title: Epigenomic analysis of Formalin-Fixed Paraffin-Embedded samples by CUT&Tag

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary: For more than a century, Formalin Fixed Paraffin Embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our single-tube CUTAC protocol are sufficient to produce high-resolution maps of paused RNA Polymerase II (RNAPII) at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumor specimens and identify regulatory element markers with high confidence and precision. Our simple workflow is suitable for automation, making possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

overall_design:
We used FFPE-CUTAC, a modification of the CUT&Tag chromatin profiling strategy, whereby antibody-targeted controlled integration of DNA sequencing adapters at sites of paused RNA Polymerase II or between nucleosomes flanking open chromatin sites produces libraries for paired-end sequencing from Formalin-Fixed Paraffin-Embedded (FFPE) sections.

Grant information: T32CA009515 - Ronald M. Paranal - National Cancer Institute

first: Steven

first: Kami

last: Ahmad

first: Ronald

last: Paranal

first: Derek

last: Janssens

first: Zachary

middle: R

last: Russell

first: Frank

last: Szulzewsky

first: Sita

last: Kugel

first: Eric

last: Holland


title: Enteric glial hub cells coordinate intestinal motility

status: Public on Sep 08 2023

city: Cleveland

first: Marissa

last: Scavuzzo

state: OH

address: 2109 Adelbert Rd

department: Genetics

country: USA

institute: Case Western Reserve University

type: Expression profiling by high throughput sequencing

summary: Enteric glia are the predominant cell type in the enteric nervous system yet their identities and roles in gastrointestinal function are not well classified. Using our optimized single nucleus RNA-sequencing method, we identified distinct molecular classes of enteric glia and defined their morphological and spatial diversity. Our findings revealed a functionally specialized biosensor subtype of enteric glia that we call “hub cells.” Deletion of the mechanosensory ion channel PIEZO2 from adult enteric glial hub cells, but not other subtypes of enteric glia, led to defects in intestinal motility and gastric emptying in mice. These results provide insight into the multifaceted functions of different enteric glial cell subtypes in gut health and emphasize that therapies targeting enteric glia could advance the treatment of gastrointestinal diseases.

overall_design: Nuclei from the whole adult duodenum of C57Bl/6 mice were isolated using CitraPrep and analyzed using snRNAseq

middle: A

first: Paul

middle: J

last: Tesar


title: AHR-TFAP2A regulates epidermal differentiation in response to environmental cues

city: Nijmegen

first: Jo Huiqing

last: Zhou

address: Geert Grooteplein 26/28

country: Netherlands

institute: Radboud University

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: In Vitro Expanded Cord Blood γδ T Cells Acquired Unique Cell State Composition to Mediate Sustained Human AML Suppression [5GEX]

city: Singapore

first: Wee Kiat Jeremy

last: Ng

address: 20 College Road

country: Singapore

institute: Singapore General Hospital

summary: Single cell RNA-seq of cultured gamma-delta  T-cells from cord blood were expanded over 2 weeks to identify cell states following in-vitro expansion. Cells were expanded in a feeder co-culture with the EBV-LCL feeder line. After 2 weeks, cells were challenged with the AML cell line K562. Single-cell RNA-sequencing was then performed to identify transcriptomic changes. RNA-seq was paired  with single cell TCR sequencing to identify clonotypes mapping to specific cell states.

overall_design: Cultured gamma-delta T-cells were sorted with FACS, cultured in the presence of in RPMI + 10% FBS + 170 U/mL recombinant human interleukin-2 (rhIL-2), 25U/ml rhIL-15 as well as 0.7U/ml rhIL-21, with EBV-LCL feeder over a period of  2 weeks and analyzed using scRNAseq before/after antigen challenge with K562.

first: Jeremy

middle: W

first: Kar

last: Tan

first: Alice

middle: M

last: Cheung


title: AHR-TFAP2A regulates epidermal differentiation in response to environmental cues [ChIP-seq]

summary: The Aryl Hydrocarbon Receptor (AHR) is an environmental sensor and an indispensable regulator of epithelial homeostasis. To investigate AHR-mediated gene regulation, we performed a comprehensive transcriptomic and epigenomic analysis using human primary epidermal keratinocytes. Our results showed that AHR activation led to the induction of canonical transcription factors involved in epidermal differentiation as an early response, i.e. Transcription Factor AP-2α (TFAP2A), while epidermal differentiation-related genes, such as filaggrin and keratins, were activated as late responsive genes. The identified AHR-TFAP2A axis and its role in keratinocyte terminal differentiation was confirmed through AHR and TFAP2A knockout using CRISPR/Cas9. Our findings demonstrate that AHR regulates epidermal differentiation through the transient activation of specific transcription factors, such as TFAP2A, in response to environmental cues. The herein identified AHR-TFAP2A axis provides a promising target for the treatment of diseases related to skin barrier dysfunction.

overall_design: To characterize the gene expression pattern upon AHR activation in keratinocytes, we performed RNA-seq on either TCDD or CT treated keratinocytes. In addition, to identify AHR-responsive enhancers, we performed ChIP-seq of AHR and H3K27ac that marks active enhancers in the genome of keratinocytes after TCDD treatment for 30 and 90 minutes. With integrated analyses of  RNA-seq, AHR, and H3K27ac ChIP-seq data, we identified that TFAP2A is a direct AHR target and is an important transcription factor involved in epidermal differentiation. To further test our hypothesis that AHR controls epidermal differentiation via TFAP2A expression, we knocked down TFAP2A in monolayer primary keratinocyte cultures and performed RNA-seq analysis.

first: Jieqiong

last: Qu

first: Jos

middle: P

last: Smits

first: Felicitas

last: Pardow

first: Noa

last: van den Brink

first: Huiqing

first: Ellen

middle: H

last: van den Bogaard


title: RNA-seq results in the kidney of male tubule-specific Apela knockout mice treated with renal I/R injury

city: Wuhan

first: Mingrui

last: Xiong

address: Hangkong Road 13

country: China

institute: School of Pharmacy, Tongji Medical College, Huazhong University of Science & Technology

summary: To understand key pathways and regulatory genes in male tubule-specific Apela knockout mice treated with renal I/R injury

overall_design: male tubule-specific Apela knockout mice and their age-, sex- matched control mice were treated with bilateral I/R injury. For each experimental group, two biological replicates of RNA samples were sent for RNA-seq. Each sample consists of a pool of RNAs extracted from inguinal adipose tissue or brown adipose tissue of 3 different mice.

first: Ling

last: Zheng

first: Kun

last: Huang


title: Resistance and Aerobic Exercise Intervention Induces Differential DNA Methylation in Women with Polycystic Ovary Syndrome: a clinical trial study

city: Provo

first: Timothy

middle: G

last: Jenkins

state: UT

address: 4005 Life Sciences Building

department: Cell Biology and Physiology

institute: Brigham Young University

summary: This is a study to identify the effects of exercise on the epigenome of women diagnosed with Poly Cistic Ovarian Syndrome (PCOS), to understand if Resistence training, or continuous training had any positive effect on disease outcome.

overall_design: Whole blood samples were collected from 112 females with PCOS. 60 were placed on a Resistence Training workout, and 52 were placed on an Aerobic Continuous workout. Differences in methylation patterns were subsequently assesed, as well as the overall state of PCOS symptoms.


title: CD4 T cells in Giant Cell Arteritis (RNA-Seq)

city: Rochester

first: Yuki

last: Sato

state: MN

address: 200 1st ST SW

department: Rheumatology

institute: Mayo Clinic

summary: CD4 T cells are important in GCA

overall_design: Human ascending aortic aneurysm tissue samples derived from aortitis patients and disease control patients

first: Abhinav

last: Jain

first: Cornelia

last: Weyand


title: The bone transcription factor Osterix controls extracellular matrix and node of Ranvier related gene expression in oligodendrocytes

city: Chicago

first: Benayahu

last: Elbaz

state: Illinois

address: 303 E. Chicago Ave. Ward 7-641. Chicago, IL 60611

department: Neurology

institute: Northwestern University


title: Profiling of RNA-binding protein LSM14B target sites in oocyte by LACE-seq

city: Qingdao

first: Li

last: Hui

address: 72 Binhai Road, Jimo District, Qingdao, Shandong

institute: Shandong University

summary: RNA-binding proteins (RBPs) have essential functions during oocyte development. In order to explore the regulatory role of RNA-binding protein LSM14B in oocyte, we determined the binding sites and regulatory mechanisms for LSM14B by LACE-seq.

overall_design: Cells are first irradiated with ultraviolet C (UV-C) light to crosslink RBPs and their interacting RNAs. We next use LSM14B antibody to specifically pull-down protein–RNA complexes from the cell lysate. To cut RNAs into individual RBP-associated short fragments, we treat the RBP–RNA complexes on the beads with micrococcal nuclease (MNase). The 3′ ends of fragmented RNAs are then dephosphorylated and ligated with a 5′ pre-adenylated linker containing four randomized nucleotides. Next, a biotinylated primer containing the T7 promoter is used for reverse transcription on beads. Since reverse transcriptase would efficiently stop before the intact RBP–RNA crosslinking sites, sequencing these truncated cDNA ends enables us to identify 3′ sequences downstream of the bound element of a defined RBP. As immunopurified RNAs from single or dozens of cells are very limited, we adopted a T7 RNA polymerase-based in vitro transcription (IVT) approach to amplify trace amounts of truncated cDNAs linearly. The cDNAs are tailed with poly(A) and subsequently enriched using streptavidin beads via the biotin moiety at the 5′ end of the T7 primer. Next, we synthesize second-strand cDNA on beads with an adaptor containing oligo-(dT). After pre-amplifying the double-stranded DNA (dsDNA) fragments by PCR for 14–18 cycles, the products are purified and used for IVT. Subsequently, the transcribed RNAs are converted into libraries for single-end deep sequencing (~180bp).

first: You

middle: Q

last: Su

first: Yuan

middle: C

last: Xue

first: Hui

last: Li

first: Hai-lian

last: Zhao


title: Prmt5-dependent regulation of gene expression and TAD boundary strength during early adipogenesis

city: Farmington

first: Sabriya

last: Syed

state: CT

address: 10 Discovery Dr

institute: The Jackson Laboratory for Genomic Medicine


title: In Vitro Expanded Cord Blood γδ T Cells Acquired Unique Cell State Composition to Mediate Sustained Human AML Suppression.

first: Wee Kiat Jeremy

last: Ng


title: Comparing the Transcriptomes of WT oocytes  with Lsm14b KO oocytes by RNA-Seq Analysis

summary: The success of human reproduction relies on high quality oocytes. Oocyte quality is manifested by the competence to complete meiosis, to be fertilized, and to support embryonic development. This meiotic and developmental competence is gradually established during the course of oocyte and follicle development, and is determined in large part by the autonomous gene expression program intrinsic to the oocyte. In order to explore the regulatory role of LSM14B in oocyte, we analyzed the effect of Lsm14b KO on gene expression in GV-stage fully-grown oocytes (FGOs) in mice by comparing the corresponding transcriptomes via RNA-Seq Analysis.

