title: Schwann cell-secreted PGE2 promotes sensory neuron excitability during development [scRNAseq_Dataset1]

status: Public on Jun 03 2024

city: Austin

first: Husniye

last: Kantarci

state: TX

address: NHB Building,, 100 E 24TH ST, Room 4.358

department: Neuroscience

institute: UT Austin

summary: Electrical excitability—the ability to fire and propagate action potentials—is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons or requires signaling from glial cells remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage gated sodium channels, and to fire action potential trains. In this scRNAseq study, we found that inactivating PGE2 synthesis in Schwann cells in vivo impaired somatosensory neuron maturation, with the most dramatic effects on nociceptor and proprioceptor somatosensory neuron subtypes.

overall_design: DRG ganglia from all axial levels were dissected from Ptges3-Flox and Ptges3-cKO mice at P4 stage and DRG neurons were analyzed using scRNASeq.

first: Pablo

last: Elvira

first: Arun

last: Thottumkara

first: Emma

last: O’Connell

first: Manasi

last: Iyer

first: Lauren

last: Donovan

first: Micaela

last: Quinn Dugan

first: Nicholas

last: Ambiel

first: Alejandro

last: Granados

first: Hong

last: Zeng

first: Nay

last: Saw

first: Amanda

last: Brosius Lutz

first: Steven

last: Sloan

first: Erin

last: Gray

first: Khanh

last: Tran

first: Aditi

last: Vichare

first: Ashley

middle: K

last: Yeh

first: Alexandra

last: Münch

first: Max

last: Huber

last: Agrawal

first: Maurizio

last: Morri

first: Haining

last: Zhong

first: Mehrdad

last: Shamloo

last: Anthony Anderson

first: Vivianne

last: Tawfik

first: J

last: Du Bois

last: Bradley Zuchero


title: Epigenetic priming induces cancer testis antigens and human endogenous retroviruses in glioma for enhanced T cell responses at single cell resolution (scRNA-Seq)

city: Los Angeles

first: Thomas

middle: Jasper

last: Lai

state: CA

address: Gonda Room 1554 695 Charles E. Young Dr.

department: Neurosurgery

country: USA

institute: University of California; Los Angeles

type: Expression profiling by high throughput sequencing

summary: Glioblastoma (GBM) is the most common malignant primary brain tumor and remains incurable. Previous work has shown that systemic administration of Decitabine (DAC) induces sufficient expression of NY-ESO-1 in GBM for targeting by adoptive T-cell therapy in vivo. However, the mechanisms by which DAC enhances immunogenicity in GBM remain to be elucidated. Using patient tissue, immortalized glioma cells, and primary patient-derived gliomaspheres, we demonstrate in vitro that basal NY-ESO-1 expression is restricted by promoter hypermethylation in gliomas. DAC treatment of glioma cells specifically inhibits DNA methylation silencing and renders NY-ESO-1 an inducible tumor antigen. Targeting of DAC-induced NY-ESO-1 in primary GBM cells promotes specific and polyfunctional NY-ESO-1 TCR-T cell responses. DAC further upregulates other tumor-associated cancer testis antigens concomitantly with tumor-intrinsic reactivation of human endogenous retroviruses (hERV) and type I interferon. Overall, we demonstrate that DAC promotes an inducible tumor antigen and enhances T cell functionality against GBM.

overall_design: To characterize the effects of Decitabine on primary and immortalized glioma cell lines.

middle: J

first: Lu

last: Sun

first: Terry

last: Prins

first: Janet

last: Treger

first: Tie

last: Li

first: Matthew

middle: Z

first: David

middle: A

last: Nathanson

first: Linda

middle: M

last: Liau

first: Albert

first: Robert

first: Richard

middle: G

last: Everson


title: Epigenetic priming induces cancer testis antigens and human endogenous retroviruses in glioma for enhanced T cell responses at single cell resolution

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Epigenetic priming induces cancer testis antigens and human endogenous retroviruses in glioma for enhanced T cell responses at single cell resolution (methylation)

status: Public on Jun 04 2024


title: Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity [Visium]

city: Toronto

first: Amy

last: Wong

state: Ontario

address: 686 Bay Street, Toronto

department: Developmental & Stem Cell Biology

country: Canada

institute: The Hospital for Sick Children

summary: While animal models have provided key insights into conserved mechanisms of how the lung forms during development, human-specific developmental mechanisms are not always captured. To fully appreciate how developmental defects and disease states alter the function of the lungs, studies in human lung models are important. Here, we sequenced >150,000 single single-cells from 19 healthy human fetal lung tissues from gestational weeks 10-19 and identified at least 58 unique cell types/states contributing to the developing lung. We captured novel dynamic developmental trajectories from various progenitor cells that give rise to ciliated, pulmonary neuroendocrine cells, and other specialized cell types. We also identified four CFTR-expressing progenitor cell types and pinpointed the temporal emergence and spatial localization of these cell types. These developmental dynamics reveal broader epithelial cell plasticity and novel lineage hierarchies that were not previously reported. Combined with spatial transcriptomics, we identified both cell autonomous and non-cell autonomous signalling pathways that may dictate the temporal and spatial emergence of cell lineages. Finally, we showed that human pluripotent stem cell (hPSC)-derived fetal lung models capture similar cell lineage trajectories specifically through progenitor cells that express abundant levels of the CFTR gene. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development and benchmarks fetal lung cultures from hPSC differentiations to similar developmental window.

overall_design: Two archived FFPE fetal lung tissues (GW 15 and 18) were freshly sectioned and analyzed through 10X Visium.

first: Henry

last: Quach

first: Spencer

last: Farrell

first: Ming

last: Wu

first: Kayshani

last: Kanagarajah

first: Joseph

middle: W

last: Leung

first: Xiaoqiao

last: Xu

first: Prajkta

last: Kallurkar

first: Andrei

middle: L

last: Turinsky

first: Christine

middle: E

last: Bear

first: Felix

last: Ratjen

first: Brian

last: Kalish

first: Sidhartha

last: Goyal

first: Theo

last: Moreas

middle: P


title: Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity [scRNAseq-hPSC]

overall_design: CA1 and BU3 hPSCs were differentiated into fetal lung cells and organoids (Ngan et al., 2022) and analyzed through scRNAseq.


title: A novel STING variant triggers endothelial toxicity and SAVI disease

city: Milan

first: Monah

last: Abou Alezz

address: Via Olgettina 60

country: Italy

institute: SR-TIGET

summary: Gain-of-function mutations in STING cause STING-associated vasculopathy with onset in infancy (SAVI) characterized by early-onset systemic inflammation, skin vasculopathy, and interstitial lung disease. Here, we report and characterize a novel STING variant (F269S) identified in a SAVI patient. single-cell transcriptomics of patient bone marrow revealed spontaneous activation of interferon (IFN) and inflammatory pathways across cell types and a striking prevalence of circulating naïve T cells was observed. Inducible STING F269S expression conferred enhanced signaling through ligand-independent translocation of the protein to the Golgi, protecting cells from viral infections but preventing their efficient immune priming. Additionally, endothelial cell activation was promoted and further exacerbated by cytokine secretion by SAVI immune cells, resulting in inflammation and endothelial damage. Our findings identify STING F269S mutation as a novel pathogenic variant causing SAVI, highlight the importance of the crosstalk between endothelial and immune cells in the context of lung disease and contribute to a better understanding of how aberrant STING activation can cause pathology.

overall_design: To study the phenotype of blood cells carrying a novel identified STING variant (F269S) causing SAVI disease, we interrogated the transcriptome of different bone marrow (BM)-derived mononuclear cell (MNC) subsets performing single-cell RNA-Seq (scRNASeq). SAVI patient cells were compared to pediatric healthy donor control cells.

first: Erika

last: Valeri

first: Ivan

last: Merelli

first: Anna

last: Kajaste-Rudnitski


title: Methylation status of CD4+ and CD11b+ cells from Dnmt3a+/+ or Dnmt3aR878/+ mice

city: Chengdu

first: Hui

last: WANG

state: Sichuan

address: No.14,3Rd Section Of Ren Min Nan Rd

department: periodontics

country: China

institute: Sichuan University

summary: Splenic CD11b+ and CD4+ cells were isolated from Dnmt3a+/+ or Dnmt3aR878/+ mice using LS columns on the magnetic field of QuadroMACS Separator according to the manufacturer’s instructions (Miltenyi Biotec), and the High-Molecular-Weight (HMW) DNA of cells were isolated using MagAttract HMW DNA Kit according to the manufacturer’s instructions (Qiagen). WGBS was performed using the Zymo-Seq WGBS Library Kit (Zymo research) according to the manufacturer’s recommendations at the High Throughput Sequencing Core of Children’s Hospital of Philadelphia, PA, USA. The sequence was generated on Illumina NovaSeq 6000  using NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles) at 40X coverage

overall_design: Splenic CD11b+ and CD4+ cells were isolated from Dnmt3a+/+ or Dnmt3aR878/+ mice, the methylation status of these cells were detected using WGBS

last: Wang

first: Bohu

last: Pan

first: George

last: Hajishengallis


title: Transcriptomic analysis reveals sex specific genes similar to human NAFLD associated with transgenerational NAFLD

status: Public on Jun 12 2024

city: Columbia

first: Ramji

last: Bhandari

state: MO

address: 1200 University Ave

department: Division of Biological Sciences

institute: University of Missouri

summary: Comparison of bulk RNA-Seq data from the livers of male and female medaka fish, in the BPA lineage vs control lineage of f2 generation, with publicly available human NAFLD-NASH patient data revealed that 20% of DEGs were mutually upregulated with those found in human NAFLD patients. Additionally, 27.69% of DEGs were mutually downregulated, and 52.31% of ancestral BPA-specific DEGs were expressed in the livers of female fish in the BPA lineage. The activation of myc, atf4, xbp1, and stat4, as well as the inactivation of igf1, were associated with pathogenesis.

overall_design: RNA seq of the liver of biological replicates from BPA lineage and control lineage of male and female Medaka fish. Three biological replicates (3 control + 3 BPA treated) for female fish and two biological replicates (2 control + 2 BPA treated) for male fish were used.

first: Sourav

last: Chakraborty

first: Santosh

last: Anand


title: Tumor cell intrinsic type I interferon signaling dictates CD47-SIRPα blockade immunotherapy via metabolic reprograming [dataset 1]

city: Beijing

first: Mingzhao

last: Zhu

address: 15 Da Tun Rd, Chaoyang District

department: Key Laboratory of Infection and Immunity

institute: Institute of Biophysics, Chinese Academy of Sciences

summary: Innate immune checkpoint has emerging as a highly potential target for cancer immunotherapy in recent years. The CD47-SIRPα axis is the best-studied innate checkpoint in cancer. However, the transcription profile of tumor cell duiring CD47-SIRPα blockade therapy remains unclear.

overall_design: C57BL/6 mice (n=3 per group) transplanted s.c. with 5×10E5 MC38 cells were treated i.t. with 50 μg CV-1 or hIgG every three days. Two days after the third treatment, CD45- cells were sorted for the generation of RNA-seq libraries.

