title: Integrative transcriptomic analysis in human and mouse models of anaphylaxis identifies upregulation of cell movement, migration and neuroinflammatory signalling pathway

city: Ljubljana

first: Ales

last: Maver

address: Slajmerjeva 3

country: Slovenia

institute: Clinical institute of medical genetics Ljubljana Slovenia

summary: The underlying mechanisms of IgE-mediated anaphylaxis remain poorly understood. Furthermore, it remains to be determined to what extent findings from animal/mouse models reflects the pathophysiological mechanism in human. Therefore, to better characterize the mechanisms leading to potentially lethal events, analysis of global transcriptional changes (RNA-sequencing) in peripheral blood human samples in patients with anaphylaxis presenting at the emergency department and in patients with anaphylaxis during double-blind placebo-controlled food challenges (DBPCFC) to peanut, was performed. Mouse samples with different severity of IgE-mediated food-induced anaphylaxis were also included in RNA-sequencing analyses.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Emergency Department Study (ED study). We recruited 15 patients (8 female patients; age, 20-79 years) presenting with an acute episode of anaphylaxis to the ED of University Hospital Golnik, Slovenia (Table E1). Reaction severity was graded according to the Mueller criteria7. We collected blood samples at presentation to the ED and 7-14 days and/or 1 month after the anaphylactic episode (Table E1).  Peanut Allergy Study (DBPCFC study). We recruited 11 patients with peanut allergy in whom allergy was confirmed by using DBPCFCs as described in details elsewhere8. Blood samples were collected before the challenge, at the cessation of the challenge because of the onset of objective symptoms, and 2 to 5 hours after the challenge (Table E2).  Mouse oral antigen-induced IgE mediated anaphylaxis study (Mouse study). IgE-mediated anaphylaxis was induced in mice as described previously7, and whole blood samples were obtained after a challenge from 23 mice with different severity: allergic/no anaphylaxis (IgE anti-TNP+saline, 7 mice), mild anaphylaxis (IgE anti-TNP+OVA-TNP, 8 mice), severe anaphylaxis (IgE anti-TNP+OVA-TNP+IL-4 complex, 8 mice).  This study was conducted in accordance with the amended Declaration of Helsinki. Ethical approval was obtained from the Slovenian National Medical Ethics Committee (ED study) and the London Central Research Ethics Committee (DBPCFC study). All subjects provided written informed consent. All mice were maintained in a barrier facility, and animals were handled under approved protocols of the Institutional Animal Care and Use Committee from Cincinnati Children's Hospital Medical Center.

first: Matija

last: Rijavec

first: P

last: Turner

first: K

last: Hocevar

last: Kosnik

first: A

last: Yamani

first: S

middle: P

last: Hogan

last: Custovic

first: Borut

last: Peterlin

first: Peter

last: Korosec


title: RNA Sequencing Analysis of the epididymal adipose tissue from Normal Chow, HFD Control and SZ-A-treated HFD mice

status: Public on Nov 20 2022

city: Beijing

first: Qian

middle: wen

last: Sun

state: Beijing

address: 1 Xian Nong Tan Street

country: China

institute: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College

type: Expression profiling by high throughput sequencing

summary: Ramulus Mori (Sangzhi) alkaloids (SZ-A) improves lipid metabolism and adipose tissue inflammation in HFD-induced obese mice.This study compares transcriptome profiling (RNA-seq) in the epididymal adipose tissue of normal chow, high-fat diet (HFD) control and SZ-A-treated HFD mice to verify the regulatory mechanisms of SZ-A. These results demonstrated that SZ-A regulates lipid metabolism and inflammation.

overall_design: 6-week-old C57BL/6J mice were fed with normal chow or HFD for 14 weeks, and the HFD mice were treated with saline or SZ-A for another 6 weeks. We performed RNA-Sequencing of the epididymal adipose tissue of normal chow, HFD control and SZ-A-treated HFD mice (control group n=6, HFD and SZ-A groups n=7).

first: Qianwen

first: Yanfang

last: Yang

first: Yuling

last: Liu


title: Triple transgenic reprogrammable mouse model reveals no evidence of epigenetic memory in reprogrammed cells/induced pluripotent stem cells

status: Public on Nov 21 2022

city: Quebec City

first: Samer

last: Hussein

state: Quebec

address: 9 rue Mcmahon

department: Departement of medecine

country: Canada

institute: CHU de Québec - Université Laval

type: Genome binding/occupancy profiling by high throughput sequencing

type: Methylation profiling by high throughput sequencing

summary: Virtually any nucleated somatic cell can be reprogrammed to an induced pluripotent stem cell (iPSC). The process of reprogramming involves epigenetic remodelling to turn on pluripotency-associated genes and turn off lineage-specific genes. Some evidence shows that iPSC retain epigenetic marks of their cell of origin and this “epigenetic memory” influences differentiation potential, with a preference towards their cell of origin. Here, we reprogrammed proximal tubule cells (PTC) and tail tip fibroblasts (TTF), from a reprogrammable mouse, to iPSC and differentiated the iPSC to renal progenitors to understand if epigenetic memory plays a role in renal differentiation. The reprogrammable mouse model allowed us to eliminate the variability due to different cell donors and transfection of the reprogramming factors. We showed that early passage PTC iPSC and TTF iPSC expressed low levels of renal progenitor genes and high levels of pluripotency-associated genes, and the transcriptional levels of these genes were not significantly different between PTC iPSC and TTF iPSC. In agreement with this, we used ChIP-seq to demonstrate that the H3K4me3, H3K27 and H3K36me3 profiles of PTC iPSC and TTF iPSC were not different from each other for kidney developmental genes and pluripotency-associated genes. DNA methylation also demonstrated no difference between PTC iPSC and TTF iPSC. However, when the iPSC were differentiated to renal progenitors, the progenitors derived from PTC iPSC expressed higher levels of some renal progenitor genes compared to progenitors derived from TTF iPSC. This demonstrates that although PTC iPSC and TTF iPSC are epigenetically identical, there are still differences in their response and sensitivity to differentiation cues and this ultimately influences their differentiation propensity.

overall_design: iPSCs were obtained from reprogramming of either mouse proximal tubule cells (PTC) or tail tip fibroblasts (TTF) from triple transgenic reprogrammable mice. Samples were generated in duplicates from two separate mice. Epigenetic memory was assessed through Oxford Nanopore Technologies long-read DNA sequencing (lrDNAseq), treated in duplicates, followed by DNA methylation analyis using the Nanopolish software. Chromatin Immunoprecipitation sequencing (ChIP-seq) was also performed for H3K4me3, H3K36me3 and H3K27me3.

first: Gabriel

last: Khelifi

first: Ian

last: Rogers

middle: M


title: Identification of pro-metastatic pathways and molecules in mouse models with reduced expression or signaling of the insulin-like growth factor type 1 receptor

city: Newark

first: Alexander

last: Lemenze

state: New Jersey

address: 185 South Orange Ave

country: USA

institute: Rutgers

summary: We performed single-cell RNA sequencing of multiple IGF-1R loss-of function mouse mammary tumor models to uncover how IGF-1R signaling regulates intrinsic epithelial cell signaling to suppress metastasis. We identify key pathways necessary for promoting metastasis, determined IGF-1R is required to maintain a metastatic suppressive tumor microenvironment and demonstrate that attenuated epithelial IGF-1R signaling in the MMTV-Wnt1 mouse tumor model is sufficient for metastatic invasion. We further show that adherence between luminal and basal tumor cells is necessary for tumor growth at the secondary site and that reduced IGF-1R signaling in tumor epithelial cells inhibits secondary tumor epithelial cell growth due to dysregulated E-cadherin and P-cadherin and loss of cell-cell adhesion.

overall_design: To test how loss of IGF-1R alters the primary tumor phenotype, we made use of two distinct mouse models. In one model IGF-1R function is reduced through expression of an MMTV-driven dominant-negative IGF-1R transgene in the MMTV-Wnt1 (Wnt1) basal-like breast cancer tumor model (DN-Wnt1).  The second model has a mammary luminal epithelial-specific (Keratin 8-Cre) conditional Igf1r knockout  (K8iKOR) in the MMTV-Wnt1 tumor mouse line Deletion of the Igf1r in the K8iKOR line was initiated by tamoxifen injections into 8-week old female mice to avoid alterations in pubertal mammary gland development. These two lines were compared to MMTV-Wnt1 tumors with intact IGF-1R. Tumors were identified in mice between 12 weeks- 1 year of age (latency dependent on genotype and stochastic tumor development) by palpation and were harvested and dissociated for single cell RNA-Seq analysis when they reached 1.5 cm2. Three tumors (one per genotype) were subjected to initial scRNA-Seq. Additional tumors from each genotype were similarly harvested for validation studies.

first: Alison

last: Obr

first: Alex

first: Teresa

middle: L

last: Wood


title: Enhanced myogenesis through LncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter I

status: Public on Nov 19 2022

city: Baltimore

first: Supriyo

last: De

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

institute: NIA-IRP, NIH

summary: We identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. A nucleus-resident lncRNA, lncFAM was studied using chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-ms) analysis to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYPBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYPBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL promotes MYBPC2 expression transcriptionally to increase myogenesis.

overall_design: Examination of differentially expressed genes after silencing of lncFAM in human myoblasts.

first: Ming

middle: W

last: Chang

first: Jen

middle: H

first: Xiaoling

first: Jennifer

last: Martindale

first: Rachel

last: Munk

first: Dimitrios

last: Tsitsipatis

first: Poonam

middle: R

last: Pandey

first: Yulan

last: Piao

first: Krystyna

last: Mazan-Mamczarz

first: Kotb

last: Abdelmohsen

first: Gerald

last: Wilson

first: Myriam

last: Gorospe


title: Enhanced myogenesis through LncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter II

summary: We identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis.  Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process.  A nucleus-resident lncRNA, lncFAMwas studied using chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-ms) analysis to identify the molecular mechanisms whereby it might promote myogenesis.  Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYPBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYPBPC2.  These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL promotes MYBPC2 expression transcriptionally to increase myogenesis

overall_design: Validation of Illumina sequencing by Nanopore RNA-sequencing of a single human myoblast control sample

first: Ming-Wen

first: Jen-Hao

last: Yang

first: Nirad

last: Banskota

first: Brigette

last: Romero

first: Mona

last: Batish


title: The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [eCLIP]

city: Dallas

first: Nicholas

middle: K

last: Conrad

state: TX

address: 6000 Harry Hines Blvd

department: Microbiology

institute: UT Southwestern Medical Center

summary: Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5´-end of the gene.  From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.