overall_design: WT and  Lsm14b KO GV-stage FGOs were collected in lysis buffer to release all RNAs, which then were reverse transcribed into first cDNA strands by SuperScript II reverse transcriptase(Invitrogen) and random primers. These cDNAs were amplificated according to Smart-seq2 procedures for construct Illumina sequencing library. 3 replictates of each treatment, with 20 oocyte per  samples, were collected for the experiment. The library was constructed using KAPA Hyper Prep Kit for Illumina (Roche) according to the instruction manual, which includes end repair, dA-tailing, adapter ligation, post-ligation cleanup, library amplification and post-amplification cleanup. The libraries were sequenced on an Illumina HiSeq X Ten platform with 150bp pair-end reads. All reads passed filter were trimmed to remove low-quality bases and adaptor sequences. Reads were then aligned to the mm10 reference genome using tophat2 (v2.0.13), and FPKMs were calculated and normalized using cufflinks (v2.2.1). The differentially expressed genes were calculated using default parameter of cuffdiff (v2.2.1). Hierarchical clustering was carried on log2(FPKM+1) across samples. Genes used for clustering were selected by maximum FPKM≥1 and with top 10% standard deviation of log2(FPKM+1).

first: You-qiang


title: Identifying the Translatome of WT and Lsm14b KO  GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling)

summary: RNA-binding proteins (RBPs) have essential functions during oocyte development.In order to explore the regulatory role of RNA-binding protein LSM14B in oocyte, we  Identified the translatome of WT and Lsm14b KO  GV-Stage Oocytes in mice via Scarce Sample Polysome Profiling (SSP-profiling).

overall_design: Germinal vesicle (GV) oocytes were obtained from 3 weeks old WT and Lsm14b KO mice, 46 h after injection with 5 IU pregnant mare serum gonadotropin (PMSG). GV oocytes were then transferred to polyvinyl alcohol (PVA) supplemented with cycloheximide (0.1 mg/mL) and stored at –80℃ in 1.5 ml tubes. Approximately 1500 oocytes were lysed with MCB Buffer (100mM KCl,  0.1% Triton X-100 50mM HEPES, 2mM MgCl2, 10% Glycerol,1 mM DTT, 20 U/mL RNase inhibitor, 10 mg/mL cycloheximide, plus mini-EDTA-free protease inhibitor cocktai). Oocyte lysates were centrifuged at 12,000g for 10 min at 4°C. Supernatants were loaded on a 10-mL 15%–55% sucrose gradient,centrifuged at 38000rpm for 3 h at 4℃（XPN-100，Beckman-Coulter）and collected  in 28 fractions, 1ml per fraction. RNAs from the polysome(11-28 fractions) or RNP fractions(1-10 fractions) were precipitated with ethanol overnight and purified with RNeasy Plus Micro kit (Qiagen). Polysome-bound RNAs and RNAs in the RNP fractions were reverse transcribed into first cDNA strands by SuperScript II reverse transcriptase(Invitrogen) and random primers. These cDNAs were amplificated according to Smart-seq2 procedures for construct Illumina sequencing library. 3 replictates of each treatment were collected for the experiment. The library was constructed using KAPA Hyper Prep Kit for Illumina (Roche) according to the instruction manual, which includes end repair, dA-tailing, adapter ligation, post-ligation cleanup, library amplification and post-amplification cleanup. The libraries were sequenced on an Illumina HiSeq X Ten platform with 150bp pair-end reads. All reads passed filter were trimmed to remove low-quality bases and adaptor sequences. Reads were then aligned to the mm10 reference genome using tophat2 (v2.0.13), and FPKMs were calculated and normalized using cufflinks (v2.2.1). The differentially expressed genes were calculated using default parameter of cuffdiff (v2.2.1). Hierarchical clustering was carried on log2(FPKM+1) across samples. Genes used for clustering were selected by maximum FPKM≥1 and with top 10% standard deviation of log2(FPKM+1).


title: BMPER: a novel adipose progenitor marker and adipogenic modulator

city: salt lake city

first: Sihem

last: Boudina

state: Utah

department: Nutrition and Integrative Physiology

institute: University of Utah

summary: We used single cell sequencing to identify sex and diet-specific differences in visceral adipose progenitor cells (APCs) and to discover new APC markers and adipogenic modulators. We identified the major clusters that were previously identified by other studies both in humans and mice with the notable difference being in the mesothelial cluster, which was more abundant in humans than mice. We found that diet/obesity resulted in minor differences in the proportions of major clusters in humans but increased immune cells in male mice with high fat feeding. Most importantly, we identified BMPER as a novel marker of APCs both in humans and mice and revealed its pro-adipogenic property.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Single cell RNA sequencing (scRNA-seq) was performed on stromal vascular cells isolated by collagenase digestion from omental fat of 4 male and 5 female patients who underwent either a laparoscopic abdominal surgery or a bariatric surgery. scRNA-seq was also performed in stromal vascular cells isolated by collagenase digestion from gonadal fat from male and female mice (10 mice pooled/diet/sex) fed normal chow diet (NCD) or high fat diet (HFD) for 8 weeks.

first: Jacob

last: Garrittson

first: Anna

last: Ibele

first: Keren

last: Hilgendorf

first: Chris

last: Stubben


title: Single cell RNA sequencing of bovine blastocysts: Early cellular differentiation

city: Gainesville

first: Zongliang

last: Jiang

state: FL

address: 2033 Mowry Rd

department: Animal Sciences

institute: University of Florida

summary: Differentiation of totipotent embryonic cells into the first cell lineages (trophectoderm (TE) and inner cell mass (ICM), and further into epiblast (EPI) and primitive endoderm (PE)), although well studied, is only beginning to be understood at the molecular level. An in depth understanding of the regulation of these early differentiation events would enable improvements in the quality of in vitro produced embryos, as well as the study and application of bovine embryonic stem cells. The objective of this work was to perform single cell RNASeq on bovine blastocysts derived in vivo (IVV), in vitro in a conventional in vitro production system (IVC), and in vitro in reduced nutrient media (IVR). All in vitro culture was serum free.  In vivo embryonic cells could be assigned based on Leiden clustering analysis, although there was some overlap in expression of marker genes. The two TE groups identified clearly separated in a uniform manifold approximation and projection (UMAP) plot from EPI groups. Putative epiblast cells could be identified. In in vitro embryos produced in a conventional system, considerable overlap in expression of marker genes made Leiden clustering difficult, but a selected count could be used for UMAP to tentatively identify one TE, 2 EPI and 2 PE groups. In assigned cell types we observed higher expression of the identifying marker genes, but some marker genes were expressed in all cell types. In IVC embryos, TE markers clustered while PE and EPI marker genes overlapped. In IVR embryos, again there was considerable overlap between marker gene expression. Like IVC embryos, TE was easily identified by clustering in IVR, but EPI and PE were too similar to distinguish. In summary, the methods used here could partially distinguish putative cell types in bovine blastocysts, although marker gene expression was not always discreet. This may not be surprising given differentiation events are in progress in the blastocyst stage embryo. TE cells were more easily distinguished than EPI and PE cells, which would support this hypothesis.

overall_design: Single cell RNASeq analysis of bovine blastocysts derived in vivo (IVV), in vitro in a conventional in vitro production system (IVC), and in vitro in reduced nutrient media (IVR).

first: Ye

last: Yuan

first: Rebecca

last: Krisher


title: Transcriptome analysis of day 0 3T3-L1 cells transfected with scrambled and Prmt5 siRNA

summary: This  study aims to identify genes dependent on Prmt5 for expression at the onset of 3T3-L1 differentiation

overall_design: RNA-Seq of day 0 3T3-L1 cells transfected with scrambled and Prmt5 siRNA

first: Anthony

middle: N

last: Imbalzano


title: In Vitro Expanded Cord Blood γδ T Cells Acquired Unique Cell State Composition to Mediate Sustained Human AML Suppression [VDJ]

summary: Single cell TCR sequencing was performed to examine the clonal type changes in cord blood derived gamma-delta T-cells following in-vitro expansion in a co-culture with EBV-LCL feeder over 2 weeks. Following 2 weeks of expansion, the cells were challenged with the AML cell line K562. Single-cell TCR was performed after 24 hours post-challenge to identify clones that were preferentially expanded, as well as the corresponding cell states mapped using single-cell RNA-sequencing.

first: Jeremy

first: Kar

last: Tan

first: Alice

last: Cheung


title: In Vitro Expanded Cord Blood γδ T Cells Acquired Unique Cell State Composition to Mediate Sustained Human AML Suppression [fb]


title: Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (scRNA-seq)

city: Shanghai

first: Aiwei

last: Wu

state: Shanghai

address: 800 Dongchuan Road

department: School of Life Sciences and Biotechnology

institute: Shanghai Jiao Tong University

summary: HMG-box protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that although pan-hematopoietic Tox4 knockout produces minimal defects, T cell-specific knockout reduces thymic cellularity by 50% and increases cell death. In addition, single cell RNA-seq discovered that Tox4 loss impairs T cell development starting from double positive (DP) blast stage partially due to downregulation of genes critical for cell cycle progression through G2 and M phases. Moreover, Tox4 loss decreases transcriptional output of thousands of genes in DP cells, and mechanistically, Tox4 restricts transcriptional elongation and facilitates transcriptional reinitiation in a dephosphorylation-dependent manner, which is consistent with what we have found in human cells. Furthermore, small effect of Tox4 loss on chromatin accessibility suggest that it mainly regulates transcription in an HMG box independent manner. These results establish an evolutionarily conserved a role of Tox4 in transcriptional elongation and reinitiation.

overall_design: scRNA-seq expriments of thymocytes purified from Tox4 WT and cKO mice

first: Ming

last: Yu

first: Ziling

last: Liu

first: Talang

last: Wang

first: Junhong

last: Zhi


title: Hematopoietic cell-derived IL-15 is essential for group 1 innate lymphoid cell (ILC) development in the bone marrow

city: Kyoto

first: Koichi

last: Ikuta

state: Kyoto

address: 53 Shogoin-Kawaharamacho, Sakyo-ku

department: Department of Virus Research

country: Japan

institute: Institute for Frontier Life and Medical Sciences, Kyoto University

summary: This study reveals the role of IL-15 produced by hematopoeitic cells in the development of NK cells and ILC type 1 cells (ILC1s) in the bone marrow.

overall_design: Transcriptional profiling of bone marrow NK cells and ILC1s from IL-15-flox/KO (control) and Vav-Cre; IL-15-flox/KO mice

first: Shinya

last: Abe


title: Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (RNA-seq)

overall_design: RNA-seq expriments of DP cells purified from Tox4 WT and cKO mice


title: Hi-C analysis of day 0 3T3-L1 cells transfected with scrambled and Prmt5 siRNA

first: Sabriya

last: Syed

summary: This study aims to identify chromatin contact changes between cells transfected with scrambled and Prmt5 siRNA

overall_design: Hi-C of day 0 3T3-L1 cells transfected with scrambled and Prmt5 siRNA


title: Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II


title: Identification of HSPA5 targets involved in non-alcoholic fatty liver disease