first: Hang

last: Zhou

first: Wenjun

first: Jiyu

last: Ding


title: Schwann cell-secreted PGE2 promotes sensory neuron excitability during development [RNA-Seq]

first: Husniye

last: Kantarci

summary: Electrical excitability—the ability to fire and propagate action potentials—is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons or requires signaling from glial cells remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage gated sodium channels, and to fire action potential trains. In this RNA-Seq study, we discovered that treating cultured DRG neurons with Schwann cell-conditioned media or PGE2 increased the expression of several genes required for neuronal maturation and excitability, including voltage-gated sodium channels.

overall_design: DRGs were dissected from E15 embryos using timed pregnant Sprague-Dawley rats. 10–18 embryos were pooled for each experiment, and each pregnant rat was considered one biological replicate.  DRG neurons were isolated using an immunopanning protocol. At DIV 13 DRG neurons were treated with either Schwann cell conditioned media (SCCM) or 1 um PGE2 overnight and total RNA was purified on DIV14 for bulk RNA-seq analysis

first: Pablo

middle: D

last: Elvira

first: Arun

last: Thottumkara

first: Emma

last: O’Connell

first: Manasi

last: Iyer

first: Lauren

last: Donovan

first: Micaela

last: Quinn Dugan

first: Nicholas

last: Ambiel

first: Alejandro

last: Granados

first: Hong

last: Zeng

first: Nay

last: Saw

first: Amanda

last: Brosius Lutz

first: Steven

last: Sloan

first: Erin

last: Gray

first: Khanh

middle: V

last: Tran

first: Aditi

last: Vichare

first: Ashley

last: Yeh

first: Alexandra

last: Münch

first: Max

last: Huber

last: Agrawal

first: Maurizio

last: Morri

first: Haining

last: Zhong

first: Mehrdad

last: Shamloo

first: Thomas

last: Anthony Anderson

first: Vivianne

last: Tawfik

first: J

last: Du Bois

last: Bradley Zuchero


title: Schwann cell-secreted PGE2 promotes sensory neuron excitability during development [scRNAseq_Dataset2]

first: Husniye

last: Kantarci

summary: Electrical excitability—the ability to fire and propagate action potentials—is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons or requires signaling from glial cells remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage gated sodium channels, and to fire action potential trains. In this scRNAseq study, we found that inactivating PGE2 synthesis in Schwann cells, in vivo, impaired somatosensory neuron maturation, with the most dramatic effects on nociceptor and proprioceptor somatosensory neuron subtypes.

overall_design: DRG ganglia from all axial levels were dissected from Ptges3-Flox and Ptges3-cKO mice at P4 stage and DRG neurons were analyzed using scRNASeq. Actinomycin D was used during dissection steps to block new transcription during dissection

first: Pablo

last: Elvira

first: Arun

last: Thottumkara

first: Emma

last: O’Connell

first: Manasi

last: Iyer

first: Lauren

last: Donovan

first: Micaela

last: Quinn Dugan

first: Nicholas

last: Ambiel

first: Alejandro

last: Granados

last: Zeng

last: Saw


title: Enrichment and analysis of circular RNA by Induro RT-mediated circRNA sequencing (IMCR-seq)

status: Public on Jun 01 2024

city: Ipswich

first: Zhiyi

last: Sun

state: MASSACHUSETTS

address: 240 County Road

department: Research

institute: New England Biolabs

type: Non-coding RNA profiling by high throughput sequencing

summary: Circular RNA (circRNA) has recently gained attention for its emerging biological activities, relevance to disease, and potential as biomarkers. Furthermore, the growing prominence of RNA vaccines has brought circRNAs into the spotlight as a promising alternative modality due to their enhanced stability compared to linear RNA. Nevertheless, sequencing circRNAs has presented challenges. In this context, we introduce a novel circRNA sequencing method called Induro-RT mediated circRNA-sequencing (IMCR-seq), which relies on a group II intron reverse transcriptase with robust rolling circle reverse transcription activity. The IMCR-seq protocol eliminates the need for conventional circRNA enrichment methods such as rRNA depletion and RNaseR digestion yet achieved the highest circRNA enrichment and detected 6-1000 times more circRNAs for the benchmarked human samples compared to other methods. IMCR-seq is applicable to any organism, capable of detecting circRNAs of more than 7,000 nucleotides, and is effective using as little as 10 ng of total RNA. These enhancements render IMCR-seq suitable for clinical samples, including disease tissues and liquid biopsies. To demonstrate IMCR-seq's clinical relevance, we applied it to lung tumor tissue and plasma samples from a healthy individual and a lung cancer patient and detected cancer-specific circRNAs as potential biomarkers. In summary, IMCR-seq presents an efficient and versatile circRNA sequencing method with high potential for research and clinical applications.

overall_design: We developed a new circRNA sequencing method called Induro-RT mediated circRNA-sequencing (IMCR-seq) by using the group II intron reverse transcriptase Induro-RT and long read sequencing technologies. We applied IMCR-seq to profile circRNAs of human HEK293 cell line, human brain and human lung tumor tissues, and plasma samples from a healthy individual and a lung cancer patient

first: Irem

last: Unlu

first: Sean

last: Maguire

first: Shengxi

last: Guan


title: Targeted degradation of extracellular mitochondrial aspartyl-tRNA synthetase modulates immune responses in bacterial pneumonia

city: Columbus

first: Benjamin

middle: Stuart

last: Johnson

address: 473 W 12th Ave rm524

department: Department of Internal Medicine

institute: The Ohio State University

summary: The severity of bacterial pneumonia can be worsened by impaired innate immunity resulting in ineffective pathogen clearance. We describe a mitochondrial protein, aspartyl-tRNA synthetase (DARS2), which is released in circulation during bacterial pneumonia in humans and displays intrinsic innate immune properties and cellular repair properties. DARS2 interacts with a bacterial-induced ubiquitin E3 ligase subunit, FBXO24, which targets DARS2 for ubiquitylation and degradation, a process that is inhibited by DARS2 acetylation. During experimental pneumonia, Fbxo24 knockout mice exhibit elevated DARS2 levels with a robust increase in pulmonary cellular and cytokine levels.  In silico modeling identified an FBXO24 inhibitory compound with immunostimulatory properties which extended DARS2 lifespan in cells. Here we show a unique biological role for an extracellular, mitochondrially derived enzyme and its molecular control by the ubiquitin apparatus, which may serve as a mechanistic platform to enhance protective host immunity through small molecule discovery.

overall_design: BEAS-2B Cells stably integrated with CRSPR/Cas9 and gRNA scramble (sgCon) or against FBXO24 (sgO24). sgCon and sgO24 cells were infected with PA103 MOI1 or left uninfected for  24h. Cells were collected andprocessed for RNAseq to determine differences in cellular pathways.


title: Epigenetic priming induces cancer testis antigens and human endogenous retroviruses in glioma for enhanced T cell responses at single cell resolution (bulk RNA-Seq)

first: Thomas

last: Lai

first: Lu

first: Terry

last: Prins

first: Janet

last: Treger

first: Tie

last: Li

first: Matthew

first: David

last: Nathanson

first: Linda

last: Liau

first: Albert

first: Robert

first: Richard

last: Everson


title: Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells (WGBS)

city: Shanghai

first: Yan

last: Jiang

address: 131 Dongan rd

department: Institutes of Brain Science

institute: Fudan University

summary: Short interspersed nuclear element (SINE) B2 are a type of DNA retrotransposon that organize three-dimensional genome during development. Containing binding sites for CCCTC-binding factor (CTCF), SINE_B2 serve as domain boundary elements that control chromatin state and initiate transcription of critical developmental genes. However, it is still unclear how SINE_B2 are regulated during neurodevelopment. Our findings indicate novel mechanisms that modulate critical DNA retrotransposon activity, maintain chromosome conformation and contribute to central nervous system development.

overall_design: Examination of whole genome DNA methylation in wildtype (WT) and Setdb1-KO (KO) neural progenitor cells.

first: Daijing


title: Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells (ChIP-Seq)

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Examination of histone H3K9me3 modification and CTCF binding in wildtype (WT) and Setdb1-KO (KO) neural progenitor cells.


title: tRNA m1A modification regulate hematopoietic stem cell maintenance via mTORC1 signaling

city: Hangzhou

first: Aiwei

last: Wu

state: Zhejiang

address: 2318 Yuhangtang Road

department: School of Basic Medical Sciences

institute: Hangzhou Normal University


title: Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells


title: tRNA m1A modification regulate hematopoietic stem cell maintenance via mTORC1 signaling [mim-tRNAseq]

summary: High-resolution quantitative profiling of tRNA abundance and modification status in hematopoietic cells upon Trmt6 deletion by mim-tRNAseq

overall_design: mim-tRNAseq expriments of LSK+ cells sorted from bone marrown of Trmt6 WT and cKO mice

first: Hu

last: Wang

first: Hongna

last: Zuo


title: tRNA m1A modification regulate hematopoietic stem cell maintenance via mTORC1 signaling [scRNA-seq]

summary: Hematopoietic stem cells (HSCs) have some unique physiological adaptations to maintain blood cell production and deal with stress responses throughout life. To maintain such adaptations, HSCs are particularly dependent on maintaining a tightly controlled protein translation rate. However, how HSCs fine tune protein translation has not been fully described. In this study, we report the role of TRMT6 in regulating HSC function in adult hematopoiesis. Trmt6-null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow 4 to 8 weeks after Trmt6 deletion. Trmt6-deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Loss of TRMT6 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, mTORC1 signaling pathway was highly upregulated in HSC-enriched cell populations after Trmt6 deletion by single cell RNA-seq analysis. Furthermore, TRMT6 was required for tRNA m1A modification required for HSC quiescence and self-renewal. Our data indicate that TRMT6 promotes the expression of TSC1, fine-tuning mTORC1 signaling level which is essential for HSC maintenance and self-renewal in adult hematopoiesis.

overall_design: single-cell RNA-sequencing (scRNA-seq) analysis of total LSK (Lin- Sca-1+ c-Kit+ ) hematopoietic stem and progenitor cells (HSPCs) isolated from the bone marrow of young adult (3-month-old) wild-type and hematopoietic-specific Trmt6-knockout mice.


title: Exploring the epigenetic regulatory role of KDM1A inhibition in HCC cells [HepG2_SP2509_CUTTag_seq]

first: Boshi

address: No.800 Dongchuan Road

institute: Shanghai Cancer Institute

summary: We conducted CUT&Tag-seq experiments to investigate the impact of KDM1A inhibition on histone modifications.

overall_design: We analyzed the histone marks using CUT&Tag-seq assays in HepG2 cells treated with SP2509.

