthias

last: Leisegang

first: Ralf


title: RNA stability is regulated by both RNA polyadenylation and ATP levels, linking RNA and energy metabolisms in Escherichia coli

city: Toulouse

first: Laurence

last: Girbal

address: 135 avenue de rangueil

institute: TBI

summary: The post-transcriptional process of RNA polyadenylation sits at the crossroads of energy metabolism and RNA metabolism. RNA polyadenylation is catalyzed by poly(A) polymerases which use ATP as a substrate to add adenine residues to the 3’ end of RNAs, which can alter their stability. In E. coli, RNA polyadenylation mediated by the major poly(A) polymerase (PAP I) was previously shown to facilitate degradation of individual RNAs. In this study, we performed the first ever genome-wide study of RNA stability in the absence of PAP I. Inactivation of the pcnB gene coding for PAP I led to the stabilization of more than a thousand of E. coli RNAs in the form of full-length functional molecules or non-functional fragments, involved in essential cellular functions such as DNA replication and repair, translation, central carbon metabolism, and stress response. The absence of PAP I also altered the energy metabolism, with an almost 20 % reduction in ATP levels. To better understand how RNA and energy metabolisms are interconnected, we investigated the role of ATP levels in regulating RNA stability. When we lowered intracellular ATP levels below 0.5 mM, certain RNAs were stabilized demonstrating the causal link between ATP levels and RNA stability for the first time in E. coli. Above this concentration, changes in ATP levels had no impact on RNA stability. We also demonstrated that some RNAs were stabilized when PAP I was inactivated by low ATP availability. These results clearly demonstrate that PAP I mediates an energy-dependent RNA stabilization which may contribute to cell energy homeostasis under energy-limited conditions.

overall_design: We reported RNA-seq data for genome-wide RNA half-life determination (stabilome experiments) performed in E. coli MG1655 cells treated with 0.7 mM or 2 mM 2,4-dinitrophenol (DNP). For each treatment, stabilome experiments are  based on 2 kinetics  of 6 RNA-seq performed over the time (T0, 0.4, 1, 2, 4 and 7 minutes) after rifampicin addition.

first: Charlotte

last: Roux

first: Marvin

last: Ramos-Hue

first: Marie-Pierre

last: Duviau

first: Agamemnon

middle: J

last: Carpousis

first: Marjorie

last: Audonnet

first: Sandrine

last: Laguerre

first: Eliane

last: Hajnsdorf

first: Muriel

last: Cocaign-Bousquet


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [RIP-seq]

overall_design: To investigate RNA binding to BRG1, BRG1 RNA immunoprecipitation and sequencing (RIP-seq) was performed.

last: Brandes


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [CutAndRun_PROTAC]

overall_design: To investigate which gene regulatory elements depend on BRG1 for their chromatin accessibility, CUT&RUN-sequencing was performed in HUVEC treated with a PROTAC against BRG1 at different time points.


title: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

first: Thomas

last: Gaj

summary: An abnormal expansion of a G4C2 hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

overall_design: Analyzed the differentially expressed genes for RfxCas13d and two high-fidelity RfxCas13d variants when programmed to target human C9ORF72. Experiments were conducted in HEK293T cells and done in triplicate. Each replicate is an independent biological replicate.

first: Tristan

last: McCallister

first: Colin

last: Lim

first: Mayuri

last: Singh

first: Sijia

last: Zhang

first: Najah

last: Ahsan

first: William

last: Terpstra

first: Alisha

last: Xiong

first: Maria

last: Zeballos

first: Jackson

last: Powell

first: Jenny

last: Drnevich

first: Yifei

last: Kang


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [CutAndRun_siRNA]

first: Timothy

last: Warwick

overall_design: To investigate the effect of loss of RNA binding to BRG1 on genomic localization of BRG1, BRG1 CUT&RUN was performed after transfection of HUVEC with siRNAs against different target RNAs.

first: James

last: Oo

first: Matthias

last: Leisegang

first: Ralf


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements [ATAC-seq]

overall_design: To investigate which gene regulatory elements depend on BRG1 for their chromatin accessibility, ATAC-sequencing was performed in HUVEC treated with a PROTAC against BRG1 at different time points.


