title: Maternal IgG Affects Brain Development of Mouse Pups

city: Isehara

first: Takatoshi

last: Iijima

state: Kanagawa

address: 143 Shimokasuya

department: School of Medicine

institute: Tokai University

summary: Maternal IgG is an immunoglobulin present in breast milk and has been shown to play a role in the development of the immune system in infants. The aim of this study was to investigate the effects of maternal IgG on brain development in mouse pups. Maternal IgG immunoreactivity was observed in microglial cells of the pup brain, peaking at postnatal 8 day. In particular, strong IgG immunoreactivity was observed in the microglia in the corpus callosum and cerebellar white matter. IgG stimulation of primary cultured microglia resulted in activation of the type I interferon feedback loop by Syk. Among the IgG subclasses, IgG2a was found to have a strong influence on microglia. Analysis of FcRn KO mice that were unable to take up IgG from their mothers revealed that these mice had abnormalities in the proliferation and/or survival of microglia, oligodendrocytes, and some types of interneurons. FcRn KO mice also exhibited abnormalities in social behavior and adaptation to novel environments. This study suggests that maternal IgG affects brain development in pups.

overall_design: To comprehensively explore the molecular changes in IgG-stimulated microglia at transcriptome-wide level, the transcriptomic profiles were compared between IgG-stimulated microglia and untreated controls (n = 4 cultures/group).

first: Tetsushi

last: Sadakata

first: Mizuki

first: Keizo

last: Takao

first: Ryosuke

last: Kaneko

first: Emi

last: Hosoya

first: Hisako

last: Sugimoto

first: Reiko

last: Kawabata

first: Tetsuhiro

last: Kasamatsu


title: Berberine directly targets AKR1B10 protein to modulate lipid and glucose metabolism disorders in NAFLD

status: Public on May 24 2024

city: Tianjin

first: Sa

last: Yang

address: 10 Poyanghu Road, West Area, Tuanbo New Town, Jinghai District

country: China

institute: Tianjin University of Traditional Chinese Medicine

type: Expression profiling by high throughput sequencing

summary: RNA-seq was used to investigate the downstream pathways of the target.

overall_design: The liver mRNA profiles in AKR1B10 knockdown mice


title: Whole-blood transcriptome profile in children with sickle cell anemia before and after exercise

city: Irvine

first: ABEL

last: PLAZA FLORIDO

state: California

address: 101 Academy, Suite 150 Irvine

department: Department of Pediatrics, UC Irvine School of Medicine

country: USA

institute: Pediatric Exercise and Genomics Research Center (PERC)

summary: Patients with sickle cell anemia (SCA) show higher levels of circulating pro-inflammatory cytokines and endothelial activation markers compared to healthy peers and altered blood transcriptome profiles. However, the characterization of the whole-blood transcriptome profile in response to exercise has not been evaluated in children with SCA. Twenty-three children/adolescents with SCA (10-19 years old) and 17 healthy controls (10-22 years old) performed eight 2-min bouts of cycle ergometry interspersed with 1-min rest intervals (50% or 70% of the peak workload achieved during the cardiopulmonary exercise test). Whole-blood transcriptome profile (RNA-seq) was performed before and after the exercise protocol. Following exercise, gene pathways associated with innate immunity were altered in both children with SCA and controls. Our results shed light on the common and different immune responses to acute exercise at the molecular level in children with SCA and healthy controls.

overall_design: The exercise protocol consisted of 8 bouts (2 minutes duration each with 1-minute rest between bouts) of cycling at a constant workload (randomized to 50% or 70% of the peak workload achieved during the CPET). This type of interval exercise challenge mimics the movement patterns of children during play. Blood samples were collected before (baseline) and immediately after completing the exercise protocol using PAXgene blood RNA tubes (PreAnalytiX; PN 762165). The storage of the PAXgene tubes after blood collection was as recommended by the manufacturer. The RNA was extracted using the PAXgene blood RNA kit (PreAnalytiX; PN 762174) and automated on the QIACube (Qiagen).

first: Abel

last: Plaza-Florido

first: Robert

middle: I

last: Liem

first: Fadia

last: Haddad

first: Shlomit

last: Radom-Aizik


title: Transcriptional profiling of striatum from a zQ175 KI mouse model of Huntington's disease with or without homozygous knockout of the Msh3 gene

city: Princeton

first: Jeff

last: Aaronson

state: New Jersey

address: 155 Village Boulevard, Suite 200

department: Discovery & Systems Biology

institute: CHDI

summary: Previous studies have suggested a strong association between lower MSH3 expression, reduced somatic repeat expansion, and slower disease onset and progression. The effects of a homozygous knock-out in the Msh3 gene on transcriptome phenotypes in striatum in six-month-old WT and zQ175 mice were studied. Striatum was isolated from 6-month-old mice. Transcriptomic analysis (RNASeq) was performed on four genotypes with 8 replicates per genotype: WT (WT for Htt and Msh3), zQ175 (zQ175 for Htt, WT for Msh3), WT.Msh3.HOM (WT for Htt, Homozygous for Msh3 KO), and zQ175.Msh3.HOM (zQ175 for Htt, Homozygous for Msh3 KO).

overall_design: RNASeq analysis was performed on samples from the striatum tissues of 6-month-old mice with one of the following four genotypes: WT, WT.Msh3.HOM, zQ175, zQ175.Msh3.HOM. N = 8 per group (4M/4F except zQ175.Msh3.HOM which had 5M/3F).

first: Gillian

last: Bates

first: George

last: McAllister

first: Jian

last: Chen

first: Sarah

last: Aldous


title: Enhanced Phase Separation Mediated Oncogenicity [ChIP-Seq experiment]

city: hangzhou

first: kuang

last: junqi

address: No.18 Shilongshan Road

institute: westlake University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Chromosome translocation is known to generate oncogenes such as SS18-SSX1, but the underlying mechanism remains unknown. Here we show that enhanced phase separation acquired by translocation mediates oncogenic transformation. SSX fusion dramatically enhances the phase separation property of SS18 protein through H2AK119ub. Super condensates generated by SS18-SSX recruit acetyltransferases CBP/P300 as wild type SS18, but exclude HDACs such that H3K27ac accumulates abnormally at loci from those normally occupied by SS18, resulting aberrant silencing and activation of target genes. Consistently, we show that inhibition of H3K27ac can attenuate the tumorigenicity of the fusion oncoprotein. These results provide the first case for phase separation as a transforming event  to generate oncogene and super phase separation can be targeted for cancer therapy.

overall_design: We analyzed the H3K27me3, PRC1/2 and cohesin-CTCF occupancy during synovial sarcomagenesis triggered by SS18-SSX1 overexpression and the H2AK119ub in IMR90 cell line.

first: Junqi

last: Kuang

first: Duanqing

last: Pei


title: Impact of CTD truncation on co-transcriptional splicing.

status: Public on May 27 2024

city: San Diego

first: Yajing

last: Hao

state: CA

address: George Palade Laboratories 9500 Gilman Drive, Room 217

department: Dept. of Cellular and Molecular Medicine

institute: University of California, San Diego

summary: For overexpression of exogenous RPB1, 10 ug of purified HA-tagged RPB1 plasmids (wildtype or CTD-truncated, both α-amanitin resistant) were transfected into HeLa S3 cells per 10 cm dish using TurboFect transfection reagent (Thermo Fisher Scientific). Culture medium was changed to include 2 ug/ml of Doxycycline 12 hrs post-transfection for inducing exogenous gene expression and then treated with 2.5 ug/ml α-amanitin to block the function of endogenous RPB1. Cells were further cultured for 42 hrs before harvesting for subsequent analysis.

overall_design: We expressed a-amanitin resistant full-length and CTD truncated RBP1 and compared their impact on co-transcriptional splicing after inhibiting the endogenous Pol II in HeLa cells.

first: Changwei

last: Shao

first: Xiang-Dong

last: Fu


title: Gut Microbiota Metabolite Indole-3-Acetic Acid Maintains Intestinal Epithelial Homeostasis Through Mucin Sulfation

status: Public on May 26 2024

first: Mengfan

last: Li

address: 154 Anshan Road, Heping District, Tianjin

institute: Tianjin Medical University General Hospital

summary: The incidence and prevalence of inflammatory bowel disease (IBD) is gradually increasing. A high-fat diet (HFD) is known to disrupt intestinal homeostasis and aggravate IBD, yet the underlying mechanisms remain largely undefined. Here, a positive correlation between dietary fat intake and disease severity in both IBD patients and HFD-fed mice is observed. HFD induces a significant decrease in indole-3-acetic acid (IAA) and lead to intestinal barrier damage. Furthermore, IAA supplementation enhances the intestinal mucin sulfation and effectively alleviates colitis. Mechanistically, IAA upregulates key molecules involved in mucin sulfation, including Papss2 and Slc35b3, the synthesis enzyme and the transferase of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), via the Aryl Hydrocarbon Receptor (AHR). More importantly, AHR can directly bind to the transcription start site of Papss2. Oral administration of Lactobacillus reuteri, which can produce IAA, contributes to protecting against colitis and promoting mucin sulfation, while the modified L. reuteri strain lacking the iaaM gene (LactobacillusΔiaaM) and the ability to produce IAA fails to exhibit such effects. Overall, IAA enhances intestinal mucin sulfation through AHR, contributing to the protection of intestinal homeostasis.

overall_design: RNA-seq was performed in the colonic tissues from DSS-treated mice with or without IAA


title: Inflammatory stress-mediated chromatin changes underlie dysfunction in endothelial cells. [RNA-Seq]

status: Public on May 25 2024

city: Unionville

first: Haibo

last: Liu

state: CT

address: 164 Plantation St

department: MCCB

institute: UMass Chan

summary: Inflammatory stresses underlie endothelial dysfunction and contribute to the development of chronic cardiovascular disorders such as atherosclerosis and vascular fibrosis. The initial transcriptional response of endothelial cells to pro-inflammatory cytokines such as TNF-alpha is well established. However, very few studies uncover the effects of inflammatory stresses on chromatin architecture. We used integrative analysis of ATAC-seq and RNA-seq data to investigate chromatin alterations in human endothelial cells in response to TNF-alpha and febrile-range heat stress exposure. Multi-omics data analysis suggests a correlation between the transcription of stress-related genes and endothelial dysfunction drivers with chromatin regions exhibiting differential accessibility.

overall_design: The transcriptional response to inflammatory stresses (heat stress, pro-inflammatory cytokine TNF-alpha) in human vascular endothelial cells (HUVEC) was assesed by RNA-seq.

first: Amada

middle: D

last: Caliz

first: Heather

last: Learnard

first: Milka

last: Koupenova

first: John

middle: F

last: Keaney

first: Shashi

last: Kant

first: Lihua

middle: J

last: Zhu

first: Anastassiia

last: Vertii


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity

city: Jena

first: Bjoern

last: von Eyss

address: Beutenbergstr. 11

country: Germany

institute: Leibniz Institute on Aging

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

overall_design: Refer to individual Series


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity [01c_bulk_rna_seq_K8-CreER_shTRPS1]

summary: The GATA transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and sustains the initial branching of the mammary ducts at puberty as well as the lactogenic differentiation during pregnancy. Its function in the resting mammary gland however, remains unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy resting mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. we show that TRPS1 activity is critical to maintain a functional luminal progenitor compartment and that this involves the repression of the SRF/MRTF and the YAP/TAZ transcriptional program. Mechanistically, this repression involves Krt8-dependent RhoA modulation. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors which is intrinsically linked to mechanotransduction in the mammary gland. It also provides new insights into the oncogenic functions of TRPS1 since luminal progenitors are likely the cells of origin of many breast cancers.

overall_design: To analyze the impact of TRPS1 depletion on the transcriptome and function of the luminal cells from the murine mammary gland using two independent shTRPS1 mouse lines (shTRPS1.32; shTRPS1.34) and a controll expressing an shRenilla control (shRen).

first: Marie

last: Tollot-Wegner

first: Marco

last: Jessen

first: KyungMok

last: Kim

first: Björn


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity [01b_bulk_rna_seq_luminal_vs_basal]

overall_design: To analyze the differential gene expression of luminal vs. basal cells freshly isolated and sorted from the mouse virgin mammary gland.


