title: Tulp1 deficiency causes early-onset retinal degeneration through affecting ciliogenesis and activating ferroptosis in zebrafish

city: Wuhan

first: Danna

last: Jia

address: 1037 Luoyu Road

institute: Huazhong University of Science and Technology

summary: Mutations in TUB-like protein 1 (TULP1) are associated with severe early-onset retinal degeneration in humans. However, the pathogenesis remains largely unknown. There are two homologous genes of TULP1 in zebrafish, namely tulp1a and tulp1b. Here, we generated the single knockout (tulp1a-/- and tulp1b-/-) and double knockout (tulp1-dKO) models in zebrafish. Knockout of tulp1a results in the mislocalization of UV cone opsins and the degeneration of UV cones specifically, while knockout of tulp1b results in mislocalization of rod opsins and rod-cone degeneration. In the tulp1-dKO zebrafish, mislocalization of opsins was present in all types of photoreceptors, and severe degeneration was observed at a very early age, mimicking the clinical manifestations of TULP1 patients. Photoreceptor cilium length was significantly reduced in the tulp1-dKO retinas. RNA-seq analysis showed that the expression of tektin2 (tekt2), a ciliary and flagellar microtubule structural component, was downregulated in the tulp1-dKO zebrafish. Dual-luciferase reporter assay suggested that Tulp1a and Tulp1b transcriptionally activate the promoter of tekt2. Additionally, ferroptosis might be activated in the tulp1-dKO zebrafish, as suggested by the up-regulation of genes related to the ferroptosis pathway, the shrinkage of mitochondria, reduction or disappearance of mitochondria cristae, and the iron and lipid droplet deposition in the retina of tulp1-dKO zebrafish. In conclusion, our study establishes an appropriate zebrafish model for TULP1-associated retinal degeneration and proposes that loss of TULP1 causes defects in cilia structure and opsin trafficking through the downregulation of tekt2, which further increases the death of photoreceptors via ferroptosis. These findings offer insight into the pathogenesis and clinical treatment of early-onset retinal degeneration.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wt and tulp1a&tulp1b zebrafish.

first: Pan

last: Gao

first: Yuexia

last: Lv

first: Mugen

last: Liu

first: Xiang

last: Ren

first: Fei


title: The non-apoptotic function of Caspase-8 in negatively regulating the CDK9 mediated Ser2 phosphorylation of RNA Polymerase II in cervical cancer

status: Public on Nov 23 2022

city: Essen

first: Samuel

last: Peña-Llopis

address: Hufelandstr. 55, Augenklinik 1.UG, Rm 69-71

department: Ophthalmology

country: Germany

institute: University Hospital Essen

summary: Analysis of the gene expression profile of the cervical cancer cell line HeLa in the presence or absence of Caspase-8 expression. The result offered an useful insight into the altered gene expression of metastasis associated genes due to the negative regulation of RNAPII mediated gene transcription by Caspase-8

overall_design: Total RNA from non-synchronized and S/G2-phase synchronized HeLa WT and KO cells were extracted using the RNeasy Plus kit (Qiagen) and subjected to microarray expression profiling using the HumanHT-12 v3 arrays. Additional controls were also included in the form Caspase-8 knock-down HeLa WT cells and Caspase-8 overexpressing KO cells

first: Ranadip

last: Mandal

first: Klaus

last: Strebhardt


title: A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia [ChIP-Seq]

city: Cambridge

first: Liming

last: Tao

address: 415 Main Street

country: USA

institute: Broad Institute of Harvard and MIT

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The molecular regulation of human hematopoietic stem cell (HSC) maintenance is therapeutically important, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. Here, we have studied a rare genetic disorder due to MECOM haploinsufficiency, characterized by an early-onset absence of HSCs in vivo. By generating a faithful model of this disorder in primary human HSCs and coupling functional studies with integrative single-cell genomic analyses, we uncover a key transcriptional network involving hundreds of genes that is required for HSC maintenance. Through our analyses, we nominate cooperating transcriptional regulators and identify how MECOM prevents the CTCF-dependent genome reorganization that occurs as HSCs differentiate. Strikingly, we show that this transcriptional network is co-opted in high-risk leukemias, thereby enabling these cancers to acquire stem cell properties. Collectively, we illuminate a regulatory network necessary for HSC self-renewal through the study of a rare experiment of nature.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for CTCF in MUTZ-3 cells.

first: Richard

last: Voit

first: Fulong

last: Yu

first: Vijay

last: Sankaran


title: SIRT6-dependent gene transcriptional profile in human endothelial cells

city: Rochester

first: Cameron

last: Baker

state: NY

address: 601 Elmwood Ave

institute: University of Rochester

type: Expression profiling by high throughput sequencing

summary: Sirtuin 6 (SIRT6), a well-recognized longevity gene, regulates genome stabilization, DNA repair, inflammation, and metabolic homeostasis. SIRT6 expression is decreased in atherosclerotic lesions from apolipoprotein E deficient mice and human patients. SIRT6 expression is also decreased in endothelial cells (ECs) under chronic stimulation with lipopolysaccharide, hydrogen peroxide and high glucose, three of which are risk factors associated with endothelial dysfunction and atherogenesis. To gain mechanistic insights into the effects of SIRT6 on endothelial gene transcriptome, we performed RNA-sequencing (RNA-seq) analysis of control adenovirus and SIRT6 adenovirus infected human umbilical vein endothelial cells (HUVECs). Gene ontology (GO), pathway enrichment and functional annotation clustering analysis were performed to classify differentially expressed genes and to identify the most significantly enriched GO terms/pathways/gene clusters.

overall_design: Human Umbilical Vein Endothelial Cells (HUVEC, passage 3 to passage 5) were seeded in 0.2% gelatin-coated dishes the day before experiment. HUVECs at 100% confluence were infected with control adenoviral LacZ or adenoviral SIRT6 for 48 h.

first: Zheng

middle: G

last: Jin

first: Suowen

last: Xu

first: Xiaoqian

last: Wu

first: Jason

middle: R

last: Myers

first: John

last: Ashton


title: Human Macrophage Long Intergenic Non-coding RNA, SIMALR, Suppresses Inflammatory Macrophage Apoptosis via Netrin 1

city: New York

first: Chenyi

last: Xue

institute: Columbia University

summary: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and related inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. Through RNA-seq of human monocyte-derived macrophages, we identified Suppressor of Inflammatory Macrophage Apoptosis lncRNA (SIMALR), a human macrophage-specific long intergenic noncoding RNA, to be highly induced in LPS/IFNγ-stimulated macrophages. Treatment of LPS/IFNγ stimulated THP1 human macrophages with SIMALR antisense oligonucleotides induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP, caspase 9, caspase 3, and Annexin V+. RNA-seq of control versus SIMALR knockdown in LPS/IFNγ-stimulated human monocyte derived macrophages showed NTN1 to be significantly decreased upon SIMALR knockdown. As expected, NTN1 knockdown in LPS/IFNγ-stimulated macrophages induced apoptosis. This apoptotic phenotype was attenuated by adding recombinant NTN1 after SIMALR knockdown. Furthermore, NTN1 promoter-luciferase reporter activity was increased in HEK293T cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α. RNA immunoprecipitation and microscopy studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating apoptosis of macrophages. In translational studies, SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. SIMALR is a non-conserved, human macrophage lincRNA expressed in human atherosclerosis that regulates macrophage apoptosis.  SIMALR partners with HIF1α to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of non-conserved human lincRNAs and exploring their translational and therapeutic potential in human atherosclerosis.

overall_design: RNA-seq was performed on LPS/IFNγ-stimulated human monocyte-derived macrophages that were treated with SIMALR antisense oligonucleotides or control. Ingenuity Pathway Analysis (IPA) was next performed on differentially expressed genes identified from RNA-seq. IPA predicted that SIMALR may play a role in macrophage apoptosis. To verify this prediction, we performed Annexin V+ flow analysis and western blot with apoptotic markers on LPS/IFNγ stimulated THP1 human macrophages after SIMALR knockdown. RNA-seq data also revealed NTN1, a known macrophage survival marker, to be significantly downregulated with SIMALR knockdown. To test whether NTN1 expression was regulated by SIMALR, luciferase assay with NTN1 promoter-luciferase reporter was performed after treating HEK293T cells with lentiviral overexpression of SIMALR. As NTN1 is known to be regulated by HIF1α, we also investigated whether SIMALR interacts with HIF1α with RNA immunoprecipitation and microscopy studies in LPS/IFNγ stimulated THP1 human macrophages. Finally, to see if SIMALR can be detected in human atherosclerotic plaques, we performed RNAscope assay to probe for SIMALR, along with CD68 and ACTA2.

first: Esther

last: Cynn

first: Ying

last: Wang


title: Effect of depletion of CTR9 on gene expression during early osteogenic differentiation of human primary mesenchymal stem cells

city: Madison

first: NGAI TING

last: CHAN

state: WI

address: 4849 Sheboygan Ave

department: Oncology

institute: University of Wisconsin -- Madison

summary:
Cell-fate determination of human mesenchymal stem/stromal cells (hMSCs) is precisely regulated by lineage-specific transcription factors and epigenetic enzymes. We found that CTR9, a key scaffold subunit of Polymerase Associated Factor Complex (PAFc), selectively regulates hMSC differentiation to osteoblasts and chondrocytes, but not to adipocytes. An in vivo ectopic osteogenesis assay confirmed the essentiality of CTR9 in hMSC-derived bone formation. CTR9 counteracts the activity of EZH2, the epigenetic enzyme that deposits H3K27me3, in hMSCs. Accordingly, CTR9 knockdown (1) hMSCs gain H3K27me3 mark, and the osteogenic differentiation defects of CTR9 KD hMSCs can be partially rescued by treatment with EZH2 inhibitors. Transcriptome analyses identified Bone Morphology Protein-2 (BMP-2) as a downstream effector of CTR9. BMP-2 secretion, membrane anchorage, as well as the BMP-SMAD pathway were impaired in CTR9 KD MSCs, and the effects were rescued by BMP-2 supplementation. This study uncovers an epigenetic mechanism engaging CTR9-H3K27me3-BMP-2 axis to regulate osteochondral lineage differentiation of hMSCs.
To investigate the  function of CTR9 in the gene regulation of early osteogenic committment , we established human MSCs in which CTR9 gene has been knocked down by two individual shRNAs (shControl vs shCTR9#3/#5）.

overall_design: To identify potential downstream factors regulated by CTR9 during early osteoblast differentiation, we harvested mRNAs from shControl and shCTR9 hMSCs after short-term (7 days) osteogenic induction for bulk RNA-seq and attempt to identify differentially expressed genes (DEGs) whose expression are CTR9-dependent.

