title: Claustral cell identity is determined by Nurr1 and regulates hallucinogenic-like states

city: Stockholm

first: Behzad

last: Yaghmaeian Salmani

state: Solna, Stockholm

address: Department of Cell and Molecular Biology (CMB) Karolinska Institute,, Biomedicum, Solnavägen 9, Solna, Stockholm, P.O. Box 285 SE-171 77 Stockholm, Sweden

department: CMB

institute: Karolinska Institute

summary:
Nurr1 is a transcription factor, which is highly enriched in the claustrum (CLA), an enigmatic brain region that has been hypothesized to control hallucinations. Here, by using virus mediated deletion in conditional gene-targeted mice, we investigated the role of Nurr1 in CLA. These mice exhibited loss of the CLA transcriptional identity. To further study the role of Nurr1 in CLA we designed conditional Nurr1 knock out mice under D1 receptor promoter (D1R-Nurr1cKO). Accordingly, these mice showed similar results in CLA marker gene expression with the viral-targeted strategy. Moreover, D1R-Nurr1cKO mice exhibited loss of hallucinogen receptor signaling in CLA. Functional ultrasound imaging showed that hallucinogenic serotoninergic and opioidergic receptor agonists increased functional connectivity between prefrontal cortex and somatomotor regions in control mice but not in D1R-Nurr1cKO. Conclusively, we demonstrate that Nurr1 loss ablates CLA cell identity, which may affect hallucinogenic-like states.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at two time points.

overall_design: Despite the well-established role of Nurr1 in DAergic neurons, its relevance in the CLA neurons remains elusive. Thus, in the present studies, we generated conditional knock-out (KO) mice that to lack Nurr1 in CLA neurons. Since CLA is hypothesized to be key structure for altered consciousness, we used the above-mentioned mice to examine the role of CLA in the neural correlates of hallucinogenic-like state, as well as differential gene expression analysis of the CLA neurons between the two genotypes, D1R-Nurr1cKO vs control.

first: Ioannis

last: Mantas

first: Katarina

last: Tiklova

first: Linda

last: Gillberg Ward

first: Thomas

last: Perlmann

first: Per

last: Svenningsson


title: Gene misexpression in a Smoc2+ve/ Sox2-low population in juvenile Prop1-mutant pituitary gland

status: Public on Aug 03 2024

city: Stony Brook

first: Leonard

middle: YM

last: Cheung

state: New York

address: 101 Nicolls Road

country: USA

institute: Stony Brook University

type: Expression profiling by high throughput sequencing

summary: Mutations in the pituitary specific transcription factor Prophet of Pit-1 (PROP1) are the most common genetic etiology of combined pituitary hormone deficiency (CPHD). CPHD is associated with short stature, attributable to growth hormone deficiency and/or thyroid stimulating hormone deficiency, as well as hypothyroidism and infertility. Pathogenic lesions impair pituitary development and differentiation of endocrine cells. We performed single-cell RNA sequencing of pituitary cells from a wild-type and a Prop1-mutant P4 female to elucidate population-specific differential gene expression. We observed a Smoc2+ve population that expressed low Sox2, which trajectory analyses suggest are a transitional cell state as stem cells differentiate into endocrine cells. We also detected ectopic expression of Sox21 in these cells in the Prop1df/df mutant. Prop1-mutant mice are known to overexpress Pou3f4, which we now show to be also enriched in this Smoc2+ve population. We sought to elucidate the role of Pou3f4 during pituitary development and to determine the contributions of Pou3f4 upregulation to pituitary disease by utilizing double-mutant mice lacking both Prop1 and Pou3f4. However, our data showed that Pou3f4 is not required for normal pituitary development and function. Double mutants further demonstrated that the upregulation of Pou3f4 was not causative for the overexpression of Sox21. These data indicate loss of Pou3f4 is not a potential cause of CPHD, and further studies may investigate the functional consequence of upregulation of Pou3f4 and Sox21, if any, in the novel Smoc2+ve cell population.

overall_design: ScRNAseq RNA libraries were prepared from one P4 female Prop1+/+ pituitary gland and one P4 female Prop1df/df pituitary gland from the same litter. Single-cell RNA libraries were prepared by the University of Michigan's Advanced Genomics Core on the 10X Genomics Chromium 3’ Gene Expression v3 platform following the manufacturer's instructions and sequenced on the NovaSeq6000 platform with an S4 chip. Samples were analyzed similar to our previous study 16. Samples were demultiplexed and fastq files aligned using Cellranger Single Cell Software Suite 3.1.0. Sequence reads were aligned to a modified mm10 containing extended annotation for the Prop1 3’ untranslated region 17. Binary sequence alignment maps were converted into loom files using velocyto.py 18 and imported into R for primary analyses using Seurat 3.1.2 and 5.1.0 19,20. Core Seurat functions were used to import loom files, integrate samples, generate uniform manifold approximation and projection (UMAP), calculate clusters, and calculate differential gene expression. Expression plots used Nebulosa 1.12.1 21. Plots and images generated in R or Excel were compiled in Adobe Photoshop 2024.

first: Bailey

middle: E

last: Masser

first: Michelle

middle: L

last: Brinkmeier

first: Yuxuan

last: Lin

first: Qin

last: Liu

first: Aya

last: Miyazaki

first: Jannatun

last: Nayeem

middle: Y


title: Multi-omic analysis identifies metabolic biomarkers for early detection of breast cancer and prediction of therapeutic responses

city: beijing

first: xiaowei

last: tang

state: beijing

address: xuyuanlu 38

country: China

institute: 北医

summary: Reliable blood-based tests for identifying early-stage breast cancer remain elusive. Employing single-cell transcriptomic sequencing analysis, we illustrate a close correlation between nucleotide metabolism in breast tumor cells and activation of regulatory T cells (Tregs) in the tumor microenvironment, which show distinction in subtypes of triple-negative breast cancer (TNBC) and non-TNBC patients and likely to impact on prognosis of BC through A2AR-Treg pathway. Combining machine learning with absolute quantitative plasma metabolomics, we establish an effective diagnostic model for early-stage breast cancer, utilizing a four-metabolite panel including two nucleoside metabolites, inosine and uridine. This metabolomics study, involving 1111 participants, demonstrates high accuracy across training, test, and independent validation cohorts. Surprisingly, inosine and uridine prove predictive of the response to neoadjuvant chemotherapy (NAC) in TNBC patients. This study deepens the understanding of nucleotide metabolism in development of breast cancer and introduces a promising non-invasive approach with low radiation exposure for early breast cancer detection and predicting NAC response in TNBC patients.

overall_design: To investigate the metabolic difference between breast normal tissue and cancer tissue, we took single-cell RNA-seq of 9 samples.

first: Huajie

last: Song

first: Xiaowei

last: Tang

first: Miao

first: Guangxi

last: Wang


title: Varicella-zoster virus hijacks the type I interferon response and antigen presentation pathways in matured hiPSC-derived neurospheroids

status: Public on Aug 02 2024

city: Leuven

first: Johan

last: Van Weyenbergh

address: Herestraat 49

department: DMIT

country: Belgium

institute: KU Leuven

summary: Varicella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With innate immune signalling, and more specifically type I interferon (IFN) signalling, being an important first line cellular defence mechanism against VZV infection, we here investigated the triggering of innate immune responses in a human CNS-like environment. For this, we established and characterised a 5-month matured hiPSC-derived neurospheroid (NSPH) model containing TuJ1+ MAP2+ NeuN+ neurons and GFAP+ S100b+ AQP4+ CD49f+ astrocytes. Immune competence of these NSPHs was demonstrated by secretion of IL-6 and CXCL10 following stimulation with a cocktail of pro-inflammatory stimuli. Subsequently, NSPHs were infected with reporter strains of VZV (eGFP-ORF23 VZV) and Sendai virus (eGFP SeV). Live cell and immunocytochemical analyses demonstrated VZV infection within NSPHs, while SeV infection was limited to the outer NSPH border. Next, a transcript-level immune profiling was performed using NanoString technology to explore innate immune signatures of virus-infected NSPHs. While SeV-infected NSPHs displayed a clear Type I IFN response, in VZV-infected NSPHs no Type I IFN response was activated. Even more, in the latter a strong suppression of genes related to IFN signalling and antigen presentation was noted. Validating these opposite immune signatures in VZV- and SeV-infected NSPHs, cytokine profiling of NSPH supernatant revealed increased secretion of IL6 and CXCL10 by SeV-infected NSPHs, but not by VZV-infected NSPHs. Similarly, immunocytochemical analysis demonstrated upregulation of type I IFN activated anti-viral proteins Mx1, IFIT2 and ISG15 in SeV-infected NSPHs, but not in VZV-infected NSPHs. Furthermore, CD74, a key part of the MHC class II antigen presentation pathway was found to be suppressed in VZV-infected NSPHs. Finally, even though VZV-infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules throughout the entire NSPH. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, as well as their capability to recapitulate the strong immune evasive behaviour of VZV. Subsequently, this NSPH model has the potential to study viral neuro-immune responses and evasion strategies in a human CNS-like environment.

overall_design: At 7 days post stimulation/infection, NSPHs for RNA extraction were washed with ice cold PBS, snap frozen in liquid nitrogen and stored at -80°C. RNA extraction was performed on 2 pooled NSPHs of each condition (in triplicate) using the RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer's protocol. The RNA samples were used for nCounter (NanoString) analysis on a nCounter® MAX Analysis System. Briefly, RNA extracts were hybridized to ± 600 unique capture/reporter pairs (50bp each) targeting 585 immune transcripts and 15 housekeeping genes, as defined in the Human Immunology nCounter® panel, as well as six positive and eight negative control probes (all from NanoString). Results were sequentially corrected for background (negative control probes), technical variation (positive control probes) and RNA content (housekeeping genes) using nSolver 4.0 (NanoString), followed by differential gene expression analysis and gene set enrichment analysis (based on GO-terms) using Omics Playground (BigOmics Analytics).