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity [01d_bulk_rna_seq_lum_prog_vs_mature]

overall_design: To analyze the differential gene expression of luminal progenitors vs. differentiated luminal cells freshly isolated and sorted from the mouse virgin mammary gland.


title: Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (PD in vivo)

city: Lausanne

first: Flora

last: Brozzi

address: Rue du Bugnon 9

department: DNF

country: Switzerland

institute: University of Lausanne

type: Non-coding RNA profiling by high throughput sequencing

summary: Transfer RNAs (tRNAs) play a central and well recognized role in protein synthesis. Recent studies revealed that these molecules can be cleaved to generate tRNA fragments (tRFs) with regulatory functions. Here, we studied the contribution of tRFs to pancreatic β-cell loss during the initial phases of type 1 diabetes (T1D), an autoimmune disorder characterized by the invation of immune cells in the pancreas and progressive loss of insulin-secreting cells. Small RNA-profiling showed that the pool of tRFs present in pancreatic β-cells is altered in non-obese diabetic (NOD) mice, a mouse model used to study T1D. We found that part of these changes is triggered by the exposure of β-cells to proinflammatory cytokines released during the autoimmune reaction while others result from the direct transfer of tRFs from autoreactive T lymphocytes to insulin-secreting cells via extracellular vesicles. Indeed, using an RNA-tagging approach, we could demonstrate that a group of tRFs are transferred in vivo in from CD4+CD25- T lymphocytes to pancreatic β-cells, upon T cell adoptive transfer in NOD scid mice. Morevoer, the up-regulation of selected tRFs associated with the autoimmune reaction triggers β-cell apoptosis and gene expression changes that affect the immune regulatory capacity of β-cells. Our data point to tRFs as novel players in type 1 diabetes and potentially in other autoimmune disorders.

overall_design: Using NOD mice as a model of T1D, we detected a specific tRF signature in islet cells during the initial phases of the disease. We wanted to demonstrate that some tRFs are released by CD4+ /CD25- T lymphocytes and are transferred in vivo to islet cells during the initial phases of T1D. We used CD4+/CD25- T lymphocytes of NOD BDC2.5 mice that upon inoculation into NOD.SCID mice, promote the appearance of autoimmune diabetes within less than 10 days. CD4+/CD25- T-cells from NOD BDC2.5 were isolated and incubated with Ethynyl Uridine for 48h, together with T-cell activator factors. The cells were then washed and inoculated into NOD.SCID mice by intravenous injection (IV) into the tail vein. Control mice were injected with saline solution. Two days after IV injection, animals were sacrificed, islets isolated, and β-cells FACS-sorted. The RNA isolated from FACS-sorted β-cells was biotinylated and pulled-down with streptavidin beads, with the aim of isolating only the EU-tagged RNA coming from T-cell EVs. Pulled-down RNA was analysed by small RNAseq.

first: Cristina

last: Cosentino

first: Cécile

last: Jacovetti

first: Kejing

last: Wu

first: Bayazit

middle: M

last: Bilal

first: Claudiane

last: Guay

first: Baroj

last: Abdulkarim

first: Véronique

last: Menoud

first: Romano

last: Regazzi


title: Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (EV-treated)

overall_design: Using NOD mice as a model of T1D, we detected a specific tRF signature in islet cells during the initial phases of the disease. tRFs can be produced endogenously but also released and transferred in extracellular vesicles (EVs). Since we demonstrated that EVs from NOD T-cells contain a high level of tRFs, we verify if increased abundance of tRFs in pre-diabetic NOD islets could result after a transfer from T cell EVs to β-cells. To verify this hypothesis, mouse islet cells were incubated for 24h in the presence of EVs released by NOD T lymphocytes, and small RNA-seq analysis was performed.


title: Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (NOD islets)

overall_design: NOD mice constitute the best characterized and most widely used animal model of T1D. At four weeks of age, the islets of NOD mice display a normal morphology and function but starting from the age of 6 weeks they are progressively invaded by immune cells, resulting in β-cell dysfunction and death and, starting from weeks 12-14, in the appearance of diabetes. To assess the potential contribution of tRFs to the initial phases of T1D, we isolated the RNA of pancreatic islets of NOD mice and pretreated the samples for end repair and modifications removal. We then compared by small RNA-sequencing (RNA-seq) the level of tRFs in the islets of control (4 weeks old) and prediabetic (8 weeks old) NOD mice.


title: Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis


title: Intratumoral antigen-signalling traps CD8+ T cells to confine exhaustion to the tumour site (bulk TCR-Seq)

city: Cambridge

first: James

last: Thaventhiran

address: Tennis Court Road

country: United Kingdom

institute: MRC Toxicology Unit

summary: Immunotherapy advances have been hindered by difficulties tracking the behaviours of lymphocytes following antigen-signalling. Here we present the antigen-receptor signalling reporter (AgRSR) mouse to fate-map lymphocytes responding to antigen at specific times and locations, to determine the behaviour of T cells involved in the tumour immune response. Contrary to reports of ready egress of T cells out of the tumour, we find that intratumoral antigen-signalling traps CD8+ T cells in the tumour. These clonal populations expand and become increasingly exhausted over time. In contrast, antigen- signalled regulatory T cell (Treg) clonal populations readily recirculate out of the tumour. Consequently, intratumoral antigen-signalling acts as a gatekeeper to compartmentalize CD8+ T cell responses – even within the same clonotype, enabling immunity to confine exhaustion to a specific tissue site.

overall_design: YUMMER1.7 bearing AgRSR mice were injected with tamoxifen (intraperitoneally or intratumorally).  8 or 18 days after injection, EYFP+ and EYFP- cells were sorted from the secondary lymphoid tissues and tumour, and processed through bulk TCR-seq. Library strategy: TCR-seq, VDJ from 5' RACE total mRNA. Technical details:-Sample sorting and storage: cells sorted in Buffer RLT Plus with DDT -RNA extraction kit: RNeasy Plus Micro Kit -RNA quantification and QC: Qubit RNA HS and Tapestation High Sensitivity RNA - Library preparation kit: Takara SMARTer Mouse TCR alpha-beta Profiling Kit - Library quantification and QC: Qubit DNA HS and Tapestation High Sensitivity D5000 - Sequencing instrument: Illumina NextSeq2000 -Sequencing: P2 600 cycles, 300+300 with 10%PhiX spike-in TCR-seq analysis: MiXCR.

first: Munetomo

last: Takahashi

first: Vitalina

last: Chamberlain-Evans


title: Loss of SIRPα promotes podocytes presenting antigen to activate specific T cell immune responses in lupus nephritis via activating spleen tyrosine kinase

city: Nanjing

first: QIAN

last: BIN

state: JiangSu

address: 639 Longmian Avenue

department: School of Life Science and Technology

institute: State Key Laboratory of Natural Medicines

summary: To investigate the role of SIRPα in podocytes, we generated podocyte-specific SIRPα knockout mice, Subsequent studies have found that SIRPa is deeply involved in the adaptive immune process of podocytes, and the loss of SIRPa promotes the ability of podocytes to present antigens,furthermore, the loss of SIRPa in podocytes can promote the increase of antigen presentation ability of podocytes and promote the progression of lupus nephrin

overall_design: The wild type (WT) and podocyte specific SIRPα knockout (Sirpa-/-) mice were received a single intraperitoneal injection of 0.5 ml pristane respectively, and monitored for 4 months to construct experimental LN mice. Podocytes were isolated from pristane-treated WT and Sirpa-/- mice for further analysis.

first: Bin

last: Qian

first: Limin

last: Li


title: Microarray data, 4 week HFD/NCD, nostim or 16hrstim

city: Toronto

first: Brendan

last: Cordeiro

state: Ontario

address: 209 Victoria St.

department: Keenan Research Center

country: Canada

institute: St-michael's hospital

summary: How autoimmunity is initiated in Multiple Sclerosis (MS) is unknown, but both genetic and environmental factors have been implicated. Female physiology is associated with a three times higher risk of MS and the ratio of woman to men diagnosed with MS has been increasing over time, suggesting that environmental factors are interacting with female sex to initiate this disease. One MS risk factor that has been increasing that interacts with female sex is obesity. Being overweight or obese as an adolescent increases one’s risk of MS by 2-fold and this risk increases if one is female. To explore how obesity and female sex interact to control T helper autoimmunity, we conducted a proteomic screen on the serum of male and female healthy weight and obese participants that were affected or not affected by MS. We found that obese individuals, regardless of disease status, exhibited a prominent upregulation of proteins in pathways associated with the damage-associated S100 protein pathway, T helper 1 (Th1), IL-17 pathways and RIG-I/Interferon pathways and that this increase was more prominent in women. To explore the mechanisms of how female sex interacts with increased adiposity to regulate CNS autoimmunity, we modeled a diet-induced overweight (DIO) state in mice by placing animals on high fat diet for 4 weeks and then inducing experimental autoimmune encephalomyelitis (EAE), a model of MS. We found that DIO enhanced EAE severity in mice of both sexes compared to controls and this effect was more pronounced in female mice. The increased severity in the female DIO mice associated with a greater accumulation of pathogenic IFN-+IL-17+ CD4+ cells in the CNS and more severe tissue damage.  Further studies of mice at steady-state revealed that DIO increased the frequency of CD44hiCD4+ T cells and enhanced the potential of naïve CD4+ T cells to produce IFN-This phenotype of higher T cell IFN-was also observed in female humans who were overweight. Furthermore, IFN-α was detected at significantly increased levels in the serum of the DIO-female compared to DIO-male or sex-matched control mice. Loss of IFN-α receptor in T cells negated the increases in Th1 inflammation and EAE severity caused by DIO in female mice.  These results highlight that an overweight state may interact with female sex to selectively enhance Th1 responses and CNS autoimmunity by increasing type I and type I IFN signalling in T cells.