first: Ngai

middle: T

last: Chan


title: Differential regulation of Type 1 and Type 2 mouse eosinophil activation by apoptotic cells

city: Tel Aviv

first: Guy

last: Shapira

address: Chaim Levanon 30

country: Israel

institute: Tel Aviv University

summary:
Purpose: We aimed to characterize the role of apoptotic cells in regulating eosinophils' response to diverse stimuli.
Methods:Eosinophils were purified from the peritoneal cavity of Il5Tg mice and stimulated with IL-4 (i.e. type 2 activated eosinophils), IFN-γ, and a combination of lipopolysaccharide (LPS, i.e. type 1 activated eosinophils) and   IFN-γ in the presence of apoptotic cells. Thereafter, RNA was obtained and the transcriptome signature following each stimulation was determined using RNA sequencing. Raw sequencing reads were trimmed and filtered using fastp, then aligned to mouse genome assembly (GRCm38) using STAR 2.7.2a. Normalization and differential expression analysis were performed in R 4.0.3 using the package DESeq2. Finally, gene ontology annotations were obtained from Ensembl and pathway graphs were obtained from KEGG.
Results:RNAseq analysis revealed that apoptotic cells induce transcriptional changes in eosinophils and resulted in enrichment in pathways related to wounding and cell migration.
Conclusion:RNAseq analysis of transcripts that were downregulated by apoptotic cells revealed that apoptotic cells inhibit pathways as defense responses and inflammatory responses and  that apoptotic cells augmented eosinophils' response to IL-4.

overall_design: RNAseq of eosinophils following Type 1 and Type 2 activation in the presence of apoptotic cells


title: scRNAseq dataset of PDGFRβ+ cells in fibrosing skin and kidney with or without ZFYVE9/SARA overexpression

city: Chicago

first: Matthew

middle: J.

last: Schipma

state: IL

address: 750 N. Lake Shore Drive

department: Biochemistry and Molecular Genetics

institute: Northwestern University

summary: Activated myofibroblasts play an essential role in tissue fibrogenesis by producing extracellular matrixes (ECM).  ECMs replace normal functioning tissue, reducing tissue plasticity and impairing functions of the organ.  Recent studies suggested that pericytes are a major source of myofibroblast precursors.  We previously showed that Smad Anchor for Receptor Activation (SARA) prevents cellular phenotypic transdifferentiation toward mesenchymal cells, and depletion of SARA induces transdifferentiation of epithelial cells to fibroblast-like phenotype.  Here, we generated a transgenic mice that overexpress SARA specifically pericytes by using PDGFRβ-Cre (SARATg, PDGFRβ-Cre). When subjected to either subcutaneous injection of bleomycin or intraperitoneal administration of aristolochic acid, which induces skin and kidney fibrosis, respectively, SARATg, PDGFRβ-Cre mice developed significantly less fibrosis compared to SARAWT, PDGFRβ-Cre mice.  To decipher molecular signature and pericyte trajectory under fibrotic conditions and effects of SARA overexpression, we isolated PDGFR+ cells from skin or kidney of SARATg or WT, PDGFRβ-Cre, Z/EG mice with or without fibrotic stimuli and performed scRNAseq analyses.  In both skin and kidney sample sets, we found pericytes and immune cell populations are the major cell components among the PDGFR+ GFP+ cells.  We found that pericyte populations are divided into canonical and non-canonical sub-populations and the latter has assumed myofibroblast characteristics.  Trajectory mapping revealed a single path from canonical to non-canonical pericyte sub-populations and SARA overexpression truncated the trajectory.  These results suggest that SARA prevents pericyte transdifferentiation into myofibroblasts under fibrotic condition, and therefore anti-fibrotic.

overall_design: 8-week-old SARATg or WT mice crossed with PDGFRβ-Cre and Z/EG reporter mice were subjected to either repeated subcutaneous bleomycin (20mg/kg BW 3x week for 2 weeks) or intraperitoneal injection of aristolochic acid (5mg/kg BW 3x week for 3 weeks) to induce skin or kidney fibrosis respectively, and skin an kiney were harvested.

first: Tomoko

last: Hayashida

first: Katia

middle: C

last: Scheri


title: De novo enhancer function in divergent hominin haplotypes

city: Durham

first: Craig

middle: Barrett

last: Lowe

state: North Carolina

address: 213 Research Dr

department: Molecular Genetics and Microbiology

institute: Duke University School of Medicine

summary: Identifying functional changes in gene regulatory elements in the human lineage is essential to understanding how humans emerged as a species. We performed genome-wide comparative genomic analysis of humans and great apes to identify 1581 Human Ancestor Quickly-Evolved Regions (HAQERs), which we identify as the fastest-evolved regions of the human genome (hg38). To detect how rapid sequence divergence contributed to divergent gene regulatory functionality in HAQERs, we designed in vivo STARR-seq as a multiplex single-cell assay of enhancer activity in the developing mouse brain. We tested multiple haplotypes from hominins (Human/Neanderthal/Denisova) and non-hominins (Chimpanzee and the inferred Human-Chimpanzee ancestor) for 13 HAQERs to assess their enhancer activity in the developing brain. We found significant differences in enhancer activity between hominins and non-hominins in 8 of these sequences.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: In this assay, STARR-seq plasmids along with an EGFP transfection reporter plasmid are delivered to the developing mouse cerebral cortex via in utero electroporation. Following 18 hours of incubation, mouse brains are harvested and GFP+ cells are isolated via Fluorescent Activated Cell Sorting. Single cell sequencing is then performed to recover gene expression, EGFP transfection reporter plasmid expression, and STARR-seq reporter expression. We performed two independent STARR-seq experiments (V4.2 = E14.5 injection, V4.1 = E15.5 injection)  with the same input library, which we sequenced for input normalization.

middle: B

first: Riley

middle: J

last: Mangan


title: BET Inhibitors Target the SCLC-N subtype of Small Cell Lung Cancer by Blocking NEUROD1 Transactivation

city: Bethesda

first: Haobin

last: Chen

state: MD

address: 10 Center Drive, Room 3-5888

department: Thoracic Surgery Branch

institute: National Institutes of Health

summary:
Small cell lung cancer can be divided into several molecular subtypes based on the expression of four master transcription factors (ASCL1, NEUROD1, POU2F3, and YAP1). These master factors have not been directly druggable, and we hypothesized that targeting their transcriptional coactivator(s) could provide an alternative approach. Here, we identify that BET bromodomain proteins physically interact with NEUROD1 and function as transcriptional coactivators. Using CRISPR knockout and ChIP-seq, we demonstrate that NEUROD1 plays a critical role in defining the landscapes of BET bromodomain proteins in the SCLC genome. Targeting BET bromodomain proteins by BET inhibitors leads to broad suppression of the NEUROD1-target genes, especially those associated with superenhancers, and reduces SCLC growth in vitro and in vivo. LSAMP, a membrane protein in the IgLON family, was identified as one of the NEUROD1-target genes mediating BET inhibitor sensitivity in SCLC. Altogether, our study reveals that targeting transcriptional coactivators could be a novel approach to blocking the master transcription factors in SCLC for therapeutic purposes.

The goal of this experiment was to characterize the genome-wide binding profiles of BET proteins, RPII, and H3K27Ac upon NEUORD1 knockout in H446 cells.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for NEUROD1, BRD2, BRD3, BRD4, histone H3K27Ac, and RNA polymerase II in H446 NEUROD1-KO cells (#297, cl.2-1) and control cells (NTgRNA cl.10).

first: Young

middle: K

last: Park

first: Ge

last: Kai


summary:
Small cell lung cancer can be divided into several molecular subtypes based on the expression of four master transcription factors (ASCL1, NEUROD1, POU2F3, and YAP1). These master factors have not been directly druggable, and we hypothesized that targeting their transcriptional coactivator(s) could provide an alternative approach. Here, we identify that BET bromodomain proteins physically interact with NEUROD1 and function as transcriptional coactivators. Using CRISPR knockout and ChIP-seq, we demonstrate that NEUROD1 plays a critical role in defining the landscapes of BET bromodomain proteins in the SCLC genome. Targeting BET bromodomain proteins by BET inhibitors leads to broad suppression of the NEUROD1-target genes, especially those associated with superenhancers, and reduces SCLC growth in vitro and in vivo. LSAMP, a membrane protein in the IgLON family, was identified as one of the NEUROD1-target genes mediating BET inhibitor sensitivity in SCLC. Altogether, our study reveals that targeting transcriptional coactivators could be a novel approach to blocking the master transcription factors in SCLC for therapeutic purposes.

The purpose of this part of the study was to investigate what genes are affected by BET inhibitor JQ1 in small cell lung cancer cell line COR-L279 (a SCLC-N subtype).

overall_design: Comparative gene expression profiling analysis of RNA-seq data was performed in COR-L279 cells treated with two doses of JQ1 (0.5µM and 1.5µM; 24hours) for 24 hours versus DMSO. Each sample had three biological replicates.


title: Integrated Analysis of Transcriptomics, Proteomics and Metabolomics Data Reveals the role of SLC39A1 in Renal Cell Carcinoma

city: Shenyang

first: Xiao

last: Dong

state: Liaoning

address: 155 Nanjing North Street, Heping District

department: Department of Urology

country: China

institute: The first hospital of China Medical University

summary:
To investigate the role of SLC39A1 in renal cell carcinoma, we established OS-RC-2 cell lines in which were transfected with SLC39A1 overexpressed vectors and empty vectors
We then performed transcriptomic analysis using data obtained from RNA-seq of 3 pairs of  OSRC-2 cells.

overall_design: Comparative  transcriptomic analysis of RNA-seq data for SLC39A1 overexpressed OSRC-2 cells and its negative control (ZIP, and NC).

first: Yulin

last: Yuan


title: ATR represents a therapeutic vulnerability in clear cell renal cell carcinoma

city: Freiburg

first: Kyra

last: Zodel

address: Breisacher Straße 115

department: Center for Translational Cell Research

institute: University Hospital Freiburg

summary: Metastatic clear cell renal cell carcinomas (ccRCC) are resistant to DNA damaging chemotherapies, limiting therapeutic options for patients whose tumours are resistant to tyrosine kinase inhibitors and/or immune checkpoint therapies. Here we show that mouse and human ccRCC are frequently characterised by high levels of endogenous DNA damage and that cultured ccRCC cells exhibit intact cellular responses to chemotherapy-induced DNA damage. We identify that pharmacological inhibition of the DNA damage sensing kinase ATR with the orally administered, potent and selective drug M4344 induces anti-proliferative effects in ccRCC cells due to replication stress and the accumulation of DNA damage in S phase. In some cells, DNA damage persists into subsequent G2/M and G1 phases, leading to the frequent accumulation of micronuclei. Daily single agent treatment with M4344 inhibited the growth of ccRCC xenograft tumours. M4344 synergises with chemotherapeutic drugs including cisplatin and carboplatin and the PARP inhibitor olaparib in mouse and human ccRCC cells. Weekly M4344 plus cisplatin treatment showed in vivo therapeutic synergy in ccRCC xenografts and was efficacious in an autochthonous mouse ccRCC model. These studies identify ATR inhibition as a potential novel therapeutic option for ccRCC.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA-Seq of murine and human ccRCC cell lines

first: Philipp

last: Seidel

first: Anne

last: Rubarth

first: Asin

last: Peighambari

first: Felix

last: Neumann

first: Yannick

last: Federkiel

first: Hsin

last: Huang

first: Rouven

last: Hoefflin

first: Mojca

last: Adlesic

first: Christian

last: Witt

first: David

last: Hoffmann

first: Patrick

last: Metzger

first: Ralph

last: Lindemann

first: Frank

last: Zenke

first: Christoph

last: Schell

first: Melanie

last: Boerries

first: Dominik

last: von Elverfeldt

first: Wilfried

last: Reichardt

first: Marie

last: Follo

first: Joachim

last: Albers

first: Ian

last: Frew


title: Tet2 and Tet3 mediated active cytosine hydroxymethylation in Six2 progenitor cells is critical for nephron progenitor differentiation and nephron endowment

