title: The local molecular signature of human peripheral neuropathic pain

city: Oxford

first: Georgios

last: Baskozos

address: NDCN, Level 6, West Wing, John Radcliffe Hospital

institute: University of Oxford

summary: Focal nerve injuries are often associated with neuropathic pain. Preclinical research suggests altered neuroimmune signalling underlies such neuropathic pain; however, its cause remains poorly understood in humans. In this multicentre cohort study, we describe the local cellular and molecular signature of neuropathic pain at the lesion site, using Morton's neuroma as a human model system of neuropathic pain (n = 22; 18 women) compared with nerves from participants without nerve injury (n = 11; 4 women). Immunofluorescent staining revealed demyelination and chronic infiltration of immune cells in Morton's neuroma. RNA bulk sequencing identified 3349 differentially expressed genes between Morton's neuroma and controls. Gene ontology enrichment analysis and weighted gene co-expression network analyses revealed modules specific for host defence and neurogenesis. Deconvolution analysis confirmed higher densities of macrophages and B cells in Morton's neuroma than control samples. Modules associated with defence response, neurogenesis, and muscle system development as well as macrophage cell populations identified by deconvolution correlated with patients' paroxysmal or evoked pain. Of note, we identified a consistently differentially expressed gene signature (MARCO, CD163, STAB1), indicating the presence of a specific M(GC) subset of macrophages. MARCO gene expression correlated with paroxysmal pain. Targeted immunofluorescent analyses confirmed higher densities of intraneural CD163+MARCO+ macrophage subsets in Morton's neuroma. Our findings provide detailed insight into the local molecular signature in the context of human focal nerve injury. There is clear evidence for an ongoing role of the immune system in chronic peripheral neuropathic pain in humans, with macrophages and specifically the M(GC) MARCO+ subset implicated.

overall_design: From patients with Morton’s neuroma, 22 affected plantar digital nerves were collected during routine surgical excision of the neuroma. From control participants, 11 nerves of the upper and lower limbs were collected (5 nerve grafts, 3 muscle flaps, 3 amputations). We then performed gene expression profiling using bulk RNA-seq to establish transcriptional differences between Morton’s neuroma and control nerves.

first: Oliver

last: Sandy-Hindmarch

first: Pao-Sheng

last: Chang

first: Annina

last: Schmid


title: RNA-seq data from mouse BMDMs infected with Salmonella enterica serovar Typhimurium

status: Public on Jul 31 2024

city: Hangzhou

first: Pinanong

last: Na Phatthalung

address: Department of Nutrition Yuhangtang Road 866, Hangzhou, Zhejiang Province Zijingang Campus, Zhejiang University

country: Thailand

institute: Zhejiang University

type: Expression profiling by high throughput sequencing

summary:
To investigate the gene expression profile of macrophages in response to Salmonella infection in vitro, we differentiated mouse BMDMs and the cells were used as a model for Salmonella infection
We then performed gene expression profiling analysis using data obtained from RNA-seq of BMDMs at two conditions (uninfected and Salmonella-infected cells).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Salmonella-infected BMDMs and uninfected BMDMs.

first: Fudi

last: Wang

first: Juxia

last: Min


title: Pharmacological Repositioning of the transcription factor PU.1 [ATAC-seq]

city: Bronx

first: Samuel

middle: John

last: Taylor

state: NY

address: 1300 Morris Park Ave, Chanin building 601

country: USA

institute: Albert Einstein College of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: To investigate the effects of DB2115 (PU.1 binding-site inhibitor/redistributor) upon PU.1 genomic localization, the transcriptome and chromatin accessibility.

overall_design: MOLM-13 cells grown in 10% FBS, 1% Pen Strep, RPMI media were treated with 5uM of DB2115 for 0,1,4 & 12hr before cells were harvested and Omni-ATAC sequencing pipeline was performed.

first: Ulrich

last: Steidl


title: Pharmacological Repositioning of the transcription factor PU.1

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

overall_design: Refer to individual Series


title: Pharmacological Repositioning of the transcription factor PU.1 [CUTnTag]

summary: This study aims to investigate the effects of PU.1 binding-site inhibitors/redistributors (DB2115, DB2373, DB2750, DB2826) upon the genomic occupancy of PU.1 and to understand DB2115-binding site preference amongst the PU.1 cistrome.

overall_design: MOLM-13, JURKAT & primary AML cells were treated with 5uM of DB compound for 12hr before cells were harvested for PU.1, RUNX1, GATA3, ELF1, FLI1 or GABPA CUT&Tag.


title: Hematopoiesis at single cell resolution spanning human development and maturation

status: Public on Jul 29 2024

city: Cambridge

first: Hojun

last: Li

state: MA

address: 500 Main St

department: David H. Koch Institute for Integrative Cancer Research

institute: Massachusetts Institute of Technology

summary: Hematopoiesis is a process of constitutive regeneration whereby hematopoietic stem and progenitor cells (HSPCs) constantly replenish mature blood cells. During maturation and aging, HSPCs alter their output to support the shifting demands of prenatal development and postnatal maturation both at homeostasis and in response to stress. How HSPC ontogeny changes throughout life is unknown; studies to date have largely focused on analysis of specific individual ages, particularly those done at single cell resolution. Here, we performed single cell RNA-seq of human HSPCs from early prenatal development into mature adulthood, demonstrating dynamic remodeling of HSPC transcriptional states and differentiation trajectories over time. We identified age-specific gene expression patterns of lineage commitment throughout human life, and developed an approach for identifying, prospectively purifying, and functionally validating age-specific HSPC states. Together, our findings define the temporal evolution of hematopoiesis during human development and maturation, and uncover principles applicable to age-biased blood diseases.

overall_design:
CD34+ hematopoietic stem and progenitor cells (HSPCs) from 26 human samples of fetal liver, cord blood, and bone marrow over the course of human development from early prenatal development to mature adulthood are subjected to scRNA-seq and downstream analysis.
>>> Submitter declares the raw data files cannot be uploaded through GEO as they are from human samples. A separate dbGaP submission has been made for the raw data files. <<<

first: Vincent

middle: L

last: Butty

first: Michael

last: Kuoch

first: Parker

last: Cote

first: Stella

middle: T

last: Chou

first: R

middle: G

last: Rowe


title: Pharmacological Repositioning of the transcription factor PU.1 [CUT&TAG; CLICKonCUT&TAG]

overall_design: MOLM-13, THP-1, MV411 and HL60 cell lines were treated with 5uM of DB compound for 12hr before cells were harvested for PU.1 CUT&Tag. "CLICKonCUT&TAG" was also performed on the PU.1 CUT&Tag samples to identify drug binding sites. Additionally, CRISPRd was utilized to block a specific PU.1 binding site (near the STRAP gene).


title: Chromatin-associated LTR RNA m5C oxidation by TET2 regulates chromatin state and leukemogenesis [seq_DNA-CUTandTag]

status: Public on Jul 25 2024

city: Shanghai

first: Xiaoyang

last: Dou

address: 320 Yue Yang Road

country: China

institute: CAS Center for Excellence in Molecular Cell Science, CAS

summary: Mutation of Ten-eleven translocation enzyme 2 (TET2) drives myeloid malignancy initiation and progression. TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant hematopoietic stem cell self-renewal. However, the open chromatin observed in TET2-deficient mouse embryonic stem cells, leukemic cells and hematopoietic progenitor and stem cells is inconsistent with the designated role of DNA 5mC oxidation of TET2. We show here that chromatin-associated retrotransposon RNA 5-methylcytosine (m5C) can be recognized by the methyl-CpG-binding domain protein MBD6, which guides deubiquitination of nearby histone H2AK119ub to promote an open chromatin state. TET2 oxidizes m5C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. Thereby, TET2 depletion leads to globally decreased H2AK119ub, more open chromatin, and increased transcription in stem cells. TET2 mutant human leukemia becomes dependent on this gene activation pathway, with MBD6 depletion selectively blocks proliferation of TET2 mutant leukemic cells and fully reverses the hematopoiesis defects caused by Tet2 loss, both in vitro and in vivo. Altogether, our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m5C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: H2AK119ub CUT&Tag in mESC, mHSPC and human leukemia cells

first: Zhongyu

last: Zou

first: Chuan

last: He


title: Chromatin-associated LTR RNA m5C oxidation by TET2 regulates chromatin state and leukemogenesis [seq_DNA-MeDIP]

type: Methylation profiling by high throughput sequencing

overall_design: DNA 5mC (MeDIP) in mESC and human leukemia cells


title: Energy stress-induced circEPB41(2) regulates lipogenesis through histone modifications [ncRNA-seq]

city: HEFEI

first: Yang

last: Yang

state: ANHUI

address: NO. 96, JinZhai Road

institute: University of Science and Technology of China

type: Non-coding RNA profiling by high throughput sequencing

summary: The list of circular RNAs (circRNAs) involved in tumor metabolic reprogramming is rapidly increasing, but whether circRNAs have a role in linking epigenetic modification with lipid metabolism remains elusive. Here, we demonstrate that the circRNA circEPB41(2), which expression is driven by HNRNPA1 in response to glucose deprivation, is important for de novo lipogenesis. Moreover, we uncovered a genome-wide decrease in specific histone acetylation after circEPB41(2) depletion. These changes are associated with altered gene expression and observed cellular phenotypes, suggesting their functional significance. Mechanistically, we showed that circEPB41(2) regulates histone acetylation in part by modulating the stability and polyadenylation of SIRT6 through binding and maintaining the demethylase activity of FTO. Accordingly, circEPB41(2) silencing inhibited cellular lipid accumulation, suppressed cell proliferation, and inhibited cancer xenograft growth. Thus, circEPB41(2) may consider a target for cancer treatment.

overall_design: Circular RNA sequencing data of HepG2 cells under normal and glucose deprivation status.

first: Jingjing

last: Luo

first: Yide

last: Mei


title: Chromatin-associated LTR RNA m5C oxidation by TET2 regulates chromatin state and leukemogenesis [seq_RNA-caRNA]

overall_design: chromatin-associated RNA in mESC


title: Downregulation of chemokine (C-C motif) ligand 5 (CCL5) induced by a novel 8-hydroxyquinoline derivative (91b1) suppresses tumor invasiveness in esophageal carcinoma

city: FoShan

first: yuanyuan

last: zhou

state: GuangDong

address: 33, GuangYun Road

institute: foshan university

summary:
Esophageal squamous cell carcinoma (ESCC) is a particularly aggressive form of cancer with high mortality. In this study, a novel 8-hydroxyquinoline derivative (91b1) was investigated for the anticancer activities in ESCC alongside the associated mechanisms. The in vitro cytotoxic effect of 91b1 were evaluated across five ESCC cell lines using MTS assay, with cisplatin serving as a comparative standard. Changes in gene expression profile were identified by cDNA microarray and further validated by qPCR and immunostaining. Additionally, protein levels of the most significantly downregulated target in archival ESCC samples was also studied. 91b1 demonstrated comparable anticancer effect with cisplatin. Notably, CCL5 was identified as the most substantially downregulated gene, with its suppression at both mRNA and protein expression in ESCC cells, exhibiting a dose-dependent manner. The recombinant human protein of CCL5 enhanced the invasion of ESCC cells using the trans-well assay. The upregulation of CCL5 protein was also detected in 50% of ESCC cell lines. CCL5 was also overexpressed in 76.9% of ESCC specimens. The overall results indicated that 91b1 could effectively induce anticancer effect on ESCC cells through downregulating CCL5 expression with suppression of tumor invasion. Overall, these findings suggest that 91b1 effectively inhibits ESCC cell proliferation and tumor invasion by downregulating CCL5 expression, highlighting its potential as a therapeutic agent for ESCC treatment.
We used microarrays to identify the most regulated gene expression of KYSE 150 cells treated with compound 91b1.

