title: SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer’s Disease

status: Public on Dec 10 2023

city: Champaign

first: Pablo

last: Pinera

state: Illinois

address: 2332 West John Street, Apt. D

department: Bioengineering

institute: University of Illinois at Urbana-Champaign

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: SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer’s Disease [miSeq]

country: USA

summary: Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which has broad applications in molecular and cellular biology, medicine and biotechnology. Existing exon skipping techniques include antisense oligonucleotides, targetable nucleases and base editors, which, while effective for specific applications at some target exons, remain hindered by shortcomings preventing their broader implementation including transient effects in the case of oligonucleotides or limiting PAM motifs, sequence context preferences for deaminases, and undesirable cryptic splicing in the case of gene editing tools. To overcome these limitations, we created SPLICER, a toolbox of next-generation base editors consisting of near-PAMless Cas9 nickase variants fused with different deaminases for simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing not only improves exon skipping rates but also reduces aberrant outcomes such as cryptic splicing and intron retention. SPLICER enables editing of exon splice sites with high efficiency, including many exons refractory to splicing reprogramming by the native SpCas9 BEs. To demonstrate the therapeutic potential of SPLICER, we targeted APP exon 17, which contains the amino acid residues responsible for the formation of Aβ plaques in Alzheimer’s disease. SPLICER enabled precise and highly efficient exon skipping, which reduced the formation of Aβ42 peptides in vitro while inducing DNA editing and exon skipping in vivo within a humanized mouse model of Alzheimer’s disease. Overall, SPLICER is a widely applicable and highly efficient toolbox for exon skipping with broad therapeutic applications.

overall_design: Mammalian cell transfections with Cas9 base editors to monitor DNA editing and RNA expression and alternative splicing to investigate the effect of A>G and C>T SNPs at splice donor and acceptor sites to skip exons. AAV stereotaxic injections into mice brains to moniter DNA editing and exon skipping with  base editing tools

first: Angelo

last: Miskalis

first: Shraddha

last: Shirguppe

first: Jackson

last: Winter

first: Gianna

last: Elias

first: Devyani

last: Swami

first: Ananthan

last: Nambiar

first: Wendy

last: Woods

first: Michelle

last: Stilger

first: Alejandra

last: Zeballos

first: Hayden

last: Moore

first: Sergei

last: Maslov

first: Thomas

last: Gaj

last: Perez-Pinera


title: Epithelial zonation along the mammalian small intestine defines five discrete metabolic domains  [Mouse_bulkRNA-seq]

status: Public on Nov 28 2023

city: San Francisco

first: Rachel

middle: Klapper

last: Zwick

address: 513 Parnassus Ave.

institute: University of California, San Francisco

type: Expression profiling by high throughput sequencing

summary:
Longitudinal zonation of epithelial cells across the mammalian small intestine defines five domains of nutrient absorption.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at two time points.

overall_design: Intestinal organoids derived from the distal-most domain of the small intestine from adult Lgr5creERT2; Rosa26 LSL-Cas9-eGFP/+; ROSA26tdTomato mice, in which cas9-GFP is expressed in a tamoxifen-inducible manner. Sequenced organoids are either experimental Ppard (2 mutants) or Cdx1 (1 mutant) knockout organoids lines, which were transduced with dual guide RNAs targeting either gene and CRISPR-mediated gene deletion, or control lines (uninfected with virus containing dual guide RNAs with our without 4-hydroxytamoxifen treatment, or infected with dual guide RNAs but untreated with 4-hydroxytamoxifen).

middle: K

first: Dario

last: Boffelli

first: Ophir

middle: D

last: Klein


title: SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer’s Disease [RNA-seq]

last: Pinera

overall_design: HEK293T cells were transfected via lipofectamine 2000 with plasmids encoding either a GFP control or SpRY-Cas9-ABE8e to measure the effect that SpRY-Cas9-ABE8e has on both gene expression for the targeted gene (APP exon 17) as well as overall expression on genes associated with Alzheimer's disease. Additionally, overall fold change in gene expression patterns was measured to via DEG to observe if the base editor system had any effects on off-target mRNA expression as well as effects on expression pathways involved with base-excision repair or mismatch DNA repair. Following trnasfection, RNA samples were harvested from cells via RNeasy kit and sequenced.


title: Single-cell transcriptomics uncovers the roles of mesothelial cells and microenvironmental changes in the ultrafiltration failure after long-term peritoneal dialysis

status: Public on Nov 27 2023

city: GuangZhou

first: Xiangwen

last: Diao

address: No.58 Zhongshan Second Road

country: China

institute: The First Affiliated Hospital，Sun Yat-sen University

summary: Chronic exposure to biological incompatibility of peritoneal dialysis solution is one of the principal causes of ultrafiltration failure (UF). However, the changes of peritoneal cell composition and molecular mechanism of peritoneal microenvironment after long-term PD are unclear. Single-cell RNA sequencing was performed to profile cell composition and transcriptome characteristics in effluent obtained from patients undergoing PD with short vintage (SV), long vintage without ultrafiltration failure (LV_NOT_UF), and long vintage with ultrafiltration failure (LV_UF).  A total of 6 SV patients, 6 LV_NOT_UF patients, and 4 LV_UF patients were enrolled. The median PD duration of the three groups were 5 months (range:1.0–6.0 months), 137 months (range: 92–189 months) and 132 months (range: 120–161 months), with no significant differences in age (38.3 ± 21.5 years, 45.7 ± 10.6 years and 41.0 ± 16.7 years, P>0.05) and gender (male%: 33.3%, 50.0% and 25.0%, P>0.05). scRNA-seq data annotated 8 main cell types including epithelial-like mesothelial cells (E-MCs), mesenchymal-like mesothelial cells (M-MCs), fibroblast, plasma B cells, conventional dendritic cells, plasmacytoid dendritic cells, natural killer T cells, and myeloid cells. LV_UF patients showed higher proportion of E-MCs, which exhibited activated autophagy and had properties of both anti-inflammatory and pro-inflammatory, and neutrophil- and autophagy-related DEGs of E-MCs were upregulated in the LV_UF group. In contrast, the M-MCs and the AQP1 expression in the peritoneal mesenchyme were reduced in the LV_UF group compared to the other groups. The neutrophils in PD effluent were elevated in the LV_UF group, while the M2-like MRC1-macrophages gradually decreased from the SV, LV_NOT_UF to LV_UF group. Compared with the LV_NOT_UF group, the upregulated DEGs of monocytes/macrophages in the LV_UF group not only involved in pro-inflammatory response, but also promoted EMT progress. Additionally, neutrophils and monocytes/macrophages both interacted with MCs. Our study uncovers peritoneal different individual cell populations and cellular functions in different stages of PD which indicates the dynamic changes of microenvironments of peritoneal membrane. These findings provide new insights into the novel roles of mesothelial cells and inflammatory cells in UF.

overall_design: We collected overnight effluent from 16 PD patients, including 6 SV patients, 6 patients in the LV_NOT_UF group, and 4 patients in the LV_UF group. SV group: patients received PD for less than 6 months. LV_NOT_UF group: patients received PD for more than 90 months and modified PET (Mod-PET) test showed that the ultrafiltration volume was greater than 400 mL. LV_UF group: patients received PD for more than 90 months, Mod-PET test showed that the ultrafiltration volume was less than 400 mL.

first: Cuixia

last: Zhan

first: Hongjian

last: Ye

first: Haishan

last: Wu

first: Chunyan

last: Yi

first: Jianxiong

last: Lin

first: Haiping

last: Mao

first: Wei

last: Chen

first: Xiao

last: Yang


title: Epithelial zonation along the mammalian small intestine defines five discrete metabolic domains [Mouse_scRNA-seq]

summary: Longitudinal zonation of epithelial cells across the mammalian small intestine defines five domains of nutrient absorption.

overall_design: Sorted epithelial cells and intestinal progenitor cells (intestinal stem cells and transit amplifying cells) were collected from 30 equal segments across the full length of 2 murine small intestines, barcoded using MULTIseq technology, pooled, and sequenced.


title: Epithelial zonation along the mammalian small intestine defines five discrete metabolic domains [Human_scRNA-seq]


title: Transcriptome sequencing of parasitoid wasp Muscidifurax zaraptor and its Nosema parasite

status: Public on Nov 26 2023

city: Auburn University

first: Xu

last: Wang

state: AL

address: 273 CASIC Building, Auburn Research Park, 559 Devall Dr

department: Pathobiology, College of Veterinary Medicine

institute: Auburn University

summary: Nosema is a diverse fungal genus of microsporidian unicellular, obligate symbionts of insects and other arthropods. We performed a comparative genomic analysis of N. muscidifuracis, a Nosema species infecting parasitoid wasp genus Muscidifurax, with six other genome-sequenced Nosema species. A sequence motif containing at least three consecutive Cs was significantly enriched immediately upstream of the start codon in all seven Nosema genomes. Interestingly, this motif is present in ~90% of highly expressed genes, compared to ~20% in lowly expressed genes N. muscidifuracis, which may function as a cis-regulatory element for gene expression control and regulation. Our study provides new insights into the gene regulation evolution in Nosema.

overall_design: Male and female whole adult Muscidifurax zaraptor were collected 24 hours after eclosion with three replicates for RNA sequencing to quantify the gene expression profile of M. zaraptor and its fungal pathogen Nosema muscidifuracis.


title: SLAM-seq estimates of mRNA half-life changes in S. cerevisiae strains depleted for Dcp2 and Ccr4/Pop2

status: Public on Nov 24 2023

city: Zürich

first: Mostafa

last: Zedan

address: Otto-Stern-Weg 3

country: Switzerland

institute: ETH-Zurich

summary: In eukaryotes, mRNA decay is thought to occur in an ordered manner, with an initial deadenylation step that triggers decapping and RNA degradation. We performed SLAM-seq experiments in yeast strains depleted either for the decapping or for the major deadenylase enzymes to evaluate changes in the half-life of mRNA. Our results suggest that deadenylation and decapping of mRNA are not necessarily linked for mRNA degradation, since the effects of inhibiting the enzymes on RNA stability were not correlated.

overall_design: We performed SLAM-seq to analyze changes in mRNA decay after Pop2/Ccr4 depletion, or dcp2 depletion. In short, we grew the cells in SCD media containing low uracil for two doubling times. To pulse label mRNA, we added 4TU to a final concentration of 1mM for 60 min. To deplete the targeted proteins, both Auxin and Estradiol (or only Ethanol, for the undepleted controll) were added to the media (in the presence of 4TU) and the cells were incubated for 60 min. To chase, the cells were filtered and resuspended in SCD containing excess Uracil (20 mM) and no 4TU, in the presence of Auxin and Estradiol, or Ethanol. Samples were collected every 5 mins, followed by RNA-extraction, RNA alkylation with Iodoacetamide, and library preparation using Quantseq for 3’end sequencing. The experiment was performed in two replicates.