city: Philadelphia

first: Xiujie

last: Liang

state: Pennsylvania

address: 12-188 Smilow Translational Research Center, 3400 Civic Center Blvd

department: Department of Medicine

institute: University of Pennsylvania

type: Methylation profiling by high throughput sequencing

summary: Nephron endowment is a key determinant of later life hypertension and kidney disease. Here we studied whether epigenetic changes, specifically the ten–eleven translocation (Tet) DNA demethylase family, Tet1, Tet2, and Tet3-mediated active DNA hydroxymethylation is necessary for gene expression regulation and kidney differentiation. We generated mice with deletion of Tet1, Tet2 or Tet3 in Six2 positive nephron progenitors (NP). We did not observe changes in development or kidney function in mice with nephron progenitor-specific deletion of Tet1, Tet2, Tet3 or Tet1/Tet2 or Tet1/Tet3. On the other hand, mice with combined Tet2 and Tet3 loss in Six2-positive NPCs failed to form nephrons leading to kidney failure and perinatal death. Tet2 and Tet3 loss in Six2-positive NPs resulted in defect in mesenchymal to epithelial transition and renal vesicle differentiation. Whole genome bisulfite sequencing, single cell RNA sequencing, and gene and protein expression assay identified a defect in expression in genes in the WNT-β-catenin signaling pathway in absence of Tet2 and Tet3 due to a failure in demethylation of these loci. Our results indicate the key role of Tet2 and Tet3-mediated active cytosine hydroxymethylation in NPs in kidney development and nephron endowment.

overall_design: scRNA-seq of kidneys from newborn Six2Cre and Six2CreTet2/3f/f mice. WGBS of Six2+ NP isolated from kidneys of newborn Six2Cre and Six2CreTet2/3f/f mice.

first: Katalin

last: Susztak


title: BET inhibitors target the SCLC-N subtype of small-cell lung cancer by blocking NEUROD1 transactivation

first: Haobin

last: Chen

summary:
Small cell lung cancer can be divided into several molecular subtypes based on the expression of four master transcription factors (ASCL1, NEUROD1, POU2F3, and YAP1). These master factors have not been directly druggable, and we hypothesized that targeting their transcriptional coactivator(s) could provide an alternative approach. Here, we identify that BET bromodomain proteins physically interact with NEUROD1 and function as transcriptional coactivators. Using CRISPR knockout and ChIP-seq, we demonstrate that NEUROD1 plays a critical role in defining the landscapes of BET bromodomain proteins in the SCLC genome. Targeting BET bromodomain proteins by BET inhibitors leads to broad suppression of the NEUROD1-target genes, especially those associated with superenhancers, and reduces SCLC growth in vitro and in vivo. LSAMP, a membrane protein in the IgLON family, was identified as one of the NEUROD1-target genes mediating BET inhibitor sensitivity in SCLC. Altogether, our study reveals that targeting transcriptional coactivators could be a novel approach to blocking the master transcription factors in SCLC for therapeutic purposes.

The goals for this part of the study were: 1) to identify the NEUROD1 target genes in the SCLC-N subtype small cell lung cancer. To this end, we knocked out NEUROD1 in H446 cells (SCLC-N subtype) by CRISPR and assessed gene expression alterations in three knockout and three control clones. An integrated analysis of the RNA-seq data and the NEUROD1 ChIP-seq data was performed to identify the NEUROD1 target genes; 2) to assess how NEUROD1 knockout affects the response to JQ1 (a BET inhibitor).

overall_design: Comparative gene expression profiling analysis of RNA-seq data was performed between H446 NEUROD1-KO derivatives (#297 cl. 12, cl. 15, and cl.2-1) and the control clones (NTgRNA cl.3, cl.10, and cl.13) following treatment of JQ1 (1µM; 24 hrs) or DMSO. Each sample had three biological replicates.


summary:
Small cell lung cancer can be divided into several molecular subtypes based on the expression of four master transcription factors (ASCL1, NEUROD1, POU2F3, and YAP1). These master factors have not been directly druggable, and we hypothesized that targeting their transcriptional coactivator(s) could provide an alternative approach. Here, we identify that BET bromodomain proteins physically interact with NEUROD1 and function as its transcriptional coactivators. Using CRISPR knockout and ChIP-seq, we demonstrate that NEUROD1 plays a critical role in defining the landscapes of BET bromodomain proteins in the SCLC genome. Targeting BET bromodomain proteins by BET inhibitors leads to broad suppression of the NEUROD1-target genes, especially those associated with superenhancers, and reduces SCLC growth in vitro and in vivo. LSAMP, a membrane protein in the IgLON family, was identified as one of the NEUROD1-target genes mediating BET inhibitor sensitivity in SCLC. Altogether, our study reveals that targeting transcriptional coactivators could be a novel approach to blocking the master transcription factors in SCLC for therapeutic purposes.

To investigate what genes are affected by NHWD-870 (a BET inhibitor) in SCLC-N subtype tumors in vivo, we performed an RNA-seq analysis of the tumor cells isolated from the LX22 PDX after NHWD-870 treatment at 2mg/kg via oral gavage daily for five days.

overall_design: Each group (NHWD870 vs. vehicle) contained six tumors.


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

overall_design: Refer to individual Series


title: Systematic characterization of human placenta-derived circRNAs and identification of autophagy-related circRNAs in gestational diabetes mellitus [lncRNA]

first: yin

middle: di

last: Bao

state: Hubei

address: 238 Jiefang Rd

institute: Renmin Hospital of Wuhan

type: Non-coding RNA profiling by high throughput sequencing

summary: The objective of this study was to identify and investigate the roles of circRNAs in GDM. In the current study, placental circRNA expression profiles of normal controls and GDM patients were analyzed using high-throughput sequencing. Bioinformatics analysis identified a total of 4955 circRNAs, of which 37 circRNAs were significantly deregulated in GDM placentas compared with NC placentas. GO and KEGG enrichment analyses demonstrated that metabolic process-associated terms and metabolic pathways that may be related to GDM were significantly enriched. The biological characteristics of placenta-derived circRNAs, such as their stability and RNase R resistance, were also validated.Our study indicates that aberrant expression of circRNAs may play roles in autophagy in GDM placentas, providing new insights into GDM.

overall_design: RNA-seq of placenta  from 3 pregnant women with GDM and 3 healthy pregnant women, and identification of autophagy-related circRNAs in gestational diabetes mellitus

first: Yindi

first: Jing

last: Yang


title: Transcriptom of hibernating Daurian ground squirrels

city: xi'an

first: Shanfeng

last: Jiang

state: shaanxi

address: No. 127, West Youyi Road, Beilin District

department: School of Life Sciences

institute: Northwestern Polytechnical University

summary: n this study, two types of skeletal muscle (fast muscle and slow muscle) in five periods of hibernation were studied.

overall_design: Animals are divided into five groups:summer active group (SA), pre-hibernation group(PRE), hibernation group(HIB), interbout arousal group(IBA), post-hibernation group(POST).

first: Ting

last: Qu


title: KDM6A loss recruits tumor-associated neutrophils and promotes neutrophil extracellular trap formation via the CXCL1-CXCR2 axis in pancreatic cancer

city: Ann Arbor

first: Karan

last: Bedi

state: MI

address: 1600 Huron Pkwy

department: Biostatistics

institute: University of Michigan

summary: Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, how KDM6A loss impacts PDAC tumor immune microenvironment is not known. Tumor-associated neutrophils (TANs) and neutrophil extracellular traps (NETs) in the tumor microenvironment contribute to PDAC progression. This study used genetically engineered pancreas-specific Kdm6a-knockout PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased TANs and NETs formation. Genome-wide Bru-seq analysis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), were upregulated in KDM6A-knockout PDAC cells. We confirmed that KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruits neutrophils. Furthermore, the CXCL1 neutralizing antibody blocked the chemotactic and NETs-promoting property of KDM6A-deficient PDAC cells and tumor growth in a syngeneic orthotopic xenograft mouse model, confirming that CXCL1 was the main mediator of chemotaxis and PDAC growth in this model. These findings shed light on how KDM6A regulates tumor immune microenvironment and PDAC progression and suggest that the CXCL1-CXCR2 axis may be a candidate target for treating PDACs with KDM6A loss.

overall_design: Three PANC-1 cell line samples were analyzed, including a control PANC-1 cell line without gRNA and two PANC-1 clones with UTX/KDM6A CRISPR/cas9 knockout.

first: Lin

last: Jin

first: Hong

middle: S

last: Kim

first: Feng

last: Tian

first: Zhujun

last: Yi

first: Mats

last: Ljungman

first: Marina

last: Pasca di Magliano

first: Howard

last: Crawford

first: Jiaqi

last: Shi


title: The thyroid hormone clearing  type 3 deiodinase protects from cardiac congenital abnormalities

city: Scarborough

first: Arturo

last: Hernandez

state: ME

address: 81 Research Drive

department: Center for Molecular Medicine

institute: Maine Medical Center Research Institute

summary: We examined the impact of thyroid hormone excess on the mouse fetal heart utilizing a model of deficiency in DIO3, the enzyme that is responsible for clearing thyroid hormones

overall_design: RNA sequencing profile of male fetal hearts at embryonic day E13.5

first: M

middle: E

last: Martinez


title: Comparison of gene expression profiles between precancerous cells of SCLC (preSC) and Wnt5a-transgenic preSC

city: Tampa

first: Youngchul

address: 12902 Magnolia Dr

institute: H. Lee Moffitt Cancer Center

summary: This comparision is to determine a global change in gene expression influenced by increased Wnt5a in preSC.

overall_design: mutant cell vs control

first: Kwon-Sik

last: Park

first: Kee-Beom


title: DNA methylation biomarkers for non-invasive detection of triple negative breast cancer using liquid biopsy

city: Heidelberg

first: Dieter

last: Weichenhan

address: Im Neuenheimer Feld

department: Cancer Epigenomics

institute: Deutsches Krebsforschungszentrum

type: Methylation profiling by genome tiling array

summary: Non-invasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple negative breast cancer (TNBC) which could help clinicians for easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls, and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified, and externally validated. A machine learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs  for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606, and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC=0.74 in test set and AUC=0.77 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as non-invasive liquid biopsy markers for diagnosis of TNBC.

overall_design: DNA methylation profiling of 123 TNBC tissues was performed at the DKFZ Genomics and Proteomics Core Facility (Heidelberg, Germany) using the Illumina Infinium HumanMethylation450K BeadChip (n=52) and the EPIC array (n=71) according to the manufacturer´s instructions.