first: Jonas

last: Govaerts

first: Peter

last: Ponsaerts


title: ACSL6-activated IL18R1-NF-κB promotes IL18-mediated tumor immune evasion and tumor progression

city: Guangzhou

first: Ziyang

state: Guangdong

address: 58 Zhongshan Second Road

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

summary: RNAseq analysis of MHCC97H cells infected with shNT or shACSL6 lentivirus.

overall_design: mRNA profiles of MHCC97H cells infected with shNT or shACSL6 were generated by deep sequencing, in duplicate, using Illumina Navoseq 6000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using  SYBR Green assays.

first: Yuqin

last: Di

first: Jing

last: Xiao

first: Xiang

last: Zhang

first: Lvlan

last: Ye

first: Xiangqiong

last: Wen

first: Jiale

last: Qin

first: Ligong

last: Lu

first: Xiongjun

first: Weiling

last: He


title: THE EPIGENOME PLAYS A MAJOR ROLE IN TOMATO 3D CHROMATIN ORGANIZATION

status: Public on Aug 05 2024

city: Gif-sur-Yvette

first: Xiaoning

last: HE

address: 630 Rue Noetzlin

country: France

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Translation inhibition with CHX in mouse embryonic stem cells

city: Nijmegen

first: Oscar

last: Garcia-Blay

state: Gelderland

address: Heyendaalseweg 135

department: Biophysical Chemistry

country: Netherlands

institute: Institute for Molecules and Materials (Radboud University)

summary: Gene-expression noise can influence cell-fate choices across pathology and physiology. However, a crucial question persists: do regulatory proteins or pathways exist that control noise independently of mean expression levels? Our integrative approach, combining single-cell RNA sequencing with proteomics and regulator enrichment analysis, reveals 32 putative noise regulators. The approach utilizes global translation inhibition  (i.e., potential protein regulators), and single-cell RNA sequencing (scRNA-seq) to quantify the changes in noise of all transcripts (i.e., potential mRNA targets). This dataset corresponds to the aforementioned scRNA-seq experiment upon translation inhibition with cycloheximide.

overall_design: Mouse embryonic stem cells (mESCs) were seeded and growth in serum/LIF conditions. 24 hours post-seeding, cells were treated with cycloheximide for 3 or 6 hours, or EtOH as control. After treatment cells were dechached and frozen in preparation for single-cell RNA sequencing. Cells were shipped in dry ice and single-cell RNA sequencing was performed from a freshly thawed sample, through single-cel barcoding (10x Genomics'Chromium) and sequencing  (NextSeq 2000, Single Cell 3'v3 chemistry).

first: Óscar

last: García-Blay

first: Maike

middle: M

last: Hansen


title: THE EPIGENOME PLAYS A MAJOR ROLE IN TOMATO 3D CHROMATIN ORGANIZATION [ChIP-seq]

summary: Understanding the 3D genome organization in the nucleus is becoming increasingly crucial given its involvement in many processes from transcription to the integrity of the nucleus itself, which play a role in every stage of an organism's life. Beyond the linear genome defined by its nucleotide sequence, this organization is also governed by its epigenome, but how the latter controls the 3D architecture of the nucleus is poorly understood. To decipher the epigenome influence on the formation of all levels of the nuclear organization, we studied tomato mutants defective for DDM1, a chromatin remodeler determining for the constitutive heterochromatin establishment. We observed that the ddm1 mutant exhibited weakened compartmentalization with lower insulation, more inter-compartment contacts and an increase in long-range contacts. This disorganization is accompanied by a reconfiguration of the epigenome, ultimately resulting in a reprogramming of its transcriptome. By integrating all our data, we have shown that this redistribution of the epigenome is linked to the global disorganization of the nucleus notably via new interactions mediated by H3K27me3, and is also related to new interactions between genes and/or TEs which display the same epigenetic characteristics. Altogether, our results strongly suggest that epigenetic features drive the formation of distinct compartments by playing a segregating role between active and inactive chromatin, becoming an essential characteristic for the identity of genes and TEs in terms of genome organization.

overall_design: To investigate the relationship between genetics and epigenetics in shaping the 3D genome structure, we performed ChIP-seq analyses in wild type (WT) vs ddm1 of two post-translational histone modifications marks associated with constitutive heterochromatin, H3K9me2 and H3K27me1, the Polycomb-dependent facultative heterochromatin-related mark H3K27me3, and the activate mark H3K9ac.

first: Chloé

middle: D

last: Lopes

first: Leonardo

middle: I

last: Bistrain

first: Nicolas

last: Bouché

first: David

last: Latrasse

first: Cécile

last: Raynaud

first: Moussa

last: Benhamed


title: ATAC analysis of kidney cortex following the KLF6 induction in podocytes

first: Sandeep

middle: K

last: Mallipattu

address: 101 Nicolls Rd

department: Medicine-Nephrology

summary: The goal of this study was to identify genome-wide chromatin accessibility changes caused by podocyte specific overexpression of KLF6  in STZ + UNX induced diabetic mouse model.

overall_design: Four experimental groups were analyzed

first: Nehaben

middle: A

last: Gujarati

first: Bismark

middle: O

last: Frimpong

first: Malaika

last: Zaidi


title: ATAC Sequencing of Baseline and Chronically Stimulated CART19-28z Cells in the Manuscript IL-4 Drives Exhaustion of CD8+ CART Cells

status: Public on Aug 01 2024

city: Rochester

first: Saad

last: Kenderian

state: MN

address: 200 First Street SW

department: Hematology Research

institute: Mayo Clinic

summary: Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here, we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion, RNA and ATAC sequencing on baseline and exhausted CART cells, and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further, IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely, IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore, we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy.

overall_design: In an effort to investigate the epigenetic regulation of exhaustion, we first developed an in vitro model for exhaustion. In this model, CART19-28ζ cells are chronically stimulated with the CD19-expressing JeKo-1 tumor cell line by adding fresh JeKo-1 cells every other day for one week. After one week of chronic stimulation, the CART cells were isolated and assessed for changes in phenotype. Here we provide ATAC sequencing results from baseline and chronically stimulated CART19-28ζ cells from three healthy donors.

first: Stewart

last: Carli

first: Kenderian

last: Saad


title: Genome-wide CRISPR Knockout Screen of Healthy Donor CART19-28ζ Cells in the Manuscript IL-4 Drives Exhaustion of CD8+ CART Cells

overall_design: In an effort to investigate the epigenetic regulation of exhaustion, we first developed an in vitro model for exhaustion. In this model, CART19-28ζ cells are chronically stimulated with the CD19-expressing JeKo-1 tumor cell line by adding fresh JeKo-1 cells every other day for two weeks. Then, we used this in vitro model for exhaustion to complete a genome-wide CRISPR knockout screen in healthy donor CART19-28ζ cells.


title: POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/PI3K/AKT/β-catenin/FOSL1 pathway [GSCs_RNA-seq]

city: Soochow

first: Hao

last: Wang

state: Jiangsu

address: 899 Pinghai Road

department: Department of Neurosurgery & Brain and Nerve Research Laboratory

institute: The First Affiliated Hospital of Soochow University

summary: Glioblastoma (GBM), classified as a grade 4 glioma, is the most prevalent intrinsic malignancy of the central nervous system. Glioblastoma stem cells (GSCs) are small populations of GBM cells with self-renewal and multilineage differentiation capabilities and are considered responsible for the tumorigenesis and development of GBM. GSCs also exhibit radiation resistance, chemoresistance, and angiogenic and invasive properties, which are correlated with poor outcomes in GBM patients. Therefore, targeting GSCs constitutes a promising approach for treating GBM patients. Our findings revealed that POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/PI3K/AKT/β-catenin/FOSL1 pathway.

overall_design: Comparative gene expression profiling analysis of RNA-seg data of glioblastoma stem cells treated with sh-Control, sh-POSTN or human recombinant POSTN.

first: Youxin

last: Zhou


title: POSTN secreted from glioblastoma stem cells can recruit microglia and upregulate CD70 expression in microglia through the αVβ3/PI3K/AKT/NFκB pathway [HMC3_ChIP-seq]

summary: Glioblastoma (GBM), classified as a grade 4 glioma, is the most prevalent intrinsic malignancy of the central nervous system. Glioblastoma stem cells (GSCs) are small populations of GBM cells with self-renewal and multilineage differentiation capabilities and are considered responsible for the tumorigenesis and development of GBM. POSTN secreted from glioblastoma stem cells can recruit microglia and upregulate CD70 expression in microglia through the αVβ3/PI3K/AKT/NFκB pathway, which in turn promotes Treg development and functionality and supports the formation of an immunosuppressive tumor microenvironment.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChlP-seq) for NFκB in microglia cells.


title: Transcriptomic analysis of kidney cortex following the KLF6 induction

last: Owusu Frimpong

state: NY

address: 100 Nicolls Rd

summary: The goal of this study was to identify transcriptomic changes mediated by KLF6 induction in the setting of STZ-induced diabetic kidney disease

overall_design: Four experimental groups were analyzed, two replicates per group.

