title: Human heart-forming organoids recapitulate early heart and foregut development - single-cell RNA sequencing data

status: Public on Dec 17 2020

city: Hannover

first: Robert

last: Zweigerdt

address: Carl-Neuberg-Str. 1

institute: Hannover Medical School

summary: Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.

overall_design: Single-cell RNA sequencing of two HFOs derived from the HES3 NKX2.5-eGFP reporter line on day 13 of differentiation.

first: Lika

last: Drakhlis

first: Santoshi

last: Biswanath

first: Clara-Milena

last: Farr

first: Victoria

last: Lupanow

first: Jana

last: Teske

first: Katharina

last: Ritzenhoff

first: Annika

last: Franke

first: Felix

last: Manstein

first: Emiliano

last: Bolesani

first: Henning

last: Kempf

first: Simone

last: Liebscher

first: Katja

last: Schenke-Layland

first: Jan

last: Hegermann

first: Lena

last: Nolte

first: Heiko

last: Meyer

first: Jeanne

last: de la Roche

first: Stefan

last: Thiemann

first: Christian

last: Wahl-Schott

first: Ulrich

last: Martin


title: Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide [ChIP-Seq]

status: Public on Dec 22 2020

city: Shanghai

first: Fei

last: Li

address: 320 Yue-Yang Road Shanghai

country: China

institute: Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat due to the lack of effective drugs or vaccines against SARS-CoV-2. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a second second-generation antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry into host cells, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and SARS-CoV-2-infected Ad-ACE2-transduced Tmprss2 knockout (Tmprss2-KO) and wild-type (WT) mice. TMPRSS2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cells, however, such antiviral efficacy of enzalutamide was lacking in human lung cells and organoids. Although Tmprss2 knockout effectively blocked SARS-CoV-2 infection in ACE2-transduced mice, Accordingly, enzalutamide showed no antiviral activity due to the AR-independent TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19 through reducing TMPRSS2 expression in lung cells.

overall_design: AR-ChIP-seq was performed in AR-positive lung cancer cells A549, H2126 and H1437 and prostate cancer cells LNCaP


title: Inducible stem cell-derived embryos capture mouse morphogenetic events in vitro

status: Public on Dec 20 2020

city: Cambridge

first: Florian

last: Hollfelder

address: 80 tennis court road

country: United Kingdom

institute: University of Cambridge

type: Expression profiling by high throughput sequencing

summary: We report the analysis by inDrop single cell sequencing of mouse embryos at embryonic day E5.5 and E6.5 as well as the analysis of stem cell derived synthetic embryos at day 4 of development generated with the standard protocol (Sozen et al., 2018) and the new protocol reported in this study. We find that the visceral endoderm-like layer of the synthetic embryos generated with the new protocol is much more similar to the visceral endoderm of mouse embryos than the layer of the synthetic embryos generated with the standard protocol, and this new and improved visceral endoderm-like layer endows these novel synthetic embryos with broader developmental potential

overall_design: Analysis of similarities and differences in the transcriptomes among mouse embryos and stem cell derived synthetic embryos, generated with the standard protocol (Sozen et al., 2018) and the new protocol established in this study

first: Gianluca

last: Amadei

first: Joachim

last: De Jonghe

first: Berna

last: Sozen


title: Epithelial expressions of Gata4 and Sox2 regulate murine specification of the squamous–columnar junction via MAPK/ERK signaling

city: Tokyo

first: Yasuhiro

last: Yamada

address: 7-3-1 Hongo, Bunkyo-ku

department: Department of Molecular Pathology

country: Japan

institute: University of Tokyo

summary: The squamous–columnar junction (SCJ) is a boundary consisting of precisely positioned transitional epithelium between the squamous and columnar epithelium. Transitional epithelium is a hotspot for precancerous lesions, and is therefore clinically important; however, the origins and physiological properties of transitional epithelium have not been fully elucidated. Here, by using mouse genetics, lineage tracing, and organoid culture, we examine the development of the SCJ in the mouse stomach, and thus define the unique features of transitional epithelium. We find that two transcription factors, encoded by Sox2 and Gata4, specify primitive transitional epithelium into squamous and columnar epithelium. The proximal–distal segregation of Sox2 and Gata4 expression establishes the boundary of the unspecified transitional epithelium between committed squamous and columnar epithelium. Mechanistically, Gata4-mediated expression of the morphogen Fgf10 in the distal stomach and Sox2-mediated Fgfr2 expression in the proximal stomach induce the intermediate regional activation of MAPK/ERK, which prevents the differentiation of transitional epithelial cells at the SCJ boundary. Our results have implications for tissue regeneration and tumorigenesis, which are related to the SCJ.

overall_design: Analyzing transcriptional profiles in Sox2hi, Sox2midGata4mid, Gata4hi epithelial cells  at E11.5 and E13.5 and fibroblasts at E13.5, E15.5 and E17.5.

first: Takuya

last: Yamamoto

first: Yoshiya

last: Kawaguchi


title: scRNA profiles on hESC-derived lung cells

status: Public on Dec 16 2020

city: Chicago

first: Joyce

middle: huanhuan

last: Chen

state: IL

address: 5801 S Ellis Ave, Chicago

department: Pritzker School of Molecular Engineering

country: USA

institute: Uchicago

summary: Study transcriptomes on the lung cells derived from hESC at day50

first: Huanhuan

middle: J

first: Qizhou

last: Lian

first: Fuyu

last: Duan


title: Human heart-forming organoids recapitulate early heart and foregut development - microarray data

first: Robert

last: Zweigerdt

country: Germany

overall_design: Micoarray analysis of five HES3 NKX2.5-eGFP-derived HFOs on d13 of differentiation, three NKX2.5-KO HFOs on d13, three hPSC samples, three samples of cardiomyocytes from conventional 2D cardiac differentiation, and four human embryonic hearts (5 - 7 weeks of gestation)

first: Lika

last: Drakhlis

first: Santoshi

last: Biswanath

first: Clara-Milena

last: Farr

first: Victoria

last: Lupanow

first: Jana

last: Teske

first: Katharina

last: Ritzenhoff

first: Annika

last: Franke

first: Felix

last: Manstein

first: Emiliano

last: Bolesani

first: Henning

last: Kempf

first: Simone

last: Liebscher

first: Katja

last: Schenke-Layland

first: Jan

last: Hegermann

first: Lena

last: Nolte

first: Heiko


title: Whole brain transcriptome analysis of PTEN C-terminus mutant mice

city: Daejeon

first: Eunjoon

last: Kim

address: Kuseong-dong

department: Center for Synaptic Brain Dysfunctions/Department of Biological Sciences

country: South Korea

institute: IBS/KAIST

summary: PTEN contains a C-terminal motif that binds to various PDZ domain-containing proteins including PSD-95. The PTEN C-terminus is known to mediate synaptic translocation of PTEN during long-term depression (LTD) and amyloid-b-induced synaptic depression. However, it remains unknown whether it regulates other synaptic functions. We compared the transcriptomic profiles of WT and PTEN C-terminus mutant brains (male) at P21. Here, we report genes that are differentially regulated in the mutant mice. This RNA-Seq analysis revealed a total of nine major differentially expressed genes (DEGs) –three upregulated and six downregulated (P < 0.05). Further gene set enrichment analysis (GSEA) of PTEN C-terminus mutant and WT transcriptomes using 5917 gene sets in the C5 (gene ontology) category revealed two strongly enriched gene ontology functions: ribosome biogenesis (positive) and transmembrane transport (negative). Our results provide a comprehensive transcriptome characterization of PTEN C-terminus mutant mice, which would provide a framework of understanding the role PTEN c-terminus in mediating various synaptic –and in larger sense, cellular –functions, and also allow thorough comparative analysis with the results of other PTEN mutant mouse lines.

overall_design: Whole brain transcriptome of P21 wild-type and PTEN C-terminus mutant mice.

first: Mihyun

last: Bae

first: Junyeop

middle: D

last: Roh

first: Hyojin

last: Kang


title: Transcriptomic analysis of mouse RTN neurons

status: Public on Dec 15 2020

city: Charlottesville

first: Douglas

last: Bayliss

state: VA

address: 1340 Jefferson Park Ave.

department: Pharmacology

institute: University of Virginia

summary: We report data from single cell sequencing analysis of individual RTN neurons harvested from brainstem slices

overall_design: To provide a description of the transcriptome of RTN neurons

first: Yingtang

last: Shi

middle: A

first: Suna

last: Onengut

first: Stephen

last: Turner


title: Progressive accumulation of resting cells ensures the lifelong maintenance of the hippocampal stem cell population

city: Brisbane

first: Lachlan

middle: Ian

last: Harris

state: QLD

address: 300 Herston Road

department: Cancer Research Program

country: Australia

institute: QIMR Berghofer MRI

summary: Neural stem cell numbers fall rapidly in the hippocampus of juvenile mice but stabilise during adulthood, ensuring lifelong hippocampal neurogenesis. We show that this reduction in stem cell depletion rate in young adults is the result of coordinated changes in stem cell behaviour. In particular, while proliferating neural stem cells in juveniles differentiate rapidly, they increasingly return to a resting state of shallow quiescence and progress through additional self-renewing divisions in adulthood. Single-cell transcriptomic, modelling and label-retention analyses indicate that resting cells have a higher activation rate and greater contribution to neurogenesis than dormant cells, which have not left quiescence. These progressive changes in stem cell behaviour result from reduced expression of the pro-activation protein ASCL1 due to increased post-translational degradation. These cellular mechanisms help reconcile current contradictory models of hippocampal NSC dynamics and may contribute to the different rates of decline of hippocampal neurogenesis in mammalian species including humans.

overall_design: In vivo hippocampal neural stem cells were purified through a transgenic mouse line. The mouse line expressed Nestin-GFP, and was injected with tamoxifen for 6 days, before culling on the 8th day to recombine tdTomato expression in proliferating cells, using the Ki67-creERT2 transgene. Cells were analysed by 10x Genomics Chromium Single Cell 3' sequencing. 8 samples were analysed in total, across 3 age groups: 1 month, 2 months and 6-8 months old. In total dataset there are 26,036 cells of which 2,947 are hippocampal neural stem cells.

