title: β2-microglobulin maintains glioblastoma stem cells and induces M2-like polarization of tumor-associated macrophages

city: Chapel Hill

first: Daqi

last: Li

address: 450 West Dr

institute: Univerity of North Carolina at Chapel Hill

summary:
Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor immune microenvironment (TIME), prominently containing tumor-
associated macrophages (TAM) and microglia. Here, we dem-
onstrated that β2-microglobulin (B2M), a subunit of the class I
major histocompatibility complex (MHC-I), promotes the
maintenance of stem-like neoplastic populations and repro-
grams the TIME to an anti-inflammatory, tumor-promoting
state. B2M activated PI3K/AKT/mTOR signaling by interacting
with PIP5K1A in GBM stem cells (GSC) and promoting MYC-
induced secretion of transforming growth factor-β1 (TGFβ1).
Inhibition of B2M attenuated GSC survival, self-renewal, and
tumor growth. B2M-induced TGFβ1 secretion activated para-
crine SMAD and PI3K/AKT signaling in TAMs and promoted
an M2-like macrophage phenotype. These findings reveal
tumor-promoting functions of B2M and suggest that targeting
B2M or its downstream axis may provide an effective approach
for treating GBM.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 4121/3264 cells and its KD derivatives (shB2M).

first: Qian

last: Zhang

first: Lu

first: Kexin

last: Chen

first: Junlei

last: Yang

first: Deobrat

last: Dixit

first: Ryan

middle: C

last: Gimple

first: Shusheng

last: Ci

first: Chenfei

last: Lu

first: Lang

last: Hu

first: Jiancheng

last: Gao

first: Danyang

last: Shan

first: Yangqing

first: Junxia

first: Zhumei

last: Shi

first: Danling

last: Gu

first: Wei

last: Yuan

first: Qiulian

last: Wu

first: Kailin

first: Linjie

last: Zhao

first: Zhixin

last: Qiu

first: Deguang

last: Lv

first: Hui

first: Fan

last: Lin

first: Qianghu

last: Wang

first: Jianghong

last: Man

first: Chaojun

first: Weiwei

last: Tao

first: Sameer

last: Agnihotri

first: Xu

last: Qian

first: Yu

first: Yongping

last: You

first: Nu

first: Jeremy

last: Rich

first: Xiuxing


title: Human Oral Keratinocyte Responses to T. denticola infection

status: Public on Jun 30 2022

city: Richmond

first: Daniel

last: Miller

state: Virginia

address: 1101 E. Marshall St. Sanger Hall 5-009

country: USA

institute: Virginia Commonwealth University

type: Expression profiling by high throughput sequencing

summary: The purpose of this study was to determine, at a global level, the transcriptomic responses of human gingival keratinocytes to T. denticola infection to better understand the disruption of normal barrier immunity that leads to the development of periodontal disease.

overall_design: Here, human immortalized gingival keratinocyte (HIGK) cells were infected with T. denticola and RNAseq was performed to compared transcriptome responses of T. denticola infected cell to uninfected HIGK cells.

first: Bin

last: Zhu


title: Chromatin remodeler ARID1A binds IRF3 to selectively induce antiviral interferon production in macrophages

status: Public on Jul 01 2022

city: Beijing

first: ye

last: hu

address: Dongdan

country: China

institute: PUMC

summary: Transcription factor IRF3 is critical for the induction of antiviral type I interferon (IFN-I). The epigenetic regulation of IFN-I production in antiviral innate immunity need to be further identified. Here, we report that epigenetic remodeler ARID1A, a critical component of the mSWI/SNF complex, could bind IRF3 and then was recruited to the Ifn-I promoter by IRF3, thus selectively promoting IFN-I but not TNF-α, IL-6 production in macrophages upon viral infection. Myeloid cell-specific deficiency of Arid1a rendered mice more susceptible to viral infection, accompanied with less IFN-I production. Mechanistically, ARID1A facilitates chromatin accessibility of IRF3 at the Ifn-I promoter by interacting with histone methyltransferase NSD2, which methylates H3K4 and H3K36 of promoter region. Our findings demonstrate the new roles of ARID1A and NSD2 in innate immunity, providing insight to the crosstalks of chromatin remodeling, histone modification and transcription factor in the epigenetic regulation of antiviral innate immunity.

overall_design: the BMDMs from Arid1a (f/f) cre+ and Arid1a (f/f) cre- (control ) mice infected with VSV for 0h and 8h .

first: Xin

first: Xiaoqing

last: Xu

first: Xuetao

last: Cao


title: Single-cell RNA sequencing of spinal cord CD133+ DNGR-1 traced and CD133+ non-traced cells

city: London

first: Bruno

last: Frederico

address: 1 Midland Road

country: United Kingdom

institute: Francis Crick Institute

summary: Using CD133 as a pan-ependymal cell marker, we wished to understand whether CD133+ DNGR-1 traced cells constituted a distinct subset of ependymal cells by comparing these at the single cell level with CD133+ non-traced cells purified from spinal cords of DNGR-1 lineage tracer mice.

overall_design: Myelin-depleted single cell suspensions from spinal cords of DNGR-1 lineage traced mice were stained with anti-CD133 AF488, anti-CD45 AF647 and LIVE/DEAD blue dye. LIVE/DEAD- CD45- CDF133+ DNGR-1 traced or LIVE/DEAD- CD45- CDF133+ non-traced cells were FACS sorted into two different tubes and ran onto 10x Chromium. Samples were compared bioinformatically by integration of both samples after identifying 2000 anchor features.

first: Caetano

last: Reis e Sousa

first: Probir

last: Chakravarty


title: HNF4A regulation of transcription profiles in the mouse intestinal epithelial cells

city: Worcester

first: Katherine

middle: A

last: Fitzgerald

state: Massachusetts

address: 364 Plantation Street

department: Medicine

institute: Umass Chan Medical

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

overall_design: Refer to individual Series


title: HNF4A regulation of transcription profiles in the mouse ileal and colonic epithelial cells

summary:
Purpose: HNF4A is a highly conserved nuclear receptor that has been associated with inflammatory bowel diseases. The goal of this study is to determine and compare transcriptional regulation by HNF4A in the small intestine and large intestine in mice.
Methods: Intestinal epithelial cells (IECs) were flow sorted from the ileum and colon of WT and HNF4A IEC-specific KO mice at 21~22 days old and sequenced for transcriptome. Differentially expressed genes in the illeum and colon respectively comparing the WT and HNF4A IEC-KO were determined.
Results: HNF4A regulates expression of hundreds of genes in both ileal and colonic IECs. However, many of the DE genes and the magnitude of changes are location specific. In general, a more significant dependancy on HNF4A is found in the colon. HNF4A is particularly required for the expression of a set of immune signaling molecules that are critical for the IEC-intrapithelial lymphocyte crosstalk. Those molecules include members of the butyrophilin-like molecules (Btnl1, Btnl2, Btnl4, and Btnl6) and MHC-I like molecule H2-T3 etc. Expression of the immune signaling molecules was  validated by qPCR. Further comparison with HNF4A ChIP-seq datasets and validation with ChIP-qPCR confirmed that those genes are also direct HNF4A targets.

overall_design: mRNA profiling of ileal and colonic IECs from cohoused littermate controlled 3-week-old WT (Hnf4a fl/fl) and IEC-specific HNF4A KO (Hnf4a fl/fl Villin-Cre) mice. 4 biological replicates for each group.

first: Xuqiu

last: Lei


title: Tracking regulatory T cell development in the thymus using scRNA-Seq/TCR-Seq

city: Minneapolis

first: Rebecca

last: LaRue

state: MN

address: 117 Pleasant St SE

department: Department of Lab medicine and Pathology

institute: University of Minnesota

summary: Recent studies have demonstrated that mature regulatory T cells ( Tregs) develop in the thymus via two pathways involving distinct Treg progenitors (TregP) - CD25+FOXP3- (CD25+ TregP) and CD25-FOXP3lo (FOXP3lo TregP) Treg progenitors. To examine this process in more detail we carried out single-cell RNA-Seq and TCR-Seq on sorted CD4+CD8+ double-positive (DP) thymocytes, CD4+ Ssingle -positive (CD4SP)P thymocytes, CD25+ TregP, FOXP3lo TregP, mature CD25+FOXP3+ Tregs and recirculating/long-term resident Tregs (RT-Tregs). Sorted populations were individually hashtagged and then combined into one scRNA-Seq/TCR-Seq library before sequencing and subsequent analysis. We found that both CD25+ TregP and FOXP3lo TregP arise via an initial agonist activated state that gives rise to a second transitional stage before differentiating into mature Tregs. Using both scRNA-Seq and bulk RNA-Seq on sorted thymocyte subsets we demonstrate that CD25+Foxp3- TregP are significantly enriched for Il2 production, suggesting that they are the major source of IL-2 needed to convert TregP into mature Tregs. Using TCR-Seq we found that several TCRs were clearly biased in favor of the conventional or Treg lineages, but that a large fraction of TCRs were found in both these lineages. Finally, we found that recirculating/resident Tregs in the thymus are not monomorphic, but are composed of multiple distinct subsets and that these RT-Tregs express the most diverse TCR repertoire of all CD4SP thymcocytes. Thus, our studies define multiple stages of Treg differentiation within the thymus, and serve as a resource for future studies on CD4+ thymocyte development and Treg differentiation.

overall_design: This data is derived from cells sorted in a single experiment with samples from 3 biological replicates.

first: Michael

last: Farrar

first: David

middle: L

last: Owen

first: Sarah

last: Munro

middle: S


title: Crustacean leg regeneration restores cell diversity

city: Lyon

first: Mathilde

last: PARIS

address: 32-34 Av. Tony Garnier

country: France

institute: CNRS

summary: Animals can regenerate complex organs, yet this frequently results in imprecise replicas of the original structure. In the crustacean Parhyale, we examine the fidelity of leg regeneration using cellular composition and cell molecular profiles. Single-nuclei sequencing shows that regenerated and uninjured legs are indistinguishable in terms of cell type composition and transcriptional profiles.

overall_design: Legs of the Crustacean Parhyale hawaiensis were amputated and analyzed through scRNAseq after regeneration.

first: Almazan

last: Alba

first: Cevrim

last: Cagri

first: Musser

last: Jacob

first: Averof

last: Michalis

first: Paris

last: Mathilde


title: HNF4A regulation of transcription profiles in the mouse cecal epithelial cells

summary:
Purpose: HNF4A is a highly conserved nuclear receptor that has been associated with inflammatory bowel diseases. The goal of this study is to determine transcriptional regulation by HNF4A in the cecal IECs..
Methods: Intestinal epithelial cells (IECs) were sorted from the ceca of young adult WT and HNF4A IEC-specific KO mice and sequenced for transcriptome. Differentially expressed genes comparing the WT and HNF4A IEC-KO were determined.
Results: Epithelial HNF4A regulates expression of hundreds of genes. HNF4A is particularly required for the expression of a set of immune signaling molecules that are critical for the IEC-intrapithelial lymphocyte crosstalk. Those molecules include members of the butyrophilin-like molecules (Btnl1 and Btnl6) and MHC-I like molecule H2-T3 etc. Expression of the immune signaling molecules was validated by qPCR. Further comparison with HNF4A ChIP-seq datasets confirmed that those genes are also direct HNF4A targets.