title: Yin Yang 1 Deficiency in Skeletal Muscle Stem Cell Aggravates Muscle Fibrosis in Duchenne Muscular Dystrophy Mouse by Disrupting Muscle Stem Cell/Macrophage/Fibro-adipogenic Progenitor Crosstalk [scRNA-Seq]

city: Hong Kong

first: QIANG

last: SUN

address: Ngan Shing Street

department: Department of Orthopaedics and Traumatology

summary: Adult skeletal muscle stem cells (MuSCs), also known as satellite cells (SCs), are widely believed to be responsible for muscle regeneration, which is regulated by complex networks of intrinsic and extrinsic factors. Emerging evidence demonstrates extrinsic regulations from other cell types can significantly impact the regeneration process, especially in repetitive cycles of degeneration/regeneration environment. Here, we demonstrate that the transcription factor (TF) Yin Yang 1 (YY1) deletion in MuSCs of mdx mouse induces CCL5 expression by alleviating the repressive effect on 3D structural interactions on Ccl5 locus. This leads to the increased secretion of CCL5 from MuSCs to the muscle niche, which promotes the recruitment of macrophages (MPs) through the CCL5/CCR5 axis. Incremental MPs lead to continuous inflammation that promotes the survival of Fibro-adipogenic progenitors’ (FAP) through secreting elevated TGFβ1 to stimulate FAPs resisting apoptosis, resulting in aggravated dystrophic pathology manifested by exacerbated fibrosis. Inhibition of CCL5/CCR5 axis by Maraviroc injection effectively mitigates muscle dystrophy and enhances muscle performance in YY1 deletion mdx mouse. Altogether, our findings promote us to conclude that YY1 functions as a 3D structural protein to repress Ccl5 production at the transcriptional level in MuSCs, playing a critical role in orchestrating MuSC/MP/FAP crosstalk through CCL5/CCR5 and TGFβ1 signaling. Intrinsic deletion of YY1 in MuSCs disrupts the cellular interactions and leads to skewed muscle niche with aggravated muscle dystrophy upon chronic injury.

overall_design: Droplet-based scRNA-seq on hind limb muscles from Ctrl and YY1 dKO groups.

first: Huating

last: Wang

first: Yang

last: Li

first: Chuhan


title: Effect of ECP on intestinal immune cells in immune checkpoint inhibitor-induced colitis - batch2 [scRNA-Seq]

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

summary: Immune Checkpoint Inhibitors (ICI) have proven to be beneficial against tumors by boosting anti-tumor immunity, however these therapies can induce severe inflammatory side effects in all tissues, named immune-related adverse events (irAE). The colon is affected most commonly. These cause termination of treatment. Until today, patients with irAEs received systemic immunosuppressive therapies. We have found ECP as a novel therapy, which causes immunomodulation in the inflamed tissue without systemic immunosuppression. To understand which immune cells are affected by ECP in the inflamed intestinal tract, mice were treated with DSS and anti-PD1 to induce colitis, comparable to patients suffering from ICI-induced irAEs. ECP showed beneficial effects in patients. Mice were transplanted with ECP-treated cells as treatment and scSeq was performed with Colon infiltrating CD45 positive cells. Naive mice, mice receiving DSS and isotype control and mice receiving DSS in combination with anti-PD1 and untreated splenocytes, served as control.

overall_design: Mice were left untreated or treated with DSS and anti-PD1 to induce ICI-induced colitis. Mice treated with DSS and isotype served as control. As treatment, mice with ICI-induced colitis were transplanted with ECP-treated donor splenocytes. As control, mice with ICI-colitis were transplanted with untreated donor splenocytes. Colitis tissue was digested, dead cells were removed and CD45 positive cells were selected by magnetic cell sorting from the lamina propria. CD45 positive cells were processed for scRNA Seq.

first: Melanie

last: Boerries

first: Lukas

last: Braun

first: Robert

last: Zeiser


title: Yin Yang 1 Deficiency in Skeletal Muscle Stem Cell Aggravates Muscle Fibrosis in Duchenne Muscular Dystrophy Mouse by Disrupting Muscle Stem Cell/Macrophage/Fibro-adipogenic Progenitor Crosstalk [RNA-Seq]

institute: The Chinese University of Hong Kong

overall_design: Bulk RNA-seq in Ctrl and YY1 dKO treated freshly isolated MuSCs.