overall_design: Total RNA was extracted from 2×108 cells of KYSE150 treated with 91b1 at 9.5μg/ml and DMSO for 48 hours using RNeasy Mini Kit. The cDNA microarray analysis and the associated quality control using Human Genome U133 Plus 2.0 arrays (Affymetrix, USA) were performed in the Centre for Genomic Sciences of the University of Hong Kong according to the Affymetrix's protocol. The signals of the differentially expressed genes in 91b1-treated KYSE150 were compared with the DMSO-treated KYSE150 control.

first: Johnny

middle: C

first: Dessy

last: CHAN

first: Po-yee

last: CHUNG

first: Yijiang

last: LIU

first: Alfred

middle: K

last: LAM

first: Simon

last: LAW

first: Wolin

last: HUANG

first: Albert

middle: S

first: Kim-hung

first: Yuanyuan

last: ZHOU


title: Time-series spatial transcriptomic analysis of murine small intestine (SI) undergoing irradiation induced injury-repair with and without LXR activation

status: Public on Jul 19 2024

city: Stockholm

first: Jennifer

last: Fransson

address: Karolinska Institutet

country: Sweden

institute: Karolinska Institutet

summary: To unbiasedly characterize the spatial transcriptomics landscape of murine small intestine damage and repair, we performed a time-series spatial transcriptional analysis of total small intestinal tissue during the course of irradiation damage from mice receiving standard diet. Additionally, to understand the role of LXR activation during damage and repair, we also collected small intestine tissues from mice fed with GW3965 diet.

overall_design: Wild type C57BL/6J male mice were fed with either a control (standard) diet or a diet with LXR agonist GW3965. Ten days after diet, mice were exposed to 10Gy total body irradiation (TBI) and SI Swiss rolls (starting from distal SI) were collected on day3 after irradiation (3 dpi), processed and submitted for sequencing. SI Swiss rolls from mice fed with either standard or GW3965 diet but no irradiation (0 dpi) were used as control.

first: Srustidhar

last: Das

first: Eduardo

last: Villablanca


title: Chromatin-associated LTR RNA m5C oxidation by TET2 regulates chromatin state and leukemogenesis [seq_DNA-ATAC_set2]

first: Xiaoyang

last: Dou

overall_design: Chromatin accessibility (ATAC-seq) in mESC, mLK and human leukemia cells

first: Zhongyu

first: Chuan


title: Chromatin-associated LTR RNA m5C oxidation by TET2 regulates chromatin state and leukemogenesis [seq_RNA-caRNA-UBS-amplicon]

overall_design: RNA m5C in mESC

last: Zou


title: Transcriptome analysis of the opportunist pathogenic bacterium Clostridium perfringens ATCC 13124 grown in the presence of host polysaccharide hyaluronic acid and mucin

city: Uji

first: Sayoko

last: Oiki

address: Gokasho

country: Japan

institute: Kyoto University

summary: Purpose: The purpose of this study is to clarify the response of Clostridium perfringens ATCC 13124 to host polysaccharide. Methods: Clostridium perfringens ATCC 13124 cells were cultured anaerobically in a medium containing Minimal medium-like condition Poor + medium, medium in which hyaluronic acid or mucin was added to Poor + medium. Total RNA was extracted from bacterial cells by the Hot-Phenol method. Samples for RNA-seq were prepared according to the Illmina protocol available from the manufacturer. Array leads passed through quality filters were analyzed at the transcript isoform level using bowtie v 1.1.2. Results: Using the optimized data analysis workflow, we mapped about 50 million sequence leads per sample to the whole genome of Clostridium perfringens ATCC 13124. In addition, 2735 transcripts in C. perfringens ATCC 13124 were identified using a Bowtie aligner. Lead counts per genome were extracted from known gene annotations using the HTSeq program.

overall_design: RNA profile of Clostridium perfringens ATCC 13124 grown under different conditions: Minimal medium-like condition Poor + medium, medium supplemented with hyaluronic acid in Poor + medium, or medium supplemented with mucin.

first: Tomoya

last: Kumon

first: Wataru

last: Hashimoto


title: Time-series transcriptomic analysis of murine colon undergoing AOM-DSS tumorigenesis with and without LXR activation

summary: To unbiasedly characterize the differentially expressed genes and pathways during colorectal cancer initiation and progression, we performed a time-series transcriptional analysis of total colonic tissue during the course of azoxymethane (AOM)-dextran sodium sulfate (DSS) induced colorectal cancer from mice receiving standard (chow) diet. Additionally, to understand the role of LXR activation during AOM-DSS tumorigenesis, we also collected colonic tissues from mice fed with GW3965 diet.

overall_design: Wild type C57BL/6J male mice were fed with either a control (standard) diet or a diet with LXR agonist GW3965. Mice were injected with 10mg/kg body weight azoxymethane (AOM). One week after AOM injection, mice were exposed to DSS in drinking water for 7 days followed by 7 days of recovery (DSS-cycle). This DSS cycle was repeated two more times for a total of 3 times with 7 days between each cycle. Mice receiving both standard and GW3965 diet but no AOM or DSS were used as controls. Samples were collected on day22, day43 or day70 after AOM injection, processed and submitted for RNA sequencing.

first: S

first: J

first: E

middle: J


title: 10X Single cell RNAseq (scRNAseq) analysis of intestinal epithelial cells (IECs) from murine small intestinal organoids treated with vehicle (DMSO) or LXR agonist GW3965.

summary: Intestinal organoids are a good model of in vitro tissue regeneration that faithfully recapitulates majority of cell types of the intestine. To analyze the impact of LXR activation in intestinal regeneration and overall cellular composition, organoids established from WT mice were treated with either vehicle (DMSO) or GW3965 and were analyzed using 10X scRNAseq platform.

overall_design: Small intestinal crypts isolated from WT mice were used for establishing organoids. Starting from the seeding organoids were either treated with vehicle (DMSO) or 1uM GW3965 for a period of 5 days. Organoid medium was changed every 48 hours when the treatment was refreshed. On day5, organoids were removed from the matrigel and single cell suspensions were prepared by TrpLE digestion. Further, Epcam+ IECs were sorted by flow cytometry and processed according to 10x Genomics protocol for Chromium Single cell 3’ kit (version 3.1) and submitted for sequencing.


title: Long-read single-cell RNA transcriptomics reveals a signature of PIK3CA mutation in a human capillary malformation

city: Columbus

first: Katherine

middle: E

last: Miller

state: OH

address: 700 Children's Dr.

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

summary: PIK3CA-related overgrowth syndromes (PROS) are caused by somatic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, however identification of an appropriate agent can be hampered by the mosaic nature and low variant allele frequency of the causal variant. Moreover, our understanding of the molecular repercussions of these variants at the single cell level remain limited. Here we report in vitro expansion of affected tissue followed by exome sequencing and combined 3’ whole transcriptome and targeted long-read single cell RNA-sequencing in a patient with clinical symptoms consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). This approach identified a targetable PIK3CA variant restricted to a PAX3+ fibroblast population. These studies highlight the utility of novel next-generation sequencing strategies in the management of suspected syndromes of somatic mosaicism and provide insight into the underlying pathophysiology of a debilitating genetic syndrome.

overall_design: In this study, we use exome sequencing, 3’-based single cell RNA-sequencing, and long-read single cell RNA-sequencing of in vitro expansion of affected tissue from a patient with clinical symptoms consistent with MCAP to diagnose and reveal disease specific transcriptional changes.

first: Catherine

last: Cottrell


title: Identification of the role of TNFR1 on hematopoietic cells in pancreatic tumor growth.

status: Public on Jul 27 2024

city: Frederick

first: Nathan

last: Wong

state: MD

address: 8560 Progress Drive

institute: FNLCR/NCI/NIH

summary: Genetic abrogation of TNFR1 inhibits pancreatic cancer growth by increasing dendritic cell number and function, and enhanced anti-tumor immunity.

overall_design: KPC mice were lethally irradiated (9 Gy) at the age of 7 weeks. Irradiated mice were reconstituted with 10^7 bone marrow cells from either WT or TNFR1 KO bone marrow. Infiltrating cells were collected when pancreatic tumor was detected to approximately 300 mm^3

first: Ashwell

last: Jonathan

first: Alam

last: Muhammad

first: Wong

last: Nathan

first: Kelly

last: Michael


title: Single cell analysis of epithelial (EPI), immune (IMM) and stromal cells (double negative, DN) cells from murine small intestine in steady state following LXR activation.

first: Jennifer

summary: LXRs (LXRa and LXRb) are widely expresded in a variety of cell types. We addressed how transient LXR activation using GW3965 impacts the cellular composition and/or transcriptional changes in the SI of WT mice.

overall_design: WT mice were fed with either control (standard) or GW3965 containing diet for a period of 10 days. Following this, epithelial (EpCAM+CD45-, EPI), immune (EpCAM-CD45+, IMM) or stromal cells (EpCAM-CD45-, double negative, DN) cells were sorted and processed for single cell transcriptomics analysis using protocol from 10X genomics.

first: Srustidhar

first: Sanghita

last: Banerjee

first: Eduardo


title: Time-series spatial transcriptomic analysis of murine colon undergoing AOM-DSS tumorigenesis with and without LXR activation

summary: To unbiasedly characterize the spatial transcriptomics landscape of murine colorectal cancer initiation and progression, we performed a time-series spatial transcriptional analysis of total colonic tissue during the course of azoxymethane (AOM)-dextran sodium sulfate (DSS) induced colorectal cancer from mice receiving standard (chow) diet. Additionally, to understand the role of LXR activation during AOM-DSS tumorigenesis, we also collected colonic tissues from mice fed with GW3965 diet.

overall_design: Wild type C57BL/6J male mice were fed with either a control (standard) diet or a diet with LXR agonist GW3965. Mice were injected with 10mg/kg body weight azoxymethane (AOM). One week after AOM injection, mice were exposed to DSS in drinking water for 7 days followed by 7 days of recovery (DSS-cycle). This DSS cycle was repeated two more times for a total of 3 times with 7 days between each cycle.  Colonic Swiss rolls were collected on day22, day43 or day70 (only for STD Diet) after AOM injection, processed and submitted for sequencing. Colonic Swiss rolls from mice fed with standard (for 43 days, defined as d0 as they were untreated) diet but no AOM or DSS were used as control.