first: Mehdi

last: Manoochehri

first: Ute

last: Hamann

first: Yassen

last: Assenov

first: Clarissa

last: Gerhäuser


title: Articular Fibrocartilage-Targeted Therapy by Microtubule Stabilization

city: Nanjing

first: Jiawei

last: Li

state: Jiangsu

address: Zhongshan road No.321

department: State Key Laboratory of Pharmaceutical Biotechnology, Division of Sports Medicine and Adult Reconstructive Surgery, Department of Orthopedic Surgery

institute: Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School Nanjing

summary: To investigate the fibrotic effect on BMSCs by CTGF and TGFβ1 treatment, and the anti-fibrotic effect of docetaxel on fibrotic chondrocytes. We established BMSCs primary culture and treated CTGF and TGFβ1, then treated by docetaxel.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for control Bone marrow-mesenchymal stem cells (BMSCs) and  fibrotic BMSCs (treated with CTGF and TGF-β1), induced fibrotic-chondrocytes (iFCs)and iFCs treated with docetaxel

first: Huiming

last: Jiang

first: Zhongyang

last: Lv

first: Dongquan


title: Mineralocorticoid Receptor Antagonists and Glucocorticoids Differentially Affect Skeletal Muscle Inflammation and Pathology in Muscular Dystrophy

city: Columbus

first: Jill

last: Rafael-Fortney

state: OH

address: 370 W. 9th Avenue

department: Physiology and Cell Biology

institute: The Ohio State Universtiy

summary: We report the RNA Sequencing of myleoid cells isolated from 4.5-week-old mdx mice that were administered spironolactone, prednisolone, or vehicle for 7 days. We find that both spironolactone and prednisolone downregulated expression of genes involved in inflammation and fibrosis, while prednisolone further perturbed cell cycle genes.

overall_design: Single cell suspensions were prepared from quadriceps from 4.5-week-old mdx mice treated with spironolactone, prednisolone, or vehicle for 7 days. Cells from 3 pairs of mdx quadriceps were pooled for each biological replicate. Myeloid cells were isolated and RNA was extracted from myeloid cells.

first: Zachary

middle: M

last: Howard

first: Chetan

last: Gomatam

first: Charles

middle: P

last: Rabolli

first: Jeovanna

last: Lowe

first: Shyam

last: Bansal

first: Federica

last: Accornero

middle: A


title: The single cell atlas of human endometrial stromal cells

city: RTP

first: Franco

last: Demayo

state: NC

address: 111 TW Alexander Drive

institute: NIEHS

summary: Primary human endometrial stromal cells (HESCs) from three patients and the Telomerase-immortalized endometrial stromal cell line (THESC) were cultured in the cluture medium and collected for scRNA-Seq. 13 distinct cell clusters were identified among all the cells, which not only reflect the differences between the four samples, but also the heterogeneity within each sample divided into mature, proliferative and active fibroblast. The conserved transcriptomic changes from the three primary HESC were observed in comparison to the THESCs. Cell clustering with batch correction further indicate that the intrinsic cellular distribution were similar among all the primary HESCs and THESCs. These intrinsic cell clustering showed varaiable stages of cell cycle and a cell development lineage between proliferative and mature or active fibroblast.

overall_design: Primary human endometrial stromal cells (HESCs) from three patients and the Telomerase-immortalized endometrial stromal cell line (THESC) were cultured in the culture medium and collected for scRNA-Seq.

first: Rong

first: Tianyuan

last: Wang

first: Xin

last: Xu

first: San-Pin

last: Wu

first: Francesco

last: DeMayo


title: Olig2 ablation in immature oligodendrocytes does not enhance CNS myelination and remyelination

city: Cincinnati

first: Richard

last: Lu

address: 3333 Burnet Ave

department: CBDI

institute: Cincinnati Children's Hospital Medical Center

summary: The oligodendrocyte (OL) lineage gene Olig2 persistently expresses throughout oligodendroglial development and required for oligodendroglial specification and differentiation. Here,  Together, by leveraging multiple immature OL-expressing Cre lines at different stages, we demonstrate that Olig2 is required for differentiation and myelination of immature OL and myelin repair and raise fundamental questions about the previously proposed role for Olig2 in opposing OL myelination, while highlighting the importance of using Cre-dependent reporter for lineage tracing in studying cell fate transition.

overall_design: 3 Control Samples and 3 Mutant

first: Jiajia

first: Lijun

last: Yang

first: Qing


title: Single cell RNA sequence of liver tumors in hepatocyte-specific Kras mutant mice.

city: Suita

first: Yuki

last: Makino

state: Osaka

address: 2-2, Yamadaoka

department: Department of Gastroenterology and Hepatology

country: Japan

institute: Osaka University Graduate School of Medicine

summary: Tumor consisting cells in hepatocyte-specific Kras mutant mice were classified as tumor cells and stromal cells including macrophages, hepatic stellate cells, endothelial cells, and so on. Positive cells for STAT3-target genes, Il-6 family cytokines, and connective tissue growth factor were elucidated.

overall_design: Two liver tumors in hepatocyte-specific Kras mutant mice (Alb-Cre +/- Kras G12D/+) were dissociated into a single-cell suspension and subjected to single cell RNA-sequence (scRNA-seq) to elucidate the mRNA expression profile of tumor-consisting cells.

first: Hayato

last: Hikita

first: Seiya

last: Kato

first: Masaya

last: Sugiyama

first: Minoru

last: Shigekawa

first: Takahiro

last: Kodama

first: Ryotaro

last: Sakamori

first: Nobuyuki

last: Takemura

first: Norihiro

last: Kokudo

first: Hideki

last: Yokoi

first: Masashi

last: Mukoyama

first: Tomohide

last: Tatsumi

first: Tetsuo

last: Takehara


title: Landscape of immune-related signatures induced by targeting of epigenetic regulators in melanoma III

city: Milan

first: Andrea

last: Anichini

address: Via Venezian 1

department: Research

country: Italy

institute: Fondazione IRCCS Istituto Nazionale dei Tumori

summary:
Epigenetic drugs exert a wide range of immune-related effects, but have strong drug-specific heterogeneity in immunomodulation, thus hampering selection of the most promising agent for innovative cancer immunotherapy approaches. Here we identified immune-related signatures induced by four classes of epigenetic drugs in melanoma cells to define the most active agent and to understand its biological activity in -vitro, in a pre-clinical model and in clinical samples. Gene modulation, induced by inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), bromodomain and extraterminal domain proteins (JQ1 and OTX-015) and enhancer of zeste homolog 2 (GSK126), was assessed in human melanoma cell lines. All drugs modulated genes belonging to 20 families. Guadecitabine, followed by givinostat, was the most active drug and upregulated >160 immune-related genes characterized by low expression and high methylation. JQ1 and OTX-015 showed predominant inhibitory effects, GSK126 was the least active. A dominant immunomodulatory effect of guadecitabine and JQ1 was observed in combinatorial treatments.
This experiment deals with the characterization of gene expression in tumor nodules removed from immunodeficient mice treated or not with guadecitabine (as described in Covre A et al. Seminars in Oncology 2015;42:506-513) after s.c. tranplant of human melanoma  cells from two cell lines 313 and 195 (cell lines described in Coral S et al. J. Cell Physiol 2006;207:58-66).

overall_design: Whole genome gene expression was assessed by Clariom S arrays in two melanoma cell lines treated or not with guadecitabine and in 20 tumor nodules removed from immunodeficient mice injected s.c. with such cell lines and treated or not with guadecitabine.

first: ANDREA

last: ANICHINI


title: Landscape of immune-related signatures induced by targeting of epigenetic regulators in melanoma IV

summary: Epigenetic drugs exert a wide range of immune-related effects, but have strong drug-specific heterogeneity in immunomodulation, thus hampering selection of the most promising agent for innovative cancer immunotherapy approaches. Here we identified immune-related signatures induced by four classes of epigenetic drugs in melanoma cells to define the most active agent and to understand its biological activity in -vitro, in a pre-clinical model and in clinical samples. Gene modulation, induced by inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), bromodomain and extraterminal domain proteins (JQ1 and OTX-015) and enhancer of zeste homolog 2 (GSK126), was assessed in human melanoma cell lines. All drugs modulated genes belonging to 20 families. Guadecitabine, followed by givinostat, was the most active drug and upregulated >160 immune-related genes characterized by low expression and high methylation. JQ1 and OTX-015 showed predominant inhibitory effects, GSK126 was the least active. A dominant immunomodulatory effect of guadecitabine and JQ1 was observed in combinatorial treatments.

overall_design: Gene modulation in melanoma cell lines treated with epigenetic drugs was assessed by NanoString nCounter PanCancer Immune profiling panel. The following cell lines were evaluated for immune-related gene modulation by Guadecitabine, Decitabine, Givinostat, JQ1, GSK126 and an unrelated drug abemaciclib: VRG100, BRM17, FRN39, CST30, BNV13, MNT59, GRD43, GML41, CPN20, SLN91.  The following six cell lines were treated also with OTX-015: BRM17, VRG100, GRD43, FRN39, CPN20 and SLN91. The following six cell lines were also treated with combinations of the epigenetic drugs: GRD43, BNV13, FRN39, CPN20, SLN91 and MNT59. Overall the experiment includes 107 arrays.


title: Landscape of immune-related signatures induced by targeting of epigenetic regulators in melanoma II

summary:
Epigenetic drugs exert a wide range of immune-related effects, but have strong drug-specific heterogeneity in immunomodulation, thus hampering selection of the most promising agent for innovative cancer immunotherapy approaches. Here we identified immune-related signatures induced by four classes of epigenetic drugs in melanoma cells to define the most active agent and to understand its biological activity in -vitro, in a pre-clinical model and in clinical samples. Gene modulation, induced by inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), bromodomain and extraterminal domain proteins (JQ1 and OTX-015) and enhancer of zeste homolog 2 (GSK126), was assessed in human melanoma cell lines. All drugs modulated genes belonging to 20 families. Guadecitabine, followed by givinostat, was the most active drug and upregulated >160 immune-related genes characterized by low expression and high methylation. JQ1 and OTX-015 showed predominant inhibitory effects, GSK126 was the least active. A dominant immunomodulatory effect of guadecitabine and JQ1 was observed in combinatorial treatments.
This experiment deals with the characterization of gene modulation by epigenetic drugs (guadecitabine, givinostat, JQ1, GSK-126) and by the unrelated drug Abemaciclib in two melanoma cells lines CST30 and VRG100

overall_design: Whole genome gene expression was assessed by Clariom S arrays in two cell lines CST30 and VRG100 treated or not with epigenetic drugs.


title: Landscape of immune-related signatures induced by targeting of epigenetic regulators in melanoma I

summary:
Epigenetic drugs exert a wide range of immune-related effects, but have strong drug-specific heterogeneity in immunomodulation, thus hampering selection of the most promising agent for innovative cancer immunotherapy approaches. Here we identified immune-related signatures induced by four classes of epigenetic drugs in melanoma cells to define the most active agent and to understand its biological activity in -vitro, in a pre-clinical model and in clinical samples. Gene modulation, induced by inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), bromodomain and extraterminal domain proteins (JQ1 and OTX-015) and enhancer of zeste homolog 2 (GSK126), was assessed in human melanoma cell lines. All drugs modulated genes belonging to 20 families. Guadecitabine, followed by givinostat, was the most active drug and upregulated >160 immune-related genes characterized by low expression and high methylation. JQ1 and OTX-015 showed predominant inhibitory effects, GSK126 was the least active. A dominant immunomodulatory effect of guadecitabine and JQ1 was observed in combinatorial treatments.
This experiment deals with the characterization of gene expression in untreated human melanoma cell lines used throughout the project.