first: Piero

last: Rigo

first: Thomas

last: Stiehl

first: Zachary

last: Gaber

first: Sophie

last: Austin

first: Maria

last: del Mar Masdeu

first: Amelia

last: Edwards

first: Noelia

last: Urban

first: Anna

last: Marciniak-Czochra

first: Francois

last: Guillemot


title: Mexican Ganoderma lucidum extracts decrease lipogenesis modulating transcriptional metabolic networks and gut microbiota in C57BL/6 mice fed with a high-cholesterol diet from early Drosophila embryo

city: Mexico

first: Sandra

middle: Lorena

last: Romero-Cordoba

state: Mexico

address: Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan. México, D.F.

department: Cancer Genomics

country: Mexico

institute: National Instute of Genomic Medicine

summary:
Background: Prevention of hyperlipidemia and associated diseases is a health priority. Complementary medicine based on scientific evidence has recently recognized the potential of natural products for modulating lipid metabolism, such as the medicinal mushroom Ganoderma lucidum (Gl), which possesses hypocholesterolemic, prebiotic and antidiabetic properties.
Methods: Whole-transcriptomic changes in liver and kidney from a mouse model (C57BL/6), under a high-cholesterol diet and standardized Gl extracts (Gl-1, Gl-2) or simvastatin administration, were analyzed to determine Gl hypocholesterolemic activity. Further effects of Gl extracts on lipid metabolism were evaluated using an in vitro hepatic-like macrophage model. Additionally, correlations among hepatic gene expression, microbiota and serum lipid profiles in vivo established by Gl extracts were evaluated.
Results: Based on the hepatic and renal mRNA profiles of mice treated with Gl extracts and high-cholesterol diet, we identified relevant metabolic pathways modulated by Gl involving the restriction of lipid biosynthesis and the enrichment of lipid degradation and secretion. We further showed that Gl extracts induce a significant decrease of macrophage lipid storage and cholesterol biosynthesis, which occurs concomitantly by the down-modulation of Fasn and Elovl6. We also determined that prebiotic effects of Gl extracts modulating gut microbiota are correlated with the gene expression portraits.
Conclusions: Our high-throughput analysis allowed to identify key transcriptomic nodes established by Gl extracts and their interaction with microbiome composition related to lipid catabolic signaling. Our results indicated that our Gl extracts have a robust potential to be used as transcriptome modulators and prebiotic agents to prevent metabolic disorders associated to hypercholesterolemia.

overall_design: We used male 7-week-old C57BL/6 mice (26 g ± 0.5 g of weight) purchased from the animal facility of the INCMNSZ. Animals were housed (4/cage) in a 12-h light–dark cycle at a constant temperature (23 ± 2 °C) and relative humidity (45-55%). Mice were assigned to one of following experimental groups (N=40, n=8 per experimental group) in a complete randomization strategy: 1) Control: Control diet (AIN-93); 2) HCD: High-cholesterol diet (0.5% cholesterol) (Sigma-Aldrich, New Zealand); 3) HCD+Sim: High-cholesterol diet (0.5%) plus simvastatin (0.03 g/100 g); 4) HCD+Gl-1: High-cholesterol diet (0.5%) plus the Gl-1 extract (1.0%); 5) HCD+Gl-2: High-cholesterol diet (0.5%) plus the Gl-2 extract (1.0%). Sample size was calculated in accordance with the 3R's model with G*power software (two tail test, power = 0.95 and effect size=2). Allocation and treatment was performed with a not blinded strategy. Animals were fed with AIN-93 standard diet (average intake: 3.47 g/day of 9.5 g available per mouse) and water ad libitum. Each treatment (Simvastatin and lyophilized Gl extracts) were added to food pellet daily. At day 43, mice were deprived of food and water for 8 h and blood samples from the portal vein were collected for serological analysis. Mice were sacrificed at this end point, and liver and kidney tissues were collected and stored at -80ºC for further processing. Confounders were not controlled on this experiment.

middle: L

last: Romero-Córdoba

first: Ivan

last: Salido-Guadarrama

first: María

middle: E

last: Meneses

first: Giulia

last: Cosentino

first: Marilena

middle: V

last: Iorio

first: Elda

last: Tagliabue

first: Nimbe

last: Torres

first: Mónica

last: Sánchez-Tapia

first: Myrna

last: Bonilla

last: Castillo

first: Beatriz

last: Petlacalco

first: Armando

middle: R

last: Tovar

first: Daniel

last: Martínez-Carrera


title: TgIF2K-B is an eIF2α kinase in Toxoplasma gondii that responds to oxidative stress and optimizes pathogenicity

city: Indianapolis

first: Ronald

middle: C

last: Wek

state: IN

address: 635 N. Barnhill Dr. MS4067

department: Biochemistry and Molecular Biology

institute: Indiana University School of Medicine

summary: Here we report on gene expression changes in T. gondii in response to oxidative stress induced by sodium arsonite in wild-type and TgIF2K-B knockout intracellular parasites.

overall_design: total RNA seq conducted on purified wild-type and TgIF2K-B KO parasites following sodium arsonite or no treatment

first: Leonardo

last: Augusto

first: Jennifer

last: Martynowicz

first: Parth

middle: H

last: Amin

first: Kenneth

last: Carlson

first: William

last: Sullivan Jr


title: Transcriptome profiling (Whole RNA sequencing) to study the funciton of clk-2 in NMD pathway in C.elegans

city: Oslo

first: Rafal

last: Ciosk

state: Oslo

address: Blindernveien 31

department: Department of Biosciences

country: Norway

institute: University of Oslo

summary: We identified clk-2 as a novel player for NMD pathway by forward genetic screen, the goal of the whole RNA sequencing experiment was to study the genome-wide effect of clk-2 in NMD

overall_design: mRNA profiling of C.elegans young adults of clk-2, smg-1, smg-2 and N2 wild type worms used as control.

first: Yanwu

last: Guo


title: SNPC-1.3 is a sex-specific transcription factor drives male piRNA expression in C. elegans

city: Baltimore

first: Margaret

last: Starostik

state: MD

address: 3400 Charles Street

department: Department of Biology

institute: Johns Hopkins University

type: Non-coding RNA profiling by high throughput sequencing

summary: Piwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Nearly one-third of the ~15,000 piRNA loci in the C. elegans hermaphrodite are enriched during spermatogenesis, whereas >2,000 piRNAs are enriched during oogenesis. However, the mechanism underlying this sex-specific regulation is unknown. Here we identify SNPC-1.3, a variant of a conserved subunit of the snRNA activating protein complex, as a male-specific piRNA transcription factor. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires the core piRNA transcription factor SNPC-4. Loss of snpc-1.3 leads to depletion of spermatogenic piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to the snpc-1.3 promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression of snpc-1.3 and male piRNAs during oogenesis. Thus, sexual dimorphic regulation of snpc-1.3 coordinates male and female piRNA expression during germline development.

overall_design: We performed small RNA-seq and ChIP-seq in C. elegans N2 worms and various mutants to explore the role of SNPC-1.3 and TRA-1 in regulating piRNA expression during spermatogenesis and oogenesis.

first: Charlotte

middle: P

last: Choi

first: Rebecca

last: Tay

first: Suhua

last: Feng

first: James

last: Moresco

first: Brooke

last: Montgomery

first: Emily

last: Xu

first: Maya

last: Hammonds

first: Michael

last: Schatz

first: Taiowa

first: John

last: Yates III

first: Steven

last: Jacobsen

middle: K

last: Kim


title: SHAPE analysis of RNA structure in living cells with unprecedented accuracy

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: University of Groningen

summary: Due to the mounting evidence that RNA structure plays a critical role in regulating almost any essential physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 performs comparably well to NAI on naked RNA under in vitro conditions and it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

sample_organism:
Escherichia coli
Bacillus subtilis
Homo sapiens

overall_design:
SHAPE-MaP

Definitions:
TGIRT-III/HIV RT/SSII: reverse transcriptase enzymes
I5: Indoline-5-carboxylic acid imidazolide
1M4: 1-Methylimidazole-4-carboxylic acid imidazolide
6A3: 6-Aminopyridine-3-carboxylic acid imidazolide
B5: Benzotriazole-5-carboxylic acid imidazolide
NIC: Nicotinic acid imidazolide
NAI: 2-Methylpyridine-3-carboxylic acid imidazolide
2A3: 2-Aminopyridine-3-carboxylic acid imidazolide

first: Tycho

last: Marinus

first: Adam

middle: B

last: Fessler

first: Craig

last: Ogle


title: Characterizing RNA stability genome-wide through combined analysis of PRO-seq and RNA-seq data

city: Cold Spring Harbor

first: Yixin

last: Zhao

state: NEW YORK

address: 1 Bungtown Road

institute: Cold Spring Harbor Laboratory

summary: The rate at which RNA molecules decay is a key determinant of cellular RNA concentrations, yet current approaches for measuring RNA half-lives are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On and sequencing (PRO-seq) and RNA sequencing (RNA-seq). Our method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium.

overall_design: We collected new data in multiple replicates (two for PRO-seq, four for RNA-seq), all from the same source of K562 cells.

first: Amit

last: Blumberg

first: Charles

middle: G

last: Danko

last: Siepel


title: Transcription levels of a long noncoding RNA shape a cell fate regulatory circuit (TES-seq)

city: London

first: Folkert

last: van Werven

address: 1 Midland Road

institute: Francis Crick Institute

summary: Long noncoding RNAs (lncRNA) play diverse roles in gene regulation controlling key  cellular processes, most notably, cell-fate programming {Anderson, 2016 #20;Flynn, 2014 #16;Guttman, 2011 #35;Wang, 2011 #18}. Many long noncoding RNAs (lncRNAs) act in cis through transcription-coupled chromatin alterations that drive changes in local gene expression { Martens, 2004 #47; Kim, 2012 #74;van Werven, 2012 #57;Hainer, 2011 #73;Kim, 2016 #41;Ard, 2016 #21;Latos, 2012 #7}. How transcription of some lncRNAs leads to activation of gene expression, while others inhibit and repress gene expression remains poorly understood {Kornienko, 2013 #17}. Here we investigated in S. cerevisiae the function of the lncRNA IRT2, which is expressed upstream in the promoter of the master regulator for entry into meiosis, IME1. We report the surprising finding that distinct levels of IRT2 transcription regulates opposing chromatin and transcription states in order to ensure that only diploids, and not haploids, enter meiosis and form gametes. In haploid cells, IRT2 transcription at very low levels is required for the correct induction of the adjacent lncRNA IRT1, which in turn represses the IME1 promoter and prevents meiotic entry {van Werven, 2012 #57}. Low levels of IRT2 transcription stimulates histone exchange delivering acetylated histone H3 lysine 56 (H3K56ac) to chromatin, thereby facilitating chromatin disassembly and recruitment of the transcriptional activator of IRT1, Rme1. Inhibiting IRT2 transcription, or mutations that resulted in cells lacking H3K56ac impairs Rme1 recruitment and IRT1 induction, and consequently haploid cells induce IME1 and undergo a lethal meiosis. In contrast to its function at low levels, increasing IRT2 transcription enhances transcription-coupled chromatin assembly and interferes with IRT1 expression, promoting IME1 expression and meiotic entry in diploid cells {Moretto, 2018 #10}. Thus, transcription of lncRNAs, even at very low levels, can play an important role in regulating gene expression, and changes in lncRNA transcription levels can confer distinct regulatory and cell fate outcomes.