title: Protein aggregation in plant mitochondria lacking Lon1 inhibits translation and induces unfolded protein responses

status: Public on Jul 08 2024

city: Tianjin

first: Ce

last: Song

address: Weijin road NO.94

institute: Nankai University

summary: Loss of Lon1 led to stunted plant growth and accumulation of nuclear‐encoded mitochondrial proteins including Lon1 substrates. However, an in‐depth label‐free proteomics quantification of mitochondrial proteins in lon1 revealed that the majority of mitochondrial‐encoded proteins decreased in abundance. Additionally, we found that lon1 mutants contained protein aggregates in the mitochondrial that were enriched in metabolic enzymes, ribosomal subunits and PPR‐containing proteins of the translation apparatus. These mutants exhibited reduced general mitochondrial translation as well as deficiencies in RNA splicing and editing. These findings support the role of Lon1 in maintaining a functional translational apparatus for mitochondrial‐encoded gene translation. Transcriptome analysis of lon1 revealed a mitochondrial unfolded protein response reminiscent of the mitochondrial retrograde signalling dependent on the transcription factor ANAC017. Notably, lon1 mutants exhibited transiently elevated ethylene production, and the shortened hypocotyl observed in lon1 mutants during skotomorphogenesis was partially alleviated by ethylene inhibitors. Furthermore, the short root phenotype was partially ameliorated by introducing a mutation in the ethylene receptor ETR1. Interestingly, the upregulation of only a select few target genes was linked to ETR1‐mediated ethylene signalling. Together this provides multiple steps in the link between loss of Lon1 and signalling responses to restore mitochondrial protein homoeostasis in plants.

overall_design: Arabidopsis thaliana is Columbia-0 (Col-0) background. T-DNA insertion mutant lon1-2 (AT5G26860, SALK_012797) are owned by our laboratory. Arabidopsis grown best under 16/8 hours light / dark cycle at room temperature (18-22 ° C), with light intensity of 120-150 µ mol / m2 (normal conditions). Seven-day-old seedlings were harvested in biological triplicate under normal conditions. Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Then these libraries were sequenced on the Illumina sequencing platform (DNBSEQ-T7) and 125bp/150bp paired-end reads were generated.

first: Lei

last: Li


title: Epicardial transplantation of antioxidant polyurethane scaffold based human amniotic epithelial stem cell patch for myocardial infarction treatment

status: Public on Jul 18 2024

first: Jinying

state: --- Select One ---

address: No. 866 Yuhangtang Road, Zijingang Campus, Zhejiang University

institute: zhejiang university

summary: Myocardial infarction (MI) is a leading cause of death globally. Stem cell therapy is considered a potential strategy for MI treatment. Transplantation of classic stem cells including embryonic, induced pluripotent and cardiac stem cells exhibited certain repairing effect on MI via supplementing cardiomyocytes, however, their clinical applications were blocked by problems of cell survival, differentiation, functional activity and also biosafety and ethical concerns. Here, we introduced human amniotic epithelial stem cells (hAESCs) featured with immunomodulatory activities, immune-privilege and biosafety, for constructing a stem cell cardiac patch based on porous antioxidant polyurethane (PUR), which demonstrated decent hAESCs compatibility. In rats, the administration of PUR-hAESC patch significantly reduced fibrosis and facilitated vascularization in myocardium after MI and consequently improved cardiac remodeling and function. Mechanistically, the patch facilitated a desirable immune response for cardiac repair, framing the infiltration of neutrophils and inflammatory macrophages. Our findings may provide a potential therapeutic strategy for MI.

overall_design: To investigate the effects of the PUR-hAESC patch on cardiac immune cells. We performed scRNA-seq analysis of sorted CD45+ cells from the hearts of MI and PUR-hAESC groups 7 days after the operation using the 10x Genomics platform


title: Newly diagnosed multiple myeloma patients with poor response to standard therapy

status: Public on Jul 17 2024

city: St. Louis

first: Jessica

last: Silva-Fisher

address: 425 Euclid

institute: Washington University in St. Louis

summary: In this study, we analyzed transcriptome sequencing data to compare NDMM patients who were poor responders to the standard treatment approach (progression free survival < 24 months) to patients who were standard responders (progression free survival > 24 months).

overall_design:
Transcriptome sequencing was taken from NDMM patients were obtained from the Multiple Myeloma Research Foundation (MMRF) Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profiles (CoMMpass) study (https://registry.opendata.aws/mmrf-commpass), accessed in February of 2021. We compared NDMM patients with poor (<24 months) and standard (>24 months) treatment responses, identifying differentially expressed RNAs.

***************************************************************
Submitter statement concerning raw data: "This data is coming from MMRF CoMMpass study, so it is only accessible through our university collaborators by dbgap access. We only have access to deidentified information and counts for the samples."
***************************************************************


title: Antibiotics damage the colonic mucus barrier in a microbiota-independent manner II

status: Public on Jul 01 2024

city: Safed

first: Shai

last: Bel

address: 8 Henrietta Szold St, P.O. Box 1589

department: The Azrieli Faculty of Medicine

country: Israel

institute: Bar-Ilan University

summary: Antibiotic use is a risk factor for development of inflammatory bowel diseases (IBDs). IBDs are characterized by a damaged mucus layer, which does not properly separate the host intestinal epithelium from the microbiota. Here, we hypothesized that antibiotics might affect the integrity of the mucus barrier. By systematically determining the effects of different antibiotics on mucus layer penetrability we found that oral antibiotic treatment led to breakdown of the mucus barrier and penetration of bacteria into the mucus layer. Using fecal microbiota transplant, RNA sequencing followed by machine learning and ex vivo mucus secretion measurements, we determined that antibiotic treatment induces ER stress and inhibits colonic mucus secretion in a microbiota-independent manner. This mucus secretion flaw led to penetration of bacteria into the colonic mucus layer, translocation of microbial antigens into circulation and exacerbation of ulcerations in a mouse model of IBD. Thus, antibiotic use might predispose to development of intestinal inflammation by impeding mucus production.

overall_design: We treated germ-free mice with PBS or 2.5mg of vancomycin via intraperitoneal  injection (i.p.) twice a day for three day. RNA was extracted and sequenced.

first: Amir

last: Erez


title: A Pvr–AP-1–Mmp1 signaling pathway is activated in astrocytes upon traumatic brain injury

status: Public on Jun 30 2024

city: Beijing

first: Tingting

address: Beichen road

institute: Chinese Academy of Sciences

summary: Traumatic brain injury (TBI) caused by external mechanical forces is a major health burden worldwide, but the underlying mechanism in glia remains largely unclear. We report herein that Drosophila adults exhibit a defective blood-brain-barrier (BBB), elevated innate immune responses, and appear to be hypertrophy of astrocytes upon consecutive strikes with a high-impact trauma device. RNA sequencing (RNA-seq) analysis of these astrocytes revealed upregulated expression of genes encoding PDGF and VEGF receptor-related (Pvr, a receptor tyrosine kinase (RTK)), adaptor protein complex 1 (AP-1, a transcription factor complex of the c-Jun N-terminal Kinase (JNK) pathway) composed of Jun-related antigen (Jra) and kayak (kay), and matrix metalloproteinase 1 (Mmp1) following TBI. Interestingly, Pvr is both required and sufficient for AP-1 and Mmp1 upregulation, while knockdown of AP-1 expression in the background of Pvr overexpression in astrocytes rescued Mmp1 upregulation upon TBI, indicating that Pvr acts as the upstream receptor for the downstream AP-1–Mmp1 transduction. Moreover, dynamin-associated endocytosis was found to be an important regulatory step in downregulating Pvr signaling. Our results identify a new Pvr–AP-1–Mmp1 signaling pathway in astrocytes in response to TBI, providing potential targets for developing new therapeutic strategies of TBI.

overall_design: To investigate the astrocyte function in Drosophila brain after traumatic brain injury, we sorting astrocytes from brain tissues before and  4 and 24hours after injury. Each group has 5 replications.

first: Wenwen

last: Shi

first: Margaret

last: Ho

first: Yong

middle: Q

last: Zhang


title: Transcription factors ASCL1 and OLIG2 drive glioblastoma initiation and co-regulate tumor cell types and migration [ChIP-seq]

city: Albuquerque

first: Tou Yia

last: Vue

state: New Mexico

address: 915 Camino De Salud NE, Fitz Hall 145

department: Neurosciences

institute: University of New Mexico Health Sciences Center

summary: Glioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex genetic alterations. The basic-helix-loop-helix (bHLH) transcription factors ASCL1 and OLIG2 are dynamically co-expressed in GBMs; however, their combinatorial roles in regulating the plasticity and heterogeneity of GBM cells are unclear. Here, we show that induction of somatic mutations in subventricular zone (SVZ) progenitor cells leads to the dysregulation of ASCL1 and OLIG2, which then function redundantly and are required for brain tumor formation in a mouse model of GBM. Subsequently, the binding of ASCL1 and OLIG2 to each other’s loci and to downstream target genes then determines the cell types and degree of migration of tumor cells. Single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in specifying highly migratory neural stem cell (NSC)/astrocyte-like tumor cell types, which are marked by upregulation of ribosomal protein, oxidative phosphorylation, cancer metastasis, and therapeutic resistance genes.

overall_design: ChIP-seq assays for ASCL1 and OLIG2 binding were done using two orthotopic lines of patient-derived GBM xenografts (PDX-GBMs) (R548 and R738).

first: Bianca

last: Myers

first: Tou

middle: Y


title: Porcine PARP11 is essential for the proliferation of pseudorabies Virus

city: Changchun

first: Chunyun

last: Qi

address: 5333#, Xinzhu Road, Changchun 130062, China

institute: Jilin university

summary: PRV infection in swine can cause devastating disease and pose a potential threat to humans. Advancing the interplay between PRV and host is essential to elucidate the pathogenic mechanism of PRV and identify novel anti-PRV targets. In this study, we identified PARP11 as a host factor that can significantly affect PRV infection. Inhibition of PARP11 and knockout of PARP11 can significantly promoted PRV infection. Subsequently, we further found that PARP11 knockout upregulated the transcription of NXF1 and CRM1, resulting in enhanced transcription of viral genes. Furthermore, we also found that PARP11 knockout could activate the autophagy pathway and suppress the mTOR pathway during PRV infection. These findings could provide insight into the mechanism in which PARP11 participated during PRV infection and offer a potential target to develop anti-PRV therapies.

overall_design: To further investigate the host response against PRV in PARP11-KO cells, RNA-seq was performed to compare the gene expression profiles of WT and PARP11-KO cells at 12 hpi.

first: Zicong

last: Xie


title: Spatial Transcriptomics of Meningeal Inflammation Reveals Variable Penetrance of Inflammatory Gene Signatures into Adjacent Brain Parenchyma

city: Baltimore

first: Matthew

middle: D

last: Smith

address: 600 N Wolfe Street

department: Neurology

institute: Johns Hopkins University

summary: While modern high efficacy disease modifying therapies have revolutionized the treatment of relapsing-remitting multiple sclerosis, they are less effective at controlling the progressive forms of the disease. Meningeal inflammation is a recognized risk factor for cortical grey matter pathology which can result in disabling symptoms such as cognitive impairment and depression, but the mechanisms linking meningeal inflammation and grey matter pathology remain unclear. Here, we performed MRI-guided spatial transcriptomics in a mouse model of autoimmune meningeal inflammation to characterize the transcriptional signature in areas of meningeal inflammation and the underlying brain parenchyma. We found broadly increased activity of inflammatory signaling pathways at sites of meningeal inflammation, but only a subset of these pathways active in the adjacent brain parenchyma. Sub-clustering of regions adjacent to meningeal inflammation revealed the subset of immune programs induced in brain parenchyma, notably the B cell mediated immune response and antigen processing/presentation. Trajectory analysis of spatially resolved spots confirmed variable penetration of immune signatures originating from meningeal inflammation into the adjacent brain tissue. This work contributes a valuable data resource to the field, provides the first detailed spatial transcriptomic characterization in a model of meningeal inflammation, and highlights several candidate pathways in the pathogenesis of grey matter pathology.