first: Tiantian

last: Jing


title: Loss of miR-145 promotes remyelination and functional recovery in a model of chronic centrel demyelination

status: Public on May 31 2024

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

institute: Ottawa Hospital Research Institute

summary: Strategies for treating progressive multiple sclerosis (MS) remain limited. Here, we found that miR-145-5p is overabundant uniquely in chronic lesion tissues from secondary progressive MS patients. We induced both acute and chronic demyelination in miR-145 knockout mice to determine its contributions to remyelination failure. Following acute demyelination, no advantage to miR-145 loss could be detected. However, after chronic demyelination, animals with miR-145 loss demonstrated increased remyelination and functional recovery, coincident with altered presence of astrocytes and microglia within the corpus callosum relative to wild-type animals. This improved response in miR-145 knockout animals coincided with a pathological upregulation of miR-145-5p in wild-type animals with chronic cuprizone exposure, paralleling human chronic lesions. Furthermore, miR-145 overexpression specifically in oligodendrocytes (OLs) severely stunted differentiation and negatively impacted survival. RNAseq analysis showed altered transcriptome in these cells with downregulated major pathways involved in myelination. Our data suggest that pathological accumulation of miR-145-5p is a distinctive feature of chronic demyelination and is strongly implicated in the failure of remyelination, possibly due to the inhibition of OL differentiation together with alterations in other glial cells. This is mirrored in chronic MS lesions, and thus miR-145-5p serves as a potential relevant therapeutic target in progressive forms of MS.

overall_design: RNA-seq samples aligned to RNor6, OL-miR-145 and OL-miR-ctl (derived from primary cultures treated with miRNA mimics in vitro).

first: Samantha

middle: F

last: Kornfeld

first: Sarah

last: Cummings

first: Rebecca

last: Yaworski

first: Rashmi

last: Kothary


title: MYCN-driven de-differentiation profile identifies a subgroup of aggressive retinoblastoma II

city: Essen

first: Petra

last: Ketteler

address: Hufelandstraße 55

country: Germany

institute: University Hospital Essen

summary: Retinoblastoma are childhood eye tumors arising from retinal precursor cells. Two distinct retinoblastoma subtypes with different clinical behavior have been described based on gene expression and methylation profiling. Using consensus clustering of DNA methylation analysis from retinoblastomas, we identified a MYCN-driven of subtype 2 retinoblastomas characterized by DNA hypomethylation and high expression of genes involved in protein synthesis. Subtype 2 retinoblastomas outside the MYCN-driven were characterized by high expression of genes from mesodermal development, including NKX2-5. Knockdown of MYCN expression in retinoblastoma cell models caused growth arrest and reactivated a subtype 1-specific photoreceptor signature. These molecular changes suggest that removing the driving force of MYCN oncogenic activity rescues molecular circuitry driving subtype 1 biology. The MYCN-RB gene signature generated from the cell models better identified MYCN-driven retinoblastoma than MYCN amplification and could identify cases that may benefit from MYCN-targeted therapy. MYCN drives tumor progression in a molecularly defined retinoblastoma subgroup, and inhibiting MYCN activity could restore a more differentiated and less aggressive tumor biology

overall_design:
RNA-sequencing data from primary retinoblastomas (N=50),  retinoblastoma cell lines (N=8) and MYCN-KD retinoblastoma  cell line models (N=4) were generated. The data from  primary retinoblastomas were integrated with the previously published  data from two relapse retinoblastomas ** (van Tilburg et al., 2021; Heipertz et al., 2022; Peterziel et al., 2022; Worst et al., 2016)
Pseudoalignments to hg38 (GRCh38) and quantification were performed using the kallisto/sleuth pipeline

first: Tatsiana

last: Ryl

first: Elena

last: Afanasyeva


title: Combined inhibition of EZH2 and CDK4/6 perturbs endoplasmic reticulum-mitochondrial 1 homeostasis and increases antitumor activity against glioblastoma

city: Rostock

first: Dirk

last: Koczan

address: Schillingallee 70

department: Institute for Immunology

institute: University of Rostock

summary: Background: Few druggable targets have been discovered in glioblastoma (GBM); however, increasing knowledge has uncovered two hallmarks of this disease: epigenetic modifications and cell cycle dysregulation.  Methods: Here, we investigated the effects of the EZH2 inhibitor GSK126 alone and in combination with the CDK4/6 inhibitor abemaciclib on morphology, growth, invasiveness, protein and gene expression, mitochondrial function, and induction of cell death in patient-derived GBM cells (GBM03, GBM06, GBM14, and GBM15).  Results: EZH2 expression, CDK4 amplification, and CDKN2A deletion were first confirmed. The combined use of GSK126 and abemaciclib resulted in synergistic effects in all patient-derived GBM cell  lines. After treatment, the cells appeared swollen with a large flat cytoplasm and cellular stress fibers  due to HIF1 upregulation and CalR translocation. Notably, abemaciclib-induced cellular stemness  (NANOG+, OCT3/4+, and SOX2+) was antagonized by this combination treatment. The MitoStress Test  revealed a massive impairment of mitochondrial function. The basal oxygen consumption rate, ATP  synthesis, and maximal respiration of the mitochondria decreased, confirming reduced cellular fitness  and disrupted endoplasmic reticulum-mitochondrial homeostasis. This was paralleled by massive  mitochondrial ROS production leading to mitochondrial depolarization and upregulation of the UPR  sensors PERK, ATF6, and IRE1. Spheroid invasion was reduced in 3/4 cases, again with synergistic  effects after dual EZH2 and CDK4/6 blockade. Differentially expressed genes involved mitotic  aberrations/spindle assembly (Rb, PLK1, RRM2, PRC1, CENPF, TPX2), histone modification (HIST1H1B,  HIST1H3G), DNA damage/replication stress events (TOP2A, ATF4), immuno-oncology (DEPDC1), and a  shift in the stemness profile towards a more differentiated state. Notably, the antitumor effect was  partially confirmed in ovo as well as in patient-derived organoids.  Conclusions: We propose dual blocking of GBM hallmarks as a potential treatment strategy. Biomarker-driven targeted therapies will hopefully guide the development of more refined treatments.

overall_design: Triplicates of patient derived glioblastoma cells (GBM15), treated with the EZH2 inhibitor GSK126 and the CDK4/6 inhibitor abemaciclib were compared alone and in combination (kombi) with sham controls (DMSO).

first: Thomas

last: Freitag

first: Phillip

last: Kaps

first: Justus

last: Ramtke

last: Bertels

first: Emily

last: Zunke

first: Björn

last: Schneider

first: Anne-Sophie

last: Becker

first: Daniel

last: Dubinski

first: M

middle: T

last: Freiman

first: Felix

last: Wittig

first: Burkhard

last: Hinz

first: Mike-Andrew

last: Westhoff

first: Hannah

last: Strobel

first: Franziska

last: Meiners

last: Wolter

first: Nadja

last: Engel

first: Sascha

last: Troschke-Meuer

first: Wendy

last: Bergmann-Ewert

first: Susanne

last: Staehlke

first: Annabel

last: Wolff

first: Florian

last: Gessler

first: Christian

last: Junghanss

first: Claudia

last: Maletzki


title: Co-transcriptional splicing facilitates transcription of gigantic genes

city: Cambridge

first: Yukiko

last: Yamashita

state: MA

address: 455 main ST

institute: Whitehead Institute for Biomedical Research

summary: Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.

overall_design: Total RNA-seq from control, U2af38 RNAi, and SPRK RNAi Drosophila testes with three biological replicates and two technical replicates per genotype.

first: Jaclyn

last: Fingerhut

first: Romain

last: Lannes

first: Troy

last: Whitfield

first: Prathapan

last: Thiru


title: Elevating PLK1 Overcomes BETi-Resistance in Prostate Cancer via Triggering BRD4 Phosphorylation-dependent Degradation in Mitosis [ChIP-seq]

city: Lexington

first: Yanquan

last: Zhang

state: KY

address: 760 Press Ave, Rm 352/HKRB

department: Toxicology and Cancer Biology

institute: U of Kentucky

summary: To investigate the effect of phosphorylated BRD4 at S24 and S1100 on transcription, we established C4-2 stable expressing BRD4 WT, S24/1100A, S24/1100D cell lines in which endogenous BRD4 has been knocked down by shRNA targeted to 3'-UTR. We then performed a CUT&RUN assay using anti-BRD4 or anti-H3K27Ac antibodies followed with a sequencing of ChIPed DNA from 4 different cells at 18 hour treatment of nocodazole.

overall_design: Examination of BRD4 and  H3K27Ac modification in human prostate cancer cells C4-2 cells expressing BRD4-WT, -S2A and -S2D mutants. There are 14 ChIP-Seq samples.

first: Xiaoqi

last: Liu


title: Elevating PLK1 Overcomes BETi-Resistance in Prostate Cancer via Triggering BRD4 Phosphorylation-dependent Degradation in Mitosis

summary: To investigate the effect of phosphorylated BRD4 at S24 and S1100 on transcription, we established C4-2 stable expressing BRD4 WT, S24/1100A, S24/1100D cell lines in which endogenous BRD4 has been knocked down by shRNA targeted to 3'-UTR. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at 18 hour treatment of nocodazole.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for C4-2_BRD4 WT, 2A, 2D upon nocodazole treatment.


title: The N6-methyladenosine Epitranscriptomic Landscape of Lung Adenocarcinoma [RNA-seq]

first: Housheng

middle: Hansen

last: He

address: 101 College St

institute: UHN

summary: N6-methyladenosine (m6A) modification plays a central role in both RNA and tumor biology. Yet comprehensive m6A epitranscriptome profiling of primary tumors remains uncharted. To fill this gap, we profiled the m6A epitranscriptome of 10 non-neoplastic lung (NL) tissues and 51 lung adenocarcinoma (LUAD) tumors, integrating them with corresponding transcriptome, proteome and extensive clinical annotation. A total of 8,030 genes are marked with m6A modification, with variable abundances across tumors. Distinct clusters and genes were found to be linked exclusively to disease progression through m6A, rather than mRNA or protein levels. In comparison with NL tissues, 430 transcripts were hypo-methylated in tumors, and 222 were hyper-methylated. Fully 69% of these had no influence on RNA abundances. Amongst these genes, EML4 emerged as a novel metastatic driver, displaying significant hyper-methylation in tumors. m6A modification promoted EML4 translation and led to widespread EML4 overexpression in primary tumors. Functionally, EML4 modulated cytoskeleton dynamics and cell morphology, triggering cellular protrusions and enhancing cellular motility, local invasion, and metastasis. Clinically, high EML4 protein abundance correlated with features of metastasis. A METTL3 small molecule inhibitor markedly diminished both EML4 m6A and protein abundance, and efficiently suppressed lung metastases in vivo. Our findings unveil a dynamic and functional epitranscriptomic landscape in lung adenocarcinoma, pinpointing the hyper-methylation of EML4 as a key driver of tumor metastasis.

overall_design: To further elucidate the molecular mechanism underlying the pro-metastatic role of EML4, we performed RNA-seq in EML4-modified and control A549 and H358 cells.

middle: H

first: Shiyan

last: Wang

first: Yong

last: Zeng


title: The N6-methyladenosine Epitranscriptomic Landscape of Lung Adenocarcinoma

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

overall_design: Refer to individual Series


title: The N6-methyladenosine Epitranscriptomic Landscape of Lung Adenocarcinoma [RIP-seq]

overall_design: RIP-seq was performed to identify the binding targets of 5 major m6a regulators.