overall_design: RNA profiling of cecal IECs from cohoused littermated controlled 8-week-old WT (Hnf4a fl/fl) and HNF4A IEC-specific  KO (Hnf4a fl/fl Villin-Cre) mice. 3 biological replicates for each group.


title: Histone methyltransferase DOT1L is essential for self-renewal of germline stem cells

city: Yiwu

first: keren

last: cheng

state: Zhejiang

address: 1575 chouzhou bei road

department: School of Medicine

institute: Zhejiang University

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
Spermatogonial stem cells undergo both self-renewal to maintain the stem cell population and differentiation to produce mature sperm. These processes are controlled by both stem cell-intrinsic and external niche factors. DOT1L, the sole H3K79 methyltransferase, is dispensable for mouse embryonic stem cell self-renewal but instead functions as a barrier to somatic cell reprogramming. Here we show that DOT1L is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L in the germ cells show a failure in the maintenance of spermatogonial stem cells without a block in spermatogenic cell differentiation and thus a progressive loss of germ cells, leading to a Sertoli-cell-only syndrome. Chemical inhibition of DOT1L in cultured stem cells reduces the spermatogonial stem cell activity after transplantation. RNA-seq analysis reveals downregulation of Hoxc cluster genes in DOT1L-inhibited spermatogonia stem cells. Single cell RNA-seq analysis demonstrates that inhibition of DOT1L sequesters spermatogonial stem cells in a primitive state and prevents them from transitioning to a progenitor state. These results identify a new function for DOT1L in adult stem cells and provides a paradigm for regulation of spermatogonial stem cell self-renewal.

Self-renewal of spermatogonial stem cells is vital to life-long production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli-cell-only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation, prevents spermatogonial stem cells from transitioning to a progenitor state, and sequesters them in a primitive state. Furthermore, DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.

overall_design:
Mouse testicular cells were prepared from 7-day-old ROSA mice, B6.129S7-Gt(ROSA)26Sor/J (Jackson Laboratory) that expresses a LacZ (β-galactosidase) reporter gene in all tissues, by enzymatic digestion using trypsin-EDTA (Thermo Fisher Scientific) and cultured on SNL 76/7 cell feeders in a serum-free medium with 10 ng/mL GDNF (R&D Systems) and 0.5 ng/mL FGF2 (Corning). SSCs formed clumps and expanded continuously. After two months in culture, 1 x 10^5 clump-forming cells were seeded on SNL-feeder cells in 24-well plates with 2 µM EPZ5676 (Cayman Chemical) or DMSO (Sigma-Aldrich) as a vehicle control.

EPZ5676 vs Control

first: Keren

last: Cheng

first: Huijuan

last: Lin

first: P

middle: J

last: Wang


title: Arabidopsis COILIN orchestrates transcriptional as well as post-transcriptional regulation to combat pathogens

city: Jeddah

first: Sabiha

last: Parween

address: Thuwal

department: Hirt lab

country: Saudi Arabia

institute: KAUST

summary: The objective of the study was to understand the role of Coilin in plant pathogen interactions.

overall_design: WT (col-0) and coilin mutant plans were either mock treated or with the bacteria Pst DC3000 hrcC- strain. For each sample 3 biological replicates were used.

first: Aala

last: Abulfaraj

first: Hanna

middle: M

last: Alhoraibi

first: Kiruthiga

last: Mariappan

first: Jean

last: Bigeard

first: Huoming

last: Zhang

first: Marilia

last: Almeida-Trapp

first: Olga

last: Artyukh

first: Delphine

last: Pflieger

first: Ikram

last: Blilou

first: Heribert

last: Hirt

first: Naganand

last: Rayapuram


title: Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

summary: Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B cell acute lymphoblastic leukemia. However, the mechanisms underlying disease due to ETV6 dysfunction are poorly understood. In order to address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6, which recapitulates aspects of human disease. We found defects in hematopoiesis, primarily related to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, IL-18 and IL-13 abrogated B cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with that from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

overall_design: hematopoietic progenitor populations were sorted by flow cytometry from mice with wild type (n=3 mice) and homozygous mutation in Etv6 (h.P214L) (n=4 mice)

first: C

middle: D

last: Porter

last: Zhou

first: J

last: Di Paola


title: Transcriptomics of the liver and the muscle from in vitro or in vivo-produced dairy male calves

city: Utrecht

first: Maria

middle: Belen

last: Rabaglino

address: Yalelaan 7

department: Population Health Science

country: Netherlands

institute: Utrecht University

summary: In cattle, the in vitro production (IVP) of embryos is becoming more relevant than embryos produced in vivo, i.e., after ovarian stimulation and embryo transfer (MOET). However, the effects of IVP on the developmental programming of specific organs in the postnatal calves are yet unknown. The objective of this study was to compare the hepatic and muscular transcriptomic modifications between IVP and MOET male calves of three months of age (n=4 per group). Tissue samples from liver and semitendinosus muscle were obtained at 3 months of age, and the extracted RNA was sequenced through RNAseq to determine differentially expressed genes (DEG; FDR<0.05). KEGG pathways enrichment analysis showed that DEG up-regulated in the liver and the muscle of the IVP calves were involved in oxidative phosphorylation and the tricarboxylic acid cycle. In contrast, ribosome and translation were up-regulated in the liver but down-regulated in the muscle of the IVP calves compared to the MOET calves (FDR<0.05). In conclusion, our findings indicated an altered hepatic and muscular energy regulation in phenotypically normal IVP calves compared to MOET calves.

overall_design:
Samples were obtained from the liver and muscle from three-month-old IVP calves (n=4) and control MOET calves (n=4).
The samples in this study are from the same animals as the samples in GSE176219.

middle: B

first: Jan

last: Bojsen-Møller Secher

first: Poul

last: Hyttel

first: Haja

middle: N

last: Kadarmideen


title: Development of miniBac-seq to profile bacterial transcriptome from ultra-low input RNA amount

city: Shanghai

first: Tianmin

last: Wang

address: Huaxia Middle Road, 393

department: School of Life Science and Technology

institute: ShanghaiTech University

summary: We developed miniBac-seq, a strand-specific method for high-quality bacterial RNA-seq library construction from sub-nanogram of total RNA, equivalent to a few thousand bacterial cells; we also tested its potent to be adopted as a single cell RNA-seq method. Here we provide the supporting data to the paper introducing this technology.

overall_design: We used E. coli K12 BW25113, typically cultivated in M63B1 broth and extracted total RNA at the Log phase. Fig2 Impact of customized rRNA depletion method on transcript quantification; Fig4 Overall performance of miniBac-seq; Fig 5 Detection limit of miniBac-seq, via library construction from a gradient of input RNA (5 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg); Fig 6 Impact of barcode introduced at RT on transcript quantification

first: Ping

last: Shen


title: HSP70 inhibition synergizes with MEK inhibition for the treatment of NRAS mutant melanoma

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: NRAS-mutant melanoma is currently a challenge to treat.  This is due to an absence of inhibitors directed against NRAS, along with acquired and adaptive resistance of this tumor type to inhibitors in the MAPK pathway.  Inhibitors to MEK (mitogen-activated protein kinase kinase) have shown some promise for this tumor type.  In this work we explored the use of MEK inhibitors for NRAS-mutant melanoma, and at the same time investigated the impact of the brain micro-environment, specifically astrocytes, on the response of a melanoma brain metastatic cell line to MEK inhibition.  This led to the surprising finding that astrocytes enhance the sensitivity of melanoma tumors to MEK inhibitors (MEKi).  We show that MEKi cause an upregulation of the transcription factor ID3, but this is blocked by conditioned media from astrocytes.  We show that silencing ID3 enhances the sensitivity of melanoma to MEK inhibitors, thus mimicking the effect of the brain microenvironment.  Moreover, we report that ID3 is a client protein of the chaperone HSP70, and that HSP70 inhibition causes ID3 to misfold and accumulate in a detergent-insoluble fraction in cells.  We show that HSP70 inhibitors synergize with MEK inhibitors against NRAS-mutant melanoma, and that this combination significantly enhances the survival of mice in two different models of NRAS-mutant melanoma.  These studies highlight ID3 as a mediator of adaptive resistance, and support the combined use of MEK and HSP70 inhibitors for the therapy of NRAS-mutant melanoma.

overall_design: RNA-seq experiment in WM4265-3 and WBM983B melanoma cell lines cultured in astrocyte (AM) or astrocyte conditioned (ACM) media and treated with PD901

first: Joshua

last: Parris

first: Maureen

middle: E

last: Murphy


title: A systematic survey of PRMT interactomes reveals key roles of arginine methylation in the global control of RNA splicing and translation

city: ShangHai

first: Zefeng

address: 320 YueYang Road

department: CAG-MPG Partner Institution for Computational Biology

institute: Chinese Academy of Sciences


title: A systematic survey of PRMT interactomes reveals key roles of arginine methylation in the global control of RNA splicing and translation [Ribo-Seq]

summary: We used Ribo-seq to determine how PRMT inhibition affects translation of different mRNAs, This analysis can generate a “snapshot” of all mRNAs that are occupied by active ribosomes (i.e., undergoing active translation) in a cell at a particular condition.

overall_design: Ribo-seq was used to measure the distribution of ribosome protected RNA fragments in control and PRMT inhibition samples.

first: Huan-Huan

last: Wei

first: Xiao-Juan

last: Fan


title: A systematic survey of PRMT interactomes reveals key roles of arginine methylation in the global control of RNA splicing and translation [RNA-seq]

summary: Since many proteins involved in splicing regulation were identified as PRMT substrates, we examined the impact of PRMT inhibition on alternative RNA splicing. We achieved effective gene silence with shRNAs in six different PRMTs and examined their effect on splicing using RNA-seq.

overall_design: mRNA profiles of PRMT knockdown and wild type HEK293T cells

first: Yue

last: Hu


title: Single-cell RNA sequencing analysis of non-haematopoietic DNGR-1 lineage traced cells from murine spinal cord

first: Bruno

last: Frederico

summary: Stem cells are defined by two cardinal properties: limitless self-renewal and multipotency. We have serendipitously found that non-haematopoietic DNGR-1 lineage traced cells residing in the ependymal cell layer of the central nervous system display the two cardinal properties of stem cells, both in vitro and in vivo. However, whether these properties were a feature of all DNGR-1-traced cells or were confined to a particular subset of these is unclear. To address the potential heterogeneity of DNGR-1-traced ependymal cells and caractherise their putative stem cell compartment we conducted single-cell RNA sequencing of DNGR-1-traced cells isolated from uninjured spinal cords.