first: Léna

last: Audebert

first: Frank

last: Feuerbach

first: Alexandra

last: Schürch

first: Laurence

last: Decourty

first: Abdelkader

last: Namane

first: Emmanuelle

last: Permal

first: Karsten

last: Weis

first: Gwenaël

last: Badis

first: Cosmin

last: Savean


title: Adherent reformed islets: a long-term primary cell-based platform for exploring mouse and human islet biology

status: Public on Dec 25 2023

city: London

first: Naila

last: Haq

state: London

address: Room HB4.2E, 2nd Floor, Hodgkin Building, Guy's Campus, London Bridge,

country: United Kingdom

institute: King's College London

summary: Pancreatic islets are 3-dimensional micro-organs that maintain β-cell functionality via cell-cell and cell-matrix communication. Isolated primary islets are the gold standard for in vitro models. However, native islets present experimental challenges for long-term mechanistic studies owing to their short culture life (approximately 1 week). We developed a novel long-term protocol to study the function of primary islets. The protocol employed reformed islets following dispersion and a fine-tuned culture environment. Reformed islets are highly similar to their primary counterparts across various physiological characteristics. Long-term culture of reformed islets enables high-resolution imaging, repeated functional assessment, and the study of cell-cell communication. Unlike other platforms such as stem cell-derived organoids, reformed islets retain their resident immune populations, making them ideal for studying both resident and infiltrating immune cells and their interactions with hormone-producing islet cells. Qualitative and quantitative analyses revealed that the composition and cytoarchitecture of the reformed islets mimicked those found in primary islets, including the presence of macrophages and CD4+ and CD8+ T cells, which are the key resident immune cell types. Reformed islets secrete insulin and are glucose-responsive, and their β-cells can be stimulated to proliferate using GLP-1 receptor agonism. Furthermore, a comparison of the transcriptomic landscape of isolated human islets and reformed islets generated from the same donor demonstrated a high degree of similarity. Our reformed islets provide an ideal platform to study diabetes pathology. We recapitulated both the T1DM and T2DM disease milieu and validated our model for studying islet immune trafficking and invasion using activated macrophages and T cells. Our data illustrates that reformed islets are an anatomical and functional alternative to native human and mouse islets.  Moreover, reformed islets have an advantage over mouse and human β-cell lines, including MIN6 and EndoC-βH1cells, that lack the signalling input of non-β-endocrine cells and immune cell crosstalk. In this study, we showed that reformed islets are a durable paradigm (cell-based model) for islet-based exploration and a means of target discovery/validation for diabetes research.

overall_design: Given the need for alternative methods for the long-term culture of primary islets, we aimed to refine reaggregation techniques to generate a superior adherent 3D islet culture platform using native murine or human islets as the source.  These reaggregated islets are referred to as reformed islets. They can be maintained in culture for at least 4-6 weeks, making them ideal for long-term studies, including high-resolution imaging, suitable for repeated functional assessment, and studying cell-cell communication. We found the reformed islets to be architecturally, transcriptionally, and functionally highly similar to the native islets they are generated from.

first: N

first: K

middle: W

last: Toczyska

first: M

middle: E

last: Wilson

last: Jacobs

first: Min

last: Zhao

first: Y

last: Lei

first: J

last: Pearson

first: S

middle: J

last: Persaud

first: T

last: Pullen

first: G

middle: A

last: Bewick


title: Radiomics Predicts High or Low Recurrence Risk and is Associated with LncRNAs in Breast Cancer

status: Public on Nov 21 2023

city: Guangzhou

first: Wenhao

last: Ouyang

state: GuangDong

address: Department of Medical Oncology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University

summary: The key radiomic features were found to vary after neoadjuvant chemotherapy. Moreover, lncRNAs were discovered to be significantly correlated with radiomics and recurrence-free survival (RFS). The findings indicate that the radiomic signature can be conveniently used for individualized prediction of RFS and that radiomics is associated with lncRNAs in breast cancer.

overall_design: Total RNA-seq profiles of breast cancer patients

first: Wei

last: Ren

first: Yunfang

last: Yu

first: Herui

last: Yao


title: Histone modifications mediate transcription factor regulation and contribute to neuronal subtype identity

status: Public on Nov 18 2023

city: Blacksburg

first: Xiguang

last: Xu

state: VA

address: 1015 Life Science Circle

department: Department of Biomedical Sciences and Pathobiology

institute: Virginia Tech

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To investigate the cell-type specific histone modification and transcription factor bindings, we performed cut and run of four histone modifications and the transcription factor EGR1 in excitatory and inhibitory neurons in adult mouse brain.

overall_design: CUT&RUN

first: Liduo

last: Yin

first: Hehuang

last: Xie


title: HBV-KMT2B integrated human induced pluripotent stem cells (KMT2B-Int iPSCs) vs heterozygous mutated KMT2B iPSCs (KMT2B-HT iPSCs) vs homozygous mutated KMT2B iPSCs (KMT2B-KO iPSCs) vs their original iPSCs (KMT2B-WT iPSCs)

status: Public on Nov 15 2023

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: 10-1 tebiro 6-chome

country: Japan

institute: Toray Industries,Inc.

summary: Transcriptional profiling of all KMT2B-WT, HT, KO, Int iPSCs. KMT2B-HT, KO, and Int iPSCs were generated by gene editing using CRISPR/Cas9 and Cre recombinase technologies.

overall_design: Transcriptional profiling of all KMT2B-WT, HT, KO, Int iPSCs. As the KMT2B-WT iPSCs, P11025 iPSCs were used, which were reprogrammed by the episomal vector from normal skin. KMT2B-HT, KO, and Int iPSCs were generated by gene editing using CRISPR/Cas9 and Cre recombinase technologies. KMT2B-HT iPSCs were used as control cell lines of KMT2B-Int iPSCs because KMT2B Int iPSCs were heterozygous KMT2B due to HBV integration. DNA sequences as results of gene editing such as integration of HBV sequences were determined by direct sequencing. Knockout of KMT2B also was confirmed by immunoblotting. All human iPSC lines were cultured and harvested before being fully confluent.

first: Jun

last: Tsuchiya

first: Masato

last: Miyoshi

first: Sei

last: Kakinuma


title: Epithelial zonation along the mammalian small intestine defines five discrete metabolic domains

first: Rachel

last: Zwick

relation:
SuperSeries of: GSE201855
SuperSeries of: GSE201857
SuperSeries of: GSE201858
SuperSeries of: GSE251694
SuperSeries of: GSE251695


title: Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity

status: Public on Nov 14 2023

city: Rehovot

first: Aviad

last: Ben-Shmuel

state: Other

address: Nella and Leon Benoziyo Building for Biological Sciences, Herzl

department: Biomolecular Sciences

country: Israel

institute: Wiezmann Institute

summary: Cancer associated fibroblasts (CAFs) are one of the most abundant components of the breast tumor microenvironment (TME) and major contributors to immune modulation in the TME. CAFs are well known to regulate the activity of diverse types of immune cells including T cells, macrophages and dendritic cells, however little is known about their interaction with Natural killer (NK) cells. NK cells constitute an important arm of anti-tumor immunity, yet the regulation of NK cell activity by CAFs in solid tumors is poorly understood. Here we find, using mouse models of cancer and ex-vivo cocultures, that immunosuppressive CAF subsets severely inhibit NK cell cytotoxicity towards cancer cells. We unravel the mechanism by which this suppression occurs, through CAF-mediated downregulation of the NK-surface receptors, Natural Killer Group 2D (NKG2D) and DNAX Accessory Molecule-1 (DNAM-1). Ligands for these receptors are known to be expressed by cancer cells and minimally expressed in healthy tissue. Here we find that CAFs also upregulate ligands for NKG2D and DNAM-1. Ligand-receptor engagement between NK cells and CAFs leads to CAF cytolysis, which in turn diminishes the expression of NKG2D and DNAM-1 on NK cells via a negative feedback loop, and promotes cancer escape from NK cell surveillance. These results reveal a CAF-mediated immunosuppressive mechanism with implications for treatment of solid tumors.

overall_design: To investigate the transcriptomic state of NK cells in the 4T1 TNBC model and assess how they may be influenced by CAFs, we sequenced 1) primary NK cells from the spleens of healthy BALB/C mice as controls (3 independent replicates) 2) primary NK cells from spleens of BALB/C mice harboring 4T1-GFP tumors as controls (4 independent replicates) and 3) 4T1 tumor infiltrating (TI) NK cells (4 independent replicates). NK cells were isolated using  magnetic seperation followed by FACS with the following markers: GFP-EPCAM-CD45+CD3-CD31-CD14-NKp46+. To investigate the genes in sCAFs that may influence NK cell migration and retention in stromal areas, we sequenced sCAFs (3 independent replicates) isolated from 4T1-GFP tumors. sCAFs were isoalted using magnetic seperation followed by FACS with the following markers: EPCAM-GFP-CD45-CD31-PDPN-

first: Yael

last: Gruper

first: Ruth

last: Scherz-Shouval


title: Influence of postruminal casein infusion and exogenous glucagon-like peptide 2 administration on the jejunal mucosal transcriptome in cattle

city: Lexington

first: Ronald

middle: Joseph

last: Trotta

state: KY

address: 808 W.P. Garrigus Bldg.

department: Animal and Food Sciences

institute: University of Kentucky

summary: The objective of the current study was to evaluate the effects of postruminal casein infusion and exogenous glucagon-like peptide 2 (GLP-2) administration on transcriptomic pathways and functions of the jejunal mucosa in cattle using next-generation RNA sequencing. Twenty-four Holstein steers [250 ± 23 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW for 7 d. Steers received subcutaneous injections in two equal portions daily of excipient (0.5% bovine serum albumin) or 100 µg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for collection of the jejunal mucosa.  Total RNA was extracted from jejunal mucosal tissue, strand-specific cDNA libraries were prepared, and RNA sequencing was conducted to generate 150-bp paired-end reads at a depth of 20 M reads in each direction. Differentially expressed genes (DEG), KEGG pathway enrichment, and gene ontology enrichment between treatments were determined based on the FDR-corrected P-value. Postruminal casein infusion downregulated (padj < 0.05) 7 genes of the jejunal mucosa and did not result in any enriched KEGG pathways or gene ontologies. Exogenous GLP-2 administration upregulated (padj < 0.05) 667 DEGs, 26 KEGG pathways, 198 biological processes, 56 cellular components, and 60 molecular functions. Exogenous GLP-2 downregulated (padj < 0.05) 1101 DEGs, 14 KEGG pathways, 270 biological processes, 105 cellular components, and 46 molecular functions. Enrichment analyses revealed that exogenous GLP-2 administration affected several pathways involving mucosal growth, nutrient digestion, absorption, and metabolism, and intestinal barrier function. Results of the current study identified pathways affected by exogenous GLP-2 administration that could have implications for improved animal growth and feed efficiency in cattle. This research generated novel targets for future research to elucidate mechanisms of GLP-2-mediated responses in the jejunal mucosa.