title: UBXN3B Is Crucial for B Lymphopoiesis [scRNA-seq]

city: Farmington

first: Tingting

last: Geng

state: CT

address: 263 Farmington Avenue

department: Department of Immunology

institute: Uconn Health

summary: Hematopoiesis is finely regulated to enable timely production of the right numbers and types of immune cells. Herein, we report the crucial function of UBXN3B in B lymphopoiesis. In the inducible global knockout and B cell-specific Ubxn3b knockout model, the terminal B cell number is reduced by > 90% in both Ubxn3b-/- mouse models. Transfer of wildtype bone marrows to irradiated global Ubxn3b-/- restores the B population, while reverse transplantation fails to do so. The deficiency begins from the precursor stage. The B population drops rapidly with significant apoptosis, presents a much higher level of pro-caspase-3 protein following induction of Ubxn3b knockout. RNA-sequencing reveals significantly suppressed cell cycle genes while upregulated TP53 signaling in Ubxn3b-/- B cells. Ubxn3b-/- mice are highly vulnerable to respiratory viruses, with increased lung viral loads and immunopathology, reduced B population and virus-specific IgM/IgG. This study reveals a cell-intrinsic essential role of UBXN3B in B cell survival and UBXN3B as a potential therapeutic target for B-cell related diseases.

overall_design: To investigate the function of UBXN3b in B lymphopoiesis, we established an inducible global knockout mouse model. In this model, tamoxifen administration induces the knockout of Ubxn3b.  Bone marrows from tamoxifen or oil treated mice were sorted for B subsets. HSCs/progenitors and B subsets (excluding mature B) wered pooled together for scRNAseq.Subsequent comparartive gene expression profilling analysis was conduted.

first: Penghua


title: Time‑course analysis of cisplatin induced AKI in preclinical models: implications for testing different sources of MSCs

city: Palo Alto

first: Avnesh

last: Thakor

state: California

address: 3155 Porter Dr

department: Radiology

institute: Stanford Medicine

summary: Cisplatin remains the most effective and widely used treatment option for solid tumors, however, it is also responsible for 30 - 46% incidence of AKI in cancer patients.  Thus, despite its broad anti-cancer potential, clinical use of this chemotherapeutic agent remains constrained.   In the last decade, mesenchymal stem cells (MSCs) have been proposed as a potentially useful therapeutic strategy in various diseases, including AKI, given their ability to reduce apoptosis and accelerate tubular cell regeneration.     In the present study, we looked at the dose dependent response to cisplatin in two different mouse strains, CD1 and C57BL/6 mice, and performed a transcriptome based time-series analysis of the molecular signature in both male and female mice during progression of cisplatin-induced AKI, in order to generate a robust and reproducible pre-clinical model with the proposed aim to investigate the ability of MSCs from three different sources (adult and neonatal) as a rescue therapy to reverse kidney injury.

overall_design: We performed time-based analysis of transcriptome of male and female C57Bl/6 mice at Day 0, 2, 3 and 5

first: Abantika

last: Ganguly

first: Shashank

last: Chetty

first: Rosita

last: Primevera

first: Steven

last: Levitte

first: Shobha

last: Regmi

first: Benjamin

last: Dulken

first: Scott

last: Sutherland

first: Wendy

last: Angeles

first: Jing

middle: S


title: Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells (Hi-C)

first: Yan

last: Jiang

overall_design: Examination of genome-wide chromatin conformation in wildtype (WT) and Setdb1-KO (KO) neural progenitor cells.

first: Daijing

last: Sun


title: Specific CD4+ T cell phenotypes associate with bacterial control in people who ‘resist’ infection with Mycobacterium tuberculosis

first: Meng

address: Biomedical Innovations Building 1300, 240 Pasteur Drive

institute: Stanford University

summary: A subset of individuals with a high probability of exposure to M. tuberculosis (M.tb) appears to ‘resist’ established M.tb infection, as demonstrated by serially negative tuberculin skin test (TST) or IFN-γ release assay (IGRA) results. While these ‘resisters’ (RSTR) display IFN-γ-independent T cell responses to the M.tb-specific antigens ESAT-6 and CFP-10, it is currently unknown whether unique T cell functional programs are associated with this clinical outcome. We used multi-modal single-cell RNA, TCR sequencing, multi-parameter flow cytometry, and cytokine analysis in a discovery and validation format to compare the phenotypes and functions of M.tb-specific T cells between RSTRs and matched controls with ‘latent’ M.tb infection (LTBI). M.tb-specific T cells were clonally expanded in both RSTRs and LTBIs, confirming the priming of adaptive immune responses after M.tb exposure. However, M.tb-specific T cells derived from RSTRs showed enrichment of T regulatory as well as Th17-like functional programs compared to LTBIs, which were characterized by Th1*-like effector programs. Th17-like functional programs were also associated with a lack of progression to active TB among South African adolescents with LTBI, as well as bacterial control in published non-human primate studies. Together, these data suggest that ‘resisters’ may successfully control M.tb after exposure and immune priming and establish a set of T cell biomarkers to facilitate further study of this important clinical phenotype.

overall_design: We conducted the SELECT-seq protocol, which includes stimulation and sorting procedures, as described in a recent publication: Huang et al, PNAS, 2019, Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion. Briefly, PBMCs were stimulated by the overlapping peptide pools targeting ESAT-6 and CFP-10. Notably, before the stimulation, the cells were cultured in 1 μg/mL anti-CD154 antibody for 30 mins to prevent CD154 downregulation. After stimulation, cells were first stained and index-sorted on live CD3+/TCRαβ+ cells positive for CD69 and CD154 and/or CD137 on a BD FACSAria™ Fusion. These antigen-specific T cells were FACS-sorted into individual wells of a 96-well PCR plate with lysis buffers. To assess the technical variability, we mixed the lysis buffers with the external RNA control – ERCC RNA Spike-In (a final estimate of ~5000 molecules per well, Thermo Fisher Scientific, Waltham, MA). We used the modified Smart-Seq2 protocol (Clontech Laboratories, Mountain View, CA) to generate the cDNA library. From the library, we took a small aliquot (1 μL) for the nested PCR to amplify and sequence 15 targeted RNAs (TBX21, RORC, GATA3, FOXP3, RUNX1, RUNX3, IFNG, IL2, IL17A, GZMB, PERF, IL4, IL5, IL13, TGFB) and the CDR3 regions of both TCRα and TCRβ chains. Based on the TCR sequences, the Smart-seq2 generated cDNAs of the clonally expanded activated T cells were manually selected only on the ESAT-6/CFP-10-reactive cells for the high-coverage in-depth single-cell full transcriptomic sequencing. We applied the tagmentation and indexing protocol as in the SMART-seq2 protocol and amplified the tagmented DNA. The final pooled library was prepared using the Nextera XT library prep kit (96 index primers; Illumina, San Diego, CA) protocol and was sequenced on an Illumina HiSeq 2500.

first: Jolie

last: Phan

first: Nathan

last: Kieswetter

first: Gerlinde

last: Obermoser

first: Mark

last: Davis

first: Chetan

last: Seshadri


title: RNAseq of gingival fibroblasts exposed to PRF membranes and PRF serum.

city: Vienna

first: Hannes

last: Kühtreiber

state: Vienna

address: Lazarettgasse 14

department: Department of Dermatology

country: Austria

institute: Medical University Vienna

summary: Platelet-rich fibrin (PRF) is prepared from the coagulated plasma of fractionated blood. When squeezing between two plates, PRF is separated into the solid PRF membranes and a liquid exudate, the PRF serum. The question arises regarding the extent to which the overall PRF activity remains in the membranes and what is lost in the serum. To this aim, we have exposed gingival fibroblasts to lysates prepared from PRF membranes and PRF serum, followed by bulk RNA sequencing. A total of 268 up- and 136 down-regulated genes in gingival fibroblasts exposed to PRF lysates are significantly regulated under the premise of a minimum log2 2.5-fold change and a minus log10 significance level of two, respectively. PRF serum caused 62 up- and 32 down-regulated genes when gingival fibroblasts were exposed to PRF serum, respectively. Among the 61 genes commonly up-regulated by PRF lysate and serum were CXCL1, CXCL5, CXCL6, CXCL8, IL33, and IL6 and PTGS2, STC1. PRF lysate further increased the chemokines CCL2, CCL7, CXCL2, CXCL3, and the IL1R1, IL1RL1, and IL1RN – as well as the paracrine factors IL11, LIF, IGF1, BMP2, BMP6, FGF2, CCN2/CTGF and HAS1, HAS2, HAS3. The 16 up-regulated genes by PRF serum included DKK1. Among the 122 down-regulated genes by PRF lysate were IFIT1, IFIT2, IFIT3, OSR1, OSR2. Among the 32 down-regulated genes by PRF serum were FGF18 and GDF15. Taken together, PRF lysates, compared to PRF serum, cause a more complex response of gingival fibroblasts with a chemokine with an obvious increase in chemokine expression and spectrum of paracrine factors.

overall_design: The preparation of PRF was approved by the Ethical Committee of the Medical University of Vienna (NR. 1644/2018). Glass tubes (Bio-PRF, Venice, FL, USA) supporting blood coagulation were subjected to 700 g for 8 min (Z306 Hermle, Universal Centrifuge, Wehingen, Germany). The yellow PRF clot was removed and squeezed between two metal plates. The PRF serum fraction was collected and stored frozen. One cm of the PRF membrane was used per mL of serum-free medium to prepare PRF lysates. PRF membranes underwent two freeze-thaw cycles at −80 °C, followed by 30 s sonication at room temperature (Sonopuls 2000.2, Bandelin electronic, Berlin, Germany). After centrifugation at 15,000× g for 10 min, aliquots of the PRF lysates were frozen for less than one month. PRF lysates were exposed to 72° and 95°C for 10 minutes for indicated experiments. Gingival fibroblasts of human origin were derived from small gingival tissue specimens procured during wisdom teeth extraction from three healthy individuals who provided informed consent. The protocol was endorsed by the Ethical Committee of the Medical University of Vienna (EK Nr. 631/2007). Fibroblasts obtained by explant cultures exhibited the typical spindle-shaped morphology. Gingival fibroblasts were exposed to 30% PRF lysate and 10% PRF serum for 6 hours under standard conditions at 37 °C, 5% CO2, and 95% humidity.

first: Layla

last: Panahipour

first: Reinhard

last: Gruber


title: The regulation of histone modification by KDM1A in liver cells

first: Boshi

last: Wang

summary: We performed CUT&Tag-seq to explore the epigenetic regulatory roles of KDM1A in healthy liver cells.

overall_design: CUT&Tag-seq experiments for H3K4me1 were conducted in healthy liver cell line THLE2 with down-regulation of KDM1A.

first: Tiantian

last: Jing


title: RNA sequencing of SW1990 KRAS-G12D or KRAS-G12D/K128R knockin cell lines

city: Leuven

first: Wout

last: Magits

address: Herestraat 49, bus 912

country: Belgium

institute: VIB-KULeuven Center for Cancer Biology

summary: The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not yet fully understood. K128 ubiquitination is the most prevalent modification of the GTPase core domain in both NRAS and KRAS. The ubiquitination at K128 was decreased in cancer samples compared to normal tissue. We found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAP), NF1 and RASA1, which increases RAS binding to GAP proteins and promotes GAP-mediated GTP hydrolysis. K128 ubiquitination is transiently induced by growth factors or cytokines, which restricts the extent of wild-type RAS activation in a GAP-dependent manner. In contrast, the loss of K128 ubiquitination in the KRAS-G12D mutant promotes tumor growth by suppressing RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Dysregulation of K128 ubiquitination unleashes both wild-type and mutant RAS signaling and triggers a senescence-associated secretory phenotype, driving RAS-driven tumorigenesis.