title: Yin Yang 1 Deficiency in Skeletal Muscle Stem Cell Aggravates Muscle Fibrosis in Duchenne Muscular Dystrophy Mouse by Disrupting Muscle Stem Cell/Macrophage/Fibro-adipogenic Progenitor Crosstalk

overall_design: Refer to individual Series


title: RNA polyadenylation and cellular ATP levels regulate the stability of RNAs in Escherichia coli

first: Laurence

last: Girbal

summary: To investigate the extent of the effect of poly(A) polymerase (PAP I)-mediated polyadenylation on RNA stability, we performed the first genome-wide study of RNA stability in the absence of PAP I activity. Inactivation of the pcnB gene coding for PAP I led to a global stabilization of E. coli RNAs, with 1403 stabilized transcripts and only 4 destabilized. Stabilized RNAs were involved in essential cellular functions such as DNA replication and repair, translation, RNA degradation, central carbon metabolism but also in stress responses. Because PAP I is an ATP-consuming enzyme we wondered whether the RNA stabilization observed after inactivation of PAP I could also be related to changes in intracellular ATP levels. We demonstrated for the first time in E. coli, that lowering intracellular ATP levels below 1 µM/OD stabilizes RNAs. Although the ATP level was reduced by 20 % in the pcnB mutant on glucose, the ATP level was still too high to have any role in the observed RNA stabilization. However, in experiments where the ATP level was artificially strongly decreased, inactivation of PAP I by substrate availability was implicated in the stabilization mechanism of certain RNAs. This study clearly demonstrates that PAP I is at the crossroads of the regulation of RNA stability by energy status in E. coli cells.

overall_design: We reported RNA-seq data for transriptome and genome-wide RNA half-life determination (stabilome experiments) performed in two strains: the wild-type E. coli MG1655 strain (named MG) and its mutant deleted of the pcnB gene coding for the poly(A) polymerase (PAP I) (named PCNB). Transcriptome experiments were carried out in biological triplicates for MG (3 RNA-seq) and PC (3 RNA-seq) corresponding to the time T0 of the stabilome experiments. For each strain, the stabilome experiment is based on 3 kinetics (K1, K2 and K3) of 4 RNA-seq performed over the time after rifampicin addition.

first: Charlotte

last: Roux

first: Marvin

last: Ramos-Hue

first: Marie-Pierre

last: Duviau

first: Agamemnon

last: Carpousis

first: Marjorie

last: Audonnet

first: Sandrine

last: Laguerre

first: Eliane

last: Hajnsdorf

first: Muriel

last: Cocaign-Bousquet


title: Yin Yang 1 Deficiency in Skeletal Muscle Stem Cell Aggravates Muscle Fibrosis in Duchenne Muscular Dystrophy Mouse by Disrupting Muscle Stem Cell/Macrophage/Fibro-adipogenic Progenitor Crosstalk [ChIP-Seq]

first: QIANG

overall_design: YY1 ChIP-seq in WT MuSC


title: Yin Yang 1 Deficiency in Skeletal Muscle Stem Cell Aggravates Muscle Fibrosis in Duchenne Muscular Dystrophy Mouse by Disrupting Muscle Stem Cell/Macrophage/Fibro-adipogenic Progenitor Crosstalk [Hi-C]

overall_design: Hi-C in Ctrl and YY1 dKO MuSC

first: Huating


title: Immune checkpoint inhibitor-induced colitis


title: Effect of ECP on intestinal immune cells immune checkpoint inhibitor-induced colitis [scRNA-Seq]

summary: Mice were treated with DSS and anti-PD1 to induce colitis, comparable to patients suffering from ICI-induced immune-related adverse events. ECP showed beneficial effects in patients. Mice were transplanted with ECP-tretaed cells as treatment and scSeq was performed with Colon infiltrating CD45 positive cells.

overall_design: Mice were treated with DSS and anti-PD1 to induce ICI-induced colitis. As treatment, mice were transplanted with ECP-treated donor cells. Colitis tissue was digested and CD45 positive cells were selected by Magnetic cell sorting. Cell were processed for scRNA Seq.


title: Development and validation of a gene-based classification model for pN2 lung adenocarcinoma

status: Public on Nov 26 2024

city: Xi'an

first: Yanlu

last: Xiong

state: Shaanxi

address: 569 Xinsi Road, Baqiao District

department: Tangdu Hospital

institute: Air Force Medical University

summary: Lung adenocarcinoma (LUAD) with pathological ipsilateral mediastinal lymph node (LN) involvement (pN2) exhibits strong biological and clinical heterogeneity. Thus, it is necessary to classify the biomolecular characteristics that lead to the prognostic heterogeneity of pN2-LUAD. The clinical characteristics and bulk RNA sequencing (RNA-seq) data of 75 patients with pN2-LUAD obtained from The Cancer Genome Atlas (TCGA) database were collected as the training set. The disease-free survival (DFS) and overall survival (OS) of patients with different molecular classifications were evaluated. Next, differentially expressed genes (DEGs), biology, and immune cell infiltration in the microenvironment were analysed. Finally, DEGs in the pN2-A and pN2-B groups were included using a least absolute shrinkage and selection operator (LASSO) model, and gene signatures were selected for pN2-A/B type classification. The RNA-seq and single-nucleus RNA sequencing (snRNA-seq) data from our center (n=58) and the GSE68465 dataset (n=53) were used as the validation data sets.