sample_organism:
Homo sapiens
Human gammaherpesvirus 8

platform_organism:
Homo sapiens
Human gammaherpesvirus 8

overall_design: We reactivated iSLK cells latently infected with wild-type BAC16 KSHV. The virus was reactivated with sodium butyrate and doxycycline and cells were harvested 24 hours later. After lysis and immunoprecipitation with anti-PNUTS ab (A300-439A-T; Bethyl), samples were run on an SDS-PAGE, transferred to PVDF membrate, and cut from the gel from the 115-190kD region. Total lysate was run on the gel and a size-matched input (SMI) was processed in parallel. RNA was isolated from both SMI and IP samples, libraries were made for sequencing. The protocol followed that of van Nostrand et al (2016). Two independent biological replicates were performed.

first: Anne

last: Devlin

first: Olga

middle: V

last: Hunter

first: Spencer

middle: D

last: Barnes


title: Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform

city: Ghent

first: Thomas

last: Jacobs

address: Technologiepark-Zwijnaarde, 71

country: Belgium

institute: VIB Center for Plant Systems Biology

summary: Plant biotechnology needs new methods that accelerate design-build-test-learn cycles to develop new gene editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle –from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors and used it to develop an optimized LbCas12a-ABE system. Sequential improvement of five components –NLS, crRNA, LbCas12a nuclease, adenine deaminase and linker– led to a systematic, stepwise increase in ABE activity  at extrachromosomal GFP reporter (from 0.5% to 40%) and endogenous target sites. We confirmed the activity of LbCas12a-ABE in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a  nuclease and a LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.

sample_organism:
Triticum aestivum
Zea mays

platform_organism:
Triticum aestivum
Zea mays

overall_design: Measurement of editing rates at endogenous target sites in wheat or maize

first: Christophe

last: Gaillochet


title: RNA-seq analysis of Piezo1-deficient muscle satellite cells

status: Public on Nov 18 2022

city: Shizuoka

first: Yuji

last: Hara

address: 52-1 Yada,Suruga-ku

department: Department of Integrative physiology

country: Japan

institute: School of Pharmaceutical Sciences, University of Shizuoka

summary:
Muscle satellite cells (MuSCs), skeletal muscle-resident stem cells, are crucial for regeneration of myofibers. Mechanical cues are thought to be important for activation and proliferation of muscle satellite cells, but the molecular entity that senses biophysical forces in MuSCs remains to be elucidated.
In this study, we identified PIEZO1, a mechanosensitive ion channel that is activated by membrane tension, as a critical determinant for myofiber regeneration. We investigated gene profiles of Piezo1-deficient MuSCs to understand the role of PIEZO1 during myogenesis. Our results suggest that PIEZO1 governs the cytoskeletal reorganization to regulate cellular events in MuSCs (i.e., activation, cell-division, and proliferation) during skeletal muscle regeneration.

overall_design: Wild type or Piezo1-deficient satellite cells were isolated by FACS (fluorescence-activated cell sorting) as a population of VCAM1+ve, CD31-ve, CD45-ve, Sca1-ve cells. Total RNA samples were isolated from MuSCs cultured for 3 days.

first: Kotaro

last: Hirano

first: Akifumi

last: Shiomi

first: Kaori

last: Nishikawa

first: Hirofumi

last: Shintaku


title: The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [ChIP-seq]

overall_design: A viral gene reporter was integrated into the AAVS1 locus on chr19 of HCT116 cells. Pol II ChIP-seq using antibodies against RPB3 was performed on samples treated with either a nontargeting siRNA (siNT) or one of two siRNAs targeting PNUTS (siP1 or siP2). Pellets and inputs were analyzed for each sample; two biological replicates were perforemd (Rep_A and Rep_B).

first: Ashutosh

last: Shukla

first: Ivan

last: D'Orso


title: The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [RNA-seq]

overall_design: Latently infected (BAC16) iSLK cells were treated with siRNAs to PNUTS or a nontargeting control for 48 hr, then cells were induced to undergo lytic phase. RNA was harvested at time 0, 8, 24, or 48 hrs after lytic reactivation and subjected to high throughput sequencing. Three biological repliates were performed.


title: The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication

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

overall_design: Refer to individual Series


title: Enhanced myogenesis through LncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter

first: Supriyo

last: De


title: Gene expression changes in Mettl3-deleted mouse uterus on gestational day 4

city: Guangzhou

first: Ji-Long

last: Liu

address: 483 Wushan Rd

institute: South China Agricultural University

summary: We specifically deleted Mettl3 in the female reproductive tract using the Pgr-Cre driver. To portray the molecular mechanism for Mettl3 function in uterus, uterine tissues were collected from Mettl3f/f and Mettl3d/d mice on gestational day 4 and subjected to RNA-seq analysis.

overall_design: Uterine tissues were collected from Mettl3f/f and Mettl3d/d mice on gestational day 4 and subjected to RNA-seq analysis.

first: Jia-Peng

last: He


summary: Global m6A-modified mRNAs in the uterus of wild-type mouse were analyzed by using methylated RNA immunoprecipitation sequencing (MeRIP-seq)

overall_design: We investigated global m6A-modified mRNAs in the uterus of wild-type mouse by using methylated RNA immunoprecipitation sequencing (MeRIP-seq)


title: Chimeric RNA RRM2-C2orf48 plays an oncogenic role in the development of NNK-induced lung cancer

first: qiaoyuan

last: yang

address: Xinzao Town, Panyu District

institute: Guangzhou Medical University

summary: Chimeric RNAs have been used as biomarkers and therapeutic targets for multiple types of cancers. However, less attention has been paid to their mechanism of action in neoplasia. Here, we report that chimeric RNA RRM2-C2orf48 promotes 4-(methyl nitrosamine)-1-(3-pyridinyl)-1-butanone (NNK)-induced lung cancer. We found that RRM2-C2orf48 had higher expression levels in lung cancer cell lines and in lung cancer tissues from 74 patients than in normal cells and tissues, respectively. And its expression level was correlated with smoking. Next, we demonstrate that the expression level of RRM2-C2orf48 could increase after treatment with 200 mg/L NNK for 24 h. Overexpressing RRM2-C2orf48 accelerated the process of NNK-induced lung cancer and promoted cell growth. Meanwhile, suppressing RRM2-C2orf48 inhibited cell growth. Finally, we identified miR-219a-2-3p as a potential target of RRM2-C2orf48. In summary, RRM2-C2orf48 accelerated the process of NNK-induced lung cancer and miR-219a-2-3p may be involved in this process.

overall_design: Comparative gene expression profiling of RNA-seq data from BEAS-2B cells and their NNK-induced malignant transformed cells (2B-NNK)

first: Qiaoyuan

last: Yang

first: Jiazhen

last: Zhou

first: Xinchao

last: Guan

first: Enwu

last: Xu

first: Jiaxin

first: Rui

last: Xiong


title: The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [CRISPR]

first: Nicholas

last: Conrad

overall_design: We integrated the ORF59-GFP reporter into the AAVS1 safe harbor locus of HCT116 cells. Using the Brunello knockout CRISPR lentivirus library (Doench et al. 2016; Addgene #73179), we looked for genes that, upon knockout, enhanced GFP. We performed three replicates of the screen and compared the sgRNAs found in sorted cells (those with high GFP signal) to sgRNAs found in the unselected (Input) pool of cells .The sequencing data were then analyzed using MAGeCK (Li et al. 2014).

first: Anne

last: Devlin

first: Julio

middle: C

last: Ruiz

first: Ashwin

last: Govindan


title: Gene expression changes in Mettl3-deleted mouse oviduct on gestational day 3

summary: We specifically deleted Mettl3 in the female reproductive tract using the Pgr-Cre driver. To portray the molecular mechanism for Mettl3 function in oviduct, whole oviduct were collected from Mettl3f/f and Mettl3d/d mice on gestational day 3 and subjected to RNA-seq analysis.

overall_design: Oviduct tissues were collected from Mettl3f/f and Mettl3d/d mice on gestational day 3 and subjected to RNA-seq analysis.


title: siRNAs targeting a chromatin-associated RNA induce its transcriptional silencing in human cells

city: Toulouse

first: Estelle

last: Nicolas

address: 118 route de Narbonne

country: France

institute: INSERM

summary: Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, such as plants or fission yeasts. In mammals, its extent remained somewhat debated. Previous studies showed that siRNAs targeting gene promoters can induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by nascent RNA capture and RNA Pol II ChIP, we show that siRNAs targeting a chromatin-associated non-coding RNA induce its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further show that this silencing is due to base pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we show that only the wild type allele, but not the deleted allele, is silenced by the siRNA, indicating that transcriptional silencing occurs only in cis.  Finally, we demonstrate that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.

overall_design: Strand-specific RNA-seq datasets in senescent WI38 hTERT RAF1-ER  cells-treated with siRNAs against VINK vlincRNA (2 different control siRNAs, siCtle-1 and siCtle2, and 2 different VINK siRNAs, siVINK-1 and siVINK-2; 2 replicates) and in  senescent WI38 hTERT RAF1-ER in which the region (210 bp) covering the siVINK1 target sequence was deleted on the two alleles using CRISPR-Cas9 (control siRNA, siCtle-1, and VINK siRNA, siVINK-1; 2 replicates).

first: Julien

last: Ouvrard

first: Marion

last: Aguirrebengoa

first: Didier

last: Trouche


title: Maternal diet induces persistent DNA methylation changes in the muscle of beef calves

status: Public on Nov 17 2022

city: Madison

first: Francisco

last: Peñagaricano

state: Wisconsin

address: 1675 Observatory Drive

department: Animal and Dairy Sciences

institute: University of Wisconsin-Madison

summary: Maternal nutrition during pregnancy can induce epigenetic alterations in the fetal genome, such as changes in DNA methylation. It remains unclear whether these epigenetic alterations due to changes in maternal nutrition are transitory or persist over time. As such, this study was specially designed to determine whether DNA methylation modifications induced by maternal nutrition persist over time.

overall_design: Beef cows were randomly assigned to either a control or a methionine-rich diet from -30 to +90 d, relative to the beginning of the breeding season. The methylome of loin muscle from the same bull calves (n=9-10 per maternal diet) at 30 and 200 days of age were evaluated using whole-genome bisulfite sequencing.