overall_design: Steers were transferred to metabolism stalls (1.2 m x 2.4 m) for the 7-d treatment period. Due to limited stall space and infusion apparatus, steers were stratified by BW into 6 replicate blocks. Steers were then randomly assigned to one of four treatments within each replicate block. The experimental design was a randomized complete block design with a 2  2 factorial arrangement of treatments. All steers were abomasally infused with 3.94  0.245 g raw corn starch  kg BW-1  d-1. Along with raw corn starch, steers were abomasally infused with either water or 10% (wt:wt) sodium caseinate solution to provide 0 or 1.30  0.299 g sodium caseinate  kg BW-1  d-1. Additionally, steers received subcutaneous injection treatments in two equal portions daily. Subcutaneous injection treatments were either vehicle (5 g/L bovine serum albumin (BSA) diluted in 9 g/L NaCl) or 100 µg GLP-2 in vehicle  kg BW-1  d-1. This resulted in four treatments: 1) abomasal water + vehicle injection (control), 2) abomasal water + GLP-2 injection (GLP-2), 3) abomasal 10% sodium caseinate solution + vehicle injection (casein), and 4) abomasal 10% sodium caseinate solution + GLP-2 injection (casein + GLP-2). All steers received abomasal infusion and subcutaneous injection treatments for 7 d.

first: Kendall

middle: C

last: Swanson

first: James

middle: L

last: Klotz

first: David

last: Harmon


title: Contributions of chondrocyte senescence and menopause to the increased incidence of osteoarthritis in older women

status: Public on Nov 06 2023

city: Rochester

first: Jessica

last: Hohenstein

state: MN

address: 200 1st St SW

institute: Mayo Clinic

summary: Osteoarthritis (OA) is a degenerative joint disease and a leading cause of disability worldwide. Aging is one of the major risk factors for OA, with the disease prevalence dramatically increasing after age 50. While OA is strongly associated with aging, the specific mechanisms underlying this connection remain unclear. In this pilot study, we performed bulk RNA sequencing on human knee articular chondrocytes to identify transcriptomic changes underlying OA development in the context of aging. We compared the gene expression profiles of chondrocytes isolated from osteoarthritic cartilage of older individuals (age 70-80 years) to those of chondrocytes from the healthy cartilage of a young adult (age 26 years). To model aging in vitro, chondrocytes were serially passaged until they reached the replicative senescence.

overall_design: osteoarthritic chondrocytes and healthy chondrocytes were used. The cells from each biospecimen were subjected to replicative senescence by passaging until they reached their Hayflick limit,which designated as old cells.

first: Aysegul

last: Zeybek

first: Chris

last: Evans

last: Nagelli


title: Illumina RNAseq of ccRCC tumour cell line RCC4

status: Public on Oct 30 2023

city: York

first: Joshua

last: Lee

address: University Road

department: Hull York Medical School

institute: University of York

summary: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer.  Here, we used Illumina RNAseq to profile the transcriptomes of a ccRCC cell line RCC4 treated with pro-inflammatory cytokines. Our results  here validates the existence of novel isoforms and genes that were discovered in the archival tumour samples by long-read sequencing. In addition, results here revealed potential tumour origin of these novel isoforms and genes.

overall_design: RCC4 cells previously transduced with lentiviral Cas9 expression vector  were harvested for total RNA after being treated with pro-inflammatory cytokines IFNg&TNF for 24h . Poly(A)+ RNA molecules were isolated and used as input for sequencing library preparation (NEB Ultra III directional RNA library prep kit) according to manufacturer's instructions. Sequencing libraries were sequenced on an Illumina HiSEq 3000 sequencer.

first: Dimitris

last: Lagos


title: PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression

status: Public on Nov 01 2023

city: Buffalo

first: Henry

middle: G.

last: Withers

state: New York

address: Elm and Carlton Streets

department: Biostatistics and Bioinformatics

institute: Roswell Park Comprehensive Cancer Center

summary: PTEN loss, one of the most frequent mutations in prostate cancer (PC), is presumed to drive disease progression through AKT activation. However, two transgenic PC models with Akt activation plus Rb loss exhibited different metastatic development: Pten/RbPE:-/- mice produced systemic metastatic adenocarcinomas with high AKT2 activation, whereas RbPE:-/- mice deficient for the Src-scaffolding protein, Akap12, induced high-grade prostatic intraepithelial neoplasias and indolent lymph node dissemination, correlating with upregulated phosphotyrosyl PI3K-p85α. Using PC cells isogenic for PTEN, we show that PTEN-deficiency correlated with dependence on both p110β and AKT2 for in vitro and in vivo parameters of metastatic growth or motility, and with downregulation of SMAD4, a known PC metastasis suppressor. In contrast, PTEN expression, which dampened these oncogenic behaviors, correlated with greater dependence on p110α plus AKT1. Our data suggest that metastatic PC aggressiveness is controlled by specific PI3K/AKT isoform combinations influenced by divergent Src activation or PTEN-loss pathways.

overall_design: Four high-grade PIN lesions from Akap12/Rb-null mice  (Pr_14273, Pr_14345, Pr_14354, Pr_14433).  Two PC tumors from Pten/Rb-null mice (PT_T57, PT_173PT) are available in GSE90891 as samples GSM2417050 and GSM2417051, respectively.

first: Karina

last: Miller

first: Irwin

middle: H

last: Gelman


title: LARP1 senses free ribosomes to coordinate supply and demand of ribosomal proteins

status: Public on Oct 28 2023

city: Baltimore

last: Saba

state: MD

address: 725 N Wolfe St, 714 PCTB

department: Molecular Biology and Genetics

institute: Johns Hopkins

summary: Terminal oligopyrimidine motif-containing (TOP) mRNAs encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies implicate LARP1 in 40S- or 80S-ribosome complexes that repress and stabilize TOP mRNAs. However, a mechanistic understanding of how LARP1 and TOP mRNAs interact with ribosomes to coordinate TOP mRNA outcomes is lacking. Here, we show that LARP1 senses the cellular supply of ribosomes by directly binding non-translating 80S ribosomes. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the 40S mRNA channel and mutations at the LARP1-ribosome interface block formation of the 40S/80S-LARP1-TOP complexes. Free cytosolic ribosomes induce sequestration of TOP mRNAs in repressed 80S-LARP1-TOP complexes independent of alterations in mTOR signaling. Together, this work demonstrates a ribosome-sensing function of LARP1 that allows it to tune ribosome protein synthesis to the availability of free ribosomes.

overall_design: To quantitatively evaluate the number of ribosomes associated with different mRNA species upon inhibiton of the mammalian target of rapamycin (mTOR), we treated U266B1 cells with Torin1, separated cell lysates across sucrose gradients, and performed cDNA-PCR nanopore sequencing (with spike-in eGFP mRNA) of mRNAs extracted from each fraction

first: Zixuan

last: Huang

first: Kate

last: Schole

first: Xianwen

last: Ye

first: Shrey

last: Bhatt

first: Yi

last: Li

first: Winston

last: Timp

first: Jingdong

last: Cheng

last: Green


title: Modeling early phenotypes of Parkinson’s disease by age-induced midbrain-striatum assembloids

city: Esch-sur-Alzette

first: Jens

middle: Christian

last: Schwamborn

address: 6 Avenue du Swing

country: Luxembourg

institute: Univeristy of Luxembourg

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

overall_design: Refer to individual Series


title: Modeling early phenotypes of Parkinson’s disease by age-induced midbrain-striatum assembloids [scRNA-Seq]

summary: Parkinson’s disease, an aging-associated neurodegenerative disorder, is characterised by nigrostriatal pathway dysfunction caused by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Human in vitro models are enabling the study of the dopaminergic neurons’ loss, but not the dysregulation within the dopaminergic network in the nigrostriatal pathway. Additionally, these models do not incorporate aging characteristics which potentially contribute to the development of Parkinson’s disease. Here we present a nigrostriatal pathway model based on midbrain-striatum assembloids with inducible aging. We show that these assembloids can develop characteristics of the nigrostriatal connectivity, with catecholamine release from the midbrain to the striatum and synapse formation between midbrain and striatal neurons. Moreover, Progerin-overexpressing assembloids acquire aging traits that lead to early neurodegenerative phenotypes. This model shall help to reveal the contribution of aging as well as nigrostriatal connectivity to the onset and progression of Parkinson’s disease.

overall_design: Single-nuclei RNA sequencing of midbrian-striatum assembloids, midbrain organoids and striatum organoids, generated from the same iPSC line. Sequencing was performed on assembloids at D30 of differentiation, which is the equivalent time point for midbrain organoids at D50 and striatum organoids at D65.

first: Kyriaki

last: Barmpa


title: Modeling early phenotypes of Parkinson’s disease by age-induced midbrain-striatum assembloids [RNA-Seq]

overall_design: RNA sequencing analysis of assembloids generated from a healthy male cell line (WT) and its isogenic genetically engineered line with the progerin transgene (PROG), under the control of the TetOn system. Assembloids from both cell lines were either untreated (UNTR) or treated with doxycycline (DOX). Doxycycline is used to induce the expression of the progerin transgene in the PROG assembloids.


title: A cross-sectional study of gene expression in different strains of Nothobranchius furzeri

status: Public on Oct 24 2023

city: Jena

first: Philipp

last: Koch

address: Beutenbergstraße 11

department: Core Facility Life Science Computing

country: Germany

institute: Leibniz Institute on Aging - Fritz Lipmann Institute

summary: We quantified gene expression in three different strains of the short-lived killifish Nothobranchius furzeri during aging.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for brain and skin samples at different life times in short (GRZ-D) and longer-lived strains ( MZCS-0403 and MZM-0403).

first: Kathrin

last: Reichwald

first: Marco

last: Groth

first: Alessandro

last: Cellerino


title: Aminoglycoside heteroresistance in Enterobacter cloacae is driven by the cell envelope stress response.

status: Public on Oct 18 2023

first: Avinash

middle: R

last: Shenoy

address: Rm 4.40A Flowers Bldg, Armstrong Road

department: Dept of Infectious Disease

institute: Imperial College London

summary: Enterobacter cloacae is a Gram-negative nosocomial pathogen of the ESKAPE (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter spp.) priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, E. cloacae can also display forms of antibiotic refractoriness, such as heteroresistance and tolerance. Here, we report that E. cloacae displays transient heteroresistance to aminoglycosides, which is accompanied with the formation of small colony variants (SCVs) with increased minimum inhibitor concentration (MIC) of gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene-expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the cpxRA two-component system abrogated aminoglycoside heteroresistance and SCV formation, pointing to its indispensable role in these processes. The introduction of a constitutively active allele of cpxA led to high aminoglycoside MICs, consistent with cell envelope stress response driving these behaviours in E. cloacae. Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild-type, but not in the ΔcpxRA mutant. Moreover, copper exposure also elevated the gentamicin MICs of clinical isolates from bloodstream infections, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in E. cloacae strains. Altogether, we establish that E. cloacae relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic.

sample_organism: Enterobacter cloacae subsp. cloacae ATCC 13047

overall_design: Two comparisons were made in this study. 1. Differentially expressed genes in exponentially growing "Control" E. cloacae vs small colony variants of E. cloacae ("SCV") 2. SCV untreated or treated with gentamicin (8 mg/L) for 2 h ("SCV Genta").


