title: The miR-28-5p targetome discovery identified SREBF2 as one of the mediators of the miR-28-5p tumor suppressor activity in prostate cancer cells

city: Copenhagen

first: Francesco

last: Russo

address: Blegdamsvej 3b

country: Denmark

institute: Novo Nordisk Foundation Center for Protein Research, University of Copenhagen

summary: miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have a tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) cells affecting cell proliferation/survival as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing we collected the complete repertoire of miR-28-5p targets obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22 we found that the 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with increasing importance in PCa. We validated miR-28-5p/SREBF2 interaction demonstrating that SREBF2 inhibition affects almost all the tumor processes affected by the miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development and potential new targets for anti-tumor therapy.

overall_design: The miRNA pull out assay was performed as described in Rizzo et al. [ref: doi:10.7150/jca.18396]. DU-145 were transfected using Lipofectamine 2000 (Thermo Fisher) with 60 nM of either miR-28-5p duplex (ds-miR-28CT) or a mix of 3’ biotin-tagged miR-28-5p 8tU (nucleotide 8 was a thiouridine) and miR-28-5p 18tU duplexes (ds-miR-28BIO). All oligos were synthetized by Bio-Synthesis Inc. 24 hours after transfection cells were irradiated with UV (365nm, 2J/cm2) using the Bio-Link crosslinking (BLX) (Ambrose Lourmat) and total RNA extracted with TRIzol (Thermo Fisher) directly on adherent cells following the manufacturer’s protocol. 15 μg of RNA was incubated 4 hours at 4°C with 100 μl of streptavidin-conjugated beads (Streptavidin Sepharose high performance, GE Healthcare) and the RNA complexed with the beads was recovered using TRIzol. We performed two biological replicates obtaining three miR-28CT (control) and two miR-28BIO (miR-28-5p) pull out samples consisting of background RNA and ds-miR-28BIO/interacting mRNA complexes respectively. The RNA isolated after the miRNA pullout procedure was used for the construction of the cDNA libraries using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina) according to the manufacturer’s suggestions. cDNA libraries were sequenced by HiSeq2000 (Illumina) in single-reads mode (50bp) by IGA Technology Service, Udine, Italy, obtaining about 20 million of reads for each samples.

first: Sofia

last: Fazio

first: Gabriele

last: Berti

first: Monica

last: Evangelista

first: Romina

last: D'Aurizio

first: Alberto

last: Mercatanti

first: Marco

last: Pellegrini

first: Milena

last: Rizzo


title: Transcriptomic and epigenetic profiling of genome control in the germline stem cell cycle in mice

status: Public on Feb 06 2020

city: Kyoto

first: Shinichiro

last: Chuma

state: Kyoto

address: 53 Kawahara-cho, Shogoin, Sakyo-ku

department: Institute for Frontier Life and Medical Sciences

country: Japan

institute: Kyoto university

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Transcriptomic and epigenetic profiling of genome control in the germline stem cell cycle in mice [ChIP-seq]

summary: The faithful transmission of genome information is fundamental to the germline stem cell cycle. One exception to this is meiosis when active recombination takes place. This study aims to understand how dna damage responses and meiotic recombination mechanisms are properly coordinated with developmental programs of germ cells by comparing transcriptomic and epigenetic profiles of germline competent pluripotent stem cells, germline stem cells and somatic cells etc. in mice.

overall_design: Histone modification and transcription factor binding profiles of mouse germline stem (GS) cells treated with or without retinoic acid were generated by Illumina deep sequencing.

first: Mihoko

last: Hosokawa


title: Sexually dimorphic methylation of CD3+ T-lymphocyte DNA in offspring of overweight and obese mothers in a high risk, minority population in the Bronx

status: Public on Feb 07 2020

city: Bronx

first: Maureen

middle: J

last: Charron

state: NY

address: 1300 Morris Park Ave

department: Biochemistry

country: USA

institute: Albert Einstein College of Medicine

type: Methylation profiling by high throughput sequencing

summary: Maternal obesity impacts the health of offspring, increasing the risk of developing obesity and/or other metabolic dysregulation in childhood or later in life. Using a genome-wide methylation assay, we identified sex-dependent dysregulation of the methylome of CD3+ T-lymphocytes, a cell type that plays an important role in obesity and inflammatory diseases, in newborn offspring of overweight and obese mothers. Furthermore, the differentially methylated loci were targeted to regulatory regions of the genome, in imprinted genes and genes identified from GWAS as being related to type 2 diabetes and obesity.

overall_design: DNA methylation was assessed globally at specific CpG sites in CD3+ T cell from 76 consenting women who delivered healthy, non-anomalous singleton AGA term neonates following an uncomplicated intrapartum course at the Weiler Division of Montefiore Medical Center.

first: Yoshinori

last: Seki

first: Lyda

last: Williams

first: Mamta

last: Fuloria

first: Masako

last: Suzuki


title: Gene expression profiling for control and high temperature treated Japanese medaka

status: Public on Feb 05 2020

city: Québec

first: Valerie

last: Langlios

address: 490, rue de la Couronne

country: Canada

institute: Institut national de la recherche scientifique

summary: the experiment was designed to investigate the temperature effect on gene expression

overall_design: RNA-seq of single stage 39 embryos grown at 24 C (control) or 32 C (high temperature).

last: Langlois


title: circRNA Microarray for human colorectal adenocarcinoma and normal mucosa

city: Beijing

first: Rui

last: Chen

address: No.10 Xitoutiao, You An Men

country: China

institute: Capital medical university

summary: Total RNA from each sample was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols. Briefly, total RNA from each sample was amplified and transcribed into fluorescent cRNA utilizing random primer according to Arraystar’s Super RNA Labeling protocol (Arraystar Inc.). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8x15K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C.

overall_design: Examination of circRNA expression in CRC

first: Xiaobo

last: Li


title: Widespread prion-based control of growth and differentiation strategies in Saccharomyces cerevisiae

city: Stanford

first: Anupam

middle: K.

last: Chakravarty

state: CA

address: 269 Campus Dr, CCSR3120

department: Chemical & Systems Biology

institute: Stanford University

summary: Theory and experiment suggest that organisms would benefit from pre-adaptation to future stressors based on reproducible environmental fluctuations experienced by their ancestors. Yet mechanisms driving pre-adaptation remain enigmatic. We report that the [SMAUG+] prion allows yeast to anticipate nutrient repletion after periods of starvation, providing a strong selective advantage. By transforming the landscape of post-transcriptional gene expression, [SMAUG+] regulates the decision between two broad growth and survival strategies: mitotic proliferation or meiotic differentiation into a stress-resistant state. [SMAUG+] is common in laboratory yeast strains, where standard propagation practice produces regular cycles of nutrient scarcity followed by repletion. Distinct [SMAUG+] variants are also widespread in wild yeast isolates from multiple niches, establishing that prion polymorphs can be utilized in natural populations. Our data provide a striking example of how protein-based epigenetic switches, hidden in plain sight, can establish a transgenerational memory that integrates adaptive prediction into developmental decisions.

overall_design: Two biological replicates were analyzed for each of 4 conditions (two independent transient overexpression of VTS1 gene for wild type genotype, one 'no transient overexpression' control for wild-type genotype, one vts1 deletion genotype)

middle: K

first: Alan

last: Itakura

first: Daniel

middle: F

last: Jarosz


title: Transcriptomic and epigenetic profiling of genome control in the germline stem cell cycle in mice [RNA-seq]

overall_design: Transcriptome profiles of mouse germline stem (GS) cells, embryonic stem (ES) cells, and primary embryonic fibroblast (PEF) cells were generated by Illumina deep sequencing.


title: Validation of a gene signature in histologically normal surgical margins predictive of oral squamous cell carcinoma recurrence

city: Toronto

first: Igor

last: Jurisica

state: Ontario

address: 60 Leonard Avenue

department: Krembil Research Institute

institute: University Health Network

summary:
The aim of this study is to provide an extended, retrospective validation of a previously described 4-gene signature (MMP1, COL4A1, P4HA2, and THBS2) predictive of oral carcinoma recurrence.  We assessed an independent cohort including 245 histologically normal surgical margins from 62 patients with OSCC. Gene expression was determined using a color-coded probe assay for quantitative gene expression analysis.
All margins and tumor blocks were cut (4-5µm tick) and stained with Hematoxylin-Eosin and tissues were subjected to needle microdissection using the stereo microscope Leica EZ4 (Leica Microsystems, Wetzlar, Germany) before RNA extraction, in order to isolate pure tumor or normal cell populations. RNA from FFPE samples was isolated using the RecoverAll Total Nucleic Acid Isolation kit (Ambion/Life Technologies, Carlsbad, CA, USA), following our previously reported protocol with modifications to improve RNA yield [PMID:23835716]. RNA was stored at -80ºC until RNA extraction and gene expression validation. In addition, tumor tissues from the same patients were collected for further analysis (not in scope of the presented study). Probes were designed for the 4-gene signature (MMP1, COL4A1, P4HA2 and THBS2) and contained one capture probe linked to biotin and one reporter probe attached to a color-coded molecular tag for each gene sequence, according to the nCounterTM code-set design. RNA samples were randomized using a numerical ID and were then subjected to NanoString nCounterTM analysis by the Princess Margaret Genomics Centre (https://www.pmgenomics.ca), Toronto, ON, Canada. Patient samples were analyzed in two batches consisting of 125 and 152 margins tissue samples. The detailed protocol for mRNA transcript quantification analysis, including sample preparation, hybridization, detection and scanning followed the manufacturer’s recommendations and was described in [PMID: 21549012]. We used 400 ng of total RNA isolated from FFPE tissues for detection of probe signals. Raw data were analyzed using the nCounterTM digital analyzer software (http://www.nanostring.com/support/ncounter/) and gene expression levels were subjected to further bioinformatic and statistical analyses.

overall_design: From the originally 277 margin samples obtained from 67 patients, 8 samples were removed as technical replicates, along another 24 samples from the five patients with insufficient follow up (follow up < 3 years). This study was thus conducted on 245 margins samples obtained from 62 OSCC patients. The number of margins, per patient, ranged between 1-8.