overall_design: DNGR-1 lineage traced cells pooled from a spinal cords dissected from DNGR-1 lineage tracer mice were FACS sorted and subjected to single-cell RNA sequencing

first: Caetano

last: Reis e Sousa

first: Probir

last: Chakravarty


title: A systematic survey of PRMT interactomes reveals key roles of arginine methylation in the global control of RNA splicing and translation [Poly-Seq]

summary: To examine how PRMT inhibitions affect the translation of different mRNAs, we sequenced the mRNA population associated with different ribosomal fractions after treatment of PRMT1 and PRMT4 inhibitors

overall_design: DMSO or Inhibitor-treated HEK293T cells were subjected to polysome profiling. Different polysome profiling fractions bound with indicated ribosomes were analyzed with RNA-seq to test translation alteration under PRMT inhibition.


title: Bulk transcriptomic comparison between in vitro propagated DNGR-1-lineage traced cells and hippocampus derived neural stem cells

summary: We compared by bulk RNA sequencing the transcriptomic profile of in vitro cultured DNGR-1-traced neural stem cells or their differentiated astrocytic progeny with similar cell fates generated from bonafide neural stem cells derived from the hippocampus.

overall_design: Passage 10 NSCs adherent cultures were generated from tdTomato+, EYFP+ sorted from neurospheres grown from spinal cords of DNGR-1CreROSALSLtdTomato ot DNGR-1CreROSALFLEYFP mice, respectively. Hippocampus derived adherent NSCs cultures were generated from the hippocampus of GlastCreHuweflROSALSLEYFP mice. NSCs  seeded in adherent conditions (+eGFP +bFGF+laminin)  were recovered in StemPro accutase after 4 days or induced to differentiate into astrocytes (+BMP4) for 6 days. Cells from either condition were lysed with RLT buffer supplemented with 2-mercaptoethanol and cells lysates stored at -80C. Three replicates for each cell fate (3x10_6 NSCs or astrocytes) generated from passages 10, 11 and 12 were sequenced..


title: Interactomics Analyses of Wild-type and Mutant A1CF Reveal Diverged Functions in Regulating Cellular Lipid Metabolism

city: Boston

first: Yu-Xin

last: Xu

state: United States

address: 185 Cambridge Street Simches Research Building, CPZN 5.500

department: Center for Genomic Medicine

institute: Massachusetts General Hospital

summary: Population genetic studies highlight a missense variant (G398S) of A1CF that is strongly associated with higher levels of blood triglycerides (TGs) and total cholesterol (TC). Functional analyses suggest that the mutation accelerates the secretion of very low-density lipoprotein (VLDL) from the livers by an unknown mechanism. Here, we used multi-omics approaches to interrogate the functional difference between the WT and mutant A1CF. Using metabolomics analyses, we captured the cellular lipid metabolite changes induced by the transient expression of the proteins, confirming that the mutant A1CF is able to relieve the TG accumulation induced by WT A1CF. Using a proteomics approach, we obtained the interactomic data of WT and mutant A1CF. Networking analyses show that WT A1CF interacts with three functional protein groups, RNA/mRNA processing, cytosolic translation and, surprisingly, mitochondrial translation. The mutation diminishes these interactions, especially with the group of mitochondrial translation. Differential analyses show that the WT A1CF-interacting proteins most significantly different from the mutant are those for mitochondrial translation, whereas the most significant interacting proteins with the mutant are those for cytoskeleton and vesicle-mediated transport. RNA-seq analyses validate that the mutant, but not the WT, A1CF increases the expression of the genes responsible for cellular transport processes. On the contrary, WT A1CF affected the expression of mitochondrial matrix proteins and increased cell oxygen consumption. Thus, our studies confirm the previous hypothesis that A1CF plays broader roles in regulating gene expression. The interactions of the mutant A1CF with the vesicle-mediated transport machinery provide mechanistic insight in understanding the increased VLDL secretion in the A1CF mutation carriers.

overall_design: Mutant A1CF vs. wild type

first: Sekar

last: Kathiresan


title: Transcriptome comparison between TCR γδ T subsets reveals their exclusive and shared markers

status: Public on Jun 29 2022

city: Bron

first: Hector

last: Hernandez-Vargas

state: Rhone

address: 36 bis Rue de la Batterie

institute: Genomics Consulting

summary:
Intestinal intraepithelial gamma-delta T cells (iIEL gd T cells) constitute a peculiar subset of T lymphocytes located in the gut epithelial barrier involved in intestinal homeostasis. To date, type 1 and type 17 TCR gamma-delta T cells have been well characterized into two functional categories based on phenotypic and functional markers e.g. CD44 and CD27. However, It remains to be determined how to classify iIEL gamma T cells based on commonly used markers.
We addressed this question by comparing the transcriptomes of mouse iIEL gamma-delta T cells with distinct functionally well-described gamma-delta T cells present in the periphery.

overall_design: Different subsets of gamma-delta T cells were isolated from mice in biological triplicates and profiled using whole genome transcriptome (Affymetrix genechip 2.0 St arrays).

first: Apostol

last: Apostolov

first: Saidi

last: Soudja


title: Tfap2e/AP-2ε has both activating and repressive roles in controlling genetic programs of vomeronasal sensory neurons that underly sociosexual behavior in mice

city: Ithaca

first: Megan

last: Rothstein

state: NY

address: 526 Campus Road Rm 433

institute: Cornell University

summary: Neuronal identity dictates the position in an epithelium, and the ability to detect, process, and transmit specific signals to specified targets. Transcription factors (TFs) determine cellular identity via direct modulation of genetic transcription and recruiting chromatin modifiers. However, our understanding of the mechanisms that define neuronal identity and their magnitude remains a critical barrier to elucidate the etiology of congenital and neurodegenerative disorders. The rodent vomeronasal organ provides a unique system to examine in detail the molecular mechanisms underlying the differentiation and maturation of chemosensory neurons. Here we demonstrated that the identity of postmitotic/maturing VSNs and vomeronasal dependent behaviors can be reprogrammed through the rescue of AP-2ε expression in the AP-2εNull mice and by inducing ectopic AP-2ε expression in mature apical VSNs. We suggest that the transcription factor AP-2ε can reprogram VSNs bypassing cellular plasticity restrictions, and that it directly controls the expression of batteries of vomeronasal genes.

overall_design: CUT&RUN for TFAP2E in mouse P10 VNO tissue

first: Jennifer

last: Lin

first: Tyler

last: Mitchell

first: Alison

last: Pehl

first: Ed

last: Taroc

first: Raghu

middle: R

last: Katreddi

first: Katherine

last: Parra

first: Damian

middle: G

last: Zuloaga

first: Marcos

last: Simoes-Costa

first: Paolo

last: Forni


title: Overlapping transcriptional programs promote survival and axonal regeneration of injured retinal ganglion cells [2]

status: Public on Jun 28 2022

city: Cambridge

first: Wenjun

last: Yan

state: MA

address: 52 Oxford Street

department: Department of Molecular  and Cellular Biology

institute: Harvard University

summary: Neurons of the central nervous system (CNS) display only a limited ability to survive and regenerate their axons after an injury. In mice, 85% of retinal ganglion cells (RGCs) die within 2 weeks of axotomy by optic nerve crush (ONC) and only few survivors regenerate axons. In the past years, a multitude of interventions have been identified to improve RGC survival and regeneration after an injury, however, each only protects a subset of neurons and stimulates axon regrowth in an even smaller set.. Here, we sought out to elucidate the molecular mechanisms underlying this selective responsiveness and investigated genes regulated by three well established survival and regeneration-promoting interventions – activation of the MTOR pathway via deletion of its inhibitor Pten, activation of the Jak/Stat-pathway by deletion of its endogenous inhibitor Socs3, and overexpression of the neurotrophic cytokine CNTF. Analysis of the transcriptomes from >125,000 single RGCs at various time points after ONC showed that while broad survival of all RGC types could be induced with each intervention, type-independent axon regeneration was only overcome with the manipulation of multiple pathways. Those RGCs were able to mitigate the injury response and simultaneously upregulated survival and regeneration associated programs (prior and after injury). Four independent ways of analysis identified these programs to be differentially regulated among RGCs, with distinct signatures for degenerating, surviving and regenerating cells. Finally, testing some genes associated with the regeneration-program in vivo identified potential future therapeutic targets to promote neuroprotection and axonal regeneration.

overall_design: To separate RGCs that regenerated from those that survived but did not regenerate, we used a retrograde labeling method in which we injected a small fluorescently labeled dextran (micro-Ruby, MR) into the nerve stump ~1.5mm distal to the crush site at 20dpc. The dextran was taken up by regenerating axons and retrogradely transported to RGC somata, which were also labeled with YFP. We used FACS to isolate regenerating (MR+YFP+) and non-regenerating (MR-YFP+) RGCs 24hrs after tracer injection, collected single cells in individual wells, and performed scRNA-seq using SmartSeq2 (SS2).

first: Anne

last: Jacobi

first: Nicholas

last: Tran

first: Inbal

last: Benhar

first: Feng

last: Tian

first: Rebecca

last: Schaffer

first: Zhigang

last: He

first: Joshua

last: Sanes


title: Adenovirus Overexpression of human ApoF in C57BL6/J mice

city: Lille

first: Joel

last: Haas

address: 1 Rue du Professeur Calmette

institute: Institut Pasteur de Lille

summary: Background: Non-alcoholic fatty liver disease (NAFLD) affects nearly 25% of the global population. Cardiovascular disease (CVD) is the most common cause of death among patients with NAFLD, in line with highly prevalent dyslipidemia in this population. Increased plasma triglyceride (TG)-rich lipoprotein (TRL) concentrations, an important risk factor for CVD, are closely linked with hepatic TG content. Therefore, it is of great interest to identify regulatory mechanisms of hepatic TRL production and remnant uptake in the setting of hepatic steatosis.  Approach and results: To identify liver-regulated pathways linking intra-hepatic and plasma TG metabolism, we performed transcriptomic analysis of liver biopsies from two independent cohorts of obese patients. Hepatic APOF (encoding apolipoprotein F) expression showed the 4th strongest negatively correlation with hepatic steatosis and the strongest negative correlation with plasma TG levels. The effects of adenoviral-mediated human ApoF (hApoF) overexpression on plasma and hepatic TG were assessed in C57BL6/J mice. Surprisingly, hApoF overexpression increased both hepatic VLDL-TG secretion and hepatic lipoprotein remnant clearance, associated a ~25% reduction in plasma TG levels. Conversely, reducing endogenous ApoF expression reduced VLDL secretion in vivo, and reduced hepatocyte VLDL uptake by ~15% in vitro. Transcriptomic analysis of APOF-overexpressing mouse livers revealed a gene signature related to enhanced ApoB-lipoprotein clearance, including increased expression of Ldlr and Lrp1, among others.   Conclusion: These data reveal a previously undescribed role for ApoF in the control of plasma and hepatic lipoprotein metabolism by favoring VLDL-TG secretion and hepatic lipoprotein remnant particle clearance.

