title: Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1b stimulation

status: Public on Jun 15 2023

city: TROMSOE

first: Casper

middle: Jan Elis

last: Wahlund

address: Hansine Hansens veg 18

department: Clinical Medicine

country: Norway

institute: UiT - The Arctic university of Norway

summary: We replicated the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermitted hypoxia alone or in combination with IL-1b to mimic VTE-associated inflammatory stimuli on primary monocytes. Our transcriptome analysis revealed that sustained hypoxia limited the pro-inflammatory response induced by IL-1b, and triggered a metabolic shift in the monocyte transcriptome. On the other hand, intermittent hypoxia alone had a modest effect, but in combination with IL-1b it significant altered the expression of 2207 genes. Notably, intermittent hypoxia enhanced the IL-1b -stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1. The RNA seq data are paired with validations on protein level in the published paper. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis.

overall_design:
Setup of intermittent hypoxia combined with inflammatory IL-1b stimulation of primary monocytes. RNA isolated from nine donors was pooled into three pools of 3 donors each before sequencing. DGE and GSEA analyses were performed to map the effects of inflammation and hypoxia separately and combined.
**RAW data not provided due to Norwegian laws on GDPR prevent us from publicly sharing the raw RNA sequences**

middle: J

first: Safak

last: Caglayan

first: Paulo

last: Czarnewski

first: John-Bjarne

last: Hansen

first: Omri

last: Snir


title: Cardiomyocyte progenitors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodelling after experimental myocardial infarction

status: Public on Jun 20 2023

city: Berlin

first: Karsten

last: Jürchott

address: Luisenstraße

department: Rahel Hirsch Center

country: Germany

institute: BIH at Charité

type: Expression profiling by high throughput sequencing

summary: Both naturally occurring and artificially engineered multipotent cardiac progenitor cells (CPCs) are associated with highly variable cardiomyocyte (CM) differentiation efficiencies, resulting in unpredictable and/or low CM yields. Therefore, CM-committed progenitors may be a more reliable cell product for therapeutic use. Here, we demonstrate that unipotent progenitors are part of the phenotype spectrum arising during direct reprogramming of neonatal cardiac fibroblasts towards CMs using lentiviral transduction of Gata4, Mef2c, Tbx5, and Myocd. Induced CM progenitors (iCMPs) simultaneously express CPC- and CM-specific proteins and can be selected via hairpin oligonucleotide molecular beacons targeting Myh6/7-mRNA–expressing cells. Purified iCMPs can be expanded extensively with preserved phenotype using ascorbic acid and give rise to CM-like cells in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevent CM loss, attenuate fibrotic scarring, and preserve ventricular function. Given the clinical failure of conventional cardiac cell therapy using adult stem cells, further translational development of this iCMP approach is warranted.

overall_design: Three sample groups were analyzed: (1) murine neonatal cardiac fibroblasts (CF, starting cells), (2) murine induced cardiomyocyte progenitors (iCMP, purified GMTMy-reprogrammed cells), (3) murine adult left ventricular heart tissue (AHT). Two biological replicates were included for each sample group.

first: Dipthi

middle: B

last: Somesh

first: Ulrike

last: Krüger

first: Marten

last: Jäger

first: Christof

last: Stamm


title: scRNA-seq, snRNA-seq and RNA-seq of mouse hepatocytes during liver maturation

status: Public on Jun 26 2023

city: Beijing

first: Cheng-ran

last: Xu

address: NO.5 YIHEYUAN ROAD HAIDIAN DISTRICT, BEIJING, P.R.CHINA

department: School of Basic Medical Sciences

country: China

institute: Peking University

summary: Hepatocytes are the predominant cell type in the liver and execute numerous essential biological functions. However, the crucial events and putative regulators during hepatocyte maturation require in-depth investigation. In this study, we performed single-cell and single-nucleus RNA-seq (scRNA-seq and snRNA-seq) to explore the developmental process of hepatocytes. We defined three maturation stages of postnatal hepatocytes, each of which establishes specific metabolic functions and exhibits distinct strategies for regulating proliferation. Hepatic zonation is gradually formed during hepatocyte maturation. The cells or nuclei of different ploidy exhibit zonation preferences in distribution, and asynchrony in the hepatocyte maturation pathway. In addition, combining gene regulatory network analysis and in vivo genetic manipulation, we identified critical maturation- and zonation-related transcription factors. This study not only delineates comprehensive transcriptomic profiles of hepatocyte maturation, but also presents a paradigm to identify functional genes in the development of hepatocyte maturation and zonation by combining genetic manipulation and measuring coordinates in a single-cell developmental trajectory.

overall_design: To investigate the precise developmental process of hepatocyte maturation, we performed mSTRT scRNA-seq of hepatocytes isolated from the Alb-Cre; Rosa-tdTomato mouse strain at embryonic day (E) 17.5, E19.5, P0, P3, P6, P9, P12, P15, P18, P24, P30 and P60. We also performed mSTRT scRNA-seq of 2N, 4N and 8N hepatocytes at P60 to investigate the heterogeneity of hepatocytes with different ploidy. To explore the maturation process of the nucleus with various ploidy, we isolated nuclei from hepatocytes at P0, P9, P18 and P60 and performed snRNA-seq. To validate the functions of TFs during hepatocyte maturation, we knocked out Nr2f2, Usf1, Xbp1 and Tef in hepatocytes and performed scRNA-seq using the Smart-seq 3 method. To validate that CD324 and CD73 are zonated cell surface markers for PV and CV hepatocytes, we performed bulk-cell RNA-seq to CD324+ cells and CD73+ cells, respectively.

first: Li

last: Yang

first: Xin

last: Wang

first: Jia-Xi

last: Zheng

first: Zi-Ran

first: Lin-Chen

last: Li

first: Yu-Long

last: Xiong

first: Bi-Chen

last: Zhou

first: Jie

last: Gao

first: Cheng-Ran


title: The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

city: Rehovot

first: Alexander

last: Maman

address: Herzl St 234

department: Molecular Genetics

country: Israel

institute: Weizmann Institute of Science

summary:
This SuperSeries is composed of the SubSeries listed below.
Abstract:
N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTC m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

overall_design: Refer to individual Series


title: RNA-seq of tissue biopsies obtained from metastatic lesions of patients with treatment-naïve pancreatic carcinoma at pre- and post-treatment with gemcitabine/nab-paclitaxel/indoximod.

status: Public on Jun 22 2023

city: Philadelphia

first: Gregory

middle: L

last: Beatty

state: PA

country: USA

institute: University of Pennsylvania

summary: To investigate the impact of first-line treatment with gemcitabine/nab-paclitaxel/indoximod on the transcriptional profile of metastatic lesions in patients with pancreatic carcinoma, we collected tissue biopsies at pre- and post- (8wks) treatment and performed RNAseq.

overall_design: Gene expression profiling analysis using data obtained from RNA-seq of tissue biopsies obtained from metastatic lesions of patients with treatment-naïve pancreatic carcinoma at pre- and post-treatment (8 wks) with gemcitabine/nab-paclitaxel/indoximod.

first: Beatty

last: Gregory


title: The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles  [m6A-Seq]

summary: N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTC m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

overall_design: poly-A- enriched RNA from SK1 S.cerevisiae during synchronized sporulation induction, for different deletion strains. Strains with induced auxin targeted protein degradation are indicated and were treated with either DMSO or IAA.

first: Imke

last: Ensinck

first: Schraga

last: Schwartz

first: Folkert

last: van Werven


title: Loss of PBAF Promotes Effector Differentiation and Limits CD8+ T Cell Exhaustion in Chronic Viral Infection and Cancer

status: Public on Jun 24 2023

city: Chicago

first: Weiguo

last: Cui

state: IL

address: 303 E Chicago Ave

department: Pathology

institute: Northwestern University

type: Genome binding/occupancy profiling by high throughput sequencing

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


title: The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles [RNA-Seq]

summary: N6-methyladenosine (m6A), the most abundant mRNA modification, is  deposited in mammals/drosophila/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast  MTC is critical for meiosis but was known to comprise three proteins, of which  two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The MTC’s mRNA interacting core, comprising Ime4, Mum2, Vir1, and Kar4, is critical for the m6A-independent function in meiosis, while the MTC’s catalytic activity is enabled by Slz1 during meiosis. Kar4 also has a mechanistically separate function from the MTC during mating. Our findings demonstrate that the yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and-independent functions, highlighting its complexity and paving the path to dissecting the multi-layered functions in mammals.

overall_design: poly-A- enriched RNA from SK1 S.cerevisiae during synchronized sporulation induction, for different deletion strains.


title: Paired single-cell host profiling with multiplex-tagged bacterial mutants reveals intracellular virulence-immune networks V

status: Public on Jun 21 2023

first: Noa

last: Bossel Ben-Moshe

address: Hertzl Street

institute: Weizmann

summary: Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA-sequencing (scRNA-seq) are increasingly used to study the host factors underlying diverse cellular phenotypes. But current approaches do not permit the simultaneous unbiased study of both host and bacterial factors during infection. Here, we developed scPAIR-seq, an approach to analyze both host and pathogen factors during infection by combining multiplex-tagged mutant bacterial library with scRNA-seq to identify mutant-specific changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of Salmonella Typhimurium secretion system effector mutants. We developed a pipeline to independently analyze redundancy between effectors and mutant-specific unique fingerprints, and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome.