first: Jessica

last: Vasquez

first: Yiqing

last: Guo

first: Monica

middle: P

last: Revelo


title: Post-prandial cardiac hypertrophy is sustained by mechanics, epigenetic, and metabolic reprogramming in pythons [RNA-Seq]

status: Public on Aug 04 2024

city: Boulder

first: Mary

middle: Ann

last: Allen

state: Co

address: UCB 596  JSCBB

department: BioFrontiers

institute: University of Colorado

summary: Constricting pythons, known for their ability to consume infrequent, massive meals, exhibit rapid and reversible cardiac hypertrophy following feeding. Our primary goal was to investigate how python hearts achieve this adaptive response after feeding. Isolated myofibrils increased force after feeding without changes in sarcomere ultrastructure and without increasing energy cost. Ca2+ transients were prolonged after feeding with no changes in myofibril Ca2+ sensitivity. Feeding reduced titin-based tension, resulting in decreased cardiac tissue stiffness. Feeding also reduced the activity of sirtuins, a metabolically-linked class of histone deacetylases, and increased chromatin accessibility. A transcription factor enrichment analysis on transposase-accessible chromatin with sequencing (ATAC-Seq) revealed the prominent role of transcription factors YY1 and NRF1 in post-feeding cardiac adaptation. Gene expression was also changed in favor of translation and metabolism. Finally, metabolomics analysis and ATP production assay demonstrated that cardiac adaptation after feeding not only increased energy demand but also energy production. These findings have broader implications for our understanding of cardiac adaptation across species and hold promise for the development of innovative approaches to address cardiovascular diseases.

overall_design:
To determine the gene expression profile of post-prandial python hearts, the hearts of fasted and 24 post fed Python regius were collected for RNA-sequencing analysis.
Three fasted and three fed pythons were included in the study.
Gene expression analysis was performed between fasted and 24 hour post fed python hearts.

first: Claudia

last: Crocini

first: Kathleen

middle: C

last: Woulfe

first: Christopher

last: Ozeroff

first: Stefano

last: Perni

first: Joseph

last: Cardiello

first: Cierra

middle: J

last: Walker

first: Kristi

middle: S

last: Anseth

first: Leslie

last: Leinwand


title: POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/PI3K/AKT/β-catenin/FOSL1 pathway [GSCs_CHIP-seq]

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChlP-seq) for β-catenin in glioblastoma stem cells.


title: mRNA Profiling of Chronically Stimulated CART19-28z Cells in the Manuscript IL-4 Drives Exhaustion of CD8+ CART Cells

first: Saad

last: Kenderian

overall_design: In an effort to investigate the epigenetic regulation of exhaustion, we first developed an in vitro model for exhaustion. In this model, CART19-28ζ cells are chronically stimulated with the CD19-expressing JeKo-1 tumor cell line by adding fresh JeKo-1 cells every other day for one week. After one week of chronic stimulation, the CART cells were isolated and assessed for changes in phenotype. Here we provide mRNA profiling of baseline and chronically stimulated CART19-28ζ cells from three healthy donors.

last: Carli


title: Mitochondrial Rewiring with Small-Molecule Drug-Free Nanoassemblies Unleashes Anticancer Immunity

city: Hangzhou

first: Lulu

last: Ren

address: 3 East Qingchun Road

institute: Sir Run Run Shaw Hospital, Zhejiang University School of Medicine

summary: The immunosuppressive tumor microenvironment (TME) remains a major obstacle to tumor control and causes suboptimal responses to immune checkpoint blockade (ICB) therapy. Thus, developing feasible therapeutic strategies that trigger inflammatory responses in the TME could improve the ICB efficacy. Mitochondria play an essential role in inflammation regulation and tumor immunogenicity induction. Herein, we report the discovery and characterization of a new class of previously unappreciated small molecules that can recapitulate aqueous self-assembly behavior, specifically target cellular organelles (e.g., mitochondria), and invigorate tumor cell immunogenicity. Mechanistically, this nanoassembly platform dynamically rewires mitochondria, induces endoplasmic reticulum stress, and causes apoptosis/paraptosis-associated immunogenic cell death. After treatment, stressed and dying tumor cells can act as prophylactic or therapeutic cancer vaccines. In preclinical mouse models of cancers with intrinsic or acquired resistance to programmed death-1 (PD-1) blockade, the local administration of nanoassemblies inflames the immunologically silent TME and synergizes with ICB therapy, generating potent antitumor immunity. This chemically programmed small-molecule immune enhancer acts like no other reported cytotoxic therapeutics and offers a promising new strategy for synchronous and dynamic tailoring of innate immunity to achieve traceless cancer therapy and overcome immunosuppression in cancers.

overall_design: Mice bearing anti-PD-1-resistant MC38 tumors received intratumoral injections of vehicle (saline containing 5% DMSO) or mtDSN-2 (1 mg kg−1) every three days. After a total of three doses, tumors were collected, minced and dissociated (n = 3 mice or samples were pooled to control for biological variability).


title: Urinary cytokeratin 20 (encode by Krt20) as a predictor for chronic kidney disease following acute kidney injury

first: FAN FAN

last: HOU

address: 1838 North Guangzhou Ave, Guangzhou

institute: Nanfang Hospital, Southern Medical University

summary: Kidney transcriptome sequencing was applied to identify the top up-regulated genes in mice with AKI. The product of the top-ranked gene was identified in the tubular cells and urine both in mouse and human AKI. Data from two cohorts of patients with a prehospitalization estimated glomerular filtration rate (eGFR) ≥ 45ml/min/1.73m2 who survived for at least 90 days after AKI were used to derive and validate multivariable prediction models. AKI to CKD progression was defined as a persistent eGFR < 60ml/min/1.73m2 and with a minimum 25% reduction from baseline eGFR 90 days after AKI in patients with prehospitalization eGFR ≥ 60ml/min/1.73m2. AKI to advanced CKD was defined by a sustained reduction of eGFR < 30 ml/min/1.73m2 90 days after AKI in those with prehospitalization eGFR 45-60ml/min/1.73m2. Kidney cytokeratin 20 (CK20) was significantly up-regulated in injured proximal tubular cells and detectable in urine within 7 days after AKI. High concentrations of urinary CK20 (uCK20) were independently associated with the severity of histological AKI and the risk of AKI to CKD or advanced CKD progression. In Test set, the AUC of uCK20 for predicting AKI to CKD or advanced CKD was 0.80, outperformed currently used biomarkers for detecting kidney tubular injury. Addition of uCK20 to an established clinical model significantly improved the ability for predicting AKI-CKD progression with an AUC of 0.90, and largely improved the risk reclassification.In conclusion This finding highlighted uCK20 as a novel and useful predictor for AKI-CKD progression, and may provide a tool to early identify patients at high risk of CKD following AKI.

overall_design: Male C57BL/6 mice aged 6-8 weeks (20-24g) were purchased from Beijing SpePharm Biotechnology Co. Ltd. IRI was induced by clamping the left renal pedicle for 20-min (mild AKI) or 40-min (severe AKI) by using the microaneurysm clamps, genetic change between 20-min IRI (left kidney) and self-control kidneys (right kidney) or between 40-min IRI and self-control kidneys at 1d and 3d after AKI were analyzed.  n = 6 for each group of mice. Overall, we used RNA sequencing to identify the top up-regulated genes in the kidney of mice with tubular epithelial cell injury.

first: Rui

last: Ma

first: Han

last: Ouyang

first: Xiaobing

last: Yang

first: Fan

middle: F

last: Hou


title: Bulk RNA-sequencing of transduced colorectal cancer cell line HCT-116 and breast cancer cell line MCF-7 overexpressing full-lenght p53β or p53β-stop-lost.

city: Leiden

first: Tom

last: van Wezel

address: Albinusdreef 2

department: Pathology

institute: Leiden University Medical Center

summary: Whole-transcriptome analysis of full-lenght p53β or p53β-stop-lost-overexpression cell lines.

overall_design: Bulk RNA-sequencing of transduced colorectal cancer cell line HCT-116 and breast cancer cell line MCF-7 overexpressing full-lenght p53β or p53β-stop-lost cultured under identical conditions.


title: single cell sequencing of mouse lung single cell suspensions

city: HENNINGSDORF

first: Jörg

last: Kaufmann

state: Brandenburg

address: NEUNDORFSTR 20B

country: Germany

institute: Pantherna Therapeutics GMBH

summary: Dysfunction of endothelial cells lining the inner surface of blood vessels are causative for a number of diseases. Hence, the ability to therapeutically modulate gene expression within endothelial cells is of high therapeutic value in treating diseases such as those associated with lung edema. mRNAs formulated with lipid nanoparticles (LNPs) have emerged as a new drug modality to induce transient protein expression for modulating disease relevant signal transduction pathways. In the study presented here we tested the effect of a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76) formulated into a cationic lipid nanoparticle (cLNP) on attenuating inflammation-induced vascular leakage. After intravenous injection, the respective mRNA was found to be delivered almost exclusively to the endothelial cells of the lung, while sparing other vascular beds and bypassing the liver. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies in vitro and in vivo in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in an LPS-challenging model.

overall_design:
To investigate the specific cellular uptake of two different mRNAs in mice lung following intravenous delivery of encapsulated mRNA in the same lipid nanoparticle delivery system following 15 and 24 hours of administration.
Gene expression profiling was performed to identify lung cell types and the two target mRNAs: one encoding a potent tie2 agonist COMP-ANG1 (mRNA-76; PAN004) and the other control mRNA encoding for a non-secreted firefly luciferase (mRNAFLUC; PAN93).
Comparative gene expression profiling anlaysis of RNA-seq data for mouse lung cells, mRNA-76 and mRNAFLUC.