overall_design: C57BL6/J male mice, age 8-10wks old, housed in 12h/12h day/night cycle at 22°C. Mice were injected by tail vain with Ad5 adenovirus constructs and mice were sacrificed 4 days after injection at 3 pm following a 6 hour fast. Livers were collected and flash frozen in liquid nitrogen until sample processing and RNA extraction. Total RNA was extracted from 20 mg of liver tissue using the Qiagen RNEasy column kit.


title: RNA sequencing of HECTD3 knockdown during TNFalpha stimulation in primary HUVEC cells

city: Kunming

first: Huichun

last: Liang

address: No.17, Longxin Road, Panlong District

institute: Kunming Institute of Zoology. CAS

summary: We isolated and validated primary HUVECs from the neonatal umbilical cord vein, and performed genome-wide expression analysis to profile differentially expressed genes in control and HECTD3-KD HUVECs treated with or without TNFα for 2 hours. Compared with control HUVECs, 59 genes showed lower expression in HECTD3-KD HUVECs in response to TNFα. Most of them are NF-κB target genes, including adhesion molecules SELE (E-selectin), ICAM-1, and VCAM-1.

overall_design: Examination of differentially expressed genes in control and HECTD3-KD with or without TNFalpha stimulation in primary HUVEC cells.

first: Fubing

last: Li


title: Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids [array]

city: Armilla

first: Bioinformatics Unit

last: IPBLN-CSIC

state: GRANADA

address: Avda. del Conocimiento, 17. Instituto de Parasitología y Biomedicina Lopez-Neyra. Bioinformática

country: Spain

institute: IPBLN-CSIC

summary: Giant cell arteritis (GCA) is a complex systemic vasculitis mediated by the interplay between both genetic and epigenetic factors. Monocytes are crucial players of the inflammation occurring in GCA. Therefore, characterization of the monocyte methylome and transcriptome in GCA would be helpful to better understand disease pathogenesis. We performed an integrated epigenome- and transcriptome-wide association study in CD14+ monocytes from 82 patients with GCA, cross-sectionally classified into three different clinical status (active, in remission with or without glucocorticoid (GC)-treatment), and 31 healthy controls. We identified a global methylation and gene expression dysregulation in GCA monocytes. Specifically, monocytes from active patients showed a more pro-inflammatory phenotype compared with healthy controls and patients in remission. In addition to inflammatory pathways known to be involved in active GCA, such as response to interleukin (IL)-6 and IL-1, we identified a significant number of chemokines, such as CCRL2, and integrins. Furthermore, monocytes from patients in remission with treatment showed downregulation of genes involved in inflammatory processes as well as overexpression of GC receptor-target genes. Finally, we identified changes in DNA methylation correlating with alterations in expression levels of genes with a potential role in GCA pathogenesis, such as ITGA7 and CD63, as well as genes mediating the molecular response to GC, including FKBP5, ETS2, ZBTB16, and ADAMTS2. Our results revealed profound alterations in the methylation and transcriptomic profiles of monocytes from GCA patients, uncovering novel genes and pathways involved in GCA pathogenesis and in the molecular response to GC treatment.

overall_design: Five hundred nanograms of each DNA sample was bisulfite-converted with the EZ DNA MethylationTM kit (Zymo Research, Irvine, CA, USA), following the manufacturer's instructions. Samples were hybridized onto an Infinium MethylationEPIC Bead Chip array (Illumina, Inc., San Diego, CA, USA).

first: Elkyn

last: Estupiñán-Moreno

first: Lourdes

last: Ortiz-Fernández

first: Tianlu

first: José

last: Hernández-Rodríguez

first: Laura

last: Ciudad

first: Eduardo

last: Andrés-León

last: Terron-Camero

first: Sergio

last: Prieto-González

first: Georgina

last: Espígol-Frigolé

first: Maria

last: Cid

first: Ana

last: Márquez

first: Esteban

last: Ballestar

first: Javier

last: Martín


title: Transcriptome of cytokines-polarized mouse bone marrow-derived macrophages

city: Cincinnati

first: Yuting

last: Tang

state: OH

address: 3333 Burnet Ave

institute: Cincinnati Children's Hospital Medical Center

summary: To understand the molecular signature of the IL-10/IL-18 polarized macrophages, we performed transcriptome analysis for mouse BMDMs polarized with PBS (Naïve), IFN-γ (classic M1 stimulator, M1, 20ng/ml), IL-4 (alternative M2 stimulator, M2, 20ng/ml), IL-10 (M10, 2000ng/ml), IL-18 (M18, 2000ng/ml) or IL-10 and IL-18 (M1018, 2000ng/ml each). These data suggest that IL-10 and IL-18 cooperatively modulate a set of gene expressions and pathway activities in macrophages and contribute to a distinct polarization state.

overall_design: Transcriptome of mouse bone marrow-derived macrophages polarized with six different groups of cytokines.

first: Qian

last: Xu

first: Gang

last: Huang


title: High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects

first: Hui

last: Yang

state: Shanghai

address: 320 Yueyang Road

institute: CAS Center for Excellence in Brain Science and Intelligence Technology

summary:
We found that several variants with 2-4 mutations on Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains showed undiminished on-target activity but markedly reduced collateral effects.
Furthermore, transcriptome-wide off-target effects and cell growth arrest induced by wild-type Cas13d were found to be absent for a Cas13d variant.
Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for the targeted degradation of RNAs in basic research and therapeutic applications.

overall_design: We have designed a dual-fluorescent reporter system that revealed substantial collateral effects of Cas13-mediated RNA knockdown of both exogenous and endogenous genes in mammalian cells. This reporter system was used to screen over 200 Cas13 variants obtained by structure-guided mutagenesis. To study transcriptome-wide off-target effect induced by wild-type Cas13d (RfxCas13d) and its absence in cfCas13d ( named hfCas13d in the publication) variant, we designed a three groups experiment using HEK293 cell lines, including an inactive dead Cas13d control (dCas13d with gRNA targeting endogenous genes), a wild-type Cas13d treatment groups (Cas13d with the corresponding gRNA targeting endogenous genes) and a "collateral cleavage-free" Cas13d treatment groups (cfCas13d  with the corresponding gRNA targeting endogenous genes). We chose 2 gRNAs targeting PPIA, and 1 gRNA each targeting RPL4, CA2, or PPARG, respectively. Three replicates were prepared for each group for whole transcriptome sequencing. We finally have 44 RNAseq samples in total, three for each groups except one sample missing in cfCas13d for targeting PPIA with gRNA g1.

first: Huawei

last: Tong

first: Qingquan

last: Xiao

first: Yuanhua

last: Liu


title: Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids

first: Bioinformatics Unit

last: IPBLN-CSIC

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

overall_design: Refer to individual Series


title: Single Cell RNA-seq data of parental HMLER cells and HMLER-derived clones C1 and C2

status: Public on Jun 27 2022

city: Jerusalem

first: Matan

last: Hofree

address: The Edmond J. Safra, Givat Ram Campus

department: School of Computer Science and Engineering | The Lautenberg Center for Immunology and Cancer Research

country: Israel

institute: The Hebrew University of Jerusalem

summary: Analysis of gene expression patterns at single cell level of parental HMLER cells and HMLER-derived non-convertible clone C1 and convertible clone C2. Two conditions were used including independently cultured or cultured together in vitro.

overall_design: Parental HMLER cells and HMLER-derived clones C1 and C2 cells were separately labelled by stably expressing RNA barcodes (indicated in the sample characteristics field). Cells were collected from either independently cultured or co-cultured conditions and then processed with 10x genomics single-cell RNA sequecing.

first: Yun

last: Zhang

first: Robert

last: Weinberg


title: Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids [RNA-Seq]

overall_design: DNA methylation and gene expression profiling analysis of Illumina Infinium EPIC array and RNA-seq data sets for CD14+ monocytes in healthy individuals and giant cell arteritis patients within three different clinical status (active (ACT), in remission with glucocorticoid (GC) treatment (RT), and in remission without treatment(RNT)).

first: Elkyn

last: Estupiñán-Moreno

first: Lourdes

last: Ortiz-Fernández

first: Tianlu

last: Li

first: José

last: Hernández-Rodríguez

first: Laura

last: Ciudad

first: Eduardo

last: Andrés-León

last: Terron-Camero

first: Sergio

last: Prieto-González


title: FOXP3 exon 2 controls Treg stability and autoimmunity [ChIP-seq]

city: Indianapolis

first: Sheng

state: IN

address: 1044 W Walnut Street, Room R4-272

department: Pediatrics

institute: Indiana University School of Medicine

summary: Differing from the mouse Foxp3 gene that encodes only one protein product, human FOXP3 encodes two major isoforms through alternative splicing – a longer isoform (FOXP3 FL) containing all the coding exons and the other shorter isoform lacking the amino acids encoded by exon 2 (FOXP3 ΔE2). The two isoforms are naturally expressed in humans yet their differences in controlling regulatory T cell phenotype and functionality remains unclear. Here we show that patients expressing only the shorter isoform failed to maintain self-tolerance and developed IPEX syndrome. Mice with Foxp3 exon 2 deletion developed excessive TFH and GC B cell responses and systemic autoimmune disease with anti-dsDNA and anti-nuclear autoantibody production and immune-complex glomerulonephritis. Regulatory T cells expressing FOXP3 ΔE2 were unstable and sufficient to induce autoimmunity when transferred into Tcrb-deficient mice.  Mechanistically, FOXP3 ΔE2 isoform allows increased expression of selected cytokines but decreased expression of a set of Foxp3 positive regulators without altered binding to these gene loci. We demonstrate that exon 2 of FOXP3 is required to maintain Treg stability and immune homeostasis.

overall_design: Global DNA binding by FOXP3 FL and FOXP3 ΔE2 isoforms.

first: Jianguang

last: Du

first: Yongyao

last: Fu

first: Baohua

last: Zhou


title: FOXP3 exon 2 controls Treg stability and autoimmunity


title: Identification of a shared disease-associated oligodendrocyte signature among multiple CNS pathologies

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

institute: Weizmann Institute of Science

summary: The role of oligodendrocytes in Alzheimer’s disease (AD) has never been fully elucidated. To dissect their possible contribution to the disease we perform single-cell RNA-sequencing (scRNA-seq) on the brains of healthy and 5XFaD (genetic model of AD) at different ages.

overall_design: We performed massively parallel single-cell RNA sequencing (MARS-seq2.0) on non-immune cells (CD45-) or specifically on oligodendrocytes (GalC+) from brains of WT and 5XFaD mice.