overall_design: Female SJL/J mice aged 7-8 weeks were divided into naïve (aka.control) (N=5) and EAE (N=6) groups. After 12 weeks of EAE, MRI-guided dissection of coronal brain sections containing meningeal inflammation were analyzed with spatial transcriptomics and compared to homologues brain regions from control mice

first: Sachin

last: Gadani

first: Saumitra

last: Singh

first: Sophia

last: Kim

first: Peter

last: Calabresi

first: Pavan

last: Bhargava


title: Chromatin-associated LTR RNA m5C oxidation by TET2 regulates chromatin state and leukemogenesis [seq_RNA-caRNA-m5C-MeRIP]

first: Xiaoyang

last: Dou

summary: Mutation of Ten-eleven translocation enzyme 2 (TET2) drives myeloid malignancy initiation and progression. TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant hematopoietic stem cell self-renewal. However, the open chromatin observed in TET2-deficient mouse embryonic stem cells, leukemic cells and hematopoietic progenitor and stem cells is inconsistent with the designated role of DNA 5mC oxidation of TET2. We show here that chromatin-associated retrotransposon RNA 5-methylcytosine (m5C) can be recognized by the methyl-CpG-binding domain protein MBD6, which guides deubiquitination of nearby histone H2AK119ub to promote an open chromatin state. TET2 oxidizes m5C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. Thereby, TET2 depletion leads to globally decreased H2AK119ub, more open chromatin, and increased transcription in stem cells. TET2 mutant human leukemia becomes dependent on this gene activation pathway, with MBD6 depletion selectively blocks proliferation of TET2 mutant leukemic cells and fully reverses the hematopoiesis defects caused by Tet2 loss, both in vitro and in vivo. Altogether, our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m5C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies.

overall_design: RNA m5C in mESC, mLK and human leukemia cells

first: Zhongyu

last: Zou

first: Chuan

last: He


title: Transcription factors ASCL1 and OLIG2 drive glioblastoma initiation and co-regulate tumor cell types and migration

overall_design: Fluorescently labeled tumors were induced in the brains of immunocompetent transgenic mice. Control tumors had endogenous expression of Ascl1 and experimental tumors overexpressed Ascl1 (Ascl1-OE). Tumor cells were isolated by fluorescence activated cell sorting (FACS) and then analyzed using scRNA-seq.


title: REGULATION OF WHOLE-TRANSCRIPTOME SEQUENCING EXPRESSION IN COPD AFTER PERSONALIZED PRECISE EXERCISE TRAINING: A PILOT STUDY

city: shanghai

first: panpan

last: liu

address: No219,MiaoPU Road

institute: shanghai pudong new area gongli hospital

summary: Aerobic exercise, the cornerstone of pulmonary rehabilitation, improves prognosis of COPD patients; however, few studies have comprehensively examined the changes in RNA transcript levels and the crosstalk between various transcripts in this context. This study identified the expression of RNA transcripts in COPD patients who engaged in aerobic exercise training for 12 weeks, and further constructions of the possible RNAs networks were made.

overall_design: Peripheral blood white blood cell samples for all four COPD patients who benefited from 12 weeks of PR were collected pre- and post-aerobic exercises and evaluated for the expression of mRNA, miRNA, lncRNA, and circRNA with high-throughput RNA sequencing followed by GEO date validation. In addition, enrichment analyses were conducted on different expressed mRNAs. LncRNA-mRNA and circRNA-mRNA coexpression networks, as well as lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA competing expression networks (ceRNAs) in COPD were constructed.

first: meilan

last: zhang

first: lei

last: zhao

first: xinguo

last: sun


title: STARR-seq of artifical enhancer seqeunces consisting of homotypic and heterotypic TF binding site repeats

city: Lausanne

first: Bart

last: Deplancke

address: SV Station 19

country: Switzerland

institute: EPFL

summary: STARR-seq libraries of artificial enhancer sequences created by combining one or two transcription factor (TF) motifs in various combinations were used to assess cooperativity between TF motif pairs.

overall_design: We designed STARR-seq libraries that contained combinations of one or two different motifs with varying motif numbers (1 to 5) and using two different spacer lengths between motif instances (5bp or 10bp). A total of 8 different TF motifs were included and three different seqeunce contexts were used as flanking seqeunces for each motif combination. 12bp random barcodes were added to the insert libraries by PCR before cloning the fragments into the STARR-seq vector. We designed STARR-seq libraries that contained combinations of one or two different motifs with varying motif numbers (1 to 5) and using two different spacer lengths between motif instances (5bp or 10bp). A total of 8 different TF motifs was included and three different seqeunce contexts were used as flanking seqeunces for each motif combination. 5bp spacer libraries were also stably integrated into MEC1 cells using CRE-recombinase-mediated cassette exchange as part of  an endogenous version of the STARR-seq assay.

first: Judith

last: Kribelbauer


title: Targeting transcriptional factor YY1 is synthetic lethal with loss of the histone demethylase KDM5C

status: Public on Jun 28 2024

city: Wuhan

first: Jie

last: Xiong

address: 185, Donghu road

institute: Wuhan University

summary: In this study, we screened KDM5C-binding proteins and found that Yin Yang 1 (YY1) interacts with KDM5C. Interestingly, a synergistic antitumor effect was exerted when both KDM5C and YY1 were depleted, and targeting YY1 appeared to be a vulnerability in KDM5C-deficient cancer cells. Mechanistically, KDM5C is essential for global YY1 chromatin recruitment, especially at promoters. Moreover, an intact KDM5C JmjC domain but not KDM5C histone demethylase activity is required for KDM5C-mediated YY1 chromatin binding.Transcriptional profiling revealed that dual inhibition of KDM5C and YY1 led to significantly increased transcriptional repression of many cell cycle- and apoptosis-related genes. In summary, our work demonstrates a synthetic lethal interaction between YY1 and KDM5C and suggests a therapeutic vulnerability that can be targeted using combination therapies.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for YY1 and H3K4me3 in ACHN cells or HK2 cells.

first: Qian

last: Zheng

first: Feng

last: Li


title: Targeting transcriptional factor YY1 is synthetic lethal with loss of the histone demethylase KDM5C [RNA-Seq]

overall_design: Comparative gene expression profiling analysis of RNA-seq data from shNC, shKDM5C, shYY1 and double-knockdown of KDM5C and YY1 cell lines in ACHN or HK2 cells


title: Evidence for a role of human blood-borne factors in mediating age-associated changes in molecular circadian rhythms (preliminary experiment)

status: Public on Jun 26 2024

city: Philadelphia

first: Antonijo

last: Mrčela

state: Pennsylvania

address: 3400 Civic Center Boulevard

department: Institute for Translational Medicine and Therapeutics

institute: University of Pennsylvania

summary: Aging is associated with a number of physiologic changes including perturbed circadian rhythms; however, mechanisms by which rhythms are altered remain unknown. To test the idea that circulating factors mediate age-dependent changes in peripheral rhythms, we compared the ability of human serum from young and old individuals to synchronize circadian rhythms in culture.We collected blood from apparently healthy young (age 25-30) and old (age 70-76) individuals and used the serum to synchronize cultured fibroblasts. We found that young and old sera are equally competent at initiating robust ~24h oscillations of a luciferase reporter driven by clock gene promoter. However, cyclic gene expression is affected, such that young and old sera promote cycling of sets of different genes. Genes that lose rhythmicity with old serum entrainment are associated with oxidative phosphorylation and Alzheimer’s Disease as identified by STRING and IPA analyses. Also, the expression of cycling genes associated with cholesterol biosynthesis increased in the cells entrained with old serum. Genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions, We did not observe a global difference in the distribution of phase between groups, but found that peak expression of several clock-controlled genes (PER3, NR1D1, NR1D2, CRY1, CRY2, and TEF) lagged in the cells synchronized ex vivo with old serum. Taken together, these findings demonstrate that age-dependent blood-borne factors affect peripheral circadian rhythms in cells and have the potential to impact health and disease via maintaining or disrupting rhythms respectively.

overall_design: mRNA profiles of fibroblasts synchronized in vitro by sera from two subjects (one old, one young) at two-hour intervals, from 12 to 58 hours post serum addition, with two replicates per subject per time point.

first: Jessica

last: Schwarz

first: Nicholas

middle: F

last: Lahens

first: Yongjun

first: Cynthia

last: Hsu

first: Gregory

last: Grant

first: Carsten

last: Skarke

first: Shirley

last: Zhang

first: Amita

last: Sehgal


title: Evidence for a role of human blood-borne factors in mediating age-associated changes in molecular circadian rhythms

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

overall_design: Refer to individual Series


title: Inflammation Impacts Androgen Receptor Signaling in Basal Prostate Stem Cells Through Interleukin 1 Receptor Antagonist

city: West Lafayette

first: Majid

last: Kazemian

state: IN

address: 201 S. University Steet

department: Biochemistry and Computer Science

institute: Purdue University

summary: Chronic prostate inflammation in patients with benign prostate hyperplasia (BPH) correlates with the severity of symptoms. How inflammation contributes to prostate enlargement and/or BPH symptoms and the underlying mechanisms remain unclear. In this study, we utilized a unique transgenic mouse model that mimics chronic non-bacterial prostatitis in men and investigated the impact of inflammation on androgen receptor (AR) in basal prostate stem cells (bPSC) and their differentiation in vivo. We found that inflammation significantly enhanced AR levels and activity in bPSC. More importantly, we identified interleukin 1 receptor antagonist (IL-1RA) as a crucial regulator of AR in bPSC during inflammation. IL-1RA was one of the top genes upregulated by inflammation, and inhibiting IL-1RA reversed the enhanced AR activity in organoids derived from inflamed bPSC. Additionally, IL-1RA appeared to activate AR by counteracting IL-1's inhibitory effect. Furthermore, using a lineage tracing model, we observed that inflammation induced bPSC proliferation and differentiation into luminal cells even under castrate conditions, indicating that AR activation driven by inflammation is sufficient to promote bPSC proliferation and differentiation. Taken together, our study uncovered novel mechanisms through which inflammation modulates AR signaling in bPSC and induces bPSC luminal differentiation that may contribute to prostate hyperplasia.

overall_design: ATAC-seq of basal stem cells isolated from naive or inflamed mouse prostates


title: Evidence for a role of human blood-borne factors in mediating age-associated changes in molecular circadian rhythms (main experiment)

overall_design: mRNA profiles of fibroblasts synchronized in vitro by sera from eight different subjects (four old, four young) at two-hour intervals, from 32 to 58 hours post serum addition.