first: Tomas

last: Tokar

first: Patricia

middle: P

last: Reis

first: Rashmi

middle: S

last: Goswami

first: Levi

last: Waldron

first: Yali

last: Xuan

first: Mahadeo

last: Sukhai

first: Bayardo

last: Perez-Ordonez

first: Colleen

last: Simpson

first: David

last: Goldstein

first: Dale

last: Brown

first: Patrick

last: Gullane

first: Jonathan

last: Irish

first: Suzanne

last: Kamel-Reid


title: MiR-210 impact on the transcriptome suggests regulation of inflammation and regeneration pathways

city: Milan

first: Fabio

last: Martelli

address: via morandi 30

country: Italy

institute: IRCCS-Policlinico San Donato

summary:
In order to understand the consequences of miR-210 blocking on the ischemia response, the transcriptomic changes were investigated by microarray technology in gastrocnemius muscles of ANTI-210 and SCR treated mice, 7 days after ischemia.
Identified differentially expressed genes were used to perform gene-ontology enrichment-analysis, allowing to identify several terms falling into the biological macro areas of angiogenesis and blood vessel development, as well as related categories, such as cell adhesion, migration and proliferation. These findings are in agreement with the identified positive role played by miR-210 in the neo-vascularization process. Furthermore, many terms were also related to metabolism and mitochondrial organization, in keeping with the role of miR-210 in the regulation of oxidative phosphorylation.

overall_design: MiR-210 was blocked in ischemic gastrocnemius muscles by injecting ANTI-210 LNA-GAPmers (Exiqon) and transcriptomic changes were measured by lllumina BeadChip platform.

first: Matteo

last: Carrara

first: Germana

last: Zaccagnini

first: Biagina

last: Maimone

first: Christine

last: Voellenkle


title: Genome-wide maps of chromatin state for LAG-1 in germline

status: Public on Feb 17 2020

city: St. Louis

first: Jian

last: Chen

state: MO

address: 4523 Clayton Ave.

department: Genetics

institute: Washington University School of Medicine

summary: We report the application of conventional ChIP-seq analysis for LAG-1 chromatin state in germline. We identified 137 genes putatively regulated by LAG-1 that's overlapped by three independent biological replicates.  These genes included previously known targets lst-1, sygl-1, and mir-61/250.

overall_design: Three independent biological replicates were performed for ChIP-seq analyses. ChIP-seq peaks were called by MACS2.0 with fold enrichment (IP / input) greater than 2.0 and False discovery rate less than 0.05.

first: Chen

last: Jian

first: Schedl

last: Tim


title: Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability

city: Oxford

first: Aino

middle: Inkeri

last: Järvelin

address: South Parks Road

department: Department of Biochemistry

country: United Kingdom

institute: University of Oxford

summary: The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by known extrinsic signals. However, the intrinsic mechanisms that control the characteristic proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, while heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates.

overall_design: RNA-seq and RIP-seq in Drosophila L3 larval brain.

first: Tamsin

last: Samuels

middle: I

first: Ilan

last: Davis


title: A Non-Amyloid Prion Particle that Activates a Heritable Gene Expression Program.

first: Anupam

last: Chakravarty

summary: Spatiotemporal gene regulation is often driven by RNA-binding proteins that harbor long intrinsically disordered regions in addition to folded RNA-binding domains. We report that the disordered region of the evolutionarily ancient developmental regulator Smaug drives selfassembly into gel-like condensates. These proteinaceous particles are not composed ofamyloid. Yet they are infectious, allowing them to act as a protein-based epigenetic element: a prion. In contrast to many amyloid prions, condensation of Smaug enhances its function in mRNA decay, and its self-assembly properties are conserved over large evolutionary distances. Yeast cells harboring the [SMAUG+] prion downregulate a coherent network of mRNAs and exhibit improved growth under nutrient limitation. Smaug condensates formed from purified protein can transform naïve cells to acquire the prion. Our data establish that non-amyloid selfassembly of RNA-binding proteins can drive a form of epigenetics beyond the chromosome, instilling adaptive gene expression programs that are heritable over long biological timescales.

first: Daniel

last: Jarosz


title: Genome-wide transcriptomic analysis reveals the gene regulatory network that controlled by SRL1 in regulating rice leaf rolling

city: nanning

first: wang

last: jun

state: guangxi

address: DaXue road100

institute: guangxi university

summary: To understand the gene regulatory network that controlled by SRL1, we analyzed the transcriptomic data that generated from the mature leaves (60 days after germination) of the SRL1 knockout mutants srl1-KO and the control non-KO. We carried out the RNA-seq with three biological replicates of srl1-KO and non-KO respectively.

overall_design: comparison of the trancriptome of the srl1-KO and the control

last: Jin

first: Jun

last: Wang


title: High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

city: Biberach an der Riß

first: Holger

last: Klein

state: Baden-Württemberg

address: Birkendorfer Straße 65

department: Global Computational Biology and Data Sciences

country: Germany

institute: Boehringer-Ingelheim Pharma GmbH & Co KG

summary:
Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8x104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.

DOI:10.1038/s41467-020-14491-x

overall_design: Identification of tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices from plasmid libraries containing up to 1.8x10^4 constructs by tetracycline or guanine stimulation at increasing dosages and subsequent cDNA amplicon sequencing.

first: Benjamin

last: Strobel

first: Maike

last: Spöring

first: Sergi

last: Sayols Puig

first: Jörg

last: Hartig

first: Sebastian

last: Kreuz


city: Paris

first: Franck

last: Letourneur

address: 22 rue mechain

country: France

institute: INSERM

summary: The antiretroviral drugs, dolutegravir, maraviroc and ritonavir-boosted atazanavir, affect inflammation and insulin sensitivity in human coronary endothelial cells through different pathways.

overall_design: Human coronary endothelial cells treated with the antiretroviral drugs dolutegravir, maraviroc and ritonavir-boosted atazanavir.

first: Jacqueline

last: Capeau

first: Florent

last: Dumont


title: UVR8-mediated changes in gene expression in response to UV-B, compared to genes misexpressed in pif4pif5 mutants

city: Geneva

first: Celine

last: Delucinge

address: 1 rue Michel Servet

department: iGE3 Genomics Platform

country: Switzerland

institute: University of Geneva

summary: We analysed global gene expression changes in Arabidopsis thaliana in response to 6h UV-B, applied at the same low level that was seen to promote subsequent UV-B stress tolerance, in order to elucidate the transcriptional reprogramming that leads to UV-B acclimation.

overall_design: mRNA profiles generated by deep sequencing from triplicate replicate Arabidopsis thaliana 4-day-old seedling samples sourced from independent seedling plates either protected from UV-B or exposed to 6h acclimation-level UV-B.

first: E

last: Tavridou

first: M

last: Pireyre

first: R

last: Ulm


title: Integration of ATAC-seq and RNA-seq identifies key genes in light induced primordium formation of Sparassis latifolia

city: Fuzhou

first: Chi

last: Yang

address: No.15 Shangxiadian Road, Cangshan District

institute: Fujian Agriculture and Forestry University

summary: Differential expression profiling of light induction and primordium differentiation was established by integrating ATAC-seq (assay for transposase accessible chromatin with high through put sequencing) and RNA-seq technology. Key genes in light induced the primordium formation of S. latifolia were identified by bioinformatics methods.

overall_design: primordia and mycelia

first: Lu

last: Ma

first: Donglai

last: Xiao

first: Zhenghe

last: Ying

first: Xiaoling

last: Jiang

first: Yanquan

last: Lin


title: DNA:cytoplasm ratio defines an upper limit to cell size [array]

city: Cambridge

first: Charles

middle: Arthur

last: Whittaker

state: MA

institute: Koch Institute

summary: Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important is not understood. Here we show that growing beyond a certain size has wide-ranging effects on cell physiology. Large cells are defective in gene induction, cell cycle progression and cell signaling. We further show that these defects are caused by the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. Finally, we determine why nucleic acid and protein biosynthesis do not scale with cell volume beyond a certain critical size. DNA becomes limiting. We conclude that the correct DNA to cytoplasm ratio is vital for many perhaps all cellular functions and that the range where this ratio supports optimal cell function is remarkably narrow.

overall_design:
Two-color microarray experiment comparing gene expression of S. cerevisiae cells of different cell size before and after 40 minutes of alpha factor exposure.
The cell sizes are:
WT: 50 fL
2h: 250 fL
6h: 815 fL
6h CHX: 325 fL

first: Gabriel

middle: E

last: Neurohr

first: Angelika

last: Amon


title: PEG project: effect of fluid flow on stromal populations in the peyer's patch

city: South San Francisco

first: Shannon

last: Turley

state: California

address: 1 DNA Way, Building 15, MS 231b

department: Cancer Immunology

institute: Genentech

summary: Here, we set out to delineate the effect of fluid flow on stromal populations in the peyer's patch.

overall_design: Mice were unperturbed or treated with PEG to diminish fluid flow for three days. Then fibroblastic reticular cells and high endothelial venules were FACS sorted for RNAseq.

first: Matthew

last: Buechler

first: Suchit

last: Jhunjhunwala

first: Nicolas

last: Lounsbury


title: Global transcriptome analysis identifies genes regulated by FT in the leaf

city: Cologne

first: Fernando

last: Andres

address: Carl von Linne 10

department: Plant Developmental Biology

institute: Max Planck Institute for Plant Breeding Research

summary: Flowering of Arabidopsis thaliana is accelerated by several environmental cues, including exposure to long days. The photoperiod-dependent promotion of flowering involves the transcriptional induction of FLOWERING LOCUS T (FT) in the phloem of the leaf. FT encodes a mobile protein that is transported from the leaves to the shoot apical meristem, where it forms part of a regulatory complex that induces flowering. Whether FT also has biological functions in leaves of wild-type plants remains unclear. In order to address this issue, we first studied the leaf transcriptomic changes associated with its over expression in the companion cells of the phloem. To this end, transgenic A. thaliana plants that misexpress FT from the pGAS1 promoter in a ft-10 tsf-1 double mutant background were employed (pGAS1:FT ft-10 tsf-1). In these transgenic plants, the use of the pGAS1 promoter ensures that the FT transgene is expressed in phloem companion cells of the minor veins, recreating the spatial pattern of expression described for the native gene. In this studuy, the transcriptome of leaves of  pGAS1:FT ft-10 tsf-1 transgenic plants was compared to that of ft-10 tsf-1 and Col-0 plants using Tiling Arrays.