title: CAPRIN1 links embryonic stem cell differentiation with RNA metabolism

city: Jerusalem

first: Michal

last: Rabani

address: Hebrew University, Edmond J. Safra Campus, Silberman building

country: Israel

institute: Hebrew University of Jerusalem

summary: Embryonic stem cells (ESCs) are self-renewing and pluripotent. In recent years, factors controlling pluripotency, mostly nuclear, have been identified. To reveal non-nuclear regulators of ESCs, we screened an endogenously-labelled fluorescent fusion-protein library in mouse ESCs. One of the more compelling hits was the cell cycle-associated protein, CAPRIN1. CAPRIN1, a Stress Granule (SG) component, exhibited a strikingly cyclical localization pattern in sync with mitosis, and localized to SGs, in response to stress. CAPRIN1 knockout had little effect in ESCs, but dramatically skewed differentiation and gene expression programs. RIP-seq and SLAM-seq revealed that CAPRIN1 associates with, and promotes the degradation of, thousands of RNA transcripts. CAPRIN1 interactome identified XRN2 as the likely ribonuclease. Upon early differentiation or stress, XRN2 colocalizes with CAPRIN1 inside SGs in a CAPRIN1-dependent manner. We propose that CAPRIN1 regulates an RNA degradation pathway operating during early ESC differentiation, eliminating undesired spuriously transcribed transcripts in ESCs.

overall_design: 1. RIP-seq for CAPRIN1-pulldown followed by RNA-seq of Total RNA.  2. RNA-seq in WT and Caprin1-KO partially differentiated ESCs (4d RA) treated with α-amanitin for 0, 2, 4 and 8 hrs.

first: Juliane

middle: O

last: Viegas

first: Gajendra

last: Kumar Azad

first: Eran

last: Meshorer


title: miRNAs in patients with Alström and Bardet-Biedl syndromes

city: Lodz

first: Urszula

last: Smyczynska

address: 15, Mazowiecka Street

department: Department of Biostatistics and Translational Medicine

country: Poland

institute: Medical University of Lodz

summary: We report miRNA profiling in patients with Alström syndrome (ALMS) and Bardet-Biedl syndrome (BBS). The aim of this study was to determine if the expression of circulating miRNAs in patients with ALMS and BBS differs from that in healthy and obese individuals and determine if miRNA levels correlate with metabolic tests, BMI-SDS and patient age.

overall_design: MiRNA was isolated from 2 ml of serum from blood samples collected during routine testing at patients’ medical centers. Study group included 13 ALMS patients, 7 BBS patients, 19 individuals with obesity and 23 healthy controls. Assays were performed in 2 batches (marked as A and B in sample Characteristics).

first: Agnieszka

last: Zmysłowska

last: Smyczyńska

first: Marcin

last: Stańczak

first: Krzysztof

last: Jeziorny

last: Szadkowska

first: Wojciech

last: Fendler

first: Maciej

last: Borowiec


title: Chromatin accessibility map of rice root tips at single-cell resolution [bulkATAC-seq]

first: Xiaofeng

last: Gu

address: Zhongguancun south street

institute: Chinese Academy of Agricultrual Sciences

summary: Background: Single-cell reconstruction of gene regulatory programs provides an important tool to understand the cellular phenotypic variation in complex tissues and their response to endogenous and environmental stimuli. While the single-cell transcriptomes of several plant organs have been elucidated, the underlying chromatin landscapes remain largely unknown.   Results: To comprehensively delineate chromatin accessibility during root development of an important crop, we applied single-cell ATAC-seq to 46,758 cells from rice root tips under normal and heat stress conditions. Our data revealed cell-type-specific accessibility variance across most of the major cell types and allowed us to identify sets of transcription factors which associate with accessible chromatin regions (ACRs). Using root hair differentiation as a model, we demonstrate that chromatin dynamics and gene expression dynamics during cell type differentiation correlate in pseudotime analyses. In addition to developmental trajectories, we describe chromatin responses to heat, and identify cell type specific accessibility changes to this key environmental stimulus.   Conclusions: Our work provides a framework for the integrative analysis of regulatory dynamics in an important plant organ at single-cell resolution.

overall_design: Single-cell ATACseq of rice root tips

first: Dan

last: Feng

first: Zhe

last: Liang


title: Therapeutically enabled mammalian kidney repair and regeneration

city: Singapore

first: Sonia

last: Chothani

address: 8 College Road

country: Singapore

institute: Duke-NUS school of medicine

summary: The kidney has large regenerative capacity that is impeded when injured renal tubular epithelial cells (TECs) undergo SNAI1-driven partial epithelial mesenchymal transition (pEMT). Here we investigate the role of IL11 in TEC pEMT and kidney repair. Wild-type mice with acute kidney injury (AKI) upregulate IL11 in TECs triggering an ERK/P90RSK/GSK3β axis of SNAI1 expression leading to impaired renal function, which is abrogated in Il11 null mice. In mouse models of AKI, a neutralizing IL11 antibody promotes kidney regeneration, while attenuating pEMT, fibrosis and kidney dysfunction. In TECs, TGFβ1 induces autocrine IL11/ERK-dependent pEMT leading to paracrine, IL11-mediated fibroblast activation. Mice with TEC-specific deletion of Il11ra1 are protected from pEMT, inflammation, fibrosis and renal failure. In a mouse model of chronic kidney disease, administration of anti-IL11 reverses fibrosis, regenerates kidney parenchyma and restores renal function. Therapeutic inhibition of IL11 signaling appears permissive for promoting kidney regeneration and improving kidney function.

overall_design: 24 samples

first: Anissa

last: Widjaja

first: Stuart

last: Cook


title: Chromatin accessibility map of rice root tips at single-cell resolution


title: Characterisation of iPSC derived Sensory neurons with Single Cell RNA-seq

city: Cambridge

first: William

last: Plumbly

address: Island Research Building, Cambridge Biomedical Campus

department: Clinical Neurosciences

country: United Kingdom

institute: University of Cambridge

summary: Despite the rapid development of protocols to differentiate human pluripotent stem cells (hPSCs) into neurons, those used to produce sensory neurons remain difficult to replicate and result in heterogenous populations. Moreover, there is a growing clinical burden of chronic pain conditions, highlighting a need for relevant human cellular models. This study presents a hybrid differentiation method to produce nociceptive sensory neurons from hPSCs. Cell lines harbouring an inducible NEUROG2 construct were patterned towards precursors with small molecules, followed by NEUROG2 overexpression. We profilled cells with single cell RNA sequencing, which revealed populations of nociceptors, demonstrated by the expression of key developmental and fucntional genes including volatge gated sodium and TRP channels. Overall, this study represents an optimisation of existing methods to efficiently produce nociceptive sensory neurons and provides a tool for chronic pain research and the development of treatments.

overall_design: Pools of neurons were processed and analysed at two time points in the differentiation protocol = D11 (precursors) and D40 (final time point). A population of iPSC derived cortical neurons produced with an iNeuron protocol was also analysed.

first: Emmanouil

last: Metzakopian

first: Nikolaos

last: Patikas

first: Sarah

last: Field


title: Modelling pain states with hiPSC derived sensory neurons

summary: We report the development of a sensory neuron differentiation protocol which can be used to produce populations of nociceptive sensory neurons. Following transcriptomic and electrophysiological characterisation, we used cultures to investigate molecular pain pathways by exposing cells to three pain related stimuli: heat, cold and a migraine model. Samples were taken at four time points, up to 18 hours after stimulus exposure. Bulk RNA sequencing of samples showed that for temperature models, gene expression changes were dominated by those involved in cell stress and cell death mechanisms. For the migraine model, we obseved a complex pattern of exposure-associated gene expression changes, including the up-regulation of several genes and pathways involved in neuronal and synaptic function.

overall_design: We processed 32 samples in total. Three replicates were taken for each treatment type at each exposure time point. Heat stimulus = 45degC; cold stimulus = 4degC; migraine stimulus = 10uM bradykinin, 1uM histamine, 1uM prostaglandin E2 and 500nM serotonin. Exposure times were Short = 20 mins, Mid = 50 mins, Long = 90 mins and Overnight = 18 hours.

middle: F


title: Tissue-specific imprinting and heterogeneity of natural killer cells and group 1 innate lymphoid cells

status: Public on Nov 16 2022

city: Marseille

first: Bertrand

last: Escaliere

address: 163 avenue de Luminy

institute: CIML

summary: We report here the identification, by CITE-seq and scRNA-seq, of distinct NK cells, ILC1s and intermediate subsets across 4 tissues at steady state. We found that unlike NK cells, which had a signature well conserved between organs, ILC1s exhibited a great heterogeneity depending on the tissue microenvironment, at both the phenotypic and transcriptomic levels. Finally, we performed a multidimensional comparison of these cells across tissues and identified sets of genes expressed by each related subsets.

overall_design: Single-cell RNA sequencing of murine sorted CD45+ Lin– NKp46+ NK1.1+ cells from small intestine lamina propria, liver, salivary gland and spleen using 10x Genomics.

first: Noella

last: Lopes

first: Justine

last: Galluso

last: Escalière

first: Sabrina

last: Carpentier

first: Eric

last: Vivier

first: Yann

last: Kerdilès


title: Single cell gene expression of lung cells from the lungs of PLG-nanoparticle treated mice bearing 4T1 tumors

status: Public on Nov 15 2022

city: Ann Arbor

first: Jeffrey

last: Ma

state: Michigan

address: 1600 Huron Parkway

department: Biomedical Engineering

institute: University of Michigan

summary: Poly(lactide-co-glycolide) (PLG) nanoparticles are known to modulate immune cell behavior. We performed single-cell RNA sequencing (scRNAseq) to analyze the effects of nanoparticle immunomodulation on the immune microenvironment of the lung in the 4T1 model of murine metastatic triple-negative breast cancer.

overall_design: Lungs were isolated from 4T1-tumor bearing mice receiving 5 doses of nanoparticles or saline over the course of 14 days starting immediately after tumor inoculation. Lungs were processed into a single cell suspension and analyzed by scRNAseq.

middle: A

first: Ravi

last: Raghani

first: Sophia

last: Orbach

first: Lonnie

last: Shea


title: Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run92]

city: Berlin

first: Frederik

middle: Richard

last: Heinrich

state: Berlin

address: Chariteplatz 1

department: Bioinformatics

country: Germany

summary: The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37 °C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome.