overall_design: C57BL6 mice were placed on a normal chow diet (NCD) or a High fat-diet (HFD) for 4 weeks, then naïve CD4+ T cells (CD4+ CD44low-int) were magnetically isolated by negative selection from spleens and lymph nodes and stimulated with 2ug/ml plate-bound anti-CD3 and anti-CD28 antibodies for 16hrs. Total RNA was then isolated (RNEAsy, Qiagen). Total RNA quality was analyzed by RNA 6000 Pico LabChip on a 2100 BioAnalyzer (Agilent). RNA integrity numbers for samples were > 5.2. 10 ng of total RNA was reverse transcribed using GeneChip WT Pico Kit (Affymetrix). Each sample was hybridized to a GeneChip Mouse Gene 2.0 ST array (Affymetrix) for 16- 18 h at 45 °C. The arrays were scanned using a GeneChip Scanner 3000 (Affymetrix).RNA integrity analysis  and microarray probing and detection were performed by the Microarray Facility of the Centre for Applied Genomics at the Hospital for Sick Children, Toronto, Canada.

first: Jeeyoon

last: Ahn

first: Saurabh

last: Gawde

first: Carmen

last: Ucciferri

first: Nuria

last: Alvarez-Sanchez

first: Xavier

last: Revelo

first: Natalie

last: Stickle

first: Kaylea

last: Massey

first: David

middle: G

last: Brooks

first: Joel

last: Guthridge

first: Gabriel

last: Pardo

first: Daniel

middle: A

last: Winer

first: Robert

middle: C

last: Axtell

first: Shannon

middle: E

last: Dunn


title: Enhanced Phase Separation Mediated Oncogenicity [RNA-Seq, 2]

first: kuang

last: junqi

overall_design: We analyzed the transcriptome expression profiles (RNA-seq) in human stem cells H9 upon the knockin of C-SSC1-EGFP  at SS18 locus.

first: Junqi

last: Kuang

first: Duanqing

last: Pei


title: Enhanced Phase Separation Mediated Oncogenicity [HiC]

overall_design: We analyzed the chromatin interaction by  HiC experiment in BJ cells with expression of physiological SS18, SS18(YA)-SSX mutant and pathological SS18-SSX onco-fusion, respectively


title: Enterococcus-derived tyramine hijacks α2A-adrenergic receptor in intestinal stem cells to exacerbate colitis

city: Hefei

first: Wen

last: Pan

address: 443 Huangshan Road

department: School of Life Sciences, USTC Life Sciences and Medicine

institute: University of Science and Technology of China

summary: Inflammatory bowel disease (IBD) is characterized by dysbiosis of the gut microbiota and dysfunction of in testinal stem cells (ISCs). However, the direct interactions between IBD microbial factors and ISCs are unde scribed. Here, we identify α2A-adrenergic receptor (ADRA2A) as a highly expressed GPCR in ISCs. Through PRESTO-Tango screening, we demonstrate that tyramine, primarily produced by Enterococcus via tyrosine decarboxylase (tyrDC), serves as a microbial ligand for ADRA2A. Using an engineered tyrDC deficient  Enterococcus faecalis strain and intestinal epithelial cell-specific Adra2a knockout mice, we show that Enterococcus-derived tyramine suppresses ISC proliferation, thereby impairing epithelial regeneration and  exacerbating DSS-induced colitis through ADRA2A. Importantly, blocking the axis with an ADRA2A antagonist, yohimbine, disrupts tyramine-mediated suppression on ISCs and alleviates colitis.Our findings highlight  a microbial ligand-GPCR pair in ISCs, revealing a causal link between microbial regulation of ISCs and colitis exacerbation and yielding a targeted therapeutic approach to restore ISC function in colitis.

overall_design: After dissociated into single cell with TripLE for 10min,intestine organoids were treated with 100uM tyramine for 16h (n=3) or with control medium(n=3).

first: Chaoliang

last: Li

first: Panrui

last: Zhang

first: Yadong

last: Xie

first: Shishan

last: Wang

first: Meng

last: Guo

first: Xiaowei

last: Wei

first: Kaiguang

first: Dan

last: Cao

first: Rongbin

last: Zhou

first: Sheng

first: Xinyang

last: Song

first: Shu

last: Zhu


title: Transcriptional changes consistent with altered neuronal differentiation, angiogenesis, and tumor plasticity induced in human subpallial telencephalic organoid-glioblastoma chimeras

city: Salt Lake City

first: Simone

last: Chiola

department: Neurobiology

institute: University of Utah

summary: Glioblastoma multiforme (GBM) is one of the most aggressive and therapy-resistant brain tumors prevalent in both adults and children. Despite extensive research to understand GBM pathology, it remains unclear how neural cells in the human brain interact with GBM cells to support their brain propagation and therapy resistance and whether GBM cells exert any influence on the properties of human neural cells. In this study, we co-culture human stem cell-derived subpallial telencephalic organoids with patient-derived proneural or mesenchymal GBM spheroids to investigate their reciprocal interactions. We show that both proneural and mesenchymal GBM spheroids readily fuse and propagate with human organoids, forming organoid-GBM chimeras, without the need for exogenous growth factors. GBM cells within the chimeras adapt by modulating gene expression profiles consistent with diminished proliferation, heightened hypoxia, increased angiogenesis, and proneural-to-mesenchymal transition in proneural GBM. Both proneural or mesenchymal GBMs also exert an impact on the properties of neural cells in the chimeras, leading to the suppression of neuronal genes and an upregulation expression of genes associated with hypoxia and angiogenesis. Collectively, this study identifies specific genes and molecular pathways that can be altered in GBM and neural cells by reciprocal interactions in a human developing brain-like environment for an increased understanding of GBM pathology and future therapy development.

overall_design: Subpallial telencephalic organoids, GBM spheroids, and organoids-spheroids chimeras

first: Jingye

last: Yang

first: HMArif

last: Ullah

first: Kandy

last: Napan

first: Qiju

last: Huang

first: Nicholas

last: Gamboa

first: Osama

last: Youssef

first: Howard

last: Colman

first: Samuel

middle: H

last: Cheshier

first: Alex

last: Shcheglovitov


title: Translocational attenuation regulated by PERK-SRP14 axis is a novel protective mechanism of UPR

city: NOVATO

first: Yaofu

last: Liu

address: 1599 S NOVATO BLVD

institute: Buck Institute for Research on Ag

summary: Slowing down protein synthesis, increasing protein folding capability, and enhancing misfolded protein degradation are the main mechanisms of unfolded protein response (UPR) to lessen ER stress. It has also been shown that substrate-specific translocational attenuation may be another protective measure during ER stress. Nevertheless, the underlying mechanism is still obscure. Through a multi-omics analysis, we found the protein SRP14, a subunit of SRP, was markedly decreased in ER-stressed cells. Given its critical function in the selective translocation of proteins, we investigated the mechanisms of SRP14 down-regulation and its role in ER stress. It was found that UPR, mainly the activation of PERK, induced SRP14 ubiquitination and proteasomal degradation. Furthermore, enforced expression of SRP14 in stressed cells augmented the substrate's translocation, aggravated ER stress, and reduced cell viability. Taken together, these results suggest the specific translocational attenuation regulated by the PERK-SRP14 axis is a protective measure to mitigate ER stress.

overall_design: To investigate the effects of ER stress induced by HRD1 knockdown on gene expression, we constructed a LoVo cell line that expresses a Tet-On shRNA against HRD1. After exposure to doxycycline (Dox) for 0, 3, and 5 days to induce HRD1 knockdown, the cells were harvested and processed for RNA-Seq.

first: Yili


title: Effect of specific G-quadruplex stabilization on gene expression and chromatin accessibility at gene promoters in Hela cells.

city: changchun

first: Gen

last: Qin

address: renmin street 5625

department: Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization

institute: Changchun Institute of Applied Chemistry, Chinese Academy of Sciences

summary: To investigate specific G-quadruplex stabilization in the regulation of gene expression and chromatin accessibility, we established HeLa cell lines in which each target g-quadruplexes has been stabilized.

overall_design: Comparative gene expression and chromatin accessibility profiling analysis of RNA-seq and ATAC-seq data for HeLa cells in which each target g-quadruplexes has been stabilized.

first: Geng

first: Zhenqi

first: Jinsong

last: Ren

first: Xiaogang

last: Qu


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity [04_cut_run_TRPS1]

first: Bjoern

last: von Eyss

overall_design: To identify genomic targets of TRPS1 in mammary gland luminal cells, primary luminal cells were freshly isolated from virgin wildtype mice by flow cytometry (CD49f_high, EpCAM_high) and analyzed by CUT&RUN

first: Marie

last: Tollot-Wegner

first: Marco

last: Jessen

first: KyungMok

last: Kim

first: Björn


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity [03_scatac_shTRPS1_organoids]

overall_design: To analyze the impact of TRPS1 depletion on chromatin accessibility, primary mouse mammary gland organoids from shTRPS1 transgenic mice (n=4) were cultured and shRNA expression was induced by doxycycline treatment (DOX; 0.1 µg/ml). Ethanol-treated organoids (EtOH) served as a control.


title: Su(Hw) interacts with Combgap to establish long-range chromatin contacts

city: Moscow

first: Marina

middle: Yu.

last: Mazina

address: Vavilova, 34/5

country: Russia

institute: Institute of Gene Biology RAS

summary: Insulator-binding proteins (IBPs) play a critical role in genome architecture by forming and maintaining contact domains. While the involvement of several IBPs in organising chromatin architecture in Drosophila has been described, the specific contribution of the Suppressor of Hairy wings (Su(Hw)) IBP to genome topology remains unclear. In this study, we provide evidence for the existence of long-range interactions (LRIs) between Su(Hw) and Combgap ChIP-Seq peaks, reflected in the indirect binding of these proteins to chromatin in ChIP experiments. Loss of Su(Hw) binding results in the disappearance of Su(Hw)-Combgap LRIs and a decrease in spatial self-interactions among a subset of Su(Hw) sites. Our findings suggest that Su(Hw)-Combgap LRIs are associated with active chromatin rather than Polycomb-directed repression. Furthermore, we observe that the majority of transcription start sites that are down-regulated upon loss of Su(Hw) binding to chromatin are located within 2 kb of Combgap peaks and exhibit Su(Hw)-dependent changes in Combgap and transcriptional regulators’ binding.

overall_design: All flies were raised at 25°C on standard agar medium. Ovaries were dissected from recently eclosed 15 hour-old flies (contain only egg chamber stages 1-8).


title: Small-molecule-induced epigenetic rejuvenation overcomes myelinogenic barriers and stimulates myelin repair [RNA-seq]

city: cincinnati

first: XUEZHAO

last: LIU

state: OH

institute: cincinnati children's hospital medical center

summary: Failure of oligodendrocytes to remyelinate underlies diseases such as multiple sclerosis (MS). We found that epigenetic silencing prevents oligodendrocytes from producing myelin sheaths in demyelinating MS lesions. Here, we developed a transgenic reporter system to identify a small-molecule epigenetic modulator that stimulates oligodendrocyte maturation and myelin ensheathment in vitro. This compound promoted remyelination in animal models of MS and myelination of regenerated axons and increased myelin sheath lengths in human iPSC-derived organoids. Multi-omics analyses revealed that the compound induced an enhancer/super-enhancer landscape that upregulates crucial myelinogenesis-associated pathways, including RRAS2-AKT signaling, driving actin depolymerization necessary for myelin ensheathment. Strikingly, the compound also induced phase-separated nuclear condensates of SREBP1/2, which concentrate transcriptional co-activators to drive lipid and cholesterol biosynthesis. Silencing expression of a potential target of the small molecule, HDAC3, facilitated robust myelination and remyelination. Our findings suggest that small-molecule-modulated epigenome rejuvenation relieves epigenetic silencing barriers and promotes myelin repair.