overall_design: Col-0, ft-10 tsf-1 and pGAS1:FT ft-10 tsf-1 plants were grown under LD conditions during 9 days. Leaves of each genotypes were harvested at the end of the light period (ZT16). Biological triplicates of each genotype were used for the Tiling Array experiments.

last: Andrés

first: Seonghoe

last: Jang

first: Bruno

last: Heuttel

first: George

last: Coupland


title: Dual threshold optimization and network inference reveal convergent evidence from TF binding locations and TF perturbation responses

status: Public on Feb 04 2020

first: Yiming

last: Kang

address: 1 Brookings Dr

department: Computer Science and Engineering

institute: Washington University in St. Louis

summary: A high-confidence map of the direct, functional targets of each transcription factor (TF) requires convergent evidence from independent sources. Two significant sources of evidence are TF binding locations and the transcriptional responses to direct TF perturbations. Systematic data sets of both types exist for yeast and human. Standard analysis of the genes whose regulatory DNA is bound by a TF, assayed by ChIP-chip/seq, and the genes that respond to a perturbation of that TF, shows that these two data sources rarely converge on a common set of direct, functional targets. Even taking the few genes that are both bound and responsive as direct functional targets is not safe -- when there are many non-functional binding sites and many indirect targets, non-functional sites are expected to occur in the cis-regulatory DNA of indirect targets by chance. To address this problem, we introduce Dual Threshold Optimization, a new method for setting significance thresholds on binding and response data, and show that it improves convergence. It also enables comparison of binding data to perturbation-response data that has been processed by network inference algorithms, which further improves convergence. Next, we analyze a comprehensive new data set measuring the transcriptional response shortly after inducing overexpression of a yeast TF. We also present a new yeast binding location data set obtained by transposon calling cards and compare it to recent ChIP-exo data. The combination of dual threshold optimization and network inference greatly expands the high-confidence TF network map in both yeast and human.  In yeast, measuring the response shortly after inducing TF overexpression and measuring binding locations by using transposon calling cards or ChIP-exo improve the network synergistically.

overall_design: "Calling card" assays for transcription factors were performed in S. cerevisiae.

first: Nikhil

middle: R

last: Patel

first: Christian

middle: A

last: Shively

first: Pamela

last: Recio

first: Xuhua

last: Chen

first: Robi

middle: D

last: Mitra

first: Michael

last: Brent


title: Next generation sequencing of the mammary gland and pituitary of  WT and CXCR2 KO animals in SOPF or conventional conditions

city: Montpellier

first: Gwendal

last: Lazennec

address: 1682 rue de la valsiere

institute: CNRS

summary: We report the comparison of gene expression in the mammary gland and pituitary of WT Balb and CXCR2 KO balb animals which were housed in conventional or SOPF conditions

overall_design: mRNA profile was generated by deep sequencing using NextSeq 500  Illumina.


title: Effect of systemic adipsin overexpression on db/db mouse pancreatic islets

city: New York

first: Gregory

last: Putzel

address: 430 East 29th Street

department: Microbiology

institute: NYU Langone Health

summary: RNAseq of pancreatic islets of db/db mice treated with AAV- Adipsin vs AAV-GFP 4 weeks post AAV injection

overall_design: Isolated pancreatic islets from db/db-GFP and db/db-Adipsin mice were used. Unbiased transcriptomic profiling was performed at 1 month post-transduction, when serum glucose levels were similar between both groups. RNA samples from pancreatic islets isolated from 4 db/db mice treated with AAV-GFP and 3 db/db mice treated with AAV-Adipsin were included in the analysis. Each mouse was a separate biological replicate.

first: Nicolás

last: Gómez-Banoy


title: A pan-cancer transcriptome analysis to identify the molecular mechanism of prexasertib resistance_RNASeq

city: Indianapolis

first: Ricardo

last: Martinez

state: IN

address: 307 E Merrill St

institute: Eli Lilly and Company

summary:
The combined influence of oncogenic drivers, genomic instability, and/or DNA damage repair deficiencies increases replication stress in cancer. Cells with high replication stress rely on the upregulation of checkpoints like those governed by CHK1 for survival. Previous studies of the CHK1 inhibitor prexasertib demonstrated activity across multiple cancer types. Therefore, we sought to (1) identify markers of prexasertib sensitivity and (2) define the molecular mechanism(s) of intrinsic and acquired resistance using preclinical models representing multiple tumor types. Our findings indicate that while cyclin E dysregulation is a driving mechanism of prexasertib response, biomarkers associated with this aberration lack sufficient predictive power to render them clinically actionable for patient selection. Transcriptome analysis of a pan-cancer cell line panel and in vivo models revealed an association between expression of E2F target genes and prexasertib sensitivity and identified innate immunity genes associated with prexasertib resistance. Functional RNAi studies supported a causal role of replication fork components as modulators of prexasertib response. Mechanisms which protect cells from oncogene-induced replication stress may safeguard tumors from such stress induced by a CHK1 inhibitor, resulting in acquired drug resistance. Furthermore, resistance to prexasertib may be shaped by innate immunity.
We included RNASseq data here as part of the study. Microarray data was uploaded separately.

overall_design: Prexasertib resistant cell lines were generated using a long-term drug concentration escalation protocol. With 3 biological replicates in both resistant and parental lines, except OV-90 parental line with 2 biological replicates, RNAseq were used to identify differentially expressed genes, which helped to understand molecular mechanism of acquired resistance.

first: Xi

last: Rao


title: Differential RNA expression profile in antimony resistant and antimony sensitive clinical isolates of Leishmania donovani

city: Kolkata

first: Syamal

last: Roy

state: West Bengal

address: Chunilal Bhawan 168, Maniktala Main Road, Kol

department: Infectious Disease and Immunology

country: India

institute: Former: CSIR-IICB, Present: NIPER, Kolkata

summary: Infection with antimony resistant (SbR) but not with sentitive (SbS) Leishmania donovani (LD) gives rise to aggressive pathology and higher organ parasite load in infected patients, the cause of which is far from clear. The pentose phosphate pathway (PPP) is a key route of glucose metabolism in most organisms, providing NADPH for use as a cellular reductant and various carbohydrate intermediates used in cellular metabolism. Alteration in PPP is primary reason behind aggressive pathology and multi drug resistance in several types of carcinoma and other infectious disease model. A time kinetic study with equal inoculum of SbRLD and SbSLD promastigotes and host cells infected with equal number of SbR and SbSLD, resulted in remarkable higher growth rate in SbRLD which is also reflected in terms of their DNA content. Our result showed an activated PPP pathway in response to oxidative stress in SbRLD. Our result suggest that SbRLD have adapted to a higher nucleotide metaboism that provide them a selective survival advantage in presence of drug mediated oxidative stress and explains, in part, the cause of higher organ parasite load in SbRLD infected patients.

overall_design: Three clinical isolates representing SbRLD (BHU575, BHU814 and BHU138) and three clinical isolates representing SbSLD (BHU77, BHU581 and BPK206) along with sensitive reference strain Ag83 were grown in 75cm2 tissue culture flask. Total RNA was isolated from each isolate and two biological replicates were prepared and submitted for microarray profiling. The main objective of the microarray analysis was to compare differential RNA expression profile between antimony resistant and antimony sensitive Leishmania donovani isolates with particular focus to identify those RNAs which are involved in carbohydrate metabolism

first: Rupkatha

last: Mukhopadhyaya

first: Piyush

last: Nanda

first: Anupam

last: Gautam

first: Sandip

last: Paul

first: Amit

last: Ghosh

first: Budhaditya

last: Mukherjee


title: Genome-wide Analysis of Transcription Factors among Differentially Expressed Genes Induced by Adding Exogenous Nitrogen in Poplar Plants under Cadmium Stress

city: Chengdu

first: Jinliang

last: Huang

state: Sichuan

address: 211 Huimin Road, Wenjiang District

institute: Sichuan Agricultural University

summary:
Purpose:  In order to explore the detoxification mechanism of nitrogen against cadmium stress in plants
Methods:Cultivation and treatment for four months of P.yunnanensis seedlings by soil culture, in triplicate, qRT–PCR validation was performed using TaqMan and SYBR Green assays
Results:Exogenous nitrogen induced the expression of resistance genes
Conclusions: Our study represents that exogenous nitrogen addition under Cd stress induced more differential expressed TFs.

first: Huang

last: Jinliang

first: Zhang

last: Fan


title: Transgenerational effects of water-deficit and heat stress on germination and seedling vigour – new insights from durum wheat microRNAs

city: Urrbrae

first: Haipei

last: Liu

state: South Australia

address: PMB1 Waite Building, School AFW

country: Australia

institute: The University of Adelaide

type: Non-coding RNA profiling by high throughput sequencing

summary: Water deficiency and heat stress can severely limit crop production and quality. Stress imposed on the parents during reproduction could have transgenerational effects on their progeny.  Seeds with different origins can vary significantly in germination time-course and early growth. Here, we investigated how water-deficit and heat stress on parental durum wheat plants affected seedling establishment of the subsequent generation. One stress-tolerant and one stress-sensitive Australian durum genotype were used. Seeds were collected from parents with or without exposure to stress during reproduction. Generally stress on the previous generation negatively affected seed germination and seedling vigour, but to a lesser extent in the tolerant variety. Small RNA sequencing utilising the new durum genome assembly has revealed significant differences in microRNA (miRNA) expression in the two genotypes. A bioinformatics approach was used to identify multiple miRNA targets which have critical molecular functions in stress adaptation and plant development and could therefore contribute to the phenotypic differences observed. Our data provides the first confirmation of the transgenerational effects of reproductive-stage stress on germination and seedling establishment in durum wheat. New insights gained on the epigenetic level indicate that durum miRNAs could be key factors in optimising seed vigour for superior breeding germplasm and/or varieties.

overall_design: Examine the seedling miRNA population from two seed source of two genotypes

first: Amanda

last: Able

first: Jason


title: Effect of C3a treatment over cell death  of pancreatic islets ex vivo

first: Gregory

summary: RNAseq of islets from WT mice treated in vehicle or palmitate conditions, wit or without recombinant C3a

overall_design: Islets from 10-12 week old male WT mice were isolated and cultured in batches of approximmately 120 islets in each the following conditions: 1) vehicle 2) vehicle +C3a 3) Palmitate 4) Palmitate + C3a.