title: Inflammation Impacts Androgen Receptor Signaling in Basal Prostate Stem Cells Through Interleukin 1 Receptor Antagonist [Cut & Tag]

summary: Chronic prostate inflammation in patients with benign prostate hyperplasia (BPH) correlates with the severity of symptoms. How inflammation contributes to prostate enlargement and/or BPH symptoms and the underlying mechanisms remain unclear. In this study, we utilized a unique transgenic mouse model that mimicschronicnon-bacterial prostatitis in men and investigated the impact of inflammation on androgen receptor (AR) in basal prostate stem cells (bPSC) and their differentiationin vivo. We found that inflammation significantly enhanced AR levels and activity in bPSC. More importantly, we identified interleukin 1 receptor antagonist (IL-1RA) as a crucial regulator of AR in bPSC during inflammation. IL-1RA was one of the top genes upregulated by inflammation, and inhibiting IL-1RA reversed the enhanced AR activity in organoids derived from inflamed bPSC. Additionally, IL-1RA appeared to activate AR by counteracting IL-1a's inhibitory effect. Furthermore, using a lineage tracing model, we observed that inflammation induced bPSC proliferation and differentiation into luminal cells even under castrate conditions, indicating that AR activation driven by inflammation is sufficient to promote bPSC proliferation and differentiation. Taken together, our study uncovered novel mechanisms through which inflammation modulates AR signaling in bPSC and induces bPSC luminal differentiation that may contribute to prostate hyperplasia.

overall_design: CUT/Tag for Androgen Receptor and H3k27ac in basal stem cells isolated from naive or inflamed mouse prostates


title: Regulation of stochastic gene bursting by chromatin-independent acetylation

city: Bethesda

first: Varun

last: Sood

state: Maryland

address: 41 Medlars Drive

department: Laboratory of receptor biology and gene expression

institute: National Institues of Health, National Cancer Institute, Center for Cancer Research

summary: Stochastic transcriptional bursting is now recognized as a universal property of gene expression.  While different genes exhibit distinct bursting patterns, the molecular basis for differences in bursting are largely unknown.  We have developed and applied a high-throughput-imaging based screening strategy to identify cellular factors and mechanisms that determine the bursting behavior of human genes.   Focusing on epigenetic regulators, we find that global acetylation is the strongest acute modulator of burst frequency, size and heterogeneity of bursting. Acetylation similarly affected the Off-time but On-time changes were gene-specific.  These effects were not strongly linked to promoter acetylation, which did not correlate with bursting properties and forced promoter acetylation altered bursting for only some genes. Instead, we identified acetylation of the Integrator complex as a key determinant of gene bursting with elevated Integrator acetylation leading to faster bursting. Taken together our results suggest a dominant role of non-histone proteins in determining gene bursting properties and they identify non-epigenetic acetylation of a transcription cofactor as an allosteric modulator of bursting via a pause-release related bursting checkpoint

overall_design:
Spike in calibrated ChIP-seq for H3K27ac in DMSO and Panobinostat treated HBEC cells
Spike-in and allele normalized PRO-seq in HBEC cells overexpressing ectopic INTS4 mutants at lysine 26 and 27 that are changed to either glutamine (acetylation-mimic) or alanine (acetylation-null)

first: Dan

last: Larson

first: Tom

last: Misteli


title: Epigenetic regulation of cell state by H2AFY governs immunogenicity in high-risk neuroblastoma [CUT&RUN]

status: Public on Jun 25 2024

city: Badalona

first: David

last: Corujo

address: Ctra de Can Ruti, Camí de les Escoles

country: Spain

institute: Josep Carreras Leukaemia Research Institute

summary: Childhood neuroblastoma with MYCN-amplification is classified as high-risk and often relapses after intensive treatment regimen. Immune checkpoint blockade therapy shows limited efficacy in neuroblastoma patients and the cancer intrinsic immune regulatory network is poorly understood. Here, we leveraged genome-wide CRISPR/Cas9 screens in a human co-culture system and identified H2AFY as a resistance gene to nivolumab. Genetic deletion of H2afy in MYCN-driven neuroblastoma cells reverted in vivo resistance to PD-1 blockade by eliciting concurrent activation of the adaptive and innate immunity. Analysis of single-cell RNA sequencing datasets revealed that H2AFY mRNA was enriched in adrenergic cancer cells and was associated with patient survival. Mapping of the epigenetic and translational landscape demonstrated that H2afy deletion promoted cell transition to a malignant mesenchymal-like state. With a multi-omics approach, we uncovered H2AFY-associated genes that were functionally relevant and prognostic in patients. Altogether, our study elucidates the role of H2AFY as an epigenetic gatekeeper for cell state and immunogenicity in high-risk human neuroblastoma.

overall_design: H2AFY may serve as an epigenetic gatekeeper for cell state transition and malignant behavior in human NB. To validate this hypothesis, we mapped the genomic dstibution of H2AFY in 9464D neuroblastoma cells using CUT&RUN and a H2afy KO cell line as negative control.

first: Rebeca

last: Parracho

first: Marcus

last: Buschbeck

first: Yumeng

last: Mao


title: Insights into RNA‐mediated pathology in new mouse models of Huntington's disease

status: Public on Jun 23 2024

city: Poznan

first: Agnieszka

last: Fiszer

address: Noskowskiego Str. 12/14

department: Department of Medical Biotechnology

country: Poland

institute: Institute of Bioorganic Chemistry Polish Academy of Sciences

summary: Huntington's disease (HD) is a neurodegenerative polyglutamine (polyQ) disease resulting from the expansion of CAG repeats located in the ORF of the huntingtin gene (HTT). The extent to which mutant mRNA‐driven disruptions contribute to HD pathogenesis, particularly in comparison to the dominant mechanisms related to the gain‐of‐function effects of the mutant polyQ protein, is still debatable. To evaluate this contribution in vivo, we generated two mouse models through a knock‐in strategy at the Rosa26 locus. These models expressed distinct variants of human mutant HTT cDNA fragment: a translated variant (HD/100Q model, serving as a reference) and a nontranslated variant (HD/100CAG model). The cohorts of animals were subjected to a broad spectrum of molecular, behavioral, and cognitive analysis for 21 months. Behavioral testing revealed alterations in both models, with the HD/100Q model exhibiting late disease phenotype. The rotarod, static rod, and open‐field tests showed some motor deficits in HD/100CAG and HD/100Q model mice during the light phase, while ActiMot indicated hyperkinesis during the dark phase. Both models also exhibited certain gene deregulations in the striatum that are related to disrupted pathways and phenotype alterations observed in HD. In conclusion, we provide in vivo evidence for a minor contributory role of mutant RNA in HD pathogenesis. The separated effects resulting from the presence of mutant RNA in the HD/100CAG model led to less severe but, to some extent, similar types of impairments as in the HD/100Q model. Increased anxiety was one of the most substantial effects caused by mutant HTT RNA.

overall_design: Transcriptomic analysis was performed on the nCounter system using a Mouse Neuropathology Panel (measuring the expression of 760 neuropathology-related mouse genes and 10 reference genes, nCounter Analysis System FLEX, NanoString Technologies) according to the manufacturer’s instructions (nCounter XT Assay User Manual, July 2016, MAN-10023-11). For the NanoString nCounter XT CodeSet Gene Expression Assay, we used 100 ng of total RNA (per sample) purified from 21-month-old WT, HD/100Q and HD/100 CAG mice (4 animals per group).

first: Magdalena

last: Wozna-Wysocka

last: Jazurek-Ciesiolka

first: Lukasz

last: Przybyl

first: Dorota

last: Wronka

first: Julia

middle: O

last: Misiorek

first: Joanna

last: Suszynska-Zajczyk

first: Grzegorz

last: Figura

first: Adam

last: Ciesiolka

first: Paula

last: Sobieszczanska

first: Anna

last: Zeller

last: Niemira

first: Pawel

middle: M

last: Switonski


title: Single cell multiome profiles of bone marrow hematopoietic stem and progenitor cells from young and aged donors.

status: Public on Jun 24 2024

city: Toronto

first: Andy

last: Zeng

state: ON

address: 101 College Street

country: Canada

institute: University of Toronto

summary: To investigate molecular differences between HSPCs from young and aged donors, we performed 10x Single Cell Multiome on bone marrow HSPCs from  Young, Middle Aged, and Older Aged donors.

overall_design: Bone marrow cells from young donors (26-year-old female, and 24-year-old male) were purchased from Lonza, while bone marrow samples from aged donors (70 and 77-year-old females) undergoing hip replacement surgery were collected at the Traumatology and Orthopedics Hospital Lomas Verdes (IMSS), Mexico. These elderly donors were confirmed to have no dysplasia of any hematopoietic lineages by histological and CBC analysis. Ethical approval was obtained from the Institutional Review Board (R-2012-785-092). Patient consent was obtained verbally, and as determined by the Institutional Ethical Board.  BM samples were thawed via slow dropwise addition of X-VIVO 10 media (LONZA) with 50% FBS and 100μg/mL DNaseI (Roche). Cells were centrifuged at 400g for 10 min, then dead cell depleted using a commercial kit (EasySep Dead Cell Removal (Annexin V) Kit, STEMCELL) per the manufacturer’s instructions. Cells were resuspended in PBS + 5% FBS and stained for 15 min at RT for fluorescence-activated cell sorting with the following antibodies: anti-CD45RA-FITC (1:50, BD, clone HI100), anti-CD90-PE (1:50, BD, clone 5E10), anti-CD19-BV711 (1:50, BD, clone SJ25C1), anti-CD49f-PE-Cy5 (1:50, BD, clone GoH3), anti-CD271-APC (1:100, BD, ME20.4-1.H4), anti-CD34-APC-Cy7 (1:200, BD, clone 581), anti-CD38-PE-Cy7 (1:200, BD, clone HB7), anti-CD10-AlexaFluor700 (1:50, BD, clone HI10a), anti-CD14-BV605 (1:200, BD, clone M5E2), anti-CD45-V500 (1:50, BD, clone HI30) and anti-CD33-BV421 (1:100, BioLegend, clone WM53). Cells were washed following staining and resuspended in PBS + 2% FBS containing propidium iodide and filtered through 40μm nylon mesh for cell sorting. Lin–CD34+CD38– and Lin–CD34+CD38+ populations were sorted into PBS + 0.04% BSA + EDTA on a BD FACSAria Fusion or BD FACSAria III. Cells were counted and Lin–CD34+CD38– and Lin–CD34+CD38+ cells mixed in the following manner (1:0.33 for 24yM, 1:1 for 26yF, 1:1 for 70yF, and 1:0.5 for 77yF) for downstream 10x Genomics multiome sample preparation by the Princess Margaret Genome Centre.