overall_design: RNA-seq profiling of pN2 LUAD.


title: Transcriptomic analysis of methylmercury effects on Drosophila flight muscle and ventral nerve cord development

first: Matthew

last: Rand

state: NY

address: 601 Elmwood Ave Box EHSC

department: Environmental Medicine

institute: University of Rochester

summary: The greatest concern for exposure to methylmercury (MeHg) lies with the fetus and young children, as exemplified in historic cases of Congenital Minamata Disease (CMD) resulting from prenatal exposure. Children that experience CMD characteristically present with various degrees of cognitive and motor symptoms and signs, much like cerebral palsy.  MeHg has thus been characterized as a neurotoxicant, where motor deficits are ascribed to central nervous system targets. Skeletal muscle as a post-synaptic MeHg target and contributor to the etiology of CMD has garnered far less attention.  Prior studies using Drosophila to model CMD revealed that developmental exposure of MeHg in the larval/pupal stages can elicit graded and latent dose responses affecting adult flight behavior at lower doses (0.4-2.5 ppm in food) and eclosion (emergence from the pupa case) at higher doses (>2.5 ppm in food).  The latter phenotype is accompanied by dysmorphogenesis of skeletal muscles. Here, we investigate respective roles for muscle and neural targets in MeHg toxicity. Using RNA-seq analysis, we find that developmental MeHg exposure produces 10-times as many differentially expressed transcripts in indirect flight muscle (IFM) compared to the VNC. Among known MeHg response genes, Nrf2 antioxidant response pathway genes showed muscle-specific MeHg-induced expression changes. Within the muscle transcriptome, the most enriched and significant Gene Ontology terms identified genes required for mitochondrial ribosomal translation at the pupa stage and mitochondrial function (respiratory chain complex I) and vesicle trafficking (ESCRT III) pathways in adults, all showing decreased expression with MeHg exposure. By using an intact, whole-animal developmental model, we identify preferential candidates to evaluate a novel role for muscle-specific mitochondria and intercellular vesicular communication mechanisms as targets in MeHg toxicity and the etiology of CMD.

overall_design: The sample types are total RNA isolated from Drosophila indirect flight muscles (IFMs) and from ventral nerve cord (VNC). Tissues were collected at two stages of development: 30 hours after pupa formation (APF) and 15 day old adults. Sexes were mixed. In addition, treatments were performed ahead of sample collection which included feeding at the larval stage on food containing 0µM (control) or 5 µM (experimental) methylmercury (MeHg).  Larval feeding results in MeHg accumulation that reaches a steady state that is maintained in the pupal stages, where there is no further uptake or excretion.  Adults were moved to MeHg-free food upon eclosion (~96 hr APF) to clear MeHg. Tissues (IFMs and VNCs) were dissected from pupae at 30h after pupa formation and from adults at 15 days post eclosion. Treatments and sample collection were carried out in triplicate. The variables being tested are MeHg exposure, developmental stage and tissue type.

first: Jennifer

last: Becker

first: Catherine

middle: R

last: Beamish


title: LncRNA adapters determine SWI/SNF complex occupancy at gene regulatory elements

first: Timothy

last: Warwick

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


title: Inflammatory crosstalk impairs phagocytic receptors and aggravates atherosclerosis in clonal hematopoiesis in mice

first: Chenyi

last: Xue

institute: Columbia University

summary: Low frequency Jak2VF mutations promote atherosclerosis via IL-1 mediated cross-talk. Therapeutic approaches that increase MERTK or TREM2 could promote plaque stabilization in inflammasome-driven atherosclerosis.

overall_design: To determine if activating TREM2 could improve atherosclerosis in LAB Jak2VF CH mice, we administered 4D9 or isotype control IgG antibodies by i.p. injection beginning 2 weeks after initiation of the WD and continued injections with the diet for an additional 14 weeks. Digested live aortic cells were used for the Singe cell RNA-Seq analysis.

first: Wenli

last: Liu

first: Eunyoung

last: Kim


title: ECP as a treatment for immune checkpoint inhibitor-induced colitis [array]

first: Geoffroy

last: Andrieux

summary: Mice were treated with DSS and anti-PD1 to induce colitis, comparable to patients suffering from ICI-induced immune-related adverse events. ECP showed beneficial effects in patients. Mice were transplanted with ECP-tretaed cells as treatment and RNA was extracted from colitis tissue to anravel the mechanisms causing the reduction of colitis severity.