overall_design: Analyzing transcriptional profile of BMDMs infected with S.Tm SPI-2 mutants

first: Ori

last: Heyman

first: Dror

last: Yehezkel

first: Neta

last: Blumberger

first: Gili

last: Rosenberg

first: Camilla

middle: C

last: Mattioli

first: Aryeh

last: Solomon

first: Dotan

last: Hoffman

last: Ben-Moshe

first: Roi

last: Avraham


title: Mediator 1 ablation induces enamel-to-hair lineage conversion through enhancer dynamics

city: San Francisco

first: Yuko

last: Oda

state: CA

address: 111N-MB 360 1700 Owens St, Suite 360

department: Endocrine Research

institute: VA Medical Center UCSF

summary:
Postnatal cell fate has been postulated to be primarily determined by the local tissue microenvironment. Here, we found that Mediator 1 (Med1) dependent epigenetic mechanisms dictate tissue-specific lineage commitment and progression of dental epithelia. Deletion of Med1, a key component of the Mediator complex linking enhancer activities to gene transcription, provokes a tissue extrinsic lineage shift, causing hair generation in the dental environment. Med1 deficiency gives rise to unusual hair growth via primitive cellular aggregates on incisors. Mechanistically, we found that Med1 establishes super-enhancers that control enamel lineage transcription factors in dental stem cells and their progenies. However, Med1 deficiency reshapes the enhancer landscapes and causes a switch from the dental epithelial transcriptional program towards hair and epidermis on incisors in vivo, and in dental epithelial stem cells in vitro. Med1 loss also provokes an increase in the number and size of enhancers. Interestingly, control dental epithelia already exhibit enhancers for hair and epidermal key transcription factors; these expand in size and transform into super-enhancers upon Med1 loss suggesting that these epigenetic mechanisms cause the transcriptomic and phenotypic shift towards epidermal and hair lineages. Thus, we propose a role for Med1 in safeguarding lineage specific enhancers, highlight the central role of enhancer accessibility and usage in lineage reprogramming and provide new insights into ectodermal regeneration.
Duplicate Chip-seq for dental tisses from head region of cervical loop (CLH) containing dental epithelial stem cells and tail region (CLT) including progenies that were dissected from mandible of 4 week old conditional Krt14CreMed1 knockout (cKO) mice or littermate control (Ctrl) mice. Chip-seq was conducted by using antibodies against Mediator 1 (Med1) and H3K27ac (H3).

overall_design: Yuko, Oda

first: Roman

last: Thaler


title: Chip-exo and Chip-seq of  human KRAB-ZNFs transduced in HEK 293T cells

city: Lausanne

first: Jonas

last: de Tribolet-Hardy

address: Bâtiment SV 0405

country: Switzerland

institute: EPFL

summary: Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) are one of the largest groups of transcription factors encoded by tetrapods, with 378 members in human alone. KZFP genes are often grouped in clusters reflecting amplification by gene and segment duplication since the gene family first emerged more than 400 million years ago. Previous work has revealed that many KZFPs recognize transposable element (TE)-embedded sequences as genomic targets, and that KZFPs facilitate the co-option of the regulatory potential of TEs for the benefit of the host. Here, we present a comprehensive survey of the genetic features and genomic targets of human KZFPs, notably completing past analyses by adding data on more than a hundred family members. General principles emerge from our study of the TE-KZFP regulatory system, which point to multipronged evolutionary mechanisms underlaid by highly complex and combinatorial modes of action with strong influences on human speciation.

overall_design: We transduced HEK 293T cells with doxycyclin-inducible plasmids encoding for human KRAB-ZFPs fused with 3 HA tags at the C-terminus. After verifying the correct integration and expression of the KZFP by Sanger sequencing and Western blot, we performed ChIP-exo and ChIP-seq and prepared a barcoded Illumina library for each sample. For the ChIP-exo, the libraries were pooled and sequenced 12 per lane on an Illumina Hiseq 2500 to a minimum depth of around 15 million 100 bp single-end reads. For the ChIP-seqs, all samples were pooled and sequenced on the same lane on an Hiseq 4000 to a depth of around 30 Million 75 bp paired-end reads. The reads were mapped to the hg19 assembly of the human genomes and peaks were identified using MACS. For the ChIP-exos we used a total input derived from random sampling from all samples in the study, as it was found to perform better than either the standard total input or the exonuclease treated total input. For the ChIP-seqs, either individual inputs or a pooled input for one sequencing lane was sequenced.

first: Christian

middle: W

last: Thorball

first: Romain

last: Forey

first: Evarist

last: Planet

first: Julien

last: Duc

first: Alexandre

last: Coudray

first: Bara

last: Khubieh

first: Sandra

last: Offner

first: Cyril

last: Pulver

first: Jacques

last: Fellay

first: Michael

last: Imbeault

first: Priscilla

last: Turelli

first: Didier

last: Trono


title: Modulation of the endocytic, bioenergetic and transcriptomic pathways subserve the neuroprotective, anti-amyloidogenic action of cleavage-specific tau 12A12mAb in Alzheimer’s Disease mouse model

status: Public on Jun 14 2023

city: Catania

first: Sebastiano

last: Cavallaro

address: Via Paolo Gaifami,18

country: Italy

institute: IRIB-CNR

summary: Beyond deficits in hippocampal-dependent episodic memory, Alzheimer’s Disease (AD) features sensory impairment in visual cognition consistent with extensive neuropathology in the retina. 12A12 is a monoclonal cleavage specific antibody (mAb) which in vivo selectively neutralizes the AD-relevant, harmful N-terminal 20-22kDa tau fragment(s) (i.e NH2htau) without altering the full-length normal protein. When systemically-injected into Tg2576 mouse model overexpressing a mutant form of Amyloid Precursor Protein (APP), APPK670/671L linked to early-onset familial AD, this conformation-specific tau mAb successfully neutralizes the NH2htau accumulating both in their brain and retina and, thus, markedly alleviates the phenotype-associated signs. By means of combined biochemical and metabolic and transcriptomic experimental approach, we report that 12A12mAb downregulates the steady state expression levels of APP and Beta-Secretase 1 (BACE-1) and, thus, limits the Amyloid beta (Aβ) production both in hippocampus and retina from this AD animal model. The endocytic (BIN1, RIN3) and bioenergetic (glycolisis and mitochondria) pathways controlling the cellular fate of APP processing towards the amyloidogenic route subserve the local, antibody-mediated anti-amyloidogenic action in vivo. Our results show for the first time that similar molecular and metabolic retino-cerebral pathways are modulated in coordinated fashion in response to 12A12mAb treatment to tackle the neurosensorial Aβ accumulation in AD neurodegeneration.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for hippocampi and retinas of wild-type, naive Tg-AD, Tg-AD+mAb mice.

first: Maria

last: Guarnaccia

first: Giovanna

last: Morello


title: Transciptomic analysis in HAV infected Huh7.5 cells

status: Public on Jun 13 2023

city: CHAPEL HILL

first: Jason

last: Whitmire

state: NC

address: 5062 Genetic Medicine Bldg

institute: UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL

summary: This study is to analyze the transcriptomic changes induced by hepatitis A virus infection in cells.

overall_design: Huh7.5 cells were mock infected or infected with HM175/p16 virus for 7 or 14 days. Total RNAs were extracted using mirVana kit. Libraries were prepared with Illumina TruSeq RNA Library Prep Kit and sequenced on Illumina Hi-Seq4000 sequencer. Three biological replicates were done for each condition.

first: You

last: Li

first: Tomoyuki

last: Shiota

first: Stanley

last: Lemon


title: Expanding the list of sequence-agnostic enzymes for chromatin conformation capture assays with S1 nuclease

city: Novosibirsk

first: Andrey

last: Popov

address: Academician Lavrentiev Avenue, 10

country: Russia

institute: Institute of Cytology and Genetics, SB RAS

summary: This study presents a novel approach for mapping global chromatin interactions using S1 nuclease, a sequence-agnostic enzyme. We develop and outline a protocol that leverages S1 nuclease's ability to effectively introduce breaks into both open and closed chromatin regions, allowing for comprehensive profiling of chromatin properties. Our S1 Hi-C method enables the preparation of high-quality Hi-C libraries, marking a significant advancement over previously established DNase I Hi-C protocols. Moreover, S1 nuclease's capability to fragment chromatin to mono-nucleosomes suggests the potential for mapping the three-dimensional organization of the genome at high resolution. This methodology holds promise for an improved understanding of chromatin state-dependent activities and may facilitate the development of new genomic methods.

overall_design:
Human K562 cells were grown in RPMI-1640 medium with 10% FBS and a penicillin/streptomycin mix. Human peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood samples, which were collected in tubes with EDTA. RBC lysis buffer (BioLegend) was used for erythrocyte removal according to the manufacturer’s instructions. Isolated PBMCs were processed immediately for crosslinking. We then obtained the Hi-C libraries following our own protocol.
*** Raw data not provided for the patient samples (PBMC) due to privacy concerns. ***

last: Gridina

first: Artem

last: Shadskiy

first: Nikita

last: Torgunakov

last: Kechin

first: Maxim

last: Filipenko

first: Evgeny

last: Khrapov

first: Oxana

last: Ryzhkova

first: Veniamin

last: Fishman


title: Paired single-cell host profiling with multiplex-tagged bacterial mutants reveals intracellular virulence-immune networks II

first: Noa

last: Bossel Ben-Moshe

overall_design: Examination of differntially expressed genes between uninfected and WT/∆sifA infected BMDMs

first: Ori

last: Heyman

first: Dror

last: Yehezkel

first: Neta

last: Blumberger

first: Gili

last: Rosenberg

first: Camilla

last: Mattioli

first: Aryeh

last: Solomon

first: Dotan

last: Hoffman

last: Ben-Moshe

first: Roi

last: Avraham


title: RAGE Inhibitor TTP488 (Azeliragon) Reduces Metastasis in Triple-Negative Breast Cancer

status: Public on Jun 09 2023

city: NW Washington

first: Barry

middle: I

last: Hudson

state: District of Columbia

address: 3970 Reservoir Road,

department: Oncology

institute: Georgetown University

summary: We tested the preclinical efficacy of two small molecule RAGE inhibitors (TTP488 (Azeliragon) and FPS-ZM1) against TNBC metastasis in the orthotopic xenograft MDA-MB-231/4175 - NSG model. Both TTP488 and FPS-ZM1 impaired TNBC metastasis in this lung metastatic breast cancer model, with TTP488 affecting metastasis to a greater degree than FPS-ZM1. Transcriptomic analysis of the primary tumors revealed that TTP488 and FPS-ZM1 displayed high concordance in gene expression changes affecting metastatic pathways.

overall_design: Comparison of gene expression changes in RAGE inhibitor treated primary tumor tissues and vehicle (DMSO) treated tumors.

first: Alec

last: McIntosh

first: Melinda

last: Magna


title: 3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions.