title: Whole genome bisulfite sequencing of human spermatozoa reveals differentially methylated patterns from type 2 diabetic patients

first: Deli

state: New York

address: 1300 York Ave

institute: Weill Cornell Medicine

summary: We investigated the genome-wide DNA methylation profiles in sperm by comparing 8 individuals with T2DM and 9 non-diabetic controls using whole genome bisulfite sequencing (WGBS) method. Our study provides the first evidence that T2DM reprograms sperm DNA methylome and provides new insights into the intricate mechanisms of susceptibility to T2DM in offspring.

overall_design: Whole genome bisulfite sequencing (WGBS) genomic DNA was isolated from control and T2DM samples. One replicate was done for each sample.

first: Xiongfeng

last: Chen

first: Gang


title: Comparative transcriptome analysis of inbred lines and contrasting hybrids reveals overdominance mediate early biomass vigor in hybrid cotton

city: Anyang

first: Jianyong

last: Wu

address: Huanghe Dadao 38#

institute: Institute of Cotton Research, CAAS

summary: Through genome-wide comparative transcriptome analysis, we strongly predict that overdominance at gene expression level plays a pivotal role in early biomass vigor of hybrids. The combinational contribution of circadian rhythm and other metabolic processes may be controlled vigorous growth in hybrids. Our result provides  an important foundation for dissecting molecular mechanisms of biomass vigor in hybrid cotton

overall_design: 42 samples, triplicates

first: Chaozhu

last: Xing


title: Deposition of centromeric histone H3 variant CENP-A/Cse4 into chromatin is facilitated by its C-terminal sumoylation

status: Public on Feb 21 2020

city: Worcester

first: Richard

last: Baker

address: 55 Lake Avenue North

department: Microbiology & Physiological Systems

institute: University of Massachusetts Medical School

summary: Centromeric localization of CENP-A (Cse4 in S. cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Overexpression of CENP-A leads to its mislocalization and contributes to aneuploidy in yeast, flies, humans and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and non-centromeric regions, respectively. Ubiquitin ligases such as Psh1 and Slx5 and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to non-centromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C-terminus of Cse4 and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A (K changed to arginine (R) or alanine (A)) and reduced interaction of mutant Cse4 K215/215R/A with Scm3 and CAF-1 when compared to wild type Cse4. Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction at centromeric and non-centromeric regions were reduced. Furthermore, in contrast to GAL-CSE4 which exhibits Synthetic Dosage Lethality (SDL) in psh1Æ, slx5Æ, and hir2Æ strains, GAL-cse4 K215/216R does not exhibit SDL in these strains. Taken together, our results identify and define a role for C-terminal sumoylation of Cse4 for its incorporation into chromatin.

overall_design: Cse4 ChIP-seq was carried out with strains overexpressing wild-type Cse4 or mutant Cse4 alleles with either arginine or alanine replacing the lysines at positions 215 and 216. Replicate samples of input and immunoprecipitated DNA were analyzed.

first: Kentaro

last: Ohkuni

first: Evelyn

last: Suva

first: Wei-Chun

last: Au

first: Robert

middle: L

last: Walker

first: Reuben

last: Levy-Myers

first: Paul

last: Meltzer

first: Munira

last: Basrai


title: BubR1 allelic effects drive phenotypic heterogeneity in MVA progeria syndrome [3 month old RNA-seq]

city: Rochester

first: Hu

last: Li

state: MN

address: 200 First Street, Gonda Building G19-408

department: Molecular Pharmacology & Experimental Therapeutics

institute: Mayo Clinic

summary: Mosaic-variegated aneuploidy (MVA) syndrome is a rare childhood disorder characterized by biallelic BUBR1, CEP57, or TRIP13 aberrations, increased chromosome missegregation, and a broad spectrum of clinical features, including various cancers, congenital defects, and progeroid pathologies. To investigate the mechanisms underlying this disorder and its phenotypic heterogeneity, we mimicked the BUBR1L1012P mutation in mice (BubR1L1002P) and combined it with two other MVA variants, BUBR1X753 and BUBR1H, generating a truncated protein and low amounts of wildtype protein, respectively. Whereas, BubR1X753/L1002P and BubR1H/X753 mice die prematurely, BubR1H/L1002P mice are viable and exhibit many MVA features, including cancer predisposition and various progeroid phenotypes, including short lifespan, dwarfism, lipodystrophy, sarcopenia, and low cardiac stress tolerance. Strikingly, although these mice had a similar reduction in total BubR1 and spectrum of MVA phenotypes as BubR1H/H mice, several progeroid pathologies were attenuated in severity, which in skeletal muscle coincided with reduced senescence-associated secretory phenotype (SASP) complexity. Additionally, mice harboring heterozygous BUBR1 MVA variants developed mild MVA pathologies later in life, with alleles conferring unique phenotypic profiles. Together, these data demonstrate that subtle BUBR1 allelic effects contribute to disease heterogeneity in both MVA patients and heterozygous carriers of MVA mutations, independent of aneuploidy rates and BUBR1 protein levels.

overall_design: Skeletal muscle (skM, gastrocnemius) from 3-month-old wildtype and two BubR1 MVA allele carriers, BubR1+/L1002P and BubR1+/X753 were collected and harvested from naturally aged mice, euthanized at 3 months of age. RNA was extracted from tissues and was used for RNA sequencing.

first: Jan

last: van Deursen


title: Transcriptomic profiling of a CamKII-Dars2 neuronal knockout mouse

city: Baltimore

first: Ali

last: Fatemi

state: MD

address: 707 N Broadway

department: Neurogenetics

institute: Kennedy Krieger Institute

summary:
Purpose: Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (DARS2).  While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brain stem, and spinal cord, clinical symptoms appear quite variable and a multitude of gene variants have been associated with the disease.  Here, Dars2 deletion from CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at 5 months, and culminating by 9 months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation.
Methods: Cortical samples were collected from DARS2-Flox/CamKII-Cre(+) and DARS2-Flox/CamKII-Cre(-) at 14 weeks of age. RNA-seq libraries were prepared using purified RNA isolated from frozen tissue using the RNeasy Mini kit. RNA quality and concentration were assayed using a Fragment Analyzer instrument. RNA-seq libraries were prepared using the TruSeq Stranded mRNA PolyA (Unique Dual Index) kit.. Libraries were sequenced on an Illumina HiSeq 4000 instrument. Samples were taken from N=7 DARS2-Flox/CamKII-Cre(+)  and N=6 DARS2-Flox/CamKII-Cre(-) male mice.
Conclusions:  RNA-seq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and the defense response regulation.  Among these transcripts, Cystatin F (Cst7), a protease inhibitor recognized as a marker of neurodegenerative disease and active demyelination, is upregulated over 200-fold in mutant mice and may serve as a key regulator of disease progression.

overall_design: RNA extracted from cortex samples of 7 male DARS2_flox/CamKII_Cre(+) and 6 male Dars2_flox/CamKII-Cre(-) mice at 5 weeks of age.

first: Christina

last: Nemeth

first: Lauren

last: LeBon


title: Next Generation Sequencing Facilitated Cerebellum DNA Methylation Analysis of Essential Tremor Patients

city: Loma Linda

first: Charles

last: Wang

address: 11021 Campus Street

department: Department of Basic Sciences

institute: Loma Linda University

summary: We report the RRBS-based cerebellum DNA methylation profiling of essential tremor (ET) patients and controls. By obtaining over thirty billion bases of sequence from bisulfited-converted DNA, we generated genome-wide methylation profile of human cerebellum tissue. We identified differential DNA methylation patterns in 735 genes at various gene parts, across 12 ET patients. Many of those differentially methylated CpGs in ET patients are located in upstream regulatory regions of genes. We further found that many differentially methylated genes in ET pateints are associated with neurodegenerative disease. This study provided a framework towards understanding epigenetic alteration in cerebellum of essnetial tremor patients.

overall_design: Examination of cerebellum DNA methylation in 12 deceased essential tremor patients and 11 control patients.

first: Khashayar

last: Dashtipour


title: A system biology approach reveals neuronal and muscles developmental defects after chronic exposure to ionising radiation

city: Saint Paul lez Durance

first: olivier

last: ARMANT

address: Cadarache BP3

department: SERIS

institute: IRSN

summary: Assessment of the transcriptomics effects of chronic exposure to gamma ionizing radiation (IR) on zebrafish development  at dose rates of 0.5mGy/h, 5mGy/h and 50mGy/h. Embryos were exposed chronically to IR from fertlization to developmental stages: 24hpf, 48hpf and 96hpf.

overall_design: Chronic exposure of zebrafish fertilized eggs to an external source of gamma radiation (137Cs) at 0.5mGy/h, 5mGy/h and 50mGy/h, plus non-exposed controls. Eggs were exposed continuously to IR at the 3 dos rates until they reach 24hpf, 48hpf and 96hpf stage, and subjected to mRNAseq stranded protocol (Illumina). All conditions were performed in at least triplicated biological experiments.

first: Olivier

last: Armant

first: Sophia

last: Murat El Houdigui


title: Novel autophagy inhibitor Lys05 enhances impact of ionizing radiation on human lung cancer cells H1299

city: Hradec Kralove

first: Lucie

last: Cechakova

address: Trebesska 1575

department: Department of Radiobiology

country: Czech Republic

institute: University of Defense in Brno

summary: Autophagy inhibition through small-molecule inhibitors is one of the approaches to increasing the efficiency of radiotherapy of oncological patients. A new inhibitor, Lys05, with potential to accumulate within lysosomes and to block autophagy has been discovered recently. Several studies have described its chemosensitizing effects, but nothing is known about its impact in the context of ionizing radiation (IR). To investigate the mechanisms underlying its role in radiosensitization we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative) in combination with multiple approaches (cell biology techniques to reveal the phenotypes, and quantitative phosphoproteomics to comprehensively describe Lys05-induced changes in irradiated cells). In the presented study, we report for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in eradication of lung cancer cells.

overall_design: H1299 cells were either irradiated or left unirradiated and both of these groups were either treated with Lys05 or spautin or left untreated. This results in six experimental groups per one time interval. The cells were sampled at time one, twenty-four and fourty-eight hours post treatment. In each time & treatment group were n=3 samples. There were 6 experimental groups * 3 times * 3 biological replicates = 54 samples. These samples were hybridized in two-color fashion (portion of the three biological replicates were labeled with Cy3, part with Cy5) but were analyzed as single channel (quantile normalization across all channels).