overall_design: Whole genome gene expression was assessed by Clariom S arrays in 23 human melanoma cell lines kept in-vitro in exponential growth phase in RPMI 1640 medium supplemented with 5% FCS.


title: The dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia [ribosome profiling]

first: Christian

last: Rohde

address: Im Neuenheimer Feld 410

institute: Heidelberg University

summary: We performed ribosome profiling to determine  the effect of FBL knockdown on mRNA translation in human leukemai cell Kasumi-1.

overall_design: Kasumi-1 cells stably expressing doxcyxycline inducible control or FBL specific shRNA were used. Cells were treated with 100 ng/ml doxcyxycline for three days to induce the expression of shRNA. Cells were then collected for Ribosome profiling.

first: Fengbiao

last: Zhou

first: Carsten

last: Müller-Tidow


title: The dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia [ribomethseq_FBL_KD]

summary: We investigated the effect of FBL and SNRD127 on rRNA 2'-O-Me in human leukemai cell Kasumi-1.

overall_design: FBL and SNORD127 were  knocked down or knocked out from leukemia cell line Kasumi-1. Then we re-expressed wildtype, mutant FBL, SNORD127 or the empty control in the knockdown/knockout cells.


title: lncRNA and mRNA profiles show difference in Tetralogy of Fallot

city: Suzhou

first: Xinxin

last: Yan

address: 242 Guangji Road

department: Department of Pharmacy

institute: Affiliated Suzhou Hospital of Nanjing Medical University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: In this study,compared with normal group, there were 94 differentially expressed lncRNAs and 83 mRNA transcripts in TOF (P<0.05). Correlation analysis between lncRNA and mRNA further showed that differentially expressed lncRNA can be linked to mRNAs, suggesting the regulator role of lncRNA in mRNA expression

overall_design: In this study, we characterize differentially expressed lncRNAs and mRNAs through sequencing analysis of 3 pairs of fetal normal myocardial tissues and fetal myocardial tissue of TOF.

first: Zhongshan

last: Gou


title: The dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia [ribomethseq_primary_samples]

summary: Eukaryotic ribosomal RNA carries diverse posttranscriptional modifications, the function of which are mostly unexplored. The evolutionarily conserved 2’-O-methylation (2’-O-Me) occurs at more than 100 sites and is essential for ribosome biogenesis. Plasticity of 2´-O-Me in ribosomes and its functional consequences in human disease remain to be elucidated. We performed RiboMethSeq to  estabolish the full rRNA 2’-O-Me landscape (ribomethylome) in human acute myeloid leukemia (AML) through profiling 94 patient samples as well as 21 normal hematopoietic samples of 5 different lineages.  The gene expression in the same samples was determined by RNA-Seq.  We found that higher 2’-O-methylation on exterior dynamic sites associated with leukemia stem cell (LSC) signatures.

overall_design: 94 primary AMLs and 21 normal hematopoeitic cells of 5 different lineages was used for RiboMethSeq. The gene expression profiling in the same samples was determined by RNA-Seq. The association of rRNA 2’-O-Me pattern was confirmed with second independent AML cohort.


title: RNA-seq data on Wild type mouse and DhhCre;Nf1fl/fl natural history

first: Kwangmin

last: Choi

state: Ohio

address: 333 Burnet Avenue

department: Experimental Hematology & Cancer Biology

overall_design: RNA-seq

first: Nancy

last: Ratner

first: Jianqiang

last: Wu

first: Mitch

last: Springer


title: The dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia [mRNA_primary_samples]

summary: Eukaryotic ribosomal RNA carries diverse posttranscriptional modifications, the function of which are mostly unexplored. The evolutionarily conserved 2’-O-methylation (2’-O-Me) occurs at more than 100 sites and is essential for ribosome biogenesis. Plasticity of 2´-O-Me in ribosomes and its functional consequences in human disease remain to be elucidated. We performed RiboMethSeq to  estabolish the full rRNA 2’-O-Me landscape (ribomethylome) in human acute myeloid leukemia (AML) through profiling 94 patient samples as well as 21 normal hematopoietic samples of 5 different lineages.  The gene expression in the same samples was determined by RNA-Seq. We found that higher 2’-O-methylation on exterior dynamic sites associated with leukemia stem cell (LSC) signatures.

overall_design: The gene expression profiling in the 90 AML samples was determined by RNA-Seq. The association of rRNA 2’-O-Me pattern with gene expression signature was investigated.


title: Multiple Nf1 Schwann cell populations reprogram the plexiform neurofibroma tumor microenvironment

address: 3333 Burnet Avenue

summary: To define alterations early in tumor formation, we studied nerve tumors in neurofibromatosis 1 (NF1), a tumor predisposition syndrome in which affected individuals develop plexiform neurofibroma (PN). These benign tumors are driven by NF1 loss in Schwann cells (SC). By comparing normal nerve cells to PN cells using single cell and bulk RNA sequencing, we identified changes in five SC populations, including a de novo Schwann cell progenitor-like population. Long after Nf1 loss, SC populations developed PN-specific expression of Dcn, Postn, and CD74, and showed dramatic expansion of immune and stromal cell populations; in human PN, immune and stromal cells comprised 90% of cells. Label transfer enabled verification of each SC population and predicted tumor unique patterns of cell-cell communication in each SC population. We identified PROS-AXL, FGF-FGFR, and MIF-CD74 and its effector pathway NFkB as deregulated in NF1 SC populations, including SCP-like cells. Each receptor-ligand pair was predicted to influence surrounding cells in mouse and human. These findings highlight remarkable changes in all PN cells driven by loss of NF1 in multiple types of SCs and identify therapeutic targets for PN.

overall_design:
[Mouse] single cell mRNA profiles of paraspinal tumors (2 or 7 mo. Dhh-Cre;Nf1fl/fl) (n=6), and age-matched wild type dorsal root ganglia (DRG) with nerve roots

[Human] single cell mRNA profiles of human plexiform neurofibromas (n=4)
>>> Submitter declares that the human raw data will be deposited in dbGAP due to patient privacy concerns. <<<

first: Leah

last: Kershner

first: Xiyuan

last: Zhang

first: Melissa

last: Perrino

first: Nathan

last: Salomonis

first: Jack

middle: F

last: Shern


title: RNA profiling of Col-0 and vil1-1 1-day-old seedlings in Arabidopsis

city: Austin

first: Wei

last: Zong

state: Texas

department: Molecular Biosciences

institute: University of Texas at Austin

summary: 1-day-old seedlings of Col-0 and vil1-1 were performed  RNA-Seq to identify differentially expressed genes caused by VIL1 mutation in Arabidopsis

overall_design: 6 samples from Col-0, vil1-1 1-day-old seedlings grow under long day condition with three biological replicates

first: Sibum

last: Sung


title: The dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia

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

overall_design: Refer to individual Series


title: A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia [10X Genomics]

city: Cambridge

first: Liming

last: Tao

address: 415 Main Street

institute: Broad Institute of Harvard and MIT

summary: We uncover a key transcriptional network involving hundreds of genes that is required for HSC self-renewal.

overall_design: MECOM impact on the hematopoietic stem cells

first: Richard

last: Void

first: Fulong

last: Yu

first: Vijay

last: Sankaran

first: Regev

last: Aviv


title: The dynamic rRNA ribomethylome drives stemness in acute myeloid leukemia [mRNA_FBL_KD]

first: Christian

last: Rohde

summary: Eukaryotic ribosomal RNA carries diverse posttranscriptional modifications, the function of which are mostly unexplored. The evolutionarily conserved 2’-O-methylation (2’-O-Me) occurs at more than 100 sites and is essential for ribosome biogenesis. Plasticity of 2´-O-Me in ribosomes and its functional consequences in human disease remain to be elucidated.  The effect of rRNA 2’-O-Me on protein translation was investigated by nascent proteomcis analysis coupled with RNA-Seq in human leukemia cell line Kasumi-1 with and without knockdown of 2´-O-methyltransferase FBL.

overall_design: Human AML cell line expressing doxycycline inducible control shRNA (shLUC) or FBL specific shRNA (shFBL) was used. The cells were treated with 100 ng/ml doxycycline for 72 hours for FBL knockown. The newly newly synthesized proteins and total RNA were isolated from the control and FBL knockdown cells. Total RNA was submitted to RNA-Seq and nascent proteins were used for mass spectrometry proteomics. The changes in nascent proteins were normalized by mRNA alteration.

first: Fengbiao

last: Zhou

first: Carsten

last: Müller-Tidow


title: Single cell sequencing of gastric cancer and adjacent normal tissues

city: Jining

first: Rou

last: Zhao

address: 89 Guhuai Road

institute: Affiliated Hospital of Jining Medical College

summary: In this study, we used single-cell RNA sequencing (scRNA-seq) to further elucidate the composition of the gastric cancer and adjacent normal tissue microenvironment and found that TNF and ACTG1 may be key regulators of regulatory T cells (Tregs) in gastric cancer. A total of seven cell types were identified, including B cells, T cells, NK cells, mast cells, fibroblasts, endothelial cells, and epithelial cells. T cells were further divided into 8 subgroups. Among them, Tregs showed the greatest difference between GC and adjacent normal tissues. Next, we clarified the function and signaling pathway of Tregs in the GC tumor microenvironment, obtained the key regulatory proteins TNF and ACTG1, and explored their clinical significance and possible effects on Tregs in the GC tumor microenvironment. This scRNA-seq study may reveal the composition of tumor microenvironment and clarify the role of Tregs in the development of gastric cancer, providing new methods for GC treatment.

overall_design: The cancer tissue sample was from a GC patient with untreated, primary, nonmetastatic gastric tumor who underwent GC resection. Adjacent normal gastric tissue was collected more than 5 cm from the carcinoma tissue.

first: Bin

first: Qingli

last: Bie


title: A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia [smart-seq2]

summary: we uncover a key transcriptional network involving hundreds of genes that is required for HSC self-renewal.


title: Comprehensive Analysis of a Competing Endogenous RNA Network Identifies Five-lncRNA Nomogram as a Prognostic Model for Cervical Cancer

status: Public on Nov 22 2022

city: Taiyuan

first: Haixia

last: Luo

address: 382 Wuyi Road

institute: The Second Hospital of Shanxi Medical University

summary:
LncRNAs have been found to regulate mRNA expression levels and serve an important role in cervix carcinogenesis.
To explore the lncRNA expression profiles during the development and progression of cervical cancer, we performed microarray analysis with total RNA in with only Human papillomavirus (HPV) 16-positive, 7 stage IA-IIA SCC (following the International Federation of Gynecology and Obstetrics (FIGO) criteria (10)) and 7 matched normal controls (NC).

overall_design: In the study presented here, we designed to analyze the mRNA and lncRNA expression in  normal cervical epithelium(n=7),and HPV16 positive cervical squamous cell carcinoma tissues(n=7).