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.

summary: Background: It has been reported that HSPA5 is an endoplasmic reticulum chaperone, which regulates cell metabolism, especially lipid metabolism. There are many reports about the interaction between HSPA5 and protein regulating cell function, but there is no report about HSPA5 binding RNA and its biological function. However, it may have RNA binding ability by using interaction capture.   Method: In the present study, the ability of HSPA5 to modulate the AS of cellular genes was assessed using a high-throughput RT-PCR approach to examine AS in lipid metabolism-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNA sassessment.  Results: Upon HSPA5 expression, we detected modifications to the AS profiles of numerou genes involved in pathology of nonalcoholic fatty liver disease. Moreover, we show that HSPA5 modulates the expression levels of various splicing factors such as NEAT1, LRP1, and EGFR. Finally, RNA immunoprecipitation coupled to RIP-Seq assays demonstrate that HSPA5 immunoprecipitates specific cellular mRNAs.  Conclusion: The HSPA5 protein can modulate the AS profiles of numerous cellular genes and it binds lncRNA and mRNA, and the related genes are links to nonalcoholic fatty liver disease, which is also the innovation of this project.

overall_design: RIP-seq analysis of HSPA5 IP versus input in Hela cells.

first: Aliya

last: Rehati


title: SHP-1 regulates transcription of PCK1 to control gluconeogenesis

city: Quebec

first: Michael

last: Schwab

state: Québec

address: 2725 Chemin Ste-Foy

country: Canada

institute: CRIUCPQ / Hopital Laval

summary: The protein-tyrosine phosphatase SHP-1 (PTPN6) is an important glucose homeostasis modulator. Besides negatively regulating insulin signaling, the specific role of SHP-1 in metabolic control remains poorly understood. We show that SHP-1 acts as a co-activator for transcription of the phosphoenolpyruvate carboxykinase 1 (PCK1) gene, thereby modulating basal gluconeogenesis in hepatocytes. SHP-1 interacts with RNA polymerase II-subunits and signal transducer and activator of transcription 5 (STAT5), and localizes to the nucleus, where a sub-fraction of SHP-1 associates with chromatin. While SHP-1 binds to the PCK1-promoter, its loss affects RNA polymerase II-recruitment to this and other promoters of genes enriched for glucose metabolism-related functions. SHP-1-downregulation, and similarly STAT5 pharmacological inhibition reduce PCK1-transcript levels correlating with blunted gluconeogenesis. Overall, we identified a novel molecular SHP-1-function, that of a regulator of PCK1-transcription and subsequently hepatic gluconeogenesis, through physical interaction with the transcription machinery, mediated by an Akt-independent mechanism, but independent of STAT5 tyrosine-phosphorylation status.

overall_design: ChIP-seq analysis of HepG2 SHP-1-WT (CRISPR control) and SHP-1-KO (CRISPR KO) cells. Antibodies used: anti-Rpb1 (anti-RNAPII-CTD) and anti-Rpb1-pSer2 (anti-RNAPII-CTD-pSer2)

first: Amit

last: Kumar

first: Eric

last: Fournier

first: Maruhen

last: Silveira

first: Steve

last: Bilodeau

first: Andre

last: Marette


title: Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (TTchem-seq)

overall_design: TTchem-seq expriments of DP cells purified from Tox4 WT and cKO mice


title: BMAL1 loss in oligodendroglia contributes to abnormal myelination and sleep

status: Public on Sep 07 2023

city: Palo Alto

first: Erin

last: Gibson

state: CA

address: 3165 Porter Drive

department: Psychiatry and Behavioral Sciences

institute: Stanford University

summary: Myelination depends on maintenance of oligodendrocytes that arise from oligodendrocyte precursor cells (OPCs). We show that OPC-specific proliferation, morphology, and BMAL1 are time-of-day dependent. Knock out of Bmal1 in OPCs during development disrupts expression of genes associated with circadian rhythms, proliferation, density, morphology, and migration, leading to changes in OPC dynamics in a spatio-temporal manner. Furthermore, these deficits translate into thinner myelin, dysregulated cognitive and motor function, and increased sleep fragmentation.  OPC-specificBmal1loss in adulthood does not alter OPC density at baseline but impairs remyelination of a demyelinated lesion driven by changes in OPC morphology and migration. Lastly, we show sleep fragmentation is associated with increased prevalence of the demyelinating disorder multiple sclerosis (MS), suggesting a link between MS and sleep that requires further investigation. These findings have broad mechanistic and therapeutic implications for brain disorders that include both myelin and sleep phenotypes.

overall_design: We developed a model to specifically knock out the Bmal1 DNA-binding domain in OPCs during embryonic development by crossing clock gene knock out (Bmal1fl/fl) and cell type-specific Cre driver (NG2::Cre) mice to generate a Bmal1 transcriptional hypomorph that significantly decreased BMAL1 levels specifically in OPCs (NG2:Cre+;Bmal1fl/fl or OPC-Bmal1-KO). To test the role of BMAL1 as a transcriptional regulator in OPCs, we isolated OPCs from P6-7 OPC-Bmal1-WT and OPC-Bmal1-KO mice at 6-hr intervals for 24 hrs. OPCs were isolated at 4 different zeitgeber times: ZT0 (lights on), ZT6, ZT12 (lights off), and ZT18. For each biological replicate, 2 mice from both sexes were pooled to obtain around one million OPCs. OPCs were purified using immunopanning through 2 negative selection plates for microglia and endothelial cells with BSL1 (Vector Laboratories L1100) and one positive selection plate with an anti-PDGFRα antibody (rat anti-mouse CD140A, BD Pharmingen 558774). OPCs were immediately lysed using QIAzol Lysis Reagent. RNA extraction was performed following manufacturer’s instructions and RNA purity, integrity and concentration were assessed using a Bioanalyzer. Library prep and Bulk RNA sequencing was performed using Novogene Co., Ltd. sequencing service.

first: Daniela

last: Rojo


title: Genomic binding of wild-type and mutant Trp53 in mouse breast cancer cell lines

city: Amsterdam

first: Max

middle: D

last: Wellenstein

address: Plesmanlaan 121

institute: Netherlands Cancer Institute

summary: TP53 is one of the most frequently mutated gene in breast cancer. While p53 mutations often lead to loss of wild-type p53 activity, they can have a wide variety of gain-of-function (GOF) consequences. The impact of these GOF mutations in p53 on the anti-tumor immune response in breast cancer remains elusive. To address this, we have generated mouse mammary tumor models based on orthotopic injection of isogenic cell lines harboring the p53 mutations that most frequently occur in human breast cancer. By comparing the tumor immune landscape of these models and human breast tumors, we have uncovered that specific p53 point mutants consistently induce an immunologically ‘hot’ tumor phenotype, characterized by high infiltration of cytotoxic T cells, while other p53 mutations induce a non-T cell inflamed (‘cold’) microenvironment. In accordance with these high T cell levels, the hotp53 mutant tumors respond better to anti-PD-1immune checkpoint blockade than cold p53 mutant tumors. By comparing the different p53 mutants in terms of proteome profile, chromatin bindingproperties and protein complex formation, we have uncovered that T cell-enriched p53 mutants activate autophagy. Disruption of autophagy in an immunologically hot p53 mutant tumor abrogated the response to anti-PD-1 therapy. This work demonstrates that not all p53 mutations shape the immune microenvironment of mammary tumors in a similar fashion, and that response to immune checkpoint blockade depends on specific mutations in p53. This argues that screening for specific p53 aberrations in breast cancer may help guide immunotherapeutic strategies.

overall_design: The binding of Trp53 to the genome was examined in nine cell lines either harbouring differential Trp53 mutations or not (wild-type) . Cells were grown in DMEM + 10% FBS + 1% P/S. As a control, a TP53-KO cell line was used.  Two biological replicates were performed.

first: Stefan

last: Prekovic

first: Isabel

last: Mayayo-Peralta

first: Wilbert

last: Zwart

first: Karin

last: de Visser


title: Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (CUT&Tag)

first: Aiwei

last: Wu

overall_design: CUT&Tag of proteins  of DP cells purified from Tox4 WT and cKO mice

first: Ming

last: Yu

first: Ziling

last: Liu

first: Talang

last: Wang

first: Junhong

last: Zhi

first: Ruoyu

last: Zhao


title: BRAFV600E expression in intestinal tissue promotes a cholesterol metabolic gene signature that sustains hyperplasia and characterizes serrated colorectal neoplasia

city: Leicester

first: Nicolas

last: Sylvius

address: University Road

department: CBS-Genetics

country: United Kingdom

institute: University of Leicester

summary: BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis are poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice.  Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

overall_design: 20 samples

first: Alessandro

last: Rufini

first: Pooyeh

last: Farahmand

first: Catrin

last: Pritchard

first: Paulina

last: Rzasa

first: Nic


title: Genome-wide profiling of Prmt5 binding during 3T3-L1 adipogenesis

first: Sabriya

last: Syed

summary: This  study aims to determine the unique role of Prmt5 during adipogenesis by profiling Prmt5’s chromatin binding sites genome-wide at three time points during 3T3-L1 differentiation.

overall_design: Prmt5 ChIP-Seq at day 0, day 1, and day 2 of adipogenesis using 3T3-L1 cells, a mouse fibroblast-derived preadipocyte cell line.

first: Anthony

last: Imbalzano


title: Extracellular vesicles promote activation of pro-inflammatory cancer-associated fibroblast in oral cancer

city: Vancouver

first: Rebecca

last: Towle

state: BC

department: Integrative Oncology

institute: British Columbia Cancer Research Centre

summary: Introduction: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer and has a survival rate of ~50% over five years. New treatment strategies are sorely needed to improve survival rates – and a better understanding of the mechanisms underlying tumorigenesis is needed to develop these strategies. The role of the tumor microenvironment (TME) has increasingly been identified as crucial in tumor progression and metastasis. One of the main constituents of the TME, cancer-associated fibroblasts (CAFs), plays a key role in influencing the biological behavior of tumors. Multiple mechanisms contribute to CAF activation, such as TGFβ signaling, but the role of extracellular vesicles (EVs) in CAF activation in OSCC is poorly understood. Assessing the impact of oral cancer-derived EVs on CAF activation will help to better illuminate OSCC pathophysiology and may drive development of novel treatments options.  Materials and Methods: EVs were isolated from OSCC cell lines (Cal 27, SCC-9, SCC-25) using differential centrifugation. Nanoparticle tracking analysis was used for EV quantification and size characterization, and Western blot to confirm the presence of EV protein markers. Oral fibroblasts were co-cultured with enriched EVs, TGFβ, or PBS over 72 hours to assess activation. Flow cytometry was used to evaluate the difference in CAF markers. RNA collected from fibroblasts was extracted and the transcriptome was sequenced. Conditioned media from the co-cultures was evaluated with cytokine array profiling.   Results: OSCC-derived EVs can activate oral fibroblasts into CAFs that are different from those activated by TGFβ, suggesting different mechanisms of activation and different functional properties. Gene set enrichment analysis using expression data from activated CAFs showed several upregulated inflammatory pathways in those CAFs exposed to OSCC-derived EVs. Marker genes for inflammatory CAF subtypes were also upregulated. This was not seen in CAFs activated by TGFβ. Finally, cytokine array analysis on secreted proteins from CAFs revealed elevated levels of several pro-inflammatory cytokines from CAFs activated by EVs, for instance IL-8 and CXCL5.   Conclusions: Taken together, our results reveal the ability of OSCC-derived EVs to activate fibroblasts into CAFs. These CAFs seem to have unique properties, differing from TGFβ-activated CAFs. Gaining an understanding of the interplay between EVs and stromal cells such as CAFs could lead to further insights into OSCC tumorigenesis and potential novel therapeutics.