overall_design: To investigate the effect of a KRAS G12D/K128R mutation on the transcriptome, we generated single cell clones of the SW1990 cell line (KRAS G12D) harbouring an inducible K128R knock-in mutation (using a Cre-inducible CRISPR-Cas9 knock-in approach). 3 independent single cell clones were used for RNA-seq, both before and after induction of the K128R mutation.

first: Anna

last: Sablina


title: Endothelial Overexpression protects Metabolic Homeostasis and  the Development of Obesity and cardiovascular disease

city: Boca Raton

first: Claudia

last: Rodrigues

state: Florida

address: 777 Glades Rd, BC-71, Rm. 224

department: Biomedical Science

institute: Florida Atlantic University

summary: Introduction: Obesity is a major risk factor associated with multiple pathological conditions including diabetes and cardiovascular disease. Endothelial dysfunction is an early predictor of obesity.  However, little is known regarding how early endothelial changes trigger obesity. In the present work we report a novel endothelial-mediated mechanism essential for regulation of metabolic homeostasis, driven by c-Myc. Methods: We used conditional overexpression (EC-Myc OE) mouse models to investigate the role of endothelial-specific c-Myc in metabolic homeostasis during high-fat diet exposure. Body weight, metabolic parameters and cardiovascular function were collected over time and tissue samples collected at endpoint for biochemical, pathology and RNA-sequencing analysis.  Results: In the present study we demonstrate that Overexpression of endothelial c-Myc attenuated diet-induced obesity and visceral fat accumulation and prevented the development of glucose tolerance and cardiac dysfunction. Transcriptome analysis of skeletal muscle suggests that the protective effects promoted by endothelial c-Myc overexpression are associated with the expression of genes known to increase weight loss, energy expenditure and glucose tolerance. Discussion: Our results show a novel important role for endothelial c-Myc in regulating metabolic homeostasis and suggests its potential targeting in preventing obesity and associated complications such as diabetes type-2 and cardiovascular dysfunction.

overall_design: Gene expression analysis of skeletal muscle samples of endothelial-specific c-Myc overexpression and its potential implications for obesity and associated complications such as diabetes type-2 and cardiovascular dysfunction.

first: Jacqueline

last: Machi

first: Isabella

last: Altilio

first: Yue

last: Qi

first: Alejo

last: Morales

first: Diego

last: Silvestre

first: Diana

middle: R

last: Hernandez

first: Nicolas

last: Da Costa-Santos

first: Mehrnoosh

last: Neghabi

first: Parisa

last: Nategh

first: Thiago

last: Castro

first: João

last: Werneck-de-Castro

first: Mahsa

last: Ranji

first: Fabiana

last: Evangelista

first: Roberto

middle: I

last: Vazquez-Padron

first: Ernesto

last: Bernal-Mizrachi

middle: O


title: The epigenetic regulatory role of KDM1A in HCC cells

first: Boshi

last: Wang

summary: We performed CUT&Tag-seq to explore the epigenetic regulatory roles of KDM1A in HCC cells.

overall_design: CUT&Tag-seq experiments for H3K4me1 were performed in HepG2 cells treated with KMD1A inhibitors

first: Tiantian

last: Jing


title: CUT&Tag-seq Assays in Healthy and Cancerous Liver Cells

summary: We performed CUT&Tag-seq assays for KDM1A using the healthy liver cell lines (THLE2, THLE3) and HCC cell lines (HepG2, Huh7).

overall_design: CUT&Tag-seq assays using the KDM1A antibody were performed in healthy liver cell lines (THLE2, THLE3) and HCC cell lines (HepG2, Huh7).


title: ATAC-seq Assays in Healthy and Cancerous Liver Cells

summary: We performed ATAC-seq assays using the healthy liver cell lines (THLE2, THLE3) and HCC cell lines (HepG2, Huh7).

overall_design: ATAC-seq assays were performed in healthy liver cell lines (THLE2, THLE3) and HCC cell lines (HepG2, Huh7).


title: RNA-sequencing of matched samples from rhabdomyosarcoma samples, obtained before therapy and mid-treatment resection

city: Memphis

first: Michael

last: Dyer

state: TN

address: 262 Danny Thomas Pl MS 324

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

summary: Eighteen samples were obtained from 9 patients who underwent delayed resection. The purpose of this study is to identify transcriptomic shifts in samples during therapy.

overall_design: Scrolls from formalin-fixed paraffin-embedded blocks underwent automated RNA extraction using the Maxwell RSC RNA extraction kit. RNA-seq libraries were then generated using the Takara SMARTer Stranded Total RNA-seq Kit v2 – pico input. Libraries then underwent sequencing on an Illumina NovaSeq sequencer.

first: Anand

last: Patel


title: Elf1 promotes TC-NER in yeast by using its C-terminal domain to bind TFIIH

city: Pullman

first: John

middle: J.

last: Wyrick

state: WA

address: Biotechnology Life Sciences 241

department: School of Molecular Biosciences

institute: Washington State University

summary: Transcription coupled-nucleotide excision repair (TC-NER) repairs DNA lesions that stall RNA polymerase II (Pol II) transcription. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD is required for efficient Rad26-dependent and Rad26-independent TC-NER across the yeast genome. The Elf1-CTD is also important for TC-NER in rad16∆ cells deficient in GG-NER. Finally, we show that a mutant in the Elf1-CTD (elf1-Y99A) that disrupts binding to a subunit of TFIIH affects Rad26-independent repair in a rad26∆ mutant background.

overall_design: We used the CPD-seq method to map UV-induced cyclobutane pyrimidine dimers (CPDs) a single nucleotide resolution across the yeast genome, both immediately after UV-radiation (UV 0hr) and following 2 hours of repair (UV 2hr). The no UV control represents unirradated sample. These experiments were performed in different mutant backgrounds, including mutants in Elf1.

middle: J

first: Kathiresan

last: Selvam


title: The regulation of chromatin accessibility in HCC cells by KDM1A

summary: We investigated the impact of KDM1A knockdown on chromatin accessibility in HCC cells using ATAC-seq assays.

overall_design: We analyzed chromatin accessibility profiles using ATAC-seq assays in HepG2 cells with downregulated KDM1A.


title: The transcription factor IRF4 impedes human CD8 T-cell function and promotes cells proliferation and PD-1 expression

city: Woluwe Waint Lambert

first: Pierre

last: van der Bruggen

address: Avenue Hippocrate 75

institute: de Duve institute

summary: The transcription factor IRF4 is well known to be quickly upregulated after TCR activation, and magnitude of IRF4 expression is proportional to the strength of TCR activation. However, contradictory reports have been published as to the role of IRF4 in mouse T-cells during acute and chronic viral infections. Furthermore, no data have been published as to the role of IRF4 in human CD8 T-cells, both in normal and pathological conditions. In this work, we subjected CD8 T-cells from healthy donors to IRF4 overexpression or knock-out and assessed the impact of these modifications on T-cell functions and transcriptome.

overall_design: CD8 T-cells isolated from PBMC of hemochromatosis donors were subjected to lentiviral transduction or CRISPR Cas9 to induce or delete IRF4 expression, respectively. Cells were then activated or not in vitro for 5h with coated anti-CD3. IRF4 overxpressing or IRF4 KO CD8 T-cells were FACS sorted before RNA sequencing. IRF4 overexpressing cells were compared to RFP (ctrl gene) overexpressing cells, while IRF4 KO cells were compared to CD8 T-cells nucleofected with a ctrl gRNA.

first: Thibault

last: Hirsch

first: Damien

last: Neyens

first: Céline

last: Duhamel

first: Alexandre

last: Bayard

first: Christophe

last: Vanhaver

last: Dauguet


title: MYCN-driven de-differentiation profile identifies a subgroup of aggressive retinoblastoma

first: Petra

last: Ketteler

type: Methylation profiling by genome tiling array

summary: Retinoblastoma are childhood eye tumors arising from retinal precursor cells. Two distinct  retinoblastoma subtypes with different clinical behavior have been described based on gene  expression and methylation profiling. Using consensus clustering of DNA methylation analysis from  retinoblastomas, we identified a MYCN-driven cluster of subtype 2 retinoblastomas characterized by  DNA hypomethylation and high expression of genes involved in protein synthesis. Subtype 2  retinoblastomas outside the MYCN-driven cluster were characterized by high expression of genes from  mesodermal development, including NKX2-5. Knockdown of MYCN expression in retinoblastoma cell  models caused growth arrest and reactivated a subtype 1-specific photoreceptor signature. These  molecular changes suggest that removing the driving force of MYCN oncogenic activity rescues  molecular circuitry driving subtype 1 biology. The MYCN-RB gene signature generated from the cell  models better identified MYCN-driven retinoblastoma than MYCN amplification and could identify  cases that may benefit from MYCN-targeted therapy. MYCN drives tumor progression in a molecularly  defined retinoblastoma subgroup, and inhibiting MYCN activity could restore a more differentiated and  less aggressive tumor biology

overall_design:
Samples from primary retinoblastomas (N=59), retinoblastoma cell lines (N=9) were profiled by either Illumina 450K or EPIC beadchip methylation arrays.
The data from  primary retinoblastomas were integrated with the previously published data from two relapse retinoblastomas ** (van Tilburg et al., 2021; Heipertz et al., 2022; Peterziel et al., 2022; Worst et al., 2016): INF_R_1024 and INF_R_1673.

first: Tatsiana

last: Ryl

first: Elena

last: Afanasyeva


title: N-terminal acetylation of the Set1-COMPASS fine-tunes H3K4 methylation patterns

city: Chuncheon

first: Junsoo

last: Oh

country: South Korea

institute: Kangwon National University

overall_design: Refer to individual Series


title: N-terminal acetylation of the Set1-COMPASS fine-tunes H3K4 methylation patterns (ChIPseq for ard1 deletion strain)

summary: H3K4 methylation is a conserved histone modification crucial for gene regulation, yet the post-translational modifications of the Set1-COMPASS complex remain largely unexplored. This study elucidates the significance of N-terminal acetylation in modulating H3K4 methylation patterns. Firstly, loss of NatA complex resulted in a significant decrease in H3K4me3 levels and a shift of H3K4me2 from 5' transcribed regions to promoters. Importantly, NatA physically interacted with the Set1-COMPASS complex and facilitated N-terminal acetylation of the Shg1 subunit. Surprisingly, deletion of SHG1 or mutation of the acetylation site (A2P) in Shg1 led to reduced H3K4 methylation in cells lacking Spp1. Additionally, NatB complex also contributed to H3K4 methylation regulation, potentially through N-terminal acetylation of Swd1. These findings highlight the novel role of N-terminal acetylation in fine-tuning the function of the Set1-COMPASS complex and shaping H3K4 methylation patterns. This study sheds light on the intricate regulation of H3K4 methylation and emphasizes the significance of N-terminal acetylation as a regulatory mechanism in this process.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for histone modifications H3K4me2 and H3K4me3 as well as histone H3 in WT and ard1 deletion strains in Saccharomyces cerevisiae.