title: Growth-phase dependent control of rRNA synthesis in Saccharomyces cerevisiae [NET-seq]

status: Public on Jun 20 2024

city: Birmingham

first: Catarina

middle: Alexandra

last: Mendes Felgueira

state: AL

address: 1720 University Blvd

institute: University of Alabama at Birmingham

summary: Saccharomyces cerevisiae is one of the most well-studied model organisms used in the scientific community. Its ease of manipulation, accessible growth conditions, short life cycle, and conserved eukaryotic metabolic pathways make it a useful model organism. Consequently, yeast has been used to investigate a myriad of phenomena, from microbial to human studies. Most of the research performed using this model organism utilizes yeast cell populations when they are growing exponentially, a growth phase aptly termed exponential or log phase. However, log phase encompasses several yeast generations and ranges several hours of yeast growth, meaning that there is a potential for variability during this “homogenous” growth phase. Cells in log phase require robust ribosome biogenesis to support their rapid growth and cell division. Interestingly, during log phase, ribosomal RNA (rRNA) synthesis (which is the first and rate limiting step in ribosome biosynthesis) has been shown to decrease prior to growth rate decline in stationary phase. In this study, we utilized several genomic and biochemical methods to elucidate the relationship between subphases of log phase and rRNA synthesis. Our results indicate that as yeast cells progress through subphases of log growth both of Pol I transcription and rRNA processing are repressed. Overall, this study establishes a growth phase dependent control of rRNA synthesis that unexpectedly begins prior to the switch to stationary phase (i.e. pre-diauxic shift) as a putative mechanism of anticipating nutrient starvation.

overall_design: WT yeast strain was grown at 30oC and harvested at the indicated OD600. For each growth timepoint three biological replicates were obtained. NET-seq libraries were then prepared for each sample.

middle: A

last: Schneider


title: Exposure of  bronchial airway epithelial cells to elexacaftor/tezacaftor/ivacaftor, Pseudomonas aeruginosa strain PA14 and outer membrane vesicles

city: Hanover

first: Thomas

middle: H.

last: Hampton

state: NH

address: Dewey Field Road, HB 7550

department: Microbiology and Immunology

institute: Geisel School of Medicine at Dartmouth

summary: Elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) is highly effective treatment for many cystic fibrosis patients, at least partly because it increases CFTR mediated Cl- and HCO3- secretion by airway epithelial cells leading to improved lung function and less frequent exacerbations and hospitalizations. However, little is known about how ETI affects airway epithelial cells in ways not related to CFTR mediated Cl- and HCO3- secretion, for example how ETI affects the expression of genes other than CFTR or how ETI might affect airway cells’ response to infection. It is established that CF airway cells bearing the delta F 508 mutation the CFTR gene respond characteristically differently from wild type CFTR cells, and we hypothesized that, as a highly effective CFTR  modulator, ETI might make airway cells from CF donors respond to pathogen stimulation (Pseudomonas aeruginosa PA14 or outer membrane vesicles isolated from these bacteria) in more the same way that cells from wild type, healthy control cells do. We tested this hypothesis by measuring gene expression responses in polarized primary CF airway cells exposed to ETI alone or ETI in the presence of a pathogen challenge (PA14 or outer membrane vesicles). Responses of CFTR wild type  primary CF airway cells to PA14 or outer membrane vesicles was also measured for comparison

overall_design:
Polarized primary airway epithelial cells from donors with wild type CFTR were conditionally exposed to either Pseudomonas aeruginosa strain PA14 at a multiplicity of infection of 20:1 or 2e10 outer membrane vesicles (OMV) isolated from Pseudomonas aeruginosa strain PA14 for 6 hours, or media only as a control.
Polarized primary airway epithelial cells from donors homozygous for the delta F 508 CFTR mutation were conditionally exposed to elexacaftor (3 micromolar) tezacaftor (3 micromolar) ivacaftor (10 nanomolar)  dissolved in DMSO or DMSO as a control for 48 hours and then conditionally exposed to either Pseudomonas aeruginosa strain PA14 at a multiplicity of infection of 20:1 or 2e10 outer membrane vesicles (OMV) isolated from Pseudomonas aeruginosa strain PA14 for 6 hours, or media only as a control.

first: Roxanna

last: Barnaby

middle: H

first: Bruce

last: Stanton


title: Transcriptomic response in osteoarthritis determined by single-cell RNA sequencing in a naturally occurring equine model

status: Public on Jun 21 2024

city: Fort Collins

first: Dylan

middle: T

last: Ammons

state: CO

address: 200 W Lake St

department: Clinical Sciences

institute: Colorado State University

summary: We used single-cell RNA sequencing to characterize the heterogeneity of equine synovial fluid and synovial tissue in a healthy and osteoarthritic joints.

overall_design: The complete dataset consists of 150bp paired end sequencing data obtained using a 10x Genomics single-cell RNA sequencing platform (Chromium Next GEM Single Cell 3ʹ Kit v3.1 protocol). The final dataset consists of data from 4 biologically distinct horses. Synovial fluid samples were collected from an osteoarthritis (OA) affected joint and the contralateral normal joint for the following library pairs: sf_normal_1+sf_oa_1 and sf_normal_2+sf_oa_2. The remaining two synovial fluid samples were obtained from biologically distinct horses, one from an OA affected joint (sf_oa_3) and one from an unaffected normal joint (sf_normal_3). The final two libraries (syn_normal_1+syn_oa_1) were synovial tissue samples obtained from the same horse from which the sf_normal_2+sf_oa_2 libraries were prepared; syn_normal_1 was collected from the unaffected normal joint while syn_oa_1 was collected from the OA affected joint.

first: Lynn

last: Pezzanite


title: Senescent cells cluster CTCF on nuclear speckles to sustain their splicing program  [e-CLIP]

status: Public on Jun 17 2024

city: Göttingen

first: Argyris

last: Papantonis

address: Robert-Koch-Str. 40

department: Institute of Pathology

country: Germany

institute: University Medical Center Göttingen

summary: Senescence —the end-point of the replicative life span of normal cells— is characterized by a complex sequence of molecular and biochemical events. One of these is the dramatic reorganization of CTCF into large senescence-induced CTCF clusters (SICCs). However, the molecular determinants, genomic consequences, and functional purpose of SICCs remained unknown. Here, we combine super-resolution imaging, 3D genomics, and functional assays with computational modelling to address all aspects of SICC emergence. First, we used modelling and data from the overexpression of nuclear factors lost upon senescence entry to explain how changes in the nuclear environment allow for CTCF clustering. Second, we discovered that cells entering senescence repurpose SRRM2 —a key component of nuclear speckles— and BANF1 —a protein known to act as ‘molecular glue’ for chromosomes during mitosis— to cluster CTCF on the surface of speckles, while rewiring higher-order genome architecture. Last, we could show that SICCs are required for the faithful execution of the senescence splicing program as their disruption by SRRM2 or BANF1 depletion reverts splicing choices. Together, the senescence-specific clustering of CTCF on nuclear speckles is a new paradigm of a change in the biochemical environment of the nucleus being translated into its architectural reorganization to allow for the deployment of a splicing program driving homeostatic deregulation.

overall_design: To detect the transcripts directly bound by CTCF, we conducted enhanced cross-linking with immunoprecipitation (eCLIP) experiments on the wild type and the ICM-induced senescent IMR90 cell lines.

first: Spiros

last: Palikyras

first: Yajie

last: Zhu


title: Growth-phase dependent control of rRNA synthesis in Saccharomyces cerevisiae [RNA-seq]

overall_design: WT yeast strain was grown at 30oC and harvested at the indicated OD600. For each growth timepoint three biological replicates were obtained. mRNA-seq libraries were prepared for each sample using NEBNext kit.

first: David


title: Senescent cells cluster CTCF on nuclear speckles to sustain their splicing program [RNA-seq]

overall_design: To invstigate the function of SICCs in the senescence splicing program, we performed RNA-seq on the IMR90 cell lines of wild type (WT), ICM-induced senescence (ICM), siRNA-mediated HMGB2 knockdown on the wild type background (siHMGB2), SRRM2 knockdown and BANF1 knockdown on the background of ICM (siSRRM2 and siBANF1). Next, we compared the expression profile and the splicing profile of ICM vs WT, siHMGB2 vs WT, siSRRM2 vs ICM, and siBANF1 vs ICM.


title: Senescent cells cluster CTCF on nuclear speckles to sustain their splicing program [Chipmentation]

overall_design: We performed ChIPmentation of the Nuclear Speckle component SON and the transcription factor HMGB2, in order to indentify the interaction with CTCF as well as their contribution towards genome architecture and gene expression.

first: Shyam

last: Ramasamy

first: Vassiliki

last: Varamogianni Mamatsi


title: Long Terminal Repeats of Gammaretroviruses Retain Stable Expression After Integration Retargeting or Knock-In into the Restrictive Chromatin of Lamina-Associated Domains

city: Prague

first: Dalibor

last: Miklik

address: Videnska 1083

country: Czech Republic

institute: Institute of Molecular Genetics of the Czech Academy of Sciences

summary: Retroviruses integrate their genomes into the genomes of infected host cells and form a genetic platform for stable gene expression. Epigenetic silencing can, however, hamper the expression of integrated provirus. As gammaretroviruses (γRVs) preferentially integrate into sites of active promoters and enhancers, the high expression activity of γRVs can be attributed to the integration preference. Long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters for gene expression. Here, we investigate the capacity of different γRV LTRs to drive stable expression inside a non-preferred epigenomic environment using MLV-derived BET-independent (Bin) vectors. We demonstrate that different γRV LTRs are either rapidly silenced or long-term active with active proviral population prevailing under normal and retargeted integration.

overall_design: The K562 cell line was transduced at low multiplicity of infection with MLV-derived vectors expressing GFP under the control of MLV long terminal repeat. The MLV vectors were produced with wt or BET-independent (Bin) integrase; Bin vectors carried integrase with W390A mutation or W390A-mutated integrase fused with CBX chromodomain. The GFP-expressing K562 cells were bulk sorted at three days post infection (3 dpi) and harvested for DNA extraction at 14 dpi. Amplicons with integration sites were prepared separately for each transduced population and samples were pooled prior the sequencing.

first: Martina

last: Slavkova

first: Jiri

last: Hejnar


title: γ-Glutamylcyclotransferase is transcriptionally regulated by c-Jun and controls proliferation of glioblastoma stem cells through Notch1 levels

status: Public on Aug 30 2024

city: Kyoto

first: Kozue

last: Nose

address: Misasagi-Nakauchicho 5, Yamashina-ku

institute: Kyoto Pharmaceutical University

summary: Glioblastoma stem cells (GSCs) have been reported to cause poor prognosis of glioblastoma by contributing to therapy-resistance. γ-Glutamylcyclotransferase (GGCT) is highly expressed in various cancer types, including glioblastoma, and its inhibition suppresses cancer cell growth. However, the mechanism of GGCT overexpression and its function in GSCs are unknown. In this study, we show that GGCT is highly expressed in GSCs established from a mouse glioblastoma model and its knockdown suppresses their proliferation. We also found that c-Jun transcription factor at the downstream of NRas regulates GGCT promoter activity via its consensus sequence. Gene expression analysis revealed that GGCT knockdown showed a repressive effect on the Delta-Notch pathway and decreased Notch1 expression. Notch1 knockdown alone inhibited the GSC proliferation, confirming that Notch1 is functional in this model. Forced expression of the Notch1 intracellular domain restored the growth inhibitory effect of GGCT knockdown. Moreover, GGCT knockdown inhibited GSC tumorigenic potential in vivo. These results indicate that GGCT, whose expression is promoted by c-Jun, plays an important role in the proliferation and tumorigenic potential of GSCs, and that the phenotype caused by its knockdown is contributed by a decrease in Notch1. Thus, GGCT may represent a novel therapeutic target for attacking GSCs.