title: Regulation of muscle stem cell function by Mpp7, Amot, Yap and Taz

status: Public on Oct 07 2023

first: Anwen

last: Shao

address: 3520 San Martin Drive

department: Department of Embryology

institute: Carnegie Instituition for Science

summary: Coordinating mechanical sensing and transcription activation is essential for muscle stem cell (MuSC) function during muscle regeneration. Here we report that MPP7, localized at the apical side of quiescent MuSCs, is an important regulator in MuSC activation, including proliferation and self-renewal. Mechanistically, MPP7 translocates to cell nucleus in response to the change of actin polymerization state with its binding partner AMOT (an actin binding protein) upon MuSC activation and acts to enhance the transcription activity of YAP and TAZ in the MuSC.  RNA-seq of MuSCs isolated from control (Con), Mpp7 conditional knockout (cKO), Amot cKO, and Yap and Taz double cKO (YapTaz cKO) after 48 hrs in culture (for activation) in 2 replicas was used to determine the downtream genes regulated by Mpp7, Amot, Yap and Taz.  Carm1 was one of the commonly shared down-regulated genes in all 3 cKOs and has been shown to be critical for MuSC self-renewal. We further demonstrated that the MPP7-PDZ domain interacts with TAZ indirectly via AMOT, and the MPP7-L27 domain cooperates with TAZ and another trsncription factor YY1 to regulate a select set of donwstream genes, including Carm1, for MuSC proliferation and self-renewal. Our data identified a coordinated molecular network from mechanical sensing to transcription regulation for MuSC function during muscle regeneration.

overall_design:
To investigate the relationship of MPP7/AMOT/YAP and TAZ in regulating satellite cell (SC) activation, Mpp7 cKO, Amot cKO and YapTaz cKO mice were generated and SCs from the mice were sorted and cultured to activate SCs.
Gene expression profiling analysis was then performed using RNA seq data from control and conditional knockout SCs at 48 hrs after culture.
Cross comparison of differentially expressed genes from RNA seq for control and Mpp7 cKO, Amot cKO and YapTaz cKO SCs

first: Chen-Ming

last: Fan


title: Intercellular communication atlas reveals Oprm1 as a neuroprotective factor for retinal ganglion cells

status: Public on Oct 01 2023

first: Ying

last: Xin

address: 400 N Broadway (Smith Building)

institute: Johns Hopkins University

summary: The neuronal cell death results in neurodegenerative diseases. The extracellular environment plays a critical role in regulating cell viability. In this study, we explore how intercellular communication contributes to the survival of retinal ganglion cells (RGCs) following the optic nerve crush (ONC). By performing single-cell RNA-seq on whole retinal cells, we observed transcriptomic responses in non-RGC retinal cells to the injury, with astrocytes and Müller glia having the most interactions with RGCs. By comparing the RGC subclasses showing distinct resilience cell death, we identified top 47 interactions that are stronger in the high-survival RGCs, likely representing neuroprotective interactions. We performed functional assays on one of the receptors, Mu-opioid receptor (Oprm1). Although Oprm1 is preferentially expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), its neuroprotective effect could be transferred to multiple RGC subclasses by specific overexpressing Oprm1 in pan-RGCs. Our study provides an atlas of cell-cell interactions in both intact and post-ONC retina and an effective strategy to predict molecular mechanisms in neuroprotection, underlying the principal role played by extracellular environment in supporting neuron survival.

overall_design: With 12-hour, 24-hour, 48-hour post-ONC, or sham conditions, the mice were euthanized. Each time point had two biological replicates, except the 12-hour condition had one sample. Retinas were quickly and carefully dissected, and whole retinal cells were dissociated using the Papain enzyme system (LK003150, Worthington). 30mM actinomycin-D was added to the Papain system to prevent post-enucleation transcription. The tissues then underwent manual trituration in neurobasal medium supplemented with 3% BSA, 10 mg/mL Ovomucoid, 1U/uL RNase inhibitor, and 3mM actinomycin D. Cells were filtered through a 40-μm strainer and centrifuged under 500´g. The scRNA-seq libraries were prepared using the 10X Genomics Chromium 3’ Single-Cell Gene Expression V3 Kit following the standard protocol by the manufacturer. Approximately 8,000-12,000 cells were loaded onto the platform. Libraries were sequenced with the NovaSeq S2 100 platform.

first: C

last: Qian

first: Y

last: Qi

first: H

last: Wang

first: B

last: Dong

first: D

last: Zack

first: S

last: Blackshaw

last: Hattar

first: F

middle: Q

last: Zhou

first: J


title: MUC1-C integrates induction of aerobic glycolysis with suppression of oxidative phosphorylation in cancer stem cells

status: Public on Oct 11 2023

first: Henry

last: Withers

summary: The MUC1-C protein evolved in mammals for adaptation of barrier tissues to loss of homeostasis. Prolonged activation of MUC1-C in settings of chronic inflammation promotes lineage plasticity, epigenetic reprogramming and the cancer stem cell (CSC) state. The effects of MUC1-C on the metabolism of CSCs remain unexplored. The present studies performed on purified populations of triple-negative breast cancer (TNBC) CSCs demonstrate that MUC1-C integrates the capacity for self-renewal with the activation of aerobic glycolysis. We show that MUC1-C is essential for (i) CSC mammosphere formation and tumorigenicity and (ii) activation of GLUT1, HK2 and other glycolytic genes. We further show that MUC1-C activates nuclear genes that encode components of mitochondrial Complexes II-V of the electron transfer chain (ETC). Of importance, MUC1-C also represses mitochondrial DNA (mtDNA) genes encoding components of Complexes I-V. The observed involvement of MUC1-C in the suppression of mtDNA genes is explained by MUC1-C-mediated (i) downregulation of the mitochondrial transcription factor A (TFAM), which is required for mtDNA transcription, and (ii) induction of the mitochondrial transcription termination factor 3 (mTERF3). In support of this pathway to suppress mitochondrial ROS production, targeting MUC1-C increases (i) mtDNA gene transcription, (ii) superoxide levels and (iii) loss of self-renewal capacity. These findings indicate that MUC1-C regulates CSC self-renewal and redox balance by integrating activation of glycolysis with suppression of oxidative phosphorylation.

overall_design: Human TNBC cells (BT-549) were grown in 2D or 3D (mammospheres) culture. In triplicate experiments, MUC1-C or MYC was silenced using doxycycline inducible shRNAs and total RNA collected to assess effects on global transcriptional profiles. BT-549 mammospheres were additionally treated with GO-203, an inhibitor of MUC1-C function.

first: Donald

last: Kufe

first: Nami

last: Yamashita

middle: G


title: Expression data of tonsills of chronic tonsillitis, and peri-tumoral tonsillar tissues and lymph nodes in oropharyngeal cancer

status: Public on Oct 03 2023

city: Kanazawa

first: Naohiro

last: Wakisaka

state: Ishikawa

institute: National Hospital Organization Kanazawa Medical Center

summary:
Secondary lymphoid organs (SLOs) serve as an interface between tumor cells and immune system as an initial site of antigen presentation which is critical for the development of an effective anti-tumor immune response.
We used microarray to analyze the gene expression profile of tonsills from chronic tonsillitis, and peri-tumoral tonsillar tissues and lymph nodes of oropharyngeal cancer.

overall_design: We analyzed and compared the profiles of these organs.


title: cohesin spreads bi-directionally to form chromatin jets in C. elegans

status: Public on Sep 30 2023

city: New York

first: Jun

last: Kim

state: NY

address: 100 Washington Square East 1009 Silver Center

department: Biology

institute: NYU

summary: Inspection of Hi-C and ChIP-seq data suggests that cohesin spreads from putative loading sites form chromatin jets

overall_design: ChIP-seq and Hi-C data in auxin treated control, SMC-3 and WAPL-1 depletion conditions. All experiments were performed in two or more biological replicates. L2/L3 stage worms were used.

first: Sevinc

last: Ercan


title: ChEC-seq2: an improved chromatin endogenous cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein–DNA interactions

status: Public on Nov 07 2023

city: Evanston

first: Jason

last: Brickner

state: IL

address: Hogan 1-500, 2205 Tech Drive

institute: Northwestern University

summary: We validated ChEC-seq2 with MNase-fusions to transcription factors with well-documented motifs and binding sites in S. cerevisiae.

overall_design:
ChEC-seq2 with Rap1-MNase, Gcn4-MNase, and Ino2-MNase in S. cerevisiae.  Soluble MNase was used as a control.  Biological triplicates are provided.

***********************************************
Grant for this project:
Grant ID: R35 GM136419
Grant Title: The molecular mechanisms and functional significance of gene positioning
PI: Jason Brickner

first: Daniel

last: VanBelzen

first: Chengzhe

last: Duan

first: Donna


title: TRIM28-dependent developmental heterogeneity determines cancer susceptibility through distinct epigenetic states [DNA methylation array - d10earclips]

status: Public on Sep 07 2023

city: Grand Rapids

first: Luca

last: Fagnocchi

state: MI

address: 333 Bostwick AVE NE

institute: VAI

type: Methylation profiling by genome tiling array

summary: Mutations in cancer risk genes increase susceptibility, but not all carriers develop cancer. Indeed, while DNA mutations are necessary drivers of cancer, only a small subset of mutated cells go on to cause the disease. To date, the mechanisms underlying individual cancer susceptibility remain unclear. Here, we took advantage of a unique mouse model of intrinsic developmental heterogeneity (Trim28+/D9) to investigate whether early-life epigenetic variation influences cancer susceptibility later in life. We found that heterozygosity of Trim28 is sufficient to generate two distinct early-life epigenetic states associated with differing cancer susceptibility. These developmentally primed states exhibit differential methylation patterns at typically silenced heterochromatin, detectable as early as 10 days of age. The differentially methylated loci are enriched for genes with known oncogenic potential, frequently mutated in human cancers and correlated with poor prognosis. This study provides genetic evidence that intrinsic developmental heterogeneity can prime individual, lifelong cancer susceptibility.