first: Hyunjoo

last: Woo

first: Jung-Shin

last: Lee

first: TaeSoo

last: Kim


title: N-terminal acetylation of the Set1-COMPASS fine-tunes H3K4 methylation patterns (ChIPseq for shg1 and spp1 deletion strains)

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for histone modifications H3K4me2 and H3K4me3 as well as histone H3 in WT, shg1Δ, spp1Δ and shg1Δspp1Δ strains in Saccharomyces cerevisiae.


title: Longitudinal biopsies of fusion-positive rhabdomyosarcoma orthotopic xenografts

first: Michael

last: Dyer

summary: In this experiment, 3 mice were implanted orthotopically with a patient-derived xenograft, SJRHB013759_X14. After tumor engraftment and visible nodule formation, all 3 mice underwent therapy. One mouse received vehicle control (saline), while two mice were treated with a combination of vincristine and irinotecan, to mimic salvage therapy for relapsed rhabdomyosarcoma. Sedated needle biopsies were performed before (time = 0),  during therapy (time = 3, 7, and 14 days), and after recurrence.

overall_design: Mice bearing orthotopic xenografts underwent sedated biopsies before, during and after completion of therapy. Tissue biopsies underwent Trizol RNA extraction, followed by automated RNA-sequencing library construction.

last: Patel

first: Elizabeth

last: Stewart


title: ISL1 and POU4F1 directly interact to regulate the differentiation and survival of inner ear sensory neurons

city: Augusta

first: Lin

last: Gan

state: Geogia

address: 1120 15th Street

department: Department of Neuroscience & Regenerative Medicine

institute: Augusta University

summary: The inner ear sensory neurons play a pivotal role in auditory processing and balance control. Though significant progresses have been made, the underlying mechanisms controlling the differentiation and survival of the inner ear sensory neurons remain largely unknown. During development, ISL1 and POU4F transcription factors are co-expressed and are required for terminal differentiation, pathfinding, axon outgrowth and the survival of neurons in the central and peripheral nervous systems. However, little is understood about their functional relationship and regulatory mechanism in neural development. Here, we have knocked out Isl1 or Pou4f1 or both in mice of both sexes. In the absence of Isl1, the differentiation of cochleovestibular ganglion (CVG) neurons is disturbed and with that Isl1-deficient CVG neurons display defects in migration and axon pathfinding. Compound deletion of Isl1 and Pou4f1 causes a delay in CVG differentiation and results in a more severe CVG defect with a loss of nearly all of spiral ganglion neurons (SGNs). Moreover, ISL1 and POU4F1 interact directly in developing CVG neurons and act cooperatively as well as independently in regulating the expression of unique sets of CVG-specific genes crucial for CVG development and survival by binding to the cis-regulatory elements including the promoters of Fgf10, Pou4f2, and Epha5 and enhancers of Eya1 and Ntng2 These findings demonstrate that Isl1 and Pou4f1 are indispensable for CVG development and maintenance by acting epistatically to regulate genes essential for CVG development.

overall_design: Single nuclei from mouse cochlea at E12.5 was applied to prepare sequencing library with Chromium Single Cell Multiome ATAC + Gene Expression kit (10X Genomics).

first: Shuchun

last: Li

first: Mei

last: Xu


title: ZMYM3 is required for KDM1A-mediated histone de-methylation in HCC cells

city: Shanghai

first: Boshi

last: Wang

address: No.800 Dongchuan Road

institute: Shanghai Cancer Institute

summary: To investigate the relationship between KDM1A and ZMYM3, we conducted CUT&Tag-seq experiments to detect the KDM1A-mediated histone de-methylation in HCC cells with ZMYM3 downregulation.

overall_design: CUT&Tag-seq experiments for H3K4me1 were performed in three groups of 97H cells: Vector control + sh-control, KDM1A+sh-control, and KDM1A+sh-ZMYM3.

first: Tiantian

last: Jing


title: The epigenetic regulatory role of KDM1A in HCC cells [HepG2_shKDM1A_CUTTag_seq]

overall_design: CUT&Tag-seq experiments for H3K4me1, H3K4me2, H3K9me2, and H3K27ac were performed in HepG2 cells stably expressing sh-control or sh-KDM1A.


title: Single-cell transcriptomics reveals transcriptional programs underlying male and female cell fate during Plasmodium falciparum gametocytogenesis

city: Stockholm

first: Mubasher

last: Mohammed

state: Stockholm lan

address: Svante Arrhenius Väg 20c, MBW, Stockholms University

department: Department of Molecular Biosciences, the Wenner-Gren Institute

country: Sweden

institute: Stockholm University

summary: The Apicomplexa constitute a large phylum of parasitic protozoans with complex life cycles that typically include meiotic sex. The life cycle of the malaria parasite, Plasmodium falciparum, includes obligate transition and stage development between a human and mosquito host. Asexual parasite replication in the human erythrocytes is followed by differentiation which leads to the formation of a precursor gamete stage, referred to as gametocytes. The gametocyte stage is solely responsible for malaria transmission into the mosquito vector where gamete fusion followed by meiosis occurs. How the parasite differentiates into male and female gametocytes in the absence of sex chromosomes largely remains an open question. Here, we combine FACS-based cell enrichment of a gametocyte reporter line followed by single-cell RNA-seq, to enable targeted characterization of the entire gametocyte developmental stage. Our data defines differential transcription programs during male and female gametocyte development and highlights a bifurcation point for sexual cell fate. We perform prediction analyses of novel candidate driver genes underlying P. falciparum sexual cell fate. Additionally, we delineate the timing of expression of members of the ApiAP2 family of transcription factors and predict their specificity in male or female P. falciparum gametocyte development.

overall_design: Using P. falciparum NF54-tdTomato, we established five consecutive time points (Day 1, 3, 5, 7, and 9) for time courses scRNA-seq to cover a broad range of gametocytes throughout their developmental period. The culture samples were then purified using Magnetic cell separation (MACs) sorter MidiMACS™ from day 3 to day 9 for contaminating asexual forms removal. The gametocytes were then counted using a hemocytometer and the gametocytaemia was set to 400.000 cells/ml. On day one, 2 μg/ml of Hoechst 33342 was added to the gametocytes sample, incubated for 15 mins, and FACs-sorted, single-cell sorting was performed with a MoFlo Astrios EQ (Beckman Coulter, USA) according to tdTomato fluorescence into a total volume of 4ul of sterile-filtered 1 x PBS. Hoechst 33342 was detected using the 488 nm laser with the 450/50 nm filter and tdTomato (RFP) was detected at the 561 nm laser with a 586/16 nm filter. Using a targeted population strategy, we sorted single cells from each time point into a defined bulk population to ensure enough targeted cells per time point. We then pooled all of the defined bulk population into a single container before single-cell RNA-sequencing was performed using a chromium single-cell barcoding system instructions to recover 5000 to 8000 cells/ul for each time point.

first: Mohammed

last: Mubasher

first: Ankarklev

last: Johan


title: Generation of a novel iPSC-based model to explore the molecular and functional phenotype of a rare genetic immunodeficiency, the ICF syndrome type 2.

city: Caserta

first: Francesco

last: Cecere

state: Caserta

address: Via A. Vivaldi, n. 43

department: DiStABiF

institute: University of Campania "Luigi Vanvitelli"

summary: ICF syndrome (Immunodeficiency, Centromeric instability and Facial anomalies) is a rare genetic disorder characterized by severe immunodeficiency that leads to lethal infections in early childhood. In addition, more than half of affected individuals show mild to severe intellectual disability at very early onset. Two genes explain the majority of the ICF cases, DNMT3B and ZBTB24, defining ICF1 and ICF2 subtypes. Despite unifying clinical features, ICF patients display variability in molecular and phenotypic defects with no clear genotype/phenotype correlation. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome.  Among the disease models, patient-derived iPSCs were acknowledged as a powerful tool enabling the study of ICF syndrome etiology during early development.   Here, we show the generation of a novel ICF2 disease model by reprogramming CD34+ cells derived from patient peripheral whole blood into iPSCs. We provide the molecular and phenotypical characterization of the disease model.  In particular, we examined the DNA methylation profiles at genome-wide level, demonstrating that iPSCs recapitulate the methylation defects specifically associated with ZBTB24 deficiency. We discuss these results and compare them with the molecular features identified in ICF1-patient derived iPSCs caused by DNMT3B loss of function.

overall_design: DNA samples extracted from ICF2 pYM_patient and Control iPSCs, as well as pYM_patient peripheral  blood were subjected to methylation analysis using Infinium MethylationEPIC 850K Bead Chip Array. We assessed the methylation levels at both genome-wide and satellite repetitive elements. 4 Controls from the same batch were downloaded from GSE237676 (GSM7635690 to GSM7635693). The GSM is also reported as colname in the Matrix_processed_blood file.

first: Maria

last: Matarazzo

last: Strazzullo

first: Vincenzo

last: Lullo


title: CUT&Tag-seq Analysis of HCC-PDX Samples Treated with SP2509

summary: Nude mice harboring HCC-PDX were subjected to treatment with either vehicle or SP2509, followed by analysis of tumor samples using CUT&Tag-seq assays.

overall_design: Tumor sample fragments (20-30 mm3) derived from an HCC patient into male nude mice (6 weeks old). After one month, twelve mice with tumors reaching an average size of approximately 163 mm3 were selected and randomly divided into 2 groups: six mice in the control group received intraperitoneal injection of 10μl/g vehicle (10% DMSO, 90% corn oil), and six mice in the test group received SP2509 treatment (i.p., 10μl/g, 25 mg/kg BW). Subsequently, tumor xenograft volumes and mice body weights were measured every three days for three weeks. The formula for calculating tumor volume was V = 0.5 × a × b2, where a and b represent the long and short diameters of the tumor, respectively. On the 21st day, mice were euthanized. The dissected tumor samples from PDX models were then subjected to the following real-time PCR assays and CUT&Tag-seq assays. This experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai GeneChem Organism.


title: ZMYM3 is required for the role of KDM1A in HCC cells [97H_KDM1A_shZMYM3_CUTTag_seq]

summary: To investigate the relationship between KDM1A and ZMYM3, we conducted CUT&Tag-seq experiments to detect the DNA binding regions of KDM1A in HCC cells with ZMYM3 downregulation.

overall_design: KDM1A CUT&Tag-seq experiments were performed in three groups of 97H cells: Vector control + sh-control, KDM1A+sh-control, and KDM1A+sh-ZMYM3.


title: The regulation of transcriptome in HCC cells by KDM1A [HepG2_shKDM1A_RNAseq]

summary: We investigated the impact of KDM1A knockdown on transcriptome in HCC cells through RNA-seq assays

overall_design: we analyzed gene expression profiles using RNA-seq assays in HepG2 cells with downregulated KDM1A.


title: High-throughput sequencing for analysis of macropahges from old and young mice.

city: Pittsburgh

first: Daniel

last: Clark

state: PA

address: 335 Sutherland Dr.