overall_design: 3’ end RNA sequencing analysis of Saccharomyces cerevisiae SK1 strains. The SPO (low nutrient) condition correspond to strains grown in YPD to saturation, then shifted to pre-sporulation media for 16h before being transfered to sporulation media. the u6bsD mutant harbours a clean deletion (no marker) of the Ume6 binding site present 2306 bp upstream IME1 ATG. Two biological replicates of each condition were analysed. Details for library preparation are described below.

first: Fabien

last: Moretto

first: Ezgi

last: Wood

first: Minghao

last: Chia

first: Cai

last: Li


title: Transcription levels of a long noncoding RNA shape a cell fate regulatory circuit (TSS-seq)

overall_design: 5’ end RNA sequencing analysis of Saccharomyces cerevisiae SK1 strains. The SPO (low nutrient) condition correspond to strains grown in YPD to saturation, then shifted to pre-sporulation media for 16h before being transfered to sporulation media. the u6bsD mutant harbours a clean deletion (no marker) of the Ume6 binding site present 2306 bp upstream IME1 ATG. Two biological replicates of each condition were analysed. Details for library preparation are described below.


title: Transcription levels of a long noncoding RNA shape a cell fate regulatory circuit (PolII associated RNA-seq)

overall_design: POLII associated RNA sequencing. The SPO (low nutrient) condition correspond to strains grown in YPD to saturation, then shifted to pre-sporulation media for 16h before being transfered to sporulation media. the u6bsD mutant harbours a clean deletion (no marker) of the Ume6 binding site present 2306 bp upstream IME1 ATG. Two biological replicates of each condition were analysed. Details for library preparation are described below.


title: Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution

status: Public on Dec 14 2020

city: Oxford

first: Zhiyuan

last: Hu

address: John Radcliffe Hospital/Headley Way

department: MRC Weatherall Institute of Molecular Medicine

institute: University of Oxford

type: Methylation profiling by high throughput sequencing

summary: Although various methods have been developed for sequencing cytosine epigenetic modifications, specific and quantitative sequencing of the two major epigenetic modifications of cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at base-resolution is still challenging. Most times it requires subtraction of two methods to obtain both the true 5mC and 5hmC information, which increases noise and requires high sequencing depth. Recently we developed TET assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of DNA methylation, which provides the sum of 5mC and 5hmC. Here we extend it to two sister methods, TAPSβ and CAPS (Chemical-Assisted Pyridine borane Sequencing), for whole-genome subtraction-free and specific sequencing of 5mC and 5hmC, respectively. We also demonstrated Pyridine borane Sequencing (PS) of whole-genome 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), the further oxidized derivatives of 5mC and 5hmC. This completes the versatile borane reduction chemistry-based methods as a comprehensive suite for direct and quantitative sequencing of all four individual cytosine epigenetic modifications.

overall_design: Four samples in total for TAPSbeta, CAPS, PS and PS-c.

first: Yibin

last: Liu

first: Jingfei

last: Chen

first: Paulina

last: Siejka-Zielińska

first: Jinfeng

first: Masato

last: Inoue

first: Ahmed

last: Ahmed

first: Chun-Xiao

last: Song


title: Transcription levels of a noncoding RNA orchestrate opposing regulatory and cell fate outcomes in yeast

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

overall_design: Refer to individual Series


title: Transcription levels of a long noncoding RNA shape a cell fate regulatory circuit (mRNA-seq)

overall_design: mRNA sequencing analysis of Saccharomyces cerevisiae SK1 strains. The SPO (low nutrient) condition correspond to strains grown in YPD to saturation, then shifted to pre-sporulation media for 16h before being transfered to sporulation media. the u6bsD mutant harbours a clean deletion (no marker) of the Ume6 binding site present 2306 bp upstream IME1 ATG. Two biological replicates of each condition were analysed. Details for library preparation are described below.


title: Joint profiling of histone modifications and transcriptome in single cells from mouse brain

city: New York

first: Zhu

last: Chenxu

email: cxzhu@pku.edu.cn, czhu@nygenome.org

state: New York

address: 101 Avenue of the Americas

institute: New York Genome Center

summary: Genome-wide profiling of histone modifications can reveal not only the location and activity state of regulatory elements, but also the regulatory mechanisms involved in cell-type-specific gene expression during development and disease pathology. Conventional assays to profile histone modifications in bulk tissue lack resolution due to the cellular heterogeneity. Here, we describe an ultra-high throughput method for single-cell joint profiling of histone modifications and transcriptome that could produce cell-type-resolved maps of chromatin state and transcriptome in complex tissues. We used this method to profile five histone modifications jointly with transcriptome in the adult mouse frontal cortex and hippocampus. Integrative analysis of the resulting maps identified distinct groups of genes subject to divergent epigenetic regulatory mechanisms. Our results demonstrate a general approach for comprehensive analysis of chromatin state and gene regulation in complex tissues and provide insights into the gene regulatory processes in major neuron cell types in the mouse.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Combined analysis of RNA and histone modifcations in single-cells.

first: Chenxu

last: Zhu

first: Bing

last: Ren


title: Different mutant RUNX1 oncoprotein classes program alternate haematopoietic differentiation trajectories [ChIP-seq2]

city: Birmingham

first: Peter

last: Keane

address: Vincent Drive

department: Institute for Cancer and Genomic Sciences

institute: University of Birmingham

summary: Mutations of the hematopoietic master regulator RUNX1 cause acute myeloid leukaemia, familial platelet disorder and other haematological malignancies whose phenotypes and prognoses depend on the class of RUNX1 mutation. The biochemical behaviour of these oncoproteins and their ability to cause unique diseases has been well studied but the genomic basis of their differential action is unknown. To address this question we compared integrated phenotypic, transcriptomic and genomic data from cells expressing four types of RUNX1 oncoproteins in an inducible fashion during blood development from embryonic stem cells. We show that each class of RUNX1 mutation rapidly deregulates endogenous RUNX1 function by different mechanisms, leading to specific alterations in developmentally controlled transcription factor binding and chromatin programming. The result are distinct perturbations in the trajectories of gene regulatory network changes underlying blood cell development that are consistent with the nature of the final disease phenotype. The development of novel treatments for RUNX1-driven diseases will therefore require consideration beyond RUNX1 haploinsufficiency.

overall_design: ChIP-Seq targeting GATA1 and PU1 in mouse hematopietic progenitor cells expressing a doxycyclin inducible Runx1 oncoprotein construct. Samples include cells with and without doxycycline treatment.

first: Sophie

last: Kellaway

first: Benjamin

last: Edginton-White

first: Regha

last: Kakkad

first: Ella

last: Kennett

first: Constanze

last: Bonifer


title: Invalidation of a novel marker for therapy-resistant leukemic stem cells responsible for relapse in patients and PDX

city: Strassen

first: Tony

last: KAOMA

address: 1 A-B Rue Thomas Edison

department: Quantitative Biology Unit

country: Luxembourg

institute: Luxembourg Institute of Health

summary: Drug tolerant leukemic cell stem (LSC) subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these Relapse-Initiating Cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncover that the G-protein coupled receptor CALCRL is expressed in RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM) and not CGRP correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency and sensitizes to cytarabine in patient-derived xenograft models.

overall_design: Transfection of MOLM14 cells using genetic approach with shRNA constructs

last: Kaoma

first: Nathalie

last: Nicot

first: Clément

last: Larrue

first: Jean-Emmanuel

last: Sarry


title: Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories [RNA-Seq]

overall_design: RNA-Seq of hematopietic progenitor cells expressing a doxycyclin inducible Runx1 oncoprotein construct. Samples include cells with and without doxycycline treatment. Two biological replicates are included per sample


title: Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories [ATAC-Seq]

summary: Mutations of the hematopoietic master regulator RUNX1 cause acute myeloid leukaemia, familial platelet disorder and other haematological malignancies whose phenotypes and prognoses depend on the class of RUNX1 mutation. The biochemical behavior of these oncoproteins and their ability to cause unique diseases has been well studied but the genomic basis of their differential action is unknown. To address this question we compared integrated phenotypic, transcriptomic and genomic data from cells expressing four types of RUNX1 oncoproteins in an inducible fashion during blood development from embryonic stem cells. We show that each class of RUNX1 mutation rapidly deregulates endogenous RUNX1 function by different mechanisms, leading to specific alterations in developmentally controlled transcription factor binding and chromatin programming. The result are distinct perturbations in the trajectories of gene regulatory network changes underlying blood cell development that are consistent with the nature of the final disease phenotype. The development of novel treatments for RUNX1-driven diseases will therefore require consideration beyond RUNX1 haploinsufficiency.

overall_design: ATAC-Seq of mouse hematopietic progenitor cells expressing a doxycyclin inducible Runx1 oncoprotein construct. Samples include cells with and without doxycycline treatment.