status: Public on Jun 07 2023

city: Sapporo

first: Tatsuya

last: Sato

state: Hokkaido

address: South-1, West-17, Chuo-ku

department: Department of Cellular Physiology and Signal Transduction

country: Japan

institute: Sapporo Medical University School of Medicine

summary: Adipose tissues are closely related to physiological functions and pathological conditions in most organs. Although differentiated 3T3-L1 preadipocytes have been used for in vitro adipose studies, the difference in cellular characteristics of adipogenic differentiation in two-dimensional (2D) culture and three-dimensional (3D) culture remain unclear. In this study, we evaluated gene expression patterns using RNA sequencing and metabolic functions using an extracellular flux analyzer in 3T3-L1 preadipocytes with and without adipogenic induction in 2D culture and 3D culture. In 2D culture, 565 up-regulated genes and 391 down-regulated genes were identified as differentially expressed genes (DEGs) by adipogenic induction of 3T3-L1 preadipocytes, whereas only 69 up-regulated genes and 59 down-regulated genes were identified as DEGs in 3D culture. Ingenuity Pathway Analysis (IPA) revealed that genes associated with lipid metabolism were identified as 2 out of the top 3 causal networks related to diseases and function in 3D spheroids, whereas only one network related to lipid metabolism was identified within the top 9 of these causal networks in the 2D planar cells, suggesting that adipogenic induction in the 3D culture condition exhibits a more adipocyte-specific gene expression pattern in 3T3-L1 preadipocytes. Real-time metabolic analysis revealed that the metabolic capacity shifted from glycolysis to mitochondrial respiration in differentiated 3T3-L1 cells in the 3D culture condition but not in those in the 2D cultured condition, suggesting that adipogenic differentiation in 3D culture induces a metabolic phenotype of well-differentiated adipocytes. Consistently, expression levels of mitochondria-encoded genes including mt-Nd6, mt-Cytb, and mt-Co1 were significantly increased by adipogenic induction of 3T3-L1 preadipocytes in 3D culture compared with those in 2D culture. Taken together, the findings suggest that induction of adipogenesis in 3D culture provides a more adipocyte-specific gene expression pattern and enhances mitochondrial respiration, resulting in more adipocyte-like cellular properties.

overall_design: Two independently prepared samples of 2D cultured 3T3-L1 cells or 3D cultured 3T3-L1 spheroids with or without adipogenic induction were subjected to RNA isolation, and total RNA was initially extracted from each sample. After checking the RNA content and quality, cDNA libraries were prepared, and their quality and quantity were validated. Thereafter, sequential steps including cluster generation, sequencing the multiplexed samples, image analysis, base calling, and quality filtering were carried out. The differentially expressed genes (DEGs, fold-change 2.0 and FDR adjusted P < 0.05 and q < 0.2) were statistically determined by using mapping of sequence data (Qiagen, Redwood City, CA, USA). To elucidate possible gene functions, gene ontology (GO) enrichment analysis as well as Ingenuity Pathway Analysis (IPA, Qiagen, USA) were performed.

first: Fumifito

last: Hikage

first: Hiroshi

last: Ohguro

first: Masato

last: Furuhashi


title: BRG1 defines a genomic subset of inflammatory genes transcriptionally controlled by the glucocorticoid receptor

status: Public on Jun 23 2023

city: Freising

first: Franziska

last: Greulich

address: Gregor-Mendel-Str. 2

department: Metabolic Programming

institute: TU München


title: Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone

city: Suita

first: Noriyuki

last: Tsumaki

state: Select One

address: 2-2 Yamadaoka

department: Department of Biochemistry and Molecular Biology

institute: Osaka University, Graduate School of Medicine

summary: The nucleus pulposus (NP) is composed of notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). CLCs and chondrocytes have been reported to be similar. However, the interactions between CLCs and NCs remain unclear. In this study, to clarify how cells in NP and chondrocytes are regulated, we performed single-cell RNA sequencing (scRNA-seq) analysis of articular cartilage (AC) and NP of cynomolgus monkeys and found that part of CLCs and articular chondrocytes had similar gene expression profiles. These cells were enriched for genes related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell–cell interaction analysis revealed sonic hedgehog (SHH) expression in NCs, resulting in hedgehog signaling to CLCs, whereas no chondrocytes in our AC samples expressed hedgehog ligands.

overall_design: We harvested the nucleus pulposus (NP) from the lumbar IVD (L3/4) and the articular cartilage (AC) from the left femoral condyle of a cynomolgus monkey (3 years old). Additionally, we harvested the AC from the left femoral condyle of another cynomolgus monkey (3 year old). We subjected these samples to scRNA-seq analysis

first: Hiroki

last: Hagizawa

first: Saeko

last: Koyamatsu


title: Unravelling the genetic determinants of sinusoidal obstruction syndrome: an integrative multi-omics approach

city: Geneva

first: Isabelle

last: Dupanloup

address: Rue Michel-Servet, 1

institute: University of Geneva

summary: We used bulk RNAseq to analyze gene expression changes in six lymphoblastoid cell lines before and after incubation with busulfan

overall_design: A set of six immortalized non-malignant lymphoblastoid cell lines (LCLs; GM7056, GM12239, GM12762, GM12057, GM12489, and GM12546), acquired in 2012 from the International HapMap Consortium’s CEPH Families Reference Panel 142011/147712 (Coriell Cell Repository, Camden, NJ, USA), were used for assessing the baseline and busulfan-induced gene expression profiles.

first: N

last: Waespe

first: S

last: Jurkovic Mlakar

first: I

first: T

last: Nava

first: M

last: Ansari


title: Transcriptomic profile in in vitro produced mouse late two-cell stage embryos treated with MYC inhibitor 10074-G5

status: Public on Jun 06 2023

city: KYOTO-SHI

first: Takuto

last: Yamamoto

state: (None)

address: Faculty of Agriculture main Bldg., Oiwake-tyo, Saikyo-ku

institute: Kyoto University

overall_design:
We obtained RNA-Seq transcriptomic profiles in mouse late two-cell stage embryos (36 h post-insemination) which had been in vitro treated with a myelocytomatosis oncogene (MYC) inhibitor 10074-G5 from one-cell stage (6 hpi).
The experiments were duplicated using ten embryos per group.

first: Haoxue

last: Wang

first: Hana

first: Shinnosuke

last: Honda

first: Shuntaro

last: Ikeda

first: Naojiro

last: Minami


title: Targeting KAT6A/KAT6B dependencies in breast cancer with a novel selective, orally bioavailable KAT6 inhibitor, CTx-648/PF-9363

status: Public on Jun 05 2023

city: San Diego

first: Chi-Yeh

last: Chung

address: 10770 Science Center Drive

institute: Pfizer Inc.


title: Prefrontal D2- dopamine receptor deactivates top-down inhibitory control by regulating striatal cholinergic interneurons

status: Public on Jun 17 2023

city: Toronto

first: Martin

last: Beaulieu

state: Ontario

address: 1 king's college circle room 4336

department: Pharmacology and Toxicology

country: Canada

institute: University of Toronto

summary: Inhibitory control is a core cognitive function preventing the expression of maladaptive behaviors. This function is overridden in mental illnesses including bipolar disorder, autism spectrum disorders and schizophrenia, causing maladaptive psychomotor agitation. We developed intersectional CRISPR/Cas9 targeting approaches in adult mice to identify receptor/circuit selective mechanisms responsible for inactivation of inhibitory control in two models of amphetamine-induced psychomotor agitation. Inactivation of dopamine D2-receptors in the medial-prefrontal cortex (mPFC) abolished amphetamine-induced: psychomotor agitation, motor sensitization, and cAMP-associated transcriptome changes in the striatum. Further characterization using intersectional projection preparations indicate that these deficits implicate D2-receptors expressed on projection neurons innervating cholinergic interneurons of the dorsomedial striatum (DMS). Trans-synaptic targeting of acetylcholine production in DMS neurons receiving these projections restored psychomotor agitation in mice lacking mPFC D2- receptor expression. These findings identify a circuit by which activation of cortical D2-receptor circumvent an acetylcholine-mediated inhibition of DMS functions by the mPFC to promote psychomotor activation.

overall_design:
To investigate the impact of mPFC D2R deficit on striatal responses to amphetamine, we performed RNA-seq analysis using mice that D2R was targeting using CRISPR/Cas9 strategy and received an acute administration of amphetamine (3mg/kg, i.p).
D2RCre_floxCas9-EGFP mice received virus injection in mPFC of AAV5 D2R-sgRNA (PFC_D2R-KO mice) or AAV5 syn-mCherry (control mice).
Treatment: 5 days of saline injection (ip) followed by a single injection of amphetamine (3mg/kg, ip) on the challenge day (day9).
4 groups: VV-Control = vehicle treatment in control mice; VA-Control = amphetamine treatment in control mice; VV-KO = vehicle treatment in PFC_D2R-KO mice; VA-KO = amphetamine treatment in PFC_D2R-KO mice.

first: Clementine

last: Quintana

first: Jean

middle: M


title: R-loop homeostasis and cancer mutagenesis promoted by the DNA cytosine deaminase APOBEC3B

status: Public on Jun 10 2023

city: Leicester

first: Michael

last: Tellier

state: Select State

address: Lancaster Road

department: Department of Molecular and Cell Biology

country: United Kingdom

institute: University of Leicester

summary: The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here, APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in human cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts overexpressed genes and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B mediates R-loop homeostasis and contributes to R-loop mutagenesis in cancer.

overall_design: In MCF10A cells: ChIP-seq of eGFP-tagged APOBEC3B (A3B-eGFP) in non-induced (DMSO), induced (DMSO + Doxycycline), and induced + PMA 2h (Doxycyline + PMA) conditions. DRIP-seq in WT or A3B-KO cells treated with DMSO, PMA 2h, or PMA 6h.