overall_design: RNA sequing on human oral fibroblast (HOrF) cell line after exposure to PBS, Evs derived from SCC9, Evs derived from SCC25 or treated with TGFb.

first: Julia

last: Arebro

first: Cathie

last: Garnis


title: Tox4 is an evolutionarily conserved regulator of elongation and recycling of RNA polymerase II (ATAC-seq)

first: Aiwei

overall_design: ATAC-seq expriments of DP cells and lymphatic CD8+ T cells purified from Tox4 WT and cKO mice


title: Multimodal characterization of murine gastruloid development

city: LAUSANNE

first: ALEXANDRE

last: MAYRAN

state: Waadt

address: Station 19

country: Switzerland

institute: EPFL

summary: Gastruloids are highly scalable, three-dimensional assemblies generated from pluripotent stem cells that recapitulate fundamental principles of embryonic pattern formation in vitro. Using single cell RNA and multiome sequencing we provide a comprehensive resource mapping cellular states and cell types found during gastruloid development and compare them to the in vivo embryo. We further develop a high throughput gastruloid handling and imaging pipeline to spatially monitor cell type emergence and unfolding of symmetry breaking during gastruloid development. We report spatial variability of pluripotency states in early gastruloids that determines a binary cell response to Wnt activation. While cells situated in the core of the gastruloid revert to an ectopic pluripotent state, peripheral cells differentiate to a primitive streak like state. These two populations then cause gastruloids to break radial symmetry, allowing axial elongation and commitment to the three embryonic germ layers. Finally by performing a phenotypic compound screen, we perturb thousands of gastruloids at relevant developmental time points deriving a phenotypic landscape and inferring molecular regulator networks underlying gastruloid development. Employing this resource, we improve the formation of anterior structures in the existing gastruloid model, using a dual Wnt modulation approach to differentiate an anterior ectopic pluripotent core to anterior ecto- and endodermal structures. This work gives is a resource to understand how gastruloids develop and, more generally, how homogenous cell populations can generate complex patterns in vitro.

overall_design: RNAseq from different timing of Wnt Pulse

first: Alexandre

last: Mayran

first: Simon

last: Suppinger

first: Nadim

last: Aizarani

first: Prisca

last: Liberali


title: Targeting TCA cycle through Cuproptosis confers Synthetic Lethality to ARID1A Deficiency Hepatocellular Carcinoma

city: Beiing

first: TAO

last: XING

state: Beiing

address: Beiing

institute: Peking University Cancer Hospital

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

sample_organism:
Homo
Homo sapiens

platform_organism:
Homo
Homo sapiens

overall_design: Refer to individual Series


title: Gene expression profile of cells from  Hey2 enhancer-LacZ transgenic (Tg) mouse embryos at embryonic day (E) 7.5.

city: Bunkyo-ku

first: Akiyasu

last: Iwase

state: Tokyo

department: Graduate School of Medicine

institute: The University of Tokyo

summary: We used single cell RNA sequencing (scRNA-seq) to analyze cells that activate the Hey2 enhancer in mouse embryos at E7.5.

overall_design: E7.5 Hey2 enhancer-LacZ Tg mouse embryos were collected, and distal part of extraembryonic tissue and posterior half of the embryos were removed. The remaining part of the embryos were used for single-cell (sc) RNA-seq.

first: Yusuke

last: Watanabe


title: Targeting TCA cycle through Cuproptosis confers Synthetic Lethality to ARID1A Deficiency Hepatocellular Carcinoma [ATAC-Seq]

summary: To further explore the mechanisms of  glucose metabolic reprogramming in ARID1A deficiency HCC, we conducted multi-omics profilings in ARID1A deficient and wild type Hep3B cells.

overall_design: ATAC-seq was performed to determine chromatin accessibility in ARID1A deficient and wild type Hep3B cells.

first: Tao

last: Xing


title: Impaired PPARɣ activation by cadmium exacerbates infection-induced lung injury

city: Birmingham

first: David

last: Crossman

state: AL

address: 720 20th Street South

institute: University of Alabama at Birmingham

summary: Emerging data indicates an association between environmental heavy metal exposure and lung disease, including lower respiratory tract infections (LRTIs). Here, we show by single cell RNA-sequencing an increase in Pparg gene expression in lung macrophages from mice exposed to cadmium and/or infected with S. pneumoniae. However, the heavy metal cadmium or infection mediated an inhibitory post-translational modification of peroxisome proliferator-activated receptor ɣ (PPARɣ) to exacerbate LRTIs. Cadmium and infection increased ERK activation to regulate PPARɣ degradation in monocyte-derived macrophages. Mice harboring a conditional deletion of Pparg in monocyte-derived macrophages had more severe S. pneumoniae infection after cadmium exposure, showed greater lung injury, and had increased mortality. Inhibition of ERK activation with BVD-523 protected mice from lung injury after cadmium exposure or infection. Moreover, subjects residing in areas of high air cadmium levels had increased cadmium concentration in their BAL fluid and showed PPARɣ inhibition that was mediated, at least in part, by ERK activation in isolated BAL cells. These observations suggest that impaired activation of PPARɣ in monocyte-derived macrophages exacerbates lung injury and the severity of LRTIs.

overall_design: 8- to 12-week-old male and female WT C57BL/6J mice were intratracheally administered 100 ng/kg of CdCl2 or saline, as a vehicle control. Mice were administered sterile saline or 10^3 Streptococcus pneumoniae (strain A66.1, type 3) i.t and were euthanized after 15 days. Bacterial infections were performed 5 days after cadmium exposure.

first: Jennifer

middle: L

last: Larson-Casey

first: Shanrun

last: Liu

middle: K

first: A

middle: B

last: Carter


title: Lactobacillus johnsonii N6.2 phospholipids induce immature-like dendritic cells with a migratory-regulatory-like transcriptional signature

first: Alexandra

middle: E.

last: Cuaycal

address: 1275 Center Drive

department: Infectious Diseases and Immunology/Biomedical Engineering

summary: Shifts in the gut microbiota composition, called dysbiosis, have been directly associated with acute and chronic diseases. However, the underlying biological systems connecting gut dysbiosis to systemic inflammatory pathologies are not well understood. Phospholipids (PLs) act as precursors of both, bioactive inflammatory and resolving mediators. Their dysregulation is associated with chronic diseases including cancer. Gut microbial-derived lipids are structurally unique and capable of modulating host’s immunity. Lactobacillus johnsonii N6.2 is a Gram-positive gut symbiont with probiotic characteristics. L. johnsonii N6.2 reduces the incidence of autoimmunity in animal models of Type 1 Diabetes and improves general wellness in healthy volunteers by promoting, in part, local and systemic anti-inflammatory responses. By utilizing bioassay-guided fractionation methods with bone marrow-derived dendritic cells (BMDCs), we report here that L. johnsonii N6.2 purified lipids induce a transcriptional signature that resembles that of migratory (mig)DCs. RNAseq-based analysis showed that BMDCs stimulated with L. johnsonii N6.2 total lipids upregulate maturation-mig related genes Cd86, Cd40, Ccr7, Icam1 along with immunoregulatory genes including Itgb8, Nfkbiz, Jag1, Adora2a, IL2ra, Arg1, and Cd274. Quantitative reverse transcription (qRT)-PCR analysis indicated that PLs are the bioactive lipids triggering the BMDCs response. Antibody-blocking of surface Toll-like receptor (TLR)2 resulted in boosted PL-mediated upregulation of pro-inflammatory Il6. Chemical inhibition of the IKKα kinase from the non-canonical NF-κB pathway specifically restricted upregulation of Il6 and Tnf. Phenotypically, PL-stimulated BMDCs display an immature like-phenotype with significantly increased surface ICAM-1. This study provides insight into the immunoregulatory capacity of Gram-positive, gut microbial-derived phospholipids on innate immune responses.

overall_design: mRNA profiles of bone marrow-derived dendritic cells stimulated with Lactobacillus johnsonii N6.2 purified lipids, alpha-galactosylceramide or the vehicle control were generated by paired-end RNA sequencing on a NovaSeq 6000 Illumina platform (instrument HWI-ST1276).

middle: E

first: Leandro

last: Teixeira

first: Graciela

last: Lorca

first: Claudio

middle: F

last: Gonzalez


title: The role of dietary fiber in microglial transcriptome

city: Clayton

first: Jan

last: Schroeder

state: VIC

address: Building 75 Innovation Walk

department: Anatomy & Developmental Biology

country: Australia

institute: Monash University

summary: To understand how dietary fiber affects microglial transcriptome at early stage, we fed mice diets releasing different amount of fiber and profiled microglia at 3 weeks. We found that fiber significantly altered the transcriptomic signature of microglia.

overall_design: Microglial RNA profile of WT and 5xFAD mice fed zero fiber, control or HAMSAB (high-amylose maize resistant starch modified with acetate and butyrate) diet

first: Yichen

last: Zhou

last: Schröder


title: Sleep-active hypothalamic MCH-system modulates hippocampal synaptic plasticity and is vulnerable in early Alzheimer’s Disease

city: Leuven

first: Kristofer

middle: James

last: Davie

address: Herestraat 49

department: Center for Brain and Disease Research

country: Belgium

institute: Vlaams Instituut voor Biotechnologie

summary: In Alzheimer’s disease (AD), pathophysiological changes in the hippocampus cause deficits in episodic memory formation, leading to cognitive impairment. Hippocampal hyperactivity and decreased sleep quality are associated with early AD, but their basis is poorly understood. We find that homeostatic mechanisms transiently counteract increased excitatory drive of hippocampal CA1 neurons in AppNL-G-F mice, but fail to stabilize it at control levels. Spatial transcriptomics (ST) analysis identifies the Pmch gene encoding Melanin-Concentrating Hormone (MCH) as part of the adaptive response in AppNL-G-F mice. Hypothalamic MCH peptide is produced in sleep-active lateral hypothalamic neurons that project to CA1 and modulate memory. We show that MCH downregulates synaptic transmission and modulates firing rate homeostasis in hippocampal neurons. Moreover, MCH reverses the increased excitatory drive of CA1 neurons in AppNL-G-F mice. Consistent with our finding that a reduced fraction of MCH-neurons is active in AppNL-G-F mice, these animals spend less time in rapid eye movement (REM) sleep. In addition, MCH-axons projecting to CA1 become progressively impaired in both AppNL-G-F mice and AD patients. Our findings identify the MCH-system as vulnerable in early AD and suggest that impaired MCH-system function contributes to aberrant excitatory drive and sleep defects, which can compromise hippocampal-dependent functions.