title: Expression data profile of A498 cells infected with E. coli 83972, mutants or stimualted with rNlpD

status: Public on Dec 11 2020

city: Lund

first: Ines

last: Ambite

address: Klinikgatan 28

department: Laboratory Medicine

country: Sweden

institute: Lund University

summary: Unlike pathogens, which attack the host, commensal bacteria create a state of friendly coexistence. Here, we identify a new mechanism of bacterial adaptation to the host niche, where they reside. Asymptomatic carrier strains are shown to inhibit RNA Polymerase II (Pol II) in host cells by targeting Ser2 phosphorylation; a step required for productive mRNA elongation. Assisted by a rare, spontaneous loss-of-function mutant from a human carrier, the bacterial NlpD protein is identified as a Pol II inhibitor. After internalization by host cells, NlpD is shown to target constituents of the Pol II phosphorylation complex (RPB1 and PAF1C), attenuating host gene expression. Therapeutic efficacy of the rNlpD protein is demonstrated in a urinary tract infection model, by reduced tissue pathology, accelerated bacterial clearance and attenuated Pol II-dependent gene expression. The findings suggest an intriguing, evolutionarily conserved mechanism for bacterial modulation of host gene expression, with a significant therapeutic potential.

overall_design: A498 kidney epithelial cells were infected with 10^5 CFU/ml of E. coli 83972, SN25 or SN25-pRH320 or treated with bacterial supernatants or recombinant NlpD protein for 4 hours. Cells with culture medium with PBS were used as a control. Isolated RNA was subjected to Affymetrix whole genome transcriptome analysis.

first: Catharina

last: Svanborg


title: Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories


title: Resolution of genome-wide molecular signals in canine mammary tumor model

first: Kiley

last: Graim

state: NY

address: 162 Fifth Ave

institute: Flatiron Institute

summary: Modeling molecular changes in breast cancer with naturally occurring canine mammary tumors and cross-organism analysis enables resolution of malignancy-specific processes.

overall_design: 89 mammary tissue samples from 16 dogs of diverse breeds, where each dog was required to provide a minimum of one sample from each histological group: normal, pre-malignant, and malignant.  Sequencing was performed using the Illumina HiSeq2000 platform.

first: Chandra

last: Theesfeld

first: Karin

last: Sorenmo

first: Olga

last: Troyanskaya


title: NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis

city: Montpellier

first: Amal

last: Makrini

address: 1919 route de mende

country: France

institute: IGMM

summary: The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RuvBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.

overall_design: RNAseq from HEK NOPCHAP1 KO cells compared to control cells

first: Yoann

last: Abel

first: Ana

last: Paiva

first: Jonathan

last: Bizarro

first: Marie-Eve

last: Chagot

first: Paulo

last: Santo

first: Marie-Cécile

last: Robert

first: Marc

last: Quinternet

first: Franck

last: Vandermoere

first: Pedro

middle: M

last: Sousa

first: Philippe

last: Fort

first: Bruno

last: Charpentier

first: Xavier

last: Manival

first: Tiago

last: Bandeiras

first: Edouard

last: Bertrand

first: Céline

last: Verheggen


title: Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories [ChIP-Seq]

first: Peter

last: Keane

overall_design: ChIP-Seq targeting RUNX1 and H3K27ac in mouse hematopietic progenitor cells expressing a doxycyclin inducible Runx1 oncoprotein construct. Samples include cells with and without doxycycline treatment.

first: Sophie

last: Kellaway

first: Benjamin

last: Edginton-White

first: Regha

last: Kakkad

first: Ella

last: Kennett

first: Constanze

last: Bonifer


title: Tailoring the resolution of single-cell RNA sequencing for primary cytotoxic T cells

status: Public on Dec 13 2020

city: Freising

first: Patrick

last: Roelli

address: Weihenstephaner Berg 3

department: Tierphysiologie & Immunologie

institute: TUM

summary: Single-cell RNA sequencing in principle offers unique opportunities to improve the efficacy of contemporary T-cell based immunotherapy against cancer. The use of high-quality single-cell data will aid our incomplete understanding of molecular programs determining the differentiation and functional heterogeneity of cytotoxic T lymphocytes (CTLs)  allowing for optimal therapeutic design. So far  a major obstacle to high depth single-cell analysis of CTLs is the minute amount of RNA available  leading to low capturing efficacy. To overcome this  we tailor a droplet-based approach for high-throughput analysis (tDrop-seq) and a plate-based method for high-performance in-depth CTL analysis (tSCRB-seq). The latter gives  on average  a 15-fold higher number of captured transcripts per gene compared to droplet-based technologies. The improved dynamic range of gene detection gives tSCRB-seq an edge in resolution sensitive downstream applications such as graded high confidence gene expression measurements and cluster characterization. We demonstrate the power of tSCRB-seq by revealing the subpopulation-specific expression of co-inhibitory and co-stimulatory receptor targets of key importance for immunotherapy.

overall_design: This study groups a total of 27 samples of single-cell RNA sequencing with two different protocols (Drop-seq and SCRB-seq) and their modifications.

first: Kristiyan

last: Kanev

first: Ming

last: Wu

first: Christine

last: Wurmser

first: Mauro

last: Delorenzi

first: Michael

middle: W

last: Pfaffl

first: Dietmar

last: Zehn


title: Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

city: Marseille

first: Delphine

last: Potier

address: 27 boulevard Lei Roure

institute: CRCM

summary: Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

overall_design: We used single-cell RNA sequencing (scRNAseq) approach to investigate leukemic cells of one B-ALL patient at two-time points; before (T1) and after (T2) anti-CD19 CAR-T cell therapy. At T2 leukemic cells were CD19 negative (CD19neg). Therefore, we asked whether these CD19neg B-ALL clones were present before CAR-T cell therapy. To address this issue, T1 and T2 samples were sorted according to forward scatter and side scatter, a cell viability marker, CD3 and CD19 surface expression. We obtained 4 tubes of sorted cells: T1-CD19pos, T1-CD19neg, T2-CD19pos and T2-CD19neg. Each of those samples were marked with a distinct anti-CD45-hashtag oligonucleotide (HTO) antibody, then mixed and analyzed by scRNAseq using the 5’ 10X Genomics technology.

first: Tracy

last: Rabilloud

first: Saran

last: Pankaew

first: Mathis

last: Nozais

first: Marie

last: Loosveld

first: Dominique

last: Payet-Bornet


title: Hopeful monsters: Unintended sequencing of famously malformed mite mitochondrial tRNAs reveals widespread expression and processing of sense-antisense pairs

status: Public on Dec 10 2020

city: Fort Collins

first: Jessica

middle: Marie

last: Warren

state: Colorado

address: Biology Building, 251 W Pitkin St.

department: Biology

institute: Colorado State University

summary: While performing mitochondrial isolations and recently developed tRNA-seq methods (AlkB treatment and YAMAT-Seq) in plant tissue, we inadvertently sequenced the mitochondrial tRNAs from a common plant pest, the acariform mite Tetranychus urticae, to a high enough coverage to detect all previously annotated T. urticae tRNA regions. The results not only confirm expression, CCA-tailing and post-transcriptional base modification of these highly divergent tRNAs, but also revealed paired sense and antisense expression of multiple T. urticae mitochondrial tRNAs.

overall_design: Analysis of full-length tRNA reads from  the mitochondria of Tetranychus urticae.

first: Daniel

last: Sloan


title: Stereotactic body radiotherapy is effective in modifying the tumor genome and tumor immune microenvironment in non-small cell lung cancer or lung metastatic carcinoma [RNA-seq]

city: Chongqing

first: Guanghui

last: Li

state: Chongqing

address: Xinqiao Street No. 2r

department: Xinqiao Hospital

institute: Army Medical University

summary: Background and purpose: To directly reveal the change in genome mutation, RNA transcript of tumor cells, and tumor microenvironment (TME) after stereotactic body radiotherapy (SBRT) in paired human lung tumor specimens.

overall_design:
Materials and methods: Paired tumor samples were collected from 10 patients with non-small cell lung cancer (NSCLC) or lung metastatic carcinoma within a week before and after SBRT. DNA and RNA of tumor tissues was extracted from the paired samples. Whole-exome and RNA sequencing assays were performed by next-generation sequencing. Gene mutation, genomic expression, T-cell receptor (TCR) repertoire, and profiling of tumor-infiltrating immune cells were analyzed through bioinformatics analysis in paired tumor samples.
Case numbers are matched between the RNA-seq data set (this series) and the exome-seq data set (GSE162944). For example, case 2 in exome seq is from the same patient as case 2 in the RNA-seq study.

first: Pu

last: Zhou

first: Diangang

last: Chen

first: Bo

last: Zhu

first: Wei


title: The cyanobacterium, Nostoc punctiforme can protect against programmed cell death and induce defence genes in Arabidopsis thaliana.

city: Dublin

first: Samuel

middle: Patrick

last: Belton

state: Leinster

address: UCD, Belfield, Dublin 4

department: School of Biology and Environmental Science

country: Ireland

institute: University College Dublin

summary: Many cyanobacteria can form nitrogen-fixing symbioses with a broad range of plant species. Unlike other plant-bacteria symbioses, little is understood about the immunological responses induced by plant cyanobionts (symbiotic cyanobacteria). Here, we used Arabidopsis thaliana suspension cell cultures as a model system to demonstrate that the model plant-symbiotic cyanobacteria, Nostoc punctiforme is capable of protecting against plant programmed cell death (PCD). We also profiled the early transcriptomic changes that were induced in response to conditioned medium (CM) from N. punctiforme cell cultures. Interestingly, the PCD reduction was preceded by the induction of genes associated with defence and immunity, the most striking of which were a number of WRKY-family transcription factors. Down-regulated included genes involved in the regulation of cell growth and differentiation. This work is the first to show that a cyanobacteria can regulate plant PCD and provides a useful transcriptome resource for studying early plant cell responses to symbiotic cyanobacteria.

overall_design: Arabidopsis thaliana suspension cell cultures were incubated with a 20% volume/volume of conditioned medium (CM) from Nostoc punctiforme liquid cultures. As a control treatment, cells were instead incubated with a 20% volume of fresh N. punctiforme growth medium (BG) (growth medium in which no cells were previously grown). After incubation for 1 hour, A. thaliana cells were harvested and RNA extracted for transcriptomic profiling.

first: Carl

last: Ng

first: Paul

middle: F

last: McCabe


title: Stereotactic body radiotherapy is effective in modifying the tumor genome and tumor immune microenvironment in non-small cell lung cancer or lung metastatic carcinoma


title: Effect of DMF and MEF on gene expression in select mouse organs:  Multi-dosing regimen

city: Cambridge

first: Suzanne

last: Szak

state: MA

address: 14 Cambridge Ctr

department: Drug Discovery

institute: BiogenIdec, Inc.