overall_design: Mice were treated with DSS and anti-PD1 to induce ICI-mediated colitis. As treatment, mice were transplanted with ECP-treated donor cells. Colitis tissue of control or ECP treated mice were harvested and total RNA was extracted for Microarray analysis.

first: Braun

last: Lukas

first: Zeiser

last: Robert

first: Andrieux

last: Geoffroy

first: Boerries

last: Melanie


title: H. pylori SlyD Enhances TPT1 Stability via hnRNPK to Promote OCT1-Mediated CDX2 Transcriptional Activation Triggering Gastric Intestinal Metaplasia

city: Shenyang

first: Shuwen

last: Zheng

state: --- Select One ---

address: No.155 NanjingBei Street, Heping District

institute: the First Hospital of China Medical University

summary: Gastric intestinal metaplasia (GIM) is an important precancerous lesion in intestinal-type gastric cancer, with persistent Helicobacter pylori (H. pylori) infection inducing the condition. In a previous study, we identified a new H. pylori virulence factor, SlyD. However, its mode of action between GIM was unknown. Our findings offer clues to reveal the molecular mechanism of H.pylori infection-associated gastric intestinal metaplasia, and provides the mechanism of a novel H. pylori virulence factor SlyD in gastric precancerous disease.

overall_design: The H. pylori 26695 was acquired from the American Type Culture Collection (ATCC). A SlyD inactivated mutant strain, where chloramphenicol-resistant fragments replaced slyD fragments (ΔSlyD), was generated based on H. pylori 26695. AGS cells were exposed to H. pylori 26695 or △SlyD for 24 hours (MOI = 100). Total RNA was extracted for RNA sequencing. RNA sequencing was used to screen for differentially expressed genes associated with H. pylori SlyD infection.

first: Yingying

last: Wang

first: Chuxuan

last: Ni

first: Yuehua

last: Gong


title: STK19 accelerates the clearance of lesion-stalled RNAPII to facilitate transcription-coupled DNA repair

city: London

first: Richard

middle: James

last: Mitter

state: LONDON

address: 1 Midland Road

department: Bioinformatics & Biostatistics

country: United Kingdom

institute: The Francis Crick Institute

summary: Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modelling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

overall_design: HEK293 FlpIn TRex cells wild type, STK19 KO or CSB KO were used in order to assess transcription recovery after UVC irradiation. Cells were either left untreated or treated with 15J/m2 UVC and left to grow for the speficied time (2h, 5h, 12h, 24h or 36h). Cells were then harvested and submited to the TTchem-seq (Gregersen et al., 2020) to look at the effects in nascent transcription of STK19 knock out in HEK293 FlpIn TREx cells with CSB KO as control cell line .

first: Marta

last: Rodríguez-Martínez

first: Jesper

middle: Q

last: Svejstrup


title: Sitravatinib in combination with nivolumab plus ipilimumab in patients with advanced clear cell renal cell carcinoma: a phase 1 trial

city: Houston

first: Pavlos

last: Msaouel

address: 1155 Pressler Street, Unit 1374

department: Genitourinary Medical Oncology

institute: The University of Texas MD Anderson Cancer Center

summary: Sitravatinib is an immunomodulatory tyrosine kinase inhibitor that can improve responses when combined with immune checkpoint therapy (ICT). We conducted a phase I trial to determine the optimal dose of triplet therapy with sitravatinib plus nivolumab plus ipilimumab in 22 previously untreated patients with advanced clear cell renal cell carcinoma (ccRCC). The primary endpoint was safety. The addition of even a low sitravatinib dose of 35 mg daily to nivolumab 3 mg/kg and ipilimumab 1 mg/kg resulted in high frequency of immune-related adverse events (irAEs). Subsequent dose reduction of ipilimumab to 0.7 mg/kg in combination with nivolumab 3 mg/kg allowed safe escalation of sitravatinib up to 100 mg daily. Key secondary endpoints were objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). Overall, the triplet combination in the dose-finding setting achieved an ORR of 45.5%, DCR of 86.4%, and a median PFS of 14.5 months with 72.7% of patients alive after a median follow-up of 15.7 months. Single-cell RNA-seq performed in longitudinally collected tumor biopsies from 12 patients treated with the triplet therapy identified a tumor cell-specific epithelial-mesenchymal transition (EMT)-like program associated with treatment resistance and poor outcomes in patients of this trial and in patients of the TCGA ccRCC cohort. Within the tumor microenvironment (TME), the emergence of treatment resistance was characterized by a transition from cytotoxic to exhausted T cell state and enrichment for M2-like myeloid cells. The observed changes in gene expression dynamics and cellular states in tumor cell and TME may help inform future strategies to optimize ICT efficacy.