first: Martin

last: Ondrej

first: Vojtech

last: Pavlik

first: Petr

last: Jost

first: Dana

last: Cizkova

first: Ales

last: Bezrouk

first: Jaroslav

last: Pejchal

first: Ravi

last: Amaravadi

first: Jeffrey

last: Winkler

last: Tichy


title: Library selection with a randomized repertoire of (βa)8-barrel enzymes results in unexpected induction of gene expression

city: Regensburg

first: Gerhard

last: Lehmann

address: Universitaetsstrasse 31

department: Biochemistry 1

institute: University of Regensburg

summary: The (βa)8-barrel enzyme fold is frequently encountered in nature and catalyzes a multitude of different reactions. It is therefore interesting to evaluate the evolutionary potential of this fold by laboratory experiments. Along these lines, we tested the ability of the (βa)8-barrel to acquire new catalytic functions by an approach combining random mutagenesis and selection in vivo.  The genes encoding 52 different phosphate-binding (βa)8-barrel proteins were randomized by error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli (E. coli) strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same variants of point-mutated E. coli genes nanE (encoding a putative N-acetlymannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5’-phosphate synthase) were responsible for rescuing both DserB and DserC. Remarkably, all selected PdxJ variants have acquired a 10-residue C-terminal extension from the cloning vector, that is essential for the rescue of DserB and DserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or Serc reactions in vitro. However, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicates that HisC is promiscuous for the SerC reaction, as well. The successful rescue of DserB and DserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (βa)8-barrel fold. To our knowledge, this is the first example of (βa)8-barrels functioning not as enzymes, but by activating transcription via an unknown mechanism.

overall_design: Comparison of the expression pattern of cells expressing modified (ba)8-barrel proteins to cells expressing a negative control

first: Bettina

last: Rohweder

first: Norbert

last: Eichner

first: Gunter

last: Meister

first: Reinhard

last: Sterner


title: Laminin-binding integrins are essential for the maintenance of functional secretory epithelium in lactation

city: Paris

first: Pierre

last: de la Grange

address: 29, rue du Faubourg Saint-Jacques

department: Paris Biotech Santé

institute: GenoSplice technology

summary: Integrin dimers α3/β1, α6/β1 and α6/β4 are the mammary epithelial cell receptors for laminins, which are major components of the basement membrane, a specialized extracellular matrix surrounding the mammary epithelium. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated by the Cre-Lox approach. During pregnancy, mutant mice displayed low levels of luminal progenitor activity and retarded lobulo-alveolar development, whereas their mammary glands seemed to be functional at the onset of lactation. Myoepithelial cell morphology was markedly altered in mutant glands, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. However, lactation was not sustained in mutant mice, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. This study reveals an essential role for laminin-binding integrins in functional mammary gland development.

overall_design: Ctrl basal cells (4 biological replicates) /a3a6KO basal cells (3 biological replicates)

first: Marisa

middle: M

last: Faraldo

first: Mathilde

last: Romagnoli

first: Sylvain

last: Baulande

first: Marina

last: Glukhova


title: A single cell atlas of the developing Drosophila ovary identifies follicle stem cell progenitors

first: Maija

last: Slaidina

address: 540 1st ave

department: Skirball Institute

institute: New York University School of Medicine

summary: We use single cell RNA sequencing approach to identify and characterize all cell types in developing Drosophila ovaries at late third instar larval stage (LL3).

overall_design: Single-cell RNA-sequencing libraries of developing Drosophila ovaries

first: Ruth


title: Transcriptome (RNA-seq) of statin-treated human primary myotubes

status: Public on Feb 24 2020

city: Berlin

first: Stefanie

last: Grunwald

address: Lindenberger Weg 80

department: Muscle Research Unit

institute: Charité Medical Faculty - Experimental and Clinical Research Center

summary: Statin-induced myopathy is a common side-effect but its mechanisms in human skeletal muscle have never been explained satisfyingly. We exposed 25 different primary human muscle cell lines to either a lipophilic or a hydrophilic statin, quantified cholesterol, mevalonate, proliferation, differentiation, and performed a comprehensive transcriptome and proteome analysis. We found reduced mevalonic acid (MVA) and cholesterol levels demonstrating incorporation of statins into the cells. Statins also significantly impaired proliferation and differentiation. Using an integrated pathway analysis approach with transcriptome and proteome data from statin-treated human primary myotubes, we modelled a novel statin-induced metabolism network for human muscle cells. This analysis confirmed the effect of statins on cholesterol biosynthesis but also uncovered alterations of acetyl CoA-dependent lipid and fatty acid metabolism

overall_design: Human primary human myoblasts were treated with 2 different statins under fusion conditions.

first: Simone

last: Spuler

last: Haafke

first: Giannino

last: Patone

first: Kathrin

last: Saar


title: Laminin-binding integrins are essential for the maintenance of functional secretory epithelium in lactation [luminal cells]

overall_design: Ctrl luminal cells/a3a6KO luminall cells from 15-day pregnant mice - 4 biological replicates each


title: Sporadic activation of an oxidative stress-dependent NRF2–p53 signaling network in breast epithelial spheroids and premalignancies

city: Charlottesville

first: Kevin

middle: A.

last: Janes

state: VA

address: Box 800759 Health System

department: Department of Biomedical Engineering

institute: University of Virginia

summary: Breast–mammary epithelial cells experience different local environments during tissue development and tumorigenesis.  Microenvironmental heterogeneity gives rise to distinct cell-regulatory states whose identity and importance are just beginning to be appreciated.  Cellular states diversify when clonal 3D spheroids are cultured in basement membrane, and prior transcriptomic analyses identified a state associated with stress tolerance and poor response to anticancer therapeutics.  Here, we examined the regulation of this state and found that it is jointly coordinated by the NRF2 and p53 pathways, which are co-stabilized by spontaneous oxidative stress within the 3D cultures.  To identify common adaptive programs downstream of NRF2 deficiency, we inducibly knocked down NRF2 and profiled 3D spheroids by RNA sequencing.

overall_design: Bulk RNA from MCF10A-5E and MCF10DCIS.com Day 10 spheroids with or without NRF2 knockdown. MCF10A-5E shControl, shNRF2 #1, shNRF2#2 (denoted shNRF2v4) and MCF10DCIS.com shControl, shNRF2#1. Each cell line has 4 replicates.

first: Elizabeth

last: Pereira


title: BubR1 allelic effects drive phenotypic heterogeneity in MVA progeria syndrome [10 month old RNA-seq]

first: Hu

last: Li

overall_design: Skeletal muscle (skM, gastrocnemius) and fat (IAT) from 8-10-month-old wildtype and progeroid BubR1 MVA mutant mice (BubR1H/L1002P and BubR1H/H) were collected and harvested from naturally aged mice, euthanized at 8-10 months of age. RNA was extracted from tissues and was used for RNA sequencing.

first: Jan

last: van Deursen


title: RNA helicase DDX21 mediates nucleotide stress responses (iCLIP)

city: Boston

first: Leonard

last: Zon

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: The RNA helicase DDX21 acts as trancriptional sensor for nucleotide lowering

overall_design: irCLIP-seq analysis of DDX21 and CDK9 in human A375 melanoma cells treated with DMSO, 25 uMA77 1726 and 25uM A771726 plus nucleotides

first: Cristina

last: Santoriello

first: Yang

last: Song

first: Ryan

last: Flynn


title: Deep sequencing and automated histochemistry of human tissue slice cultures improve their usability as preclinical model for cancer research

city: Leipzig

first: Susann

last: Haehnel

address: Liebigstrasse 13

department: Institute of Anatomy

institute: University of Leipzig

summary:
Cancer research requires models closely resembling the tumor in the patient. Human tissue cultures can overcome interspecies limitations of animal models or the loss of tissue architecture in in vitro models. However, analysis of tissue slices is often limited to histology. Here, we demonstrate that slices are also suitable for whole transcriptome sequencing and present a method for automated histochemistry of whole slices.
Tumor and peritumoral tissue from a patient with glioblastoma was processed to slice cultures, which were treated with standard therapy including temozolomide and X-irradiation. Then, RNA sequencing and automated histochemistry was performed.
RNA sequencing was successfully performed with a sequencing depth of 243 to 368 x 106 reads per sample. Comparing tumor and peritumoral tissue, we identified 1888 genes significantly downregulated and 2382 genes upregulated in tumor. Treatment significantly downregulated 2017 genes, whereas 1399 genes were upregulated. Pathway analysis revealed changes in the expression profile of treated glioblastoma tissue pointing towards downregulated proliferation. This was confirmed by automated analysis of whole tissue slices stained for Ki67.
In conclusion, we demonstrate that RNA sequencing of tissue slices is possible and that histochemical analysis of whole tissue slices can be automated which increases the usability of this preclinical model.

overall_design: total RNA of glioblastoma and peritumoral brain tissue slices from one patient were treated with temozolomide and x-irradiation or left untreated as control slices, experiment was done in duplicates

first: Kristin

last: Reiche

first: Dennis

last: Loeffler

first: Andreas

last: Horn

first: Conny

last: Blumert

first: Sven-Holger

last: Puppel

first: Nicole

last: Kaiser

first: Felicitas

last: Rapp

first: Michael

last: Rade

first: Friedemann

first: Juergen

last: Meixensberger

first: Ingo

last: Bechmann

first: Frank

last: Gaunitz

first: Karsten

last: Winter


title: RNA helicase DDX21 mediates nucleotide stress responses (RNA-Seq A375)

first: Leonard

last: Zon

overall_design: RNA Seq human A375 melanoma cells treated with DMSO, 25 uMA77 1726 and 25uM A771726 plus nucleotides

first: Cristina


title: RNA helicase DDX21 mediates nucleotide stress responses (RNA-Seq drLef)

overall_design: RNA seq  analysis from Sox10 sorted cells from embryos injected with Ctr9Mo and treated with 32uMProgesterone

last: Santoriello

first: Yang


title: Cardiovascular consequences of K(ATP) dysfunction in Cantu Syndrome arising from in mutations Kir6.1 (KCNJ8) and SUR2 (ABCC9)

city: Indianapolis

first: Scot

last: Matkovich

address: Lilly Corporate Center

institute: Eli Lilly and Company

summary: Cantu Syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) K(ATP) channel subunits. We generated mice carrying CS-associated SUR2[A478V] and Kir6.1[V65M] mutations, knocked in to the endogenous loci using CRISPR/Cas9 engineering. Mirroring human CS, both of these animals have low systemic blood pressure and dilated, compliant blood vessels, as well as dramatically enlarged hearts with increased contractility. Whole-cell patch-clamp recording reveal enhanced basal K(ATP) conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure. Echocardiography confirms in situ cardiac enlargement, with increased contractility and maintained ejection fraction. Whole-cell voltage clamp of ventricular myocytes reveals increased basal L-type Ca2+ current (LTCC), explaining enhanced contractility. Cardiac hypertrophy and enhanced contractility may arise as secondary compensation, to maintain tissue perfusion in the presence of marked vascular dilation. All of the above features are more prominent in Kir6.1[V65M] animals than in SUR2[A478V] animals, and exacerbated in homozygous animals of each genotype. Compensatory mechanisms are likely to be limiting, since survival is inversely correlated with severity of the phenotype, with very early death in homozygous Kir6.1[V65M] animals. The SUR2[A478V] and Kir6.1[V65M] animals reiterate and explain cardiovascular features in human CS, and raise concerns regarding the long-term consequences of CS specifically, and reduced smooth muscle tone in general.