overall_design: Comparison of DNA methylation profiles by array of 10 days old mouse ear clips, between 4 genotypes (WT, Trim28, Tp53 and Trim28/Tp53) and 2 developmental trajectories ('light' and 'heavy')

first: Ilaria

last: Panzeri

last: Pospisilik


title: Promiscuous CRE-CRE interaction that acts as a causative force driving de novel methylation in cis-regulatory elements in plants

status: Public on Sep 21 2023

city: Kearneysville

first: Zongrang

last: Liu

state: WV

address: 2217 Wiltshire Road

department: Appalachian Fruit Research Station

institute: USDA-ARS

type: Methylation profiling by high throughput sequencing

type: Non-coding RNA profiling by high throughput sequencing


title: Promiscuous CRE-CRE interaction that acts as a causative force driving de novel methylation in cis-regulatory elements in plants [AGeRdDM ssRNAseq]

summary: Cis-regulatory elements (CREs) dictate spatiotemporal expression and tissue specificity of proximal genes. However, when in a transgenic state, many of them become highly vulnerable to RNA-Directed DNA Methylation (RdDM) that is often transcriptionally deleterious and biologically detrimental. This transgene-specific RdDM vulnerability suggests the existence of anti-RdDM elements (AREs) to defend CREs against de novo methylation in vivo.  In this work, we identify such an ARE at the Arabidopsis AGAMOUS (AG) locus, which includes a physically separated enhancer and promoter, both of which are highly vulnerable to transgene silencing.  We demonstrate that this ARE effectively represses RdDM activity at its cognate and heterologous Cis-regulatory elements (CREs) control gene expression amplitude, tissue specificity and timing, whereby they directly impact phenotypic variation. However, in plants, CREs frequently become prone to RNA-directed DNA methylation (RdDM) when in a transgenic state (particularly in a tandem-repeat configuration), and the mechanism driving this phenomenon is currently unknown. In this study, we demonstrate that three of six flower-specific CREs, including the AGAMOUS enhancer (AGe), undergo transcriptional silencing when in a transgenic state, leading to the production of mutant flowers in 28-44% of transgenic lines. This silencing is dependent on the transgene self-transcribed non-coding RNAs (ncRNAs) that feed into biogenesis of 24-nt small interfering RNAs (siRNAs) and the canonical RdDM pathway, but not RNAs transcribed by the RNA polymerase II promoter. Strikingly, this ncRNA-dependent, siRNA-driven RdDM is prone to the enhancing action of an adjacent CaMV35S enhancer (35Se), which was shown to promiscuously interact with, and ectopically activate, numerous CREs including the AGe. This enhancement corresponded with an increase in ncRNAs specifically transcribed from the antisense strand of the transgene. We further demonstrate that the same 35Se inserted approximately 3.5 kb upstream of the native AGe within a genome promiscuously interacts with and activating de novo RdDM activity in the AGe, resulting in a mutant flower. These findings provide the first evidence that promiscuous CRE-CRE interactions serve as a causative force driving RdDM/silencing, and novel insight into the mechanism underlying the methylation-vulnerablity of the CRE transgenes, duplicated genes and repetitive genomic regions rich in transposon elements, where promiscuous CRE-CRE interactions are common.

overall_design: Analyses of ncRNA expression and profile in various Arabidopis lines

first: Yazhou

last: Yang

first: Jia

first: Stacy

last: Singer

first: Guohua

last: Yan

first: Dennis

last: Bennet

first: Yue

first: Jean-Michel

last: Hily

first: Weirong

last: Xu

first: Yingzhen

first: Xiping

last: Wang

first: Gan-Yuan

last: Zhong

first: Zhongchi

first: Yong-Chiang

last: An

first: Huawei


title: Expression profile of multipotent basal cells and basal-derived luminal cells isolated from the tip region (last 100µm) of the ductal tree of mouse prostate at P10-P13.

status: Public on Sep 06 2023

city: Bruxelles

first: Yura

last: Song

address: 808, route de Lennik

country: Belgium

institute: Université Libre de Bruxelles

summary: To investigate the molecular reprogramming of epithelial cells of the prostate during basal to luminal differentiation in vivo, we took advantage of the spatial restriction of multipotent basal cells at the distal region (tip - 100μm) of the developing prostate at postnatal day (P)10 (Tika et al., Development, 2019). The distal tips (100μm) of P10-P13 prostate glands were manually dissected from the main ducts under the stereoscope in order to enrich for multipotent basal cells and basal-derived luminal cells. Cell populations were isolated for RNA sequencing based on EpCAM and Cd49f expression via FACS.

overall_design: Prostate tissue of mice at P10-P13 was microdissected under a stereoscope to separate the different lobes. The ventral lobe was used to further separate (by cutting) the tips from the main ducts. Tissues were digested and living cells were isolated using fluorescence-activated cell sorting and analysis.  Sorted cells were collected in lysis buffer for RNA extraction. Extracted RNA was sequenced using the bulk RNA-seq strategy.

first: Elisavet

last: Tika

first: Cedric

last: Blanpain

first: Andrew

middle: S

last: Goldstein


title: Transcriptomic profiling of ccRCC nephrectomy samples by PCR-cDNAseq

status: Public on Sep 04 2023

first: Joshua

last: Lee

summary: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. Following primary tumour resection approximately 30% of patients experience disease recurrence associated with metastasis. To date, long-read RNA sequencing has not been applied to kidney cancer. Here, we used ONT long-read PCR-cDNAseq to profile the transcriptomes of ccRCC archival tumours, 6 of which were from patients who went on to relapse. Our results revealed a loss of immune infiltrate in tumours of patients who relapse. Moreover, thousands of novel isoforms were discovered, including a novel PD-L1 transcript encoding for the soluble version of the protein but having a longer 3'UTR than the currently annotated transcript. Finally, we have identified a novel non-coding gene that was over-expressed in patients who experience recurrence. Our data shows that ONT long-read PCR-cDNAseq can be used in archival tumour samples to comprehensively characterise tumour transcriptomes, and to reveal novel features that would have been missed by short-read RNAseq.

overall_design: RNA was extracted from nephractomy samples, with 2 μg of total RNA per sample used as input for sequencing library preparation (PCS111). Each library was loaded on individual PromethIon flow cell (R9) and sequenced for 72h. Reads with Q >7 were used for subsequent bioinformatic analysis.

first: Elizabeth

last: Snell

first: Joanne

last: Brown

first: Rosamonde

last: Banks

first: Naveen

last: Vasudev

first: Dimitris

last: Lagos


title: Transcriptomic profiling of ccRCC nephrectomy samples by Direct RNAseq

summary: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. Following primary tumour resection approximately 30% of patients experience disease recurrence associated with metastasis. To date, long-read RNA sequencing has not been applied to kidney cancer. Here, we used ONT long-read Direct RNA sequencing to profile the transcriptomes of ccRCC archival tumours, 6 of which were from patients who went on to relapse. Our results revealed a loss of immune infiltrate in tumours of patients who relapse. Moreover, thousands of novel isoforms were discovered, including a novel PD-L1 transcript encoding for the soluble version of the protein but having a longer 3'UTR than the currently annotated transcript. Finally, we have identified a novel non-coding gene that was over-expressed in patients who experience recurrence. Our data shows that DRS can be used in archival tumour samples to comprehensively characterise tumour transcriptomes, and to reveal novel features that would have been missed by short-read RNAseq.

overall_design: RNA was extracted from nephractomy samples, with 2 μg of total RNA per sample used as input for sequencing library preparation (RNA002). Each library was loaded on individual PromethIon flow cell (R9) and sequenced for 72h. Reads with Q >7 were used for subsequent bioinformatic analysis.


title: Long read transcriptome sequencing of ccRCC tumour cell line RCC4

summary: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. To date, long-read RNA sequencing has not been applied to kidney cancer. Here, we used ONT long-read Direct RNA sequencing to profile the transcriptomes of ccRCC cell line RCC4, with and without exposure to pro-inflammatory cytokines. Our results revealed differentially expressed genes induced by the pro-inflammatory cytokines. Moreover, results here revealed potential tumour origin of novel isoforms and genes that were discovered in the archival tumour samples by long-read sequencing.

overall_design: RCC4 cells (with and without exposure to pro-inflammatory cytokines IFNg&TNF for 24h) were harvested for total RNA. Poly(A)+ RNA molecules were isolated, with 500ng used as input for sequencing library preparation (RNA002). Each library was loaded on individual PromethION flow cell (R9) and sequenced for 72h. Reads with Q>7 were used for subsequent bioinformatic analysis.


title: Arabidopsis thaliana gene expression changes upon chitosan and CAPE treatments

status: Public on Sep 01 2023

city: Praha 6

first: Karel

last: Muller

address: Rozvojová 263

department: Lab. of hormonal regulations in plants

country: Czech Republic

institute: Institute of Experimenal Botany of the Czech Academy of Sciences

summary: The phytopathological studies often deal with approaches involving the employment of high elicitor concentrations necessary to provoke responses that could be easily observed and quantified. We tested the range of gene expression changes for a low concentration chitosan elicitor treatment of seedlings, 0.001%, and we compared it with the higher one, 0.01%. We uncover both the quantitative and qualitative changes in gene expressions between the two treatments. Further on, we tested if the gene expression can be modified by the treatment with the defence-related CAPE peptide (10 uM). We discovered several interesting candidate genes specific for each of treatments, and combination of treatments, that would be worth of further more focused genetic studies.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from A. thaliana seedlings, treated for 24 h with the two different chitosan elicitor concentrations (0.001% and 0.01%), CAPE peptide derived from AtPR1 (10 uM), and combination of both. The seven-day-old whole seedlings (ca 50 seedlings) were used as a source of RNA, always as three replicates.

first: Tamara

last: Pečenková

first: Matěj

last: Drs


title: Promiscuous CRE-CRE interaction that acts as a causative force driving de novel methylation in cis-regulatory elements in plants [AGeRdDM BSseq]

first: Zongrang

last: Liu

summary: Cis-regulatory elements (CREs) control gene expression amplitude, tissue specificity and timing, whereby they directly impact phenotypic variation. However, in plants, CREs frequently become prone to RNA-directed DNA methylation (RdDM) when in a transgenic state (particularly in a tandem-repeat configuration), and the mechanism driving this phenomenon is currently unknown. In this study, we demonstrate that three of six flower-specific CREs, including the AGAMOUS enhancer (AGe), undergo transcriptional silencing when in a transgenic state, leading to the production of mutant flowers in 28-44% of transgenic lines. This silencing is dependent on the transgene self-transcribed non-coding RNAs (ncRNAs) that feed into biogenesis of 24-nt small interfering RNAs (siRNAs) and the canonical RdDM pathway, but not RNAs transcribed by the RNA polymerase II promoter. Strikingly, this ncRNA-dependent, siRNA-driven RdDM is prone to the enhancing action of an adjacent CaMV35S enhancer (35Se), which was shown to promiscuously interact with, and ectopically activate, numerous CREs including the AGe. This enhancement corresponded with an increase in ncRNAs specifically transcribed from the antisense strand of the transgene. We further demonstrate that the same 35Se inserted approximately 3.5 kb upstream of the native AGe within a genome promiscuously interacts with and activating de novo RdDM activity in the AGe, resulting in a mutant flower. These findings provide the first evidence that promiscuous CRE-CRE interactions serve as a causative force driving RdDM/silencing, and novel insight into the mechanism underlying the methylation-vulnerablity of the CRE transgenes, duplicated genes and repetitive genomic regions rich in transposon elements, where promiscuous CRE-CRE interactions are common.

overall_design: Comparison of DNA methylation profile among different lines

first: Yazhou

last: Yang

first: Jia

first: Stacy

last: Singer

first: Guohua

last: Yan

first: Dennis

last: Bennet

first: Yue

first: Jean-Michel

last: Hily

first: Weirong

last: Xu

first: Yingzhen

first: Xiping

first: Gan-Yuan


title: miR-22 gene therapy as a novel option for HCC treatment in part via augmentation of retinoic acid but silencing IL17 signaling in orthotopic murine models

status: Public on Aug 21 2023

city: Sacramento

first: Ying

last: Hu

email: yinghu0821@gmail.com, yighu@ucdavis.edu

state: CA

address: 4645 2nd Ave, Research III building, 3400 Suite

department: Department of Pathology and Laboratory Medicine

institute: UC Davis

summary:
miR-22 expression is reduced in hepatocellular carcinoma (HCC), a deadly cancer with very limited treatment options. Our published data showed that retinoic acid (RA) via its receptor RARβ induces miR-22. Moreover, miR-22 increase the expression of RARβ, which signifies the significance of RA signaling in tumor suppression. Using orthotopic HCC mice models including β-catenin positive HCC, we studied the anti-HCC effect of miR-22 for the first time focusing on the potential roles of RA signaling in regulating tumor immunity based on transcriptomic profiling data found in both mouse and human HCC.
Our data showed that miR-22 treated HCC without toxicity, with improved survival compared with Lenvatinib. Reduced RA signaling and increased IL17 signaling found in mouse HCC as well as isolated T cells were consistently found in miR-22 low human HCC, whereas miR-22 treatment of mouse HCC reversed both pathways verified in β-catenin positive HCC. Moreover, in isolated T cells, miR-22 silenced the Il17a gene, which lead to Th17 cell reduction, by reducing the recruitment of HIFα to the Rorc gene as well as the RORγT, HIF1α, and STAT3 to the Il17a gene. Further, IL23-driven IL17 signaling expansion attenuated the anti-HCC effects of miR-22. Additionally, CD8 blockade diminished the therapeutic effects of miR-22. Together, via regulating tumor immunity, miR-22 can be a novel option for HCC treatment.