department: Periodontics and Preventive Dentistry

institute: University of Pittsburgh

summary: Immune cells are essential for proper fracture healing. The goal of this study was to compare the transcriptional profiles of the CD45+ immune cells isolated from the healing fracture callus of old (24 month) and young (3 month) mice (C57BL/6). Analysis of all CD45+ cells was completed via single cell RNAseq. Additional analysis of the macrophages involved in fracture healing in old and young mice was performed via bulk RNAseq. We show that there were multiple subpopulations of macrophages that were transcriptionally heterogenous and present simultaneously within the fracture callus tissue. Attenuated healing in the old mice was associated with a transcriptional profile that shifted towards more pro-inflammatory phenotypes.

overall_design: Transcriptional profiles of macrophages from the fracture callus of old and young mice

first: Mary

last: Nakamura

first: Ralph

last: Marcucio


title: CD4+ T cells exhibit distinct transcriptional phenotypes in the lymph nodes and blood following mRNA vaccination in humans

city: St. Louis

first: Julian

last: Zhou

address: 425 S. Euclid Ave.

department: Pathology & Immunology

institute: Washington University in St. Louis

summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mRNA vaccination induce robust CD4+ T cell responses. Using single-cell transcriptomics, here, we evaluated CD4+ T cells specific for the SARS-CoV-2 spike protein in the blood and draining lymph nodes (dLNs) of individuals 3 months and 6 months after vaccination with the BNT162b2 mRNA vaccine. We analyzed 1,277 spike-specific CD4+ T cells, including 238 defined using Trex, a deep learning-based reverse epitope mapping method to predict antigen specificity. Human dLN spike-specific CD4+ follicular helper T (TFH) cells exhibited heterogeneous phenotypes, including germinal center CD4+ TFH cells and CD4+IL-10+ TFH cells. Analysis of an independent cohort of SARS-CoV-2-infected individuals 3 months and 6 months after infection found spike-specific CD4+ T cell profiles in blood that were distinct from those detected in blood 3 months and 6 months after BNT162b2 vaccination. Our findings provide an atlas of human spike-specific CD4+ T cell transcriptional phenotypes in the dLNs and blood following SARS-CoV-2 vaccination or infection.

overall_design: Peripheral blood and/or drainging lymph node immune cells were across time points for infected or vaccinated individuals underwent single-cell RNA and T cell receptor sequencing to analyze the adaptive immune response.

first: Nicholas

last: Borcherding

first: Wooseob

last: Kim

last: Quinn

first: Fangjie

last: Han

middle: Q

first: Alexandria

last: Sturtz

first: Aaron

last: Schmitz

first: Tingting

last: Lei

first: Stefan

middle: A

last: Schattgen

middle: K

last: Klebert

first: Teresa

last: Suessen

first: William

last: Middleton

first: Charles

middle: W

last: Goss

first: Chang

last: Liu

first: Jeremy

middle: C

last: Crawford

first: Paul

middle: G

last: Thomas

first: Sharlene

last: Teefey

first: Rachel

middle: M

last: Presti

first: Jane

last: O'Halloran

first: Jackson

last: Turner

first: Ali

last: Ellebedy

first: Philip

last: Mudd


first: Lin

last: Gan

summary: To investigate the downstream genes of Isl1 and Pou4f1 in CVGs, we examined the gene expression level of each single knockout and double knockout compared with control.

overall_design: We performed gene expression profiling analysis using data obtained from RNA-seq of 4 different genotypes at E11.5.  Comparative gene expression profiling analysis of RNA-seq data for control, Isl1CKO, Pou4f1KO and DKO CVGs.

first: Mei

last: Xu

first: Shuchun

last: Li


title: UPF1 governs early B cell development through regulation of V(D)J recombination

status: Public on May 30 2024

city: Kyoto

first: Kotaro

last: Akaki

address: Sakyo-ku, Yoshidakonoecho

department: Department of Medical Chemistry

country: Japan

institute: Kyoto University

summary: The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, particularly nonsense-mediated mRNA decay (NMD). Here, we unveil a novel role of UPF1, showcasing its upregulation during the early stages of B cell development, a phenomenon that is integral to the promotion of early B cell differentiation in the bone marrow. B cell-specific Upf1 deletion in mice severely impeded early to late LPre-B cell transition when VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 proved indispensable for VH-DHJH recombination without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene managed to rescue the differentiation defect in early LPre-B cells under Upf1 deficiency. However, the differentiation ceased after the light chain recombination, leading to the failure to develop into immature B cells. Significantly, UPF1 was shown to interact with and govern the expression of genes linked to immune responses, cell cycle regulation, NMD, and unfolded protein response in B cells. Collectively, our data underscores the critical roles of UPF1 in VH-DHJH recombination at early LPre-B cell stage and enabling the gene expression transition from early Lpre- to sPre-B cell stage, thereby establishing proper B cell development.

overall_design: To investigate the effect of Upf1-deficiency Igh-recombined B cell progenitor cells, we isolated early LPre and sPre B cells from Upf1-conditional knockout mice with recombined Igh knonk-in mutation known as B1-8hi, which is enables eLPre to sPre differentiation of Upf1-deificient progenitor B cells.

first: Iwai

last: Noriki

first: Akaki

last: Kotaro

first: Mino

last: Takashi

first: Takeuchi

last: Osamu


title: The origins of IgA-secreting cells in the acinar structures of the nasal turbinates

city: Rehovot

first: Jingjing

last: Liu

address: Herzl 234

country: Israel

institute: Weizmann Institute of Science

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


title: Homo Sapiens Nasal Inferior Turbinates of house dust mite-induced allergic rhinitis and controls

first: Tine

last: Wils

state: Vlaams-Brabant

address: Herestraat 49 - box 811

department: Department of Microbiology, Immunology and Transplantation

institute: KU Leuven

summary: To elucidate the epithelial cell diversity within the nasal inferior turbinates, a comprehensive investigation was conducted comparing control subjects to individuals with house dust mite-induced allergic rhinitis. This study aimed to delineate the differential expression profiles and phenotypic variations of epithelial cells in response to allergic rhinitis. This research elucidated distinct subpopulations and rare cell types of epithelial cells within the nasal turbinates, discerning alterations induced by allergic rhinitis. Furthermore, by interrogating transcriptomic signatures, the investigation provided novel insights into the cellular dynamics and immune responses underlying allergic rhinitis pathogenesis

overall_design: Single cell suspension of surgically collected inferior turbinates were obtained by enzymatic digestion using liberase (87.5 µg/ml, 5401127001, Roche) and DNase I (300 µg/ml, 10104159001, Roche) in RPMI with 10% FBS for 30’ with additional mechanical disruption using a 16G needle after both 15’ and 30’ of enzymatic digestion. Digestion was stopped by adding EDTA (20 mM, 15575020, Invitrogen), whereafter red blood cells were lysed using 9.9% NaCl solution. Negative selection for immune, myeloid and fibroblast was performed using anti-CD45 (11153D, Invitrogen), anti-CD15 (11137D, Invitrogen) and anti-CD31 (11155D, Invitrogen) dynabeads, respectively. Cells were resuspended in PBS and filtered through a 70 μm cell strainer (542070, Greiner). Cells were loaded on the 10X Genomics Chromium 3’ device (3’ V2, 10X Genomics, Pleasanton, California, USA), and afterwards RNA libraries were constructed and sequenced using the NovaSeq 6000 S4 Reagent Kit V1.5 (20028312, Illumina) and using the NovaSeq 6000 device (Illumina, San Diego, California, USA). Raw sequencing files were cleaned, mapped to the human reference genome, filtered, normalized, demultiplexed and counted using fastaq-mcf, fastQC, Cell Ranger and fastMnM, after which data was analyzed using different R tools, such as Seurat, Harmony and Bioconductor in R 4.2.1 (36–41).

first: Emma

last: Ruysseveldt

first: Katleen

last: Martens

first: Brecht

last: Steelant

first: Peter

last: Hellings


title: Spatial transcriptomic in synovial tissue of  psoriasis (Ps) and psoriatic arthritis (PsA) humanized  mice.

city: Rochester

first: Soumyaroop

last: Bhattacharya

state: NY

address: 601 Elmwood Ave Box 703

department: Pediatrics

institute: University of Rochester

summary: Spatial transcriptomics was performed in areas enriched with CD45+ immune cells or CD8+ T cells. Data was generated with 12 mice: 4 NSG-SGM3 mice reconstituted with blood fo 4 healthy individuals, 4 NSG-SGM3 mice reconstituted with blood from 4 PsA patients and 4 NSG-SGM3 mice reconstituted with blod cells of 4 Ps patients.

overall_design: In the study  we enrolled 4 healthy individual, 4 patients with psoriasis and 4 patients with Psoriatic Arthritis . Skin lesions and artritis were evaluated with the Psoriasis Area Severity Index (PASI) Score and the extent of PsA with the Disease Assessment in Psoriatic Arthritis (DAPSA) score. We identified 1813 genes using  the GeoMx platform in mouse synovail tissue collected from NSG-SGM3 mice engrafted with human PBMC and sera from healthy individuals, psoriais and Psoriatic arthritis patients.

first: Brian

last: Isset

first: Ernest

last: Meyer


title: CUT&TAG analysis of R-loops in Mcm8 knockout MEFs and CUT&TAG analysis of R-loops and MCM8-binding sites in HEK293 cells

city: Jinan

first: Can

middle: xin

last: Wen

address: 44 West Wenhua Rd

department: Center for Reproductive Medicine

institute: Shandong University

summary: To reveal the role of MCM8 in suppressing R-loop accumulation, we performed the CUT&TAG assay using the S9.6 antibody to map genome-wide R-loops in Mcm8 wildtype MEFs and Mcm8 knockout MEFs. We also conducted the CUT&TAG assay to detect genome-wide R-loops in Ddx5 downregulated MEFs by adenovirus infection and in control MEFs. To investigate the underlying molecular mechanism of MCM8 suppressing R-loops, we conducted the DNA sequencing of libraries from CUT&TAG assay using the antibody against FLAG in HEK293 cells transfected with FLAG-MCM8 plasmid and using the S9.6 antibody in HEK293 cells. Besides, an IgG control and control of RNH1 overexpression were included.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: CUT&TAG-seq for R-loops using the S9.6 antibody in Mcm8 wildtype and MCM8 knockout MEFs. CUT&TAG-seq for R-loops using the S9.6 antibody in DDX5 downregulated MEFs and control MEFs. CUT&TAG-seq for R-loops using the S9.6 antibody in HEK293 cells with or without RNH1 overexpression.  CUT&TAG-seq for MCM8-binding sites using the anti-FLAG antibody in HEK293 cells with or without FLAG-MCM8 overexpression.CUT&TAG-seq using the no-specific antibody IgG was used as a control.