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: RNAseq of rat iOLs treated with veh or 26C or KD

first: Xuezhao

first: Xuelian

last: He

first: Qing

middle: R

last: Lu


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity [02_citeseq_shTRPS1_mmg]

overall_design: To analyze the differential gene expression of luminal progenitors vs. differentiated luminal cells freshly isolated and sorted from the mouse virgin mammary gland.


title: TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity [01a_bulk_rna_seq_PCX_Cre_shTRPS1]

overall_design: To analyze the impact of TRPS1 depletion on the transcriptome and function of the murine mammary gland using two independent shTRPS1 mouse lines (shTRPS1.32; shTRPS1.34) and a controll expressing an shRenilla control (shRen).


title: Enhanced Phase Separation Mediated Oncogenicity [ChIP-Seq]

first: kuang

last: junqi

summary: Chromosome translocation is known to generate oncogenes such as SS18-SSX1, but the underlying mechanism remains unknown. Here we show that enhanced phase separation acquired by translocation mediates oncogenic transformation. SSX fusion dramatically enhances the phase separation property of SS18 protein through H2AK119ub. Super condensates generated by SS18-SSX recruit acetyltransferases CBP/P300 as wild type SS18, but exclude HDACs such that H3K27ac accumulates abnormally at loci from those normally occupied by SS18, resulting aberrant silencing and activation of target genes. Consistently, we show that inhibition of H3K27ac can attenuate the tumorigenicity of the fusion oncoprotein (we do not have the data in your figures?). These results provide the first case for phase separation as a transforming event  to generate oncogene and super phase separation can be targeted for cancer therapy.

overall_design: We analyzed the H3K27ac, H3K119ub, SS18 and SS18-SSX1  occupancy  during synovial sarcomagenesis triggered by SS18-SSX1 overexpression.

first: Junqi

last: Kuang

first: Duanqing

last: Pei


title: Small-molecule-induced epigenetic rejuvenation overcomes myelinogenic barriers and stimulates myelin repair [HiChIP]

overall_design: Hichip of rat iOLs treated with veh or 26C with H3K27ac antibody


title: Blood DNA methylation altered by Mycobacterium tuberculosis infection in mice

city: Columbus

first: Russell

last: Ault

address: 370 W 9th Ave

institute: The Ohio State University College of Medicine

type: Methylation profiling by high throughput sequencing

summary: Knowing when a person was infected with Mycobacterium tuberculosis (M.tb) is critical as recent infection is the strongest clinical risk factor for progression to TB disease in immunocompetent individuals. However, time since M.tb infection is very difficult to determine in routine clinical practice. We determined whether DNA methylation patterns in the blood correlate with time since M.tb infection or exposure to develop a biomarker for recent exposure. As proof of concept, we found a DNA methylation signature that is present during early infection of mice that persists for at least 5 months post-infection.

overall_design: Blood was collected from 54 C57BL/6 mice and 46 BALB/c mice infected with a low dose of M.tb (Erdman), as well as 23 C57BL/6 mice and 28 BALB/c mice that served as uninfected, age-matched controls. Blood was collected at days 7, 14, 30, 62, 90, 120 and 153 post-infection. Whole blood was subjected to reduced representation bisulfite sequencing. We identify stable DNA methylation changes associated with infection.

first: Colwyn

last: Headley

first: Tucker

last: Piergallini

first: Shalini

last: Gautam

first: Varun

last: Dwivedi

first: Melanie

last: Carless

first: Joanne

last: Turner


title: Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (NOD EVs)

first: Flora

last: Brozzi

overall_design: Using NOD mice as a model of T1D, we detected a specific tRF signature in islet cells during the initial phases of the disease. tRFs can be produced endogenously but also released and transferred in extracellular vesicles (EVs). Since immune cells are known to release EVs, we investigated whether tRFs are part of their cargo and if increased abundance of tRFs in pre-diabetic NOD islets could result from a transfer from T cell EVs to β-cells. Small RNA profiling was performed on EVs produced by activated CD4+/CD25- T lymphocytes of 8 weeks-old NOD mice.

first: Cristina

last: Cosentino

first: Cécile

last: Jacovetti

first: Kejing

last: Wu

first: Bayazit

last: Bilal

first: Claudiane

last: Guay

first: Baroj

last: Abdulkarim

first: Véronique

last: Menoud

first: Romano

last: Regazzi


title: Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (tRF overexpression)

overall_design: Using NOD mice as a model of T1D, we detected a specific tRF signature in islet cells during the initial phases of the disease. Through an RNA-tagging approach we were able to show both in vitro and in vivo that a fraction of the tRFs displaying elevated levels during insulitis are directly transferred from autoreactive T cells to β-cells via EVs. To gain more insights into the role of the tRFs transferred in β-cells, we performedbulk RNA-seq analysis in mouse islet cells overexpressing mimic oligonucleotides of LysCTT-5’, PheGAA-5’ and SerGCT-3’, for 48h. A scramble sequence of the mimic tRFs was used in the control condition (CT).


title: Role of tRNA-derived fragments in the cross-talk between immune cells and beta cells during type 1 diabetes pathogenesis (Cyt-treated)

overall_design: Using NOD mice as a model of T1D, we detected a specific tRF signature in islet cells during the initial phases of the disease. Part of the changes in the tRF profile of pre-diabetic NOD mice is likely to be triggered by chronic exposure of islet cells to proinflammatory factors released by the invading immune cells. To determine the impact of pro-inflammatory cytokines on tRF levels, mouse islet cells were treated with IL-1β, TNF-α and IFN-γ for 24h, prior to tRF profiling by small RNA-seq.


title: Distinctive roles for the chromatin remodeling ATPases Brg1 and Brm in early zebrafish embryos

first: Bradley

last: Cairns

state: UT

address: 2000 Cir of Hope Dr

department: oncological Sciences

institute: Huntsman Cancer institute/HHMI

overall_design: Refer to individual Series


title: The 8p11-p12 amplicon oncogene ASH2L regulates expression of genes involved in tumorigenic processes via promoter H3K4me3 in luminal breast cancer. (SUM-44 ASH2L knockdown H3K4me3 ChIP-seq)

city: Charleston

first: Gary

last: Hardiman

state: SC

address: 135 Cannon Street

department: Medicine

institute: Medical University of South Carolina

summary: Amplification of the 8p11-p12 genomic region occurs in 30% of luminal B breast cancers and results in coordinate overexpression of several oncogenes, including ASH2L, which upregulates gene expression via promoter tri-methylation of lysine 4 on histone 3 (H3K4me3). Since 8p11-p12 amplification is associated with endocrine resistance, the major cause of breast cancer mortality, understanding the underlying biology is essential to improving treatment options for patients. To explore the role of amplified and overexpressed ASH2L on epigenetic regulation of gene expression, we performed H3K4me3 ChIP-seq and RNA-seq in breast cancer cells with ASH2L amplification and overexpression following ASH2L knockdown and assessed the biological effects of the suite of genes identified in this manner. We discovered that ASH2L-regulated genes are implicated in cell cycle processes and response to the CDK4/6 inhibitor palbociclib and confirmed these effects in additional breast cancer cell lines. We also discovered that ASH2L regulates expression of another 8p11-p12 amplicon oncogene NSD3, also an epigenome modifier, which has previously been shown to result in overexpression and estrogen-independent activation of ERα. Together, these data establish ASH2L as a breast cancer oncogene and an important component of the 8p11-p12 amplicon that acts as an oncogenic unit in breast cancer.

overall_design: Twelve total samples: three biological replicates of H3K4me3 ChIP and input paired samples for LacZ control knockdown and ASH2L knockdown (shRNA-mediated) SUM-44 cells

first: Jamie

middle: N

last: Mills

first: E

middle: S

last: Hazard

first: Antonio

last: Giordano

first: Yueying

last: Liu

first: Robert

last: Wilson

first: Dongjun

last: Chung

first: Stephen

middle: P

last: Ethier


title: Distinctive roles for the chromatin remodeling ATPases Brg1 and Brm in early zebrafish embryos [RNA-seq]

summary: Brg1 and Brm function as alternative core enzymatic ATPases of SWI/SNF complex in vertebrates, with clear homologs in zebrafish. Brg1 (but not Brm) is required for early cleavage-stage transcription in mice, but where and how Brg1 or Brm impact chromatin in early embryos remains unexplored. Here, we utilize zebrafish embryos to provide the first simultaneous genome-wide profiling of Brg1 and Brm occupancy in any organism/cell type, alongside our profiling of open chromatin and RNA polymerase II. We reveal shared and unique loci for Brg1 and Brm, their shared occupancy of open chromatin and particular developmental promoters, and higher transcription at co-occupied genes. Interestingly, only Brg1 is strongly associated with active histone modifications. Strikingly, only Brm commonly occupies gene bodies, overlapping precisely with Pol II. Functional experiments involving Pol II elongation inhibition alongside bioinformatic analyses suggest that Brm travels with Pol II into gene bodies, primarily at short, very highly-transcribed genes with few introns. Taken together, our results support roles for Brg1 primarily at promoters and enhancers, and for Brm in association with Pol II in elongation through chromatin within gene bodies during genome-wide transcriptional activation

overall_design: RNAseq data from MBT and postMBT stages were generated.

first: Mengyao

last: Tan

first: Candice

middle: L

last: Wike

first: Yixuan

last: Guo

first: Christian

last: Pflueger


title: DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells

status: Public on May 23 2024

city: Oslo

first: Adam

last: Sharples

address: 220 Sognsveien

country: Norway

institute: Norwegian School of Sport Sciences

summary: Skeletal muscle tissue demonstrates global hypermethylation with aging. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 hours, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; ‘pathways-in-cancer’ (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), ‘rap1-signaling’, ‘axon-guidance’ and ‘hippo-signalling’. Aged cells also demonstrated a hypermethylated profile in pathways; ‘axon-guidance’, ‘adherens-junction’ and ‘calcium-signaling’, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alteration in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in the ‘focal adhesion’ and ‘PI3K-AKT signalling’ pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells (on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P). Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3.