first: Mor

last: Kenigsbuch

first: Pierre

last: Bost


title: Transcriptome sequencing was performed on Gossypium barbadense fibers at different developmental stages

city: Urumqi

first: Yajie

last: Duan

state: Xinjiang

address: Nongda East Road No. 311

department: College of Agronomy

institute: Xinjiang Agricultural University

summary:
The purpose of this study was to find candidate genes related to fiber strength by comparing the differences in gene expression levels of different samples at different stages of fiber development.
Methods: PimaS-7 and 5917, two Gossypium barbadense materials with significant difference in fiber strength, were used as experimental materials. Transcriptome sequencing was performed on the fibers at 0,5,10,15,20,25,30,35 dPA after flowering, with three biological replicates。Sequence reads that were quality-screened were analyzed at the transcriptional subtype level using two methods :Burrows-Wheeler Aligner (BWA), then Aova (Aova) and Tophat, then Cufflinks.
Results: Transcriptome analysis of 48 samples was completed, and a total of 317.57Gb of Clean Data was obtained. The Clean Data of all samples reached 5.78Gb, and the percentage of Q30 bases was 92.78% or above.The Clean Reads of each sample were sequenced with the designated reference genome, and the alignment efficiency ranged from 85.56% to 94.79%.Based on the comparison results, alternative splicing prediction analysis, gene structure optimization analysis and discovery of new genes were performed. 7,726 new genes were discovered, of which 6,612 were functional annotations.
Conclusion: In this study, RNA-seq technology was used to analyze the changes of gene expression during the development of  Gossypium barbadense  fiber.Our results suggest that NGS provides a comprehensive and more accurate quantitative and qualitative assessment of RNA content in  tissues.we conclusion that based on RNA-seq technology, the genes related to target traits can be obtained quickly.

overall_design: Two cotton materials PimaS-7 and 5917, in eight fiber development stages, were generated by sequencing, Three biological replicates, using Illumina.

first: Yanying

last: Qu

first: Quanjia

last: Chen


title: JAK-STAT Signaling Confers Lineage Plasticity and AR Targeted Therapy Resistance in Prostate Cancer

city: Dallas

first: Tao

last: Wang

state: Texas

address: 5323 Harry Hines Blvd.

institute: University of Texas Southwestern Medical Center

summary: Emerging evidence indicates that various cancers, including prostate, breast, melanoma and lung cancers, could gain resistance to targeted therapies by acquiring lineage plasticity. Although various genomic and transcriptomic aberrations correlate with lineage plasticity-driven resistance, the molecular mechanisms and kinetics of acquiring lineage plasticity are not fully elucidated. Through integrated transcriptomic and single cell RNA-seq (scRNA-seq) analysis of more than 80,000 cells, we show that the ectopic activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway drives lineage plasticity and Androgen Receptor (AR) targeted therapy resistance in PCa with TP53/RB1-deficiency. Ectopic activation of JAK-STAT signaling enables Heterogeneous and AR-independent subclones to emerge upon the selective pressure of AR targeted therapy, including subclones expressing multi-lineage, progenitor-like and epithelial to mesenchymal transition (EMT)-like lineage survival transcriptional programs. Both genetic and pharmaceutical inactivation of key components of the JAK-STAT signaling pathway significantly re-sensitizes resistant PCa tumors to AR targeted therapy. In summary, these results show for the first time that JAK-STAT signaling pathway is a key effector in driving lineage plasticity and represents a potential therapeutic target for overcoming AR targeted therapy resistance.

overall_design: 1) Examination of transcriptomic changes in LNCaP/AR prostate cancer cell lines with modifications of TP53/RB1/SOX2 genes. There is total 8 different conditions with 3 biological replicates each. 2) Single cell RNA-seq examine the transcrptomic changes in LNCaP/AR prostate cancer cell lines with various CRISPR guides targeting TP53/RB1/JAK1 and treated with enzalutamide or vehicle. There is total 6 different conditions, with more than 10,000 cells being sequenced each.

first: Ping

last: Mu


title: Expression data from recombinant HSP90α (rHSP90α) treated human monocytes

city: Mannheim

first: Feyza Gul

middle: Ozbay

last: Kurt

address: Theodor-Kutzer-Ufer 1-3

department: Skin Cancer Unit

country: Germany

institute: German Cancer Research Center/ Mannheim University Medical Center

summary:
Tumor cells modulate host immunity by secreting extracellular vesicles (EV) and soluble factors in circulation. Their interactions with myeloid cells could lead to the generation of myeloid-derived suppressor cells (MDSC), which strongly inhibit the anti-tumor function of T and NK cells. We demonstrated previously that EV derived from mouse and human melanoma cells induced such immunosuppressive activity via upregulating the expression of programmed cell death ligand 1 (PD-L1) on myeloid cells that was dependent on the heat-shock protein 90a (HSP90a) in EV and on the toll-like receptor (TLR) on myeloid cells. Here, we investigated whether soluble HSP90α could convert monocytes into immunosuppressive MDSC.
CD14+ monocytes were isolated from the peripheral blood of healthy donors, incubated with human rHSP90α alone or in the presence of inhibitors of TLR4 signaling and analyzed by flow cytometry. Inhibition of T cell proliferation assay was applied to assess immunosuppressive function of rHSP90α-treated monocytes. The concentration of HSP90α was measured by ELISA in plasma of advanced melanoma patients and correlated with clinical outcome. We found that the incubation of monocytes with rHSP90α for 16 h resulted in a strong upregulation of PD-L1 expression, whereas ROS and NO production as well as the expression of arginase-1, adenosine producing ectoenzymes CD39 and CD73 remained unchanged. The PD-L1 upregulation can be blocked by anti-TLR4 antibodies and an NF-κB inhibitor. After longer incubation (for 24h), rHSP90α-treated monocytes downregulated HLA-DR expression and acquired an augmented viability and resistance to apoptosis. Moreover, these monocytes were converted into MDSC indicated by their capacity to inhibit T cell proliferation mediated by TLR4 signaling as well as PD-L1 and indolamin-2,3-Dioxygenase (IDO) 1 expression. Higher levels of HSP90α in plasma of melanoma patients correlated with augmented PD-L1 expression on circulating monocytic (M) MDSC. Furthermore, melanoma patients with high levels of HSP90α displayed shorter progression-free survival (PSF) upon the treatment with immune checkpoint inhibitors (ICI).

overall_design: Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors by density gradient centrifugation using Biocoll (Biochrom). CD14+ monocytes were isolated by MACS (Miltenyi Biotec) according to the manufacturer’s instructions.  Monocytes were cultured at a concentration of 1x106/mL in RPMI-1640 medium supplemented with 10 mM HEPES, 1mM Sodium pyruvate, 1mM non-essential amino acids, 50µM β-mercaptoethanol, 10% FBS, 1% Penicillin/Streptomycin (all Gibco). Monocytes were treated with rHSP90a at a concentration of 2 μg/mL/106 cells or with PBS (control) for 24 hours. To obtain a more comprehensive view on the effect of rHSP90α on human monocytes, we performed a microarray analysis with Affymetrix Human Clariom S array, comparing rHSP90α-treated and control monocytes.

first: Feyza

last: Ozbay Kurt

first: Ihor

last: Arkhypov

first: Thomas

last: Hielscher

first: Viktor

last: Umansky


title: Reversal of the renal hyperglycemic memory by targeting sustained tubular p21 expression

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary: A major obstacle in diabetes is the metabolic or hyperglycemic memory, which lacks specific therapies. Here we show that glucose-mediated changes in gene expression largely persist in diabetic kidney disease (DKD) despite reversing hyperglycemia. The senescence-associated cyclin-dependent kinase inhibitor p21 (Cdkn1a) was the top hit among genes persistently induced by hyperglycemia and was associated with induction of the p53-p21 pathway. Persistent p21 induction was confirmed in various animal models, human samples and in vitro models. Tubular and urinary p21-levels were associated with DKD severity and remained elevated despite improved blood glucose levels in humans. Mechanistically, glucose-induced and sustained tubular p21 expression is linked to demethylation of its promoter and reduced DNMT1 expression. Exploiting signaling of the disease resolving protease activated protein C (3K3A-aPC, parmodulin-2) reversed sustained tubular p21 expression, tubular senescence, and DKD. Thus, p21-dependent tubular senescence is a pathway contributing to the hyperglycemic memory, which can be therapeutically targeted.

overall_design: In wild-type mice (C57BL/6, age 8-10 weeks) mice hyperglycemia was induced using low dose streptozotocin (STZ) injection (intraperitoneally, 60 mg/kg body weight, for 5 consecutive days). Subsets of mice were treated with a sodium-glucose co-transporter 2 inhibitor (SGLT-2i, Dapagliflozin®, 25 mg/kg body weight, in the drinking water .  Mice were sacrificed at the end of study period and kidney samples were isolated and RNAseq was performed.

first: B

last: Isermann

first: R


title: Single cell analysis of innate lymphoid cells and T lymphocytes in the small intestinal resection of Crohn’s disease patient versus those of adjacent non-inflamed tissue.

city: New York

first: Gregory

last: Putzel

address: 430 East 29th Street

department: Microbiology

institute: NYU Langone Health

summary: The focus of this study is to define the full spectrum of T cells and innate lymphocytes in the context in human inflammatory bowel disease. Results from this study may yield novel findings on the heterogeneity of these lymphocytes and determine how they change in the inflamed human intestine.

overall_design: CD45+, CD19/CD14/CD123/ Fc-epsilon-RI-alpha/CD34/CD94-, CD4-, CD127+ILCs (7,000) and CD45+CD3+ T cells (28,000) were sorted from the resection of inflamed Crohn’s disease patient versus adjacent tissue.

first: Mengze

last: Lyu

last: Sonnenberg


title: FOXP3 exon 2 controls Treg stability and autoimmunity [RNA-seq]

first: Sheng

last: Liu

overall_design: Differential regulation of target genes by FOXP3 FL and FOXP3 ΔE2 isoforms

first: Jianguang

last: Du

first: Baohua

last: Zhou


title: Nacent transcription profiling of cell-cycle synchronised U2OS cells using SLAM-seq

city: Copenhagen N

first: Mads

last: Lerdrup

address: Blegdamsvej 3C

department: Department of Cellular and Molecular Medicine

country: Denmark

institute: University of Copenhagen

summary: To assess changes in the nascent  and steady state transcriptomes occuring in a cell-cycle dependent manner, we performed SLAM-seq in the U2OS osteosarcoma cell line after FACS sorting according to cell cycle stage

overall_design: U2OS cells from G1, Early S, Late S, and G2/M cell cycle phases

first: Rahul

last: Bhowmick

first: Sampath

last: Gadi

first: Giacomo

last: Rossetti

first: Manika

middle: I

last: Singh

first: Ying

first: Thanos

last: Halazonetis

first: Ian

last: Hickson


title: A novel gene signature unveils three distinct immune-metabolic rewiring patterns conserved across diverse tumor types and associated with outcome