overall_design: Cartilage tissues were transferred into a hypoxia chamber (4% O2) immediately after being removed from OA patients and minced finely. Collagenase II at concentrations of 0.02%, 0.1%, 0.25%, 0.5%, 1%, and 2% was applied for 0.5, 1, 2, 4, and 18 hours to digest the minced tissue. Actinomycin D (ActD) was added to test its capacity in stabilizing the transcriptome. Cell yield, viability, cell size, and transcriptome were determined using counter chamber, flow cytometry, and RNA sequencing (RNA-seq). Collagenase II at 2% concentration released small chondrocytes from cartilage matrix during the first digestion hour and started to release large cells thereafter, reaching a complete release at 4 hours. During 4-hour digestions, collagenase II at 2% and 1% but not at lower concentrations yielded maximal release also of the large chondrocyte population. RNA-seq analysis revealed that a 4-hour digestion period with 1% or 2% collagenase II plus ActD optimally preserved the transcriptome. Thus, this study provides an isolation protocol for single chondrocytes from human articular cartilage optimized for transcriptome preservation and RNA-seq analysis.

first: Ping

last: Shen

first: Pawel

last: Durek

first: Mir-Farzin

last: Mashreghi

first: Max

last: Löhning


title: Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run54]


title: Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome

overall_design: Refer to individual Series


title: The transcriptomic landscape of elderly acute myeloid lekiemia identifies B7H3 and BANP as a new favorable signature in high-risk patients

city: Pamplona

first: Beñat

last: Ariceta

address: Av. de Pío XII, 55

department: Hemato-Oncology

country: Spain

institute: CIMA

summary: Acute myeloid leukemia (AML) in the elderly remains a clinical challenge, with a five-year overall survival less than 10%. The current ELN 2017 genetic risk classification considers cytogenetic and mutational characteristics to stratify fit AML patients into different prognostic groups. However, this classification is not validated for elderly patients treated with a non-intensive approach, and its performance seems to be suboptimal in this context. Indeed, the transcriptomic landscape of AML in the elderly has been less explored and it might help stratify this group of patients. In the current study, we analyzed the transcriptome of 224 AML patients > 65 years-old at diagnosis treated in the Spanish PETHEMA-FLUGAZA clinical trial and tried to define new prognostic groups in this population. We identified a specific transcriptomic signature for high-risk patients with mutated p53 or complex karyotype, revealing that low expression of B7H3 gene with high expression of BANP gene distinguish a subset of AML patients surviving more than 12 months. This specific signature highlights the potential of transcriptomics to identify biomarkers of good prognosis in elderly AML and recognizes these two genes as possible therapeutic targets in this malignancy.

overall_design: Gene expression analysis of RNAseq data of elderly patients with AML at diagnosis treated in the Spanish PETHEMA-FLUGAZA clinical trial.

first: Sara

last: Villar

first: Xabier

last: Agirre

first: Daniela

last: Uribarri

first: Rosa

last: Ayala

first: David

last: Martínez-Cuadrón

first: Juan

last: Bergua

first: Susana

last: Vives

first: Lorenzo

last: Algarra

first: Mar

last: Tormo

first: Pilar

last: Martínez

first: Josefina

last: Serrano

first: Catia

last: Simoes

last: Herrera

first: María

middle: J

last: Calasanz

first: Ana

last: Alfonso-Pierola

first: Bruno

last: Paiva

first: Joaquín

last: Martínez-López

first: Jesús

last: San Miguel

first: Felipe

last: Prósper

first: Pau

last: Montesinos


title: RNA-Targeting CRISPR/Cas13d System Alleviates Disease-Related Phenotypes in Pre-clinical Models of Huntington’s Disease (Mouse).

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

summary: Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Although the pathogenesis of HD remains complex, the CAG-expanded (CAGEX) HTT mRNA and protein ultimately causes disease through a toxic gain-of-function mechanism. As the reduction of pathogenic mutant HTT mRNA is beneficial as a treatment, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d/CAGEX) that eliminates toxic CAGEX RNA in HD patient-derived fibroblasts and iPSC-derived striatal neurons. We show that intrastriatal delivery of Cas13d/CAGEX via a single adeno-associated viral vector, serotype 9 (AAV9) mediates significant and selective reduction of mutant HTT mRNA and protein levels within the striatum of heterozygous zQ175 mice, an established mouse model of HD. Moreover, the reduction of mutant HTT mRNA renders a sustained partial reversal of HD phenotypes, including improved motor coordination, attenuated striatal atrophy, and reduction of mutant HTT protein aggregates. Importantly, phenotypic improvements were durable for at least 8 months without gross or behavioral adverse effects, and with minimal off-target interactions of Cas13d/CAGEX in the mouse transcriptome. Taken together, we demonstrate a proof-of-principle of an RNA-targeting CRISPR/Cas13d system as a therapeutic approach for HD, a strategy with broad implications for the treatment of other dominantly inherited neurodegenerative disorders.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HD and WT mice treated with Cas13d/NT or Cas13d/CAGEX

first: Kathryn

last: Morelli

first: Maya

last: Gosztyla


title: RNA-Targeting CRISPR/Cas13d System Alleviates Disease-Related Phenotypes in Pre-clinical Models of Huntington’s Disease (Human).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HD and control iPSC-derived neurons treated with Cad13/NT or Cas13d/CAGEX


title: Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription

city: Pasadena

first: Isabel

last: Goronzy

address: 1200 E California Blvd

department: Biology and Biological Engineering

institute: California Institute of Technology

summary: The genome is highly organized, yet whether DNA structures, such as A/B compartments, represent locations that are permissive/impermissive for transcription or whether these structures are simply correlated with locations of active/inactive genes remains unknown. This is because current methods cannot simultaneously measure DNA organization and transcription. Recently, we developed RNA&DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of DNA and RNA. Here, we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and their spatial contacts within the nucleus. We find that transcription occurs within B compartments – with multiple, simultaneously active genes colocalizing within them – and at genes localized proximal to nucleoli. Together, these results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.

overall_design: RD-SPRITE was used to simultaneously map genomic structure (DNA-DNA interactions) and nascent transcription (RNA-RNA interactions and RNA-DNA interactions).

first: Sofia

last: Quinodoz

first: Mitchell

last: Guttman


title: RNA-Targeting CRISPR/Cas13d System Alleviates Disease-Related Phenotypes in Pre-clinical Models of Huntington’s Disease

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


title: Immunoediting instructs tumor metabolic reprogramming to support immune evasion

city: Epalinges

first: Ping-Chih

last: Ho

address: Ch. des Boveresses 155

country: Switzerland

institute: Ludwig Cancer Institute, UNIL

type: Genome variation profiling by high throughput sequencing


title: B cell fate mapping reveals their contribution to the memory immune response against helminths

city: Erlangen

first: Simon

last: Schaefer

state: Bavaria

address: Glueckstr. 6

department: Genetics

institute: FAU Erlangen

summary: To identify B cell memory in the response against helminths we used a fate-mapping aproach using inducible Cre to label B cells participating in the immune response against Nippostrongylus brasiliensis. We transferred our marked cells into wild-type recipient mice and compared the cells before and after the transfer.

overall_design:
Immunoglobulin specific repertoire analysis of murine B cell receptor heavy chain
Samples from pooled from 3-4 mice, technical controls were included

first: Paul

last: Haase

last: Schäfer

first: Roman

last: Gerlach

first: Thomas

last: Winkler

first: David

last: Voehringer


title: CRISPR-based targeted haplotype-resolved assembly of a megabase region

city: Shanghai

first: Yun

last: Liu

address: 130 Dong An Road

institute: Fudan University


title: CHD2 maintains spermatogonial self-renewal by promoting chromatin activity and mRNA stability

city: Changsha

first: Ziqian

last: Min

state: Hunan

address: Tianma road

institute: Hunan University

summary: Chromodomain Helicase DNA-binding Domain 2 (CHD2), as a chromatin remodeling factor, was shown to be involved in the regulation of gene expression in embryonic development, neurodevelopment and myelopoiesis. However, its role in male germ cell development has not been elucidated. Here, we confirmed that CHD2 is abundantly expressed throughout the male germ cells with the highest expression in the spermatocytes of meiosis I. By constructing a heterozygous gene knockout mouse model of Chd2 (Chd2+/-), we demonstrated that CHD2 haploinsufficiency resulted in testicular developmental delay and increased rate of abnormal sperm in mice. DNA damage repair, synapsis and cell proliferation during spermatogenesis are impaired in Chd2+/- mice. In vitro experiments in C18-4 and GC-1 spg cells showed that CHD2 knockdown inhibits spermatogonial self-renewal. Mechanically, CHD2 maintained the enrichment of H3K4me3 in Ccnb1 and Ccnd2 promoter consequently promoting the transcription of Ccnb1 and Ccnd2. In addition, by interacting with cleavage stimulation factor CSTF3, CHD2 binds Oct4, Plzf mRNA and upregulates the expression of OCT4 and PLZF by improving mRNA stability. This is the first time to reveal the role and mechanism of CHD2 in maintaining spermatogonial self-renewal by promoting chromatin activity and mRNA stability in spermatogenesis.

overall_design: Testicular tissues mRNA profiles of Chd2+/+ and Chd2+/- mice

first: Huan

last: Xin


title: CRISPR-based targeted haplotype-resolved assembly of a megabase region [WGBS]

summary: Constructing high-quality haplotype-resolved genome assemblies has substantially improved the ability to detect and characterize genetic variants. A targeted approach providing readily access to the rich information from haplotype-resolved genome assemblies will be appealing to groups of basic researchers and medical scientists focused on specific genomic regions. Here, using the 4.5 megabase, notoriously difficult-to-assemble major histocompatibility complex (MHC) region as an example, we demonstrated an approach to construct haplotype-resolved assembly of the targeted genomic region with the CRISPR-based enrichment. Compared to the results from haplotype-resolved genome assembly, our targeted approach achieved comparable completeness and accuracy with reduced computing complexity, sequencing cost, as well as the amount of starting materials. Moreover, using the targeted assembled personal MHC haplotypes as the reference both improves the quantification accuracy for sequencing data and enables allele-specific functional genomics analyses of the MHC region. Given its highly efficient use of resources, our approach can greatly facilitate population genetic studies of targeted regions, and may pave a new way to elucidate the molecular mechanisms in disease etiology.