overall_design: See published paper: https://www.nature.com/articles/s41598-020-72730-z Note that for Young Adult Human Skeletal Muscle Tissue Samples used in this publication can be found deposited in GSE114763 & GSE162288


title: The NK cell stress response status modulates anti-tumor immunity [ChIPmentation]

city: New York

first: Kathryn

middle: Grace

last: Hockemeyer

state: New York

address: 550 1st Avenue

department: Pathology

institute: NYU Langone Health

summary: The tumor microenvironment (TME) comprises numerous forms of cellular stress, which can activate Heat Shock Factor 1 (HSF1), the master transcription factor of the proteotoxic stress response. We profiled HSF1 activity across tumor-infiltrating immune populations, revealing the highest activity in CD8+ T cells and the lowest in natural killer (NK) cells. To elucidate the mechanisms through which HSF1 regulates immune surveillance, we generated an in vivo model of augmented HSF1 activity. Tumor challenge revealed that accumulation of HSF1 dampens NK-mediated tumor immunity and impairs both cytotoxicity and production of interferon gamma (IFN-γ) in NK cells. Single-cell transcriptional profiling identified a loss of anti-tumor signaling pathways, including interferon signaling, within the HSF1-enhanced TME. In NK cells, integration of chromatin accessibility with transcriptomics revealed that HSF1 deregulates accessibility and expression of genes encoding for NK receptors, leading to an inhibitory bias.

overall_design: HSF1 ChIPmentation was performed on primary NK cells extracted from 5 WT or 5 HSF1-S303/7A animals. Naïve NK cells were used immediately after isolation.

first: Iannis

last: Aifantis


title: rhBMP-2 synergizes with other signals to induce hBM-MSC multiple phenotype differentiation

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

institute: Max Planck Institute for Molecular Biomedicine

summary:
The Recombinant human bone morphogenetic protein 2 (rhBMP-2) and the human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied with research and translational bone regenerative purposes. rhBMP-2 induces in vivo the endochondral ossification and bone formation processes, while hBM-MSCs are considered its target, bone forming cells. The in vitro ability of rhBMP-2 to induce hBM-MSC osteogenic differentiation has been previously tested. However, bone formation implies mesenchymal cell differentiation to multiple phenotypes, including chondrogenesis, adipogenesis and also osteogenesis. In this article, we wondered whether rhBMP-2 may drive not only osteogenic, but also multilineage differentiation of hBM-MSCs, both in vivo and in vitro. aiming to model all the differentiation processes observed in the rhBMP-2 induced in vivo bone formation. First, the rhBMP-2 and hBM-MSCs were tested in an in vivo implantation model to assess their ability to induce mature bone formation and mutilineage differentiation of implanted cells. Then, hBM-MSCs were in vitro treated with rhBMP-2 at short- and long-term cell culture periods, alone or in combination with osteogenic, adipogenic and chondrogenic media, aiming to define the role of rhBMP-2 in these differentiation processes. Data indicate that hBM-MSCs respond to rhBMP-2 at short term, but fail to differentiate in long term cultures, which was linked to the induction of rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, when rhBMP-2 is used in combination with other differentiation signals in in vitro long term cell cultures, the mentioned negative feedback loop mechanism disappears and rhBMP-2 acts as potentiator of multilineage differentiation, not only osteogenesis, but also adipogenesis and chondrogenesis. Altogether, data indicate rhBMP-2 alone is unable to induce hBM-MSC terminal differentiation in in vitro settings, but synergizes with other signals to potentiate multiple differentiation phenotypes. When using rhBMP-2 and hBM-MSCs for translational bone formation applications, they should be considered that these regenerative agents are acting in a system together with other signals, triggering multiple phenotypes depending on the signaling environment, to yield the formation of mature bone and bone marrow tissues.
In vivo implantation assay and sample testing: All animals were maintained and handled in accordance with the Guidelines for Accommodation and Care of Animals (European Convention for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes) and internal guidelines. Experimental procedures were approved by the ethical committee and local competent authorities (Project number PRO-AE-SS-171). All animals were housed in ventilated cages and fed with standard diet ad libitum. E. coli produced recombinant BMP-2 (rhBMP-2) was obtained from commercial providers (Noricum SL, Cat.# BMP2). RNA Sequencing and data processing : The quantity and quality of the RNAs were evaluated using Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat.# Q32855) and Agilent RNA 6000 Nano Chips (Agilent Technologies, Cat.# 5067-1511), respectively. Sequencing libraries were prepared following “TruSeq Stranded mRNA Sample Preparation Guide (Part # 15031058 Rev. E)” using the “TruSeq Stranded mRNA Library Prep” kit (Illumina Inc. Cat. # 20020594) and TruSeq RNA CD Index Plate (96 Indexes, 96 Samples) (Illumina Inc. Cat. # 20019792). Starting from 700 ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific, Cat. # 18064-014) for 10 min at 25°C, 15 min at 42°C, 15 min at 70°C and pause at 4°C. cDNA second strand was synthesized with Illumina reagents at 16°C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 sec at 98°C, 15 cycles of 10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C, 5 min at 72°C and pause at 4°C). Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat. # 5067-4626 ) and quantified using Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific, Cat. # Q32854).

overall_design: The data BMP2 (BMP2), is performed with 3 replicates. The data CTL (CTL), is performed with 3 replicates.

first: Neda

last: Kathami

first: Carolina

last: Moreno-Vicente

first: Pablo

last: Martín

first: Jhonatan

last: Vergara-Arce

first: Raquel

last: Ruiz-Hernández

first: Ana

last: Aranzay

first: Daniela

last: Gerovska

first: Sandra

last: Camarero

first: Ander

last: Abarrategi


title: Enhanced Phase Separation Mediated Oncogenicity [ChIP-Seq, 2]

city: hangzhou

first: kuang

last: junqi

address: No.18 Shilongshan Road

institute: westlake University

summary: Chromosome translocation is known to generate oncogenes such as SS18-SSX1, but the underlying mechanism remains unknown. Here we show that enhanced phase separation acquired by translocation mediates oncogenic transformation. SSX fusion dramatically enhances the phase separation property of SS18 protein through H2AK119ub. Super condensates generated by SS18-SSX recruit acetyltransferases CBP/P300 as wild type SS18, but exclude HDACs such that H3K27ac accumulates abnormally at loci from those normally occupied by SS18, resulting aberrant silencing and activation of target genes. Consistently, we show that inhibition of H3K27ac can attenuate the tumorigenicity of the fusion oncoprotein. These results provide the first case for phase separation as a transforming event  to generate oncogene and super phase separation can be targeted for cancer therapy.

overall_design: We analyzed the endogenous SS18 protein cupancy  during synovial sarcomagenesis triggered by SS18-SSX1 overexpression and the location of LLPS deficiency mutant SS(YA).

first: Junqi

last: Kuang

first: Duanqing

last: Pei


title: Single-molecule RNA-FISH analysis reveals stochasticity in reactivation of latent HIV-1 regulated by Nuclear Orphan Receptors NR4A and cMYC

first: Ganjam

middle: V

last: Kalpana

state: NY

address: 1300, Morris Park Ave.,

department: Genetics

institute: Albert Einstein Collge of Medicine

summary: HIV-1 eradication strategies require reactivation of all latent cells. Current methods lack effectiveness due to incomplete knowledge of proviral reactivation. We employed a single-molecule RNA-FISH (smRNA-FISH) analysis and found that proviral reactivation is highly variable, stochastic, and occurs in bursts and waves, with different kinetics in response to diverse LRAs. Approximately 1-5% of latent cells displayed stochastic reactivation even without stimulation. Through single-cell RNA-seq analysis, we identified NR4A3 and cMYC as extrinsic factors selectively associated with HIV-1 reactivation. Concomitant with HIV-1 reactivation cMYC was downregulated and NR4A3 was upregulated in both latent cell lines and primary CD4+ T-cells from aviremic patients. By inhibiting cMYC using SN-38, an active metabolite of irinotecan, we induced NR4A3 and HIV-1 expression. Our results suggest that inherent stochasticity in proviral reactivation contributes to cell-to-cell variability, which could potentially be modulated by drugs targeting cMYC and NR4A3.

overall_design: scRNA-seq analysis of uninduced ACH2, J1.1, or J-Lat cells. The frozen cells were revived and allowed to grow for two passages and then the ongoing cultures were submitted to genomics facility for scRNA-Seq (1 million cells/ml).

first: Rajiv

last: Pathak

first: Masako

last: Suzuki


title: Multi temperal single-cell profiling decodes cross-talk of γδ17 T cells and neutrophils in radiation pneumonitis

city: Beijing

first: Ziming

last: Jiang

address: 17 Panjiayuan Nanli Chaoyang District

department: Department of Thoracic Surgery

institute: National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital

summary: Radiation pneumonitis (RP) is a common and fatal complication in thoracic radiation therapy. The immune microenvironment plays an important role in the RP development. However, the full scope of radiation immune changes over time remains unclear. In this study, a murine model of RP was established, and distinct early (10 days) and late phases (100 days) were defined. We conducted single-cell RNA sequencing and related RNA-seq and proteomics to explore the underlying mechanisms of persistent inflammation during RP. By examining the immunological effects of RP over time at the single-cell level, we shed light on its dynamic immunological effects of RP over time at single-cell resolution and provided potential therapeutic targets for clinical interventions.

overall_design: C57BL/6N female mices were analysed after being treated either with a single dose of 20 Gy of 9 MeV electron-ray or with no treatment from 10 days or 100 days post-radiation.

first: Wenting

last: Ren

first: Xiaoxiang

last: Zhou

first: Shiqi

last: Li

first: Yibo

last: Gao

first: Jie

last: He


title: Enhanced Phase Separation Mediated Oncogenicity [RNA-Seq]

overall_design: We analyzed the transcriptome expression profiles (RNA-seq) during synovial sarcomagenesis triggered by SS18-SSX1 overexpression and by SS18-SSX1 overexpression with treatment by CBP/P300 inhibitor A485.


title: Role of growth plate Nox2 in alcoholic osteopenia

city: New Orleans

first: Kim

middle: B

last: Pedersen

state: LA

address: 1901 Perdido St., Rm 5206

department: Pharmacology and Exp. Therapeutics

institute: LSU Health Sciences Center, Dept. of Pharmacology

summary: To study the role of Nox2 in ethanol toxicity on the bone growth plate chondrocytes, we generated Nox2 conditional knockout mice (CKO), in which the catalytic subunit of Nox2, Cybb, is deleted in chondrocytes using a Cre-lox system, where Cre is expressed from the Col2a1 promoter.  CKO mice and floxed control mice were fed an ethanol-containing Lieber De-Carli-based diet or pair-fed a control diet for 8 weeks starting at 5-6 weeks of age. As both the Nox2 genotype and ethanol diminished the number of chondrocytes in the growth plates, we conducted an RNA-Seq analysis of the growth-plate containing regions of the femurs.

overall_design: Femurs were separated into femoral shaft, femoral marrow and the femoral end pieces containing the growth plates (Pedersen et al. Bone Rep. 2019 May 31;11:100211. doi: 10.1016/j.bonr.2019.100211.). RNA was isolated from femoral end pieces from female mice. Each sample of RNA subjected to RNA-Seq was a pool of RNA from three mice. The design is a 2 X 2 factorial design with two genotypes (CKO and floxed controls) and two diets (ethanol-fed and pair-fed) with three samples per group. The RNA-Seq libraries and initial analyses were performed by the commercial vendor Azenta/GeneWiz.