status: Public on Jun 26 2022

city: BARCELONA

first: Leire

last: Pedrosa

state: Barcelona

address: Carrer del Villarroel 170

institute: IDIBAPS

summary: Existing immune markers and tumor mutational burden signatures have modest predictive accuracies of immune-check-point inhibitors (ICI) therapeutic efficacy. In this study, we developed an immune metabolic signature suitable for personalized ICI therapies.  A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 75 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here we reveal that IMMETCOLS signature classifies tumors into 3 distinct immune-metabolic clusters. Cluster 1 displays enhanced glycolysis-Warburg, hexosamine biosynthesis and epithelial-to-mesenchymal biomarkers. On multivariate analysis, Cluster 1 tumors were enriched in pro-immune signature but not Immunophenoscore, and were associated with the poorest median survival. It’s predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival  and preferential use of alternative carbon sources, including glutamine and lipid uptake/β-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic, but also has a solid mitochondrial function, with concomitant up-regulation of glutamine and essential amino acids transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together these findings suggest that IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches

overall_design: 77 FFPE tissue samples from metastatic colorectal cancer (mCRC) tumours

first: Carles

last: Foguet

first: Helena

last: Oliveres

first: Iván

last: Archilla

first: Marta

last: García

first: Adela

last: Rodríguez

first: Antonio

last: Postigo

first: Daniel

last: Benítez-Ribas

first: Jordi

last: Camps

first: Miriam

last: Cuatrecasas

first: Antoni

last: Castells

first: Aleix

last: Prat

first: Timothy

last: Thomson

first: Joan

last: Maurel

last: Cascante


title: Regulation of nuclear actin levels and MRTF/SRF target gene expression during PC6.3 cell differentiation

city: Helsinki

first: Maria

last: Sokolova

address: Viikinkaari 5

department: Institute of Biotechnology

country: Finland

institute: University of Helsinki

summary: Actin has important functions in both cytoplasm and nucleus of the cell, with active nuclear transport mechanisms maintaining the cellular actin balance. Nuclear actin levels are subject to regulation during many cellular processes from cell differentiation to cancer. Here we show that nuclear actin levels increase upon differentiation of PC6.3 cells towards neuron-like cells. Photobleaching experiments demonstrate that this increase is due to decreased nuclear export of actin during cell differentiation. Increased nuclear actin levels lead to decreased nuclear localization of MRTF-A, a well-established transcription cofactor of SRF. In line with MRTF-A localization, transcriptomics analysis reveals that MRTF/SRF target gene expression is first transiently activated, but then substantially downregulated during PC6.3 cell differentiation. This study therefore describes a novel cellular context, where regulation of nuclear actin is utilized to tune MRTF/SRF target gene expression during cell differentiation.

overall_design: RNA-seq was performed in four biological replicates from undifferentiated PC6.3 cells and cells treated with 50 ng/ml NGF for 1-5 days.

first: Alise

last: Hyrskyluoto


title: Single-cell gene expression reveals the stage-specific roles of TCF1 in T cell development and T cell malignancy

city: Beijing

first: Shuyang

last: Yu

address: 2 Yuanmingyuan West Road

department: College of Biological Sciences

institute: China Agricultural University

summary: T cell factor 1 (TCF1; encoded by Tcf7) plays a critical role in T cell development and functions as a tumor suppressor by repressing the expression of LEF1 in the thymus. Here we reported the stage-specific regulation of TCF1 in early T cell differentiation and T cell malignancy despite the broad understand on the functions of TCF1. To illustrate the stage-specific functions of Tcf7 in T cell development and tumorigenesis, we applied single-cell transcriptional analysis to identify the precise trajectory of gene expression changes between the surface receptor-defined stages of T cell development.

overall_design: To systematically investigate the gene expression pattern in WT and stage-specific deletion of Tcf7, we sequenced 768 single cells from four genotype mice (WT, Tcf7-/-, Tcf7fl/flVav-cre, Tcf7fl/flLck-cre). The cell type of WT is DN1 (96 single cell) and DN2-3 (96 single cell). The cell type of Tcf7-/- is DN1 (96 single cell) and DN2-3 (96 single cell). The cell type of Tcf7fl/flVav-cre is DN1 (96 single cell) and DN2-3 (96 single cell). The cell type of Tcf7fl/flLck-cre is DN2-3 (192 single cell).

first: Fang

last: Wang


title: Heterotypic Influenza Infections Mitigate Susceptibility to Secondary Bacterial Infection

city: Pittsbrugh

first: John

last: Alcorn

address: 4401 Penn Ave

institute: UPMC Children's Hospital of Pittsburgh

summary: Influenza associated bacterial super-infections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory prior to adulthood. Due to this we sought to understand the role of immune memory during bacterial super-infections. An influenza heterotypic immunity model was established using influenza A/PR/8/34 and A/X31. We report here that influenza experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naïve animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer specific CD8+ T cells. The protection that was seen in memory experienced mouse model is associated with the reduction in inflammatory mechanisms making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity re-shapes the lung environment and the immune response to a re-challenge event, which is highly relevant to the context of human infection.

overall_design: Bulk-RNA Seq Transcriptomic analysis of mice at day 7 acute influenza and secondary MRSA infection or at Day 61 memory influenza and secondary MRSA infection.

first: Ellyse

last: Cipolla

middle: F


title: The Transcription Factor ThPOK Regulates ILC3 Lineage Homeostasis and Function during Intestinal Infection II

city: hangzhou

first: lie

last: wang

state: zhejiang

address: Zijingang Campus, Zhejiang University，Yuhangtang Road 866

department: immunology

institute: zhejiang universiry

summary: To investigate the role of ThPOK in the regulation of ILC3 development and function, we established conventional ThPOK knockout mice and carried out single-cell RNA sequencing  (BD Rhapsody) on intestinal ILC3s.

overall_design: ILC3s from 6-week-old male C57BL/6 mice were isolated from the small intestine and analyzed by single-cell RNA sequencing technology (BD Rhapsody).

first: Xianzhi

last: Gao

first: Lie


title: Single cell analysis of ROR-gamma-t-expressing immune cells in the mesenteric lymph nodes of healthy mice

first: Gregory

last: Putzel

summary: The focus of this study is to define the full spectrum of ROR-gamma-t-expressing immune cells in the mouse mesenteric lymph nodes of healthy mice. Results from this study may yield novel findings on the role of innate and adaptive lymphocytes in orchestrating immune tolerance, immunity and inflammation.

overall_design: ROR-gamma-t+ immune cells were sorted from the mesenteric lymph nodes of ROR-gamma-t-EGFP mice and mixed at a 1:1 ratio of TCRb+ cells to TCRb- cells, to enrich the non-T cell population. Cells were pooled from two mice.

first: Mengze

last: Lyu

last: Sonnenberg


title: TGF-b promotes stem-like T cells via enforcing their lymphoid tissue retention

status: Public on Jun 25 2022

city: San Antonio

first: Nu

last: Zhang

address: 7703 Floyd Curl Drive

institute: UTHSA

summary: Stem-like CD8+ T cells sustain the antigen-specific CD8+ T cell response during chronic antigen exposure. However, the signals that control the maintenance and differentiation of these cells are largely unknown. Here, we demonstrated that TGF-b was essential for the optimal maintenance of these cells and inhibited their differentiation into migratory effectors during chronic viral infection. Mechanistically, stem-like CD8+ T cells carried a unique expression pattern of a4 integrins (i.e., a4b1hi and a4b7lo) controlled by TGF-b. In the absence of TGF-b signaling, greatly enhanced expression of migration-related markers, including altered expression of a4 integrins led to enhanced egress of stem-like CD8+ T cells into the circulation accompanied by further differentiation into transitional states. Blocking a4 integrin significantly promoted their lymphoid tissue retention and therefore partially rescued the defective maintenance of Tcf-1+ subset in the absence of TGF-b signaling. Thus, TGF-b promotes the maintenance and inhibits the further differentiation of stem-like T cells at least partially via enforcing their lymphoid tissue residency.

overall_design:
Wild type and TGF-b receptor 2 conditional knockout (KO) P14 TCR transgenic CD8+ T cells were adoptively cotransferred into C57BL/6 recipients followed by LCMV Clone 13 infection. Day 15 post infection, pooled spleen and lymph node donor WT and KO P14 T cells were FACS sorted into 4 subsets based on the expression of Ly108 and CD69 (i.e., Ly108+CD69+, Ly108+CD69-, Ly108-CD69- and Ly108-CD69+). Total RNA was extracted from sorted T cells and subjected to RNA-seq analysis.

Please note that one of the samples (CL_WT_1) did not pass quality control and was excluded from data processing. Thus, the *genename.txt processed data files contain total 15 data columns.

first: Liwen

first: Chaoyu

last: Ma

first: Wei

last: Liao

first: Guo

last: Li


title: SF3B1 promotes glycolysis and tumor malignancy through splicing-independent activation of HIF1α [RNA-Seq murine organoids]

city: Zurich

first: Patrik

last: Simmler

address: Otto-Stern-Weg 7

country: Switzerland

institute: ETH Zurich

summary: Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated spliceosomal components in cancer. Recent studies additionally imply an involvement of the wildtype protein in tumor progression, but the underlaying mechanism remains elusive.  Here we report that SF3B1 acts as a coactivator for hypoxia-inducible factor (HIF) 1α. SF3B1 directly interacts with HIF1a, augments HIF1a-HIF1b heterodimer binding to hypoxia response elements in vivo and enhances transactivation of HIF target genes. In human patients, primary patient cells and in a mouse model of pancreatic ductal adenocarcinoma (PDAC), efficient HIF1a-signalling relies on SF3B1. Monoallelic deletion of Sf3b1 in this mouse tumor model severely delays tumor initiation and development and diminishes HIF target gene transcription, glucose uptake and proliferation in corresponding tumor organoids in hypoxia, while splicing remains functional. Here, we define a subset of aggressive chromophobe renal cell carcinomas (chRCCs) able to transcriptionally compensate monoallelic SF3B1 loss, a feature that correlates with poor prognosis and lymph node spread and we show that the oncogenic hotspot mutation SF3B1K700E has reduced co-activation efficiency. Also, we report increased sensitivity of cells with low levels of SF3B1 to the HIF-inhibitor PX-478. These findings reveal a splicing-independent, tumor promoting function of SF3B1, important in the pathogenesis of PDAC and other cancers.

overall_design: Multiple organoid lines (5x KPC, 4x KPCS) were seeded as described, where each line was derived from an individual mouse. For more informations on the establishment of organoid lines, please refer to the methods part of the manuscript. After 48 hours of exposure to 1% O2 or 21% O2, organoids were harvested and RNA was extracted using the NucleoSpin® RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. For each organoid line, 2-3 wells of organoids from a 24 well plate were harvested in lysis buffer RA1 (RNA extraction kit, MN) supplemented with TCEP (Tris(2-carboxyethyl)phosphine hydrochloride).