summary: Delayed-release dimethyl fumarate (DMF) is approved in the United States, European Union, Canada, and Australia for the treatment of multiple sclerosis. DMF is also a component in a defined-mixture product with three salts of monoethyl fumarate (MEF) that is approved in Germany for the treatment of psoriasis. Characterizing common or distinct pharmacodynamic properties of DMF and MEF would provide insights into the mechanisms of action of delayed-release DMF versus fixed combination products containing DMF and MEF salts. In this study we evaluated the pharmacodynamic effects and pharmacokinetics of DMF and MEF in central nervous system and peripheral tissues of naïve mice following a single dose or 10 daily doses of DMF, MEF, or a combination of the two. DMF and MEF exhibited similar pharmacokinetic profiles, but differences were noted in biodistribution: monomethyl fumarate (MMF, the primary metabolite of DMF) exhibited a higher degree of brain penetration, whereas MEF was preferentially partitioned into kidney. Both common and distinct pharmacodynamic responses were observed in all assessed tissues for DMF and MEF alone or in combination. These findings indicate that all fumaric acid esters cannot be considered equivalent, and combinations of compounds may exert effects not observed when agents are used individually.

overall_design: C57BL/6 mice were administered vehicle (0.8% hydroxypropyl methylcellulose), DMF (100 mg/kg or 179.2 mg/kg), a mixture of MEF salts (Ca2+, Mg2+, Zn2+; 79.2 mg/kg), or a combination of DMF (100 mg/kg) and MEF salts (79.2 mg/kg)once daily for 10 days (n = 6−10 mice per dose group). Global transcriptional profiling was performed on whole blood and a panel of tissues (brain, inguinal lymph node, mesenteric lymph node, jejunum and spleen) to identify genes that were significantly modulated relative to vehicle controls 12 hours after the final dose on day 10.

first: Brian

middle: T

last: Wipke

first: Kristopher

last: King

first: Melanie

middle: S

last: Brennan

first: Norm

last: Allaire

first: Patrick

last: Cullen

first: Alice

last: Thai

first: Ankur

last: Thomas

first: Davide

last: Gianni

first: Alex

last: Chou

first: Theresa

middle: A

last: Hillery

first: Kejie

first: Liyu

last: Yang

first: John

last: Carulli

first: Chaoran

last: Huang

first: Mark

last: Rogge

first: Norman

last: Kim

first: Kenneth

middle: J

last: Rhodes

first: Robert

last: Scannevin


title: HEK-293 cells: Control cells transfected with  hRIZ2 and rRIZ1

status: Public on Dec 09 2020

city: Caserta

first: Monica

last: Rienzo

address: Viale  Lincoln

department: Department of Environmental, Biological, and Pharmaceutical Sciences and Technologies

country: Italy

institute: University of Campania “Luigi Vanvitelli”

summary: In order to evaluate the effects of RIZ2 overexpression on gene expression, a pilot expression study was performed through microarrays analysis. We compared the differential gene expression between HEK-293 cells overexpressing human RIZ2 (pEGFP-hRIZ2) and rat RIZ1 (pEGFP-rRIZ1) versus control cells transfected with the E-GFP empty vector alone (pEGFP).

overall_design: four-condition experiment, pEGFP vs pEGFP_hRIZ2 cells and rRIZ1 and rRIZ1+hRIZ2. Biological replicates: 1 control triplicates, 1 transfected triplicates.

first: Ciro

last: Abbondanza

first: Amelia

last: Casamassimi

first: Anna

last: Sorrentino

first: Caterina

last: De rosa


title: A stem cell based in vitro model of NAFLD enables the analysis of patient specific individual metabolic adaptations in response to a high fat diet and AdipoRon interference

status: Public on Dec 08 2020

city: Düsseldorf

first: Wasco

last: Wruck

address: Moorenstr. 5

institute: Universitätsklinikum Düsseldorf

summary: A stem cell based in vitro model of NAFLD recapitulates regulatory networks and suggests a steatosis associated phenotype. AdipoRon treatment influences metabolism, immune system, cell stress and signalling

overall_design: The transcriptomes of iPSC generated hepatocyte like cells with and without oleic acid induction and with and without AdipoRon treatment were compared by microarray analysis (PrimeView Human Gene Expression Array, Affymetrix, Thermo Fisher Scientific).

first: Nina

last: Graffmann

first: James

last: Adjaye


title: Effect of DMF and MEF on gene expression in select mouse organs

first: Suzanne

last: Szak

overall_design: Refer to individual Series


title: On the NF-Y regulome as in ENCODE (2019)

status: Public on Dec 07 2020

city: Milano

first: Diletta

last: Dolfini

state: MI

address: Via Celoria, 26

department: Bioscience

institute: University of Milan

summary: The ongoing ENCODE consortium represents a useful compendium of locations of TFs, chromatin marks, gene expression data. In previous reports, we identified modules of CCAAT-binding NF-Y with individual TFs. Here, we analyzed all 363 factors currently present: 68 with enrichment of CCAAT in their locations, 38 with overlap of peaks. New sequence-specific TFs, co-activators and co-repressors are reported. Co-association patterns correspond to specific targeted genes categorizations and gene expression changes, as assessed by RNA-seq after NF-Y inactivation. These data widen and better define a coherent model of synergy of NF-Y with selected groups of TFs and cofactors.

overall_design: RNA-seq of HeLa cell line after inactivation of NF-YB.

first: Andrea

last: Bernardini

first: Mirko

last: Ronzio


title: Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24

status: Public on Dec 06 2020

city: West Haven

first: Sarah

last: Slavoff

state: Connecticut

address: Yale University West Campus-Building 600

department: Chemistry

institute: Yale University- West Campus

summary: We characterize non-annotated protein alt-RPL36 which is encoded by RPL36 transcript isoform 2 and overlaps with the RPL36 open reading frame. Its function is examined by specific CRISPR deletion of the GTG start codon located  5' to the RPL36 ATG initiation site. Overall changes in transcript expression are evaluated by comparison with wild type HEK293T cells. RPL36 protein expression is not affected by the altRPL36 start codon deletion.

overall_design:
Gene expression changes between two biological replicates of control (HEK293T) cells and HEK293T cells with alt-RPL36 knockout are quantified.

The 200 kb DNA region around CRISPR/Cas9 target sites in our alt-RPL36 knockout and knock-in HEK293T cell lines was enriched using Samplix Xdrop Technology and sequenced via long-read Oxford Nanopore Technology.

first: Alexandra

last: Khitun

first: Xiongwen

last: Cao


title: Stereotactic body radiotherapy is effective in modifying the tumor genome and tumor immune microenvironment in non-small cell lung cancer or lung metastatic carcinoma [exome-seq]

first: Guanghui

last: Li

overall_design:
Materials and methods: Paired tumor samples were collected from 10 patients with non-small cell lung cancer (NSCLC) or lung metastatic carcinoma within a week before and after SBRT. DNA and RNA of tumor tissues was extracted from the paired samples. Whole-exome and RNA sequencing assays were performed by next-generation sequencing. Gene mutation, genomic expression, T-cell receptor (TCR) repertoire, and profiling of tumor-infiltrating immune cells were analyzed through bioinformatics analysis in paired tumor samples.
Case numbers are matched between the RNA-seq data set (GSE162945) and the exome-seq data set (this series). For example, case 2 in exome seq is from the same patient as case 2 in the RNA-seq study.

first: Pu

last: Zhou

first: Diangang

last: Chen

first: Bo

last: Zhu

first: Wei


title: Transcriptional responses of wild-type and zin1 mutant Arabidopsis plants to infection with Pseudomonas syringae

city: Albany

first: Jennifer

last: Lewis

state: CA

address: 800 Buchanan St

institute: University of California, Berkeley

summary: Phytopathogens use secreted effector proteins to suppress host immunity and promote pathogen virulence, and there is increasing evidence that the host-pathogen interactome comprises a complex network.  In an effort to identify novel interactors of the Pseudomonas syringae effector HopZ1a, we performed a yeast two-hybrid screen that identified a previously uncharacterized Arabidopsis protein that we designate HopZ1a Interactor 1 (ZIN1).  Additional analyses in yeast and in planta revealed that ZIN1 also interacts with several other P. syringae effectors.  We show that an Arabidopsis loss-of-function zin1 mutant is less susceptible to infection by certain strains of P. syringae, while overexpression of ZIN1 results in enhanced susceptibility.  Functionally, ZIN1 exhibits topoisomerase-like activity in vitro.  Transcriptional profiling of wild-type and zin1 Arabidopsis plants inoculated with P. syringae indicated that ZIN1 regulates a wide range of pathogen-responsive biological processes, although the list of genes more highly expressed in zin1 versus wild-type plants was particularly enriched for ribosomal protein genes.  Altogether, these data illuminate ZIN1 as a potential susceptibility hub that interacts with multiple effectors to influence the outcome of plant-microbe interactions.

overall_design: Wild-type and zin1 mutants infiltrated with either 10 mM MgCl2 (Control), 1 x 10^8 cfu/mL Pseudmonas syringae pv. tomato DC3000, or 1 x 10^8 cfu/mL P. syringae pv. tomato DC3000 ΔhrcC (three biological replicates each for a total of 18 samples)

middle: D

first: Karl

last: Schreiber


title: Hypoxia exposure and methylation

status: Public on Dec 04 2020

city: Jena

first: Ainash

last: Childebayeva

state: thuringia

address: Kahlaische Strasse 10

department: Archaeogenetics

institute: Max Planck Institute for the Science of Human History

summary: Individuals with various high-altitude exposures have been recruited in this study to research the effects of differential exposure to high altitude on the epigenome

overall_design: Individuals were recruited in Lima and Cerro de Pasco.

first: A


title: Precise and broad scope genome editing based on high-specificity Cas9 nickases

city: Stanford

first: Marie

last: Le Bouteiller

address: 269 campus drive

department: Radiation Oncology

institute: Stanford University

summary: RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off- target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by “tiptoeing” over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of genome editing procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

overall_design: We employed an saCas9:RAG1.1 gRNA bait DNA double strand break to map genome-wide repair outcomes in the presence of nucleases or nickases variants  SpCas9, eSpCas9(1.1), SpCas9D10A and eSpCas9(1.1)D10A with a VEGFA-targeting gRNA (Sp-gVEGFA) using LAM-HTGTS. Each nuclease/nickase was tested in duplicate.