overall_design: To uncover the underlying mechanism by which miR-22 treats HCC, we performed gene expression profiling using data obtained from RNA-seq of RAS/AKT-induced-HCCs treated with AAV control or miR-22 as well as WT FVB/N normal livers (n=3/groups)

first: Yu-jui

middle: Y

last: Wan


title: SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary [scRNA-seq]

status: Public on Aug 18 2023

first: Dr. Chiara

last: Giannarelli

address: 435 E 30th St

department: Cardiology

institute: NYU Langone Health

summary:
COVID-19 patients present higher risk for myocardial infarction (MI), acute coronary syndrome,
and stroke for up to 1 year after SARS-CoV-2 infection. While the systemic inflammatory response
to SARS-CoV-2 infection likely contributes to this increased cardiovascular risk, whether SARS35
CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques to locally
promote inflammation remains unknown. Here, we report that SARS-CoV-2 viral RNA (vRNA)
is detectable and the virus replicates in coronary atherosclerotic lesions taken at autopsy from
patients with severe COVID-19.

overall_design: carotid artery from atherosclerotic human samples, used scRNAseq to under the underlying immune molecular mechanism

first: Natalia

last: Eberhardt

first: Chiara


title: SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary

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

overall_design: Refer to individual Series


title: Molecular Disparities in Colorectal Cancers of White Americans, Alabama African Americans, and Oklahoma American Indians

status: Public on Aug 23 2023

city: Oklahoma City

first: Hiroshi

last: Yamada

state: Oklahoma

department: Medicine

institute: OUHSC

summary: In the US, the majority of cancer samples analyzed are from white people, leading to biases in racial and ethnic treatment outcomes. Colorectal cancer (CRC) incidence and mortality rates are high in Alabama African Americans (AAs) and Oklahoma American Indians (AIs). We hypothesized that differences between racial groups may partially explain these disparities. Thus, we compared transcriptomic profiles of CRCs of Alabama AAs, Oklahoma AIs, and white people from both states. Compared to CRCs of white people, CRCs of AAs showed (a) higher expression of cytokines and vesicle trafficking toward modulated antitumor-immune activity, and (b) lower expression of the ID1/BMP/SMAD axis, IL22RA1, APOBEC3, and Mucins; and AIs had (c) higher expression of PTGS2/COX2 (an NSAID target/pro-oncogenic inflammation) and splicing regulators, and (d) lower tumor suppressor activities (e.g., TOB2, PCGF2, BAP1). Therefore, targeting strategies designed for white CRC patients may be less effective for AAs/AIs. These findings illustrate needs to develop optimized interventions to overcome racial CRC disparities.

overall_design: RNAseq study: To investigate and compare transcriptome landscape of Colorectal cancer (CRC) patients of white, Alabama Black/African American (AA) and Oklahoma American Indian (AI)

first: Chao

first: Kenneth

last: Jones

first: Philip

last: O’Neill

first: Madka

last: Venkateshwar

first: Srikanth

last: Chiliveru

first: Hyung-Gyoon

last: Kim

first: Mark

last: Doescher

first: Katherine

middle: T

last: Morris

first: Upender

last: Manne

first: Chinthalapally

middle: V

last: Rao


title: RNA Polymerase II, the BAF remodeler and transcription factors synergize to evict nucleosomes

status: Public on Oct 12 2023

city: Omaha

first: Sandipan

last: Brahma

state: NE

address: 985805 Nebraska Medical Center, DRCI 6054

institute: University of Nebraska Medical Center

summary: Chromatin accessibility is a hallmark of active transcription and requires ATP-dependent nucleosome remodeling by Brahma-Associated Factor (BAF). However, the mechanistic link between transcription, nucleosome remodeling, and chromatin accessibility is unclear. Here, we used a chemical-genetic approach to dissect the interplay between RNA Polymerase II (RNAPII), BAF, and DNA-sequence-specific transcription factors (TFs) in mouse embryonic stem cells. By time-resolved chromatin profiling with acute transcription block at distinct stages, we show that RNAPII promoter-proximal pausing stabilizes BAF chromatin occupancy and enhances nucleosome eviction by BAF. We find that RNAPII and BAF probe both transcriptionally active and Polycomb-repressed genomic regions and provide evidence that TFs capture transient site exposure due to nucleosome unwrapping by BAF to confer locus specificity for persistent chromatin remodeling. Our study reveals the mechanistic basis of cell-type-specific chromatin accessibility. We propose a new paradigm for how functional synergy between dynamically acting chromatin factors regulates nucleosome organization.

overall_design: We used Cleavage under targets and Tagmentation (Cut-and-Tag), a chromatin profiling strategy in which antibody-targeted controlled integration of DNA sequencing adapters produces libraries for paired-end DNA sequencing.

first: Steven

last: Henikoff


title: SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary [plaque]

overall_design: Human carotid vascular explants were  infected with 105 PFU/mL of SARS-CoV-2 virus (USA-WA1/2020 strain) . Non-infected tissues were used as controls.


title: SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary [MAC_FOAM-RNAseq]

overall_design: Human primary monocytes derived from PBMCs were obtained and differentiated to macrophages in the presence of 20 nM human macrophage-colony stimulating factor (M-CSF) for 5 days. Macrophages were first treated with ox-LDL at final concentration of 10 μg/mL for 1 hour before infection. Macrophages and foam cells were cultured with SARS-CoV-2 virus (USA-WA1/2020 strain) at MOI 0.1 in infection media with 2% FBS.  Mock infected cells were used as controls. Cells were washed twice with PBS and harvested at different time points.


title: Differential chromatin accessibility and transcriptional dynamics define breast cancer subtypes and their lineages

status: Public on Aug 10 2023

city: Saint Louis

first: Li

last: Ding

state: MO

address: 660 S. Euclid Avenue

institute: Washington University in St. Louis

summary: To explore pre-cancer states during early malignancy, we evaluated tumor and normal cells in the MMTV-PyVT mouse model of luminal breast cancer using snRNAseq and snATACseq.

overall_design: B6.FVB-Tg(MMTV-PyVT) 634Mul/LellJ (Strain #:022974) female mice were purchased from the Jackson Laboratory. Mice were euthanized by carbon dioxide asphyxiation and five pairs of mouse mammary glands were collected at 12 weeks old. For each pair, the left mammary gland was flash frozen in liquid nitrogen, and the right glands were fixed in 10% neutral buffered formalin (Epredia, 5725) then embedded in paraffin. The 5 left mammary glands were pooled together for snRNA-Seq and snATAC-Seq sample preparation. The paraffin-embedded glands were used for H&E staining. All animal experiments were approved by the Washington University in Saint Louis Institutional Animal Care and Use Committee (IACUC) office.

first: Jayasinghe

last: Reyka


title: 3D genome remodeling and homologous pairing during meiotic prophase of mouse oogenesis and spermatogenesis

status: Public on Sep 23 2023

city: Beijing

first: Jing

last: He

state: Beijing

address: Room D110, Medical Science Building, School of Medicine, Tsinghua University

department: School of Medicine

institute: Tsinghua University

summary: We comprehensively compared the chromatin structures and transcriptomes in successive substages of female and male mouse meiotic prophase by using sisHi-C and RNA-Seq methods. Interestingly, the transcriptional change happened earlier than chromatin structures reprograming that chromatin structures largely maintained the pre-meiotic condition in leptotene. Also, compartments and TADs gradually disappeared and then slowly recovered in both oocytes and spermatocytes. We characterized the events of homologous chromosomes pairing and found homologues adopted two sex-conserved pairing strategies prior to and after leptotene-to-zygotene transition, which firstly contacted more frequently in LINE enriched compartment B and then switched to SINE enriched compartment A. The sexual difference of transcriptome was most obvious in late meiotic prophase, which reflected in gamete morphology and function differences. We also complemented marker genes for each substage of oocytes and spermatocytes meiotic prophase, and predicted the sex-specific meiotic functional genes, whose mutation or deletion may cause sex different effects on fertility. In summary, this study revealed the sexual similarities and dimorphic of higher-order chromatin architecture, homologous pairing and transcriptome during meiotic prophase of both oogenesis and spermatogenesis.

overall_design: By crossing female C57BL/6NJ (mVenus-P2A-HA-Sycp3 transgene mice) to male PWK-PhJ mice, we successfully purified oocytes and spermatocytes at time-points enriched of specific meiotic prophase substages, with the help of FACS of SYCP3 signal from F1 hybrid mice (B6xPWK). Then we performed sisHi-C, RNA-Seq and ATAC-Seq.

first: An

last: Yan


title: Decitabine treatment reveals a functional role of DNA hypomethylation in organising enhancer-promoter interactions [RNA-seq]

status: Public on Aug 08 2023

city: Adelaide

first: Joanna

last: Achinger-Kawecka

state: SA

address: AHMS Building, North Terrace

country: Australia

institute: South Australian Immunogenomics Cancer Institute

summary: DNA methylation is one of the most important epigenetic marks and changes in DNA methylation patterns have been associated with many different types of cancer. Recently, three-dimensional (3D) genome organisation has been demonstrated to play a fundamental role in gene regulation. Determining the interplay between DNA methylation and 3D chromatin structure and function is challenging due to the extreme complexity of epigenetic regulation. To study the impact of DNA methylation on 3D genome organization, we performed a time-course of Decitabine treatment in tamoxifen-resistant MCF7 (TAMR) cells. Decitabine was applied daily for 7 days, followed by “recovery” up to 28 days and DNA methylation (EPIC), gene expression (RNA-seq) and promoter-enhancer interactions (PCHi-C) were assessed on day 7 post-Decitabine- and Vehicle-treatment and day 28 “Recovery” and Vehicle treatment in duplicate. Our data revealed a functional link between DNA hypomethylation at distal enhancer regions, enhancer-promoter interactions, and gene regulation.

overall_design: To understand the role of Decitabine-induced DNA hypomethylation on 3D genome organisation, we used in situ Promoter Capture Hi-C to generate promoter-centric 3D genome maps of TAMR cells treated with Decitabine for 7 days and following 21 days of “recovery”. At the same time, we examined the genome-wide DNA methylation (EPIC Microarrays) and gene expression (RNA-seq) in the same samples.