overall_design: Heterozygous or homozygous mutant hearts were compared to wild-type littermate hearts.

first: Conor

last: McClenaghan

first: Colin

middle: G

last: Nichols


title: Hypoxia-induced priming of microglial inflammatory gene expression is associated with TLR4 signaling H3K4me3 enrichment

city: Madison

first: Jyoti

last: Watters

state: WI

address: 2015 Linden Drive

department: CBS

institute: University of Wisconsin-Madison

summary: We analyzed genome-wide H3K4me3 enichment and performed RNAseq analyses in N9 microglia cells after hypoxia exposure

overall_design: Examination of H3K4me3 in five experiments in which cells were exposed to either normoxia (21% O2) or hypoxia (1% O2); examination of total mRNA from three experiments in which cells were exposed to normoxia or hypoxia

middle: J

first: Elizabeth

last: Kiernan


title: A Western Dietary Pattern and Atorvastatin Induce Epicardial Adipose Tissue Interferon Signaling in the Ossabaw Pig

first: Alice

middle: H

last: Lichtenstein

address: 711 Washington St

department: Jean Mayer USDA Human Nutrition Research Center on Aging

institute: Tufts University

summary: Epicardial adipose tissue (EAT) inflammation is thought to potentiate the development of coronary artery disease (CAD). Overall diet quality and statin therapy are important modulators of inflammation and CAD progression. Our objective was to examine the effects and interaction of dietary patterns and statin therapy on EAT gene expression in the Ossabaw pig.

overall_design: Thirty-two Ossabaw miniature pigs (16 boars, 16 gilts) were randomized using a 2x2 factorial design into four groups: Hearty Healthy diet (HHD); Heart Healthy diet + atorvastatin (HHD+S); Western diet (WD); and Western diet + atorvastatin (WD+S). Heart Healthy diet (high in unsaturated fat, unrefined grain, fruits/vegetables [HHD]) or Western diet (high in saturated fat, cholesterol, refined grain [WD]), with or without atorvastatin.

first: Maura

last: Walker

first: Nirupa

last: Matthan

first: Gloria

last: Solano-Aguilar

first: Saebyeol

last: Jang

first: Sukla

last: Lakshman

first: Aleksey

last: Molokin

first: Tyler

last: Faits

first: Joseph

last: Urban Jr

first: W

last: Johnson

first: Stefania

last: Lamon-Fava


title: Lack of RAN-mediated toxicity in Huntington’s disease knock-in mice

status: Public on Feb 03 2020

city: Guangzhou

first: Su

last: Yang

address: Huangpu Avenue

institute: Jinan University

summary: Identification of repeat-associated non-AUG (RAN) translation in trinucleotide (CAG) repeat diseases leads to an emerging concept that CAG repeat diseases are caused by non-polyglutamine products. Nonetheless, the exact contribution of RAN translation to the pathogenesis of CAG repeat diseases remains elusive. Via CRISPR/Cas9-mediated genome editing, we established new knock-in mouse models that harbor expanded CAG repeats in the mouse huntingtin gene, which express RAN translated products or polyglutamine products respectively. Here we report that RAN translation is not detected in the knock-in mouse models, and that only the expanded polyglutamine products can cause neuropathology and behavioral phenotypes. Therefore, polyglutamine products, rather than RAN translated products, play a major role in the pathogenesis of CAG repeat diseases.

overall_design: We performed RNA sequencing analysis using striatal tissues from 6-month old WT, E1 and KI-96 mice, each group includes at least four biological replicates.

first: Xiao-Jiang

last: Li


title: Distinct transcriptional responses across tissue-resident macrophages to short-term and long-term metabolic challenge

city: Basel

first: Marc

last: Donath

address: Hebelstrasse 20

department: Department of Biomedicine

institute: University of Basel

summary: The innate immune system safeguards the organism from both pathogenic and environmental stressors. Even physiological levels of nutrients affect organismal and intra-cellular metabolism and challenge the immune system. In the long term, over-nutrition leads to low-grade systemic inflammation. Here, we investigate tissue-resident components of the innate immune-system –macrophages and their response to short-term and long-term nutritional challenges.  We analyze the transcriptomes of seven tissue-resident macrophage populations upon metabolic challenge and identify adipose tissue macrophages and the IL-1 pathway as early sensors of metabolic changes. Furthermore, by comparing functional responses between macrophage subtypes, we propose a regulatory, anti-inflammatory role of heat shock proteins of the HSP70 family in response to a metabolic challenge. Our data provides a resource for assessing the impact of nutrition and over-nutrition on the spectrum of macrophages across tissues with a potential for identification of systemic responses.

overall_design: To investigate the short-term and long-term metabolic challenges mice were subjected to two nutritional protocols: 12h fasting, followed by 2h refeeding (FARE) and high fat diet (HFD) for 8 weeks. In the HFD experiment mice were additionally treated with Streptozotocin (STZ) at week 4 which resulted with islet ablation and hyperglycemia. Macrophages were isolated and purified  using  fluorescence-activated cell sorting (FACS). Six tissue resident macrophages populations (adipose tissue macrophages, microglia, pancreatic islet macrophages, Kupffer cells, colonic macrophages, peritoneal macrophages) and monocytes were isolated from fasted and refed mice (six up to ten replicates per condition). Three tissue macrophage populations (microglia, pancreatic islet macrophages, peritoneal macrophages) were isolated from mice fed by normal diet, by HFD and by HFD treated with STZ (three up to five replicates per condition). PolyA RNA fraction was extracted from purified macrophage populations and subjected to RNA-Seq.

first: Urszula

last: Brykczynska

first: Marco

last: Geigges

first: Sophia

last: Wiedemann

first: Erez

last: Dror

first: Marianne

last: Böni-Schnetzler

first: Christoph

last: Hess

first: Renato

last: Paro


title: Methionine metabolism shapes T helper cell responses through regulation of epigenetic reprogramming [ChIP-Seq]

city: Grand Rapids

first: Russell

last: Jones

state: Michigan

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

summary: Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Chromatin-modifying enzymes are dependent on metabolic intermediates for chromatin remodeling, linking nutrient availability and cellular metabolism to the cellular epigenetic landscape. Here we identify methionine as a key nutrient affecting T cell epigenetic reprogramming in CD4+ T helper (Th) cells. Using metabolomic approaches, we showed that methionine is rapidly taken up by activated T cells and then serves as the major substrate for the biosynthesis of S-adenosyl-L-methionine (SAM), the universal methyl donor for cellular methyltransferases. Conversely, methionine restriction (MR) depletes intracellular SAM pools, reduces global histone H3K4 methylation (H3K4me3) in T cells, and reduces H3K4me3 levels at the promoter regions of key genes involved in CD4+ Th17 cell proliferation and cytokine production. Applied to the mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), dietary methionine restriction reduced the expansion of pathogenic Th17 cells in vivo, leading to reduced T cell-mediated neuroinflammation and disease onset. Overall our data identify methionine as a key nutritional factor that shapes T cell proliferation, differentiation, and function in part through regulation of histone methylation in T cells.

overall_design: ChIP-seq for CD4+ Th17 cells cultured under control (200 uM) or methionine restriction (3 uM) conditions for 5 days

first: Dominic

last: Roy

first: Jocelyn

last: Chen


title: Methionine metabolism shapes T helper cell responses through regulation of epigenetic reprogramming

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

overall_design: Refer to individual Series


title: RNA helicase DDX21 mediates nucleotide stress responses (PRO-Seq)

first: Leonard

last: Zon

overall_design: PRO-seq analysis of DDX21 and CDK9 in human A375 melanoma cells treated with DMSO, 25 uMA77 1726 and 25uM A771726 plus nucleotides

first: Cristina

last: Santoriello

first: Henriques

last: Telmo

first: Yang

last: Song


title: RNA helicase DDX21 mediates nucleotide stress responses

sample_organism:
Danio rerio
Homo sapiens

platform_organism:
Danio rerio
Homo sapiens


title: De novo transcriptome assembly of the five major organs of Zanthoxylum armatum and the identification of genes involved in terpenoid compounds and fatty acids metabolism

city: Beijing

first: Wenkai

last: Hui

state: Beijing

address: Qinghua East Street

institute: Beingjing Forestry University

summary: Zanthoxylum armatum (Z. armatum) is one of the most economical trees with the unique numbing taste. However, the underlying regulation mechanism involved in the numbing-taste remains scarce. Thus, uncovering the vital genes associated with numbing-taste biosythesis pathways is critical for revealing the genetic information and breeding the high-quality Z. armatum germplasms. Here, a de novo transcriptome assembly of the five major organs was performed in Z. armatum, including roots, stems, leaf buds, mature leaves and fruits. A total of 111 318 unigenes were generated with an average of 1 014 bp. Meanwhile, a large number of SSR and SNP/Indel loci were obtained to improve our understanding about the phylogenetic and genetic information in Z. armatum. The organ specific unigenes of five major samples were screened and annotated with GO and KEGG enrichment analysis, respectively. Additionally, 53 and 34 unigenes were exclusively up-regulated in fruit samples, and identified as candidate unigenes for terpenoid biosynthesis process and fatty acid biosynthesis, elongation and degradation pathways, respectively. Moreover, he forty days after fertilization (Fr4 stage) could be a important period to accumulate the terpenoid compounds in the fruit development and maturation process of Z. armatum. The Fr4 stage could be a vital point to promote the first few steps of fatty acid biosynthesis process, and the subsequent reactions could be significantly catalyzed at sixty two days after fertilization (Fr6 stage). The present study conducted the de novo transcriptome assembly of the five major organs in Z. armatum. To the best of our knowledge, this study is the first comprehensive analysis to reveal the underlying genes related to the unique numbing taste in Z. armatum. The assembled transcriptome profiles will expand the genetic information and improve our understanding for gene functional studies, which helps in engineering high-quality cultivars of Z. armatum.

overall_design: The de novo transcriptome assembly of the five major organs was performed in Z. armatum, including roots, stems, leaf buds, mature leaves and fruits. Each sample were collected from three individual plants and mixed together as a biological replicate, three independent biological replicates was performed in this study.

first: Wen

middle: K

first: Fei

middle: Y

last: Zhao

first: Jing

last: Wang

first: Xiao

first: Jue

middle: W

first: Yu

last: Zhong

first: Hong

first: Jun

middle: X

last: Zheng

first: Liang

middle: Z

last: Zhang

first: Qing

last: Que

first: Ai

last: Wu

first: Wei

last: Gong


title: Identification of genes regulated by Erythroferrone (ERFE)  in Xenopus embryos at tadpole stages.