overall_design: To test the effect of suppression of GGCT on the gene expression, murine GSC was tranducted with lentiviral non-target (control) or GGCT shRNA. GGCT knockdown GSCs were compared to control cells.

first: Keiko

last: Taniguchi

first: Mitsugu

last: Fujita

first: Chiami

last: Moyama

first: Masaya

last: Mori

first: Mizuki

last: Ishita

first: Tatsushi

last: Yoshida

first: Hiromi

last: Ii

first: Toshiyuki

last: Sakai

first: Susumu

last: Nakata


title: Wogonin inhibitsovarian cancer by activating the AMPK-TET2-5hmC axis

status: Public on Jun 16 2024

city: Minxiong Township

first: Wen

middle: Long

last: Huang

state: Chiayi County

address: No. 168. Section 1, Daxue Road

department: Biomedical Sciences

country: Taiwan

institute: CCU

summary: Ovarian cancer is one of the most common female cancers. In the quest of effective anti-cancer agents, this study explores the effects of wogonin, a naturally occurring flavonoid, on the viability and migration of A2780 and Kuramochi ovarian cancer cell lines. Our findings demonstrate that wogonin not only impedes cancer cell growth and mobility in both in vitro and in vivo but also significantly enhances the cytotoxic efficacy of cisplatin. Mechanistic investigations reveal that wogonin suppresses genes associated with proliferation and EMT and upregulates metabolic pathways, particularly the AMPK signaling, which is crucial for increasing 5hmC levels, highlighting wogonin's role in promoting DNA demethylation through the stabilization of TET2. Our findings not only highlights the therapeutic potential of wogonin but also its preventative capability against ovarian cancer in individuals with metabolic disorders, such as diabetes, who are at increased risk for ovarian cancer. This study highlights wogonin's dual functionality in ovarian cancer treatment and prevention, emphasizing its promising prospects for future oncological applications.

overall_design: we aim to investigate if wogonin can inhibit ovarian cancer growth, by affecting the DNA demethylation process.

first: Shu-Yi

last: Yang

first: Jing-Siang

last: Jhang

first: Wen-Long

first: Leah H.J.

last: Tsai

first: Ru-Inn

last: Lin

first: Chin

last: Li

first: Michael W.Y.

last: Chan


title: Global STARR-seq library profiling of open chromatin regions in livers of mice treated with the nuclear receptor CAR (Nr1i3) agonist ligand TCPOBOP, in livers of control male mice and in livers of control female mice. [G187_global]

city: Boston

first: David

middle: J.

last: Waxman

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

summary: Massively parallel reporter assays (MPRA) are widely used to discover functional enhancers but have largely been limited to transfected cell models, which are confounded by vector-induced innate immune responses and lack the physiologically relevant cellular and endogenous hormonal context and chromatin environment of complex mammalian tissues. Here, we combine hydrodynamic injection with a modified STARR-seq-based MPRA to determine condition-specific enhancer activity in mouse liver at scale. Strong liver enhancer activity was observed with STARR-seq libraries containing an Albumin minimal promoter but not when using a Super Core promoter or an origin of replication promoter. Analysis of a global STARR-seq library, comprised of ~50,000 genomic sequences released by DNase-I digestion of mouse liver nuclei, identified condition-specific enhancers and revealed strong correlations between liver enhancer activity and the chromatin state of the corresponding endogenous genomic regions.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: A global STARR-seq reporter library was prepared by cloning into a STARR-seq vector with a minimal Albumin promoter ~50,000 genomic regions released by limited DNase-I digestion of mouse liver nuclei. The global reporter library was delivered to mouse liver by hydrodynamic tail vein injection (HDI). Three types of Illumina sequencing libraries were prepared and are included in this GEO submission: the original global STARR-seq plasmid libraries used for HDI, global STARR-seq DNA libraries extracted from the transfected livers, and global STARR-seq RNA libraries prepared from STARR-seq reporter RNA extracted from each transfected liver. These libraries were all generated by PCR amplification carried out in parallel and under the same PCR conditions. The STARR-seq plasmid library is the same material used for HDI and indicates the relative abundance of each reporter sequence mapping to the ~50,000 genomic regions (DNase I-hypersensitive (DHS) regions) used for library preparation. The STARR-seq DNA plasmid library was prepared using DNA reporter-specific primers that amplify plasmid DNA extracted from the transfected livers. STARR-seq enhancer activity was calculated by dividing the Illumina sequencing read counts of each RNA reporter (numerator) by that of the corresponding STARR-seq plasmid or DNA library sequence reads (denominator). The STARR-seq RNA libraries were prepared from poly-adenylated RNA extracted from the HDI livers were reverse-transcribed followed by DNase treatment, and then amplified using RNA reporter-specific primers; these RNA libraries represent the self-transcribing activity of each DHS region included in the STARR-seq plasmid library.

first: Ting-Ya

last: Chang


title: STARR-seq library profiling 100 defined open chromatin regions  (‘focused library’) in livers of male and female mice treated with the nuclear receptor CAR (Nr1i3) agonist ligand TCPOBOP.

summary: Massively parallel reporter assays are widely used to discover functional enhancers but have largely been limited to transfected cell models, which are confounded by vector-induced innate immune responses and lack the physiologically relevant cellular and endogenous hormonal context and chromatin environment of complex mammalian tissues. Here, we combine hydrodynamic injection with a modified STARR-seq-based MPRA to determine condition-specific enhancer activity in mouse liver at scale. Strong liver enhancer activity was observed with STARR-seq libraries containing an Albumin minimal promoter but not when using a Super Core promoter or an origin of replication promoter. We prepared a focused STARR-seq library comprised of 100 PCR-amplified open chromatin regions nearby genes showing sex-biased expression or responsiveness to TCPOBOP, a xenobiotic and agonist ligand of the nuclear receptor CAR (Nr1i3). We assayed STARR-seq activity for the 100 genomic regions in male liver, in female liver and in TCPOBOP-treated male liver to quantitatively measure their intrinsic transcriptional activity under the 3 indicated biological conditions, and thereby identified enhancers whose activity is sex-dependent or xenobiotic-responsive.

overall_design: The focused plasmid library was delivered to mouse liver by hydrodynamic tail vein injection (HDI). Three types of Illumina sequencing libraries were prepared and are included in this GEO submission: the original STARR-seq plasmid libraries used for HDI, STARR-seq DNA libraries extracted from the transfected livers, and STARR-seq RNA libraries prepared from STARR-seq reporter RNA extracted from each transfected liver. These libraries were all generated by PCR amplification carried out in parallel and under the same PCR conditions. The STARR-seq plasmid library is the same material used for HDI and indicates the relative abundance of each reporter sequence mapping to the 100 open chromatin (DNase I-hypersensitive; DHS) regions used for library preparation. The STARR-seq DNA plasmid library was prepared using DNA reporter-specific primers that amplify plasmid DNA extracted from the transfected livers. STARR-seq enhancer activity was calculated by dividing the Illumina sequencing read counts of each RNA reporter (numerator) by that of the corresponding STARR-seq plasmid or DNA library sequence reads (denominator). The STARR-seq RNA libraries were prepared from poly-adenylated RNA extracted from the HDI livers were reverse-transcribed followed by DNase treatment, and then amplified using RNA reporter-specific primers; these RNA libraries represent the self-transcribing activity of each DHS region included in the STARR-seq plasmid library.


title: STARR-seq reporter activity of plasmid libraries delivered to mouse liver by hydrodynamic injection: In vivo MPRA assay for enhancer activity

overall_design: Refer to individual Series


title: Single cell sequencing of rat spinal cord

city: xi'an

first: Erliang

address: xin si street

institute: tangdu hospital

summary: Spinal cord injury (SCI) refers to the damage to the spinal cord due to trauma, disease or degeneration, and the number of new cases is increasing yearly. The changes of cells in the area of SCI are significant to tissue injury. However, the changes in cellular composition, the trajectory of cell development and the interesting relationship of intercellular communication in the injured area are still unclear. In this study, we used single-cell RNA sequencing to evaluate almost all the cell types that make up the site of SCI in rats.

overall_design: According to the injury degree of spinal cord injury, the spinal cord cells of rats were isolated and analyzed by scRNAseq.


title: ACAT1 promotes bladder tumorigenesis by inducing cell cycle via AKT/GSK3β/c-Myc signaling pathway

status: Public on Jun 12 2024

first: Yu

last: Xiao

address: Donghu Road 169

department: Department of Urology

institute: Zhongnan Hospital of Wuhan University

summary: Acetyl-CoA acetyltransferase 1 (ACAT1) played a key role in regulating gene expression and tumorigenesis. Nevertheless, the biological function of ACAT1 in bladder cancer (BLCA) has yet to be elucidated. This study aimed to elucidate the bioinformatics features and biological functions of ACAT1 in BLCA. Here, we demonstrate for the first time that ACAT1 is elevated in BLCA tissues, correlating with specific clinicopathological features and an unfavorable prognosis for survival in BLCA patients. ACAT1 emerged as an independent risk factor in BLCA. Phenotypically, ACAT1 knockdown inhibited the proliferation and migration of BLCA cells both in vivo and in vitro, whereas ACAT1 overexpression yielded the opposite phenotype. Mechanistically, ACAT1 enhances BLCA cells proliferation by activating cell cycle through AKT/GSK3β/c-Myc signaling pathway. In conclusion, this study identifies ACAT1 as an oncogene in BLCA, demonstrating its capability to promote proliferation and metastasis of BLCA. This suggests that ACAT1 may serve as a potential molecular target for the diagnosis and treatment of BLCA.

overall_design: To investigate the role of ACAT1 in bladder cancer (BLCA), we first used two kinds of siRNA with different sequences, but both with excellent knockdown efficiency, to knock down the target gene ACAT1 in UM-UC-3 and T24 BLCA cell lines. Then, in order to ensure the reliability of the results, we performed independent experiments three times for each sample. Finally, the 18 samples were analyzed by RNA sequencing. (NC = negative control, Si1 = siACAT1-1, Si2 = siACAT1-2, UC3 = UM-UC-3)


title: STARR-seq enhancer activity determined following hydrodynamic delivery of STARR-seq plasmid library to mouse liver using three different STARR-seq promoters: Super Core promoter, ORI promoter, and minimal Albumin promoter [G173]

summary: Massively parallel reporter assays (MPRA) are widely used to discover functional enhancers but have largely been limited to transfected cell models. Here, we combine hydrodynamic injection with a modified STARR-seq-based MPRA to determine condition-specific enhancer activity in mouse liver at scale, and we examine how different promoters affect STARR-seq reporter activity. Strong liver enhancer activity was observed with STARR-seq libraries containing an Albumin minimal promoter but not when using a Super Core promoter or an origin of replication (ORI) promoter. This work is part of a larger study where we prepare a global STARR-seq library, comprised of ~50,000 genomic sequences released by DNase-I digestion of mouse liver nuclei, and where we identify condition-specific enhancers with strong correlations between liver enhancer activity and the chromatin state of the corresponding endogenous genomic regions.