first: Zuzanna

last: Makowska

first: Filippos

last: Klironomos

first: Ralph

last: Lesche

first: Jorg

first: Katrin

last: Radloff


title: Cannabidiol ameliorates mitochondrial disease via PPARg activation

city: Barcelona

first: Emma

last: Puighermanal

state: Barcelona

address: Passatge de la Independència 30 2-3

country: Spain

institute: Autonomous University of Barcelona

summary: Mutations in mitochondrial energy-producing genes lead to a heterogeneous group of untreatable disorders known as primary mitochondrial diseases (MD). Leigh syndrome (LS) is the most common pediatric MD and is characterized by progressive neuromuscular affectation and premature death. Here, we show that daily cannabidiol (CBD) administration significantly extends lifespan and ameliorates pathology in two LS mouse models, and cellular function in fibroblast from LS patients. CBD delays motor decline and neurodegenerative signs, improves social deficits and breathing abnormalities, decreases thermally induced seizures, and improves neuropathology in affected brain regions. Mechanistically, we identify peroxisome proliferator-activated receptor gamma (PPARg) as a key contributor to CBD’s beneficial effects, and provide proof of dysregulated PPARg expression and activity as a common feature in both mouse neurons and fibroblasts from LS patients. Taken together, our results provide the first evidence for CBD as a potential treatment for LS.

overall_design: To assess the effect of CBD in LS patient-derived fibroblasts bearing NDUFS4 and COX15 mutations.

first: E

first: M

last: Luna-Sánchez

first: A

last: Gella

last: Urpi

last: Royo

first: P

last: Tena

first: I

last: Appiah

first: MH

last: de Donato

first: F

last: Menardy

last: Bianchi

last: Alberola

first: L

last: Rodríguez-Pascau

first: C

last: Vergara

first: G

last: van der Walt

last: Gómez-Pallarès

last: Martinell

last: Sanz

first: S

last: Jurado

last: Soriano

last: Pizcueta

last: Quintana

last: Marsicano

last: Bellocchio


title: Cohesin chromatin loop formation by an extrinsic motor (ChIP-Seq)

city: London

first: Thomas

middle: M.

last: Guerin

address: nw1 1at, 1 Midland Road

country: United Kingdom

institute: The Francis Crick Institute

summary: The ring-shaped cohesin complex topologically entraps two DNAs to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape, with wide-ranging implications for gene regulation, which cohesin is thought to achieve by actively extruding DNA loops without topologically entrapping DNA. The ‘loop extrusion’ model find motivation from in vitro observations - whether this process indeed underlies chromatin loop formation in vivo remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, likely by generating DNA substrates for topological loop capture. Transcription furthermore acts as an extrinsic motor that, by pushing cohesin along transcription units, extends chromatin loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis and point to an alternative mechanism for cohesin-dependent chromatin organisation. Loop formation by DNA-DNA capture, akin to sister chromatid cohesion establishment at replication forks, unifies cohesin’s two roles in chromosome segregation and interphase genome organisation.

sample_organism:
Saccharomyces cerevisiae
Nakaseomyces glabratus

overall_design: Cohesin calibrated ChIP sequencing and Micro-C. Two independent replicates are provided

last: Guérin

first: Christopher

last: Barringtom

first: Georgii

last: Pobegalov

first: Maxim

last: Molodtsov

first: Frank

last: Uhlmann


title: The catalytic-independent function of LSD1 modulates the epigenetic landscape of mouse embryonic stem cells

city: Badalona

first: Manel

last: Esteller

address: Ctra. de Can Ruti, Cami de les Escoles s/n

department: Cancer and Leukemia Epigenetics and Biology Program (PEBCL)

institute: Josep Carreras Leukaemia Research Institute (IJC)

summary: Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for embryonic stem cell (ESC) self-renewal but is required for ESC growth and differentiation. Reexpression of a catalytically-dead LSD1 (LSD1MUT) recovers the proliferation capability of ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, a gain of H3K4me1 in Lsd1 knockout (KO) ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with the ubiquitin-specific peptidase 7 (USP7) and, consequently, inhibiting DNMT1 and UHRF1 ubiquitylation. Our studies elucidate for the first time a novel mechanism by which the scaffolding function of LSD1 controls DNA methylation in ESCs.

overall_design: We performed genome-wide CpG methylation analysis in mESCs samples using the Infinium Mouse Methylation BeadChip that interrogates more than 285k methylation sites per sample at single-nucleotide resolution.

first: Carlos

last: Garcia-Prieto


title: Unexpected nuclear hormone receptor and chromatin dynamics regulate estrous cycle dependent gene expression [HIF2A cKO RNA-seq]

city: RTP

first: Wendy

last: Jefferson

state: NC

address: 105 T. W. Alexander Drive

institute: NIEHS

summary: Uteri from intact, untreated CD-1 mice were collected at 2 months of age at diestrus (CoD) or estrus (CoE). RNA-seq, ATAC-seq, ER alpha ChIP-seq, PgR ChIP-seq, Hif2a ChIP-seq and HiC-seq were performed as well as RIME assay using ER alpha as the immunoprecipitating antibody.

overall_design: Uteri from intact, WT and HIF2A cKO untreated FVB/N mice were collected at 2 months of age at diestrus (CoD) or estrus (CoE). RNA-seq was performed.

first: Tianyuan

last: Wang

first: Carmen

last: Williams


title: MPK4 mediated phosphorylation of PIF4 controls thermosensing by regulation of H2A.Z deposition in Arabidopsis

city: New Delhi

first: Alok

middle: Krishna

last: Sinha

state: Delhi

address: Aruna Aasaf Ali Marg

department: National Institute of Plant Genome Research

country: India

summary: Plants have the ability to perceive a slight upsurge in ambient temperature and respond by undergoing morphological changes, such as elongated hypocotyls and early flowering. The dynamic functioning of PHYTOCHROME INTERACTING FACTOR4 (PIF4) in thermomorphogenesis has been well established, although the regulatory pathway involved in thermosensing is not deciphered completely. In our study, we demonstrate that an increase in temperature from 22˚C to 28˚C induces the phosphorylation of PIF4 by MITOGEN-ACTIVATED PROTEIN   KINASE 4 (MPK4) which shows high expression and activation at 28˚C. Phosphorylated PIF4 represses the expression of ACTIN-RELATED PROTEIN 6 (ARP6) that is required for mediating histone variant H2A.Z deposition at its target gene loci. We demonstrate that variation of ARP6 expression in PIF4 phosphor -null and phosphor-mimetic seedlings affects hypocotyl growth and flowering at 22˚C and 28˚C. Further, we show that change in ARP6 expression affects H2A.Z deposition at the loci of genes involved in hypocotyl elongation using PIF4 phosphor -null and phosphor-mimetic seedlings. Interestingly, the expression of MPK4 is also controlled by H2A.Z deposition in temperature dependent manner. Taken together, our findings highlight the cumulative molecular interplay between MPK4, PIF4, and chromatin modification by ARP6-mediated H2A.Z deposition as a regulatory mechanism of thermosensing.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for studying histone variant H2A.Z deposition in WT, arp6, pif4 and phosphor-null PIF4 seedlings at 22 and 28℃.

first: N

last: Verma

first: D

last: Singh

last: Banerjee

last: Mittal

last: Noryang

first: A


title: Deep starvation induces loss of cell wall and dormancy in Listeria

city: Jouy-en-Josas

first: Pierre

last: Nicolas

address: INRAE - Domaine de Vilvert

institute: INRAE - Université Paris-Saclay

summary:
Bacteria have developed multiple strategies, such as sporulation, to cope with environmental stress.
Non-sporulating bacteria, however, may “hibernate” into a so-called viable but non-culturable (VBNC) state, where they are no longer able to grow in standard culture media and thus become undetectable by conventional growth-based methods.
VBNC pathogens pose a significant risk for human and animal health as they can “wake up” back into a vegetative and virulent state.
Although hundreds of bacterial species have been reported to enter a VBNC state in response to various stresses (e.g. thermal, osmotic, starvation, antibiotics), the molecular mechanisms governing this phenotypic switch remains largely elusive.
Here, we report an in-depth characterization of the VBNC state transition process in the bacterial pathogen Listeria monocytogenes in response to nutritional deprivation.
We found that starvation in mineral water drives L.monocytogenes into a VBNC state via a unique mechanism of cell wall shedding that generates cellwall-deficient coccoid forms.
Transcriptomic and gene-targeted approaches revealed the stress response regulator SigB and the autolysin NamA as major mediators of cell wall loss and VBNC state transition.

overall_design: Comparison between cells in the first day and after 7 days of incubation in mineral water, with biological triplicates.

first: Filipe

last: Carvalho

first: Hélène

last: Bierne

first: Alessandro

last: Pagliuso


title: Amniotic fluid modifies esophageal epithelium differentiation and inflammatory responses

city: Cincinnati

first: Mark

last: Rochman

state: OH

address: 3333 Burnett Ave

department: Allergy and Immunology

institute: CCHMC

summary: The interplay between genetic and environmental factors during pregnancy can predispose to inflammatory diseases postnatally, including eosinophilic esophagitis (EoE), a chronic allergic disease triggered by food. Herein, we examined the effects of amniotic fluid (AF) on esophageal epithelial differentiation and responsiveness to proallergic stimuli. Multiplex analysis of AF revealed the expression of 66 cytokines, whereas five cytokines including IL-4 and thymic stromal lymphopoietin (TSLP) were not detected. Several proinflammatory cytokines including TNFα and IL-12 were highly expressed in the AF from women who underwent preterm birth, whereas EGF was the highest in term birth samples. Exposure of esophageal epithelial cells to AF resulted in transient phosphorylation of ERK1/2 and the transcription of early response genes, highlighting the direct impact of AF on esophageal epithelial cells. In a three-dimensional spheroid model, AF modified the esophageal epithelial differentiation program and enhanced the transcription of IL-13-target genes, including CCL26 and CAPN14, which encodes for a major genetic susceptibility locus for eosinophilic esophagitis. Notably, CAPN14 exhibited upregulation in spheroids exposed to preterm but not term AF following differentiation. Collectively, our findings call attention to the role of AF as a potential mediator of the intrauterine environment that influences subsequent esophageal disorders.