overall_design: 10x linked-reads, CCS reads and WGBS for Enriched MHC, RNA-seq sequencing and Illumina Human Methylation 850 BeadChip for NA12878

first: Taotao

last: Li

first: Duo

last: Du

first: Dandan

last: Zhang


title: Cell type-specific changes in Wnt signaling and neuronal differentiation in the developing mouse cortex after prenatal alcohol exposure during neurogenesis

city: Rockville

first: Danielle

last: Sambo

state: MD

address: 5625 Fishers Lane

institute: National Institute on Alcohol Abuse and Alcoholism

summary: Fetal Alcohol Spectrum Disorder (FASD) encompasses an array of effects of prenatal alcohol exposure (PAE), including physical abnormalities and cognitive and behavioral deficits. Disruptions of cortical development have been implicated in multiple PAE studies, with deficits including decreased progenitor proliferation, disrupted neuronal differentiation, aberrant radial migration of pyramidal neurons, and decreased cortical thickness. While several mechanisms of alcohol teratogenicity have been explored, how specific cell types in the brain at different developmental time points may be differentially affected by PAE is still poorly understood. In this study, we used single nucleus RNA sequencing (snRNAseq) to investigate whether moderate PAE from neurulation through peak cortical neurogenesis induces cell type-specific transcriptomic changes in the developing murine brain. Cluster analysis identified 25 neuronal cell types, including subtypes of neural epithelial cells (NECs), radial glial cells (RGCs), intermediate progenitor cells (IPCs), projection neurons, and interneurons. Only Wnt-expressing NECs showed a significant decrease in the percentage of cells after PAE, with no cell types showing PAE-induced apoptosis as measured by caspase expression. Cell cycle analysis revealed only a subtype of RGCs expressing the downstream Wnt signaling transcription factor Tcf7l2 had a decreased percentage of cells in the G2/M phase of the cell cycle, suggesting decreased proliferation in this RGC subtype and further implicating disrupted Wnt signaling after PAE at this early developmental timepoint. An increased pseudotime score in IPC and projection neuron cell types indicated that PAE led to increased or premature differentiation of these cells. Biological processes affected by PAE included the upregulation of pathways related to synaptic activity and neuronal differentiation and downregulation of pathways related to chromosome structure and the cell cycle. Several cell types showed a decrease in Wnt signaling pathways, with several genes related to Wnt signaling altered by PAE in multiple cell types. As Wnt has been shown to promote proliferation and inhibit differentiation at earlier stages in development, the downregulation of Wnt signaling may have resulted in premature neuronal maturation of projection neurons and their intermediate progenitors. Overall, these findings provide further insight into the cell type-specific effects of PAE during early corticogenesis.

overall_design: 2 pregnant mice per treatment were treated with either saline or EtOH (S1, S2, E1, E2). Embryos were harvested, and embryo brains for snRNAseq were selected at random (2 from S1 and E1, 4 from S2 and E2 for a total of 6 per treatment). Nuclei isolation and library preparaiton was performed over 2 batches (S1+E1 and S2+E2). All libraries were sequenced on the same chip.

first: Chiraag

last: Gohel

first: Qiaoping

last: Yuan

first: Gauthaman

last: Sukumar

first: Camille

last: Alba

first: Clifton

last: Dalgard

last: Goldman


title: A SRC-3 gene signature in predecidual T-HESC

city: HOUSTON

first: Vineet

middle: Kumar

last: Maurya

address: ONE BAYLOR PLAZA

department: Molecular & Cell Biology

institute: BAYLOR COLLEGE OF MEDICINE

summary: Steroid Receptor Coactivator-3 (SRC-3) knockdown in human endometrial stromal cells (HESCs) blocks their decidualization. This result provides translational support for recent studies in the mouse in which conditional SRC-3 knockout in progesterone receptor-positive cells of the endometrium results in early pregnancy loss due to a defect in normal decidualization. RNAseq was performed on the telomerase-immortalised endometrial stromal cell line T-HESC (CRL-4003; American Type Culture Collection) with or without SRC-3 knockdown to identify the transcriptome that is dependent on SRC-3 prior to hormone-dependent HESC decidualization.

overall_design: SRC-3 was silenced  in T-HESC cells by siRNA. Total RNA was isolated from control and SRC-3 silenced cells and subjected to the RNAseq assay.

first: San-Pin

last: Wu

first: Tianyuan

last: Wang

first: Francesco

last: DeMayo

first: John

last: Lydon


title: Single-cell RNA-seq of the mouse Peyer's patches blood endothelium

status: Public on Nov 14 2022

city: Stanford

first: Menglan

last: Xiang

address: 450 Serra Mall

institute: Stanford University

summary: Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.

overall_design: Endothelial cells from the Peyer’s patches of EC-iCOUPOE and control mice were isolated 14-19 days post tamoxifen treatment. Single cell gene expression was assayed using the 10x Chromium v3 platform.

first: Thanh

middle: T

last: Dinh

first: Kevin

last: Brulois

first: Junliang

last: Pan

first: Eugene

last: Butcher


title: RNA-seq analysis of gene expression in normoxic and hypoxic cancer cell lines

city: Oxford

first: David

middle: Robert

last: Mole

address: Roosevelt Drive, Headington

department: Nuffield Department of Medicine

institute: University of Oxford

summary: Pan-genomic analysis of poly-adenylated transcript levels in normoxic and hypoxic cancer cell lines

overall_design: PolyA RNA-seq analysis of  gene expression in PC3, T47D, A549 and HCT-116 incubated in 21% oxygen and 0.5% oxygen for 16 hours

first: Olivia

last: Lombardi

first: Silvia

last: Halim


title: Chromatin accessibility map of rice root tips at single-cell resolution [scATAC-seq]

city: Beijing

first: Xiaofeng

last: Gu

address: Zhongguancun south street

summary: Background: Single-cell reconstruction of gene regulatory programs provides an important tool to understand the cellular phenotypic variation in complex tissues and their response to endogenous and environmental stimuli. While the single-cell transcriptomes of several plant organs have been elucidated, the underlying chromatin landscapes remain largely unknown.   Results: To comprehensively delineate chromatin accessibility during root development of an important crop, we applied single-cell ATAC-seq to 46,758 cells from rice root tips under normal and heat stress conditions. Our data revealed cell-type-specific accessibility variance across most of the major cell types and allowed us to identify sets of transcription factors which associate with accessible chromatin regions (ACRs). Using root hair differentiation as a model, we demonstrate that chromatin dynamics and gene expression dynamics during cell type differentiation correlate in pseudotime analyses. In addition to developmental trajectories, we describe chromatin responses to heat, and identify cell type specific accessibility changes to this key environmental stimulus.   Conclusions: Our work provides a framework for the integrative analysis of regulatory dynamics in an important plant organ at single-cell resolution.

overall_design: Single-cell ATACseq of rice root tips

first: Dan

last: Feng

first: Zhe

last: Liang


title: HIF-1a and HIF-2a ChIP-seq and RNA-seq of hypoxic cancer cell lines


title: ChIP-seq analysis of HIF-1a and HIF-2a binding in hypoxic cancer cell lines

summary: Pan-genomic analysis of hypoxic HIF-1a and HIF-2a binding by ChIP-seq in cancer cell lines

overall_design: Genome-wide assays of HIF binding in PC3, T47D, A549 and HCT-116 in 0.5% hypoxia for 16 hours


title: Identifying the differentially expressed circRNAs and mRNAs in the subacute stage of spinal cord injury in mice by RNA-sequencing

first: Yongjin

last: Li

state: China

address: Department of Orthopedics, Xuanwu Hospital, Capital Medical University, No. 45 Changchun Street, Xicheng District, Beijing

department: Department of Orthopaedics

institute: Department of Orthopaedics, Xuanwu Hospital, Capital Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary: Spinal cord injury (SCI) is a pathological condition that leading to serious nerve damage、disability and even death. Some scholars have divided the pathophysiology of SCI into primary mechanism and secondary mechanism. Secondary SCI can be divided into acute, subacute, and chronic stages, with the subacute stage lasting 2 days to 2 weeks after SCI. The subacute stage of SCI is a potentially critical time point for further study of SCI repair, which may play a significant role in the secondary SCI. Increasing evidence have revealed that circular RNAs (circRNAs) and mRNA are widely involved in the regulation of the pathological process of neurological diseases by sponging microRNAs (miRNAs). Nevertheless, the potential biological functions and regulatory mechanisms of circRNAs in the subacute stage of SCI remain unclear. We analyzed the expression and regulatory patterns of circRNAs and mRNAs in SCI mice models using RNA-sequencing. A total of 24 circRNAs and 372 mRNAs were identified to be differentially expressed, and found that circRNA02778, circRNA02370, and Ctsd were top up-regulated as well as circRNA05219 and Huwe1 were top down-regulated after SCI. Meanwhile, we identified multiple immune-related genes (Ptprc, Fcgr2b, Ccl2, Tlr7, Cxcl10, Cd28, Il10ra, Cd68, and Tlr3) related to SCI and found they were significantly upregulated after SCI, but the specific mechanisms by which they regulate SCI by mediating neuronal inflammatory and immune responses need to be further explored. These findings will contribute to elucidate the pathophysiology of SCI and provide effective therapeutic targets for SCI patients.

overall_design: CircRNAs and mRNAs expression profiling analysis of RNA-sequencing data generated from six C57BL/6 male mice samples under two conditions: control (n = 3), and spinal cord injury (n = 3). Three control mice underwent a laminectomy at the T10 level without injury to the spinal cord, the other three experimental mice were placed on the NYU IMPACTOR MODEL-II spinal cord hitter platform after laminectomy at the T10 level, and the T10 spinal cord was hit with a hitting head weighing 10 grams to construct a SCI model. All the six mice were killed on the 4th day after surgery, and the spinal cord tissue about 4 cm long around the T10 level were collected for high-throughput RNA-sequencing.