last: Wang

first: Yuanxing

last: Li

first: Xiaochen

last: Niu


title: Whole-Genome Bisulfite Sequencing of Nasopharyngeal Carcinoma and Nasopharyngeal Epithelial Tissues [WGBS]

city: Hong Kong

first: Ka Yue Larry

last: Chow

address: 21 Sassoon Road

department: Clinical Oncology

country: Hong Kong

institute: The University of Hong Kong

summary: Nasopharyngeal carcinoma (NPC) is enedemic in Southeast Asia but is uncommon worldwide. In Hong Kong, approximately 95% of NPC cases are associated with Esptein-Barr Virus (EBV). EBV has been shown to induce changes in the epigenetics of EBV-malignancies. Epigenetics in NPC may thus play an important role in NPC pathogensis. We performed whole-genome bisulfite sequencing (WGBS) to profile the methylation changes in NPC and investigate the role of abberant methylation pattern in NPC.

overall_design: Bisulfited-converted DNA of adjacent non-cancerous tissues from nine patients was pooled into three independent libraries for sequencing. Bisulfite-converted DNA of NPC tissues from 15 NPC patients was independently sequenced.

first: L

first: W

last: Dai

first: M

middle: L

last: Lung


title: Whole-Genome Bisulfite Sequencing of Nasopharyngeal Carcinoma and Nasopharyngeal Epithelial Tissues


title: Transcriptome analysis of pine wood decaying mechanism of Sparassis latifolia

city: Fuzhou

first: Chi

last: Yang

address: No.15 Shangxiadian Road, Cangshan District

institute: Fujian Agriculture and Forestry University

summary: To explore the pine wood decaying mechanism of Sparassis latifolia, we use RNA-seq to detect the gene expression.

overall_design: The cultured mycelium of Sparassis latifolia were treated by pine wood for different time, 0 h, 1 h, 6 h, 1 d, 5 d, and 10 d.

first: Lu

last: Ma

first: Yanquan

last: Lin


title: miRNAs expression profiling analysis of exosomes from serum of C57BL/6 mice infected with or without P. berghei ANKA (namely inf-Exos or un-Exos)

city: Guangzhou city

first: Bo

last: Huang

state: Guangdong Province

address: No.280, East Ring Road

institute: Guangdong Pharmaceutical University

summary:
To explore the potential exosomal miRNAs regulating macrophage M1 polarization,  we isolated and characterized inf-Exos or un-Exos, ad the miRNA profiling between inf-Exos and un-Exos was then compared by using miRNA-seq.
We then performed miRNAs expression profiling analysis using data obtained from RNA-seq of inf-Exos (n = 5) and un-Exos  (n = 5)

overall_design: Comparative miRNAs expression profiling analysis of inf-Exos (n = 5) and un-Exos  (n = 5)

first: Wenbing

last: Liu


title: TAp63 in pancreas cancer predicts the squamous subtype and promotes a suppressive immune micromilleu

city: Suwon

first: Su Bin

last: Lim

state: Gyeonggi-do

address: Worldcup-Ro 164, Yeongtong-Gu

country: South Korea

institute: Ajou University School of Medicine

summary: To evaluate the role of aberrant TAp63 in PDAC, we analyzed transcriptomics by RNAseq in the TAp63 and deltaNP63 over-expressing pancreatic cancer cells.

overall_design: A transcriptomic analysis by over-expression of TAP63 and deltaNP63  in the pancreatic cancer cells

first: Seokhwi

last: Kim

first: Su

first: Dakeun

last: Lee


title: A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia [amplicon-seq]

first: Liming

last: Tao

first: Richard

last: Void

first: Fulong

last: Yu

first: Vijay

middle: G

last: Sankaran

first: Regev

last: Aviv


title: T-cell receptor repertoire in rectal cancer

city: Koto-ku

first: Osamu

last: Gotoh

state: Tokyo

address: Ariake 3-8-31

institute: Japanese Foundation For Cancer Research

summary: Emerging evidence suggests that an increased density of pre-treatment CD8+ tumor-infiltrating lymphocytes (TILs) is associated with good response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer. However, the significance of T-cell complexity in the clinical setting remains unknown. High-throughput T-cell receptor (TCR) β sequencing was applied to quantify the TCR repertoire of pre-treatment biopsies from 67 patients with advanced rectal cancer receiving preoperative CRT. Changes in TCR repertoire before and after CRT were also analysed in 23 patients.

overall_design: T-cell receptor repertoire was analyzed using total RNA (pre-treatment: 67 samples; post-treatment: 23 samples), according to the manufacturer’s instructions.

first: Takashi

last: Akiyoshi

first: Seiichi

last: Mori


title: Cell atlas of mouse colon tissues of liver cirrhosis mouse

city: Jinan

first: Jing

last: Shen

address: Wenhua West Road 44

institute: Shandong University

summary: Liver disease alters the gut microenvironment by liver-gut axis. To investigate the composition and transcriptome changes of various intestinal cell populations in liver cirrhosis, we delineated a single-cell atlas of the colon from mice treated CCl4 for 6 weeks.

overall_design: 6-week-old SPF mice were treated with CCl4 for 6 weeks, and the mild-colon tissues were harvested for single-cell RNA sequencing.

first: Zhen

last: Li

first: Xiaoyu

first: Shiyang


title: Targeting cell cycle and apoptosis to overcome chemoresistance in AML

city: Brisbane

first: Jasmin

last: Straube

state: QLD

address: 300 Herston Rd

country: Australia

institute: QIMR Berghofer

summary: A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockout of cyclin dependent kinase inhibitor 2A (CDKN2A) as contributing to resistance. To investigate the therapeutic potential of upregulating CDKN2A expression using WM-1119 and further elucidate its mechanism of action, we performed RNA-seq on WM-1119- (or vehicle control) treated OCI-AML3 cells with or without CRISPR mediated genetic depletion of CDKN2A.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for OCI-AML3 cells with and without CDKN2A depletion treated with WM-1119 at 1 uM or vehicle control for 7 days.

first: Victoria

middle: Y

last: Ling

first: Steven

middle: W

last: Lane


title: RNA-seq of control and FK866-treated ILC2s in vitro

summary: To investigate the effect of NAD+ metabolism on ILC2s function, sorted ILC2s from the gut were treated with NAMPT inhibitor  FK866 for bulk RNA sequcecing.

overall_design: Sorted ILC2s from the gut were cultured with IL-2, IL-7, IL-25 and IL-33, and treated with NAMPT inhibitor  FK866  for 16 hours.

last: Liu


title: Secondary infections regenerate the intestinal CD103+ tissue-resident memory T-cell pool

city: Freising

first: Gustavo

middle: P.

last: de Almeida

state: Bavaria

address: Liesel-Beckmann-Str. 1

department: Division of Animal Physiology and Immunology

institute: Technical University of Munich

summary: Using a novel fate-mapping approach, we identified that resident intestinal CD103 expressing T-cells lack secondary expansion potential and are replaced by de novo formed cells during re-infection.

overall_design: mRNA profiles of small intestine lamina propria transferred INTEGRA CD8 T cells that either express CD103 and the stable fluorescent marker zsGreen (DP) or that do not express CD103 or zsGreen (DN). The cells were sampled without reinfection, day 2 and day 8 after either VSV-OVA or VSV-wt reinfection.

first: M

last: von Hoesslin

last: Kuhlmann

first: G

first: D

last: Zehn


title: Effect of Nrf2 knockout on gene expression in mouse bone marrow stromal/stem cells

city: Nanjing

first: Renlei

last: Yang

address: Hanzhong Road 146

institute: Nanjing medical university

summary: To investigate novel downstream targets of Nrf2 in BM-MSCs, we isolated BM-MSCs from 8-week-old WT and Nrf2 knockout mice and performed gene expression profiling analysis using data obtained from RNA-seq of WT and Nrf2-deficient BM-MSCs.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT and Nrf2-deficient BM-MSCs derivatives.

first: Jie

last: Zhang

first: Dengshun

last: Miao


title: Whole-Genome Bisulfite Sequencing of Nasopharyngeal Carcinoma and Nasopharyngeal Epithelial Tissues [SeqCapEpi]

first: Ka Yue Larry

last: Chow

summary: Nasopharyngeal carcinoma (NPC) is enedemic in Southeast Asia but is uncommon worldwide. In Hong Kong, approximately 95% of NPC cases are associated with Esptein-Barr Virus (EBV). EBV has been shown to induce changes in the epigenetics of EBV-malignancies. Epigenetics in NPC may thus play an important role in NPC pathogensis. We performed targeted bisulfite sequencing to accurately profile the methylation changes in NPC and investigate the role of abberant methylation pattern in NPC.

overall_design: EpiTect Bisulfite Kit (Qiagen) was used for bisulfite conversion of DNA. SeqCapEpi Enrichment System (Roche) was used for enriching regions of interest.

first: L

first: W

last: Dai

last: Lung


title: Screening of differentially expressed microRNAs in MSCexo and TSA-MSCexo

city: Tianjin

first: Sheng

state: Xiqing District

institute: The First Teaching Hospital of Tianjin University of Traditional Chinese Medicine

summary: To explore how TSA-MSCexo improves myocardial ischemia-reperfusion injury, miRNA microarray analysis was used to screen differentially expressed miRNAs in MSCexo and TSA-MSCexo.

overall_design: Three samples of MSCexo and TSA-MSCexo, respectively. Three independent experiments were performed.

first: Lan

first: Weilong

last: Cao

first: Rui

last: Guo

first: Zhihao

first: Jing

first: Qianqian

first: Yuting

last: Huang

first: Chuanrui

last: Ma

first: Guanwei

last: Fan


title: Spiral ganglion subtype specification occurs prior to birth in the mouse

city: Bethesda

first: Robert

last: Morell

state: MD

address: 35A Convent Drive, Room 1F-103

institute: National Institute on Deafness and Other Communication Disorders

summary: Little is known about the timing and factors which regulate the specification of neuronal subtypes in the cochlear spiral ganglion. Here we collect Spiral Ganglion Neurons (SGNs) from the mouse across four developmental timepoints (E14, E16, E18 and P1) in order to transciptionally profile their development.

overall_design: We dissected mouse cochleas at E14,E16, E18 and P1, removing as much non-neuronal tissue as possible, and pooling samples from 5 different embryos/pups. The tissue was dissociated and single SGNs captured and transcriptionally profiled using 10x Chromium  and Illumina sequencing platforms. At E16 the spiral ganglion was cut in half and collected in apical and basal sections. Three individuall biological replicates were collected at each age, except P1 where 2 replicates were collected

first: Tessa

middle: R

last: Sanders

first: Matthew

last: Kelley


title: Genome wide identification of BysR binding sites in Burkholderia sp. JP2-270

city: Hangzhou

first: Lijuan

last: Wu

state: Zhejiang

address: 28 Shui Dao Suo Road, Fuyang District

institute: China national rice research institute

summary: To verify genes that are directly regulated by BysR, we used DNA affinity purification sequencing analysis (DAP-seq) for genome-wide recognition of BysR binding sites in vitro. The HALO-fusion BysR protein was successfully expressed and purified. After affinity purification and sequencing, at least 22 million double-end reads per sample were generated and with > 99 % of reads uniquely mapped to the JP2-270 genome. A total of 470 enriched common peaks of two replicates with –log10(P-value) ≥ 2 were called . The mean width of DAP-seq peaks was < 1,000. In total, 367 (78%) of these peaks were found to locate in the -1 kbp to 1 kbp regions by the analysis of peak summit positions relative to the start codons of JP2-270 open reading frames.