first: Qian

last: Wang

first: Jin

last: Liu

first: Josephine

last: Janssen

first: Richard

last: Frock

first: Manuel

last: Goncalves


title: The genome-wide impact of trisomy 21 on DNA methylation and its implications for hematopoiesis

city: oxford

first: Thomas

last: Jackson

address: weatherall institute of molecular medicine

department: paediatrics

institute: University of Oxford

summary:
Background: Down syndrome (DS) is associated with a wide range of phenotypes. To address the hypothesis that the genome-wide perturbation of gene regulation in DS is modulated by epigenetic changes, we performed an epigenome-wide association study (EWAS) on neonatal bloodspots comparing 196 newborns with DS and 439 newborns without DS.
Results: We identified 652 epigenome-wide significant CpGs (P<7.67x10-8) and 1,052 differentially methylated regions (DMRs) across the genome. Differential methylation at promoters/enhancers correlated with gene expression changes in DS versus non-DS fetal liver hematopoietic stem/progenitor cells (HSPC) (P<0.0001). Two of the top 3 DS-associated CpGs overlapped RUNX1, and were highly significantly hypermethylated in DS newborns (P<1.0x10-21). The top two DMRs overlapped promoters of RUNX1 and FLI1, both important regulators of megakaryopoiesis. FLI1 expression was markedly reduced in DS fetal liver HSCs (P<0.0001) and myeloid progenitors (P<0.0001). By contrast, RUNX1 expression was increased in DS fetal liver myeloid progenitors (P<0.0001), consistent with selective hypermethylation of the RUNX1 P2 promoter, which dominates in embryonic development, but sparing the P1 promoter that drives definitive hematopoiesis.  Targeted sequencing of GATA1 revealed somatic, preleukemic mutations in 16.3% DS newborns, consistent with the high frequency of these mutations in DS newborns. Removal of DS newborns with GATA1 mutations had minimal impact on the EWAS results.
Conclusions: DS has profound, genome-wide effects on DNA methylation in hematopoietic cells in early life, which may contribute to the high frequency of hematological problems, including leukemia, in children with DS.  Blood-detectable DS-related epigenetic changes may underlie an array of DS outcomes, including neurologic and immune-related morbidities.

overall_design: RNA-seq

first: Ivo

last: Muskens

first: Shaobo

first: Natalina

last: Elliot

first: Helen

last: Hansen

first: Swe

last: Myint

first: Priyatama

last: Pandey

first: Jeremy

last: Schraw

first: Ritu

last: Roy

first: Joaquin

last: Anguiano

first: Katerina

last: Goudevenou

first: Kimberly

last: Siegmund

first: Philip

last: Lupo

first: Marella

last: de Bruijn

first: Kyle

last: Walsh

first: Paresh

last: Vyas

first: Xiaomei

last: Ma

first: Anindita

first: Irene

last: Roberts

first: Joseph

middle: L

last: Wiemels

first: Adam

last: de Smith


title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

status: Public on Dec 05 2020

city: Oxford

first: Lucy

last: Garner

address: Experimental Medicine, Level 5, John Radcliffe Hospital

department: Nuffield Department of Medicine

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


title: Wolbachia and virus alter the host transcriptome at the interface of nucleotide metabolism pathways

city: Saint Paul

first: Amelia

middle: Ryan Isis

last: Lindsey

email: amlind@iu.edu, alindsey@umn.edu

state: MN

address: 1500 Gortner Ave

department: Department of Entomology

institute: University of Minnesota

summary: Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for Dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila melanogaster model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.

overall_design: To determine the effect of Wolbachia and virus infection on fly gene expression, we established in vivo systemic viral infections in adult Drosophila, using a block design with a time series. Flies with or without Wolbachia (W+/W-), were injected with either virus or saline (SINV+/SINV-), and collected at 6, 24, and 48 hours post-injection (hpi). For each unique condition of W-SINV-time, we generated four biological replicates (A-D), with each replicate consisting of a pool of five virgin females. In total, 48 libraries were generated.

middle: R

first: Tamanash

last: Bhattacharya

first: Richard

last: Hardy

last: Newton


title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets I

summary: Tissue-resident memory T (TRM) cells provide key adaptive immune responses in infection, cancer, and autoimmunity. However transcriptional heterogeneity of human intestinal TRM cells remains undefined. Here, we investigated transcriptional and functional heterogeneity of human TRM cells through study of donor-derived intestinal TRM cells from intestinal transplant recipients. Single-cell transcriptional profiling identified two distinct transcriptional states of CD8+ TRM cells, delineated by ITGAE and ITGB2 expression. We defined a transcriptional signature discriminating these two CD8+ populations, including differential expression of cytotoxicity- and residency-associated genes. Flow cytometry of recipient-derived cells infiltrating the graft and lymphocytes from healthy gut confirmed the two CD8+ TRM phenotypes. CD103+ CD8+ TRM cells produced IL-2, and demonstrated greater polyfunctional cytokine production, while β2-integrin+ CD69+ CD103- TRM cells had higher granzyme expression. Phenotypic and functional analysis of intestinal CD4+ T cells identified many parallels, including a distinct β2-integrin+ population. Together, these results describe the transcriptional, phenotypic, and functional heterogeneity of human intestinal CD4+ and CD8+ TRM cells.

overall_design: Donor-derived CD103- and CD103+ CD8+ T cells were sorted from the small intestine of two intestinal transplant recipients for plate-based single-cell RNA sequencing using the Smart-Seq2 protocol (Picelli et al., Nat Meth, 2013).

middle: C

first: Michael

last: FitzPatrick

first: Nicholas

last: Provine

first: Paul

last: Klenerman

last: Allan


title: Heat Shock Proteins and Small Nucleolar RNAs are Dysregulated in a Drosophila Model for Feline Hypertrophic Cardiomyopathy

city: Raleigh

first: Mary

middle: Anna

last: Carbone

state: NC

address: 112 Derieux Place, Thomas Hall room 3543

department: Biological Sciences

institute: North Carolina State University

summary: We report the application of RNA-sequencing technology for high-throughput profiling of Drosophila that express clinical variants of feline-MyBPC3 associated with Hypertrophic Cardiomyopathy (HCM).

overall_design: Differential expression analyses comparing transcriptome-wide effects of wild-type feline-MyBPC3 compared to the clinical variants (A31P and R820W) in a Drosophila model for feline Hypertrophic Cardiomyopathy (HCM).


title: Polymerase recycling contributes to transcriptional noise

city: Coventry

first: Daniel

last: Hebenstreit

address: Gibbet Hill Campus

institute: University of Warwick

summary: RNA-seq samples used to examine crosstalk between processes at the 5’ and 3’ ends of a transgene

overall_design: Expression analysis of transgene

first: Massimo

last: Cavallaro

first: Mark

first: Matt

last: Jones

first: James

last: Teahan

first: Simone

last: Tiberi

first: Bärbel

last: Finkenstädt

last: Hebestreit


title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets II

last: Garner

overall_design: CD3+ T cells (TRM cells) were sorted from the small intestine of a single subject one year post-transplant. Single-cell RNA sequencing and single-cell TCR sequencing were performed using the 10x Genomics Chromium V(D)J Reagents Kit (v1 Chemistry).

middle: B

first: Philip


title: Cardiac transcriptome in iron-deficiency anemia

status: Public on Dec 03 2020

city: London

first: YuJin

last: Chung

address: Westminster Bridge Rd

institute: Kings College London

summary:
Purpose: The goal of this study was to probe for the effects of iron-deficiency anemia on the cardiac transciprtome using RNA-seq
Methods: C57B6 mice were weaned onto a control or iron-deficient diet for 6 weeks. Hearts were removed and total mRNA was submitted for RNA-seq. Sequencing data was aligned using STAR and differetial gene expression analysis conducted in R using EdgeR and DESeq2. qRT–PCR validation for genes of interest was performed using TaqMan and SYBR Green assays.
Results: We mapped about 24 million sequence reads per sample to the mouse genome (build mm10) and identified 13,590 transcripts in the hearts of control and iron-deficient mice after removing lowly expressed genes and PCR duplicates. Differential gene expression analysis showed approximately 78% downregulated and 22% upregulated genes in iron-deficiency anemia compared to controls. PCA plot showed control and iron-deficient hearts clustering in two distinct and separate clusters.
Conclusions: Our study represents the first whole-transcriptomic study on cardiac samples obtained from iron-deficient and anemic mice, with biologic replicates, generated by RNA-seq technology. The RNA-seq data presented here can be used by others to explore which pathways are affected by iron-deficiency anemia

overall_design: Cardiac mRNA from control and iron-deficient anemic mice (males)

first: Yu

first: Pawel

last: Swietach

first: Peter

last: Robbins

first: Samira

last: Lakhal-Littleton


title: Stearoyl-CoA desaturase mediated monounsaturated fatty acid availability supports humoral immunity

status: Public on Dec 02 2020

city: Rochester

first: Hu

last: Zeng

address: 200 First ST SW

institute: Mayo Clinic

summary: Immune cells can metabolize fatty acids (FAs) to generate energy. The source of different fatty acid species, and their impacts on humoral immunity remains poorly understood. Here we report that proliferating B cells require increased amount of monounsaturated fatty acids (MUFA) to maintain mitochondrial metabolism and mTOR activity, and to prevent excessive autophagy and endoplasmic reticular (ER) stress. Furthermore, B cell extrinsic Stearoyl-Coa desaturase (SCD) activity generates endogenous MUFA to support early B cell development and germinal center (GC) formation in vivo during immunization and influenza infection. Thus, SCD-mediated MUFA production is critical for humoral immunity.

overall_design: Splenic follicular B cells were flow sorted.They were activated by LPS/IL-4 for 72 hours. Total RNA was extracted using Rneasy micro kit (Qiagen).