overall_design:
We performed single cell RNA sequencing (scRNA-seq) in 19 tissue samples collected from 12 patients enrolled in the trial. Baseline samples were collected from all 12 patients, while 4 patients also had samples collected prior to the second infusion of nivolumab plus ipilimumab (C2, Post timepoint) and 3 patients also had samples at the end of treatment (EOT timepoint) due to disease progression. Whenever possible, longitudinally collected samples were biopsied from the same organ site.                             contributor: Moon Shots Cancer Genomics Laboratory (CGL) contributor: MD Anderson institutional Advanced Genomics Technology Core (AGTC).              *************************************************************** Raw data is not provided due to patient privacy concerns. ***************************************************************

***************************************************************
Submitter states that missing raw files are due to patient privacy concerns.
***************************************************************

first: Kai

last: Yu

first: Ying

last: Yuan

first: Jianfeng

last: Chen

first: Linghua

first: Jianjun

last: Gao


title: Single cell gene expression profile of peripheral whole blood cells from long COVID patients

city: Suwon

first: Karolina

middle: Hanna

last: Prazanowska

address: Worldcup-Ro 164

institute: Ajou University School of Medicine

summary: In the post COVID-19 pandemic era, long COVID has emerged as a serious health concern. We generated single-cell RNA sequencing (scRNA-seq) data of peripheral whole blood cells from 18 patients involved in clinical trial studying the effectiveness of comercially available herbal medications in long COVID.

overall_design: The collected blood samples were processed to remove red blood cells via lysis. Processed samples were prepared for a multiplexed sequencing analysis. Obtained reads were pre-processed according to BD Rhapsody protocol.

first: Su

last: Lim


title: Chemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells [ATAC-seq]

city: Guangzhou

first: Yage

last: Nie

address: No.74 Zhongshan Rd.2, Guangzhou

institute: Sun Yat-sen University

summary: Definitive endoderm (DE) derived from human pluripotent stem cells (hPSCs) holds great promise for cell-based therapies and drug discovery. However, current DE differentiation methods required undefined components and/or expensive recombinant proteins, limiting their scalable manufacture and clinical use. Homogeneous DE differentiation in defined and recombinant protein-free conditions remains a major challenge. Here, by systematic optimization and high-throughput screening, we report a fully synthetic, small molecule-based defined system that contains only four components (4C), enabling highly efficient and cost-effective DE specification of hPSCs in the absence of recombinant proteins. 4C-induced DE can differentiate into functional hepatocytes, lung epithelium, and pancreatic β cells in vitro and multiple DE-derivatives in vivo. Genomic accessibility analysis reveal that 4C reconfigures chromatin architecture to allow key DE transcription-factor binding and identify TEAD3 as a novel key DE regulator. This system may facilitate mass production of mature DE-derivatives for drug discovery, disease modelling, and cell therapy.

overall_design: Bulk ATAC-seq  was performed on human embryonic stem cells (hESCs) at various time points (Day 0, 1, 2, and 3) following their treatment with Vitamin C (Vc) or LDN.


title: Chemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells [RNA-seq]

overall_design: Total RNA of cells was isolated using RNAzol. Sequencing libraries were constructed using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, NR605-1) according to the manufacturer’s instruction.ATAC-seq sequencing libraries were constructed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) according to the manufacturer’s instruction. Quality control of the libraries was performed using an Agilent 2100 Bioanalyzer and sequencing was performed using an Illumina NovaSeq 6000 Sequencer.


title: Protein kinase N1 mediates phosphorylation of H3.3 serine 31 for rapid gene activation

city: Bad Nauheim

first: Carsten

last: Kuenne

address: Ludwigstrasse 43

department: Bioinformatics

institute: Max Planck Institute for Heart and Lung Research


title: Protein kinase N1 mediates phosphorylation of H3.3 serine 31 for rapid gene activation (ATAC-Seq I)