overall_design: Identification of the binding sites of BysR in vitro in the genom of Burkholderia sp. JP2-270.


title: ChIP after EBF1 degradation

city: Freiburg

first: Nikolay

last: Zolotarev

address: Stübeweg, 51

department: Cellular and Molecular Immunology

institute: Max Planck Institute of Immunobiology and Epigenetics

summary: We used the dTAG protein degradation system to rapidly deplete transcription factor EBF1 in the B cell culture.  After treatment with dTAG-13 for 6 hours, we performed ChIP-seq, ATAC-seq, and RNA-seq. After the treatment, EBF1 is completely absent from the chromatin. This leads to the closure of EBF1 binding sites and changes in the target gene expression. This approach allowed us to identify direct targets of EBF1 and show the importance of EBF1 for open chromatin maintenance.

overall_design:
ChIP-seq with a-EBF1 (1C) antibody in EBF1-dTAG_C proB cells before and after 6 hours of treatment with dTAG-13.
ChIP-seq with a-Brg1 (ab110641) antibody in EBF1-dTAG_N proB cells before and after 6 hours of treatment with dTAG-13.
There are ChIP sample and input for each condition.


title: Tuning the 3D microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease

city: Zurich

first: Soeren

middle: S.

last: Lienkamp

address: Winterthurerstrasse 190

department: Institute of Anatomy

country: Switzerland

institute: University of Zurich

summary: Embedding cells in 3D environments can enhance their physiological properties and tissue architecture can be controlled using bioprinting approaches.  One cellular source for in vitro reconstituted kidney tubules are directly reprogrammed induced renal tubular epithelial cells (iRECs). Here, we investigated the compatibility of iRECs with different biomaterials as self assembly or bioprinting dependent guided growth approaches. We assess cell viability, morphology, their transcriptional changes and protein expression properties. iRECs showed high viability and biocompatibility with dispensing methods and bioinks. However, the morphology of multicellular aggregates was dramatically influenced by the microenvironment. Transcriptomic analyses revealed differentially expressed gene-signatures specific to each of the used biomaterials and to bioprinted tubule-like structures. In conclusion, we find that the 3D environment elicits a strong influence on the morphology and expression profiles of iRECs that unmasks disease phenotypes and can be harnessed for and tailored to experimental demands. Combining directly reprogrammed cells with the appropriate biomaterials will facilitate optimal construction of biomimetic kidney tubule and disease models at scale.

overall_design: iRECs were 2D cultured and at 3 timepoints (passage p35, p48 and p53) the experiment was performed as biological replicates. Experiment: harvest 2D iRECs and prepare 2D culture, 3D cultures (in Matrigel, Collagen I and Fibrin) and bioprinted (Collagen I, Fibrin) samples, in parallel from the same pool of cells. Renal Epithelial Growth Medium (REGM) was changed 3 times per week, until harvesting for RNA extraction and sequencing at day 7 in culture. Transcriptomic data analysis was performed to detect primary influences of the four microenvironments with respect to the control (2D plates iRECs).

first: Ludovica

last: Rizzo

first: Kevin

last: Troendle


title: EBF1 degradation in B Cells

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

overall_design: Refer to individual Series


title: An ancestral Wnt-Brachyury feedback loop and vertebrate-specific recruitment of mesoderm-determining target genes revealed by comparative Brachyury target screens

status: Public on Nov 30 2022

city: Vienna

first: Rohit

middle: Anil

last: Dnyansagar

address: Althanstrasse 14 (UZA1) A-1090 Wien

department: Department of Molecular Evolution and Development

country: Austria

institute: University of Vienna

summary: Transcription factor brachyury plays a key role during mesoderm development in vertebrates. In this study we study study the effect of this transcription factor in diploblastic cnidarian model organism N. vectensis

overall_design: Brachyury binding sites were identified with ChIP-seq and effect of brachyury binding on gene expression was studied with RNA-seq

first: Michaela

last: Schwaiger

first: Carmen

last: Andrikou

first: Ulrich

last: Technau


title: Characterization  of normal fibroblast transcriptomes upon transient silencing of LTBP2 by RNAseq

city: Berlin

first: Ambre

middle: Manon

last: Giguelay

state: Berlin

address: Hannoversche Str. 28

department: Cell Biology/Single Cells

institute: Berlin Institute for Health

summary:
Purpose: Our single-cell RNAseq data show that LTBP2 is a marker of a cancer associated fibroblast subpopulation in colorectal cancer liver metastases. Moreover, MTT assays of cultured  fibroblasts treated with anti-LTBP2 antibodies suggest that LTBP2 is essential for their survival. The goal of this study is to understand the role of LTBP2 in fibroblasts.
Methods: Normal olon fibroblasts (CCD18Co) were transfected 48h with siRNA containing an anti-LTBP2 or a non target sequence. Cell monolayer total RNA of 3 biological replicates was then extracted and submitted to Illumina RNAseq using NovaSeq6000.
Results: Transcriptomics revealed massive changes in gene expression when LTBP2 is knocked down: 496 genes were differentially expressed between the colon fibroblasts treated with siRNA anti-LTBP2 and NT. Those genes are implicated in diverse biological processes such as extracellular matrix organization, angiogenesis or cell adhesion.
Conclusions: Our study explored LTBP2 functions in normal fibroblasts. RNAseq data support its important role. After 48h, LTBP2 silencing induces changes in gene expression of numerous genes involved in fibroblast functions.

overall_design: Total RNA profiles of normal colon fibroblasts (CCD18Co) 48 hours after transfection with siRNA anti-LTBP2 or non target sequence (siNT)

first: Jacques

last: Colinge

first: Andrei

last: Turtoi


title: A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia

city: Cambridge

first: Liming

last: Tao

address: 415 Main Street

institute: Broad Institute of Harvard and MIT


title: Changes in gene expression after EBF1 degradation

overall_design: Stranded RNA-seq of total RNA in EBF1-dTAG_N proB cells before and after 6 hours of treatment with dTAG-13. There are 4 biological replicates for each condition. Reads were counted for exons and genes to obtain intronic counts.


title: Construction of a T cell receptor signaling range for spontaneous development of autoimmune disease

city: Suita

first: Mara

middle: Anais

last: Llamas-Covarrubias

address: 3-1 Yamadaoka

department: Department of Genome Informatics

institute: Osaka University


title: RNA-seq analysis of wild type and ΔbysR in Burkholderia sp. JP2-270

summary: Purpose: In order to identify the differentially expressed genes between wild type and ΔbysR, we performed RNA-seq analysis.Methods：All the isolates were cultured in CM medium for 24 h at 28℃. Three repetitions were performed for each treatment. The samples were sequenced on an Illumina Hiseq 2000 platform.Clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Results：A total of 9-13 million clean reads were obtained per replicate, each read with an average length of 150 bp. The square (R2) of Pearson correlation coefficient is greater than 0.903 between samples (supplementary Fig. S1) indicated that the data had a good reproducibility. Differential expression analysis of samples from WT and ΔbysR was performed, and revealed 350 differentially expressed genes (DEGs) consisting of up-regulated (302) and down-regulated genes (48) (with p-adj<0.05, |log2(FoldChange)|> 1) in ΔbysR compared to the WT.

overall_design: Total mRNA profiles of 24 h old wild type (WT) and ΔbysR of Burkholderia sp. JP2-270


title: Tconv and Treg gene expression and repertoire analysis of WT mice

summary: Single cell VDJ and RNAseq of Tconv and Treg cells obtained from BALB/c FoxP3-GFP reporter mice

first: Atsushi

last: Tanaka

first: Daron

last: Standley


title: ATAC-seq after EBF1 degradation

overall_design: ATAC-seq in EBF1-dTAG_N and _C proB cells before and after 6 hours of treatment with dTAG-13. There are 2 biological replicates for each condition.


title: p63/p73 maintain cancer stem cells in a quasi-mesenchymal state and drive metastatic colonization via EGFR signaling [ChIP-Seq]

first: Matt

last: Guenther

state: MA

address: 35 Cambridge Park Dr 4th floor

institute: Syros Pharmaceuticals

summary: p63/p73 are master transcriptional regulators of ITGB4-hi cancer stem cells and are required to maintain CSCs in a quasi-mesenchymal state and for metastatic colonization

overall_design: ChIP-seq of SUM159 ITGB4-hi or SUM159 ITGB4-low cells. ChIP-seq of rescue experiment in ITGB4-hi cells with knockout of p63/p73 (double KO, dKO).  dKO cells were engineered to ectopically express a control empty vector (EV), dNp63, TAp73, or both dNp63/TAp73 (dual).  ITGB4-lo cells were engineered to express a control empty vector (EV) or dNp63.

first: Arthur

last: Lambert

first: Chris

last: Fiore

first: Bruce

last: Wollison

first: Matthew

first: Robert

last: Weinberg


title: p63/p73 maintain cancer stem cells in a quasi-mesenchymal state and drive metastatic colonization via EGFR signaling [ATAC-Seq]

overall_design: ATAC-seq of SUM159 ITGB4-hi or SUM159 ITGB4-low cells.


title: Analysis of gene expression and polysome profiling in the presence and absence of glh-1 in C. Elegans

city: Bar Harbor

first: Joel

middle: H

last: Graber

state: ME

address: 159 Old Bar Harbor Rd

department: Bioinformatics Core

institute: MDI Biological Laboratory

summary: This experiment compares gene expression and polysome profiling between a strain where glh-1 is present - DUP64 glh-1(sam24[glh-1::gfp::3xFlag]) I and a strain where the glh-1 coding sequence has been deleted DUP144 glh-1(sam65[Δglh-1::gfp::3xFlag]) I

overall_design: For each of three replicates, 25 plates of each strain were used to inoculate 1L of S Media containing 5 grams of freeze-dried OP50 (LabTie, Leiden, The Netherlands).  Liquid cultures were incubated in 4 1L beveled flasks at 20° C until worms were gravid, at which point embryos were harvested, washed, bleach-treated (Montserrat et al. 2012) and hatched overnight. The synchronized L1-staged worms were used to inoculate another 1L of S Media with OP50 and were grown for approximately 40 hours until the majority reached the young adult stage, where they were harvested, washed, pelleted and flash frozen in 1-ml aliquots.

first: Jesse

last: Rochester

first: Catherine

last: Sharp

first: Nathaniel

middle: J

last: Maki

first: Dustin

last: Updike


title: p63/p73 maintain cancer stem cells in a quasi-mesenchymal state and drive metastatic colonization via EGFR signaling


title: Tuning the 3D-microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease.

first: Roman

last: Pichler

address: Hugstetter Str. 55

department: Nephrology

institute: University clinic Freiburg

summary: Mutations in PKD1 cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). To further investigate the impact of Pkd1 knockout on renal tubular cells, a direct reprogramming approach was applied. After direct reprogramming of mouse embryonic fibroblasts to induced renal tubular epithelial cells (iRECs), Pkd1 knockout iREC clones were generated by Cre-mediated recombination of floxed Pkd1 alleles. The knockout clones were compared to their corresponding wild type clones by RNA Sequencing and transcriptome profiling.

overall_design: Three knockout clones (Cre) were compared to their corresponding wild type clones (Flp): clone 4 Cre vs. Flp, clone 7 Cre vs. Flp, clone 21 Cre vs. Flp.