overall_design: 3D esophageal epithelial spheroids were grown from EPC2 cells in Matrigel for 11 days. Spheroids were left untreated or were treated with IL-13 starting from D3. Toatl RNA was extracted from EPC2 cells at D0 (monolayer), D3, and D11 of the differentiation protocol. Spheroids from 2 independent experiments were collected and sequenced.

first: Marc

last: Rothenberg


title: The H3.3K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation

city: STANFORD

first: Joydeb

state: CA

address: 443 VIA ORTEGA

institute: Stanford University

summary: Histone H3.3 is frequently mutated in tumors, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the epigenetic landscape, its effects on gene expression dynamics remain unclear. Here, we use a synthetic reporter to measure the effects of H3.3K36M on silencing and epigenetic memory after recruitment of the ZNF10 Krüppel-associated box (KRAB) domain, part of the largest class of human repressors and associated with H3K9me3 deposition. We find that H3.3K36M, which decreases H3K36 methylation and increases histone acetylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a model for establishment and maintenance of epigenetic memory, where the H3K36 methylation pathway is necessary to maintain histone deacetylation and convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.

overall_design:
CUT&RUN for the histone modifications H3K9me3, HA-tag, H3.3, H3K36me3, H3K4me3,H3K27me3, in HEK293T cells with synthetic reporter system
EMseq DNA methylation profiling of the synthetic reporter after KRAB or HDAC4 recruitment

first: Abby

middle: R

last: Thurm

first: Connor

middle: H

last: Ludwig

first: Lacramioara

last: Bintu


title: Decidual-tissue resident memory T cells protect against nonprimary human cytomegalovirus infection at the maternal-fetal interface

city: Haifa

first: Tamar

last: Lahav

address: Zor

department: Biology

country: Israel

institute: Technion

summary: Congenital cytomegalovirus (cCMV) is the most common intrauterine infection, leading to infant neurodevelopmental disabilities. An improved knowledge of correlates of protection against cCMV is needed to guide prevention strategies. Here, we employed a unique ex vivo model of human CMV (HCMV) infection in decidual tissues of women with and without preconception immunity, recapitulating nonprimary versus primary infection at the authentic maternofetal transmission site. We showed that decidual tissues of women with preconception immunity exhibited intrinsic resistance to HCMV, mounting a rapid activation of tissue resident memory CD8+ and CD4+ T cells upon nonprimary infection. We further revealed the role of HCMV-specific decidual-tissue resident CD8+ T cells in local protection against nonprimary HCMV infection.  The findings could inform the development of vaccine against cCMV, and provide insights for further studies of the integrity of immune defense against cCMV and other pathogens in the human maternal-fetal interface.

overall_design: First-trimester decidual tissues from ten HCMV-seronegative women and ten HCMV-seropositive women, each HCMV- or mock-infected and subjected to transcriptome analysis.

first: Or

last: Alfi

last: Hashimshony

first: Rina

last: Ben-El

first: Yael

last: Mandel-Gutfreund

first: Dana

middle: G

last: Wolf


title: The fork protection complex promotes symmetric histone recycling to maintain heterochromatin [SCAR-seq]

city: Copenhagen

first: Anja

last: Groth

address: Blegdamsvej 3B

country: Denmark

institute: Novo Nordisk Foundation Center for Protein Research

summary: The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (Claspin in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging strand recycling while another histone-binding mutation impaired leading strand recycling. We propose Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

sample_organism:
Schizosaccharomyces pombe
Mus musculus

platform_organism:
Schizosaccharomyces pombe
Mus musculus

overall_design: xSCAR-seq (crosslinked Sister Chromatids After Replication) series measuring histone partition of H3K36me3 and H4K20me0 in wildtype (WT), MRC1 dHBS, MCM2 histone-binding mutant (MCM2-2A), or double mutant Mrc1dHBSMcm2-2A strains. 2 replicates per strains with corresponding stranded input (clicked_Input) controls.

first: Sebastian

last: Charlton

first: Valentin

last: Flury

first: Yutaka

last: Kanoh

first: Aitana

middle: V

last: Genzor

first: Wantong

last: Ao

first: Peter

last: Brøgger

first: Hisao

last: Masai

first: Geneviève

last: Thon


title: Preventing Site-specific Calpain Proteolysis of Junctophilin-2 Protects Against Stress-induced E-C Uncoupling and Heart Failure

city: Iowa City

first: Long-Sheng

last: Song

state: IA

address: 285 Newton Rd

department: Internal Medicine

institute: University of Iowa

summary: Junctophilin-2 (JP2) is an essential component of the excitation-contraction coupling apparatus in cardiomyocytes and becomes proteolytically cleaved in response to stress. We previously mapped the primary calpain cleavage site to JP2 residues 565-566 using in vitro assays. To determine whether this site is responsible for JP2 cleavage in vivo, we generated calpain resistant JP2 (JP2CR) knock-in mice lacking this site. We find JP2 is not cleaved in JP2CR mice during pressure overload stress resulting in better cardiac outcomes and an attenuated transcriptional response relative to wildtype mice.

overall_design:
Calpain resistant JP2 knockin mice (JP2CR) were generated by deleting codons encoding residues 563-568 in the Jph2 gene.
Sham or trans-aortic constriction (TAC) surgery was performed on 9–10-week-old JP2CR and wildtype littermate male mice.
Ventricular tissue was collected 5 weeks after surgery for RNA analysis (n=4 per group).

first: Duane

last: Hall


title: Intestinal ILC progenitors differentially generate ILC subsets dependent on the recipient tissue microenvironment

city: Cambridge

first: Ana

last: Ferreira

address: Francis Crick Avenue, Cambridge Biomedical Campus

institute: MRC - Laboratory of Molecular Biology

summary: Innate lymphoid cells (ILC) are critical in maintaining tissue homeostasis, and during infection and inflammation.  Using combinatorial reporter mice we demonstrate the existence of rare, small intestinal lamina propria (siLP)- resident, ILC progenitors (siLP-ILCP) in adult mice.  Transfer of siLP-ILCP into recipients generated ILC1/NK cells, ILC2 and ILC3 within the siLP microenvironment, but only ILC1/NK cells in the liver, lung and spleen. Single cell gene expression analysis confirmed the phenotype of the siLP-ILCPs and ILC progeny and indicated that siLP-ILCP-derived ILC1/NK cells from the siLP have a more tissue-resident phenotype than those from the lungs. Thus, in contrast to bone marrow-derived ILCPs, a local pool of siLP-ILCP can contribute to pan-ILC production in the intestinal microenvironment but has restricted potential in other tissues. Therefore, ILCP potential is influenced by both tissue of origin and the microenvironment during development. This may provide additional flexibility during the tuning of immune reactions.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for newly identified siLP ILC progenitors compared to their in vivo progeny from the siLP and lung of immunocompromised recipients (Rag2-/-Il2rg-/-)

first: Paula

last: Clark

first: Mayuri

last: Gogoi

first: Noe

last: Rodriguez-Rodriguez

first: Jane

middle: E

last: Murphy

first: Jennifer

last: Walker

first: Alastair

last: Crisp

first: Helen

last: Jolin

first: Andrew

middle: N

last: McKenzie


title: The fork protection complex promotes symmetric histone recycling to maintain heterochromatin [ChIP-Seq]

first: Anja

last: Groth

overall_design: ChIP-Seq measuring chromatin occupancy for BrdU in WT and mrc1dHBS and H3K9me2 in WT, mrc1dHBS, mcm2-2A, and mrc1dHBSmcm2-2a cells. With corresponding Inputs and replicates

first: Sebastian

last: Charlton

first: Valentin

last: Flury

first: Yutaka

last: Kanoh


title: Transcription factor KLF2 regulates cell fate decision of stem cells in infantile hemangioma

city: Shanghai

first: Qian

last: Bian

address: Jinzun Road No.115

department: Shanghai Institute of Precision Medicine, Ninth People’s Hospital

institute: Shanghai Jiao Tong University School of Medicine

summary: As the most common vascular tumor during infancy, infantile hemangioma (IH) is clinically featured by a rapid proliferation phase of disorganized blood vessels and a subsequent spontaneous involution phase. Infantile hemangioma arises from a unique type of multipotent stem cells called hemangioma stem cells (HemSCs), which could differentiate into endothelial cells, pericytes and adipocytes in IH. However, the underlying mechanisms that regulate the cell fate determination of HemSCs are not well elucidated. Here, we identified KLF2 as a candidate transcription factor involved in the control of HemSCs differentiation. KLF2 was expressed in endothelial cells in proliferating IH and its expression diminished in adipocytes in involuting IH. KLF2 regualtes the proliferation, apopotosis and cell cycle progression in HemSCs. Moreover, KLF2 is a critical regulator in HemSCs that control their differentiation direction between endothelial cells and adipocytes. Knockdown of KLF2 inhibited the formation of blood vessels in vivo while accelerated the progress of adipogenesis. RNA-seq analysis suggested an induction of pro-adipogenic transcriptome in HemSCs upon KLF2 knockdown. Our data showed that KLF2 exhibited pleiotropic effects in regulating the biological behaviours of HemSCs, and was involved in the progression and involution of IH via determining the cell fate of HemSCs.

overall_design: In this study ,we performed RNA-seq in  KLF2 knockdown HemSCs (shKLF2) and control HemSCs (shControl) to dissect the transcriptional alterations in response to KLF2.