overall_design: Bulk RNA-seq on Hippocampal neurons cultured from E18 C57/Bl6 wild type mice treated with either MCH or vehicle.

first: Sara

last: Calafate

first: Gökhan

last: Özturan

first: Nicola

last: Thrupp

first: Jeroen

last: Vanderlinden

first: Luísa

last: Santa-Marinha

first: Rafaela

last: Morais-Ribeiro

first: Antonella

last: Ruggiero

first: Ivan

last: Bozic

first: Thomas

last: Rusterholz

first: Blanca

last: Lorente-Echeverría

first: Marcelo

last: Dias

first: Wei-Ting

last: Chen

first: Mark

last: Fiers

first: Ashley

last: Lu

first: Ine

last: Vlaeminck

first: Eline

last: Creemers

first: Katleen

last: Craessaerts

first: Joris

last: Vandenbempt

first: Luuk

last: van Boekholdt

first: Suresh

last: Poovathingal

first: Dietmar

middle: R

last: Thal

first: Keimpe

last: Wierda

first: Tiago

last: Gil-Oliveira

first: Inna

last: Slutsky

first: Antoine

last: Adamantidis

first: Bart

last: De Strooper

last: de Wit


title: 3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages

city: St Louis

first: Sabine

last: Dietmann

state: MO

address: 4565 McKinley Avenue

institute: Washington University School of Medicine

summary: Naïve human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures (“blastoids”) that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick 3D extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

overall_design: Single cell RNA seq profiles of blastoids generated from 5i/L/A naïve hPSCs at preimplantaiton (7 days) and post implantation (14 and 21 days)

first: Rowan

last: Karvas

first: Ali

last: Syed

first: Thorold

last: Theunissen


city: Tsu

first: Mikiya

last: Ishihara

state: Mie

address: 2-174 Edobashi

institute: Mie University Hospital

summary: RNA expression of unstimulated γδ-T cells,γδ-TCR-stimulated γδ-T cells, unstimulated αβ-TCR transduced γδ-T cells and αβ-TCR-stimulated αβ-TCR transduced γδ-T cells.

overall_design: RNA expression of γδ-T cells and αβ-TCR transduced γδ-T cells with or without TCR stimulation was compared.

first: Hiroshi

last: Fujiwara


title: RNA sequencing of circular RNAs, long non-coding RNAs ,and mRNAs in human keloid dermal tissues

first: Yunhan

last: Liu

address: 639 Zhizaoju Road,

department: Department of Plastic and Reconstructive Surgery

institute: Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: Keloid is a dermal fibroproliferative disease with various etiologies and unclear pathogenesis. Recent studies have revealed that circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) exerted regulatory functions through a competing endogenous RNA (ceRNA) pathway in keloid progression. However, the expression profiles of circRNAs and lncRNAs in keloid dermal tissues (KDTs) remain unknown. This study aimed to identify differentially expressed circRNAs, lncRNAs and genes in KDTs.

overall_design: Examination of RNA sequencing comparing the center of the keloid dermal tissue [C,n=4],  the margin of the keloid dermal tissue [M,n=3], and normal skin from the same patients [N,n=4].

first: Tao

last: Zan


title: Single-cell multi-omics sequencing reveals the functional regulatory landscape of STAT3-V637M Hyper-IgE syndrome

city: Shenzhen

first: Jiacheng

last: Zhong

address: Longmen north rd

institute: shenzhen people's hospital

summary: Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by, among others, the excessive production of IgE and repetitive bacterial/fungal infections. Although STAT3 is a known causative factor of HIES, the biological consequences of STAT3 mutation are not completely understood, especially at the epigenetic level. Here, we used multi-omic approaches to decipher the multidimensional immune disturbance in a male HIES patient harboring STAT3-V637M. In his peripheral blood mononuclear cell (PBMC) we found significant clonal expansion of CD8 T cells (whose expression of CD8 subunits was increased, resulting in enhanced responsiveness to MHC I molecules), but not CD4 T cells and B cells in his. The lack of T cells-B cells interaction could be responsible for the limited number of his B cells, although they exhibited a higher potential in producing immunoglobulin, elevated SPIC binding might bias this process toward IgE isotype production. Immune checkpoint inhibitors, including CTLA4, LAG3, were overexpressed in his PBMC-CD4 T cells, accompanied by reduced CD28 and IL6ST (gp130) expression. For his CD4 T cells, integrative analyses predicted upstream transcription factors (ETV6, KLF13, and RORA) for LAG3, IL6ST, and CD28, respectively. The down-regulated phagocytic, and nitric oxide synthetic genes in his PBMC-monocytes seem to be the culprit of his disseminated bacterial/fungal infection. In his bronchoalveolar lavage fluid (BALF), we identified two macrophages with anti-bacterial properties, which were characterized by CXCL8/S100A8/S100A9, or SOD2, respectively. Together, this study comprehensively described how the immune cell landscape was disturbed in STAT3-V637M HIES and provided a resource for further studies on STAT3 biology.

overall_design:
We collected PBMC and BALF from an 18-year-old male harboring STAT3-V637M. Paired PBMC and BALF samples collected from the patient were subjected to GEX/V(D)J-seq and scRNA-seq according to the manufacturer’s protocol, respectively. For PBMC, single cell suspension was loaded into 10x Genomics Chromium system using Chromium Next GEM Single Cell 5' v1.1 to generate TCR/BCR libraries and 5' GEX libraries. For BALF sample, Single Cell 3' v3 Reagent Kits (10X Genomics, Pleasanton, CA, USA) were used during library preparation. Next, libraries were sequenced on a NovaSeq 6000 sequencing system (Illumina, San Diego, CA, USA). Sequencing data were processed using the Cell Ranger 6.0.1 pipeline and aligned to GRCh38-1.1.0 reference genome to generate cell-by-gene count matrix (scRNA/GEX) or filtered contig annotations (scVDJ). For the patient’s PBMC, we additionally performed scATAC-seq using 10x Genomic single cell ATAC reagent v1.1 kit in accordance with the manufacturer’s protocol. Briefly, nuclei were isolated from PBMC and counted by Trypan Blue, scATAC-seq libraries were prepared following the 10X Genomics Chromium Single Cell ATAC Reagent Kits User Guide. Next, scATAC-seq libraries were sequenced using PE50 sequencing on NovaSeq 6000. Sequencing reads were processed through the Cell Ranger ATAC 1.1.0 pipeline and aligned to GRCh38-1.1.0 reference genome for mapping, de-duplication, and identification of Tn5 cut sites.

**Submitter declares that the raw data will be submitted to another designated repository due to the patient's privacy and national legislation**

first: Lingwei

last: Wang

first: Minzhi

last: Qiu

first: Shanze

last: Chen


title: METTL3-dependent m6A methylation facilitates uterine receptivity and female fertility via balancing estrogen and progesterone signaling

city: Guangzhou

first: Meixiang

last: Yang

state: Guangdong

address: No.601, West Huangpu Avenue

institute: Jinan University

summary: To investigate the effect of Mettl3 in the uterus, we generated mice with conditional ablation of Mettl3 in progesterone receptor (PR)–positive cells (PgrCre Mettl3 fl/fl). And the uterus of Mettl3 cKO and control mice on GD4 were obtained for RNA-seq analysis.

overall_design: We performed gene expression profiling analysis using data from RNA-seq of the uterus from 3 Mettl3 cKO mice and 3 wild type control mice on GD4.


title: Identification of differentially expressed genes in skeletal muscle endothelial cells during distal tumor growth

city: Chicago

first: Jalees

last: Rehman

state: IL

address: 909 S. Ashland Ave. MBRB 2072, Mailcode 669

department: Biochemistry and Molecular Genetics

institute: UIC

summary: To investigate whether muscle ECs contribute muscle atrophy, we investigated differentially expressed genes in muscle ECs from tumor bearing mice dependent on tumor growth compared with control mice by bulk RNAseq. We then performed gene expression profiling analysis using data obtained from RNA-seq of two different groups at four time points (0, 1-, 2-, or 3- weeks).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for skeletal muscle ECs from control and tumor bearing mice.

first: Young-Mee

last: Kim

first: Xinge


title: Uncovering the role of FOXA2 in the development of human serotonin neurons [RNA-seq]

city: Shanghai

first: Ting

last: Xu

address: 1239 Siping Road

institute: Tongji University

summary: Serotonin neurons (SNs) play a pivotal role in regulating brain development and function. Directed differentiation of SNs from human pluripotent stem cells (hPSCs) provides a useful tool for uncovering the mechanism of human SN development and the associated neuropsychiatric disorders. Previous studies reported that Foxa2 was expressed by serotonergic progenitors and functioned as a serotonergic fate determinant in mouse. However, the role of FOXA2 in human SNs development is unknown. Herein, we uncovered the role of FOXA2 through direct differentiation of FOXA2 lineage-tracing, FOXA2-knockout or inducible FOXA2-overexpression hPSCs into SNs. We found that SNs were not derived from FOXA2-lineage cells and FOXA2-knockout cells could still differentiate into mature and functional SNs, whereas FOXA2-overexpression enhanced the differentiation of floor plate (FP) cells rather than SNs. Coactivation of Sonic Hedgehog (SHH) and Retinoic acid (RA) signaling pathways promoted the differentiation towards SNs. In conclusion, FOXA2 is not intrinsically required for human SNs differentiation and simultaneous activation of SHH and RA signaling pathways could improve human SN differentiation by suppressing the differentiation towards FOXA2-positive FP cells. This study innovatively uncovers the role of FOXA2 in human SN development and provides a more efficient strategy to generate human SNs.

overall_design: Comparative gene expression profiling of RNA-seq data from cells differentiated from hESCs with different treatment. (C, CP, CR,CPR)

first: Jianfeng

last: Lu

first: Lining

last: Cao


title: R274X mutation in Phf6 enhances self-renewal and repopulation capacity of hematopoietic stem cells

city: Tianjin

first: Yanjie

last: Lan

address: 288 Nanjing Road

institute: Chinese Academy of Medical Sciences & Peking Union Medical College

summary: Purpose: Next-generation sequencing (NSG) has revolutionized system-based analysis of cellular pathways. The goals of this study are to compare Phf6R274X mice BM LSK cells transcription profiling (RNA-seq)  Method: BM LSK cells mRNA profiles of Phf6R274X mice and control were generated by deep sequencing, in triplicate, using illumine GAIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays.  Results: Using an optimized data analysis workflow, we identified 613 genes up-regulated and 133 genes down-regulated (P < 0.05) in the BM LSK cells from Phf6R274X and control mice. Altered expression of 7 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes may contribute to analyze PHF6R274X mutation function.  Conclusion: Our study represents the first detailed analysis of BM LSK cell transcriptomes with PHF6R274X mutation. Our results suggested that Phf6R274X mutation enhanced the regeneration capacity of hematopoietic cells mainly by promoting the activity of cell proliferation and differentiation-related signaling pathways.