first: Guowang

first: Xiaolong

last: Chen

first: Shibao

last: Lu


title: TREM2hi Resident Macrophages Protect the Septic Heart by Maintaining Ccardiomyocytes Homeostasis

city: Hangzhou

first: Yang

last: Wang

state: Zhejiang

address: 866 Yuhangtang Rd

institute: Zhejiang University

summary: Sepsis-induced cardiomyopathy (SICM) is common in septic patients with a high mortality and displays an abnormal immune response. Owing to cellular heterogeneity, understanding the roles of immune cell subsets in SICM has been challenging. By combining scRNA-seq with fate-mapping, we identified a unique CD163+RETNLA+ subpopulation (Mac1) of cardiac resident macrophages, which underwent self-renewal during sepsis. The restoration of Mac1 numbers was associated with the improvement of cardiac function. The Mac1 subpopulation had distinct transcriptomic signatures enriched in endocytosis and showed high expression of TREM2 (TREM2hi). In addition, we observed that TREM2hi Mac1 cells actively scavenged cardiomyocyte-ejected dysfunctional mitochondria. Trem2 deficiency in macrophages impaired self-renewal capability of the Mac1 subpopulation and consequently resulted in defective elimination of damaged mitochondria, excessive inflammatory response in cardiac tissue, exacerbated cardiac dysfunction and decreased survival. Intrapericardial administration of TREM2hi Mac1 cells prevented SICM. Our findings suggest that the modulation of TREM2hi Mac1 cells could be a potential therapeutic strategy for SICM.

overall_design: Single-cell transcriptomic data are presented from 10 samples. Each sample contains a mixture of cardiac cells from 3 or 4 mice. 4 samples were obtained from WT mice at steady state and 3,7,21 days after CLP. The other 6 samples were from Trem2-/- mice and WT littermate controls at steady state and 3,7 days after CLP, respectively.

first: Kai

last: Zhang

first: Xiangming

last: Fang

first: Shiyu

first: Jiali

last: Mao

first: Yue

last: Jin

first: Hui

last: Ye

first: Yan

first: Xiwang

last: Liu

first: Chenchen

last: Gong

first: Xuejun

last: Cheng

first: Xiaoli

last: Huang

first: Andreas

last: Hoeft

first: Qixing

first: Xuekun


title: Hemocytes and fat body cells, the only professional immune cell types in Drosophila, show strikingly different responses to systemic infection

city: Kiel

first: Vaibhvi

last: Vaibhvi

state: Germany (DEU)

address: Am Botanischen Garten 3-9

department: Zoology

institute: University of Kiel

summary:
The fruit fly Drosophila is an excellent model to study the response of different Q7
immunocompetent organs during systemic infection. In the present study, we
intended to test the hypothesis that the only professional immune organs of
the fly, the fat body and hemocytes, show substantial similarities in their
responses to systemic infection. However, comprehensive transcriptome
analysis of isolated organs revealed highly divergent transcript signatures,
with the few commonly regulated genes encoding mainly classical immune
effectors from the antimicrobial peptide family. The fat body and the
hemocytes each have specific reactions that are not present in the other
organ. Fat body-specific responses comprised those enabling an improved
peptide synthesis and export. This reaction is accompanied by transcriptomic
shifts enabling the use of the energy resources of the fat body more efficiently.
Hemocytes, on the other hand, showed enhanced signatures related to
phagocytosis. Comparing immune-induced signatures of both cell types with
those of whole-body responses showed only a minimal correspondence,
mostly restricted again to antimicrobial peptide genes. In summary, the two
major immunocompetent cell types of Drosophila show highly specific
responses to infection, which are closely linked to the primary function of
the respective organ in the landscape of the systemic immune response.

overall_design: Comparative transcriptomic analysis of adult Drosophila fat body and hemocytes after sterile injury and bacterial infections

first: Sven

last: Künzel

first: Thomas

last: Roeder


title: Single-cell RNA sequencing of myeloid cells, hematopoietic stem and progenitor cells, and mesenchymal stromal cells isolated from bone marrow of C57BL/6J WT and IL-1rn-KO mice

city: Oslo

first: Lorena

last: Arranz

address: Forskningsveien 1

department: CRESCO - Centre for Embryology and Healthy Development

country: Norway

institute: University of Oslo

summary: Interleukin-1β (IL-1β) drives hematopoietic stem cell (HSC) differentiation into the myeloid lineage, and enhanced IL-1β signaling plays a key role in hematological malignancies. However, little is known on the role of its endogenous regulatory cytokine, IL-1 receptor antagonist (IL-1rn), on both healthy and malignant hematopoiesis. Here, characterize transcriptomic changes at the single cell level in myeloid cells, hematopoietic stem and progenitor cells, and CD63+ mesenchymal stromal cells between C57BL/6J and IL-1rn-KO mice.

overall_design: We profiled bone marrow (BM) hematopoietic and non-hematopoietic cells by scRNA-seq from females C57BL6/J WT (n = 1) and IL-1RN KO (n = 1) aged 13-15 weeks. We sorted myeloid cells as CD11b+, hematopoietic stem and progenitor cells (HSPC) as Lineage- Sca-1+ cKit+ (LSK) and mesenchymal stromal cells (MSC) as CD45- CD31- Ter119- CD63+ by FACS. Sorted CD63+ MSCs were pooled from two animals to reach the desired number of cells for scRNA-seq, after separation of one femur and one tibia from one of the mice dedicated to CD11b+ and LSK cell sorting. Cells were counted and their viability checked using the Countess III cell counter (Thermofisher). Single cells were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics). Each cell suspension was loaded into one port of a Chromium Next GEM Chip G (10x Genomics) with a target output of 4000-10000 cells.

first: Itziar

last: Cossio

first: Andrea

last: Rubio

first: Alberto

last: Benguria

first: Vincent

last: Cuminetti

first: Enrique

last: Vazquez

first: Ana

last: Dopazo

first: Andres

last: Hidalgo


title: Expression data from regenerating human airway epithelium in vitro.

city: Pittsburgh

first: Kambez

middle: Hajipouran

last: Benam

state: Pennsylvania

address: 200 Lothrop St, Rm E1144

institute: University of Pittsburgh

summary:
To identify transcriptomic signature of human airway epithelium as it undergoes full differentiation into mucociliated epithelial cells under air-liquid interface.
We used microarrays to detail the global gene expression pattern from day 0 through day 28 following air-liquid interface in human airway epithelial cells and identified distinct clusters of gene expression during this process.

overall_design: In a time-course study, human small airway epithelial cells (hSAEpCs) were cultured in vitro and at successive points (days 0, 2, 4, 6, 8, 10, 14, 21 and 18 [labeled D0, D2, D4, and so forth] following air-liquid interface [ALI]) were lysed in situ, their RNA were extracted and hybridized on Affymetrix microarrays.  Our objective here was to identify molecular signatures, as gene expression states for individual genes, that correlate with stage of hSAEpCs differentiation at various days post-ALI.


title: PEAC-seq: an unbiased in vivo CRISPR off-target detection technique

first: Lijia

last: Ma

address: Shilongshan Road No.18, Cloud Town, Xihu District

department: School of Life Sciences

institute: Westlake university

summary: Development of PEAC-seq for unbiased in vivo off-target detection

overall_design: We conducted PEAC-seq to detect off-targets for two different on-target sites (Pnpla3 and Pcsk9). Each site had 2 replicates (embryos), and the wildtype adult mouse genomic DNA was set as control.


title: Multiomics analysis of a DNAH5-mutated PCD organoid model revealed the key role of the TGF-β/BMP and Notch pathways in epithelial differentiation and the immune response in DNAH5-mutated patients

status: Public on Nov 12 2022

city: chengdu

first: Wenhao

last: Yang

address: Renmin Rode 17

institute: West China Second University Hospital, Sichuan University

summary: Dynein axonemal heavy chain 5 (DNAH5) is the most mutated gene in primary ciliary dyskinesia (PCD), leading to abnormal cilia ultrastructure and function. Few studies have revealed the genetic characteristics and pathogenetic mechanisms of PCD caused by DNAH5 mutation. Here, we established a child PCD airway organoid directly from the bronchoscopic biopsy of a patient with DNAH5 mutation. We found abnormal ciliary function and a decreased immune response caused by DNAH5 mutation through single-cell RNA sequencing (scRNA-seq).

overall_design: The child PCD airway organoids, directly from the bronchoscopic biopsy of a patient with DNAH5 mutation, were constructed according to the airway organoid culture protocol. The normal airway organoids were derived from the suspected patient who excluded the PCD in follow-up examinations,The airway organoid with DNAH5 mutation and normal airway organoid pellets, derived from the biopsy, were resuspended with 1 mL of TrypLE 1× (Gibco, A1217701), separately, incubated for 15-30 min at 37 °C, and mechanically sheared with 1-ml pipettes. After washing twice with AdDF+++, the cell pellets were filtered with a 40-μm strainer. Cellular suspensions (∼6000 cells) were loaded on a Chromium Single Cell Instrument (10X Genomics) to generate single-cell GEMs. Sample processing and single-cell RNA-seq library preparation were performed using Chromium Single Cell 3′ v2 reagents. Sequencing was performed on an Illumina NextSeq500 following the manufacturer’s instructions.

first: Hanmin

last: Liu


title: Slowest possible replicative life at frigid temperatures for yeast

status: Public on Nov 13 2022

city: Delft

first: Diederik

last: Laman Trip

address: Van der Maasweg 9

department: Bionanoscience

country: Netherlands

institute: Delft University of Technology

summary: Microbes and animals often inhabit frigid environments. Little is known about the design principles that govern a cell’s slow progression in life – how it proliferates, ages and dies – and potential constraints on slowly progressing life. Challenges lie in monitoring slow, intracellular processes and quantifying their combined effects on cells. Here we use budding yeast to quantitatively establish principles that govern cell proliferation and survival at near-freezing temperatures (0C – 5C). By monitoring individual cells for months at near-freezing temperatures, we found small numbers of cells dividing with weeks-long doubling times, while most others died. We found that cells die and cannot divide primarily from having high levels of Reactive Oxygen Species (ROS). Reducing ROS levels or enabling G1 exit with abundant ROS largely prevented deaths and enabled cell divisions with shortened doubling times. These perturbations yielded a comprehensive view of cell's life at near-freezing temperatures that integrated our measurements of single cells’ lifespans, days-to-months-long cell-cycle durations, protein-synthesis rates, and genome-wide transcription rates. With a mathematical model, this systems-level view revealed that protein-synthesis dynamics becomes more rate-limiting for cell duplication while mRNA-synthesis dynamics becomes less rate-limiting as temperature approaches 0C. Consequently, the protein-synthesis rate and ROS together establish low-speed and high-speed limits for life at near-freezing temperatures: shortest and longest possible doubling times. Progressing through cell cycle more slowly than the low-speed limit ensures death. This work establishes a quantitative, systems-level foundation for engineering organisms to live in frigid environments and elucidating fundamental limits to slowing down life.