first: Canxin

first: Shidou

last: Zhao

first: Yajuan

last: Yang


title: N-terminal acetylation of the Set1-COMPASS fine-tunes H3K4 methylation patterns (ChIPseq for Set1 occupancy in ard1 deletion strain)

first: Junsoo

last: Oh

summary: H3K4 methylation is a conserved histone modification crucial for gene regulation, yet the post-translational modifications of the Set1-COMPASS complex remain largely unexplored. This study elucidates the significance of N-terminal acetylation in modulating H3K4 methylation patterns. Firstly, loss of NatA complex resulted in a significant decrease in H3K4me3 levels and a shift of H3K4me2 from 5' transcribed regions to promoters. Importantly, NatA physically interacted with the Set1-COMPASS complex and facilitated N-terminal acetylation of the Shg1 subunit. Surprisingly, deletion of SHG1 or mutation of the acetylation site (A2P) in Shg1 led to reduced H3K4 methylation in cells lacking Spp1. Additionally, NatB complex also contributed to H3K4 methylation regulation, potentially through N-terminal acetylation of Swd1. These findings highlight the novel role of N-terminal acetylation in fine-tuning the function of the Set1-COMPASS complex and shaping H3K4 methylation patterns. This study sheds light on the intricate regulation of H3K4 methylation and emphasizes the significance of N-terminal acetylation as a regulatory mechanism in this process.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for TAP tagged (SET1-TAP) or 3HA tagged (SET1-3HA)Set1 in WT and ard1 deletion strains in Saccharomyces cerevisiae.

first: Hyunjoo

last: Woo

first: Jung-Shin

last: Lee

first: TaeSoo

last: Kim


title: Effect of MCM8 deficiency on gene expression of mouse embryo fibroblasts

summary: MCM8 is essential in the replication stress response to maintain genome stability during mitosis. The deficiency of MCM8 results in impaired proliferation and a prolonged cell cycle, which can decrease cell populations. To investigate the effect of MCM8 deficiency on gene expression, we conducted the RNA-seq in wildtype and Mcm8 knockout mouse embryo fibroblasts. We found that the majority of genes were co-expressed and the gene expression distribution was comparable between WT and KO MEFs, suggesting that the global transcription activity was not obviously affected after MCM8 deficiency. Analysis of differentially expressed genes (DEGs) showed they were enriched in pathways including stem cell differentiation and cell fate commitment. In addition, to determine whether MCM8 is potentially involved in regulating splicing, as strongly suggested by the MCM8 interactome, we also performed alternative splicing (AS) analysis on RNA-seq data. Our data suggested that MCM8 may not function in splicing.

overall_design:
To investigate the function of MCM8 in the regulation of cell growth, we acquired and cultured wildtype and Mcm8 knockout mouse embryo fibroblasts from embryo at embryonic day 13.5.
Comparative gene expression profiling analysis of RNA-seq data for wildtype and Mcm8 knockout mouse embryo fibroblasts.


title: ISL1 and POU4F1 Directly Interact to Regulate the Differentiation and Survival of Inner Ear Sensory Neurons

first: Lin

last: Gan

summary: Isl1 and Pou4f1 regulate a unique set of CVG gene cooperatively or individually. GO term analysis shows these genes are crucial for CVGs development. To estimate Isl1 and Pou4f1 directly or indirectly regulate these downstream genes and how they act to affect these genes, we performed the CUT&Tag of ISL1 and POU4F1 with the histone modifications, H3K4me3, H3K27ac and H3K27me3 in CVGs. The identity of the peaks were characterized and big chromatin profiles were obtained.

overall_design: CUT&Tag of ISL1 and POU4F1 with the histone modifications, H3K4me3, H3K27ac and H3K27me3, in E11.5 CVGs.

first: Mei

last: Xu

first: Shuchu

last: Li

first: Dongliang

last: Yu


title: Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria

city: Geneve

first: George

last: Allen

state: Geneve

address: Rue Michel-Servet 1

country: Switzerland

institute: University of Geneva

summary: RNA helicases—central enzymes in RNA metabolism— often feature intrinsically disordered regions (IDRs) that enable phase separation and complex interactions with other proteins and/or RNA molecules. IDRs are varied and fast evolving, which makes their function hard to predict. In the bacterial pathogen Pseudomonas aeruginosa, two non-redundant RNA helicases, RhlE1 and RhlE2, share a conserved REC catalytic core but have different C-terminal extensions (CTEs) composed of IDRs of diverse length and amino acid composition. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both CTEs facilitate RNA binding and phase separation in vitro, leading to the in vivo localization of proteins in clusters within the cytoplasm. However, the CTE of RhlE2 is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping CTEs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-CTERhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-CTERhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

overall_design: PAO1 wild type and mutants were grown in twelve 12-cm square LB plates at 16°C with 0.5% arabinose. Samples from 6 plates were pooled and three replicates of each strain were therefore obtained. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit (Illumina). Then, libraries were prepared using the Illumina TruSeq stranded mRNA kit and validated on the Bioanalyzer 2100 (Agilent). Samples were sequenced using the Illumina HiSeq 2000, 150 bp double end read at Novogene.

first: Stéphane

last: Hausmann

first: Johan

last: Geiser

middle: E

first: Sandra

last: Geslain

first: Martina

last: Valentini


title: Effect of knockout of UPF1 on gene expression during differentiation in early LPre-B stage.

first: Kotaro

last: Akaki

summary: To investigate the role of UPF1 in the early Lpre-B cells (Fixable Viability Dye-, CD19+, B220+, sIgM-, CD43+, FSChi, CD2-), we isolated early Lpre-B cells from bone marrow derived from control or Upf1-cKO mice.

overall_design:
We then performed gene expression profiling analysis using data obtained from RNA-seq of Control or Upf1-cKO early Lpre-B cells.
Comparative gene expression profiling analysis of RNA-seq data for early Lpre-B cells derived from control or Upf1-cKO mice.

first: Noriki

last: Iwai

first: Takashi

last: Mino

first: Osamu

last: Takeuchi


title: Determination the direct binding target transcripts of UPF1 in B cell.

summary:
To determine the direct binding targets of UPF1, we performed RNA-immunoprecipitation sequencing (RIP-seq), in which mRNAs bound to phosphorylated (NMD-active) UPF1 were immunoprecipitated.
And then, we analyzed the pUPF1-bound RNAs by RNA sequencing.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for immunoprecipiated with anti pUPF1 Ab or normal rabbit IgG from B cell.

first: Fabian

last: Hia


title: Role of UPF1 in B cells.

overall_design: Refer to individual Series


title: Different gene expression between normal and cancer-associated fibroblasts from diffuse-type gastric cancer

city: Suwon-si, Gyeonggi-do

first: hoon

last: hur

state: State...

address: 164, World cup-ro, Yeongtong-gu

department: Department of Surgery

institute: Ajou University

summary: To identify a cancer-associated fibroblast (CAF)-specific molecule that contributes to tumor-promoting effect, we utilized trnascriptome analysis. We performed RNA-sequencing with three normal fibroblast (NF)-CAF pairs derived from diffuse-type gastric cancer patients’ tissues. RNA-sequencing revealed several commonly increased CAF-specific genes among each pair.

overall_design: RNA-sequencing of paired normal fibroblast and cancer associated-fibroblast cells from three diffusse-type gastric cancer patients.

first: Hoon

last: Hur

first: In-Hye

last: Ham

first: Dagyeong


title: Small RNA-seq of liver of DICER1 syndrome model mouse

city: Shinjuku-ku

first: Shin-ichiro

last: Ohno

state: Tokyo

address: 6-1-1 Shinjuku

department: Molecular Pathology

institute: Tokyo Medical University

summary: In order to identify miRNAs involved in tumor development of DICER1 syndrome model mouse, next-generation sequencing analysis of RNA extracted from liver tissue was performed.

overall_design: Small RNA profiles of mouse normal liver and liver cancer were generated by deep sequencing, using Illumina HiSeq 4000.

first: Masahiko

last: Kuroda


title: Decipher immune response in early recovery phase of respiratory virus-induced disorder [Ly6GMacs scRNA-seq]

city: Liège

first: Thomas

last: Marichal

address: Avenue de l'Hopital 11

department: GIGA Institute

institute: University of Liege

summary: Appropriate lung repair responses are essential to restore functional integrity and prevent severe disease phenotypes after injury. While macrophages are thought to contribute to repair, whether distinct subsets occupy particular niches and assume dedicated tasks to mediate host recovery post- injury remains unknown. Using an in vivo model of influenza A virus (IAV) infection in combination with single-cell and spatial transcriptomic analyses, bone marrow chimeras and monocyte fate-mapping, we found the transitional appearance of Ccr2-dependent monocyte-derived Ly6G+ macrophages (Ly6G Macs) after viral clearance. Ly6G Macs inhabited the alveolar spaces of regenerating perilesional areas, exhibited a unique ultrastructural morphology as well as a high metabolic potential. Using in vivo gene targeting and ex vivo wound healing assays, we found that Ly6G Macs could limit disease severity and promote alveolar regeneration via interleukin-4 receptor- and arginase-1-dependent mechanisms acting on type 2 alveolar epithelial cells. We also found evidence that similar macrophages existed in other models of lung injury and in the airways of virally-infected humans. Our study thus identifies perilesional alveolar Ly6G Macs as a spatially-restricted short-lived macrophage subset engaging a crosstalk with type 2 alveolar epithelial cells to promote alveolar regeneration and recovery post-injury, thus representing an attractive therapeutic target.

overall_design: To investigate the role of myeloid cells in early recovery phase of respiratory virus-induced disorders we used a clinically relevant model of flu (H1N1 PR8 strain) and analysed immune cell populations 10 days post infection, when virus is completely cleared from the lung. Live CD45+, F4/80 and/or CD11b cells were FACS-sorted and their gene expression profile was analysed by RNA-seq.

first: Cecilia

last: Ruscitti


title: The origins of IgA-secreting cells in the acinar structures of the nasal turbinates [scRNA-seq]

first: Jingjing

last: Liu

summary: Nasal vaccination elicits a humoral immune response that provides protection from airborne pathogens, yet the origins and specific immune niches of antigen-specific IgA-secreting cells in the upper airways remain unknown. Here, we define glandular acinus structures of the nasal turbinates as an immunological niche that recruits IgA-secreting plasma cells from the nasal-associated lymphoid tissues (NALT) in response to intranasal vaccination. Using intact organ imaging, we investigated the cellular events that support antibody-mediated immune responses in the mouse upper airways. Through visualization of antigen-specific T and B cells in the upper airways, we demonstrate that nasal vaccination induced extensive B cell expansion in the subepithelial dome (SED) of the NALT, followed by invasion into commensal bacteria-driven chronic germinal centers (GCs) in a T cell-dependent manner. Antigen-specific B cell response in the NALT required pre-expansion of cognate T cells, which initiate the immune response in the inter-follicular regions of the NALT, and occurred effectively in the presence of Monophosphoryl-Lipid A (MPLA), a synthetic, non-toxic TLR-4 agonist. NALT ablation and blockade of PSGL-1 demonstrated that intranasal vaccination generates IgA+ plasma cells that home to the nasal turbinates through the blood circulation where they are positioned primarily around glandular acinus structures. Thus, the glandular part of the nasal turbinate is an immunological niche that hosts NALT-derived IgA-secreting cells. These cellular events can be manipulated to design vaccines against inhaled pathogens or in the treatment of upper airway allergic responses.

overall_design: Single-cell RNAseq of GFP B1-8hi B cells, five days after NP-OVA+MPLA intranasal immunization.

first: Hadas

last: Hezroni

first: Ziv

last: Shulman