overall_design: BM LSK cells from  Phf6R274X mouse model and controls .

first: Shengnan

last: Yuan


title: Gene expression profile at single cell level of muscle endothelial cells (ECs) and  muscle non-ECs from wild type and tumor bearing mice

summary: Endothelial cells are considered as a heterogeneous population dependent on vascular bed or patho/physiological conditions.  We used single cell RNA sequencing (scRNA-seq) to analyze the subpopulations of muscle ECs and non-ECS during cancer growth.

overall_design: muslce ECs and non-ECs of EC specific tdTomato mice with or without tumor were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of tdTomato signal and analyzed using scRNAseq.

first: Mark

last: Sanborn


title: Enhancer grammar of liver cell types and hepatocyte zonation states [cell_lines_ATAC]

first: Carmen

last: Bravo Gonzalez-Blas

institute: VIB-KU Leuven Center for Brain & Disease Research

summary: Cell type identity is encoded by gene regulatory networks (GRN), in which transcription factors (TFs) bind to enhancers to regulate target gene expression. In the mammalian liver, lineage TFs have been characterized for the main cell types, including hepatocytes. Hepatocytes cover a relatively broad cellular state space, as they differ significantly in their metabolic state, and function, depending on their position with respect to the central or portal vein in a liver lobule. It is unclear whether this spatially defined cellular state space, called zonation, is also governed by a well-defined gene regulatory code. To address this challenge, we have mapped enhancer-GRNs (eGRNs) across liver cell types at high resolution, using a combination of single-cell multi-omics, spatial omics, GRN inference, and deep learning. We found that zonated variation in gene expression in hepatocytes, liver sinusoidal endothelial cells and hepatocellular stellate cells corroborate cell state changes in transcription and chromatin accessibility with spatial transcriptomics. eGRN mapping suggests that zonation states in hepatocytes are driven by the repressors Tcf7l1 and Tbx3, that modulate the core hepatocyte GRN, controlled by Hnf4a, Cebpa, Hnf1a, Onecut1 and Foxa1, among others. To investigate how these TFs cooperate with cell type TFs, we performed an in vivo Massively Parallel Reporter Assay (MPRA) on 12,000 hepatocyte enhancers and used these data to train a hierarchical deep learning model (called DeepLiver) that exploits both enhancer accessibility and activity. DeepLiver confirms Cebpa, Onecut, Foxa1, Hnf1a and Hnf4a as drivers of enhancer specificity in hepatocytes; Tcf7l1/2 and Tbx3 as regulators of the zonation state; and Hnf4a, Hnf1a, AP-1 and Ets as activators. Finally, taking advantage of in silico mutagenesis predictions from DeepLiver and MPRA, we confirmed that the destruction of Tcf7l1/2 or Tbx3 motifs in zonated enhancers abrogates their zonation bias. Our study provides a multi-modal explanation of the regulatory code underlying hepatocyte identity and their zonation state, that can be exploited to engineer enhancers with desired activity levels and zonation patterns.

overall_design: 10x single cell multiome on the mouse liver

last: González-Blas

first: Stein

last: Aerts


title: Effects of dietary folic acid on methylation status of colon non-neoplastic epithelium and dysplasia in mice with colitis

city: Camden

first: Jaroslav

last: Jelinek

state: NJ

address: 403 Haddon Avenue

department: Research Labs

institute: Coriell Institute for Medical Research

type: Methylation profiling by high throughput sequencing

summary: Folic acid supplementation (8 mg/kg diet) promotes colon tumor formation in mice with established colitis induced by carcinogen azoxymethane (AOM) and dextran sulfate sodium sulfate (DSS). This induction of colon tumors was associated with hypomethylation of DNA cased by folic acid supplementation.

overall_design: Mice were fed a diet containing folic acid 2 mg/kg chow (basal level), followed by carcinogen azoxymethane injection. Colitis was induced in all mice using 4 cycles of DSS. Immediately after the first DSS treatment, mice were randomized to receive folic acid 0 mg/kg chow (folic acid deficient), 2 mg/kg chow (folic acid basal level) or 8 mg/kg chow (folic acid supplemented) for 16 weeks. At the end of study, mice were euthanized and colons were collected and process for gene expression analyses.

first: Wen

last: Chang

first: Jayashri

last: Ghosh

first: Harry

last: Cooper

first: Lisa

last: Vanderveer

first: Bryant

last: Schultz

first: Yan

last: Zhou

first: Esther

last: Kaunga

first: Karthik

last: Devarajan

first: Yuesheng

last: Li

last: Sapienza

first: Margie

last: Clapper


title: Transcription factor stoichiometry, motif affinity and syntax regulate single-cell chromatin dynamics during fibroblast reprogramming to pluripotency

status: Public on Sep 06 2023

city: Palo Alto

first: Surag

last: Nair

address: 240 Pasteur Dr

department: Computer Science

institute: Stanford University

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

overall_design: Refer to individual Series


title: Antigen perception in T cells by long-term Erk and NFAT signaling dynamics

city: Seattle

first: Hao

middle: Yuan

last: Kueh

state: WA

address: 3720 15th Ave NE

department: Bioengineering

institute: University of Washington


title: Antigen perception in T cells by long-term Erk and NFAT signaling dynamics [scRNAseq]

summary: Immune system threat detection hinges on T cells’ ability to perceive varying peptide major-histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs, but diverge only over longer (9+ hrs) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception, and establish a framework for understanding T cell responses under diverse contexts.

overall_design: CD8+ T cells were isolated from splenocytes and stimulated with 5 different pMHC conditions, or rested, for 30 hrs.

first: Matthew

middle: J

last: Wither

first: William

last: White

first: Sriram

last: Pendyala

first: Paul

last: Leanza

first: Doug

last: Fowler

middle: Y


title: The retinol-binding protein receptor STRA6 contributes to the pathogenesis of adenomyosis via Wnt/β-catenin pathway depression

city: Beijing

first: Qiu

last: Xie

state: Beijing

address: No.1 Shuaifuyuan Wangfujing Dongcheng District

institute: Peking Union Medical College Hospital


title: Transcription factor stoichiometry, motif affinity and syntax regulate single-cell chromatin dynamics during fibroblast reprogramming to pluripotency [multiome]

summary: Ectopic expression of OCT4, SOX2, KLF4 and MYC (OSKM) transforms differentiated cells into induced pluripotent stem cells. To refine our mechanistic understanding of reprogramming, especially of the early stages, we profiled chromatin accessibility and gene expression at single-cell resolution across a finely sampled time course of human fibroblast reprogramming. Using neural networks that map DNA sequence to ATAC-seq profiles at base-resolution, we annotated cell-state-specific predictive transcription factor (TF) motif syntax in regulatory elements, inferred affinity- and concentration-dependent dynamics of Tn5-bias corrected TF footprints, linked peaks to putative target genes, and elucidated rewiring of TF-to-gene cis-regulatory networks. Our models reveal that early in reprogramming, OSK, at supraphysiological concentrations, rapidly open transient regulatory elements by occupying non-canonical low-affinity binding sites. As OSK concentration falls, the accessibility of these transient elements decays as a function of motif affinity. We find that these OSK-dependent transient elements sequester the somatic TF AP-1. This redistribution is strongly associated with the silencing of fibroblast-specific genes within individual nuclei. Together, our integrated single-cell resource and models reveal insights into the cis-regulatory code of reprogramming at unprecedented resolution, connect TF stoichiometry and motif syntax to diversification of cell fate trajectories, and provide new perspectives on the dynamics and role of transient regulatory elements in somatic silencing.

overall_design:
In this study, we profiled chromatin accessibility and gene expression at single cell resolution across a finely sampled, 14 day time course of OSKM induced reprogramming of human fibroblasts. In addition, we jointly profiled chromatin accessibility and gene expression at single nucleus resolution at days 1 and 2.
**Note from submitter: RAW DATA not provided due to patient privacy**

first: Mohamed

last: Ameen

first: Kevin

last: Wang

first: Anshul

last: Kundaje


title: Transcription factor stoichiometry, motif affinity and syntax regulate single-cell chromatin dynamics during fibroblast reprogramming to pluripotency [scATAC]

overall_design:
In this study, we profiled chromatin accessibility and gene expression at single cell resolution across a finely sampled, 14 day time course of OSKM induced reprogramming of human fibroblasts. In addition, we jointly profiled chromatin accessibility and gene expression at single nucleus resolution at days 1 and 2.
**note from submitter: RAW DATA not provided due to patient privacy**

first: Ioannis

last: Karakikes


title: Hedgehog signaling is required for the maintenance of mesenchymal nephron progenitors

city: Cincinnati

first: Hee-Woong

last: Lim

state: Ohio

address: 3333 Burnet Ave. MLC 7024

department: Division of Biomedical Informatics

institute: Cincinnati Children's Hospital Medical Center

summary: The goal of our study is to identify the genes regulated by Hedgehog signaling in mesenchymal nephron progenitors.

overall_design: Six2GFPcre+ nephron progenitors with different Smo doses at E18.5 were isolated by FACS and subjected to RNA-seq. Smo KO = Smo c/c ; Six2GFPcre, Smo Het = Smo c/+ ; Six2GFPcre, Smo WT = Smo +/+ ; Six2GFPcre, Smo Transgenic = Rosa26 SmoM2/+ ; Smo +/+ ; Six2GFPcre

first: Joo-Seop

last: Park

first: Eunah

last: Chung


title: Antigen perception in T cells by long-term Erk and NFAT signaling dynamics [RNAseq]

overall_design: CD8+ T cells were isolated from splenocytes and stimulated with high affinity antigen stimulation (2 pmol M9C). After 9 hours, cells were treated with either 1 uM Trametinib (MEK inhibitor), 1 uM Cyclosporin A (Calcineurin inhibitor), both, or DMSO (0.1%), and cultured for an additional 21 hours.


title: SF3B1 mutation and ATM deletion co-drive leukemogenesis via centromeric R-loop dysregulation

city: Monrovia

first: Lili

state: CALIFORNIA

address: 1218 South Fifth Ave.

department: Systems biology

institute: City of Hope

summary: RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematological malig-nancies. We previously reported that co-expression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrate that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segrega-tion, altered spindle architecture and aneuploidy, which can be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM.  Aberrant splicing of key genes involved in R-loop processing underlies augmentation of cen-R-loop as overexpression of the normal isoform, but not the altered form, mitigates mitotic stress in SF3B1 mutant cells. Our study underscores the critical role of novel splice variants in link-ing RNA splicing dysregulation and CIN, and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.