first: Jennifer

last: McCann

first: Agnese

last: Cristini

first: Emily

middle: K

last: Law

first: Seo

last: Yun Lee

middle: A

last: Carpenter

first: Chiara

last: Beghe

first: Jae

last: Jin Kim

first: Matthew

last: Jarvis

first: Bojana

last: Stefanovska

first: Nuri

last: Temiz

first: Erik

middle: N

last: Bergstrom

first: Daniel

last: Salamango

first: Margaret

middle: R

last: Brown

first: Shona

last: Murphy

first: Ludmil

last: Alexandrov

first: Kyle

last: Miller

first: Natalia

last: Gromak

first: Reuben

middle: S

last: Harris


title: Targeting KAT6A/KAT6B dependencies in breast cancer with a novel selective, orally bioavailable KAT6 inhibitor, CTx-648/PF-9363 [ATAC-Seq]

first: Chi-Yeh

last: Chung

summary: KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and regulate diverse biological processes including transcription, cell-cycle progression, and stem cell development.  KAT6A exerts an oncogenic role in several tumor types including breast cancer where it is amplified in 10-15% of patients. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a novel benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

overall_design: Human breast cancer cell lines were treated with DMSO or 30 nM PF9363 for 2 or 5 days in triplicates, and subjected to ATAC-seq analysis

first: Shikhar

last: Sharma

first: Showkhin

last: Khan

first: Thomas

last: Paul


title: Gene expression profile at single cell level of bone marrow cells in 5TGM1 multiple myeloma mouse model of NEK2 deficiency

status: Public on Jun 01 2023

city: Little Rock

first: Yan

last: Cheng

state: Arkansas

address: 4301 W. Markham St.

department: Internal Medicine

institute: University of Arkansas for Medical Sciences

summary: To determine how the lack of NEK2 in the tumor microenvironmental cells contributes to tumor inhibition and immune regulation, we used single cell RNA sequencing (scRNA-seq) to analyze the diversity of immune cells in the bone marrow of 5TGM1 MM mouse model with and without NEK2 deficiency.

overall_design: Nek2 knockout mice had been backcrossed with C57BL/KaLwRij mice for 10 generations to allow for 5TGM1 MM cell growth. One million 5TGM1 MM cells were injected into the Nek2 wild-type and knockout mice via tail vein. Bone marrow cells were harvested on day 30 post-5TGM1 cell injection and processed for scRNA-seq analysis.

first: Cheng

last: Yan

first: Zhan

last: Fenghuang


title: Effect of NEK2 deficiency on gene expression of CD19-selected spleen B cells of Eμ-myc mouse model

summary: NEK2 overexpression in the B cell lineage affects B cell development. To determine whether deficiency of NEK2 inhibits B cell malignancies, we crossed Nek2 knockout mice with Eμ-myc transgenic mice, a well-established Myc-driven preclinical model for B cell malignancies. Three genotyping offsprings were obtained: Eμ-myc/Nek2+/+ (NEK2 wild type), Eμ-myc/Nek2+/- (NEK2 heterozygotes), and Eμ-myc/Nek2-/- (NEK2 homozygotes). Bulk RNA-seq was performed on CD19-selected spleen B cells to view the transcriptome differences.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CD19-selected spleen B cells from 6-8 weeks old Eμ-myc mice with indicated NEK2 deficiency.


title: scRNA-seq data from two macaques in AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T-cells infected in vivo

city: Silver Spring

first: Rasmi

last: Thomas

state: MD

address: 503 Robert Grant Avenue

department: MHRP

institute: Walter Reed Army Institute of Research

summary: Persistent HIV-1 reservoirs of infected CD4 T-cells are a major barrier to HIV-1 cure, though the mechanisms by which they are established and maintained in vivo remain poorly characterized. To study host properties that govern productive cellular infection, we analyzed viral mRNA+ (vRNA) CD4 T-cells of untreated SIV-infected macaques by single-cell RNAseq. Transcriptionally active infected cells, defined by spliced vRNA, were highly enriched for vRNA: 10.3% and 7.5% of all mRNA in two animals. These cells expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells.

overall_design: Plate-based single cell expression profiles from sorted CD4 memory cells of two macaques during acute SIV infection

first: Aviva

last: Geretz

first: Phil

last: Ehrenberg

first: Diane

last: Bolton


title: Transcriptomic analysis of thoraces after overexpression of an active allele of REPTOR in muscle

city: Boston

first: Yanhui

last: Hu

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary:
Purpose: identify global changes in gene expression in thorax tissues caused by an increase in activity of REPTOR in muscles. Note that the thorax of adult flies is composed mainly by muscle tissue but fat body is also present. Changes in gene expression do not exclusively reflect the muscle transcriptome
Methods: To extract total RNAs for RNA-Seq experiment, we used 5-6 thoraces dissected out from both tub-Gal80ts/+ ; dMef2-GAL4/+ (Con) and tub-Gal80ts/UAS-REPTOR[ACT] ; dMef2-GAL4/+ (REPTOR), making sure the gut of these flies was completely removed. Crosses were kept at 18°C to avoid expression of REPTOR during development. Adult males were collected every 24-48 hours and incubated 3-4 days at 18°C before being shifted to 29°C. Flies were then incubated for 4 days at 29°C. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using. We multiplexed samples in each lane, which yields targeted number of single-end 75 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were mapped back to the Drosophila genome (flybase genome annotation version r6.30) using STAR. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, differentially expressed genes between experimental and control data were analyzed with DESeq2.
Results: Gene list enrichment analysis of the downregulated thoracic transcriptome by REPTOR overexpression revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, mitochondria, glycolysis and oxidative metabolism. Also, Thor, a well-characterized target of REPTOR was upregulated and it was validated with qPCR.
Conclusions: Our study indicates that REPTOR is a strong regulator of muscle metabolism in adult flies.

overall_design: Thoraces mRNA profiles of both tub-Gal80ts/+ ; dMef2-GAL4/+ (Con) and tub-Gal80ts/UAS-REPTOR[ACT] ; dMef2-GAL4/+ (REPTOR) flies incubated for 4 days at 29°C were generated by deep sequencing, in replicate, using Illumina HiSeq2000.

first: Pedro

last: Saavedra

first: Perrimon

last: Norbert


title: Transcriptomic analysis of World Trade Center particulate Matter-induced pulmonary inflammation and drug treatments

status: Public on May 31 2023

city: Hsinchu

first: Jinn-Moon

last: Yang

address: 75 Po-Ai Street

country: Taiwan

institute: National Chiao Tung University

summary: Over 400,000 people are estimated to have been exposed to World Trade Center particulate matter (WTCPM) since the attack on the Twin Towers in Lower Manhattan on September 11, 2001. Epidemiological studies have found that exposure to dust may cause respiratory ailments and cardiovascular diseases. However, limited studies have performed a systematic analysis of transcriptomic data to elucidate the biological responses to WTCPM exposure and the therapeutic options. Here, we developed an in vivo mouse exposure model of WTCPM and administered two drugs (i.e., rosoxacin and dexamethasone) to generate transcriptomic data from lung samples. WTCPM exposure increased the inflammation index, and this index was significantly reduced by both drugs. We analyzed the transcriptomics derived omics data using a hierarchical systems biology model (HiSBiM) with four levels, including system, subsystem, pathway, and gene analyses. Based on the selected differentially expressed genes (DEGs) from each group, WTCPM and the two drugs commonly affected the inflammatory responses, consistent with the inflammation index. Among these DEGs, the expression of 31 genes was affected by WTCPM exposure and consistently reversed by the two drugs, and these genes included Psme2, Cldn18, and Prkcd, which are involved in immune- and endocrine-related subsystems and pathways such as thyroid hormone synthesis, antigen processing and presentation, and leukocyte transendothelial migration. Furthermore, the two drugs reduced the inflammatory effects of WTCPM through distinct pathways, e.g., vascular-associated signaling by rosoxacin, whereas mTOR-dependent inflammatory signaling was found to be regulated by dexamethasone. To the best of our knowledge, this study constitutes the first investigation of transcriptomics data of WTCPM and an exploration of potential therapies. We believe that these findings provide strategies for the development of promising optional interventions and therapies for airborne particle exposure.

overall_design: The lung samples from 16 mice  belonging to four groups including WTCPM, WTCPM+ros, WTCPM+dex, and control (n = 4)

first: Yun-Ti

last: Chen

first: Jinhui

last: Li

first: Jen-Ning

last: Chang

first: Yong-Chun

last: Luo

first: Wuyue

last: Yu

first: LungChi


title: Osteoinductive biodegradable intramedullary implant accelerates bone healing and mitigates complications in a rat bone transport model

city: Stanford

first: Hirotsugu

last: Maekawa

address: 240 Pasteur Dr

institute: Stanford university

summary: Bone transport distraction osteogenesis (DO) is one of the most successful surgery-driven endogenous tissue regeneration approaches for the treatment of large bone defects. However, prolonged consolidation, docking site nonunion, and pin tract infection remain challenging complications. Here, we engineered an osteoinductive, biodegradable intramedullary (IM) implant for sustained release of bone morphogenetic protein-2 (BMP-2) as an adjunctive therapy of bone transport to address the clinical challenges. A hybrid tissue engineering construct (HyTEC) technique was developed to enable sustained release of BMP-2 in a broad range. 100% bony fusion was achieved in the IM implants incorporating with 2 μg and 6 μg BMP-2 as early as 34 days after bone transport surgeries in the management of 8-mm femoral defect. Load bearing was restored 55 days after surgeries when the fixator was removed. Eluting BMP-2 from the IM implants accelerates bone formation and angiogenesis at early phase, and increase mineralization at late phase, especially at the docking sites, leading to early bony bridging. Moreover, no pin tract infection but seamless integration could be found in the 2 μg and 6 μg BMP-2 treated groups. Surgical control and IM implant showed high proportion of non-union and pin tract infections. 2 μg BMP-2 delivered by collagen sponge or 0.5 μg BMP-2-laden IM implant did not induce bone regeneration effectively, resulting in some non-unions and infections. A presence of bacteria of fecal origin in the infection sites was identified. In conclusion, this osteoinductive IM implant holds great promise in revolutionizing bone transport DO technique in the management of bone defect by accelerating bone regeneration and mitigating complications.