first: Patricia

last: Galliker

first: Cédric

last: Cortijo

first: Moritz

last: Schaefer


title: Overlapping transcriptional programs promote survival and axonal regeneration of injured retinal ganglion cells

city: Cambridge

first: Wenjun

last: Yan

address: 52 Oxford Street

institute: Harvard University

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

overall_design: Refer to individual Series


title: Gene expression profile of monocytes from healthy donor and triple negative breast cancer (TNBC) co-cultured with FAP and CD29 [bulk RNA-seq]

status: Public on Jun 24 2022

city: Paris

first: Paul

last: Gueguen

address: 26 rue D'Ulm

department: Inserm U932

institute: Institut Curie

summary:
These samples were either treatment naive or samples co-cultured with FAP and CD29.
Myeloid cell subpopulation comprises denditric cells (DC), monocytes and macrophages. We used RNA sequencing to analyze the response of monocytes to FAP/CD29 co-culture.

overall_design: Magnetically-enriched circulating CD14+ monocytes (CD14 Microbeads-Miltenyi) from HD (N=3) and TNBC (N=3) patients were co-culture with FAP+CD29+. After 3 hours of culture FACS-sorted monocytes were isolated by FACS-ARIA III (BD) cell sorter directly collected on lysing TCL buffer (Qiagen) containing 1%-mercatpoethanol before storage at -80°. RNA extraction was performed from ex vivo circulating isolated CD14+ and FAP+ CD29+ co-cultured monocytes by Single Cell RNA purification Kit (Norgen#, 51800) according to the manufacturer’s protocol

first: Timperi

last: Eleonora

first: Emanuela

last: Romano


title: Gene expression profile at single cell level of myeloid cells from juxta tumor (JT) and tumor (T) samples from treatment naive triple negative breast cancer (TNBC) patients

summary: Myeloid cell subpopulation comprises denditric cells (DC), monocytes and macrophages. We used single cell RNA sequencing (scRNA-seq) to analyze the heterogeneity of the myeloid cells, particularly to study tumor-assocaited macrophages in TNBC patients.

overall_design: JT and T myeloid cells from TNBC patients were isolated by FACS-sorting, collecting HLADR+CD11C+ double positive cells, excluding from the sorting strategy CD3, CD19, CD56 and dead cells. The collected cells were analyzed using scRNAseq.


title: Osteogenic transdifferentiation of primary human fibroblasts to osteoblast-like cells with human platelet lysate

city: Amsterdam

first: Chen

address: Meibergdreef 9

institute: Amsterdam UMC

summary: Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNA-seq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for human fibroblasts undergone osteogenic transdifferentiation

first: Ferdy

middle: K

last: Cayami

first: Lauria

last: Claeys

first: Ruben

last: de Ruiter

first: Bernard

last: Smilde

first: Lisanne

last: Wisse

first: Natalija

last: Bogunovic

first: Elise

last: Riesebos

first: Lyra

last: Eken

first: Irsan

last: Kooi

first: Erik

last: Sistermans

first: Nathalie

last: Bravenboer

first: Gerard

last: Pals

first: Sultana

last: Faradz

first: Daoud

last: Sie

first: Marelise

middle: W

last: Eekhoff

first: Dimitra

last: Micha


title: Transketolase in human Müller cells is critical to resist light stress through the pentose  phosphate and NRF2 pathways

city: Sydney

first: Ting

last: Zhang

state: NSW

address: 8 Macquarie Street

department: Save Sight Institute

country: Australia

institute: the University of Sydney

summary: The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic  pathway, provides protective metabolites and molecules essential for cell redox  balance and survival. Transketolase (TKT) is the critical enzyme that controls the  extent of “traffic flow” through the PPP. Here, we explored the role of TKT in  maintaining the health of the human retina. We found that Müller cells were the primary  retinal cell type expressing TKT in the human retina. We further explored the role of  TKT in human Müller cells by knocking down its expression in primary cultured Müller  cells (huPMCs), isolated from the human retina (11 human donors in total), under lightinduced  oxidative stress. TKT knockdown and light stress reduced TKT enzymatic  activities and the overall metabolic activities of huPMCs with no detectable cell death.  TKT knockdown restrained the PPP traffic flow, reduced the expression of NAD(P)H  Quinone Dehydrogenase 1 (NQO1) and impaired the antioxidative response of NRF2  to light stress. TKT knockdown also inhibited overall glucose intake, reduced  expression of Dihydrolipoamide dehydrogenase (DLD) and impaired the energy supply  of huPMCs. In summary, Müller cell-mediated TKT activity plays a critical protective  role in the stressed retina. Knockdown of TKT disrupted the PPP and impaired overall  glucose utilisation by huPMCs and rendered huPMCs more vulnerable to light stress  by impairing energy supply and antioxidative NRF2 responses.

overall_design: 12 mRNA profiles of human primary Müller cells cultured from 3 donors, including 4 groups with different treatments (DC, dim light TKT control, LC, strong light TKT control, DK dim light TKT knockdown, LK, strong light TKT knockdown)

first: Yingying

last: Chen

first: Shaoxue

last: Zeng

first: Rong

last: Xu

first: Kaiyu

last: Jin

first: Nathan

last: Coorey

first: Yekai

last: Wang

first: Meidong

last: Zhu

first: Xiaohui

last: Fan

first: Meixia

first: Jianhai

last: Du

first: Mark

middle: C

last: Gillies

first: Ling


title: Distinct cell adhesion signature defines glioblastoma myeloid-derived suppressor cell subsets

city: Cleveland

first: Katreya

last: Lovrenert

address: 10900 Euclid Avenue

department: Department of Genetics and Genome Sciences

institute: Case Western Reserve University

summary: Increased myeloid-derived suppressor cell (MDSC) frequency is associated with worse outcomes and poor therapeutic response in multiple types of cancer. In the glioblastoma (GBM) microenvironment, monocytic (m) MDSCs represent the predominant subset. However, the molecular basis of mMDSC enrichment in the tumor microenvironment compared to granulocytic (g) MDSCs has yet to be determined. Here we performed first broad epigenetic profiling of MDSC subsets to define underlying cell-intrinsic differences in their behavior and found that mMDSCs and gMDSCs display differences in their tumor-accelerating ability, with mMDSCs driving tumor growth in GBM models. Epigenetic assessments revealed enhanced gene accessibility for cell adhesion programs in mMDSCs and higher integrin b1 expression and function in mouse and human mMDSCs. Integrin b1 blockage abrogated the tumor-promoting phenotype of mMDSCs and altered the immune profile in the tumor microenvironment. These findings suggest that integrin b1 expression underlies the enrichment of mMDSCs in tumors and represents a putative immunotherapy target to attenuate myeloid cell-driven immune suppression in GBM.

overall_design: Assay for transposase-accessible chromatin and sequencing (ATAC-Seq) of granulocytic and monocytic myeloid-derived suppressor cells sorted from the bone marrow of sham-injected or GL261 cell-implanted male and female C57BL/6 mice. Two biological replicates per group, cells from two mice combined for each replicate.


title: Atypical mitotic DNA synthesis sequencing (MiDAS-seq) in U2OS cells depleted for RAD51

first: Mads

last: Lerdrup

summary: DNA replication stress is an established driver of cancer-associated chromosomal rearrangements. Replication stress perturbs the duplication of late-replicating loci and activates a mitotic DNA repair pathway (termed MiDAS) for completion of replication. We here investigated RAD51-independent MiDAS.

overall_design: MiDAS-seq on RAD51-depleted U2OS cells exposed to a low dose (0.4 µM) of APH in S-phase to induce replication stress

first: Rahul

last: Bhowmick

first: Sampath

middle: A

last: Gadi

first: Giacomo

last: Rossetti

first: Manika

last: Singh

first: Ying

last: Liu

first: Thanos

last: Halazonetis

first: Ian

last: Hickson


title: Adrenergic and mesenchymal neuoblastoma cells ChIP-Seq and RNA-Seq

city: Boston

first: Rani

last: George

state: Massachusetts

address: 450 Brookline Avenue

department: Pediatric Oncology

institute: Dana Farber Cancer Institute


title: Transcriptomic analysis of adrenergic and mesenchymal neuroblastoma cells using RNA-sequencing

summary: RNA-sequencing was performed on the following human neuroblastoma cell lines: Kelly, NBL-S, CHP-212, SH-SY5Y, SH-SY5Y LDK-resistant and SH-EP.

overall_design: Gene expression profiles of 6 neuroblastoma cell lines were determined using RNA-sequencing

first: Satyaki

last: Sengupta

first: Sanjukta

last: Das


title: KSHV Topologically Associating Domains in Latent Chromatin and Regulation of Reactivation

city: Sacramento

first: Clifford

middle: G.

last: Tepper

state: CA

address: 4645 2nd Avenue, Room 2300A

department: Biochemistry and Molecular Medicine

institute: UC Davis School of Medicine

summary: Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 base pair resolution and constructed a 3D KSHV genomic structural model. The latent KSHV genome formed very similar TAD structures among three different naturally infected PEL cell lines. When KSHV reactivation was triggered, genomic loops within TADs were dramatically decreased, while contacts extending outside of TAD borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D structural model proposes that the immediate-early promoter region is localized on the periphery of the 3D viral genome, while highly inducible non-coding RNA regions moved toward the inner space of the structure, resembling the coordination of a "bird cage" during reactivation. Finally, inhibition of the initial burst of lytic gene expression by stop codon insertion in the viral transactivator reduced genomic loops, while supplementing K-Rta expression in trans during establishment of latency attenuated the defect.  Our studies suggest that the latent 3D genomic structural information is embedded in the lytic gene transcription program.

sample_organism:
Homo sapiens
Human gammaherpesvirus 8

platform_organism:
Homo sapiens
Human gammaherpesvirus 8

overall_design: Capture Hi-C was utilized to analyze the fine details of the three-dimensional structure (chromatin loops, TADs) of the KSHV genome during latent infection and lytic reactivation in three human cell lines.

first: Mel

last: Campbell

first: Chanikarn

last: Chantarasrivong

first: Yuichi

last: Yanagihashi

first: Ryan

last: Davis

first: Yuanzhi

last: Lyu

first: Kazushi

last: Nakano

first: Yoshihiro

last: Izumiya


title: Embryonic heat conditioning in chicks induces transgenerational heat/immunological resilience via methylation on regulatory elements

city: Rishon Lezion

first: Tali

last: Rosenberg

address: Hamacabim

institute: ARO

type: Methylation profiling by high throughput sequencing

summary: We used in ovo embryonic heat conditioning (EHC) of first-generation chicks and assessed the effect on their untreated offspring by measuring genome-wide alterations in DNA-methylation patterns. DNA was extracted from the APH and reduced representation bisulfite sequencing (RRBS) was performed

overall_design:
DNA was isolated by TRI Reagent (Molecular Research Center). RRBS libraries were prepared using Zymo-Seq RRBS Library Kit (Zymo) and prepared from 70–140 ng of DNA. Sequencing was done on a NovaSeq 6000 instrument (Illumina) using an SP 100 cycle kit (paired end sequencing). Trimming of Illumina adapters, as well as quality trimming, were performed with Trim Galore, using the following options: --nextseq --non_directional –rrbs. Reads were mapped to the chick genome (galGal5.0) using Bismark v. 0-22-3, bowtie mode. Methylation calls were extracted with Bismark, methylation extractor mode.  DMS was extracted and analyzed by edgeR (39), with the recommended cutoff of at least 8 reads in every sample and FDR < 0.05. DMRs were extracted and analyzed by metilene v. 0.2-8. Motif enrichment analysis and DMS/DMR annotation were performed by HOMER tools. Segregating Hyper/Hypo-TADs according to TAD methylation was achieved considering only TADs with more than three DMSs. ChromHMM was used to establish a chromatin state model from an independent study that utilized bulk hypothalamic tissue, and to determine fold enrichment of DMS/DMR over state model.