overall_design: DNA:RNA hybrids (R-loop) were profiled in Nalm-6 SF3B1-K700K and SF3B1-K700E by high troughput sequencing. DNA:RNA hybrids immunoprecipitation (DRIP) were performed with S9.6 antibody extracted from hybridoma cell line. RNA analysis were performed in SF3B1-K700K and SF3B1-K700E HEK293T cells.

first: Martina

last: Cusan


title: Embryonic exposure to aluminium chloride blocks the onset of spermatogenesis through disturbing the dynamics of testicular tight junctions via upregulating Slc25a5 in offspring

city: Nanjing

first: Chen

last: junhan

address: Nanjing University, Hunan Road Street, Gulou District, Nanjing City

institute: Nanjing University

summary: Studies reveal neurotoxicity, hepatotoxicity, and developmental and reproductive toxicity in mice exposed to aluminium (Al). However, relatively few researches are conducted to clarify the mechanism of the impact of embryonic exposure to aluminium chloride (AlCl3) on the development of the reproductive system in male offspring. Here, pregnant mice were fed AlCl3 by gavage from day 12.5 of gestation. The results showed that embryonic AlCl3 exposure disrupted testicular development and spermatogenesis by damaging the testicular structure, reducing sperm counts, and altering the expression of the tight junction (TJ) proteins between Sertoli cells (SCs). Further in vitro studies showed that AlCl3 treatment stabilized the TJ proteins Occludin and ZO-1 expression by inhibiting the ERK signaling pathway to upregulate the Slc25a5 protein, which prompted ATP production to disrupt cytoskeletal protein homeostasis. Therefore, our study provided a new mechanism for AlCl3 exposure to interfere with testicular development and spermatogenesis and implied that Slc25a5 could be a target of AlCl3 affecting cell metabolism.

overall_design: TM4 cells were incubated with AlCl3 for 30 days.The mRNAs were then validated by qPCR at the animal level, finally screening out the differentially expressed target gene Slc25a5.

first: Junhan

last: Chen

first: Yunhui

last: Xia

first: Yu

last: Ben

first: Xinyan

last: Lu


title: Optimisation of cell fate decisions for cultured muscle differentiation

city: Maastricht

first: Tobias

last: Messmer

address: Watermolen 28

department: Cell Biology

institute: Mosa Meat

summary: Production of cultured meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Whilst such formulations can drive myogenic differentiation to an extent similar to serum-starvation based protocols, these cultures are visually heterogeneous in nature, with a significant proportion of cells not participating in myofusion, limiting the maturation of the muscle. Here, we use RNA sequencing to characterise this heterogeneity at single-nucleus resolution, identifying distinct cellular subpopulations, including proliferative cells that fail to exit the cell cycle, and 'reserve cells' that do not commit to myogenic differentiation. By targeting the ERK, RXR and NOTCH pathways, we show that cell cycle exit can be promoted whilst simultaneously abrogating reserve cell formation. Under these improved culture conditions, fusion indices close to 100% can be robustly obtained in 2D culture. We further show that this translates to higher levels of myotube formation and muscle protein accumulation in animal-free bioartificial muscle (BAM) constructs, providing proof of principle for the generation of highly differentiated cultured muscle with excellent mimicry to traditional meat.

overall_design: To study transcriptional hetereogeneity during myogenic differentiation, Bovine satellite cells (SCs) were differentiated using a serum-free differentiation media, harvested on subsequent days (0 h, 24 h, 48 h, 72 h, 96 h) and analysed with single-nucleus RNA sequencing.

first: Lea

last: Melzener

first: Lieke

last: Schaeken

first: Marion

last: Fros

first: Dhruv

last: Raina

first: Annemarie

last: Kiessling

first: Guy

last: Wijler

first: Tessa

last: van Haaften

first: Sergio

last: Spaans

first: Aring

last: Dogan

first: Mark

last: Post

first: Joshua

last: Flack


title: Transcription factor stoichiometry, motif affinity and syntax regulate single-cell chromatin dynamics during fibroblast reprogramming to pluripotency [scRNA]

first: Surag

last: Nair

first: Mohamed

last: Ameen

first: Kevin

last: Wang

first: Anshul

last: Kundaje


title: The retinol-binding protein receptor STRA6 contributes to the pathogenesis of adenomyosis via Wnt/β-catenin pathway depression [microRNA]

first: Qiu

last: Xie

summary: Adenomyosis is an estrogen-dependent disease in which endometrial glands and stroma are pathologically demonstrated in the myometrium. Despite its prevalence and severity of symptoms, the precise etiology and physiopathology of adenomyosis is not well understood.Vitamin defciency increase in women with adenomyosis. Much less is known about the mechanism of their relationship. We found the retinol-binding protein receptor STRA6 upregulation in uterinespiral arteries from adenomyosis patients. The increased estrogen level promote the STRA6 expression, resulting the depressed Wnt/β-catenin signaling mediated by FASN/GSK-3β interaction. Overexpression of STRA6 induced epithelial to mesenchymal transition process. miR-1249-5p achieved its negative function by regulating the target gene through 3' UTR.The depletion of STRA6 in vivo alleviated the pathogenesis of adenomyosis. These results reveal a hitherto undesribed positive feedback loop between estrogen and STRA6 in adenomyosis pathogenesis. Components of vitamin A metabolism network have more complex activities than simply controlling or transferring VA compounds between different cells.

overall_design: To investigate the pathway through which estrogen participates in the the pathogenesis of adenomyosis, microRNA sequencing were performed on rabbit samples treated with different concentrations of estrogen.  We then performed gene expression profiling analysis of RNA-seq obtained from 3 concentrations of estrogen.


title: Effect of Streptomyces roseolus cell free supernatant on fungal development, transcriptome and aflatoxin B1 production of Aspergillus flavus

city: Castanet – Tolosan

first: Selma

middle: Pascale

last: Snini

address: Avenue Agrobiopole BP 32607

country: France

institute: Chemical Engineering Laboratory

summary: Frequently observed in tropical and sub-tropical regions, crops contamination by aflatoxin B1 (AFB1) produced by Aspergillus flavus, is emerging in Europe, due to climate change. Many alternative methods are currently developed to reduce the use of chemical inputs to prevent mycotoxin contamination, such as biocontrol agents (BCAs). Actinobacteria are known to produce many bioactive compounds and some of them are able to reduce in vitro AFB1 concentration. In this context, the present study aims to analyze the effect of a cell free supernatant (CFS) from Streptomyces roseolus liquid culture on A. flavus development, as well as on its transcriptome profile using microarray assay and its impact on AFB1 concentration. To study the impact of Streptomyces roseolus cell free supernatant on global transcriptome of Aspergillus flavus we have employed whole genome microarray expression profiling.

overall_design: A. flavus was exposed to Streptomyces roseolus cell free supernatant at 1.5 g/L during 5 days at 28°C.  Four independent experiments were performed.

first: Louise

last: Maud

first: Florian

last: Boyer

first: Florence

last: Mathieu

middle: P

first: Claire

last: Naylies

first: Yannick

last: Lippi


title: Gene expression profilings in human induced pluripotent stem cell-derived cardiomyocytes in vivo and in vitro

city: Matsumoto

first: Shin

last: Kadota

address: 3-1-1 Asahi

department: Regenerative Science and Medicine

institute: Shinshu University

summary: RNA sequencing of human induced pluripotent stem cell-derived cardiomyocytes

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: There are 8 RNA-sequencing samples

first: Yuji

last: Shiba


title: Transcriptomic profile of hypodermal adventitia fibroblasts from Csf1f/f and Tek-crexCsf1f/f mice

city: Bethesda

first: Keisuke

last: Nagao

state: MD

address: 10 Center Dr, Bldg 10, 12N240B

institute: National Institutes of Health

summary: In order to explore and compare the transcriptomic profile of hypodermal adventitia fibroblasts, fibroblasts were isolated via flow cytometry mouse hypodermis and were subjected to RNA-sequencing analysis.

overall_design: Back skins from age-matched Csf1f/f and Tek-crexCsf1f/f mice were harvested and hypodermal adventitial was isolated by mechanical force and enzymatic treatments. Cells were liberated after enzymatic digestion of the tissues. Fibroblasts (CD45- CD31- Sca-1+ CD34+) were isolated by fluorescence-activated cell sorting (FACS) and their transcriptome assessed by RNA-sequencing.

first: Benjamin

last: Voisin

first: Stephen

last: Brooks


title: Adiponectin in the mammalian host influences ticks' acquisition of the Lyme disease pathogen Borrelia

city: Hangzhou

first: Xiaotian

last: Tang

state: Zhejiang

address: Agro-Bio-Environ Building, Zhejiang University

department: Entomology

institute: Zhejiang University

summary: To understand the global gene expression profile at the tick bite site of adiponectin wildtype and knockout mice

overall_design: We utilized RNA-seq to compare the transcriptome of adiponectin wildtype and knockout murine skin during tick feeding

first: Erol

last: Fikrig


title: Thioredoxin-1 promotes the restoration of alveolar bone in periodontitis with diabetes

first: Zhi

last: Song

address: No. 56, Lingyuan West Road, Guangzhou 510055, China

institute: Sun Yat-Sen University

summary: Treatment of periodontitis in people with diabetes remains challenging. Diabetes-enhanced oxidative stress is a primary cause of aggravation of periodontitis. The present study aimed to investigate the therapeutic potential of thioredoxin-1 (TRX1), an essential endogenous antioxidant protein, in the management of periodontitis with diabetes, as well as its role in modulating osteogenic differentiation. Our findings indicated that the production of reactive oxygen species (ROS) was elevated, while the expression of TRX1 was significantly reduced in the periodontal tissues of periodontitis mice with diabetes. Furthermore, knockdown of TRX1 in periodontal ligament stem cells (PDLSCs) resulted in the inhibition of osteogenic differentiation through disrupting Wnt/β-catenin signal pathway. However, this inhibition was restored upon administration of recombinant human TRX1 (rhTRX1). Importantly, rhTRX1 treatment decreased ROS generation, activated Wnt/β-catenin signal pathway and considerably promoted the alveolar bone repair of periodontitis mice with diabetes. These findings highlighted the crucial protective role of TRX1 in periodontitis with diabetes and suggested that it may serve as a potential therapeutic target for refractory periodontitis associated with oxidative stress.

overall_design: To explore the mechanism underlying the regulatory function of thioredoxin (TRX1) in osteogenic differentiation, we used LV-sh-TRX1 vector to transfact human periodontal ligament sten cells (PDLSCs) to knock down the TRX1 expression. We then performed RNA sequencing comparing TRX1 knockdown PDLSCs and normal PDLSCs under osteogenic induction. The differentially expressed genes (DEG) were further analysed.