overall_design: 8 mm bone defect and 4 mm segmental bone was created in rat femurs. Then rats were divided into three groups. For rats in control group, segmental bone was placed in the middle of defect site. Distraction osteogenesis and BMP2-eluting implant  was not used for these rats. Rats in blank group received distraction ostegenesis without BMP2-eluting implant. Rats in BMP2 group received distraction osteogenesis with BMP-2 eluting implant.

first: Sien

last: Lin

first: Yunzhi

middle: P


title: Epicardial Placement of Human Placental Membrane Protects from Heart Injury in a Swine Model of Myocardial Infarction

status: Public on May 30 2023

city: Tucson

first: Marissa

middle: Anne

last: Lopez-Pier

state: AZ

address: 1656 E. Mabel St.

department: Physiology

institute: The University of Arizona

summary: Introduction: Cardiac ischemic reperfusion injury (IRI) is paradoxically instigated by re-establishing blood-flow to ischemic myocardium typically from a myocardial infarction (MI). Although revascularization following MI remains the standard of care, effective strategies remain limited to prevent or attenuate IRI. We hypothesized that epicardial placement of human placental amnion/chorion (HPAC) grafts will protect against IRI.  Methods: Using a clinically relevant model of IRI, swine were subjected to 45 minutes percutaneous ischemia followed with (MI+HPAC, n=3) or without (MI only, n=3) HPAC. Cardiac function was assessed by echocardiography, and regional punch biopsies were collected 14 days post-operatively. A deep phenotyping approach was implemented by using histological interrogation and incorporating global proteomics and transcriptomics in non-ischemic, ischemic, and border zone biopsies.  Results: Our results established HPAC limited the extent of cardiac injury by 50% (11.02.0% vs 22.03.0%, p=0.039) and preserved ejection fraction in HPAC-treated swine (46.8±2.7% vs 35.8±4.5%, p=0.014). We present comprehensive transcriptome and proteome profiles of infarct (IZ), border (BZ), and remote (RZ) zone punch biopsies from swine myocardium during the proliferative cardiac repair phase 14 days post-MI. Both HPAC-treated and untreated tissues showed regional dynamic responses, whereas only HPAC-treated IZ revealed active immune and extracellular matrix remodeling. Decreased endoplasmic reticulum (ER)-dependent protein secretion and increased anti-apoptotic and anti-inflammatory responses were measured in HPAC-treated biopsies.  Discussion: We provide quantitative evidence HPAC reduced cardiac injury from MI in a preclinical swine model, establishing a potential new therapeutic strategy for IRI.

overall_design: comparative gene expression profiling analysis of RNA-seq data for porcine elicited or not elicited and myocardial infarction(MI) and treated(Tx) or untreated with HPAC.

first: Rinku

last: Skaria

last: Lopez-Schultz

first: John

last: Konhilas


title: A DEAD-box helicase drives the partitioning of a pro-differentiation NAB protein into nuclear foci

status: Public on May 28 2023

city: Cambridge

first: Dunaduan

last: Ma

state: Ma

address: 77 Massachusetts Ave

department: Koch Institute

institute: MIT

summary: How cells regulate gene expression in a precise spatiotemporal manner during organismal development is a fundamental question in biology. Although the role of transcriptional condensates in gene regulation has been established, little is known about the function and regulation of these molecular assemblies in the context of animal development and physiology. Here we show that the evolutionarily conserved DEAD-box helicase DDX-23 controls cell fate in Caenorhabditis elegans by binding to and facilitating the condensation of MAB-10, the Caenorhabditis elegans homolog of mammalian NGFI-A-binding (NAB) protein. MAB-10 is a transcriptional cofactor that functions with the early growth response (EGR) protein LIN-29 to regulate the transcription of genes required for exiting the cell cycle, terminal differentiation, and the larval-to-adult transition. We suggest that DEAD-box helicase proteins function more generally during animal development to control the condensation of NAB proteins important in cell identity and that this mechanism is evolutionarily conserved. In mammals, such a mechanism might underlie terminal cell differentiation and when misregulated might promote cancerous growth.

overall_design: RNA-seq experiment characterizing the transcriptome of C. elegans lin-29, mab-10, and ddx-23 mutant adult animals and comparing their gene expression profiles to that of wild-type adult animals. Experiment was performed with three biological replicates per strain.

first: Akiko

last: Doi

first: H

last: Horvitz


title: Transcriptional repression by a secondary DNA binding surface of DNA topoisomerase I safeguards against hypertranscription

status: Public on May 29 2023

city: Singapore

first: Zhenhua

last: hu

state: singapore

address: 31 Biopolis drive

country: Singapore

institute: IMCB, A*STAR

summary: Regulation of global transcription output is important for normal development and disease but only few regulators of the process are known. Here, we describe DNA topoisomerase I (TOP1) as a novel regulator where it exerts transcriptional repression to prevent transcription overdrive. This function requires a conserved secondary DNA binding surface that is distinct from its catalytic pocket.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA-seq of 293T cells transiently overexpressing TOP1 variants (wildtype and mutants at secondary DNA binding surface) or vector control, two mutant mouse embryonic stem cell (mESC) lines homozygous for Top1(R548Q) mutation and their parental wildtype line, and neurons derived from the corresponding mESC lines. For RNA-seq in 293T cells and mESCs, ERCC spike-ins were added to enable cell number normalization. PRO-seq in mutant and wildtype mESC. ChIP-seq of TOP1 and RNAP2S2P in mutant and wildtype mESC.

first: Mei

last: LAU

last: HU

first: Wee-Wei

last: TEE


title: A global view of aging and Alzheimer’s pathogenesis-associated cell population dynamics in mammalian brain

status: Public on May 26 2023

city: New York

first: Junyue

last: Cao

state: NY

address: 1230 York Avenue

institute: The Rockefeller University

summary: Prior studies applied single-cell approaches to explore gene expression changes in aged brains; however, they were limited by relatively shallow sampling of brain cell populations, and thus failed to characterize the detailed molecular signatures and dynamics of rare cell types associated with aging and diseases. Here, we set out to investigate the age-dependent transcriptional and chromatin dynamics across diverse brain cell types. With EasySci, an extensively improved single-cell combinatorial indexing strategy, we profiled ~1.5 million single-cell transcriptomes with full gene body coverage and ~400,000 single-cell chromatin accessibility profiles across mouse brains spanning different ages, genotypes, and both sexes. With a novel computational framework designed for characterizing cellular subtypes based on the expression of both genes and exons, we identified > 300 cell subtypes and deciphered the underlying molecular programs and spatial locations of rare cell types (e.g., pinealocytes, tanycytes). Leveraging the data, we generated a global readout of age-dependent cell population dynamics with great cellular subtype resolution, providing insights into expanded cell types (e.g., rare astrocytes and vascular leptomeningeal cells in olfactory bulb, reactive microglia and oligodendrocytes) and depleted cell types (e.g., neuronal progenitors, neuroblasts, committed oligodendrocyte precursors) in development and ageing. Furthermore, we explored cell-type-specific responses to genetic perturbations associated with Alzheimer’s disease and discovered rare cell types depleted (e.g., mt-Cytb+, mt-Rnr2+ choroid plexus epithelial cells) or enriched (e.g., Col25a1+ Ndrg1+ interbrain and midbrain neurons) in both AD models. Key findings are consistent between males and females, validated across the transcriptome, chromatin accessibility, and spatial analysis. In summary, these data comprise a rich resource for exploring cell-type-specific population dynamics and the underlying molecular mechanisms in the mammalian aging process.

overall_design:
Single cell RNA-seq and single cell ATAC-seq profiling for mouse brain cells spanning three age stages, in an early-onset AD model (5XFAD) and in a late-onset AD model (APOE*4/Trem2*R47H).
Single cell RNA-seq of human brain cells from the hippocampus and the superior and middle temporal gyrus (SMTG) regions comparing Alzheimer's disease and healthy control brain samples.

**Raw data were not provided for the human scRNA-seq Sample due to patient privacy concerns**

first: Andras

last: Sziraki

first: Ziyu

last: Lu

first: Jasper

last: Lee

first: Gabor

last: Banyai

first: Abdul

last: Abdul

first: Eli

last: Metzner

first: Andrew

last: Liao

first: Alexander

last: Epstein

first: Zihan

last: Xu

first: Zehao

last: Zhang

first: Peter

middle: T

last: Nelson

first: Wei

last: Zhou


title: FOXO1 dependent transcription network is a targetable vulnerability of Mantle Cell Lymphomas

status: Public on May 25 2023

city: New york

first: Jihye

last: Paik

address: 1300 York Ave.

institute: Weill Cornell Medical College

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

overall_design: Refer to individual Series


title: Targeting KAT6A/KAT6B dependencies in breast cancer with a novel selective, orally bioavailable KAT6 inhibitor, CTx-648/PF-9363 [RNA-Seq]

first: Chi-Yeh

last: Chung

overall_design: Human breast cancer cell lines were treated with DMSO or 30 nM PF9363 for 2 or 5 days in triplicates, and subjected to RNA-seq analysis

first: Shikhar

last: Sharma

first: Showkhin

last: Khan

first: Melissa

last: Davis

first: Thomas

last: Paul

first: Chin

last: Tan


title: Targeting KAT6A/KAT6B dependencies in breast cancer with a novel selective, orally bioavailable KAT6 inhibitor, CTx-648/PF-9363 [BrU-Seq]

overall_design: Human breast cancer cell lines were treated with DMSO or 30 nM PF9363 for 4 hr, 8 hr and 24 hr in triplicates, and subjected to BrU-seq analysis

first: Sean

last: Uryu


title: Time course of transcriptomic changes of thoraces comparing wild type and flies with gut yki-tumors during a tumor induction phase and tumor shutdown phase to analyze the recovery of peripheral tissue.