first: Jiawei

last: Zheng

first: Qiming

last: Chen


title: Unexpected nuclear hormone receptor and chromatin dynamics regulate  estrous cycle dependent gene expression [RNA-seq]

first: Wendy

last: Jefferson

summary: Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.

overall_design: Uteri from intact, untreated CD-1 mice were collected at 2 months of age at diestrus (CoD) or estrus (CoE). RNA-seq, ATAC-seq, ER alpha ChIP-seq, PgR ChIP-seq, Hif2a ChIP-seq and HiC-seq were performed as well as RIME assay using ER alpha as the immunoprecipitating antibody.

first: Tianyuan

last: Wang

first: Carmen

last: Williams


title: Unexpected nuclear hormone receptor and chromatin dynamics regulate  estrous cycle dependent gene expression [Hi-C]


title: Unexpected nuclear hormone receptor and chromatin dynamics regulate  estrous cycle dependent gene expression

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

overall_design: Refer to individual Series


title: Unexpected nuclear hormone receptor and chromatin dynamics regulate  estrous cycle dependent gene expression [ChIP-seq]


title: RNA-seq of O4+ oligodendrocyte progenitors isolated from P5 and P60 mice

city: New York

first: Patrizia

last: Casaccia

address: 85 St Nicholas Terrace

department: Neuroscience

institute: ASRC-CUNY

summary: Adult oligodendrocyte progenitors (aOPCs) share with their neonatal counterpart (nOPC) the ability to give rise to myelinating oligodendrocytes, but they also display unique functional features. This study addresses the molecular mechanisms underlying the intrinsic differences between these two populations. Using RNA-sequencing and unbiased histone proteomics analysis performed on PDGFRa+ OPCs sorted from a reporter mouse, we define the unique transcriptome and histone marks of aOPCs. We describe the higher levels of genes related to lipid metabolism and myelin sheath, and lower expression of genes related to cycle and proliferation in aOPCs compared to nOPCs. We also identify greater levels of activating H4K8ac and repressive H4K20me3 histone marks in aOPCs compared to nOPCs, and increased occupancy of H4K8ac mark at chromatin locations corresponding to oligodendrocyte-specific genes. Pharmacological inhibition of histone acetylation in neonatal O4+OPCs (nO4+OPCs) and adult O4+OPCs (aO4+OPCs) followed by RNA-sequencing showed a moderate change in the transcriptome of these cells resulting in the decrease of proliferation capacity in aO4+OPCs but not nO4+OPCs. Pharmacological inhibition of H4K20me3 mark was not sufficient to change the transcriptome or functional properties of nO4+OPCs and aO4+OPC. Overall, this study identifies histone acetylation as key in regulating the proliferation of aO4+OPCs.

overall_design: Transcriptome profiles (bulk RNA-seq assay) in untreated, DMSO treated, KATi treated and SUV420i treated O4+ OPCs isolated from wild-type P5 and P60 mice.

first: Ipek

last: Selcen

first: Dennis

last: Huang


title: Fine-scale contemporary recombination variation and its fitness consequences in adaptively diverging stickleback fish [ATAC-Seq]

city: Tuebingen

first: Felicity

last: Jones

address: Max-Planck-Ring 9

institute: Friedrich Miescher Laboratory of the Max Planck Society

summary: Despite deep evolutionary conservation, recombination varies greatly across the genome, among individuals, sexes and populations and can be a major evolutionary force in the wild. Yet this variation in recombination and its impact on adaptively diverging populations is not well understood. To elucidate the nature and potential consequences of recombination rate variation, we characterized fine-scale recombination landscapes by combining pedigrees, functional genomics and field fitness measurements in an adaptively divergent pair of marine and freshwater threespine stickleback populations from River Tyne, Scotland. Through whole-genome sequencing of large nuclear families, we identified the genomic location of almost 50,000 crossovers and built recombination maps for 36 marine, freshwater, and hybrid individuals at 3.8 kilobase resolution. Using these maps, we quantified the factors driving variation in recombination rate: we find strong heterochiasmy between sexes (68% of the variation) but also differences among ecotypes (21.8%). Hybrids show evidence of significant recombination suppression, both in overall map length and in individual loci. We further tested and found reduced recombination rates both within single marine–freshwater adaptive loci and between loci on the same chromosome, suggestive of selection on linked ‘cassettes’. We tested theory supporting the evolution of linked selection using temporal sampling along a natural hybrid zone, and found that recombinants with shuffled alleles across loci show traits associated with reduced fitness. Our results support predictions that divergence in cis-acting recombination modifiers whose mechanisms are disrupted in hybrids, may have an important role to play in the maintenance of differences among adaptively diverging populations.

overall_design: This GEO data deposit is part of a multipart study that includes functional genomic data related to meiosis in stickleback fish as described in Venu et al 2024.  This particular data series includes an ATACseq dataset of chromatin accessibility in stickleback primary spermatocytes. For each ATAC-seq library, the testes of three males were pooled. A control library of naked DNA was also prepared for each pool. Three marine ecotype libraries and three freshwater ecotype libraries were analyzed.

first: Vrinda

last: Venu

first: Enni

last: Harjunmaa

first: Andreea

last: Dreau

first: Shannon

middle: D

last: Brady

first: Devin

last: Absher

first: David

middle: M

last: Kingsley


title: DELE1 mitochondrial integrated stress response to CHCHD10 G58R protein misfolding in striated muscle [RNA-seq]

status: Public on Jul 31 2024

city: BETHESDA

first: Derek

middle: Paul

last: Narendra

state: MD

address: 35 CONVENT DR, Rm. 2A-211

institute: NINDS, NIH

summary: Transcriptional mitochondrial stress response to CHCHD10 G58R protein misfolding in mouse heart, gastrocnemius, and tibalis anterior muscle the presence and absence of the DELE1 mitochondrial integrated stress responses

overall_design: Transcriptomics were measured in gastrocnemius, tibialis anterior, and heat muscle from P28 mice of the following genotypes: group 1 (Chchd10+/+; Dele1+), group 2 (Chchd10+/+; Dele1-/-), group 3 (Chchd10 G58R/+; Dele1+), and group 4 (Chchd10 G58R/+; Dele1-/-) by RNA-Seq. Dele1+ indicates either Dele1+/- or Dele1+/+. Chchd10 G58R/+ mouse model has the pathogenic mutation for Autosomal Dominant Inherited Mitochondrial Myopathy (p.G58R) knocked into the equivalent position of the mouse Chchd10 gene.

first: Hsin-Pin

last: Lin

first: Xiaoping

first: Kory

last: Johnson

first: Abdel

last: Elkahloun


title: Expression data from P28 C10 G58R Dele1 mouse hearts

city: Bethesda

first: Narendra

last: Derek

address: 35 Convent Drive, Room 2A215

department: National Institute of Neurological Disorders and Stroke

institute: National Institutes of Health

summary:
Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1’s protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.
This experiment used the "Clariom S Assay, mouse" from Affymetrix/Applied Biosystems to analyze the effect of Dele1 KO in CHCHD10 G58R mouse model of mitochondrial myopathy/cardiomyopathy.

overall_design: 16 Total samples were analyzed (4 biological replicates of each of the 4 different genotypes). Genes with an FDR≤0.05 and a fold-change ≥2 were selected.


title: Expression data from P56 Ckmm-cre Tfam Dele1 mouse hearts

summary:
Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1’s protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.
This experiment used the "Clariom S Assay, mouse" from Affymetrix/Applied Biosystems to analyze the effect of Dele1 KO in Ckmm-cre Tfam (Tfam mKO) mouse model of mitochondrial myopathy/cardiomyopathy.


title: Expression data from P140 C10 S59L Dele1 mouse hearts

summary:
Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1’s protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.
This experiment used the "Clariom S Assay, mouse" from Affymetrix/Applied Biosystems to analyze the effect of Dele1 KO in CHCHD10 S59L mouse model of mitochondrial myopathy/cardiomyopathy.

overall_design: 15 Total samples were analyzed (4 biological replicates of each of the 4 different genotypes, except C10WT Dele1KO). Genes with an FDR≤0.05 and a fold-change ≥2 were selected.


title: Expression data from P28 C10 G58R Dele1 mouse gastrocnemius muscles.

summary:
Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1’s protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.
This experiment used the Clariom_S_Mouse Microarray from Affymetrix to analyze the effect of Dele1 KO in CHCHD10 G58R mouse model of mitochondrial myopathy/cardiomyopathy.

overall_design: 16 total samples were analyzed (4 biological replicates of each of the 4 different genotypes). Genes with an FDR≤0.05 and a fold-change ≥2 were selected.


title: Effect of overexpressing a dominant negative LXRalpha mutant in HepG2 cells

first: Brian

middle: Yee Hong

last: Lam

address: Box 289 Addenbookes Hospital, Hills Road

department: Metabolic Research Laboratories

institute: University of Cambridge

summary: Dominant negative mutatons in nuclear receptors can repress target gene expression, thus having stronger effects than simple loss of gene function. We sought to characterise the effects of a dominant negative mutant (p.W443R) in the hepatoma cell line HepG2 using cell lines stably transfected with wild type and mutant LXR isoforms.

overall_design: HepG2 cells stably tranfected with a construct expressing either a doxycycline inducible wildtype (WT) LXRalpha isoform or a doxycyline inducible dominant negative (p.W443R) isoform (DN) were greated with doxycycline (Dox) to induce expression from the plasmid or vehicle (DMSO) for 24 hours to induce expression of the plasmid, before treatment with either an LXR-agonist GW3965 (GW3965) or Vehicle (Vehicle) for 24 hours.