first: Xian

last: Zhou

first: Zhenqing

last: Ye


title: RNA Sequencing and RRBS of sorted myeloid cells from tet mutant zebrafish embryos

city: New York

first: Kelly

middle: Madigan

last: Banks

address: 1300 York Ave, LC711

department: Surgery

institute: Weill Cornell Medicine


title: Extensive tissue-specific, circadian molecular variation in a Drosophila Genetic Reference Panel

city: Lausanne

first: Bart

last: Deplancke

state: Vaud

address: EPFL-SV-IBI-UPDEPLA, Station 19

country: Switzerland

institute: École polytechnique fédérale de Lausanne (EPFL)

summary: We report a large-scale transcriptomic analysis of several tissues of a reference Drosophila melanogaster strain as well as 141 Drosophila Genetic Reference Panel (DGRP) lines at high temporal resolution. Comprehensive data analysis has identified thousands of genes under clock- and tissue-specific control. By using a molecular time table approach, we uncovered that >20% of probed DGRP lines exhibit aberrant circadian expression, and the genetic dissection of one line (DGRP-796) revelled disrupted circadian gene expression in all analysed tissues, revealing a novel deletion in the cry gene. Together, this study revealed extensive variation in tissue-specific circadian expression, which acts upon tissue-specific regulatory networks to generate local oscillations in gene expression. Moreover, the many other lines identified here can be now used to better understand the mechanisms underlying the molecular clock, from tissue-specific to more central mechanisms.

overall_design: The study consists from 3 datasets: 1) reference gene expression time series by sampling the brain, gut, Malpighian ttubules and fat body of a reference line (w1118 strain) every 2h over 48h in triplicates, 2) single observationss (one time point) of brains, guts and fat bodies of 141 DGRP strains 3)  time series for DGRP-796 collected every 2h over 48h in duplicates, brain and gut

first: Antonio

last: Meireles-Filho

first: Mari

last: Litovchenko


title: RRBS of sorted myeloid cells from tet mutant zebrafish embryos

summary: We report overexpression of socs3b leading to reduced cytokine signalling but no change in DNA methylation at this site

overall_design: Neutrophils and. Myeloid progenitors were sorted based on the reporters mpx:GFP and pu.1 GFP, respectivey

first: Yahui

last: Lan

first: Todd

last: Evans


title: Comparison of WT to atfs-1(null) & WT to atfs-1(et18) in C. elegans by deep sequencing

city: Worcester

first: Julie

last: Zhu

address: 364 Plantation Street

department: Molecular Cellular and Cancer Biology

institute: UMass Medical School

summary:
Comparison of gene change in the atfs-1 (null) to WT.
Comparison of gene change in the atfs-1 (et18) to WT.

overall_design: We performed two experiments. In the first experiment, we collected 3 samples of N2 and 3 samples of atfs-1(null). In the second experiment, we collected 3 samples of N2 and 3 samples of atfs-1(et18).

first: Tomer

last: Shpilka

first: Yunguang

last: Du

first: Joshua

last: Lavelle

first: Lihua


title: Delineating copy number and clonal substructure in human tumors from single-cell transcriptomes

city: Houston

first: Nicholas

last: Navin

state: tx

address: 6767Bertner AVE

institute: UT MD Anderson

summary: Single-cell transcriptomic analysis is widely used to study human tumors. However it remains challenging to distinguish normal cell types in the tumor microenvironment from malignant cells and to resolve clonal substructure within the tumor.  To address these challenges, we developed an integrative Bayesian segmentation approach called CopyKAT (Copynumber Karyotyping of Aneuploid Tumors) to estimate genomic copy number profiles at an average genomic resolution of 5Mb from read depth in high-throughput scRNA-seq data.  We applied CopyKAT to analyze 46,501 single cells from 21 tumors, including triple-negative breast cancer, pancreatic ductal adenocarcinomas, anaplastic thyroid cancer, invasive ductal carcinoma and glioblastoma to accurately (98%) distinguish cancer cells from normal cell types.  In three breast tumors, CopyKAT resolved clonal subpopulations that differed in the expression of cancer genes such as KRAS and signatures including EMT, DNA repair, apoptosis and hypoxia.  These data show that CopyKAT can aid the analysis of scRNA-seq data in a variety of solid human tumors.

overall_design: We calculated single cell copy number profiles and inferred clonal structures of tumor cells from scRNAseq data using COPYKAT.

first: Ruli

last: Gao

first: Shanshan

last: Bai

first: Ying

last: Henderson

first: Yiyun

last: Lin

first: Aislyn

last: Schalck

first: Yun

last: Yan

first: Tapsi

last: Kumar

first: Min

last: Hu

first: Emi

last: Sei

first: Alexander

last: Davis

first: Fang

last: Wang

first: Simona

last: Shaitelman

first: Jennifer

first: Ken

last: Chen

first: Stacy

last: Moulder

first: Stephen

middle: Y

last: Lai

middle: E


title: RNA Sequencing of sorted neutrophils cells from tet mutant zebrafish embryos

first: Kelly

last: Banks

summary: We report overexpression of socs3b leading to reduced cytokine signalling

overall_design: Neutrophils were sorted based on the reporter mpx:GFP

first: Yahui

last: Lan

first: Todd

last: Evans


title: An all-to-all approach to the identification of sequence-specific readers for epigenetic DNA modifications on cytosine

status: Public on Dec 01 2020

city: Baltimore

first: Heng

last: Zhu

state: Maryland

address: MRB 340, 733 N. Broadway

department: Pharmacology and Molecular Sciences

institute: Johns Hopkins School of Medicine

summary: Epigenetic modifications of DNA play important roles in many biological processes. Identifying readers of these epigenetic marks is a critical step towards understanding the underlying mechanisms. Here, we present an all-to-all approach, dubbed Digital Affinity Profiling via Proximity Ligation (DAPPL), to simultaneously profile human TF-DNA interactions using mixtures of random DNA libraries carrying different epigenetic modifications (i.e., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine) on CpG dinucleotides. Many proteins that recognize consensus sequences carrying these modifications in symmetric and/or hemi-modified forms were identified. We further demonstrated that the modifications in different sequence contexts could either enhance or suppress TF binding activity. Moreover, many modifications can affect TF binding specificity. Furthermore, symmetric modifications showed a stronger effect in either enhancing or suppressing TF-DNA interactions than hemi-modifications. Finally, in vivo evidence suggested that USF1 and USF2 might regulate transcription via hydroxymethylcytosine-binding activity in weak enhancers in human embryonic stem cells.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
An all-to-all approach, dubbed Digital Affinity Profiling via Proximity Ligation (DAPPL) was developed to simultaneously profile human TF-DNA interactions using mixtures of barcoded TF proteins and random DNA libraries. First, in order to optimize the DAPPL approach, the 16-mer DNA library was applied to profiling ETS proteins binding consensus sequences.  Second, Many TF readers of epigenetic modifications were identified using 1,239 barcoded TF/co-factors and a mixture of four DNA libraries carrying different epigenetic modifications (i.e., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine) and one DNA library without any modification on CpG dinucleotides. Third, in vivo evidence suggested that USF1 and USF2 might regulate transcription via hydroxymethylcytosine-binding activity in weak enhancers in human embryonic stem cells.
Various groups: This study included 1,543 human TF clones, which were
randomly divided into eight groups (e.g., Group_1, Group_2, etc). Each
group of TFs was subjected to the screening assay using symmetric or
hemi-epigenetic modified DNA libraries respectively.
Cyc1 and Cyc2: two DAPPL assays using one round selection and two rounds
selection separately. Here, only the pilot experiments (two files named
with DAPPL_ETS) were optimized under two conditions, one round selection
and two round selections. All other DAPPL assay (16 files named with
DAPPL_Group) were performed using one round selection only.
Hemi_modification: only one strand of the dsDNA library used in DAPPL assay
contains epigenetic modified cytosine.
Symmetric_modification: both strands of the dsDNA library used in DAPPL
assay contain epigenetic modified cytosine.
All of the computer programs and scripts used are publicly available at
https://github.com/HitTracy/DAPPL

first: Jiang

last: Qian

first: Guang

last: Song

first: Guohua

first: Ximei

last: Luo


title: Loss of cyclin A2 in murine colonic epithelial cells disrupts colon homeostasis by triggering DNA damage and dysplasia and high cyclin A2 expression is a good-prognosis factor in patients with colorectal cancer

city: Marseille

first: Quentin

last: Da Costa

address: 232 boulevard de Sainte Marguerite

institute: Institut Paoli-Calmettes

summary: To clarify the function of cyclin A2 in colon homeostasis and colorectal cancer (CRC) we generated mice deficient for cyclin A2 in colonic epithelial cells (CEC). Colons of those mice displayed architectural changes in the mucosa, and signs of inflammation as well as an increased proliferation of CEC associated with the appearance of low- and high-grade dysplasia. The main initial events triggering those alterations in cyclin A2 deficient CEC appear to be abnormal mitoses and DNA damage. Cyclin A2 deletion in CEC promoted the development of dysplasia and adenocarcinomas in the murine colitis-associated cancer model.

overall_design: CEC were isolated from the proximal colon part from 4 weeks-old Ccna2 fl/fl vil-Cre (KO) and Ccna2 fl/fl (control) mice. RNA sequencing was performed on an Illumina NextSeq 500.

first: Bénédicte

last: Lemmers

first: Emilie

last: Mamessier


title: Whole genome transcriptome profiling identifies TIGIT as a regulator of T cell exhaustion in liver cancer

city: Tuebingen

first: Stefan

last: Czemmel

address: Auf der Morgenstelle 10

department: University of Tuebingen

institute: Quantitative Biology Center (QBiC)

summary: PD-1 checkpoint inhibition has shown promising results in patients with hepatocellular carcinoma, inducing objective responses in approximately 20% of the treated patients. The role of other co-inhibitory molecules and their individual contribution to T cell dysfunction in liver cancer, however, remain largely elusive. Here we perform for the first time a comprehensive mRNA profiling of CD8 T cells in a murine model of autochthonous liver cancer by comparing the whole genome transcriptome of naive, functional effector and exhausted, tumor-specific CD8 T cells. Our results reveal a unique transcriptome signature of exhausted T cells and demonstrate that upregulation of the inhibitory immune receptor TIGIT represents a hallmark in the process of T cell exhaustion in liver cancer. Compared to PD-1, expression of TIGIT more reliably identified exhausted, HCC-specific T cells at different stages of their differentiation. In combination with PD-1 inhibition, targeting of TIGIT with antagonistic antibodies resulted in synergistic inhibition of liver cancer growth in immunocompetent mice. Finally, we demonstrate expression of TIGIT on tumor infiltrating CD8 T cells in tissue samples of HCC patients and identify two subsets of HCC patients based on differential expression of TIGIT on HCC-specific T cells. Our transcriptome analysis therefore provides a valuable resource for the identification of key pathways involved in T cell exhaustion in patients with hepatocellular carcinoma and identifies TIGIT as a potential target in checkpoint combination therapies.