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

institute: Universitaetsmedizin Goettingen

summary: RNA-seq was employed to analyze differential gene expression upon ERFE knockdown in Xenopus laevis embryos at three different developmental stages using two non-overlapping translation blocking morpholino oligonucleotides. Only a small number of genes was affected by both morpholinos and at more than one of the developmentel stages analyzed, among them gja4, the gene coding for Gap junction junction protein Alpha 4.

overall_design:
Xenopus eight-cell stage embryos were injected with 10 ng ERFE-MO1 or 20 ng ERFE-MO2 into the four vegetal blastomeres and cultivated until stage 35/36, stage 37/38 or stage 41.
Uninjected embryos as well as embryos injected with equal amounts of standard control morpholino (cMO) served as controls.

first: Tomas

last: Pieler

first: Juliane

last: Melchert

first: Thomas

last: Lingner


title: Impact of LARP7 depletion on the pA+ transcriptome of HEK293 cells upon heat shock

first: Gerhard

last: Lehmann

summary: The La-related protein LARP7 has been mainly described as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, which negatively regulates RNA polymerase II by sequestering the positive transcription elongation factor b (P-TEFb). In our studies, we discovered a novel, 7SK snRNP-independent function of LARP7. We show that LARP7 interacts with the U6 spliceosomal RNA as well as with the small nucleolar RNAs (snoRNAs) directing the 2'-O-methylations of U6. Importantly, in the absence of LARP7, significantly less 2'-O-methylations are deposited on U6 affecting splicing fidelity. Mutations in the LARP7 gene have been associated with the Alazami syndrome, a form of primary dwarfism characterized by intellectual disability. We describe a novel loss-of-function mutation of LARP7 occurring in Alazami patients and detect reduced 2'-O-methylations of U6 in patient-derived samples. Thus, aberrant posttranscriptional RNA modifications of the spliceosomal U6 snRNA may contribute to the development of this severe disease.

overall_design: Analysis of the impact of LARP7 depletion on the expression and the alternative splicing of pA+ RNA transcripts. Two replicates each of parental HEK293 cells, HEK293 LARP7 knockout cells (clone 1-11) and HEK293 LARP7 knockout cells (clone 3-6) were used for pA+ RNA sequencing.

first: Daniele

last: Hasler

first: Maciej

last: Bak

first: Norbert

last: Eichner

first: Mihaela

last: Zavolan

first: Gunter

last: Meister


title: RNA helicase DDX21 mediates nucleotide stress responses (RNA-Seq drCTr9MO)

first: Leonard

last: Zon

first: Cristina

last: Santoriello

first: Yang

last: Song


title: neural reprogramming in head and neck cancer

status: Public on Feb 13 2020

city: Houston

first: Moran

last: Amit

state: TX

address: 1515 Holcombe Blvd

department: Head & Neck Surgery - Research

institute: The University of Texas MD Anderson Cancer Center

summary:
mRNA: profiling the neural identity of cancer associated neurons
miRNA: investigating the p53 dependant exosomal miRNA content

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of cancer associated neuron identity


title: Bystander IFN- γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment

first: Philippe

last: Bousso

address: 25 Rue du Docteur Roux

department: Immunology department

institute: Institut Pasteur

summary: The cytokine IFN-g produced by tumor-reactive T cells is a key effector molecule with well characterized pleiotropic effects during anti-tumor immune responses. While IFN-g production is a transient event targeted at the immunological synapse, how IFN-g acts in time and space within the tumor microenvironment has remained elusive. Specifically, the link between duration of exposure to the cytokine and cellular response is poorly understood. Using RNA sequencing, we show that tumor cell phenotypic alterations require several hours of exposure to IFN-g, most likely due to a positive feedback loop mediated by STAT1 and IRF8.

overall_design: Eµ-myc mouse cell line were stimulated by IFNg for various amount of time and harvested at 24 hours post stimulation. polyA mRNAs were then purified and RNAseq was performed

first: Ronan

last: Thibaut

first: Pierre

last: Bost


title: Transcriptional trajectories of anther development provide candidates for engineering male fertility in sorghum.

city: New Delhi

first: Rita

last: Sharma

state: Delhi

address: SCIS, JNU

department: School of Computational & Integrative Sciences

institute: Jawaharlal Nehru University

summary: We report transcriptome profiling from three developing stages of anthers in sorghum. Sequencing of nine libraries (three replicates from each stage) yielded >1.12 billion paired-end reads. A total of 31871 known genes expressed in all three replicates of at least one anther stage.

overall_design: Examination of 3 different stages of anther development in Sorghum bicolor variety M35-1

first: Namrata

last: Dhaka

first: Kushagra

last: Krishnan

first: Manu

last: Kandpal

first: Madan

middle: P

last: Singh

first: Manoj


title: DNA methylation profile of Korean CLL patients and normal controls

city: Daejeon

first: Seungwoo

last: Hwang

address: 52 Eoeun-dong Yuseong-gu

department: Korean Bioinformation Center

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology

summary: We performed genome-wide methylation profiling of CLL in an Asian cohort for the first time. Eight Korean patients without somatic immunoglobulin heavy chain gene hypermutations underwent methyl-CpG binding domain sequencing (MBD-seq), as did five control subjects. Gene Ontology, pathway analysis, and network-based prioritization of differentially methylated genes were also performed. We found more hypomethylated regions (2,062 windows) than hypermethylated regions (777 windows). Promoters contained the highest proportion of differentially methylated regions (DMRs; approximately 0.08%), while distal intergenic and intron regions contained the largest number of DMRs. Protein-coding genes were the most abundant, followed by long noncoding and short noncoding genes. The most significantly dysregulated signaling pathways included immune/cancer-related pathways and B-cell receptor signaling. Among the top 10 hub genes identified via network-based prioritization, four novel candidate genes (UBC, GRB2, CREBBP, and GAB2) had no known relevance to CLL while the other six (STAT3, PTPN6, SYK, STAT5B, XPO1, and ABL1) have previously been linked to CLL in Caucasians. As such, our analysis identified four novel candidate genes of potential significance to Asian patients with CLL.

overall_design: Bone marrow buffy coats were collected from eight Korean CLL patients without IGHV mytations. CD19 positive B-cells were collected from five healthy donors using magnetic bead sorting. DNA methylation profiles of the eight patients and five controls were generated by MeDIP-Seq.

first: Miyoung

last: Kim


title: Liver transcriptome profiling reveals that dietary DHA and EPA levels influence suites of genes involved in metabolism, redox homeostasis, and immune function in Atlantic salmon (Salmo salar)

city: St. John's

first: Xi

last: Xue

state: NL

address: 1 Marine Lab Road

department: Department of Ocean Sciences

institute: Memorial University of Newfoundland

summary: The optimal dietary requirement of omega-3 long-chain polyunsaturated fatty acids (ω3 LC-PUFA), namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for Atlantic salmon that promotes optimal growth and health warrants careful investigation. We used 44K microarrays to study the influence of increasing levels of dietary DHA + EPA (0, 1.0 and 1.4% of the diet, as formulated) in the presence of high linoleic acid (LA) on Atlantic salmon growth and liver transcriptome. After a 14-week feeding trial, Atlantic salmon fed diet ω3LC0 (i.e. 0% of DHA + EPA) showed significantly lower final weight and weight gain, and higher feed conversion ratio compared with ω3LC1.0 and ω3LC1.4 diet groups. The microarray experiment identified 55 and 77 differentially expressed probes (Rank Products analyses; PFP < 10%) in salmon fed diets ω3LC1.4 and ω3LC1.0 compared with those fed diet ω3LC0, respectively. The comparison between ω3LC1.4 and ω3LC1.0 revealed 134 differentially expressed probes.