APH mRNA profile of 10 day old noncoditioned F1 chicks, nonchallenged (t0)/6 hours into heat challenge/6 hours into LPS challenge,  by 3'-mRNA- sequencing (RNA-seq).

first: Asaf

last: Marco

first: Tatiana

last: Kisliouk

first: Noam

last: Meiri

first: Moran

last: Gershoni


title: MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis

city: Tampa

first: Prahathees

last: Eswara

state: Florida

address: 4202 E Fowler Ave ISA2015

institute: University of South Florida

summary:
To investigate the possible genes regulated by the DNA binding protein MraZ
The bacterial division and cell wall (dcw) cluster is a highly conserved region of the genome which encodes several essential cell division factors including the central divisome protein FtsZ. Understanding the regulation of this region is key to our overall understanding of the division process. mraZ is found at the 5’ end of the dcw cluster and previous studies have described MraZ as a sequence-specific DNA binding protein. In this article, we investigate MraZ to elucidate its role in Bacillus subtilis. Through our investigation, we demonstrate that increased levels of MraZ result in lethal filamentation due to repression of its own operon (mraZ-mraW-ftsL-pbpB). We observe rescue of filamentation upon decoupling ftsL expression, but not other genes in the operon, from MraZ control. Our data suggests that regulation of the mra operon may be an alternative way for cells to quickly arrest cytokinesis potentially during entry into stationary phase and in the event of DNA replication arrest. Furthermore, through timelapse microscopy we were able to identify that overexpression of mraZ or depletion of FtsL results in de-condensation of the FtsZ ring (Z-ring). Using fluorescent D-amino acid labelling, we also observed that coordinated peptidoglycan insertion at division site is dysregulated in the absence of FtsL. Thus, we reveal the precise role of FtsL is in Z-ring maturation and focusing septal peptidoglycan synthesis.

overall_design: Comparative analysis of RNA-seq data of wild type B. subtilis (PY79), del-mraZ (MW192), or overexpression strains of mraZ (MW189) and mutant mraZ-R15A (MW256)

first: Eswara

last: Prahathees


title: Role of FGF-2 induced non-coding RNA in cancer [II]

city: Gothenburg

first: Subazini

last: TK

address: Medicineragatan 9A

country: Sweden

institute: Gothenburg university

summary: We show that IER3 and IER3-AS1 protein coding and non-coding genes mediates oncogenic properties in cancer cell lines when they are induced by FGF-2 which is fibroblast growth factor. Comprehensive analysis of mechanisms behind the regulation of these genes by FGF-2 induction is shown in this study.

overall_design: Characterization of  FGF-2 regulated transcriptome in immortalized human embryonic kidney cells HEK293 and cancer cell lines such as HeLa and BT-549 in order to identify  common and uniquely enriched FGF-2 regulated differentially expressed genes and  biological pathways.

first: Meena

last: Kanduri

first: Chandrasekhar

last: Thankaswamy Kosalai


title: Role of hnRNPK in FGF-2 induced non-coding RNA in cancer

address: Bruna starket 16

department: Department of Laboratory Medicine

institute: Gothenburg University

summary: We show that IER3 and IER3-AS1 protein coding and non-coding genes mediates oncogenic properties in cancer cell lines when they are induced by FGF-2 which is fibroblast growth factor.  Mechanistic studies show that these two RNAs are regulated by hnRNPK. Hence, we investigated the role of hnRNPK by knocking down this gene and studying the changes in the global targets including IER3 and IER3-AS1.

overall_design: Functional characterization for hnRNPK was done using lenti viral shRNA knockdown strategies followed by RNA-seq in Hela cell lines. In this study, we sequenced total RNA, cytoplasmic RNA and Nuclear RNA for control siRNA and hnRNPKsi RNA samples.

first: Bharat

last: Prajapati


title: SHORT-ROOT Stabilizes PHOSPHATE1 to Regulate Phosphate Allocation in Arabidopsis

city: Shanghai

first: Mingguang

last: Lei

address: No. 3888, Chenhua Road

institute: Shanghai Center for Plant Stress Biology

summary: Coordinated distribution of Pi between roots and shoots is an important process that plants use to maintain Pi homeostasis. SHR (SHORT-ROOT) is well-characterized for its function in root radial patterning1-3. Here, we demonstrate a new role of SHR in controlling phosphate (Pi) allocation from roots to shoots by regulating PHOSPHATE1 (PHO1) in the root differentiation zone. We recovered a weak mutant allele of SHR in Arabidopsis which accumulates much less Pi in the shoot and shows constitutive Pi starvation response (PSR) under Pi-sufficient condition. Besides, Pi starvation suppresses SHR protein accumulation and releases its inhibition on the HD-ZIP Ⅲ transcription factor PHB. PHB accumulates and directly binds the promoter of PHO2 to upregulate its transcription, resulting in PHO1 degradation in the xylem-pole pericycle cells. Our findings reveal a previously unrecognized mechanism of how plants repress Pi translocation from roots to shoots in response to Pi starvation.

overall_design: RNA-seq reads were generated for Wild-type (3 replicates) and phod1 mutant (3 replicates) under nutrient-rich condition.

first: Xinlong

last: Xiao

first: Viswanathan

last: Satheesh


title: CSDE1 is a Post Transcriptional Regulator of the LDL Receptor

city: San Francisco

first: Angelo

last: Pelonero

address: 1650 Owens Street

department: Cardiovascular Disease Institute

institute: Gladstone Institutes (UCSF)

summary: The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current clinical therapies and known causes of familial hypercholesterolemia. We show that Cold Shock Domain-Containing Protein E1 (CSDE1) enhances hepatic LDLR mRNA decay via its 3’ untranslated region and regulates atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we found 40 specific regulators of the LDLR left unidentified by observational human genetics. Among these, we show that, in HepG2 cells, CSDE1 regulates the LDLR at least as strongly as the targets exploited by the best available clinical therapies: statins and PCSK9 inhibitors. Additionally, we show that hepatic gene silencing of Csde1 treats diet-induced dyslipidemia in mice similar to that of Pcsk9 silencing. Our results suggest the therapeutic potential of manipulating a newly identified factor in the post-transcriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. We anticipate that our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, will serve as a generalizable template for the identification of therapeutic targets in other human disease states.

overall_design: Mice were fed a high-fat (Paigen) diet or standard chow diet and treated and treated with an AAV8-delivered shRNA directed against CSDE1 or a scramble. Mice were subjected to bulk RNA-sequencing (3 CSDE1 and 3 scramble each for Paigen diet and chow diet).

first: Arun

last: Padmanabhan

first: Deepak

last: Srivastava

first: John

last: Chorba


title: The nascent transcriptome during G1, Early-s, Late-S, and G2/M cell cycle phases and atypical MiDAS-seq in RAD51 knockdown U2OS cells

city: Copenhagen N

first: Mads

last: Lerdrup

address: Blegdamsvej 3C

institute: University of Copenhagen


title: Multiplexed transcriptome-based profiling of drug combinations using deterministic barcoding in picolitre-sized droplets [dataset 3]

city: Heidelberg

first: Lukas

last: Mathur

address: Meyerhofstraße 1

department: Genome Biology

institute: EMBL

summary: K562 cells were perturbed with drug combinations for 12h in droplets. Subsequently cells were processed to generate liraries for RNA-Seq.

overall_design: Control experiments for the multiplex combinatorial drug screen in droplets. Experiments were performed in bulk, droplets or with ERCC spike ins to assess the performance of droplets-based drug screens using gene expression as a readout.

first: Bence

last: Szalai

first: Ngoc-Hien

last: Du

first: Jonathan

middle: J

last: Laundry

first: Vladimir

last: Benes

first: Christoph

last: Merten


title: Bulk RNA-Seq of cutaneous squamous cell carcinomas (cSCC) with paired normal controls

city: Stanford

first: Carolyn

last: Lee

address: 269 Campus Drive

institute: Stanford University School of Medicine

summary: RNA-Seq was performed on 8 cSCC-normal pairs

overall_design: Total RNA was then isolated from human skin organoids using QIAshredder (Qiagen) and Trizol (ThermoFisher) followed by DNA removal with the TURBO DNA-free kit (Ambion) according to the manufacturer’s instructions. RNA integrity was verified using an Agilent 2100 Bioanalyzer. RNA-Seq libraries were prepared with the mRNA Seq Sample Prep Kit (Illumina, Inc.) as recommended by the manufacturer. 101 bp paired-end sequencing reads were obtained using the Illumina HiSeq platform.

first: Kenneth

middle: Y

last: Tsai

first: Tomas

last: Bencomo

first: Kimal

last: Rajapakshe

first: Cristian

last: Coarfa


title: Systematic analysis of inflammation and pain pathways in a mouse model of gout

city: Wenzhou

first: Yi

last: Zhang

state: Zhejiang

address: Wenzhou Medical University

institute: Zhang Lab

summary: Gout is a prevalent and painful inflammatory arthritis, and its global burden continues to rise.  Intense pain induced by gout attacks is a major complication of gout. Treatment and long-term  management for gout patients remain challenging. Despite the discovery of many key immune  response and inflammatory genes and pathways in gout, the molecular mechanisms of gout  inflammation are still not entirely elucidated. Particularly, systematic studies of gout inflammation  and pain are lacking. Using a monosodium urate (MSU) crystals-induced gout model, we  performed genome-wide transcriptome analysis for the ankle joint, dorsal root ganglion (DRG),  and spinal cord of gout mice. Our results revealed transcriptional changes in both the joint and the  nervous system. Furthermore, through integrated analysis with public datasets of mouse  inflammatory pain and neuropathic pain models, human GWAS, osteoarthritis (OA), and  rheumatoid arthritis (RA), we identified common and unique features associated with acute gout  inflammation and severe pain.

overall_design: Monosodium urate (MSU) crystals were used as treatments. 1 x PBS were used as controls.  Ankle joint, dorsal root ganglion (DRG), and spinal cord (SC) were collected and RNA-sequencing (RNA-seq) was performed.

first: Yuanlan

last: Fan

first: Jiajun

last: Yang

first: Song

last: Xie

first: Jiangshuang

last: He

first: Shenghui

last: Huang

first: Jiawang

last: Chen

first: Shangying

last: Jiang

first: Lei

last: Yu

first: Yujuan

last: Zhou

first: Xuxia

last: Cao

first: Xiang

last: Ji