overall_design: Data represent time-series to determine genome-wide transcription rates for various conditions. Wild-type yeast was incubated at 1.0C, 5.0C, or 30.0C with or without 250uM GSH added (3 biological replicates for each condition). The synthetic nucleotide 4-thiouracil ("4tU") was added to medium after two weeks of incubation. The abundance of 4tU-labelled mRNA was then quantified over time using next-generation sequencing (“4tU-seq”). The series has 6 timepoints at 1C, 8 time points at 5C, and 5 time points at 30C. Time point 0 is the reference for each condition. A steady-state sample was also included for each condition. All samples contained the same amount of 4tU-labelled spike-in RNA from Schizosaccharomyces pombe to normalize samples over time and across conditions.

middle: S

first: Théo

last: Maire

first: Hyun

last: Youk


title: Loss of Linker Histone H1 in the Maternal Genome Influences DEMETER-Mediated Demethylation and Affects the Endosperm DNA Methylation Landscape (RNA-Seq)

status: Public on Nov 11 2022

city: Kannapolis

first: Tzung-Fu

last: Hsieh

state: NC

address: Tzung-Fu Hsieh, 600 Laureate Way

department: Plant and Microbial Biology

institute: North Carolina State University

summary: TheArabidopsisDEMETER (DME) DNA glycosylase demethylates the central cell genome prior to fertilization. This epigenetic reconfiguration of the female gamete companion cell establishes gene imprinting in the endosperm and is essential for seed viability.  DME demethylates small and genic-flanking transposons as well as intergenic and heterochromatin sequences, but how DME is recruited to these target loci remains unknown. H1.2 was identified as a DME-interacting protein in a yeast two-hybrid screen, and maternal genome H1 loss affects DNA methylation and expression of selected imprinted genes in the endosperm. Yet, the extent to which how H1 influences DME demethylation and gene imprinting in theArabidopsisendosperm has not been investigated. Here, we showed that unlike in the vegetative cell, both canonical histone H1 variants are present in the central cell. Our endosperm methylome analysis revealed that without the maternal linker histones, DME-mediated demethylation is facilitated, particularly in the heterochromatin regions, indicating that H1-containing nucleosomes are barriers for DME demethylation. Loss of H1 in the maternal genome has a very limited effect on gene transcription or gene imprinting regulation in the endosperm; however, it variably influences euchromatin TE methylation and causes a slight hypermethylation and a reduced expression in selected imprinted genes. We conclude that loss of maternal H1 indirectly influences DME-mediated demethylation and endosperm DNA methylation landscape but does not appear to affect endosperm gene transcription and overall imprinting regulation.

overall_design: RNA sequencing using wt (Col x Ler) and h1-hybrid (h1 triple x Ler) F1 embryo and endosperm (3 bio replicates for each genotype and tissue) were performed to examine the effect of maternal H1 loss on embryo and endosperm transcriptomes.

first: Qiang

last: Han

first: Yu-Hung

last: Hung

first: Wenyan

last: Xiao


title: Staphylococcus aureus infection induces the production of the neutrophil chemoattractants CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts

city: Charlotte

first: Morgan

middle: Brittany

last: Johnson

state: North Carolina

address: 9201 University City BLVD

department: Biological Sciences

institute: University of North Carolina at Charlotte

summary: Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage.  Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection, and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge.  In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines, CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in a murine model of post-traumatic staphylococcal osteomyelitis.  RNA-Seq gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells.  Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner.  Furthermore, we have confirmed the ability of soluble osteoblast-derived chemoattractants to elicit the migration of a neutrophil-like cell line.  As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for uninfected and S. aureus infected murine osteoblasts

first: Sophie

middle: E

last: Sipprell

first: M

middle: B

first: Whitney

last: Leach

first: Samantha

last: Suptela

first: Ian

last: Marriott


title: Loss of Linker Histone H1 in the Maternal Genome Influences DEMETER-Mediated Demethylation and Affects the Endosperm DNA Methylation Landscape (Bisulfite-Seq)

summary: The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the central cell genome prior to fertilization. This epigenetic reconfiguration of the female gamete companion cell establishes gene imprinting in the endosperm and is essential for seed viability.  DME demethylates small and genic-flanking transposons as well as intergenic and heterochromatin sequences, but how DME is recruited to these target loci remains unknown. H1.2 was identified as a DME-interacting protein in a yeast two-hybrid screen, and maternal genome H1 loss affects DNA methylation and expression of selected imprinted genes in the endosperm. Yet, the extent to which how H1 influences DME demethylation and gene imprinting in the Arabidopsis endosperm has not been investigated. Here, we showed that unlike in the vegetative cell, both canonical histone H1 variants are present in the central cell. Our endosperm methylome analysis revealed that without the maternal linker histones, DME-mediated demethylation is facilitated, particularly in the heterochromatin regions, indicating that H1-containing nucleosomes are barriers for DME demethylation. Loss of H1 in the maternal genome has a very limited effect on gene transcription or gene imprinting regulation in the endosperm; however, it variably influences euchromatin TE methylation and causes a slight hypermethylation and a reduced expression in selected imprinted genes. We conclude that loss of maternal H1 indirectly influences DME-mediated demethylation and endosperm DNA methylation landscape but does not appear to affect endosperm gene transcription and overall imprinting regulation.

overall_design: Bisulfite sequencing using h1-hybrid (h1 triple x Ler) F1 embryo and endosperm (3 bio replicates for each tissue) was performed to examine the effect of maternal H1 loss on embryo and endosperm DNA methylation.


title: RNA-seq analysis of in planta condition-dependent genes in Xanthomonas oryzae pv. oryzae KACC10331

city: Busan

first: Jungwook

last: Park

address: Pusan National University, 2, Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 609-735, Korea

department: Microbiology

country: South Korea

institute: Pusan National University

summary: Xanthomonas oryzae pv. oryzae (Xoo) causes the bacterial leaf blight of rice, which leads to as much as 50% yield losses. To understand the landscape of virulence mechanisms, we constructed in planta transcriptional profiling of Xoo KACC10331 using RNA-seq. Three in planta transcriptome of Xoo KACC10331 derived from infected rice leafs were compared to three in vitro data from rich media. To obtain differentially expressed genes, we used the DEGseq package with MA-plot-based method in the R statistical environment and identified 2,094 transcripts that were significantly altered.

sample_organism: Xanthomonas oryzae pv. oryzae KACC 10331

overall_design: Six libraries constructed: three in planta RNA-seq data from rice leafs infected by Xoo KACC10331 and three in vitro RNA-seq data from PSA media.

first: Jungwoo

first: Hyun-Hee

last: Lee

first: Young-Su

last: Seo


first: Lijia

last: Ma

summary: Development of PEAC-seq for unbiased off-target detection and translocation

overall_design: According to the screen and the tag insertion test, we attached a 21nt cytosine-depleted template to the 3’ of pegRNA. We reasoned that both the on-target and the off-target cleavage sites in the genome will incorporate the complementary sequence of the template, which could further serve as a PCR handle to enrich the unknown cleavage sites caused by Cas9. Inspired by the GUIDE-seq strategy, the genomic DNA was extracted from the cells undergone Cas9 editing and segmented by Tn5. The fragments included cleaved sites were further enriched by a pair of primers, in which one primed at the inserted PCR handle and the other primed at the Tn5 adaptor.


title: Loss of Linker Histone H1 in the Maternal Genome Influences DEMETER-Mediated Demethylation and Affects the Endosperm DNA Methylation Landscape

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

overall_design: Refer to individual Series


title: Effect of ∆Np63 deletion on plasticity of post-injury lung basal cells

city: Philadelphia

first: Andrew

last: Vaughan

state: PA

address: 3800 Spruce Street, Old Vet 370 E

institute: University of Pennsylvania

summary:
Basal cells were isolated from an influenza-infected non-tamoxifen treated Krt5CreERT2; RFP; ∆Np63flox/flox mouse and cultured, allowing for inducible ∆Np63 deletion in vitro.
These basal cells were plated as monolayers, airway organoids, and alveolar organoids and treated with 1uM 4OHT for +4OHT (∆Np63 KO)  or equivalent volume DMSO for -4OHT (solvent only, ∆Np63 WT). Technical triplicates of ∆Np63 WT and ∆Np63 KO were harvested for each growth condition for RNA sequencing.

overall_design: Comparative transcriptomics of ±∆Np63 post-injury basal cell monolayers, airway organoids, and alveolar organoids

first: Aaron

last: Weiner


title: An atlas of small RNAs from potato

city: Davis

first: Patricia

last: Baldrich Gonzalez

address: 451 Health Sciences Dr., Davis, CA 95616

department: Genome Center

institute: University of California - Davis

summary: Small RNAs, including microRNAs (miRNAs), phased secondary small interfering RNAs (phasiRNA), and heterochromatic small interfering RNAs (hc-siRNA) are an essential component of gene regulation. To establish a broad potato small RNA atlas, we constructed an expression atlas of leaves, flowers, roots, and tubers of Desiree and Eva, which are commercially important potato (Solanum tuberosum) cultivars. All small RNAs identified were observed to be conserved between both cultivars, supporting the hypothesis that small RNAs have a low evolutionary rate and are mostly conserved between lineages. However, we also found that a few miRNAs showed differential accumulation between the two potato cultivars, and that hc-siRNAs have a tissue specific expression. We further identified dozens of reproductive and non-reproductive phasiRNAs originating from coding and noncoding regions that appeared to exhibit tissue-specific expression. Together, this study provides an extensive small RNA profiling of different potato tissues that might be used as a resource for future investigations.

overall_design: Small RNA expression profile of two different potato cultivars, and four different tissues (leaf - root - flower - tuber) for each. Three biological replicates included for each sample.

last: Baldrich

first: Alexander

last: Liu

first: Blake

last: Meyers

first: Vincent

middle: N

last: Fondong


title: Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering in glioblastoma stem cells

city: Edinburgh

first: Karin

last: Purshouse

address: Crewe Rd S

institute: University of Edinburgh

summary: Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression.  In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional hubs, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harboring distinct ecDNA species (EGFR, CDK4, PDGFR). We found no evidence that ecDNA routinely cluster with one another or closely interact with transcriptional hubs. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy was similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that it is the increased copy number of oncogene-harboring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with each other or with high concentrations of the transcriptional machinery.