first: James

last: Watt

first: Martin

last: Ronis


title: Acetylcyssteine synergizes PD-1 blockers against colorectal cancer progression by promoting TCF1+PD1+CD8+ T cell differentiation in mice

city: Shanghai

first: Wenchang

address: 180# Feng-Lin Road, Shanghai, 200032, China

institute: Zhongshan Hospital, Fudan University

summary: PD-1 blockade is important in treating progressive colorectal cancer (CRC). However, some patients with CRC have a poor or no response to immunotherapy. An important reason for this problem is the exhaustion of CD8+ T cells in the tumor microenvironment. Acetylcysteine (NAC) can reduce CD8+ T cell exhaustion in vitro and induce their differentiation into long-lasting phenotypes, thus enhancing the anti-tumor effect of adoptive T cell transfer. However, whether NAC can be combined with PD-1 blockade in CRC treatment and how NAC regulates CD8+ T cell differentiation remains unclear. We constructed a mouse CRC model to study the effect of NAC on tumors. The effect of NAC on CD8+ T cell differentiation and its potential mechanism were explored by cell flow assay and other studies in vitro and ex vivo. We demonstrated that NAC synergized PD-1 antibodies to inhibit CRC progression in a mouse CRC model, mediated by CD8+ T cells. NAC induces TCF1+PD1+CD8+ T cell differentiation and reduces the formation of exhausted T cells (Tex). Moreover, NAC enhanced the expression of Glut4 in CD8+ T cells, promoting the differentiation of TCF1+PD1+CD8+ T cells. Our study provides a novel idea for immunotherapy of clinically progressive CRC and suggests that Glut4 may be a new immunometabolic molecular target for regulating CD8+ T cell differentiation.

overall_design: In order to study the effect of NAC on CD8+T cells, CD8+T cells were selected from mouse spleen and cultured in normal medium or medium containing 10mM NAC for 5 days. Then the two groups of cells were taken for RNA sequencing analysis.

first: Ying

last: Yue

first: Ziwen

last: Zhong

first: Ke

first: Changhong

last: Miao


title: Cardiomyocyte specific RXFP1-overexpression protects Relaxin-independently from pressure-overload induced cardiac dysfunction

city: Mannheim

first: Carsten

last: Sticht

address: Theodor-Kutzer-Ufer

department: ZMF

institute: University Heidelberg

summary:
Aim was to investigate the effect of cardiomyocyte-specific expression of the human Relaxin Family Peptide Receptor 1 (RXFP1) in a mouse model of pressure-overload induced cardiac dysfunction (TAC).
Mice overexpressing the hRXFP1 receptor (or non-transgenic controls) underwent TAC (or Sham) surgery and were followed up for 8 weeks. Heart was removed and RNA isolated from left ventricle to perform bulk RNAseq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for left ventricular tissue from hRXFP1tg mice (or non-tg controls) following 8 weeks of TAC (or Sham)  (4 group design).

first: Julia

last: Wingert

first: Eric

last: Meinhardt

first: Nuttarak

last: Sasipong

first: Carolina

last: de la Torre

first: Philipp

last: Schlegel


title: Small-molecule-induced epigenetic rejuvenation overcomes myelinogenic barriers and stimulates myelin repair

first: XUEZHAO

last: LIU

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


title: Small-molecule-induced epigenetic rejuvenation overcomes myelinogenic barriers and stimulates myelin repair [Cut&Run]

overall_design: cut and run of treated rat iOLs  with indicated antibody

first: Xuezhao

last: Liu

first: Xuelian

first: Qing

last: Lu


title: Gene expression profile at single cell level of total alveolar type II (AT2) cells and CD44hi AT2 cells

city: Chicago

first: Qian

last: Chen

state: IL

address: 909 S Wolcott avenue, COMRB 4160

department: Department of Pharmacology and Regenerative Medicine

institute: University of Illinois at Chicago

summary: AT2 cells are the resident progenitor cells in alveoli, capable of self-proliferation and differentiation into alveolar type I cells during homeostatic maintenance and tissue regeneration. The AT2 cell population is heterogenous. We identified a small subpopulation of AT2 cells that express high levels of CD44 (CD44hi) and display progenitor functions during alveoli homeostasis. To further analyze the heterogeneity of the AT2 cell population and characterize CD44hi AT2 cells, we performed single cell RNA-seq on the total AT2 cell population and CD44hi AT2 cells.

overall_design: Total AT2 cells and CD44hi AT2 cells were isolated from 6 SpcER/Tomato/zsGreen mice by fluorescence activated cell sorting (FACS). Cells were pooled and analyzed using scRNAseq.

first: Alessandro

last: Chan

first: Yuru


title: Enhancing transcription replication conflict targets ecDNA positive cancers [KAS-seq]

city: Stanford

first: Guiping

last: Wang

address: 256 Campus Dr

institute: Stanford University

summary:
Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. EcDNA renders tumours treatment-resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival. At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription replication conflict enables targeted elimination of ecDNA-containing cancers. Stepwise analyses of ecDNA transcription reveal pervasive RNA transcription and associated single-stranded DNA (ssDNA), leading to excessive transcription replication conflicts and replication stress (RS) compared to chromosomal loci. Nucleotide incorporation on ecDNA is markedly slower, and RS is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. pRPA2-S33, a mediator of DNA damage repair that binds ssDNA, shows elevated localization on ecDNA in a transcription dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition abrogates the DNA replication check point, causing extensive and preferential tumour cell death in ecDNA-containing tumours. We advance a highly selective, potent, and bioavailable oral CHK1 inhibitor, BBI-2779, that preferentially kills ecDNA-containing tumour cells.  In a gastric cancer model containing FGFR2 on ecDNA, BBI-2779 suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. Transcription replication conflict emerges as a target for ecDNA-directed therapy, exploiting a synthetic lethality of excess to treat cancer.

This work was delivered as part of the eDyNAmiC team supported by the Cancer Grand Challenges partnership funded by Cancer Research UK (CGCATF-2021/100012 and CGCATF-2021/100025) and the National Cancer Institute (OT2CA278688 and OT2CA278635) to H.Y.C., P.S.M., and V.B. This project was supported by NIH RM1-HG007735 (H.Y.C., W.J.G., C.C.).

overall_design: Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) in isogenic cell pair of COLO320 DM and COLO320 HSR, and under transcription inhibition by triptolide, 2 biological replicates each

first: Georgi

middle: K

last: Marinov

first: Howard

last: Chang

first: William

middle: J

last: Greenleaf


title: Rhesus macaque SIV LCV Co infection Study

city: Bethesda

first: Douglas Scott

last: Merrell

state: MD

address: 4301 Jones Bridge Road

department: Dept. of Microbiology and Immunology

institute: Uniformed Services University

summary:
Question Addressed: Does gene expression change in the buccal mucosa of Lymphocryptovirus  (LCV) infected animals when they are chronically infected with Simian immunodeficiency virus (SIV)? Oropharyngeal mucosal tissue samples were collected from rhesus macaques.   A pooled common reference was used for all hybridizations.  This reference was composed of RNA harvested from rhesus macaques not infected with either LCV or SIV.
Infection: Animals were infected with SIV and/or LCV

first: Joseph

last: Mattapallil


title: Enhancing transcription replication conflict targets ecDNA positive cancers [GRO-seq]

summary:
Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. EcDNA renders tumours treatment-resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival. At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription replication conflict enables targeted elimination of ecDNA-containing cancers. Stepwise analyses of ecDNA transcription reveal pervasive RNA transcription and associated single-stranded DNA (ssDNA), leading to excessive transcription replication conflicts and replication stress (RS) compared to chromosomal loci. Nucleotide incorporation on ecDNA is markedly slower, and RS is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. pRPA2-S33, a mediator of DNA damage repair that binds ssDNA, shows elevated localization on ecDNA in a transcription dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition abrogates the DNA replication check point, causing extensive and preferential tumour cell death in ecDNA-containing tumours. We advance a highly selective, potent, and bioavailable oral CHK1 inhibitor, BBI-2779, that preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 on ecDNA, BBI-2779 suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. Transcription replication conflict emerges as a target for ecDNA-directed therapy, exploiting a synthetic lethality of excess to treat cancer.

This work was delivered as part of the eDyNAmiC team supported by the Cancer Grand Challenges partnership funded by Cancer Research UK (CGCATF-2021/100012 and CGCATF-2021/100025) and the National Cancer Institute (OT2CA278688 and OT2CA278635) to H.Y.C., P.S.M., and V.B. This project was supported by NIH RM1-HG007735 (H.Y.C., W.J.G., C.C.).

overall_design: Profiling of nascent transcription in COLO320DM/HSR and GBM39ec/HSR - 2 biological replicates each

first: Natasha

last: Weiser


title: Transcriptomic profiling of osteoarthritis synovial macrophages reveals a tolerized phenotype compounded by a weak corticosteroid response

city: Nijmegen

first: Cheng

address: Geert Grooteplein 28

department: Molecular biology

country: Netherlands

summary: It is well-known that long-term osteoarthritis prognosis is not improved by corticosteroid treatments. Here we investigate what could underlie this phenomenon by measuring the short term corticosteroid response of osteoarthritic joint synovial macrophages (OA-Mf).

overall_design: Monocytes were purified from healthy blood donor buffy coats as described previously. Monocyte-derived Mf were differentiated for 6 days in RPMI 1640 medium with 10% human serum. Training and tolerization involved exposure to lipopolysaccharide (LPS; 5 ng/ml) or β-glucan (BG; 10 μg/ml) during the first 24 h of monocyte differentiation. Synovial tissue from end-stage OA patients was obtained after joint replacement surgery. Post-operative biopsies from patients with end-stage OA were handed over to the Experimental Rheumatology laboratory of the Radboud Institute for Molecular Life Sciences. The synovial tissue was separated from muscle, fat and ligament tissue and cultured overnight in RPMI 1640 with 10% fetal bovine serum (FBS) at 37°C to let cells from a vascular origin leach out of the synovial tissue. The next day, the synovial tissue was first cut into small pieces and then digested using plain RPMI medium containing a final concentration of 0.1 mg/ml DNase I (Roche, Mannheim, Germany, 11284932001), 1 mg/ml Collagenase D (Roche, 11088858001) and 2.4 mg/ml Dispase II (Sigma, Burlington, MA, USA, D4693-1G) on a roller at 37°C for 1 h. Afterwards, a single-cell suspension was obtained by pipetting up and down to mechanically break down the tissue. Next, the suspension was washed and run through a 70 µm cell strainer, after which the digestion reaction was stopped by adding pure FBS. Erythrocytes were removed using lysis buffer (155 mM NH4Cl, 12 mM KHCO3 and 0.1 mM EDTA) and the reaction was later stopped by phosphate-buffered saline (PBS) addition. To isolate CD14+ cells, magnetic-activated cell sorting (MACS) buffer (PBS, 1.5% acid citrate dextrose-A, 1% FBS) and CD14-coated microbeads were added to the sample, which was then run through a MACS column. After five washing steps, CD14+ cells were eluted following the manufacturer’s (Miltenyi Biotec, Bergisch Gladbach, Germany) instructions and 200 000 obtained synovial cells were resuspended per 6 cm dish in RPMI medium supplemented with 10% FBS. Treatment with a final concentration of 1 µM TA (Sigma, T6501) or only dimethyl sulfoxide (DMSO) solvent (0.1% final concentration) lasted 4 h for all cell types. As a positive control for the TA treatment, peripheral blood mononuclear cells from a healthy blood donation buffy coat were exposed to TA under the same culture conditions as the isolated CD14+ OA-Mf using the same set of reagents and equipment.