title: Decitabine treatment reveals a functional role of DNA hypomethylation in organising enhancer-promoter interactions [Promoter Capture Hi-C]


title: Single-cell transcriptomic analysis of skeletal muscle regeneration across mouse lifespan identifies altered progenitor cell states associated with senescence

status: Public on Aug 07 2023

city: Ithaca

first: Lauren

middle: D

last: Walter

address: 161 Weill Hall

department: Biomedical Engineering

institute: Cornell University

summary: Skeletal muscle regeneration is driven by the interaction of myogenic and non-myogenic cells. In aging, regeneration is impaired due to various dysfunctions of myogenic and non-myogenic cells, but this is not understood comprehensively. We collected an integrated atlas of 273,923 single-cell transcriptomes from muscles of young, old, and geriatric mice (4, 20, 26 months-old) at six time-points following myotoxin injury. We identified eight cell types, including T and NK cells and multiple macrophage subtypes that displayed accelerated or delayed dynamics between ages. Through pseudotime analysis, we observed myogenic cell states and trajectories specific to old and geriatric ages. To explain these age differences, we assessed cellular senescence by scoring experimentally derived and curated gene-lists, which pinpointed stalled senescent-like subsets specifically within the self-renewing muscle stem cells as elevated in older muscles. This resource provides a holistic portrait of the altered cellular states underlying skeletal muscle regeneration decline across mouse lifespan.

overall_design: Single-cell RNA-seq datasets for uninjured and notexin-injured mouse tibialis anterior muscles. Muscles were collected at six time points (0, 1, 2, 3.5, 5, and 7 days) and there are 4 replicates per time point.

first: Benjamin

last: Cosgrove


title: Regulation of cohesin loop extrusion underlies changes in enhancer-promoter interactions during pancreatic cell differentiation (Hi-C)

status: Public on Sep 19 2023

city: XIAMEN

first: XIAOWEN

last: LYU

state: FUJIAN

address: No.4221, Xiang'an South Road, Xiang'an District

institute: XIAMEN UNIVERSITY

summary: Transcriptional reprogramming during cell fate determination involves precise targeting of enhancers to tissue-specific genes. Enhancer-promoter interactions are regulated by different types of 3D chromatin organization, including compartmental domains and CTCF loops. To understand the contribution of these different levels of chromatin organization in cell lineage commitment, we analyzed changes in 3D organization during the differentiation of human embryonic stem cells (hESCs) into pancreatic islet organoids. We find that major events in chromatin reorganization, including decompaction of transposon-enriched heterochromatin, breakdown of poised enhancer-promoter interaction networks, gain or loss of CTCF loops and establishment of novel enhancer-promoter contacts, occur either sequentially or simultaneously. Transcriptionally activated transposons released from unstable heterochromatin compartments recruit CTCF to form new loops. In contrast, when released from poised long-range interaction networks, enhancers and genes crucial for cell differentiation recruit tissue-specific transcription factors by either direct binding or indirect 3D contacts containing RNA Polymerase II. Activation of stage-specific genes correlates with gain of CTCF binding at anchors of extended CTCF loops, suggesting changes in loop extrusion capacity. In agreement with this, we find a global increase in cohesin loading leading to longer residency on extended CTCF loops encompassing tissue-specific genes. Our findings provide new insights into how distinct types of chromatin organization affect the reprogramming of gene expression patterns to regulate cell lineage commitment.

overall_design: Hi-C

first: Xiaowen

last: Lyu

first: M

last: Rowley

first: Michael

last: Kulik

first: Stephen

last: Dalton

first: Victor

last: Corces


title: Promiscuous CRE-CRE interaction that acts as a causative force driving de novel methylation in cis-regulatory elements in plants [AGeRdDM sRNAseq]

first: Zongrang

last: Liu

overall_design: Analysis of sRNA expression  among different lines

first: Yazhou

last: Yang

first: Jia

first: Stacy

last: Singer

first: Guohua

first: Dennis

last: Bennet

first: Yue

first: Jean-Michel

last: Hily

first: Weirong

last: Xu

first: Yingzhen

first: Xiping

last: Wang

first: Gan-Yuan

last: Zhong

first: Zhongchi

first: Yong-Chiang

last: An

first: Huawei


title: Decitabine treatment reveals a functional role of DNA hypomethylation in organising enhancer-promoter interactions

first: Joanna

last: Achinger-Kawecka


title: RNAseq of CD4 T cells from female Rheumatoid arthritis patients [NeumRA_CD4]

city: Gothenburg

first: Maria

middle: I

last: Bokarewa

state: VG

address: Guldhedagatan 10A

department: Rheumatology and Inflammation Research

country: Sweden

institute: Gothenburg University

summary: RNAseq of CD4 cells from 60 female RA patients activated with aCD3 for 2 hours. Patients were collected crossectionally from the Rheumatology clinic during 2019-2020. Age md 64, range 23-76 years, Disease duration md 10, range 1-45 years.

overall_design: 60 women with  rheumatoid arthritis, CD4 cells  from human PBMC isolated by positive selection on Dynabeads (cat.11331D), stimulated with anti-CD3 (OKT3, Sigma-Aldrich)  for 2h

first: Karin

middle: M

last: Andersson

first: Malin

last: Erlandsson

first: Nina

last: Oparina

first: Eric

last: Malmhäll-Bah


title: Epigenetic therapy targets the 3D epigenome in endocrine-resistant breast cancer [CUT&RUN]

status: Public on Jul 24 2023

summary: Here we investigate the impact of epigenetic therapy with Decitabine in endocrine-resistant ER+ breast cancer by using patient-derived xenograft (PDX) models. Decitabine treatment restrained tumour growth, inhibited cell proliferation and resulted in significant loss of DNA methylation, particularly at enhancers and repetitive elements. Systematic integration of matched in situ Hi-C / PCHi-C, EPIC, RNA-seq and ChIP–seq datasets revealed widespread differences in epigenome regulation and enhancer-promoter communication with Decitabine. We find that loss of DNA methylation with Decitabine strongly affects the open (A) and closed (B) compartment structure and TAD boundary insulation. Our study identified and focused on key DNA methylation-dependent, enhancer ER binding sites that are activated in Decitabine-treated PDX tumours, enabling direct interactions between promoters and multiple distal enhancers, inducing expression of ER target genes and pathways. Overall, we demonstrate that epigenetic therapy inhibits tumour progression through to rewiring of ER-mediated 3D chromatin interactions and transcriptome programs. Our findings suggest that targeting the 3D epigenome with epigenetic therapies represents a promising strategy for anti-cancer treatment in ER+ endocrine resistant breast cancer patients.

overall_design: To understand the Decitabine-induced molecular changes driving tumour regression on a global scale, we used in situ Hi-C to generate genome-wide 3D genome maps of PDX breast tumours treated with Decitabine. At the same time, we examined the genome-wide DNA methylation (EPIC Microarrays), promoter-anchored interactome (Promoter Capture Hi-C (PCHi-C)), transcription factor binding (ER and FOXA1 ChIP-seq) and gene expression (RNA-seq) in the same tumours.


title: Epigenetic therapy targets the 3D epigenome in endocrine-resistant breast cancer [WGBS]


title: Single-nucleus RNA sequencing of human subcutaneous adipose tissue biopsies from 16 Finnish participants.

status: Public on Jul 22 2023

city: Los Angeles

first: Marcus

last: Alvarez

address: 695 Charles E Young Dr South, Gonda 6335

department: Human Genetics

institute: UCLA

summary: Single-nucleus RNA-seq was performed to characterize the transcriptomes of cell types in human subcutaneous adipose tissue. Biopsies from 16 Finnish participants from the Finnish Twin Study and CRYO study were included.

overall_design:
Nuclei were isolated from fresh-frozen subcutaneous adipose tissue biopsies using manual homogenization, washing, and straining. Libraries were prepared with the 10X Chromium platform and the 10X single cell 3' protocol. Libraries were then sequenced on an Illumina NovaSeq S2 targeting a depth of 50,000 reads per nucleus.
***Submitter delcares that no clinical information was used and provided, as the data was used only as a reference***
***Submitter delcares  that the raw data is not provided due to patient privacy concerns.***

first: Alvarez

last: Marcus

first: Pajukanta

last: Päivi

first: Pietiläinen

last: Kirsi


title: Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties

status: Public on Jul 23 2023

city: Yokohama

first: Nando

middle: Dulal

last: Das

state: -- Choose State --

address: 1-7-22 Suehiro-cho, Tsurumi-ku

department: Center for Biosystems Dynamics Research

institute: RIKEN

summary: The bromodomain and extra-terminal domain (BET) family consists of acetyllysine-binding proteins involved in many cancers, including glioblastoma multiforme. While the BET family activates transcription through binding to hyperacetylated histone H4 with simultaneous acetylation of K5 and K8 (H4K5acK8ac), so far epigenomic analysis of the BET family has mainly focused on H3K27ac, which is not their binding target. Here, we have examined the epigenomic profiles, including H4K5acK8ac and a BET family protein, BRD4, along with transcriptomics using a BET inhibitor, JQ1, in three human glial cell lines. We found that the expression of genes in which H4K5acK8ac is preferred over H3K27ac at promoters was mostly downregulated upon JQ1 in a glioblastoma stem cell (GSC) line. Knockdown of genes with the H4K5acK8ac-preferred promoters diminished the stemness of GSC. By defining super-enhancers (SEs) ranked by H4K5acK8ac, we found that 43% of the H4K5acK8ac-ranked SE were different from H3K27ac-ranked SE in GSC. CRISPR-Cas9-mediated deletion of the H4K5acK8ac-preferred SEs reduced the expression of associated genes including MYCN and NFIC in GSC and diminished its stemness. These results validate a novel strategy to identify genes involved in the regulation of glioblastoma stemness, through histone H4 hyperacetylation.

overall_design: Examination of histone modifications (H4K5acK8ac, H3K27ac, H3K4me3, & H3K4me1) and BRD4 by ChIP-seq in the JQ-treated, DMSO-treated, and untreated human glial cell lines (Glioblastoma stem cell line-GSC316, Glioblastoma cell line-U87, and Microglia cell line-C13NJ). Transcriptomic profiling through CAGE-seq of JQ-treated, DMSO-treated, and untreated of Glioblastoma stem cell line-GSC316, Glioblastoma cell line-U87, and Microglia cell line-C13NJ.

first: Aki

last: Minoda

first: Takashi

last: Umehara


title: The Time-Course of Cancer Cachexia Onset Reveals Biphasic Transcriptional Disruptions in Female Skeletal Muscle Distinct from Males

status: Public on Jun 29 2023

city: Fayetteville

first: Francielly

last: Morena da Silva

state: AR

address: 155 Stadium Dr

institute: University of Arkansas

summary: Cancer-cachexia (CC) is experienced by 80% of cancer patients, representing up to 40% of cancer-related deaths. Evidence suggests biological sex dimorphism associated with cancer-cachexia. Assessments of the female transcriptome in cancer-cachexia are lacking and direct comparisons between biological sex are scarce. The purpose of this study was to define the time course of LLC-induced CC in females using transcriptomics, while directly comparing biological sex dimorphisms.