overall_design: WGS, RNAseq and Amplicon Architect files for human glioblastoma stem cells

first: Elias

last: Friman

first: Alhafidz

last: Hamdan

first: Gillian

middle: M

last: Morrison

first: Steven

last: Pollard


title: scRNA Seq of Metrnl treated mouse muscles during regeneration

city: Durham

first: James

last: White

state: nc

address: 300 N Duke Street

institute: Duke University SOM

summary: scRNA-seq comparing muscle cell suspension gene expression between young control, old control and old Meteorin-like treated mouse muscle 4 days following injury.

overall_design: scRNA-seq comparing muscle cell suspension gene expressions between young control, old control and old Meteorin-like treated mouse muscle 4 days following injury.

first: David

middle: P

first: Lauren

middle: K

last: McKay


title: DOT1L regulates chamber-specific transcriptional networks during cardiogenesis and mediates postnatal cell cycle withdrawal

city: Milan

first: Paola

last: Cattaneo

address: Via Fantoli 16/15

department: Institute of Genetic and Biomedical Research

country: Italy

institute: National Research Council of Italy


title: DNA methylation and hydroxymethylation characterize the identity of D1 and D2 striatal projection neurons

city: Paris

first: Jean-Antoine

last: Girault

address: 17 rue du Fer à Moulin

institute: INSERM, IFM

summary: Neuronal DNA modifications differ from those in other cells, including methylation outside CpG context and abundant 5-hydroxymethylation whose relevance for neuronal identities are unclear. Striatal projection neurons expressing D1 or D2 dopamine receptors allow addressing this question, as they share many characteristics but differ in their gene expression profiles, connections, and functional roles. We compare translating mRNAs and DNA modifications in these two populations. DNA methylation differences occur predominantly in large genomic clusters including differentially expressed genes, potentially important for D1 and D2 neurons. Decreased gene body methylation is associated with higher gene expression. Hydroxymethylation differences are more scattered and affect transcription factor binding sites, which can influence gene expression. We also find a strong genome-wide hydroxymethylation asymmetry between the two DNA strands, particularly pronounced at expressed genes and retrotransposons. These results identify novel properties of neuronal DNA modifications and unveil epigenetic characteristics of striatal projection neurons heterogeneity.

overall_design: RNA-seq (translatome), strand-specific MeDIP-seq and strand-specific hMeDIP-seq in D1R and D2R striatal projection neurons (= medium spiny neurons), adult mouse brain

first: Lucile

last: Marion-Poll

first: Jean-Pierre

last: Roussarie

first: Lieng

last: Taing

first: Cloelia

last: Dard-Dascot

first: Nicolas

last: Servant

first: Yan

last: Jaszczyszyn

first: Emmauelle

last: Jordi

first: Eskeatnaf

last: Mulugeta

first: Denis

last: Hervé

first: Déborah

last: Bourc'his

first: Paul

last: Greengard

first: Claude

last: Thermes


title: Genome-wide mRNA expression analyses revealed dysregulation of genes important for cardiomyocyte function in XMlc2-Cre;Dot1L mutants and controls

summary: Epigenetic enzymes play critical roles in embryogenesis by defining higher-order chromatin structures required for the establishment of organ-specific transcriptional networks. In this study we investigated the role of the histone 3 lysine 79 methyltrasferase Dot1L in cardiomyocytes during development. Cardiomyocyte-specific ablation of Dot1L resulted in postnatal lethality  with a cardiac phenotype of enlarged, rounded heart with increased ventricular wall thickness and areas of moderate trabeculation postnatally. To understand transciptomic alterations driving the observed phenotype, we conducted RNA-seq experimens in  embryonic (E16.5) and neonatal (P1) cardiomyocytes from Dot1L conditional knock out and control mice. Overall, we identified downregulation of genes higly expressed and upregulaton of genes lowly expressed. Genes dowregulated in two critical stages of cardiac development analysed were indicative of defective cardiac patterning during embryogenesis, and defective cell cycle withdrawal in the neonatal period.

overall_design: RNA-seq analyses were performed on FACS purified cardiomyocytes from XMlc2-Cre;Dot1L mutant and controls at E16.5 and P1.

first: Nuno

last: Guimaraes-Camboa

first: Paolo

last: Kunderfranco

first: Sylvia

last: Evans


title: Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells

status: Public on Nov 10 2022

city: Bethesda

first: Pavol

last: Genzor

address: 6720A Rockledge Drive, Suite 100

department: ACESO

institute: Henry M Jackson Foundation

summary: DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transposase N-terminus and the need for asymmetric transposon ends for cellular activity. We found that phosphorylation at predicted casein kinase II sites in the transposase N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. When the N-terminus is deleted, the transposase gains the ability to carry out transposition using symmetric transposon left ends. This novel activity is also conferred by appending a second C-terminal domain. When combined, these modifications together result in a transposase that is highly active when symmetric transposon ends are used. Our results demonstrate that transposase N-terminal phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposition in mammalian cells. These novel insights into the mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

overall_design: Computational: Comparative genome-wide analysis of transposase insertion sequencing in HCT116 cells transfected with wild-type and mutant piggyBac, or wild-type piggyBat transposon systems.

first: Wentian

last: Luo

first: Alison

last: Hickman

first: Rodolfo

last: Ghirlando

first: Christopher

last: Furman

first: Anna

last: Menshikh

first: Astrid

middle: D

last: Haase

first: Fred

last: Dyda

first: Matthew

middle: H

last: Wilson


title: Extension of O-linked mannosylation in the Golgi is critical for cell wall integrity signaling and interaction with host cells in Cryptococcus neoformans pathogenesis

first: EunJung

last: Thak

address: 2530 erwin rd

institute: eunjungthak

summary:
Purpose: To identify the downstream gene sets regulated by Mpk1p in C. neoformans normal and Tunicamycin-treated conditions, we compared the RNA-seq based transcriptional profiles.
Methods: Total RNA was isolated according to the RNeasy Mini Kit (Qiagen) and all procedure for RNA sequencing were conducted by ebiogen. The mRNA was isolated by using the Poly(A) RNA Selection Kit (LEXOGEN) and isolated mRNAs were used for the cDNA synthesis.
Results: Transcriptome analysis of the wild-type strain revealed that tunicamycin significantly increased expression of a subset of calcineurin-Crz1 pathway related genes , but most of which were not upregulated in O-glycan mutant strain.
Conclusion: Our results support the essential role of O-glycan in maintaining cell wall integrity upon stress conditions of C. neoformans.

overall_design: mRNA profiles of cells grown at normal and tunicamycin-treated conditions were generated by RNA-seq using NovaSeq 6000 (Illumina).

first: Hyun

middle: A

last: Kang

first: Eun

first: Ye

last: Son


title: Loss of ∆Np63 induces epigenetic rewiring and enhances plasticity towards alveolar identity

first: Andrew

last: Vaughan


title: Genome-wide maps of chromatin state of H3K79me2 in embryonic and neonatal cardiomyocytes of XMlc2-Cre;Dot1L mutants and controls

first: Paola

last: Cattaneo

summary: Epigenetic enzymes play critical roles in embryogenesis by defining higher-order chromatin structures required for the establishment of organ-specific transcriptional networks. In this study we investigated the role of the histone 3 lysine 79 methyltrasferase Dot1L in cardiomyocytes during development. We conducted chromatin immunoprecipitation experiments for H3K79me2 in embryonic (E16.5) and neonatal (P1) cardiomyocytes from Dot1L cardiomyocyte-specific conditional knock out and control mice. Genes that bear this modification in DOT1L controls lose it in cKOs, consistent with the model of DOT1L as the sole histone K79 methyltransferase. Overall, we identified enrichment of methylation of H3K79 in gene bodies and in regulatory regions associated wih trascription factors essential for cardiac patterning at E16.5 and with genes important for cardiomyocyte proliferation and maturation in the postnatal period.

overall_design: H3K79me2 ChIP-seq profiling were performed on FACS purified cardiomyocytes from XMlc2-Cre;Dot1L mutant and controls at E16.5 and P1.

first: Nuno

first: Michael

last: Hayes

first: Dennis

last: Hecker

first: Nina

last: Baumgarten

first: Marcel

last: Schulz

first: Sven

last: Heinz


title: Genome-wide maps of chromatin state of H3K27ac in embryonic and neonatal cardiomyocytes of XMlc2-Cre;Dot1L mutants and controls.

summary: Epigenetic enzymes play critical roles in embryogenesis by defining higher-order chromatin structures required for the establishment of organ-specific transcriptional networks. In this study we investigated how the landscape of the histone 3 lysine 27 acetylation in absence of the histone methyltransferase Dot1L in cardiomyocytes during development. We conducted chromatin immunoprecipitation experiments for H3K27ac in embryonic (E16.5) and neonatal (P1) cardiomyocytes from Dot1L cardiomyocyte-specific conditional knock out and control mice. Overall, we identified alteration in the enrichment of acetylation of H3K27 in regulatory regions associated wih genes essential for cardiac patterning and cardiomyocyte maturation.

overall_design: H3K27ac ChIP-seq profiling were performed on FACS purified cardiomyocytes from XMlc2-Cre;Dot1L mutant and controls at E16.5 and P1.

last: Guimaraes-Camboa

first: Sylvia

last: Evans


title: Dickkopf1 fuels inflammatory cytokine responses

city: Dresden

first: Ismini

middle: Vasileia

last: Alexaki

state: Saxony

address: Fetscherstraße 74

institute: IKL , Carl Gustav Carus Universitätsklinikum, TU Dresden

summary: Many human diseases, including cancer, share an inflammatory component but the molecular underpinnings remain incompletely understood. We report that physiological and pathological Dickkopf1 (DKK1) activity fuels inflammatory cytokine responses in cell models, mice and humans. DKK1 maintains the elevated inflammatory tone of cancer cells and is required for mounting cytokine responses following ligation of toll-like and cytokine receptors. DKK1-controlled inflammation derives from cell-autonomous mechanisms, which involve SOCS3-restricted, nuclear RelA (p65) activity. We translate these findings to humans by showing that genetic DKK1 variants are linked to elevated cytokine production across healthy populations. Finally, we find that genetic deletion of DKK1 but not pharmacological neutralization of soluble DKK1 ameliorates inflammation and disease trajectories in a mouse model of endotoxemia. Collectively, our study identifies a cell-autonomous function of DKK1 in the control of the inflammatory response, which is conserved between malignant and non-malignant cells and may be exploited therapeutically in the future.

overall_design: Bulk RNA-Seq from DKK1 competent (siCtrl) and DKK1 deficient (siDKK1) PC3 cancer cells

first: Nikolai

last: Jaschke