summary: Atherosclerosis, which develops in the inner layer of arteries, is the major cause of myocardial infarction and stroke. Atherosclerotic plaques develop preferentially in arterial regions exposed to disturbed blood flow, such as vessel curvatures, bifurcations and branching points, where endothelial cells develop an inflammatory phenotype. How disturbed flow induces endothelial cell inflammation is incompletely understood. We show here that histone H3.3 phosphorylation at serine 31 (H3.3S31) plays a critical role in disturbed flow-induced endothelial inflammation, as it allows the rapid induction of FOS and FOSB, which are required for disturbed flow-induced inflammatory gene expression. We identified protein kinase N1 (PKN1) as the kinase responsible for disturbed flow-induced H3.3S31 phosphorylation. PKN1 becomes activated by disturbed flow in an integrin a5b1-dependent manner and then translocates to the cell nucleus. We found that PKN1 is also involved in the phosphorylation of the AP-1 transcription factor JUN. Mice with endothelium-specific loss of PKN1 or endothelial expression of S31 phosphorylation-deficient mutants of H.3.3 show reduced endothelial inflammation and disturbed flow-induced vascular remodeling in vitro and in vivo. Our data identify a novel mechanism of H3.3S31 phosphorylation which may serve as a target for preventive and therapeutic anti-atherosclerotic strategies.

overall_design: HUAECs were transfected with a control siRNA or siRNAs directed against  protein kinases N1 and were exposed to disturbed flow (DF).

first: Young-June

last: Jin

first: Mette

last: Bentsen

first: Stefan

last: Günther

last: Offermanns


title: Protein kinase N1 mediates phosphorylation of H3.3 serine 31 for rapid gene activation (RNA-Seq)


title: Protein kinase N1 mediates phosphorylation of H3.3 serine 31 for rapid gene activation (ATAC-Seq II)

overall_design: In order to identify early changes in endothelial chromatin accessibility induced by disturbed flow, we performed the Assay of Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq) in human umbilical artery endothelial cells (HUAECs) exposed to disturbed flow.


title: Proline exacerbates hepatic gluconeogenesis via paraspeckle-dependent mRNA retention [lncRNA-Seq]

city: Hangzhou

first: Yurong

last: Zhao

address: 886 Yuhangtang Road

institute: Zhejiang University

type: Non-coding RNA profiling by high throughput sequencing

summary: Type 2 Diabetes (T2D) is a global health issue characterized by abnormal blood glucose levels and often associated with excessive hepatic gluconeogenesis. Increased circulating non-essential amino acids (NEAAs) are consistently observed in T2D individuals; however, the specific contribution of each amino acid to T2D pathogenesis remains less understood. Here, we reported an unexpected role of the NEAA proline in coordinating hepatic glucose metabolism by modulating paraspeckle, a nuclear structure scaffolded by the long noncoding RNA Neat1. Mechanistically, proline diminished paraspeckles in hepatocytes, liberating the retained mRNA species into cytoplasm for translation, including the mRNAs of Ppargc1a and Foxo1, contributing to enhanced gluconeogenesis and hyperglycemia. We further demonstrated that the proline-paraspeckle-mRNA retention axis existed in diabetic liver samples, and intervening this axis via paraspeckle restoration significantly alleviated hyperglycemia in both female and male diabetic mouse models. Collectively, our results not only delineated a previously unappreciated proline-instigated, paraspeckle-dependent mRNA retention mechanism regulating gluconeogenesis, but also spotlighted proline and paraspeckle as potential targets for managing hyperglycemia.

overall_design: To investigate the role of non-essential amino acids (NEAAs) on lncRNA, the mouse primary hepatocytes (MPH) are treated with or without NEAA treatment. We than performed lncRNA expression profiling analysis using data obtained from lncRNA-seq.

first: Xinxin

last: Chai

first: Junxuan

last: Peng

first: Yi

last: Zhu

first: Rong

last: Dong

first: Junwei

last: He

first: Linghao

last: Xia

first: Sishuo

last: Liu

first: Jingzhou

first: Zhengping

last: Xu

first: Chi

last: Luo

first: Jinghao

last: Sheng


title: Proline exacerbates hepatic gluconeogenesis via paraspeckle-dependent mRNA retention [RIP-Seq]

overall_design: To investigate whether proline stimulation influences mRNA dynamics of paraspeckle in mouse primary hepatocytes (MPH), and the MPH are treated with or without proline treatment. We than used RNA-immunoprecipitation coupled with high-throughput sequencing (RIP-seq) to profile the paraspeckle-retained mRNAs using NONO as a surrogate for paraspeckles.

last: Chen


title: DNA mehtylation profiling of rhabdomyosarcoma tumors derived from genetically engineered mouse models

city: Bethesda

first: Frederic

last: Barr

state: MD

address: 10 Center Drive, Rm 3B55

institute: National Cancer Institute

type: Methylation profiling by genome tiling array

summary: Genome wide DNA methylation profiling of rhabdomuyosarcoma tumors derived from genetically engineered mouse models. The Illumina MouseMethylation285 BeadChip was used to obtain DNA methylation profiles across approximately 285,000 methylation sites in 31 freshly-frozen rhabdomyosarcoma tumors.