overall_design: Genomic DNA fragments released by DNase I digestion of mouse liver nuclei were pooled and cloned into STARR-seq reporter plasmids. Three such global-scale STARR-seq libraries were prepared (designated STARR-TYC4, STARR-TYC5 and STARR-TYC6), differing only in the promoter sequences used to drive reporter activity: Alb, corresponding to a minimal Alb promoter, and Super Core-1 (SCP1) and Origin (ORI) promoters, both commonly used in STARR-seq assays. Each library was delivered to mouse liver by hydrodynamic injection, followed by extraction of plasmid DNA and reporter RNA; extractions were carried out in parallel, using the same amount of starting material from each liver. Although the recovery of plasmid DNA was similar for all 3 promoters constructs, the yields of PCR-amplified RNA libraries from the ORI-STARR-seq and SCP1-STARR-seq libraries were 5-10-fold lower than for the pPromALB-STARR-seq library. Furthermore, after Illumina sequencing (Fastq files in this GEO record), the mapped RNA reads were 50 to 150-fold lower for the SCP1-STARR-seq and ORI-STARR-seq RNA libraries (mean mapped reads = 0.57 and 1.87 million, respectively) as compared to the pPromALB-STARR-seq RNA libraries (89.5 million mapped fragments); c.f., 41-56 million mapped recovered DNA sequence reads. Thus, the Super Core-1 and ORI promoters do not support HDI-STARR-seq reporter activity in mouse liver.


title: The Capicua C1 Domain is Required for Full Activity of the CIC::DUX4 Fusion Oncoprotein

status: Public on Jun 06 2024

city: San Francisco

first: Ross

last: Okimoto

address: 513 Parnassus Ave

institute: University of California, San Francisco

summary: Rearrangements between genes can yield neomorphic fusions that drive oncogenesis. Fusion oncogenes are made up of fractional segments of the partner genes that comprise them, with each partner potentially contributing some of its own function to the nascent fusion oncoprotein. Clinically, fusion oncoproteins driving one diagnostic entity are typically clustered into a single molecular subset and are often treated a similar fashion. However, knowledge of where specific fusion breakpoints occur in partner genes, and the resulting retention of functional domains in the fusion, is an important determinant of fusion oncoprotein activity and may differ between patients. This study investigates this phenomena through the example of CIC::DUX4, a fusion between the transcriptional repressor capicua (CIC) and the double homeobox 4 gene (DUX4), which drives an aggressive subset of undifferentiated round cell sarcoma. Using a harmonized dataset of over 100 patient fusion breakpoints from the literature, we show that most bona fide CIC::DUX4 fusions retain the C1 domain, which is known to contribute to DNA binding by wild type CIC. Mechanistically, deletion or mutation of the C1 domain reduces, but does not eliminate, activation of CIC target genes by CIC::DUX4. We also find that expression of C1-deleted CIC::DUX4 is capable of exerting intermediate transformation-related phenotypes compared with those imparted by full-length CIC::DUX4, but was not sufficient for tumorigenesis in a subcutaneous mouse model. In summary, our results suggest a supercharging role for the C1 domain in the activity of CIC::DUX4.

overall_design: To investigate the impact of deleting the C1 domain from the CIC::DUX4 oncoprotein on target gene induction, clonal NIH/3T3 cells expressing empty vector (EV), full-length CIC::DUX4 (CD), or C1-deleted CIC-DUX4 (del) were generated by retroviral transduction and FACS. Two clones per group were used for polyA-selected bulk RNA sequencing, with each clone acting as a "biological replicate" within each group. Differential gene expression analyses were performed between EV and both of the full-length and C1-deleted CIC::DUX4 expressing clones.

first: Cuyler

last: Luck

first: Kyle

last: Jacobs


title: Effects of hyperglycemia on airway epithelial barrier function in WT and CF 16HBE cells

status: Public on Jun 03 2024

city: Attlanta

first: Analia

last: Vazquez Cegla

state: GA

address: 2015 Uppergate Dr.

institute: Emory University

summary: Cystic Fibrosis Related Diabetes (CFRD), the main co-morbidity in Cystic Fibrosis (CF), is associated with higher rates of lung function decline. We hypothesize that airway epithelial barrier function is impaired in CF and is further exacerbated under hyperglycemia, worsening pulmonary outcomes. Using 16HBE cells as a model cell line, we studied the effects of hyperglycemia in airway epithelial barrier function. Results show increased paracellular dye flux in CF cells in response to insulin treatment under hyperglycemia, suggesting impaired barrier integrity. Gene expression experiments identified Claudin-4 (CLDN4) as a key tight junction protein dysregulated in CF cells. Further investigation into CLDN4 protein localization by confocal microscopy showed that CLDN4 was tightly localized at tight junctions in WT cells and localization did not change under hyperglycemia. ln contrast, CLDN4 was less well-localized in CF cells at normal glucose and localization was worsened in CF cells conditioned to hyperglycemia. Treatment with highly effective modulator compounds (ETI) reversed this trend, and CFTR rescue by ETI in CF cells was not affected by insulin treatment or hyperglycemia. Bulk RNA sequencing showed differences in transcriptional responses in CF compared to WT cells under normal or high glucose, highlighting PTPRG as a promising target for further investigation.

overall_design: 16HBE cells expressing WT or DeltaF508 CFTR were plated on Transwells (Corning, 3460) and conditioned with normal or high glucose media (5.5 mM and 17.5 mM glucose, respectively) for a period of 7 days with daily media changes.

first: Guiying

last: Cui

first: Nael

last: McCarty


title: Stimulus-Response signaling dynamics characterize macrophage polarization states

city: Los Angeles

first: Apeksha

last: Singh

state: CA

address: 570 Boyer Hall

institute: University of California, Los Angeles

summary: RNAseq data from hMPDMs (HoxB4 myeloid progenitor derived macrophages), BMDMs (bone-marrow derived macrophages), and Raw264.7 cell line in unstimulated condition and 3 hours post LPS stimulation (lipopolysaccharide). Gene expression demonstrates similarities between hMPDMs and BMDMs supporting use of hMPDMs in this study of NFkB stimulus response signaling dynamics, whereas Raw264.7 cells are more distinct from BMDMs in terms of expression LPS-inducible genes.

overall_design: Bone-Marrow Derived Macrophages (BMDMs) were cultured with standard methods, L929-conditioned medium.  Raw 264.7 cells were cultured in DMEM 10 % FBS media.  After LPS stimulation, cells were harvested at 3 hours.  For PolyA+ RNA, cells were harvested in TRIzol reagent (Life Technologies, Carlsbad, CA).  Then, DNA-free RNA was extracted from cell using DIRECTzol kit (Zymo Research, Irvine, CA) according to manufacturer’s instructions.  After RNA extraction, libraries for polyA+ RNA were prepared using KAPA Stranded RNA-Seq Kit for Illumina Platforms (KAPA Biosystems, Wilmington, MA) according to the manufacturer’s instructions.  Resulting cDNA libraries were single-end sequenced with a length of 50bp on an Illumina HiSeq 2000 (Illumina, San Diego, CA).

first: Alexander

last: Hoffmann


title: Glycolysis involved in transcription dysregulation through histone lactylation in T-lineage acute lymphoblastic leukemia

status: Public on May 31 2024

city: Shanghai

first: Wu

last: Wenyan

address: 1678 Dongfang Road, Pudong New District, Shanghai

institute: Shanghai Children's Medical Center

summary: Tumor cells are known for excessive lactate production, due to Warburg effect, and transcriptional dysregulation. However, how lactate influences the epigenetic modifications at a genome-wide level and its impact on gene transcription in tumor cell remains unclear. Addressing this gap, we analyzed genome-wide modification of H3K18 lactylation (H3K18la) in T-cell acute lymphoblastic leukemia (T-ALL). Integrated analysis with histone modifications with established functions show the H3K18la is mainly involved in active regulation of gene transcription. We report increased lactate and H3K18la modification levels in T-ALL as compared to normal T cells. We observe clusters of H3K18la modifications in patterns reminiscent of super-enhancers. We show a notable shift of genome-wide H3K18la modification from T cell immunity in normal T cells to leukemogenesis in T-ALL, correlating with altered gene transcription profiles. By disrupting H3K18la modification, we uncover both synergetic and divergent changes between H3K18la and H3K27ac modifications, suggesting the H3K18la and H3K27ac modifications may have specific regulation mechanisms correspondingly. These findings expand our understanding of metabolic disruptions involvement in transcription dysregulation through epigenetic changes in T-ALL, and emphasize the collective importance of histone modifications in maintaining oncogenic epigenetic stability.

overall_design: Chromatin Immunoprecipitation followed by DNA sequencing (ChIP-Seq) was performed to investigate the role of histone H3 lysine 18 lactylation (H3K18la) in DND41 and JURKAT cells under standard culture conditions. To assess the role of H3K18la in T-ALL cell proliferation and its relationship with other histone modifications, including H3K4me1 and H3K27ac, JURKAT cells were treated with oxamate and 2-Deoxy-D-glucose (2DG), followed by ChIP-Seq analysis.

first: Wenyan

last: Wu

last: Liu

first: Huiying

last: Sun

first: Han

last: Wang

first: Xiaoyu

first: Bowen

first: Shuang

last: Zhao

first: Rongrong

last: Fan

first: Ronghua

first: Ying

last: Zhong

first: Kefei

first: Xiaoxiao

last: Chen

first: Liang

last: Zheng

first: Shuhong

last: Shen

first: Meng

last: Yin


title: A spatiotemporal molecular atlas of mouse spinal cord injury identifies a distinct astrocyte subpopulation and therapeutic potential of IGFBP2

status: Public on Jun 19 2024

city: Guangzhou

first: Guangdun

last: Peng

address: Kaiyuan Ave 190

department: Guangzhou Institute of Biomedicine and Health

institute: Chinese Academy of Sciences

summary: Spinal cord injury (SCI) triggers a cascade of intricate molecular and cellular changes that determine the outcome. In this study, we resolve the spatiotemporal organization of the injured mouse spinal cord and quantitatively assess in situ cell-cell communication following SCI. By analyzing existing single-cell RNA-sequencing datasets alongside our spatial data, we delineate a subpopulation of Igfbp2-expressing astrocytes that migrate from the white matter (WM) to grey matter (GM) and become reactive upon SCI, termed as Astro-GMii. Further, Igfbp2 upregulation promotes astrocyte migration, proliferation and reactivity, and the secreted IGFBP2 protein fosters neurite outgrowth. Finally, we show that IGFBP2 significantly reduces neuronal loss and remarkably improves the functional recovery in a mouse model of SCI in vivo. Together, this study not only provides a comprehensive molecular atlas of SCI but also exemplifies how the rich resource can be applied to endow cells and genes with functional insight and therapeutic potential.

overall_design: 64 spinal cord sections from 9 different C57BL/6J mice were sequenced using 10x Visium, including different position and timepoints. There were 4 distances of rostral (-) and the caudal (+) sections at ±0.5 and ±1.0 mm from the transection injury epicenter and four time points 0 (uninjured), 3, 24 and 72 hpi. Four consecutive cryosections of the same distance at each time points were attached to the same Visium Gene Expression slide.

first: Zeqing

first: Zhuxia

last: Li

first: Tianle

last: Luan

first: Ang

first: Yanshan

last: Fang