first: Yanhui

last: Hu

summary:
Purpose: identify global changes in gene expression in thorax tissues caused by the presence of gut tumors generated by yki expression in intestine stem cells. Note that the thorax of adult flies is composed mainly by muscle tissue but fat body is also present. Changes in gene expression do not exclusively reflect the muscle transcriptome
Methods: To extract total RNAs for RNA-Seq experiment, we used 8-10 thoraces dissected out from both esg-Gal4, tub-Gal80ts, UAS-GFP/+ (Con) and esg-Gal4, tub-Gal80ts, UAS-GFP/UAS-yki-act (yki) flies, making sure the gut of these flies was completely removed. Crosses were kept at 18°C to avoid expression of yki during development. Adult males were collected every 24-48 hours and then shifted to 29°C to induce gut yki-tumors. To assess transcriptional changes during the recovery phase, flies were moved to 18°C after a period of 14 days at 29°C in order to shutdown yki expression in the gut and suppress tumor growth. 6-day time points were analyzed during the recovery phase and included T20R, T26R, T32R and T38R. After assessing RNA quality with Agilent Bioanalyzer, rRNA depletion was performed. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using. We multiplexed samples in each lane, which yields targeted number of single-end 75 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were mapped back to the Drosophila genome (flybase genome annotation version r6.30) using STAR. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, differentially expressed genes between experimental and control data were analyzed with DESeq2.
Results: Gene list enrichment analysis of the downregulated muscle transcriptome by yki overexpression in intestine stem cells revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, amino acid metabolism, and energy metabolism. Interestingly, target genes of Foxo, a transcription factor inhibited by insulin/IGF signaling, are enriched in the upregulated thorax transcriptome of ISCs yki overexpression flies and downregulated during recovery (T20R, T26R, T32R and T38R). In particular, induction of InR and Thor, well-characterized targets of Foxo, are validated with qPCR. Also, the transcription factor REPTOR that is modulated by mTOR signaling and regulates metabolism, was found to be upregulated in thoraces of flies with yki-tumors and downregulated during recovery.
Conclusions: Our study discovered that the generation of yki-tumors in the gut drives the upregulation of REPTOR in the thorax of adult flies.

overall_design: Thoraces mRNA profiles of both esg-Gal4, tub-Gal80ts, UAS-GFP/+ (Con) and esg-Gal4, tub-Gal80ts, UAS-GFP/UAS-yki-act (yki) adult male flies during tumor induction (29°C) recovery phase (18°C) were generated by deep sequencing, in replicate.

first: Pedro

last: Saavedra

first: Perrimon

last: Norbert


title: FOXO1 dependent transcription network is a targetable vulnerability of Mantle Cell Lymphomas [ChIP-Seq]

summary: ChIP-seq analysis of CCMCL1 MCL cells

overall_design:
ChIP of FOXO1, EBF1, PAX5, IRF4, and H3K27Ac
ChIP of H3K27Ac, H3K4me1,IgG control

first: Inah

last: Hwang

first: Heng

last: Pan


title: Genome wide gene expression profiling of Col2.3GFP+ enriched osteoblasts

status: Public on May 24 2023

city: stanford

first: Hui

last: Zhu

address: 1651 Page Mill Road

institute: Stanford Universtiy

summary: We identified 593 candidate genes that are highly enriched in Col2.3GFP+ osteoblasts. Gene Ontology analysis revealed that these 593 genes are highly enriched in endochondral ossification, collagen fibril organization, positive regulation of osteoblast differentiation, and regulation of bone mineralization. The top 3 annotation terms are all associated with endoplasmic reticulum (ER) and Golgi vesicle transport. Furthermore, we have identified several vesicle trafficking genes with roles in osteoblast differentiation and function.

overall_design: Using Col2.3GFP transgenic mice that express green fluorescent protein (GFP) in osteoblasts, we isolated Col2.3GFP+ enriched osteoblasts from three sources: 1) in vivo freshly isolated from mouse long bone; 2) ex vivo isolated from mouse bone chip culture; and 3) in vitro directed differentiation from mouse embryonic stem cells (ESCs). We performed RNA-sequencing and identified 593 overlapping genes that are highly enriched in Col2.3GFP+ osteoblasts from all three sources.

first: Joy

last: Wu


title: Transcriptomic analysis of REPTOR or FoxO increased activity in muscle tissue using single nuclear RNA-seq analysis of Drosophila thoraces

summary:
Purpose: REPTOR and FoxO are two transcription factors that regulate muscle metabolism. However, these transcription factors share around 40% of target genes. Furthermore, the thorax of adult flies is composed of several tissues including muscle and fat body, making it difficult to discover direct target genes of REPTOR and FoxO specifically in muscle tissue. This experimental approach allows the identification of REPTOR-specific and FoxO-specific target genes in muscle clusters
Methods: snRNA-seq analysis of dissected adult fly thoraces, when an active allele of REPTOR or an active allele of FoxO are overexpressed using a muscle-specific driver (dMef2-Gal4)
Results: Identification of the transcriptional signature of each tissue present in the thorax of adult flies when REPTOR or FoxO are overexpressed in muscle. Discovery of potential direct target genes of REPTOR and FoxO in muscle tissue, as well as metabolic pathways regulated by each transcription factor.
Conclusions: REPTOR and FoxO modulate distinct gene signatures in muscle tissue to regulate metabolism in adult flies.

overall_design: The following genotypes were used: i) control: tub-Gal80ts/+ ; dMef2-GAL4/+. ii) REPTOR gain-of-function: tub-Gal80ts/UAS-REPTOR[ACT]; dMef2-GAL4/+. iii) FoxO gain-of-function: tub-Gal80ts/UAS-FoxO[ACT]; dMef2-GAL4/+. Two technical replicates were collected per genotype, and 30 thoraces were collected for each replicate

first: Weihang

last: Chen

first: Norbert

last: Perrimon


title: FOXO1 dependent transcription network is a targetable vulnerability of Mantle Cell Lymphomas [RNA-Seq]

first: Jihye

last: Paik

summary: RNA_seq analysis for FOXO1 depleted MCL cells

overall_design:
CCMCL1 human MCL line was FOXO1 depleted by CRISPR/Cas9 strategy using two independent sgRNAs
CCMCL1or JEKO1  human MCL line were depleted for indicated TFs by CRISPR/Cas9 strategy using two independent sgRNAs and RNAseq analysis was performed.


title: Effect of depletion of Myl3 in primary chondrocytes from mice

status: Public on May 16 2023

city: Guangzhou

first: he

last: cao

state: 广东

address: The Third Affiliated Hospital of Southern Medical University

summary: To investigate the function of Myl3 in in primary chondrocytes, we generated Col2a1-Cre: Myl3fl/fl (Myl3-KO) conditional knockout mice.We then performed RNA sequencing analysis with primary chondrocytes from Myl3-KO or control mice.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for primary chondrocytes from Myl3-KO or control mice.

first: He

last: Cao

first: Kai

last: Li


title: Elongation roadblocks mediated by dCas9 across human genes modulate transcription and nascent RNA processing

city: Lisboa

first: Rui

last: Sousa-Luís

address: Av. Professor Egas Moniz

country: Portugal

institute: Instituto de Medicina Molecular João Lobo Antunes

summary: Non-cleaving Cas9 (dCas9) is widely employed to manipulate specific gene loci often with scant regard for unintended transcriptional effects. We demonstrate here that dCas9 mediates precise transcriptional pausing followed by transcription termination and potential alternative polyadenylation. In contrast, alternative splicing is unaffected, likely requiring more sustained alteration to RNA polymerase II elongation speed. The effect on transcription is orientation-specific, with pausing only being induced when dCas9-associated guide RNA anneals to the non-template strand. Targeting template strand induces minimal effects on transcription elongation and thus provides a neutral approach to recruit dCas9 linked effector domains to specific gene regions. In essence we evaluate molecular effects of targeting dCas9 to mammalian transcription units. In so doing we also provide new information on transcriptional elongation by RNA polymerase II and coupled pre-mRNA processing.

overall_design: Prior to libraries preparation, HeLa cells were transfected with CTRL or gene-specific CRISPRi constructs targeting TXNRD1 intron (in2), TXNRD1 downstream area (DS2) or a set of alternatively polyadenylated gene (APA).

first: Inna

last: Zukher

first: Gwendal

last: Dujardin

first: Nick

last: Proudfoot


title: Transcriptomic RNA-sequencing analysis of the hypervirulent Clostridioides difficile R20291 strain in response to trehalose

city: Belfast

first: Andrew

last: Marshall

state: Select...

address: 19 Chlorine Gardens

institute: Queen's University Belfast

summary: Clostridioides difficile, the leading cause of antibiotic-associated diarrhoea worldwide, is a genetically diverse species which can metabolise a number of nutrient sources upon colonising a dysbiotic gut environment. Trehalose, a disaccharide sugar consisting of two glucose molecules bonded by an α 1,1-glycosidic bond, has been hypothesised to be involved in the emergence of C. difficile hypervirulence due to its increased utilisation by the RT027 and RT078 strains. Using RNA-sequencing analysis, we report the identification of a putative trehalose metabolism pathway which is induced during growth in trehalose: this has not been previously described within the C. difficile species. These data demonstrate the metabolic diversity exhibited by C. difficile which warrants further investigation to elucidate the molecular basis of trehalose metabolism within this important gut pathogen.

overall_design: Comparing the transcriptomic response of C. difficile during growth in trehalose (n=3) as compared to glucose grown conditions (n=3)

first: Geoff

last: McMullan


title: Incongruence between transcriptional and vascular pathophysiological cell states II

status: Public on May 12 2023

city: Madrid

last: Benedito

state: Madrid

address: C. de Melchor Fernández Almagro, 3

country: Spain

institute: CNIC

summary: The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or the ligand Dll4 dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch, inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTor, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes, and related pathophysiology. Our findings also suggest that vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

overall_design: Different experiments were performed. In the  Experiment 1, endothelial cells from mouse heart, lung, liver and brain with Dll4 loss and control conditions  were isolated by Fluorescence-activated cell sorting (FACS) according to high CD31 expression and were used for bulkRNAseq. In the Experiment 2, mouse liver endothelial cells from livers treated with Dll4 blocking antibodies and mouse IgG (control samples) were isolated by Fluorescence-activated cell sorting (FACS) according to high CD31 expression and were used for bulkRNAseq. In the Experiment 3 Cdh5-CreERT2 + iSuRe-Cre+ mouse liver endothelial cells with and without Rbpj loss were isolated  by FACS according to CD31+ MbTomato expression and were used for bulkRNAseq.