first: Ruben

last: De Francesco

first: Martijn

last: van den Bosch

first: Colin

last: Logie


title: RORA proximal enhancer regulates its expression during early human Th17 cell differentiation [ChIP]

city: Turku

first: Sini

last: Junttila

address: Tykistökatu 6 A

department: Turku Bioscience Centre

country: Finland

institute: University of Turku

summary: Distal regulatory elements, such as enhancers, play a pivotal role in dictating cell identity by controlling the transcriptional status of specific cells. The comprehensive understanding of the epigenetic landscape, encompassing enhancer establishment and chromatin accessibility, during the differentiation of human Th17 cells remains incomplete. Leveraging ATAC-seq based chromatin accessibility profile along with the profiling of key histone marks we identified potential enhancers crucial for fate specification of Th17 cells. We found that 24 single nucleotide polymorphisms (SNPs) associated with autoimmune diseases were located near Th17-enhancers. Interestingly, these SNPs overlapped the binding sites of transcription factor active in Th17 cells. Among the Th17 specific enhancers, we identified an enhancer in the intron of RAR-related orphan receptor alpha (RORA) that was earlier predicted to regulate RORA expression. Functional validation through luciferase reporter assays confirmed that this enhancer positively regulate the transcription. Moreover, employing CRISPR-Cas9-mediated deletion of a transcription factor binding site-rich region within the identified RORA enhancer, we demonstrated its role in regulating RORA transcription. These findings provide insights into the potential mechanism by which the RORA enhancer modulates Th17 differentiation and into the role of regulatory SNPs within noncoding regions in conferring resistance or susceptibility to Th17 cell-mediated autoimmune pathologies.

overall_design: Human CD4+ T cels are isolated from cord blood. Cells were activated under Th0 and Th17 condition for 0.5 and 72 h. ChIP assay was performed using H3K4me1, H3K4me3, and H3K27ac, antibodies following protocol described earlier (Ullah et al., 2018).

first: Mohd

last: Moin Khan

first: Subhash

last: Tripathi

first: Ubaid

last: Ullah Kalim

first: Omid

last: Rasool

first: Riitta

last: Lahesmaa


title: RORA proximal enhancer regulates its expression during early human Th17 cell differentiation

overall_design: Refer to individual Series


title: MSC-small extracellular vesicles alleviated Th2-airway inflammation by regulating the metabolism of DCs in mice

city: Guangzhou

first: Long-Xin

last: Huang

address: No. 58, Zhongshan 2nd Road

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

summary:
Background: Allergic asthma is one of the chronic inflammatory diseases and is generally induced by CD4+ T helper 2 cells (Th2) in the context of persistent inhaled stimuli. Dendritic cells (DCs) are essential to mounting the Th2-mediated airway inflammation by presenting inhaled antigens to prime CD4+ T cells. Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSCs) exhibited great interest in intractable diseases. However, whether MSC-sEV play a role on DCs in airway inflammation is still unclear.
Methods: We isolate MSC-sEV using anion-exchange chromatography. Mouse bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs) were used to study the effects of MSC-sEV on dendritic cell surface molecules and their cytokine release. Mice were sensitized with house dust mites (HDM) to induce airway inflammation, and treated with MSC-sEV, The effects of sEV on murine DCs were identified. Extracellular flux analysis techniques were used to study the effects of MSC-sEV on the metabolic state of dendritic cells. RNA sequencing to study altered gene expression in BMDCs after MSC-sEV treatment.
Results: MSC-sEV mitigated the accumulation of Th2-associated moDCs in mouse lung in response to HDM. MSC-sEV also decreased the activation of moDCs induced in vitro including the expression of co-stimulatory molecules and cytokines secretion. Furthermore, we identified that DCs were able to take MSC-sEV in vitro and in vivo. Mechanistically, using bulk RNA-sequencing, we found that MSC-sEV played roles in the metabolic pathway of murine DCs. Using extracellular flux analysis, we found that MSC-sEV increased the requirement of oxidative phosphorylation on moDCs. Importantly, MSC-sEV displayed similar effects on human moDCs including decreased co-stimulatory molecular and cytokine production.
Conclusions: MSC-sEV are able to alter the metabolic state of DCs, favoring DCs to maintain OXPHOS (oxidative phosphorylation) rather than glycolysis, thereby reducing DCs-initiated inflammatory responses and attenuating Th2 lung inflammation, suggesting MSC-sEV can be a potential clinical therapy for airway inflammation.

overall_design: To identify how sEV regulated DC activation, the analysis of global genes was applied to understand the mechanism. We compared geomatic profiles of PBS or sEV-treated BMDCs.

first: Lifen

last: Wen


title: Profiling microRNA for the prediction of stress fractures in the Thoroughbred racehorse

city: Midlothian

first: SEUNGMEE

last: LEE

address: Easter Bush Campus

department: Roslin Institute

institute: University of Edinburgh

summary: Stress fractures are microcracks in bone caused by repetitive loading and these microcracks can increase the risk of developing catastrophic fractures. Early detection of stress fractures can be difficult due to the necessity of accurate clinical detection. MicroRNAs could be used as potential diagnostic and prognostic biomarkers due to their size, abundance, tissue specificity and stability in body fluids. Global profiling of circulating plasma small RNA-sequencing between lame horses (stress fractures and non-stress fractures) and non-lame control horses and the effects and role of selected stress fracture-related miRNAs on equine bone marrow derived mesenchymal stem cells (BMMSCs) were investigated. The miRNA profiles of stress fracture cases and non-lame stress fracture cases were similar. No miRNA was identified for potential usage as a biomarker for differentiating stress fracture from lame cases. However, the miRNA profiles of lame cases and non-lame control horses were significantly different. RNA-sequencing showed three miRNAs (eca-miR-486-5p, eca-miR-26a and eca-miR-23a) were most significantly differentially expressed (p value <0.05) between lame cases and non-lame controls, which was validated by qPCR. Of these, eca-miR-486-5p demonstrated the most abundant and significant increased miRNA in the lame cases compared to non-lame control cases. Cell transfection experiments using the miR-486-5p mimic treatment in equine BMMSCs showed upregulation of the osteogenic transcription factor, Runt-related transcription factor 2 (RUNX2) and insulin-like growth factor type 1 (IGF1). A significant decrease of miR-133a in the miR-486-5p mimic treated cells was also demonstrated. Since RUNX2 is the known target of miR-133a, miR 486-5p together with miR-133a would suggest the biological importance of bone turnover and osteogenesis in equine BMMSCs, but further investigation of role of miR-486-5p and miR-133a in equine bone biology is warranted.

overall_design: To investigate gobal profiling of circulating plasma, small RNA-sequencing between lame horses (stress fractures and non-stress fractures) and non-lame control horses were carried out

first: Seungmee

last: Lee

first: Melissa

last: Baker

first: Eugenio

last: Cillan-Garcia

first: Mike

last: Clinton

first: Sarah

last: Taylor


title: Differentially expressed genes in HBV related hepatic disease patients and the correlation with prognosis

city: Chengdu

first: xipeng

last: yan

state: Sichuan

address: 26 Huacai Road, Chengdu, China

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

summary: To access different gene expression between hepatic diseases patients and healthy adults, we collected peripheral blood samples to abstract PBMC. A total of 15 patients and 5 healthy adults were recruited.

overall_design: patients with HBV related liver cirrhosis (LC),  patients with HBV induced liver failure (LF), patients with liver failer combined with fungal infection (LFF) and  healthy controls (HC)were enrolled in this study, 5 blood samples collected form each type of diseases patients or healthy adults.a total of 20 samples were enclosed in this research.

first: Yujuan

last: Guan

first: Jianping

last: Li

first: Xiaoqiong

last: Duan


title: Daphnetin induces ferroptosis in ovarian cancer by inhibiting NAD(P)H: Quinone oxidoreductase 1 (NQO1)

city: Chang chun

first: Ning

last: Ma

address: Xinmin Road 71, The First Hospital of Jilin University

institute: The First Hospital of Jilin University

summary: Ferroptosis, an emerging nonapoptotic, regulated cell death process distinguished by iron accumulation and subsequent lipid peroxidation, is intricately implicated in the development and progression of multiple cancer types. Here, we aimed to reveal that triggering ferroptosis is a promising treatment strategy for ovarian cancer. In this study, we not only validated that daphnetin caused ferroptosis, but evaluated the effects of daphnetin (and/or cisplatin) in vitro and vivo.Here, we elucidated that daphnetin, a natural product isolated from Daphne Korean Nakai, can exert antitumor effects by inducing the death and suppressing the migration of ovarian cancer cells. Subsequently, transcriptome analysis and ferroptosis inhibitor (Fer-1 and DFO) experiments revealed that there is a close correlation between daphnetin and ferroptosis in ovarian cancer. We further found that daphnetin induced ferroptosis in ovarian cancer cells, as evidenced by the accumulation of intracellular ferrous iron (Fe2+), reactive oxygen species (ROS) and lipid peroxides, as well as the depletion of glutathione (GSH) and ferroptosis indicators (SLC7A11 and GPX4). In particular, daphnetin effectively reduced the mRNA and protein levels of NQO1 (a ubiquitous flavoenzyme), and a high expression level of NQO1 was significantly associated with poor prognosis and ferroptosis resistance in ovarian cancer patients. Furthermore, NQO1 activation markedly attenuated daphnetin-induced cell death, migration and ferroptotic events in vitro and vivo. Interestingly, we also found that treatment with daphnetin, a negative regulator of NQO1, in combination with cisplatin synergistically induced ovarian cancer cell cytotoxicity. This study demonstrated that daphnetin induces ferroptosis by inhibiting NQO1 in ovarian cancer cells. Our findings identified NQO1 as a new daphnetin target and suggested that targeting NQO1 might have therapeutic effects on ovarian cancer.

overall_design: To illustrate the specific mechanism that daphnetin can induce cell death and hinder the cell invasion and migration of ovarian cancer, RNA sequencing was conducted to identify the differentially expressed mRNAs between control-treated and daphnetin-treated A2780 (or SKOV3) cells, thereby identifying the closely related signaling pathways and targets.