first: Sam

last: Lockhart

first: Milan

last: Muso

first: Stephen

last: O'Rahilly


title: Effect of damaging mutations in LXRalpha on the liver transcriptome

summary: Damaging mutations in LXRalpha are associated with hepatotoxicity. We generated  mice homozygous for a dominant-negative mutation in LXRalpha (LXRalpha-W441R) and discovered they develop severe liver injury when fed a Western diet, though knockout mice develop little to no liver injury. To examine this in further detail, we performed Bulk RNA-seq of livers from LXRalpha knockout and LXRalpha p.W441R knockin mice after 8 weeks of western diet (TD.88137).

overall_design: Mice carrying a dominant negative mutation in LXRalpha (p.W441R, DN)  and LXRalpha knockout (KO) mice (WT-wildype, Het-heterozygous, Hom - homozygous) were fed a western diet for 8 weeks, following which they were euthanised, livers were collected at necropsy and flash frozen. Total RNAs was extracted from the mouse livers with RNeasy kit (QIAGEN).


title: LSD1 inhibits the invasion and migration of breast cancer through exosomes

city: Chang Chun

first: Xin

last: Hu

address: 2699 Qianjin road,china

institute: Jilin university

type: Non-coding RNA profiling by high throughput sequencing

summary: Metastasis accounts for almost 90% of breast cancer-related fatalities, making it frequent malignancy and the main reason of tumor mortality globally among women. A key player in breast cancer is the histone demethylase lysine-specific demethylase 1 (LSD1). We used LSD1 knockdown MCF7 and T47D cell exosomes to treat breast cancer cells for greatly increasing the invasion and migration of breast cancer cells for evaluating the impact of LSD1 on breast cancer invasion and migration. miR-1290 expression was downregulated in LSD1 knockdown MCF7 exosomes. Furthermore, miR-1290 could control NAT1 expression by looking through the database of miR-1290 target genes. These data provide fresh insights into the biology of breast cancer therapy by demonstrating how the epigenetic factor LSD1 stimulates the breast cancer cells’ invasion and migration via controlling exosomal miRNA.

overall_design: To investigate the effect of exosomes on invasive migration of breast cancer cells after LSD1 Knockdown, we constructed LSD1 Knockdown and Rescue MCF7 cells by transient transfection and enriched their exosomes. We performed Small RNA gene expression profiling of exosomes from the constructed cells (Control, LSD1 KD, Rescue), respectively.

last: Zhang

first: Zhongyu

last: Chen

first: Benkai

last: Xin

first: Yueru

last: Shi

first: Yutong

last: Yao

first: Jingtong

last: Yang

first: Xiaoyu

last: Wang


title: Type I IFN signaling in the absence of IRGM1 promotes M. tuberculosis replication in immune cells by suppressing T cell responses

city: St. Louis

first: Christina

last: Stallings

state: MO

address: 425 South Euclid ave

institute: Washington University in Saint Louis

summary: Polymorphisms in the IRGM gene are associated with susceptibility to tuberculosis in humans. A murine ortholog of Irgm, Irgm1, is also essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. Multiple processes have been associated with IRGM1 activity that could impact the host response to Mtb infection, including roles in autophagy-mediated pathogen clearance and expansion of activated T cells. However, what IRGM1-mediated pathway is necessary to control Mtb infecion in vivo and the mechanistic basis for this control remains unknown. We dissected the contribution of IRGM1 to immune control of Mtb pathogenesis in vivo and found that Irgm1 deletion leads to higher levels of IRGM3-dependent type I interferon signaling. The increased type I interferon signaling precludes T cell expansion during Mtb infection. The absence of Mtb-specific T cell expansion in Irgm1 -/- mice results in uncontrolled Mtb infection in neutrophils and alveolar macrophages, which directly contributes to susceptibility to infection. Together, our studies reveal that IRGM1 is required to promote T cell-mediated control of Mtb infection in neutrophils, which is essential for the survival of Mtb-infected mice. These studies also uncover new ways type I interferon signaling can impact TH1 immune responses.

overall_design: To elucidate the role of immunity-related GTPase family M member 1 (IRGM1) in Mycobacterium tuberculosis (Mtb) infection using a murine model. Wild-type (WT) and IRGM1-deficient (Irgm1-/-) mice were infected with Mtb via aerosol route, ensuring an inoculum of 150-200 colony-forming units (CFU). Lungs were harvested 21 days post-infection (dpi) to assess the impact of IRGM1 on the host response to Mtb infection.For bulk RNA sequencing (RNA-seq), total RNA was isolated from the lungs under the following experimental conditions: Irgm1-/- naïve (uninfected control), WT naïve (uninfected control), Irgm1-/- at 21 dpi (infected), WT at 21 dpi (infected).

first: Sumanta

last: Kumar Naik

first: Michael

last: McNehlan

first: Yassin

last: Mreyoud

middle: L


title: SerpinA3N limits cartilage destruction in osteoarthritis by inhibiting macrophage-derives leukocyte elastase

city: Paris

first: Augustin

last: Latourte

address: Hôpital Lariboisière

institute: Université Paris Cité

summary: Inflammatory mediators such as interleukin 6 (IL-6) are known to activate catabolic responses in chondrocytes during osteoarthritis (OA). This study aimed to investigate the downstream targets of IL-6 in murine chondrocytes. RNA-sequencing was performed in murine articular chondrocytes treated with IL-6 in hypoxia (1%O2) or normoxia (21%O2). RNA-sequencing revealed that SerpinA3N is a major target of IL-6 in articular chondrocytes. The expression of SerpinA3N was increased in OA cartilage and further investigated.

overall_design: Mouse primary chondrocytes were cultured with 21% (normoxia) or 1% (hypoxia) oxygen in 10% FCS DMEM as described, then stimulated or not with IL-6 or IL-6R (100 ng/ml each) for 12 hr in serum-free DMEM. Total RNA was isolated by using TRIzol reagent (Life Technologies) and further purified (RNeasy Mini kit, Qiagen). At each step, RNA was checked on a bioanalyzer (RNA 6000 Pico Kit, Agilent) and quantified on Qubit. Purification was performed with the Ribo-Zero Magnetic Gold kit (Tebu Bio) to remove ribosomal RNA, followed by another purification with the RNeasy MinElute Cleanup Kit (Qiagen). cDNA libraries were constructed by using the TruSeq RNA Sample Prep kit (Illumina). Three barcoded cDNA libraries were made for each condition, at each time point, and RNA-seq (50-bp single-end reads) was performed on an Illumina HiSeq sequencer. Reads were aligned to the mouse genome (mm10) with Tophat2, then reads were counted with HTSeq. The DESeq2 package was used for analysis of differential gene expression, and Benjamini-Hochberg adjusted p values were calculated to adjust for multiple testing.

first: Julien

last: Daligault

first: Thierry

last: Grange

first: Pascal

last: Richette


title: Staphylococcus aureus adapts to exploit collagen-derived proline during chronic infection

city: NEW YORK

first: ALICE

last: PRINCE

department: PEDIATRICS ID

institute: COLUMBIA UNIVERSITY

summary: Staphylococcus aureus is a pulmonary pathogen associated with substantial human morbidity and mortality. As vaccines targeting virulence determinants have failed to be protective in humans, other factors are likely involved in pathogenesis. Here we analysed transcriptomic responses of human clinical isolates of S. aureus from initial and chronic infections. We observed upregulated collagenase and proline transporter gene expression in chronic infection isolates. Metabolomics of bronchiolar lavage fluid, fibroblast infection, growth assays and analysis of bacterial mutant strains showed that airway fibroblasts produce collagen during S. aureus infection. Host-adapted bacteria upregulate collagenase, which degrades collagen and releases proline. S. aureus then imports proline, which fuels oxidative metabolism via the tricarboxylic acid cycle. Proline metabolism provides host-adapted S. aureus with a metabolic benefit enabling out-competition of non-adapted strains. These data suggest that clinical settings characterized by airway repair processes and fibrosis provide a milieu that promotes S. aureus adaptation and supports infection.

overall_design:
S. aureus was grown to OD1 in LB plus/minus 100uM proline. RNA was extracted and DNAse treatment performed. RNA was sent for sequencing.
------------------
Azenta Life Sciences

first: Andreacarola

last: Urso

first: Alice

last: Prince


title: Chemotherapy promotes antigen presentation in monocytes of patients with high-grade serous ovarian carcinoma [scRNAseq]

city: Tomsk

first: Irina

last: Larionova

state: Tomsk oblast

address: Lenin Ave, 36, Tomsk, Tomsk Oblast, Russia

country: Russia

institute: Tomsk State University

summary: The major aim of our study was to uncover tumor-specific transcriptional and epigenetic changes in peripheral blood monocytes in patients with high-grade serous ovarian cancer (HGSOC). Another key point was to elucidate how chemotherapy can reprogram monocytes and how that relates to changes in other immune subpopulations in the blood. To this end, we performed single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from patients with HGSOC who underwent neoadjuvant chemotherapeutic treatment (NACT) and in treatment-naïve patients. Monocyte cluster was significantly affected by tumor-derived factors as well as by chemotherapeutic impact. Bioinformatical analysis revealed three distinct monocyte subpopulations within PBMCs based on feature gene expression – CD14.Mn.S100A8.9hi, CD14.Mn.MHC2hi and CD16.Mn subsets. The intriguing result was that NACT induced antigen presentation in monocytes by the transcriptional upregulation of MHC class II molecules, but not by epigenetic changes. Increased MHC class II gene expression was a feature observed across all three monocyte subpopulations after chemotherapy. Our data also demonstrated that chemotherapy inhibited interferon-dependent signaling pathways, but activated some TGFb-related genes. Our results can enable personalized decision regarding the necessity to systemically re-educate immune cells to prime ovarian cancer to respond to anti-cancer therapy or to improve personalized prescription of existing immunotherapy in either combination with chemotherapy or a monotherapy regimen.