overall_design: Gastrocnemius of male and female mice (n=8/group) were collected at 1-, 2-, 3-, 4-weeks of tumor progression. Due to dichotomous effect in tumor growth in female mice, animals were reorganized into low tumor (LT; tumor mass≤1.2g) and high tumor (HT; tumor mass≥2g). Male RNA sequencing data has been previously published  (Gene Expression Omnibus accession number GSE114820).  RNA sequencing of total muscle was re-analyzed similarly to females.

first: Nicholas

middle: P

last: Greene

first: Kevin

middle: A

last: Murach


title: EED in the Polycomb Repressive Complex 2 Epigenetically Regulates Responses in Lipopolysaccharide Tolerized Macrophages

status: Public on Jun 20 2023

city: Bangkok

first: Tanapat

last: Palaga

address: Payathai Road, Pathumwan

department: Microbiology

country: Thailand

institute: Chulalongkorn University

summary: To avoid exaggerated inflammation and injury, host cells adapt to become hypo-responsive or “tolerance” in response to successive exposure to stimuli. Such tolerized response is a part of innate immune memory which is mainly regulated via epigenetics changes and metabolic reprograming. Polycomb repressive complex 2 (PRC2) mediates the transcriptional repression by catalyzing histone H3 lysine 27 trimethylation (H3K27me3) but little is known about the roles of PRC2 in tolerant macrophages. We examined the impact of PRC2 components, EED and Ezh2, on lipopolysaccharide (LPS)-induced tolerant macrophages. In Eed KO macrophages, not only the significant reduction in H3K27me3, but also the augmentation of an active histone mark, H3K27Ac. Upon EED deletion but not Ezh2, macrophages exhibited attenuated pro-inflammatory cytokines productions (TNF-α and IL-6) in LPS-tolerant cells. In addition, LPS tolerant Eed KO macrophages exhibited low glycolytic activity rather than its littermate wild-type control. RNA-Seq analyses revealed that most of differentially expressed genes are involved in oxidative phosphorylation and TGF-β signaling. Alteration of H3K27me3 and H3K27ac in the regulatory regions of some of these genes were validated. These results indicated that PRC2 via EED epigenetically suppresses these genes in response to LPS re-exposure and lacking PRC2 activity results in hypo-responsive to LPS re-stimulation. Therefore, we provide strong evidences that PRC2 via EED mediates LPS tolerance in macrophages by epigenetically suppressing proinflammatory responses with the link to dysregulated metabolic pathway.

overall_design: LPS-induced tolerance in EED deficient BMDMs

first: Atsadang

last: Boonmee

first: Salisa

last: Benjaskulluecha

first: Patipark

last: Kueanjinda

first: Benjawan

last: Wongprom

first: Thitiporn

last: Pattarakankul

first: Kittitach

last: Sri-Ngern-Ngam

first: Supawadee

last: Umthong

first: Junichiro

last: Takano

first: Haruhiko

last: Koseki


title: Compare of differential expression profiles of PEDV-susceptible and non-susceptible cell lines at the transcriptome level to identify key molecules associated with PEDV entry

status: Public on Jul 06 2023

city: shanghai

first: Lilei

last: Lv

address: No.518 Ziyue Road

institute: Shanghai veterinary research institute

summary: Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus that poses a serious threat to the global pig industry. Despite extensive research efforts, the functional receptor for PEDV remains unclear. In this study, we identified susceptible and non-susceptible cell lines to PEDV infection, and performed RNA-seq analysis on these cell lines. Using Weighted Gene Co-expression Network Analysis (WGCNA), we have identified the key pathways that correlated with the PEDV entry pathway. We found that cholesterol, sterols, and lipid transport and homeostasis were strongly correlated with PEDV entry, suggesting a potential role for cholesterol in PEDV entry. We then treated susceptible cell lines with a cholesterol transport inhibitor and found that inhibition of cholesterol transport could significantly inhibit PEDV entry in these cells. Together, our results suggest that cholesterol transport may play a critical role in the entry of PEDV into susceptible cells, and that targeting cholesterol transport may represent a potential strategy for controlling PEDV transmission. Our findings provide new insights into the mechanism of PEDV entry and may pave the way for the development of new therapeutic strategies against this economically important virus. Further studies are warranted to elucidate the detailed mechanism of PEDV entry, and to explore the potential of cholesterol transport inhibitors as a means of controlling PEDV transmission.

overall_design: To investigate the functional receptor for PEDV,we identified susceptible and non-susceptible cell lines to PEDV infection, and performed RNA-seq analysis on these cell lines. Using Weighted Gene Co-expression Network Analysis (WGCNA) to identify the key pathways that correlated with the PEDV entry pathway.

first: Changlong

last: Liu


title: Evi1 governs Kdm6b-mediated histone demethylation to finely regulate the Laptm4b-driven mTORC pathway in vivo [LK]

status: Public on Jun 27 2023

city: Charlottesville

first: Yong

last: Huang

state: VA

address: 1340 Jefferson Park Ave

institute: University of Viginia

summary:
Ecotropic viral integration site 1 (EVI1/MECOM) overexpression is common in myeloid malignancies. We present a new Evi1 transgenic mouse model with inducible expression in hematopoietic stem cells (HSCs) and progenitor cells (HPCs) at lower levels. Upon exogenous Evi1 induction, mice displayed anemia, thrombocytopenia, lymphopenia, and dysplasia in erythroid and megakaryocyte cells with a significant expansion of committed myeloid progenitor cells, resembling human Myelodysplastic syndrome/Myeloproliferative neoplasm (MDS/MPN)-like disease.
Methods: Lin-C-Kit+ cells were isolated from 3  pairs of WT and Evi1 overexpressing mice. Around 1X106 Lin-C-Kit+ cells were harvested from each mouse. Then the cells were lysated by Trizol and total RNA was extracted by phenol-chloroform.
Results: Molecular pathways altered in Lin-C-Kit+ cells from Evi1-OE mice.
Conclusions: Multiple molecular pathways changed in Evi1 overexpressing hematopoietic stem and progenitor cells.

overall_design: 3  pairs of WT and Evi1 overexpressing mice at the same age and gender were sacrificed and the BM cells were harvested.

first: Qiong

last: Wu

first: Zhijian

last: Qian


title: Evi1 governs Kdm6b-mediated histone demethylation to finely regulate the Laptm4b-driven mTORC pathway in vivo [LSK]

summary:
Ecotropic viral integration site 1 (EVI1/MECOM) overexpression is common in myeloid malignancies. We present a new Evi1 transgenic mouse model with inducible expression in hematopoietic stem cells (HSCs) and progenitor cells (HPCs) at lower levels. Upon exogenous Evi1 induction, mice displayed anemia, thrombocytopenia, lymphopenia, and dysplasia in erythroid and megakaryocyte cells with a significant expansion of committed myeloid progenitor cells, resembling human Myelodysplastic syndrome/Myeloproliferative neoplasm (MDS/MPN)-like disease.
Methods: LSK cells were sorted by Sony SH800 from 3  pairs of mice transplanted with WT BM cells and Evi1-OE BM cells after pIpC injection. Around 5X103 LSK cells were harvested from each mouse. Then the cells were lysated by Trizol and total RNA was extracted by phenol-chloroform.
Results: Molecular pathways altered in LSK cells from recipient mice transplanted with Evi1-OE BM cells.
Conclusions: Transcription profile changed in Evi1 overexpressing hematopoietic stem and progenitor cells.

overall_design: 3  pairs of recipient mice transplanted with WT or Evi1-OE BM cells were sacrificed after pIpC injection to induce Evi1 overexpression. And the BM cells were harvested. LSK cells were sorted.


title: Evi1 governs Kdm6b-mediated histone demethylation to finely regulate the Laptm4b-driven mTORC pathway in vivo [CUT&RUN]

summary:
Ecotropic viral integration site 1 (EVI1/MECOM) overexpression is common in myeloid malignancies. We present a new Evi1 transgenic mouse model with inducible expression in hematopoietic stem cells (HSCs) and progenitor cells (HPCs) at lower levels. Upon exogenous Evi1 induction, mice displayed anemia, thrombocytopenia, lymphopenia, and dysplasia in erythroid and megakaryocyte cells with a significant expansion of committed myeloid progenitor cells, resembling human Myelodysplastic syndrome/Myeloproliferative neoplasm (MDS/MPN)-like disease.
Methods: Lin-C-Kit+ cells were isolated from 2 Evi1 overexpressing mice for CUT&RUN assay using flag antibody and IgG. Lin-C-Kit+ cells were isolated from 2 pair of WT mice and Evi1 overexpressing mice for CUT&RUN assay using H3K27me3 antibody.Around 5X105 Lin-C-Kit+ cells for each group were used in CUT&RUN assay. About 10 ng of the purified CUT&RUN DNA was used for preparation of multiplexed libraries with the NEB Ultra II DNA Library Prep Kit per manufacturer's instruction (NEB #E7103). Sequencing was conducted using an Illumina NextSeq 500 Sequencing System.
Results: Molecular pathways altered in Lin-C-Kit+ cells from Evi1-OE mice.
Conclusions: Evi1 has a special binding profiling in Lin-C-Kit+ cells. The modification profile of H3K27me3 was altered in Evi1-OE hematopoietic stem and progenitor cells.

overall_design: WT mice and Evi1 overexpressing mice with the same age and same gender were sacrificed and the BM cells were harvested.


title: A key role for NLRP3 signaling in preterm labor and birth driven by the alarmin S100B

city: Detroit

first: Adi

last: Tarca

address: 3990 John R. St, 4 Brush South

institute: Wayne State University

summary: Decidual and uterine macrophages as well as neutrophils from Nlrp3+/+ and Nlrp3-/- mice injected with S100B or PBS were sorted and analyzed using pair-end BGI RNA sequencing (RNA-seq) to evaluate the effect of Nlrp3 deficiency on the transcriptome of these innate immune cells in preterm birth.

overall_design: The study included Nlrp3+/+ dams intra-amniotically injected with S100B, Nlrp3+/+ dams intra-amniotically injected with PBS, Nlrp3-/- dams intra-amniotically injected with S100B, and Nlrp3-/- dams intra-amniotically injected with PBS. Decidual and uterine macrophages and neutrophils were sorted by fluorescence activated cell sorting. The number of samples analyzed were as follows: Macrophages, n = 3 each, Neutrophils, n = 2 each.

first: Nardhy

last: Gomez-Lopez

first: Roberto

last: Romero

middle: L

first: Bogdan

last: Done


title: Non-alcoholic steatohepatitis disrupts diurnal liver transcriptome rhythms in mice

status: Public on Jun 16 2023

city: Lübeck

first: Leonardo

last: de Assis

address: Marie Curie

institute: University of Lübeck

summary: Circadian profiling improves target gene detection and pathway characterization in mouse models of NASH. Fatty livers show 3-hour advances in metabolic and clock gene transcript rhythms. Circadian profiling further reveals temporal changes in lipid, carbohydrate, and cholesterol metabolic transcripts.

overall_design: Mice were fed with choline free methionine low high fat diet (cdHFD) or normal chow during 2 weeks. Mice were then sacrificed every 6 h and liver was collected. Rnaseq was then performed and data analyzed using different methods.

first: Henrik

last: Oster