first: Macarena

last: Fernández-Chacón

first: Severin

last: Mühleder

first: Álvaro

last: Regano

first: Lourdes

last: Garcia-Ortega

first: Susana

middle: F

last: Rocha

first: Carlos

last: Torroja

first: Maria

last: Sanchez-Muñoz

first: Mariya

last: Lytvyn

first: Verónica

last: Casquero-Garcia

last: De Andrés-Laguillo

first: Lars

last: Muhl

first: Michael

last: Orlich

first: Konstantin

last: Gaengel

first: Emilio

last: Camafeita

first: Jesus

last: Vazquez

first: Alberto

last: Benguría

first: M

last: Iruela-Arispe

first: Ana

last: Dopazo

first: Fátima

last: Sánchez-Cabo

first: Hannah

last: Carter


title: Incongruence between transcriptional and vascular pathophysiological cell states

first: Rui

last: Benedito


title: Tissue confinement regulates cell growth and size in epithelia

city: Chicago

first: John

last: Devany

state: IL

address: 929 E 57th St

department: Physics

institute: The University of Chicago

summary: Cell proliferation is a central process in tissue development, homeostasis and disease. Yet how proliferation is regulated in the tissue context remains poorly understood. Here, we introduce a quantitative framework to elucidate how tissue growth dynamics regulate cell proliferation. We show that a limiting rate of tissue expansion creates confinement that suppresses cell growth; however, this confinement does not directly affect the cell cycle. This leads to uncoupling between rates of cell growth and division in epithelia and, thereby, reduces cell volume. Division becomes arrested at a minimal cell volume, which is consistent across diverse epithelia in vivo. Here, the nucleus approaches the minimum volume capable of packaging the genome. The loss of Cyclin D1-dependent cell volume regulation results in an abnormally high nuclear-to-cytoplasmic volume ratio and DNA damage. Overall, we demonstrate how epithelial proliferation is regulated by the interplay between tissue confinement and cell volume regulation.

overall_design: Epithelial monolayers were produced with cells overexpressing p27 or p27ck - this lead to a ~3 fold change in final cell volume. Transcription was assayed to look for a transcriptional response to changes in cell volume.

first: Martin

last: Falk

first: Liam

last: Holt

first: Arvind

last: Murugan

first: Maragaret

last: Gardel


title: Bap1 promotes osteoclast function by metabolic reprogramming

status: Public on May 15 2023

city: St Louis

first: nidhi

last: rohatgi

state: MO

address: 660 S Euclid Ave

department: pathology and immunology

institute: washington university in st. louis

summary: The deubiquitinase activity of Bap1 modifies osteoclast function by metabolic reprogramming. Bap1 deficient osteoclast lineage cells upregulate the cystine transporter, Slc7a11, by enhanced H2Aub occupancy of its promoter. SLC7A11 controls cellular ROS levels and redirects the mitochondrial metabolites away from the TCA cycle, both of which are necessary for osteoclast function.

overall_design: Bone marrow macrophages from Bap1flox and Bap1LysM mice were treated with MCSF and RANKL for 24 hrs. RNA was extracted for RNAseq study

first: Nidhi

last: Rohatgi

first: Steven

last: Teitelbaum


title: Mouse Thyroid Nanopore RNAseq

city: Planegg-Martinsried

first: Irina

last: Solovei

address: Großhaderner Str. 2

institute: LMU Munich

summary: In the present work, we pull attention to a remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so called transcription loop easily observable with light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both of the alleles remain highly active over the entire cell life, i.e. depending on a species, for months or years. We demonstrate that the high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We prove that Tg is not influenced by the organismal levels of T3/T4 hormones, does not oscillate round the clock and is expressed during both exocrine and endocrine phases of the thyrocyte activity. We conclude that the thyroglobulin gene represents a valuable model to study maintenance of a high transcriptional upregulation.

overall_design: We performed RNA sequencing to investigate intron retention in Tg transcripts. For this three thyroid glands were isolated and mRNAs were analyzed using Nanopore sequencing.

first: Helmut

last: Blum

first: Stefan

last: Krebs

first: Simon

last: Ullrich


title: Characterization of epigenetic regulators, HepA and LaeA, in Aspergillus luchuensis mut. kawachii

status: Public on May 22 2023

city: Kagoshima

first: Taiki

last: Futagami

address: Korimoto 1-21-24

department: Department of Agriculture

institute: Kagoshima University

summary:
To investigate the roles of epigenetic regulator homologs, heterochromatin protein 1 (HepA) and a putative methyltransferase (LaeA), in the citric acid produciton by Aspergillus luchuensis mut. kawachii, we constructed the ΔhepA and ΔlaeA strains.
We then performed gene expression profiling analysis using data obtained from RNA-seq of Aspergillus luchuensis mut. kawachii control, ΔhepA, and ΔlaeA strains.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Aspergillus luchuensis mut. kawachii control, ΔhepA, and ΔlaeA strains.

first: Atsushi

last: Nishitani

first: Kentaro

last: Hiramatsu

first: Chihiro

last: Kadooka


title: Incongruence between transcriptional and vascular pathophysiological cell states I

first: Rui

last: Benedito

summary: The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or the ligand Dll4 induces angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTor, had no effect on the vascular expansion induced by Dll4 loss, however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes, and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

overall_design:
Liver endothelial cells of the Cdh5-CreERT2 + iSuRe-Cre mice (with or without different floxed alleles) were isolated by Fluorescence-activated cell sorting (FACS) according to their CD31+ (ECs) profile and the presence of iSuRe-Cre reporter Tomato signal (induced by Cdh5-CreERT2 - expressed only in ECs). These cells were analyzed using scRNA-seq.

Liver non Endothelial Cells of the Cdh5-CreERT2 + iSuRe-Cre mice were isolated by Fluorescence-activated cell sorting (FACS) after gating on the CD31Negative population with varying levels of CD45 (Neg, Lo and Hi) and analyzed using scRNAseq.

first: Álvaro

last: Regano

first: Carlos

last: Torroja

first: Macarena

last: Fernández-Chacón

first: Severin

last: Mühleder

first: Lourdes

last: Garcia-Ortega

first: Susana

last: Rocha

first: Maria

last: Sanchez-Muñoz

first: Mariya

last: Lytvyn

first: Verónica

last: Casquero-Garcia

last: De Andrés-Laguillo

first: Lars

last: Muhl

first: Michael

last: Orlich

first: Konstantin

last: Gaengel

first: Emilio

last: Camafeita

first: Jesus

last: Vazquez

first: Alberto

last: Benguría

first: M. Luisa

last: Iruela-Arispe

first: Ana

last: Dopazo

first: Fátima

last: Sánchez-Cabo

first: Hannah

last: Carter


title: Context dependent function of transcriptional regulator Rap1 in gene silencing and activation [ChIP-seq III]

status: Public on May 09 2023

city: Berkeley

first: Eliana

middle: Rose

last: Bondra

state: California

address: 440 Barker Hall

department: Molecular and Cell Biology

institute: University of California, Berkeley

summary: As part of a study on the mechanism of Sir-based silencing at HML in S.cerevisiae, and the contribution of Rap1 to transcription of the locus, we performed ChIP-seq of multiple components of RNA Pol II machinery in different genetic backgrounds including: sir4∆, SIR+, and with or without a mutation that abbrogated Rap1 binding at the alpha promoter.

overall_design: Taf1-3xFLAG ChIPseq; Sua7-3xFLAG ChIPseq; Elf1-3xFLAG ChIPseq; Rpb3-3xFLAG ChIPseq in silenced (SIR) or unsilenced (sir4∆) cells, with or without a mutation in the alpha2 promoter.

first: Jasper

middle: D

last: Rine


title: Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development

status: Public on May 05 2023

city: York

first: harry

middle: victor

last: isaacs

state: North Yorkshire

address: wentworth way

department: biology

institute: university of york

summary: Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. We have proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. Here we investigate the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. We characterise Xenopus Cic and show that it is widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. A similar mechanism of Cic protein degradation downstream of the Torso receptor tyrosine kinase receptor negatively regulates Cic activity in Drosophila. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. We investigate two of these genes, fos and rasl11b, which, respectively, encode a leucine zipper transcription factor and a ras family GTPase, as putative targets of the proposed Fgf/Erk/Cic axis. We identify Cic consensus binding sites in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, we show that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. Our data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding.

overall_design: We undertook triplicate RNA-seq analysis on control water injected, Fgf4 overexpressing and cic knockdown Xenopus tropicalis embryos at neurula stage 14

first: Harry

middle: V

last: Isaacs


title: Context dependent function of transcriptional regulator Rap1 in gene silencing and activation [ChIP-seq II]

summary: As part of a study on the function of Rap1 at HML and MAT in Saccharomyces cerevisiae, we performed ChIP-seq on myc-tagged Sir3 in cells with and without a Rap1 binding site mutation at the promoter.

overall_design: Sir3-13xMyc ChIP-seq. Replicates are included for each experiment.


title: VprBP/DCAF1 triggers melanomagenic gene silencing through histone H2A phosphorylation

city: Los Angeles

first: Yonghwan

last: Shin

state: CA

address: 1450 Biggy St

department: Biochemistry and Molecular Medicine

institute: University of Southern California

summary: DCAF1, also known as VprBP, is a recently identified atypical kinase and plays an important role in downregulating the transcription of tumor suppressor genes as well as increasing the risk for colon and prostate cancers. Melanoma is the most aggressive form of skin cancer arising from pigment-producing melanocytes and is often associated with dysregulation of epigenetic factors targeting histones. Here we demonstrate that DCAF1 is highly expressed and phosphorylates threonine 120 (T120) on histone H2A to drive transcriptional inactivation of growth regulatory genes in melanoma cells. As is the case for its epigenetic function in other types of cancers, DCAF1 acts to induce gene silencing program dependently of H2AT120 phosphorylation (H2AT120p). The significance of DCAF1-mediated H2AT120p is further underscored by the fact that DCAF1 knockdown- or DCAF1 inhibitor-induced lockage of H2AT120p mitigates melanoma tumor growth in xenograft models. Collectively, our results establish DCAF1-mediated H2AT120p as a key epigenetic signal for melanomagenesis and suggest the therapeutic potential of targeting DCAF1 kinase activity for effective melanoma treatment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for G361 cells  (shCont, and shDCAF1).