overall_design: Expression profiling of OT-I cells  isolated from OVA+ tumor-bearing livers (exhausted T cells) or livers of tumor-free control mice  (effector T cells), respectively. As additional reference data, naive OT-I cells (naive T cells) were isolated from spleens of naive mice, that have previously received adoptive transfer of naive OT-I cells.

first: Thomas

last: Wirth

first: Oliver

last: Dittrich-Breiholz


title: RNA transcripts from WT or Bmal1-deficient blood-brain barrier throughout a 24 hr period

city: Philadelphia

first: Shirley

last: Zhang

state: Pennsylvania

address: 3400 Civic Center Blvd, Bldg 421, Rm 10-162

institute: University of Pennsylvania

summary: The blood-brain barrier (BBB) is critical for neural function as it restricts effects of peripheral molecules on the brain. We report here circadian regulation of the BBB in mammals. Efflux of xenobiotics by the BBB oscillates in mice, with highest levels during the active phase and lowest during the resting phase. This oscillation is abrogated in circadian clock mutants. To elucidate the mechanisms of circadian regulation, we profiled the transcriptome of brain endothelial cells; interestingly, we detected limited circadian regulation of transcription, with no evident oscillations in efflux transporters. We recapitulated the cycling of xenobiotic efflux using a human microvascular endothelial cell line to find that the molecular clock drives the cycling of intracellular magnesium through transcriptional regulation of TRPM7, thereby accounting for the rhythm in efflux. As many drugs are substrates of efflux transporters, these findings highlight the importance of considering circadian regulation when targeting therapeutics to the CNS.

overall_design: 2 genotypes (control endothelium and Bmal-deficient endothelium) were assessed for RNA transcript levels over 6 time points across a circadian day with 2 replicates per time point

first: Nicholas

last: Lahens

first: Zhifeng

last: Yue

first: Denice

middle: M

last: Arnold

first: Peter

middle: P

last: Pakstis

first: Jessica

last: Schwarz

first: Amita

last: Sehgal


title: Endotoxemia by P. gingivalis change endocrine in brown adipose tissue

city: Tokyo

first: Sayaka

last: Katagiri

institute: Tokyo Medical and Dental University

summary: The purpose of this study was to evaluate the effect of endotoxemia on brown adipose tissue by injection of ultrasonicated P. gingivalis to mice

overall_design: Intravenous injection of P. gingivalisin jection, and saline at brown adipose tissue were measured at 18 hours after injection.

first: Hirishi

last: Nitta


title: RNA seq analysis of GD2-B7H3 gated CAR-T cells versus conventional B7H3 CAR-T cells

status: Public on Nov 30 2020

city: Los angeles

first: Xiangming

last: Ding

state: California

address: 4650 Sunset Blvd

institute: CHLA

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized the systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) between two generations of CAR-T cells aimed at integrating their fitness to overcome exhaustion and stress in tumor microenvironments
Methods: mRNA profiles of CAR-T cells co-cultured with target cells for 48 hours were generated by deep sequencing. Human CD45 enrichment was performed to decrease tumor contamination.
Results: Gene Set Enrichment Analysis (GSEA) of the RNA expression profile of T cells obtained 48 hours post co-culture with target cells demonstrated the enrichment of genes with memory T cell phenotype in B7H3 CAR-T compared to GD2-B7H3 T cells. However, B7H3 CAR-T cells were enriched in genes associated with glycolysis and apoptosis pathways suggesting a committed terminal fate for these T cells. There was significantly greater expression of genes attributed to exhaustion in B7H3 CAR-T cells than GD2-B7H3 T cells. In particular, B7H3 CAR-T cells showed higher expression of inhibitory receptors LAG3, HAVCR2 (TIM3), and BTLA genes, along with exhaustion-related transcription factor genes TBX21 (T-bet), PRDM1 (Blimp-1), and IKZF2 (Helios). B7H3 CAR-T cells also expressed genes that encode transcription factors reported to be associated with activated and memory T cells, such as KLF6, JUN, and JUNB.
Conclusions: Our study represents the first analysis of transcriptome comparing SynNotch gated GD2-B7H3 CAR-T cells versus the conventional B7H3 CAR-T cells. GD2-B7H3 T cells exhibited superior resistance to exhaustion and greater metabolic fitness in comparison to conventional CAR-T cells. Metabolic preference (glycolytic versus oxidative) has an enormous impact on T cell fate. After eradicating NBL cells, GD2-B7H3 T cells had an oxygen consumption rate similar to UT cells, suggesting that the gated T cells can revert to their naïve metabolic state. Improved metabolic plasticity and reprogramming in favor of oxidative rather than glycolytic phosphorylation supports the hypothesis that the gated CAR-T cells likely have intact expansion potential, similar to unmanipulated naïve T cells. We observed significant enrichment of glycolytic genes in conventional B7H3 CAR-T cells compared to GD2-B7H3 T cells, supporting our metabolic studies. Our data suggest that SynNotch gated CAR-T cells have a more favorable oxidative metabolic profile than 4-1BB CAR-T cells.

first: Babak

last: Moghimi


title: Cancer-Related Mutations are Not Enriched in Naïve Human Pluripotent Stem Cells

city: Cambridge

first: Giuliano

middle: Giuseppe

last: Stirparo

state: Cambridgeshire

address: 418 Cambridge Science Park Milton Rd

institute: Cerevance ltd

summary: A recent paper reported multiple oncogenic point mutations in naïve human pluripotent stem cells (hPSC) and implicated selective culture conditions. However, we observed that those “polymorphisms” are only found when naïve hPSCs are co-cultured with mouse feeder cells.  Inspection of the reads containing the reported mutations revealed that they are identical to mouse sequences. Filtering the data sets to remove reads contributed by mouse feeder cells reveals that the actual incidence of cancer-related polymorphisms is close to zero. Therefore, culture conditions for naïve hPSCs do not select for tumorigenic point mutations.

overall_design: Total RNA was extracted from two biological replicate cultures of each cell line and time point

first: G

middle: G

first: Guo

last: Ge

first: Austin

last: Smith


title: Targeting Transcriptional Regulation of SARS-CoV-2 Entry Factors ACE2 and TMPRSS2

city: Ann Arbor

first: Arul

middle: M.

last: Chinnaiyan

state: MICHIGAN

address: 1500 E. Medical Center Dr

institute: University of Michigan

summary:
SARS-CoV-2, the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity.  Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2, TMPRSS2, and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 years of age. In males greater than 70 years old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells.  Transcriptional repression of the AR enhancesome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.
These mouse data are part of a larger investigation (data not provided here) targeting the transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2.

overall_design: 8 week old mice were either left untreated or surgically castrated for an additional 12 days. Mice were then sacrificed and their lungs were collected and flash frozen. Nuclei were then extracted for snRNAseq.

first: Sethuramasundaram

last: Pitchiaya

first: Yuping

first: Sathiya

last: Narayanan

first: Yuanyuan

last: Qiao

first: Stephanie

last: Simko

first: Fengyun

last: Su

first: Rui

last: Wang

first: Xuhong

last: Cao


title: Metabolic control of imprinted gene expression in naive pluripotent cells

summary: Naive pluripotent epiblast cells of the preimplantation embryo and their in vitro counterpart, naive murine embryonic stem (ES) cells , are characterised by global DNA hypomethylation. This is caused by elevated expression of TETs oxidases and by downregulation of DNA methyltransferases (DNMTs). However, the signal orchestrating such dynamic changes in vitro and in vivo are only partially understood. Here we report that Stat3 induces genomic hypomethylation via metabolic reconfiguration. In Stat3 -/- ES cells we observed reduced alpha-ketoglutarate (ɑKG) production from reductive glutaminolysis. This is accompanied by reduced levels of Tet2 and its product, hydroxy-methyl-cytosine, together with increased Dnmt3a/b expression and methyl-cytosine levels. Notably, genome methylation can be dynamically controlled by simply modulating αKG availability, mitochondrial activity or Stat3 activation. Stat3-/- ES cells show also general increased methylation at Imprinting Control Regions accompanied with differential expression of >60% of imprinted genes. Several of them, including Lin28a, Ndn and Peg10, are normally upregulated during differentiation, and displayed anticipated and enhanced expression in Stat3-/- cells, indicating faster differentiation kinetics. Single-cell transcriptome analysis of Stat3-/- embryos confirmed dysregulated expression of Dnmt3a/b, Tet2, imprinted genes and anticipated expression of differentiation markers. Our results reveal that Stat3 bridges the metabolic and epigenetic profiles of naive pluripotent cells and may be relevant under Stat3-dependent pathological conditions.

overall_design: Stat3-/- ES cells

first: Riccardo

last: Betto

first: Linda

last: Diamante

first: Valentina

last: Perrera

first: Matteo

last: Audano

first: Stefania

last: Rapelli

first: Andrea

last: Lauria

first: Danny

last: Incarnato

first: Mattia

last: Arboit

first: Touboul

last: David

first: Guerineau

last: Vincent

first: Tim

last: Lohof

first: Thorsten

last: Boroviak

first: Jennifer

last: Nichols

first: Nico

last: Mitro

first: Salvatore

last: Oliviero

first: Graziano

last: Martello


title: Genome-scale CRISPR screening identifies cell cycle and protein ubiquitination processes as druggable targets for erlotinib-resistant lung cancer

city: Seoul

first: AHYOUNG

last: CHOI

address: 52, Ewhayeodae-gil

institute: Ewha womans university

summary: This study aims to identify new genes and pathways associated with erlotinib sensitivity in order to develop novel therapeutic strategies. Here, we induced artificial knock-out (KO) mutations in erlotinib-resistant human lung cancer cells (NCI-H820) using a genome-scale CRISPR-Cas9 sgRNA library to screen for genes involved in erlotinib susceptibility.

overall_design: CRISPR screening

first: Jieun

last: Lee

first: Ahyoung

last: Choi

first: Sung-Yup

last: Cho

first: Charles

first: Sanghyuk