overall_design: For scRNAseq profiling, peripheral whole-blood samples were collected from patients with ovarian cancer (n=6) and healthy donors (n=6). Ovarian cancer samples were divided according to treatment plan: 3 patients received platinum-based neoadjuvant chemotherapy (NACT) [carboplatin plus paclitaxel] prior to surgery and 3 patients had debulking surgery without upfront NACT. After surgery all patients received platinum/taxane-based adjuvant chemotherapy. The peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphosep, Lymphocyte Separation Media (#L0560-500, Biowest, France), 1.077 g/ml density, at 600g for 30 minutes. PBMCs were washed twice with DPBS without calcium and magnesium at 300 g for 10 minutes and counted using CytoFLEX flow cytometer (Beckman Coulter, USA). 1 000 000 PBMCs were used to prepare cell suspension for single-cell sequencing. Cells were put into 1.5 ml cryo tubes and mixed with 500 μl of X-VIVO™ 10 Medium (#180989, Lonza, Switzerland), 400 μl of fetal bovine serum (#10500064, ThermoFisher Scientific, USA), and 100 μl of dimethyl sulfoxide (#F135, Paneco, Russia). Subsequently, the cell suspension was stored in cryo container at -80°C for 48 hours, and then stored in liquid nitrogen until further experiments (but not more than 6 months). Single-cell RNA sequencing was performed on the Chromium X platform, using the 10х Genomics Chromium Next GEM Single Cell 3′ Reagent Kit v3.1. (10х Genomics, USA). Prior to library preparation, cells were counted and quality of samples was assessed. Up to 8000 cells were used for further manipulations. cDNA amplification and library construction were conducted following the manufacturer’s protocol. Sequencing was performed with the Illumina Nextseq 2000 platform (Illumina, USA).

first: Pavel

last: Iamshchikov

first: Anna

last: Kazakova

first: Militsa

last: Rakina

first: Maxim

last: Menyalo

first: Kadriia

last: Enikeeva

first: Guzel

last: Rafikova

first: Yuliya

last: Sharifyanova

first: Valentin

last: Pavlov

first: Alisa

last: Villert

first: Larisa

last: Kolomiets

first: Julia

last: Kzhyshkowska


title: Chemotherapy promotes antigen presentation in monocytes of patients with high-grade serous ovarian carcinoma [RRBS]

type: Methylation profiling by high throughput sequencing

overall_design: For RRBS profiling, CD14+ circulating monocyte samples were collected from patients with ovarian cancer (n=8) and healthy donors (n=6). Ovarian cancer samples were divided according to treatment plan: 3 patients received platinum-based neoadjuvant chemotherapy (NACT) [carboplatin plus paclitaxel] prior to surgery and 5 patients had debulking surgery without upfront NACT. After surgery all patients received platinum/taxane-based adjuvant chemotherapy. The peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphosep, Lymphocyte Separation Media (#L0560-500, Biowest, France), 1.077 g/ml density, at 600g for 30 minutes. The isolation of monocytes from PBMCs was performed using positive magnetic selection with CD14+ MACS beads (#130-050-201, Miltenyi Biotech, Germany), resulting in 90–98% monocyte purity as confirmed by flow cytometry. After monocyte isolation, the samples were washed twice with DPBS without calcium and magnesium at 300 g for 10 minutes. Subsequently, the cell precipitate was lysed using lysis buffer RLT (#79216, Qiagen, USA) and stored at -80°C until further experiments. Lysed monocytes were used for DNA isolation and reduced representation bisulfite sequencing (RRBS-Seq). DNA were extracted from lysed monocyte samples using AllPrep DNA/RNA/miRNA Universal Kit (#80224, Qiagen, USA). The quality of DNA was assessed by TapeStation 4150 automated electrophoresis system (#RRID: SCR_019393, Agilent Technology, USA). The quantity of DNA was assessed by Qubit 4 fluorometer (#RRID: SCR_018095, ThermoFisher Scientific, USA). Samples were purified using magnetic beads AMPure XP (Beckman Coulter) and concentration was controlled using Fluoroskan. Libraries were prepared using Zymo-Seq RRBS Library Kit (#D5461) and Zymo-Seq UDI Primer Plate (#D3096) (Zymo Research, USA), according to the manufacturer’s instructions. Sequencing was performed by the Illumina NovaSeq 6000 platform (Illumina, USA).


title: RNA expression in CD4+ T cells with anti-VEGFB treatment

first: Jianli

last: He

address: 280 South Chongqing Road,

department: Department of Biochemistry and Molecular Cell Biology

summary: The RNA expression in TCR-activated CD4+ T cells with anti-VEGFB treatment was analyzed by RNA-seq.

overall_design: CD4+ naive T cells were activated by anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) plus IL-2 (100 U/mL) with the treatment of anti-VEGF-B (1 μg/mL) or normal Goat IgG control (1 μg/mL) for 3 days. Then, 5×105 cells were pelleted to extract total RNA by TRIzol reagent.


title: Allele specific expression and bacterial community composition in the Drosophila gut

city: Planegg-Martinsried

first: Amanda

last: Glaser-Schmitt

address: Grosshaderner Str. 2

institute: LMU München

summary: The pervasiveness of gene expression variation and its contribution to phenotypic variation and evolution is well known. This gene expression variation is context dependent, with differences in regulatory architecture often associated with intrinsic and environmental factors, and is modulated by regulatory elements that can act in cis (linked) or in trans (unlinked) relative to the genes they affect. So far, little is known about how this genetic variation affects the evolution of regulatory architecture among closely related tissues during population divergence. To address this question, we analyzed gene expression in the midgut, hindgut, and Malpighian tubule as well as microbiome composition in the two gut tissues in four Drosophila melanogaster strains and their F1 hybrids from two divergent populations: one from the derived, European range and one from the ancestral, African range. In both the transcriptome and microbiome data, we detected extensive tissue- and genetic background-specific effects, including effects of genetic background on overall tissue specificity. Tissue-specific effects were typically stronger than genetic background-specific effects, although the two gut tissues were not more similar to each other than to the Malpighian tubules. An examination of allele specific expression revealed that, while both cis and trans effects were more tissue-specific in genes expressed differentially between populations than genes with conserved expression, trans effects were more tissue-specific than cis effects. Despite there being highly variable regulatory architecture, this observation was robust across tissues and genetic backgrounds, suggesting that the expression of trans variation can be spatially fine-tuned as well as or better than cis variation during population divergence and yielding new insights into cis and trans regulatory evolution.

overall_design: Total RNA was extracted from female 6-day-old Drosophila melanogaster midgut (20 per replicate) and hindgut (60 per replicate) samples for 4 isofemale strains (2 from a derived Swedish population and 2 from an ancestral African population) as well as F1 hybrids between the Swedish and African strains. 3 biological replicates were performed per strain or F1 hybrid (48 samples in total). High throughput RNA sequencing (RNA-seq) and amplicon sequencing were then performed using the same RNA extractions. RNA-seq was performed using Poly-A selection and 150-bp paired reads.

first: Marion

last: Lemoine

first: Martin

last: Kaltenpoth

first: John

last: Parsch


title: Unexpected nuclear hormone receptor and chromatin dynamics regulate  estrous cycle dependent gene expression [ATAC-seq]

city: RTP

first: Wendy

last: Jefferson

address: 105 T. W. Alexander Drive

institute: NIEHS

summary: Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.

overall_design: Uteri from intact, untreated CD-1 mice were collected at 2 months of age at diestrus (CoD) or estrus (CoE). RNA-seq, ATAC-seq, ER alpha ChIP-seq, PgR ChIP-seq, Hif2a ChIP-seq and HiC-seq were performed as well as RIME assay using ER alpha as the immunoprecipitating antibody.

first: Tianyuan

last: Wang

first: Carmen

last: Williams


title: Thr4 phosphorylation on RNA Pol II occurs at early transcription regulating 3’-end processing

city: Austin

first: Yan

middle: Jessie

last: Zhang

address: 100 E. 24th Street

institute: University of Texas-Austin

summary: RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on Ser2/Ser5 of the C-terminal heptad repeats is well-established, Thr4’s role remains enigmatic. Paradoxically, Thr4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and mRNA processing. Our investigation revealed that Thr4 phosphorylation detection was obstructed by flanking Ser5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via Thr4 phosphorylation, which previously were attributed to Ser2. Importantly, loss of Thr4 phosphorylation greatly reduces Ser2 phosphorylation, revealing a crosstalk between the two marks. Finally, the function analysis of the Thr4 phosphorylation highlighted its role in alternative 3’-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the CTD.

overall_design: HEK293T cells were transiently transfected with RPB1 constructs containing the wild-type CTD sequence or a T4A mutation in every repeat. These constructs also contain resistance to alpha amanitin. Alpha amanitin was added to cells for 48 hours to kill off endogenous RPB1 and total RNA was collected. The library prep strategy  isolates polyA RNA for further analysis.

first: Rosamaria

middle: Y

last: Moreno

first: Svetlana

middle: B

last: Panina


title: Expression data from adult Dele1 and OMA1 mouse brown adipose tissues (BATs).

first: Narendra

last: Derek

summary:
Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1’s protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.
This experiment used the Clariom_S_Mouse Microarray from Affymetrix/Applied Biosystems to analyze the effect of Dele1 KO and OMA1 KO under cold stress.

overall_design: 42 total samples were analyzed (7 biological replicates of each of the 3 different genotypes each in 2 different condition). Genes with an FDR≤0.05 and a fold-change ≥2 were selected.

first: Derek

last: Narendra

first: Hsin-Pin

last: Lin