overall_design: Bisulphite converted DNA from the 31 samples were hybridised to the Illumina MouseMethylation285 BeadChip

first: Wenyue

last: Sun

middle: G


title: Proline exacerbates hepatic gluconeogenesis via paraspeckle-dependent mRNA retention

summary: Type 2 Diabetes (T2D) is a global health issue characterized by abnormal blood glucose levels and often associated with excessive hepatic gluconeogenesis. Increased circulating non-essential amino acids (NEAAs) are consistently observed in T2D individuals; however, the specific contribution of each amino acid to T2D pathogenesis remains less understood. Here, we reported an unexpected role of the NEAA proline in coordinating hepatic glucose metabolism by modulating paraspeckle, a nuclear structure scaffolded by the long noncoding RNA Neat1. Mechanistically, proline diminished paraspeckles in hepatocytes, liberating the retained mRNA species into cytoplasm for translation, including the mRNAs of Ppargc1a and Foxo1, contributing to enhanced gluconeogenesis and hyperglycemia. We further demonstrated that the proline-paraspeckle-mRNA retention axis existed in diabetic liver samples, and intervening this axis via paraspeckle restoration significantly alleviated hyperglycemia in both female and male diabetic mouse models. Collectively, our results not only delineated a previously unappreciated proline-instigated, paraspeckle-dependent mRNA retention mechanism regulating gluconeogenesis, but also spotlighted proline and paraspeckle as potential targets for managing hyperglycemia. This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: The translational landscape of HIV-1 infected cells reveals key gene regulatory principles

city: Würzburg

first: Neva

last: Caliskan

address: Josef-Schneider-Str. 2/D15

institute: Helmholtz Institute for RNA-based Infection Research (HIRI)

summary: Human immunodeficiency virus-1 (HIV-1) uses a number of strategies to modulate viral and host gene expression during its life cycle. To characterize the transcriptional and translational landscape of HIV-1 infected cells, we used a combination of ribosome profiling, disome sequencing and RNA sequencing. We show that HIV-1 messenger RNAs are efficiently translated at all stages of infection, despite evidence for a substantial decrease in the translational efficiency of host genes that are implicated in host cell translation. Our data identify upstream open reading frames in the HIV-1 5′-untranslated region as well as internal open reading frames in the Vif and Pol coding domains. We also observed ribosomal collisions in Gag-Pol upstream of the ribosome frameshift site that we attributed to an RNA structural fold using RNA structural probing and functional analysis. Antisense oligonucleotides designed to alter the base of this structure decreased frameshift efficiency. Overall, our data highlight the complexity of HIV-1 gene regulation and provide a key resource for decoding of host–pathogen interactions upon HIV-1 infection. Furthermore, we provide evidence for a RNA structural fold including the frameshift site that could serve as a target for antiviral therapy.

sample_organism:
Homo sapiens
Human immunodeficiency virus 1

overall_design: To investigate the translational landscape of HIV-1 infected T-cells, we performed ribosome profiling experiments (RiboSeq, DisomeSeq) treated either with cycloheximide or harringtonine, as well as RNASeq at different timepoints of infection (mock, 8 hpi, 16 hpi, 24 hpi). Furthermore, we analyzed the HIV transcriptome present in the cell with Nanopore sequencing. Lastly, to investigate the structure of the ribosome frameshift site, we employed RNA structural probing (DMS-MaPseq).

first: Anuja

last: Kibe

last: Buck

first: Anne-Sophie

last: Gribling-Burrer

first: Orian

last: Gilmer

first: Lukáš

last: Pekárek

first: Patrick

last: Bohn

first: Tatyana

last: Koch

first: Chiara

middle: N

last: Mireisz

first: Andreas

last: Schlosser

first: Florian

last: Erhard

first: Redmond

middle: P

last: Smyth


title: Composition profile of gut microbiota uptaking plant-derived nanoparticles

city: Louisville

first: Huang-Ge

last: Zhang

state: KY

address: 505 S Hancock Street

department: James Graham Brown Cancer Center

institute: University of Louisville

summary: Human diet emerges as a pivotal determinant of gut microbiota composition and function. Identification of the bacterial taxa targeted by diet derived factors with causal beneficial rather than detrimental effects on therapy and their mechanism of action is challenging but necessary for future clinical progress. The germ free mice colonized with human gut bacteria and four-plants derived nanoparticles uptaking bacteria were sorted with flow cytometry and identified with 16s rRNA next-generation sequencing.

overall_design: The feces DNA from mice and human gut was extracted and amplified on 16s rRNA gene for sequencing.

first: Yun

last: Teng