first: Mengwen

last: Zhang

first: Zhentong

last: Wei

first: Songling


title: Cell-free DNA Methylation Patterns in Aging and Their Association with Inflamm-aging

first: Sijia

state: Beijing

address: No. 1 Dahua Road

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

summary: Background: Liquid biopsies analyzing cell-free DNA methylation in plasma offer a non-invasive diagnostic for diseases, with aging biomarker potential underexplored. Methods: Utilizing enzymatic methyl-seq (EM-seq), this study assessed cfDNA methylation patterns in aging with blood from 35 healthy individuals. Results: It found aging signatures, including higher cfDNA levels and variations in fragment sizes, plus over 2 million age-related differentially methylated CpG sites. A biological age predictive model based on 41 CpG sites showed a strong correlation with chronological age, verified by two datasets. Age-specific epigenetic shifts linked to inflammation were revealed through differentially methylated regions profiling and Olink proteomics. Conclusion: These findings indicate cfDNA methylation as a potential biomarker for aging, and it might exacerbate immunoinflammatory reactivity in older individuals.

overall_design: The study utilized enzymatic methyl-seq (EM-seq) to explore the cfDNA methylation profiles and patterns in aging, aiming to determine cfDNA methylation's potential as an aging marker. The present study involved blood samples collected from 35 healthy individuals.

first: Si-Jia

first: Xin

last: Gao

first: Zi-Hui

last: Wang

first: Jin

first: Lv-Tao

last: Zeng

first: Ya-Min

last: Dang

first: Li-Qun

first: Qing-Yu

first: Ying-Min

first: Hong-Lei

last: Liu

first: Ruo-Mei

last: Qi

first: Jian-Ping

last: Cai


title: RORA proximal enhancer regulates its expression during early human Th17 cell differentiation [ATAC]

first: Sini

last: Junttila

overall_design: Human CD4+ T cels are isolated from cord blood. Cells were activated under Th0 and Th17 condition for 2 h, 4 h, 24 h, 48 h, and 72 h and ATAC-seq was performed on harvested nucleii.

first: Mohd

last: Moin Khan

first: Ubaid

last: Ullah Kalim

first: Omid

last: Rasool

first: Riitta

last: Lahesmaa


title: Distinctive roles for the chromatin remodeling ATPases Brg1 and Brm in early zebrafish embryos [ChIP-seq]

city: Salt Lake City

first: Bradley

last: Cairns

address: 2000 Cir of Hope Dr

institute: Huntsman Cancer institute/HHMI

summary: Brg1 and Brm function as alternative core enzymatic ATPases of SWI/SNF complex in vertebrates, with clear homologs in zebrafish. Brg1 (but not Brm) is required for early cleavage-stage transcription in mice, but where and how Brg1 or Brm impact chromatin in early embryos remains unexplored. Here, we utilize zebrafish embryos to provide the first simultaneous genome-wide profiling of Brg1 and Brm occupancy in any organism/cell type, alongside our profiling of open chromatin and RNA polymerase II. We reveal shared and unique loci for Brg1 and Brm, their shared occupancy of open chromatin and particular developmental promoters, and higher transcription at co-occupied genes. Interestingly, only Brg1 is strongly associated with active histone modifications. Strikingly, only Brm commonly occupies gene bodies, overlapping precisely with Pol II. Functional experiments involving Pol II elongation inhibition alongside bioinformatic analyses suggest that Brm travels with Pol II into gene bodies, primarily at short, very highly-transcribed genes with few introns. Taken together, our results support roles for Brg1 primarily at promoters and enhancers, and for Brm in association with Pol II in elongation through chromatin within gene bodies during genome-wide transcriptional activation

overall_design: Examination of Brg1, Brm and PolII at 3 zebrafish developmental stages. In addition, ChIP-seq of Brm, PolII and H3K36me3 were perfromed in embryos treated with flavopiridol.

first: Mengyao

last: Tan

first: Candice

last: Wike

first: Yixuan

last: Guo

first: Christian

last: Pflueger


title: Gene expression analysis of hPSC derived macrophages (iMacs) co-cultured with hepatic cells

first: Jie

address: Tsinghua University

department: Basic Medical Sciences, School of Medicine

institute: Tsinghua University

summary: Purpose: to analyze change in gene expression of hPSC derived macrophages upon co-culture with hepatic cells including Huh7 and primary human hepatocytes Methods: SMART-seq2 amplified polyA RNA from sorted iMacs in the co-cultures.  Results: the transcriptome of iMacs changed after co-culture with Huh7 cells or pHHs, and changed after the HCV infection of the co-cultured Huh7 cells.  Conclusions: the gene expression profiles of co-cultured iMacs indicated that the culture conditions and the microenvironments could reprogrammed iMacs to adopt tissue-specific features and perform specifc functions.

overall_design: Perform RNA-seq of control iMacs, and sorted iMacs from iMac-pHH co-cultures, and iMac-Huh7 co-cultures with or without HCV.

first: Yaxuan

last: Zhang

first: Xingwu

first: Hui

last: Qiu


title: Enhancing transcription replication conflict targets ecDNA positive cancers [WGS]

first: Guiping

last: Wang

overall_design: PC3 purchased from ATCC demonstrated significant amplicon heterogeneity of the Myc oncogene. To generate an isogenic system for PC3, single clone expansion was performed to generate monoclones with Myc amplified on either ecDNA (PC3-DM) or on chromosome (PC-HSR). These monoclonal lines were subjected to characterization by metaphase-FISH, and whole genome sequencing for copy number analysis and amplicon similarity analysis.

first: Ivy

middle: T

last: Wong

first: Paul

last: Mischel


title: Gene expression analysis of hPSC-derived macrophages (iMACs) encountering Streptococcus pneumoniae.

address: 30 Shuangqing Rd, Haidian District, Beijing, China

department: School of Medicine

summary:
Purpose: to analyze gene expression changes of iMACs encountering S. pneumoniae.
Methods: SMART-seq2 amplified polyA RNA from sorted iMACs in the co-cultures.
Results: the transcriptome of iMACs changed after encountering S. pneumoniae.
Conclusions: the gene expression profiles of S. pneumoniae stimulated iMACs indicated iMACs undergoing inflammatory response and the immune response was distinct with iMACs encountering other pathogens such as HCV or SARS-CoV-2.

overall_design: Perform RNA-seq of control iMACs, and  iMACs stimulated by S. pneumoniae serotype 14.

first: Duan

last: Fuyu

first: Qiu

last: Hui

last: Jie


title: Hidden RNA profiles revealed by malignant cell fraction-informed deconvolution

city: Amsterdam

first: Yongsoo

last: Kim

address: De Boelelaan 1118

institute: University Medical Center Amsterdam, Location VUmc

summary: To understand cancer biology, it is crucial to unravel malignant- and microenvironmental cell states. Deconvolution of the bulk RNA profile from tumors can provide this insight, but is severely impaired by interpatient heterogeneity of the malignant cells. Bayesian statistics enables to inform RNA-deconvolution with malignant cell fraction estimates from e.g. DNA sequencing. We developed cell fraction-informed deconvolution, and evaluated this experimentally using 46 bulk RNA profiles from blood with 19 CyTOF determined cell fractions from this repository.

overall_design: Blood was drawn from patients undergoing surgery for glioblastoma, meningioma or astrocytoma, as well as healthy donors. PBMCs were isolated using gradient density centrifugation with lymphoprep (Axis-Shield). Multiple vials of PBMCs were cryopreserved per donor, which were partly used in a previous CyTOF study (Dusowa et al.  Front Immunol. 2024). 48 Donors for which CyTOF data was available were included in the present study. In CyTOF only live cells are phenotyped, therefore only live cells were used for RNA isolation to obtain a matching dataset. First PBMCs were thawed through dropwise addition of IMDM (Gibco) with 10% fetal calf serum (FCS, Biowest), 100 IU Penicillin and 100 IU Streptomycin (Lonza) at 37 °C. Thawed cells were centrifuged at 500 g for 8 min at 4 °C. Cell pellets were taken up in cold sterile-filtered 0.1% BSA (Roche) in PBS (Fresenius Kabi) and again centrifuged. Washed cells were stained with ice cold 7-AAD in 0.1% BSA in PBS. Live cells were sorted at 5 °C using a FACS Aria Fusion (BD Biosciences) with IMDM with 20% FCS, 100 IU Penicillin and 100 IU Streptomycin as collection medium. After sorting 0.7-1 million live cells collection tubes were immediately centrifuged for 8 min at 500 g and 4 °C. The supernatant was poured off, the remaining liquid was used to resuspend the cell pellet before addition of TRIzol™ Reagent (Invitrogen; Thermo Fisher Scientific). RNA isolation was performed according to manufacturer’s protocol. Remaining TRIzol™ contaminations were removed using the buthanol extraction protocol described by Krebs et al (2009). RNA dissolved in water was mixed with water-saturated 1-buthanol (Sigma-Aldrich), briefly centrifuged, followed by removal of the upper organic layer. After repeating the previous step once with 1-buthanol, these steps were performed twice with water-saturated diethyl ether (Riedel-de Haën). The final diethyl ether layer was left to evaporate in a fume hood for 15 minutes. RNA quality was assessed using the Agilent RNA 6000 Pico Kit and the 2100 Bioanalyzer (Agilent Technologies). Samples included for library preparation had an RNA integrity number (RIN) value of at least 6.9  and a yield of at least 0.72 µg.

first: Jurriaan

last: Janssen

first: Teodora

last: Radonic

first: Mischa

middle: F

last: Steketee

first: Saskia

last: van Asten

last: Eijk

first: Febe

last: van Maldegem

first: David

last: Noske

first: Idris

last: Bahce

first: Juan

last: Garcia Vallejo

first: Mark

last: van de Wiel

first: Bauke

last: Ylstra


title: Single-cell sequencing reveals Mincle receptor mediated inflammation for resolution in ischemic kidney injury

city: Nanjing

first: Ning

last: Li

address: Nanjing

institute: Southeast University

summary: Recent studies have demonstrated a strong association between acute kidney injury (AKI) and chronic kidney disease (CKD), while the unresolved inflammation is believed to be a driving force for this chronic transition process. As a transmembrane pattern recognition receptor, Mincle (macrophage-inducible C-type lectin, Clec4e) was identified to participate in the early immune response after AKI. However, the impact of Mincle on the chronic transition of AKI remains largely unclear. We performed single-cell RNA sequencing (scRNA-seq) with the unilateral ischemia-reperfusion (UIR) murine model of AKI at days 1, 3, 14 and 28 after injury. Potential effects and mechanism of Mincle on renal inflammation and fibrosis were further validated in vivo utilizing Mincle knockout mice. The dynamic expression of Mincle in macrophages and neutrophils throughout the transition from AKI to CKD was observed. For both cell types, Mincle expression was significantly up-regulated on day 1 following AKI, with a second rise observed on day 14. Notably, we identified distinct subclusters of Mincle-high neutrophils and Mincle-high macrophages that exhibited time-dependent influx with dual peaks characterized with remarkable pro-inflammatory and pro-fibrotic functions. Moreover, we identified that Mincle-high neutrophils represented an "aged" mature neutrophil subset derived from the "young" mature neutrophil cluster in kidney. Additionally, we observed a synergistic mechanism whereby Mincle-expressing macrophages and neutrophils sustained renal inflammation by augmenting tumor necrosis factor (TNF) production. Mincle-deficient mice exhibited reduced renal injury and fibrosis following AKI.

overall_design: Unilateral ischemia-reperfusion (UIR) murine model of AKI at days 1, 3, 14 and 28.