first: Woojin

last: An


title: Copy Number Variation data from non-trasformed human fibroblasts

status: Public on May 08 2023

city: Gliwice

first: Joanna

last: Tobiasz

address: Akademicka 2A

department: Department of Data Science and Engineering

country: Poland

institute: Silesian University of Technology

type: Genome variation profiling by genome tiling array

summary:
The search for biomarkers to predict radiosensitivity is important not only to individualize radiotherapy of cancer patients but also to forecast radiation exposure risks. The aim of this study was to devise a machine learning method to stratify radiosensitivity and to investigate its association with copy number variations (CNVs) as markers of sensitivity to ionizing radiation.
We used the Affymetrix CytoScan HD microarrays to for CNV estimation. Celluar radiosensitivity was measured by the clonogenic surviving fraction at 2 Gy (SF2).

overall_design: Fibroblast cell cultures were maintained in DMEM culture medium supplemented with 15% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin and incubated at 37 °C in 5% CO2 humidified atmosphere.

first: Najla

last: Al-Harbi

first: Sara

last: Bin Judia

first: Salma

last: Majid Wakil

last: Polanska

first: Ghazi

last: Alsbeih


title: Context dependent function of transcriptional regulator Rap1 in gene silencing and activation [ChIPseq I]

summary: As part of a study on the role of Rap1 at the promoters of HML and MAT in S cerevisiae, we endogenously tagged Rap1 with a 3xV5 tag and performed ChIPseq in a variety of genetic backgrounds including: silenced (SIR); unsilenced (sir1∆/sir3∆/sir4∆); in strains witha wild type alpha2 promoter (wtpromoter); or with a point mutation at the promoter (mutpromoter).

overall_design: 3xV5-Rap1 ChIPseq. Replicates are included for every sample.


title: A multi-stem cell basis for craniosynostosis and calvarial mineralization

status: Public on May 21 2023

city: NEW YORK

first: Lingling

last: Hu

state: New York

address: 1300 York Avenue LC-902

institute: Weill Cornell Medicine

summary: FACS-sorted 2 bulk cell populations from mouse calvarial sutures (P7) were subjected to RNA sequencing. The samples were CTSK+ calvarial stem cells (labeled as PSC here) and DDR2+ calvarial stem cells isolated from CTSK-Cre;mTmG mice (n=5). The aim of this study was to obtain differentially expressed gene list for two different cell types as well as compare transcriptional profile between these populations.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for two calvarial stem cells

first: Seoyeon

last: Bok

first: Matthew

middle: B

last: Greenblatt


title: Optogenetic perturbations of RNA expression in tissue space

status: Public on May 04 2023

city: Berlin

first: Ivano

last: Legnini

address: Hannoversche Strasse 28

department: Berlin Institute for Medical Systems Biology

institute: Max Delbrueck Center for Molecular Medicine in the Helmholtz Association

summary: Quantifying gene expression in space, for example by spatial transcriptomics, is essential for describing the biology of cells and their interactions in complex tissues. Perturbation experiments, at single-cell resolution and conditional on both space and time, are necessary for dissecting the molecular mechanisms of these interactions. To this aim, we combined optogenetics and CRISPR technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatial patterns. As a proof of principle, we optogenetically induced Sonic Hedgehog (SHH) signaling at a distinct spatial location within human neural organoids. This robustly induced known SHH spatial domains of gene expression – cell-autonomously and across the entire organoid. In principle, our approach can be used to induce or knock down RNAs from any gene of interest in specific spatial locations or patterns of complex biological systems.

overall_design: Total RNA-seq for two replicates of HEK293T cells transfected with plasmids encoding: Rfx-Cas13d (CasRx), a TetON TagRFP reporter, and either a non-targeting guide RNA or an RFP-targeting RNA. 10X Visium sequencing data from 4 samples of human induced pluripotent stem cells (hiPSCs) stably expressing a light-inducible Cre/Lox system and a LoxP-dsRed-LoxP-SHH-NeonGreen cassette for light-inducible overexpression of SHH. The 4 samples are: non-induced cells and cells induced for 36, 48, 120h. Drop-seq single-cell RNA-seq data from 4 samples of 15-days old neural organoids, stably expressing a light-inducible Cre/Lox system and a LoxP-dsRed-LoxP-SHH-NeonGreen cassette for light-inducible overexpression of SHH. The samples are two replicates of controls (non-induced) and two replicates of SHH-induced organoids pools (3 organoids per pool).

first: Lisa

last: Emmenegger

first: Alessandra

last: Zappulo

first: Ricardo

last: Wurmus

first: Anna

last: Oliveras Martinez

first: Cledi

last: Cerda Jara

first: Anastasiya

last: Boltengagen

first: Talé

last: Hessler

first: Guido

last: Mastrobuoni

first: Agnieszka

last: Rybak-Wolf

first: Stefan

last: Kempa

first: Robert

last: Zinzen

first: Andrew

last: Woehler

first: Nikolaus

last: Rajewsky


title: Gene expression profiling of nasal inflammatory dysfunctions induced by diesel particles using in vivo system.

status: Public on May 18 2023

city: Daejeon

first: Su-Jin

last: Baek

address: 1672, Yuseong-daero

country: South Korea

institute: KOREA INSTITUTE of ORIENTAL MEDICINE

summary: Genome-wide gene expression profiles in 5- and 10-days KDP20 exposure (n=8) compared to control (n=4) were performed for nasal inflammatory dysfunction induced by diesel particles, one of the notorious ambient pollutants.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mouse model exposed to KDP20 (Vehicle, KDP20(Day5), KDP20(Day10)).

first: Chan-Sik

last: Kim

first: Bongkyun

last: Park


title: The secreted neuronal signal Spock1 regulates the blood-brain barrier

status: Public on May 01 2023

city: Boston

first: Natasha

last: O'Brown

address: 200 Longwood Avenue, WAB 536

department: Systems Biology

institute: Harvard Medical School

summary: The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restricts its permeability to proteins and small molecules. Classic chick-quail chimera studies showed that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally-expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1  into spock1 mutants quenches gelatinase activity, restores vascular expression of BBB genes including mcamb,  and partially restores barrier function. These analyses support a model in which neuronally secreted Spock1 induces BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression.

overall_design: Bulk RNAseq (Libraries 66-71) of leaky mutant and wild type siblings to identify the genetic lesion responsible for the leaky phenotype. These analyses revealed linkage to chr14, and more specifically to the spock1 gene. scRNAseq (Library scDRBrain) of dissected spock1 mutant and wild type brains was then used to identify all cell-type specific changes in gene expression in the mutant background.

first: O'Brown

last: Natasha

first: Patel

last: Nikit


title: Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function [RNA-Seq]

status: Public on May 02 2023

city: Madison

first: Melissa

last: Harrison

email: mharrison3@wisc.edu, ykaur@wisc.edu, torreszelada@wisc.edu, mmfreund2@wisc.edu

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

summary: While chromatin presents a barrier to the binding of many transcription factors, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type specific binding and activity. The mechanisms governing pioneer-factor occupancy and the relationship between chromatin occupancy and opening remain unclear. We studied three Drosophila transcription factors with distinct DNA-binding domains and biological functions: Zelda, Grainy head, and Twist. We demonstrated that the level of chromatin occupancy is a key determinant of activity. Multiple factors regulate occupancy, including motif content, local chromatin, and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. Our results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features.

overall_design: RNA-seq on cells ectopically expressing Drosophila transcription factors.

first: Tyler

middle: J

last: Gibson


title: Protein-intrinsic properties and context-dependent effects regulate pioneer-factor binding and function

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

overall_design: Refer to individual Series


title: MED12 Mutation Activates Tryptophan-Kynurenine-AHR pathway and Promotes Cell Growth in Human Uterine Leiomyoma [RNA-seq]

first: Priyam

last: Patel

address: 750 North Lake Shore Drive, Rubloff 11-146

department: Quantitative Data Science Core

institute: Northwestern University

summary: Uterine leiomyoma (LM) is the most common tumor in women. Estrogen and progesterone, via their receptors ERα and PR, play essential roles in LM growth. Mediator complex subunit 12 (MED12) mutations occur in 70% of all LM and are thought to drive tumor growth in a steroid hormone-dependent manner; however, the mechanisms remain unclear. Here, we performed ChIP-seq (ERα, PR, and MED12) and RNA-seq on LM expressing mutant MED12 (mut-MED12) or wild-type MED12 and matched myometrium. Mut-MED12 altered PR and chromatin interaction landscapes, with significant PR-binding site loss in proximal promoter regions in mut-MED12 LM. Integration of cistrome and transcriptome data identified tryptophan 2,3-dioxygenase (TDO2) as a PR and MED12 target gene, which was aberrantly upregulated in mut-MED12 LM. Kynurenine, the catabolic product of TDO2, was significantly elevated in mut-MED12 LM. Tryptophan or kynurenine treatment of primary LM cells activated the aryl hydrocarbon receptor (AHR) pathway, increased cell proliferation, and inhibited apoptosis; blocking the TDO2-kynurenine-AHR pathway by siRNA knockdown or pharmacologic inhibition abolished these effects. Mut-MED12 LM cells showed higher sensitivity to these treatments. These findings suggest that activation of the TDO2-kynurenine-AHR pathway in mut-MED12 LM induces tumor growth, and may inform the development of targeted treatments and precision medicine in LM.

overall_design: Transcriptome profiling of leiomyoma of different genotype and their matched myometrial tissues

first: Azna

last: Zuberi

first: Yongchao

last: Huang

first: Ariel

last: Dotts

first: John

last: Coon V

first: Shimeng

last: Liu

first: Takashi

last: Lizuka

first: Yang

last: Dai

first: Ping

last: Yin

first: Serdar

middle: E

last: Bulun


title: MED12 Mutation Activates Tryptophan-Kynurenine-AHR pathway and Promotes Cell Growth in Human Uterine Leiomyoma.


title: MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas

overall_design: ChIP-seq of PR, ERa, and MED12 of leiomyoma of different genotype and their matched myometrial tissues