title: Transcriptome analysis of CARNMT1 knockout tissue

city: Hirakata

first: Rei

last: Yoshimoto

state: Osaka

address: 45-1 Nagaotoge-cho

department: Faculty of Agriculture

institute: Setsunan University

summary: Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we showed that CARNMT1, a known His methyltransferase of dipeptide carnosine (βAla-His), is the major protein His N-position-specific methyltransferase. ProSeAM labeling and proteomic analysis revealed that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, which is a C3H zinc finger (C3H-ZF) motif. CARNMT1-deficient and catalytically-inactive mutant mice showed embryonic lethality. Among the CARNMT1-target C3H-ZF proteins, RNA degradation mediated by Roquin and TTP was affected by CARNMT1 and its enzymatic activity. Furthermore, splicing factor, U2AF1’s 3’-splice site recognition was perturbed and pre-mRNA alternative splicing (AS) was also affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H-ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and regulation of mRNA degradation.

overall_design: Examination of transcriptome of CARNMT1 knockout mice by Ribosomal RNA depleted RNA-Seq

first: Tadahiro

last: Shimazu


title: Single Cell Sequencing of a Novel Model of Neonatal Bile Duct Ligation in Mice Identifies Etiology-Specific Macrophage Function Compared to Murine Biliary Atresia

first: Deborah

last: Winter

address: 240 E. Huron Street, M300

institute: Northwestern University

summary: Background & Aims: Macrophages (MF) play a role in neonatal etiologies of obstructive cholestasis, however, the role for precise MF subsets remains poorly defined.  We developed a neonatal murine model of bile duct ligation (BDL) to characterize etiology-specific differences in neonatal cholestatic MF polarization.  Approach & Results: Neonatal BDL surgery was performed on female BALB/c mice at 10 days of life (DOL) with sham laparotomy as controls.  Comparison was made to the Rhesus Rotavirus (RRV)-induced murine model of biliary atresia (BA).  Evaluation of changes at day 7 after surgery (BDL and sham groups) and murine BA (DOL14) included laboratory data, histology (H&E, anti-CD45 and anti-CK19 staining), flow cytometry of MF subsets by MHCII and Ly6c expression, and single cell RNA-sequencing (scRNA-seq) analysis.  Neonatal BDL achieved a 90% survival rate; mice had elevated bile acids, bilirubin, and alanine aminotransferase (ALT) versus controls (p < 0.05 for all).  Histology demonstrated hepatocellular injury, CD45+ portal infiltrate, and CK19+ bile duct proliferation in neonatal BDL.  Comparison to murine BA showed increased ALT in neonatal BDL despite no difference in histology Ishak score.  Neonatal BDL had significantly lower MHCII-Ly6c+ MF versus murine BA, however, scRNA-seq identified greater etiology-specific MF heterogeneity with increased endocytosis in neonatal BDL MF versus cellular killing in murine BA MF.  Conclusions: We generated an innovative murine model of neonatal obstructive cholestasis with low mortality.  This model enabled comparison to murine BA to define etiology-specific cholestatic MF function.  Further comparisons to human data may enable development of immune modulatory therapies to improve patient outcomes.

overall_design: CITE-seq (Cellular Indexing of  Transcriptomes and Epitopes) on CD45+CD3-CD19-Siglec-F-Ly6g-  cells from neonatal BDL, BA and control mouse models.

first: Sarah

middle: A

last: Taylor


title: Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells

city: Bern

first: Oliver

last: Mühlemann

address: Freiestrasse 3

department: Chemistry, Biochemistry and Pharmaceutical Sciences

institute: University of Bern

summary: Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that targets for degradation faulty mRNAs with premature termination codons as well as many physiological mRNAs encoding full-length proteins. Consequently, NMD functions in both, quality control and post-transcriptional regulation of gene expression, and it has been implicated in the modulation of cancer progression. To investigate the role of NMD in cancer, we knocked out SMG7 in the HT1080 human fibrosarcoma cell line. SMG7 is involved in deadenylation-coupled exonucleolytic mRNA decay, one of the two main degradation pathways in mammalian NMD. Genome-wide proteomic and transcriptomic analyses confirmed that NMD is severely compromised in these SMG7-knockout HT1080 cells. We compared the oncogenic properties between the parental, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. In parallel, we tested the effect of a drug inhibiting the NMD factor SMG1 on the HT1080 cells to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays as well as a mouse xenograft tumor model, we show that the oncogenic properties of the parental HT1080 cells are severely compromised when NMD is inhibited. Molecular pathway analysis revealed a strong reduction of the matrix metalloprotease 9 (MMP9) gene expression in NMD-suppressed cells. Since MMP9 expression promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation in NMD-suppressed cells explains, at least partially, their reduced tumorigenicity. Collectively, our findings emphasize the therapeutic potential of NMD inhibition for the treatment of certain types of cancer.

overall_design: Comparative gene expression analysis of RNAseq data from HT1080 and two independent SMG7KO clones.

first: Sofia

last: Nasif

first: Martino

last: Colombo

first: Anne-Christine

last: Uldry

first: Markus

middle: S

last: Schröder

first: Simone

last: de Brot


title: Desmocollin-1 is associated with pro-metastatic phenotype of luminal A breast cancer cells and is modulated by parthenolide

city: Brno

first: Pavel

last: Bouchal

address: Kamenice 5

department: Biochemistry

country: Czech Republic

institute: Masaryk University, Faculty of Science

summary:
Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast tumors and was found to increase metastatic potential of MCF7 cells in vitro.
Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation using parthenolide.
We performed comparative gene expression analysis using data obtained from RNA-seq of DSC1-overexpressing and control cells treated and untreated with parthenolide in biological duplicates.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MCF7-DSC1-GFP cells and control cell line MCF7-GFP treated and untreated with parthenolide.

first: Petr

last: Lapcik

last: Sulc

first: Lucia

last: Janacova

first: Katerina

last: Jilkova

first: David

last: Potesil

first: Pavla

last: Bouchalova

last: Muller


title: Single-cell epigenomic analysis of hypodermal macrophages from Csf1r-cre x Igf1-flox mice.

first: Keisuke

last: Nagao

summary: To explore the effect of cell-autonomous Igf1 expression on hypodermal macrophages, these cells were isolated from Csf1r-cre x Igf1-flox mice and subjected to single-cell ATAC-sequencing.

overall_design: Skins from Csf1r-cre x Igf1-flox and Igf1-flox mice were harvested and the hypodermal layers isolated by mechanical force and enzymatic treatments. Cells were liberated after enzymatic digestion of the tissues. Macrophages were isolated  by fluorescence-activated cell sorting (FACS) and their chromatin accessibility assessed by single-cell ATAC-sequencing using the 10X genomics plateform.

first: Benjamin

last: Voisin

first: Shubham

last: Goel


title: HUB genes transcriptionally regulate lipid metabolism in alve-olar type II cells under the condition of LPS stimulation

city: Guiyang city

first: xianjun

last: chen

state: 贵州

address: 28 Guiyi Street, Yunyan District, Guiyang city, Guizhou Province, China

department: Critical care medicine

institute: Affiliated Hospital of Guizhou Medical University

summary: Pulmonary surfactant (PS) produced by alveolar type II (ATII) cells is necessary in maintaining normal lung function, and a decrease or change in composition of PS is the main cause of alveolar collapse in acute respiratory distress syndrome (ARDS). But the mechanism of decrease or com-position change of PS is still unknown.

overall_design: We used transcriptional RNA-seq to investigate the mechanism of PS disorders in normal and LPS-injured ATII cells, the key location of ARDS pathogenesis, hoping to explore the mechanism of PS reduction or component change.

first: Xianjun

last: Chen

first: Chuan

last: Xiao

first: Ying

last: Liu

first: Qing

last: Li

first: Yumei

last: Cheng

first: Shuwen

first: Wei

first: Jia

last: Yuan

last: Wang

first: Feng

last: Shen


title: Atlas of miRNome variations in different dyslipidemic conditions: a study in C57Bl/6, Ldlr-KO, and Pcsk9-KO mice.

city: Milan

first: Matteo

last: Chiara

address: via celoria 26

department: Biosciences

country: Italy

institute: University of Milan

summary: In this study, mice with different genotypes and fed diets with different lipid content were enrolled, aiming to set up an atlas of miRNA expression levels in different organs with a relevant role in lipid/lipoprotein metabolism. Specifically, three genotypes were investigated: C57Bl/6 mice as controls, together with mice knock-out (KO) for LDLr (low-density lipoprotein receptor) and for PCSK9 (proprotein convertase subtilisin/kexin type 9). LDLr and PCSK9 are both involved in LDL turnover, the former mediating LDL clearance [PMID: 19299327], the latter causing the degradation of the LDLr protein [PMID: 17080197]. As a result, LDLrKO mice, because of their impaired LDL catabolism, are hypercholesterolemic and prone to atherosclerosis development, particularly when fed high-fat, cholesterol-containing diets [PMID: 8349823; PMID: 8182121]. On the contrary, PCSK9KO mice, characterized by an accelerated LDL catabolism, are hypocholesterolemic and atherosclerosis resistant [PMID: 15805190]. miRNA expression was investigated in liver, intestine, aorta, white adipose tissue and brain of mice on both standard and Western diet.

overall_design: short RNA sequencing of 7 organs, in  3 different genetic backgrounds, under 2 different diets

first: Marco

last: Busnelli

first: Stefano

last: Manzini

first: Alice

last: Colombo

first: Gaia

last: Zaffaroni

last: Horner

first: Giulia

last: Chiesa


title: The retinol-binding protein receptor STRA6 contributes to the pathogenesis of adenomyosis via Wnt/β-catenin pathway depression [RNA]

first: Qiu

last: Xie

overall_design: To investigate the pathway through which estrogen participates in the the pathogenesis of adenomyosis, mRNA sequencing were performed on rabbit samples treated with different concentrations of estrogen.  We then performed gene expression profiling analysis of RNA-seq obtained from 3 concentrations of estrogen.


title: Transcriptomic analysis reveals the dynamic changes of immune responses during the development of creeping fat in Crohn's disease

city: Seoul

first: SoJung

last: Park

state: Songpa-gu

address: 88, Olympic-ro 43-gil

country: South Korea

institute: Ulsan medical university

summary: In Crohn's disease, creeping fat is the characteristic expansion of mesenteric adipose tissue wrapping around the inflamed intestine. Through a comparative transcriptomic analysis of creeping fat and normal-looking mesenteric adipose tissues from patients with Crohn's disease and non-Crohn's disease, we found that a dynamic transcriptional and cell compositional change occurs during the progression from non-Crohn's disease to Crohn's disease, and finally to creeping fat.

overall_design:
Comparative gene expression profiling analysis of RNA-seq data for patients with CD CF, CD MAT and Con MAT.

**Submitter declares that the raw data cannot be deposited due to hospital policy**

first: Kyuwon

last: Kim

first: Sojung

first: Yoonho

last: Lee