first: Ludovica

last: Rizzo

first: Soeren

middle: S

last: Lienkamp


title: Tconv and Treg gene expression and repertoire analysis of ZAC mice

first: Mara

summary: Autoimmune diseases, such as rheumatoid arthritis (RA), are believed to be caused, in part, by a defect in the selection of T cells. Developing T cells undergo a process known as “negative selection” where self-derived proteins are presented to the cells; those that react to the self-peptides are either eliminated, or develop into regulatory T cells (Tregs), which suppress immune responses by conventional T cells (Tconvs). In order to study self-reactive T cell receptors (TCRs), and their likely target antigens, we performed single cell repertoire analysis in ZAC mice with impaired TCR signaling. We sequenced TCR and transcriptomes of Treg and Tconv single cells from arthritic ZAC mice spleen (n=2). We discovered a cluster of pro-inflammatory Th17 Tconv cells in ZAC spleens that was absent in WT mice. ZAC Th17 TCRs showed clonal expansion. Different mice had different TCR sequences, which is expected.

overall_design: Single cell gene expresion and repertoire data from splenic Tconvs from two arthritic ZAC mice. These mice are also Foxp3 GFP reporter

middle: M


title: Tamoxifen induces cleft palate in mice

city: chengdu

first: Jue

last: Xu

address: 18# 3rd section, Renmin South Road

institute: Sichuan University

summary: Tamoxifen (TAM), a widely-used drug in treating breast cancer, has been reported to be associated with craniofacial defects including micrognathia and cleft palate in humans. However, the exact effects of TAM on the developing palate remain unclear. In the present study, we conclude that excess TAM exposure causes cleft palate defect in mice by regulating MAPK pathways, which implicates the importance of tightly regulated MAPK signaling in palate development and provides a basis for further exploration of the molecular etiology of cleft palate defects caused by environmental factors.

overall_design: The anterior and posterior portions of the palatal shelves were dissected out from E13.5 TAM-treated mice and controls (n = 3 for each group), respectively, and subjected to total RNA extraction (RNeasy Micro Kit, cat. 74004; Qiagen). The quality and quantity of RNAs were determined by using a Qubit 2.0 Fluorometric Quantitation system (Life Technologies). Finally, the RNAs were subjected to RNA-Seq analysis. Gene Ontology and pathway categories were grouped according to functional theme and proportion of cases that were affected by individual pathway alterations was plotted per subgroup, and across series.

first: Zhen

last: Huang

first: YiPing

last: Chen


title: ANT2 accelerates cutaneous wound healing in aged skin by reverse senescence

status: Public on Nov 21 2022

city: Daegu

first: Ji Hwan

last: Park

state: Gyeongbuk

address: 333, Techno jungang-daero, Hyeonpung-eup, Dalseong-gun

department: Newbiology

institute: DGIST

summary: An effective healing response  is critical to healthy aging. Thus, the connection of regeneration and aging is needed to understand the complicated age-related healing process. Energy metabolism  has been a common hallmark of both studies. In recent years, it become an emerging factor of skin homeostasis. Adenine nucleotide translocase-2 (ANT2) is a known cell proliferation marker and mediator of ATP import into mitochondria for energy homeostasis. Although energy homeostasis and the maintenance of mitochondrial function are critical for wound healing, the role of ANT2 in wound healing has not been elucidated. We found that ANT2 expression decreased during aging in mouse skin as well as during cellular senescence. Interestingly, overexpression of ANT2 in aged mouse skin promoted the healing of full-thickness cutaneous wounds. In addition, upregulation of ANT2 in replicative senescent human diploid dermal fibroblasts (HDFs) induced cell proliferation and migration, which are critical for the wound healing process. Furthermore, overexpression of ANT2 increased ATP production rate by activating the glycolysis pathway and also increased mitophagy, both of which are involved in energy homeostasis. Notably, ANT2-mediated upregulation of HSPA6 in aged HDFs inhibited the expression of pro-inflammatory genes that mediate cellular senescence and mitochondrial damage. This study demonstrates a new physiological role of ANT2 in skin wound healing via regulation of cell proliferation, energy homeostasis, and inflammation. Thus, our study  links energy metabolism to skin homeostasis and identifies a genetic factor for improving wound healing with aging model.

overall_design: Examination of ANT2 role in wounded HDF

first: Seung-Hwa

last: Woo

first: Young-Sam

last: Lee

first: Daehee

last: Hwang


title: A comparison between novel 3D human renal proximal tubule organoids and 2D monolayer cells utilizing RPTEC-TERT1 cells

city: San Diego

first: John

last: Walker

state: CA

address: 10675 John Jay Hopkins Dr

institute: NIBR

summary: This experiment seeks to identify differences in human proximal tubule cells in 2D and 3D in response to nephrotoxins, identify early indications to predict toxicity

overall_design: 2D v 3D RPTEC-TERT1 cells compared with respect to time and temporal exposure to nephrotoxins cisplatin and cadmium chloride

first: Sarah

last: Vidal Yucha

first: Doug

last: Quackenbush

first: Tiffany

last: Chu

first: Frederick

last: Lo

first: Jeffrey

last: Sutherland

first: Guray

last: Kuzu

first: Christopher

last: Roberts

first: Fabio

last: Luna

first: S

last: Barnes

first: Pia

last: Kuss


title: Multi-omics Analyses Reveal Sex Differences in Mouse Renal Proximal Tubules

first: Lihe

address: 10 center drive MSC 1603, Building 10, Room 6N228

institute: NHLBI

summary: Sex differences have been increasingly recognized to play an important role in kidney physiology and pathophysiology, but limited resources are available for comprehensive interrogation of sex differences. Here, we coupled renal tubule microdissection with RNA-seq and ATAC-seq with a focus on proximal tubules that exhibited the greatest sex differences and carried out whole-kidney proteomics to build a comprehensive sex difference landscape between male and female mouse kidneys. The transcriptomic data indicate that the major sex differences are in S2 and S3 of proximal tubule segments, where signaling pathways including monocarboxylic acid metabolic process, organic anion transport, and organic acid transport display the greatest differences. We develop an ATAC-seq method on microdissected tubules to capture chromatin accessibility differences between sexes. The analysis reveals that major sex differences are in autosomes instead of sex chromosomes, and many of them are in S2/S3 segments related to the signaling pathways identified by transcriptomic analysis. Motif analysis identifies several transcription factors (Tead1, Nfia/b, and Pou3f3) whose interplay with other transcription factors (e.g. Hnf1b, Hnf4a) may contribute to sex differences. Finally, whole-kidney quantitative proteomics analysis correlates with the transcriptome analysis and identifies a large number of proteins (Cyp2e1, Acsm2/3) that are sex-dependent. We develop a knowledge portal to promote our understanding of sex differences in kidneys in kidneys at https://esbl.nhlbi.nih.gov/MRECA/PT/.

overall_design: Here, we combined renal tubule microdissection with RNA-seq and ATAC-seq to characterize gene expression and chromatin accessibly in proximal tubule S1, S2, and S3. We provide insights into specific genes and pathways that differ between male and female mouse kidneys. We identify sex-dependent cis-regulatory elements and reveal the interplay between transcription factors, which may result in sex-biased gene expression. We report sex differences in protein expression in kidneys to complement our gene expression data. Finally, we develop a web-based portal that allows users to download and promote understanding of the sex differences in kidneys.

first: Chun-Lin

last: Chou

first: Chin-Rang

last: Yang

first: Mark

last: Knepper


title: IL-4 receptor blockade is a global repressor of naïve B cell development and responses in a dupilumab-treated patient

city: Birmingham

first: Min

last: Gao

state: AL

address: 1900 University Blvd, THT Building Suite #130G

institute: University of Alabama at Birmingham

summary: Here, we report a case of atopic dermatitis (AD) in a patient who received biweekly doses of dupilumab, an antibody against the IL-4 receptor α chain (IL-4Rα). Single cell RNA-sequencing showed that naïve B cells expressed the highest levels of IL4R compared to other B cell subpopulations. Compared to controls, the dupilumab-treated patient exhibited diminished percentages of IL4R+IGHD+ naïve B cells and down-regulation of IL4R, FCER2 (CD23), and IGHD. Dupilumab treatment resulted in upregulation of genes associated with apoptosis and inhibition of B cell receptor signaling and down-regulation of class-switch and memory B cell development genes. The dupilumab-treated patient exhibited a rapid decline in COVID-19 anti-spike and anti-receptor binding domain antibodies between 4 and 8 and 11 months post COVID-19 vaccination. Our data suggest that intact and persistent IL-4 signaling is necessary for maintaining robust survival and development of naïve B cells, and maintaining a long term vaccine response.

overall_design: We carried out single cell RNAseq for four individuals. Subjects recruited for this study: bi-weekly dupilumab-treated subject is a 34-year-old white male with AD and celiac disease. Control #1 (Con#1) is a 57-yr-old white healthy male and Con#2 is a 53-yr-old white healthy male. Con#3 is a 57-year-old, Asian female with AD controlled by intermittent treatment with topical 0.05% Fluocinonide.

first: Shanrun

last: Liu

first: Hui-Chen

last: Hsu

middle: D

last: Mountz


title: Endothelial Rap1B mediates VEGF immunosuppression

city: Milwaukee

first: Magdalena

last: Chrzanowska

state: WI

address: 8733 W. Watertown Plank Road

department: Blood Research Institute

institute: Versiti

summary: Overcoming vascular immunosuppression: lack of endothelial cell (EC) responsiveness to inflammatory stimuli in the proangiogenic environment of tumors, is essential for successful cancer immunotherapy. The mechanisms through which Vascular Endothelial Growth Factor (VEGF) modulates tumor EC response to exclude T cells are not well understood. The goal was to determine the role of EC Rap1B, a small GTPase that positively regulates VEGF-angiogenesis during development, in tumor growth in vivo. Using mouse models of Rap1B deficiency, Rap1B+/- and endothelial-specific Rap1B KO (Rap1BiΔEC) we demonstrate Rap1B restricts tumor growth and angiogenesis. More importantly, EC-specific Rap1B deletion leads to an altered tumor microenvironment with increased recruitment of leukocytes and increased activity of tumor CD8+ T cells. We find that tumor growth, albeit not angiogenesis, is restored in Rap1BiΔEC mice by depleting CD8+ T cells.  Mechanistically, global transcriptome analysis indicated upregulation of the TNF-α signaling and cytokine-induced NFκB transcriptional activity in Rap1B-deficient ECs. In particular, endothelial Rap1B deletion led to upregulation of Cell Adhesion Molecules  (CAMs) expression in response to proinflammatory cytokines, and increased interaction with leukocytes in vitro. Importantly, deletion of Rap1 abrogated VEGF-mediated inhibition of CAM expression, demonstrating that Rap1B is essential for mediating VEGF-suppressive signaling. Thus, our studies identify a novel endothelial-endogenous mechanism underlying VEGF-dependent desensitization of EC to pro-inflammatory stimuli. Significantly, they identify EC Rap1 as a potential novel vascular target in cancer immunotherapy.

overall_design: Bulk RNAseq of siRap1B- and siControl-transfected HUVECs

first: Robert

last: Burns