overall_design:
Atlantic salmon were fed the three experimental diets (ω3LC0, ω3LC1, and ω3LC1.4; four tanks per diet) for 14 weeks. Liver samples were taken from 5 fish from each tank at week 16. A universal reference design was used for the microarray experiment. For the test samples, RNA was used from individual livers of fish from the 3 treatment groups. For each treatment group we used 8 biological replicates (2 fish from each of 4 tanks). All test samples were labeled with Cy5. The common reference was a pool of 24 RNA samples from livers of fish from all individuals invovled in microarray experiment. The common reference was labeled with Cy3. Each individual test sample was hybridized together with the common reference sample on an array, so the experiment consisted of 24 arrays. Please note that sample titles represent 'diet_tank number_individual fish number' for each sample.
variable: other: ω3LC0: ω3LC0_T8_2, ω3LC0_T8_3, ω3LC0_T23_1, ω3LC0_T23_3, ω3LC0_T31_2, ω3LC0_T31_4, ω3LC0_T35_1, ω3LC0_T35_3
variable: other: ω3LC1: ω3LC1_T7_1, ω3LC1_T7_3, ω3LC1_T20_1 ,ω3LC1_T20_2, ω3LC1_T34_1, ω3LC1_T34_3, ω3LC1_T38_2, ω3LC1_T38_3
variable: other: ω3LC1.4: ω3LC1.4_T3_3, ω3LC1.4_T3_4, ω3LC1.4_T19_2, ω3LC1.4_T19_3, ω3LC1.4_T26_1, ω3LC1.4_T26_2, ω3LC1.4_T33_1, ω3LC1.4_T33_2
repeat: biological replicate: ω3LC0_T8_2, ω3LC0_T8_3, ω3LC0_T23_1, ω3LC0_T23_3, ω3LC0_T31_2, ω3LC0_T31_4, ω3LC0_T35_1, ω3LC0_T35_3
repeat: biological replicate: ω3LC1_T7_1, ω3LC1_T7_3, ω3LC1_T20_1 ,ω3LC1_T20_2, ω3LC1_T34_1, ω3LC1_T34_3, ω3LC1_T38_2, ω3LC1_T38_3
repeat: biological replicate: ω3LC1.4_T3_3, ω3LC1.4_T3_4, ω3LC1.4_T19_2, ω3LC1.4_T19_3, ω3LC1.4_T26_1, ω3LC1.4_T26_2, ω3LC1.4_T33_1, ω3LC1.4_T33_2

first: Jennifer

last: Hall

first: Albert

last: Caballero-Solares

first: Khalil

last: Eslamloo

first: Richard

last: Taylor

first: Christopher

middle: C

last: Parrish

first: Matthew

last: Rise


title: Chromatin accessibility profiling in STAG2 mutant K562 cells

city: Dunedin

first: Jisha

last: Antony

state: Otago

address: 58 Hanover Street

department: Pathology

country: New Zealand

institute: University of Otago

summary: K562 (female chronic myelogenous leukemia) cells were CRISPR-Cas9 edited to contain STAG2 R614* mutation. STAG2 is encoded on the X-chromosome and normally mutation on one allele is sufficient for complete loss of STAG2 protein due to the wild type allele being mosaically silenced. However, we obtained both heterozygous and homozygous mutant lines that showed partial and complete loss of STAG2 protein respectively. We here examined the impact of STAG2 R614* mutation on gene expression and chromatin acessibilty.

overall_design: ATAC-sequencing, in triplicate, on the parental or wild type K562 cells (K562WT), one K562 cell lines that are heterozygous (STAG2heterozygous1) for STAG2 R614* mutation and one K562 cell lines that are homozygous for STAG2 R614* (STAG2homozygous1) mutation.

first: Gregory

last: Giminez

first: Julia

last: Horsfield


title: Chromatin accessibility and expression profiling in STAG2 mutant K562 cells


title: Impact of LARP7 depletion on the transcriptome of HEK293 cells

city: Regensburg

first: Gerhard

last: Lehmann

address: Universitaetsstrasse 31

department: Biochemistry 1

institute: University of Regensburg

summary: The La-related protein LARP7 has been mainly described as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, which negatively regulates RNA polymerase II by sequestering the positive transcription elongation factor b (P-TEFb). In our studies, we discovered a novel, 7SK snRNP-independent function of LARP7. We show that LARP7 interacts with the U6 spliceosomal RNA as well as with the small nucleolar RNAs (snoRNAs) directing the 2'-O-methylations of U6. Importantly, in the absence of LARP7, significantly less 2'-O-methylations are deposited on U6 affecting splicing fidelity. Mutations in the LARP7 gene have been associated with the Alazami syndrome, a form of primordial dwarfism characterized by intellectual disability. We describe a novel loss-of-function mutation of LARP7 occurring in Alazami patients and detect reduced 2'-O-methylations of U6 in patient-derived samples. Thus, aberrant posttranscriptional RNA modifications of the spliceosomal U6 snRNA may contribute to the development of this severe disease.

overall_design: Analysis of the impact of LARP7 depletion on the expression and the alternative splicing of RNA transcripts. Two replicates each of parental HEK293 cells and HEK293 LARP7 knockout cells (clone 1-11) were used for total RNA sequencing.

first: Daniele

last: Hasler

first: Maciej

last: Bak

first: Norbert

last: Eichner

first: Mihaela

last: Zavolan

first: Gunter

last: Meister


title: Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas

city: Bethesda

first: Pavol

last: Genzor

address: 6720A Rockledge Drive, Suite 100

department: ACESO

institute: Henry M Jackson Foundation

summary: Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids that are encountered, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that casposase integration in vitro recapitulates several properties of CRISPR-Cas integrases. The X-ray structure of Methanosarcina mazei casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization and the separation of Cas1 dimers. This, in turn, led to preferred integration of single spacers over two transposon ends.

overall_design: Next generation sequencing experiments following casposon integration assay in order to identify / characterize integrations site, motifs and any poteintial off-target sites

first: Alison

middle: B

last: Hickman

first: Shweta

last: Kailasan

first: Astrid

last: Haase

first: Fred

last: Dyda


title: Identification of small RNAs interacting with LARP7

overall_design: Identification of small RNAs interacting with endogenous LARP7 by immunoprecipitations and comparison of the enrichment over an input sample.


title: Endothelial Heterogeneity Across Distinct Vascular Beds During Homeostasis and Inflammation Using RiboTag Strategy

city: Chicago

first: Jalees

last: Rehman

state: IL

address: 840 S Wood St

institute: University of Illinois at Chicago

summary: Several studies have suggested that endothelial cells which line the blood vessels in each organ significantly differ from each other, but little is known about their distinct and characteristic signatures. We assessed the brain, lung, and heart endothelial translatome using the RiboTag mouse model which allows for the direct isolation of cell-type specific ribosomes and the mRNA undergoing translation.

overall_design: Endothelial mRNA translatome profiles of adult Cdh5CreERT2/+; Rpl22HA/+ mice were generated by RNA-Seq using Illumina HiSeq 4000


title: Differences in genetic changes in brain tissue between wild-type mice and miR-7 knockout mice in the presence of brain tissue inflammation

city: zunyi

first: xu

last: lin

address: zunyi medical University

institute: zunyi medical University

summary: Detection of genetic differences in brain tissue in wild-type mice and miR-7 knockdown mice under brain tissue inflammation

overall_design: Examination of gene expression in 2 mice.

first: Lin

last: Xu


title: Loss of Kat2a enhances transcriptional noise and depletes acute myeloid leukemia stem-like cells [RNA-Seq]

city: Cambridge

first: Cristina

last: Pina

address: Long Road

department: Department of Haematology Cambridge Biomedical Campus NHS - Blood and Transplant

institute: University of Cambridge

summary: Identification of histone acetylation targets of Kat2a activity in murine models of acute myeloid leukaemia (AML) driven by the MLL-AF9 oncogenic fusion

overall_design: Single-cell RNA-seq analysis of 2 mouse bone marrow (BM) samples obtained from pooled terminal primary MLL-AF9 AMLs established from Kat2a WT or Kat2a KO cells. AML cells were sorted as live MLL-AF9-expressing cells by Hoechst 22358 exclusion and YFP reporter expression, respectively, and libraries were prepared upon single-cell droplet-based barcoding using 10X Genomics technology.

first: Rashmi

last: Kulkarni


title: Expression profiling in STAG2 mutant K562 cells

first: Jisha

last: Antony

overall_design: RNA-sequencing, in triplicate, on the parental or wild type K562 cells (K562WT), two K562 cell lines that are heterozygous (STAG2heterozygous) for STAG2 R614* mutation and two K562 cell lines that are homozygous for STAG2 R614* (STAG2homozygous) mutation.

first: Gregory

last: Giminez

first: Julia

last: Horsfield


title: EPIGENETICS OF ALM

city: New York

first: Varshini

last: Vasudevaraja

state: NY

address: 550 1st Avenue

institute: NYU Langone Health

summary:
Epigenetic signatures of alm and assoication with outcome.
Background: Acral melanoma (AM) is a rare, aggressive type of cutaneous melanoma (CM) with a distinct genetic profile for which comprehensive genome-wide methylation analysis (GWMA) is lacking. We aimed to identify a methylome signature distinguishing primary acral lentiginous melanoma (PALM) from primary non-lentiginous AM involving the acral sites (NALM), metastatic ALM (MALM), primary non-acral CM (PCM), and acral nevus (AN).  Methods: A total of 22 PALM, 9 NALM, 10 MALM, 9 PCM, and 3 AN cases were subjected to GWMA using the Illumina Infinium Methylation EPIC array interrogating 866,562 CpG sites, and their methylomes were compared.    Results: A prominent finding was that the methylation profiles of PALM and NALM were distinct, although both are considered canonical AMs. Four of the genes most differentially methylated between PALM and NALM or PALM and MALM were HHEX, DIPK2A (formerly C3orf58), NELFB (formerly COBRA1), and TEF. However, when primary AMs (PALM+NALM) were compared with MALM, IFITM1 and SIK3 were the most differentially methylated, highlighting their pivotal role in the metastatic potential of AMs. Patients with NALM had significantly worse disease-specific (DSS) and overall survival than patients with PALM. Aberrant methylation was significantly associated with aggressive clinicopathologic parameters, including greater Breslow thickness, ulceration, increased mitotic rate, and lymph node metastasis, and with worse DSS.  Conclusion: Our study emphasizes the importance of distinguishing the two epigenetically distinct subtypes of AM. We also identified novel epigenetic prognostic biomarkers that may serve to risk-stratify patients with AM. These epigenetic alterations may be leveraged for development of targeted therapies.

overall_design: 53 samples

first: George

last: Jour


title: The Alazami Syndrome-associated protein LARP7 guides U6 small nuclear RNA modification and contributes to splicing robustness

first: Gerhard

last: Lehmann

overall_design: Refer to individual Series


title: Loss of Kat2a enhances transcriptional noise and depletes acute myeloid leukemia stem-like cells [ChIP-Seq]

summary: Identification of histone acetylation targets of Kat2a activity in murine models of acute myeloid leukemia (AML) driven by the MLL-AF9 oncogenic fusion

overall_design: Pooled mouse bone marrow (BM) samples obtained from terminal primary MLL-AF9 AMLs established from Kat2a WT (Mx1-Cre NEG = label NEG) or Kat2a KO (Mx1-Cre POS = label POS) cells were analysed for H3K9 and H3K27 acetylation marks, as well as for H3K4me3 (marking active promoters) and H3K4me1 (marking enhancers). Experiments were performed in duplicate (R1 and R2), as independent cell samples and chromatin preparations from the same pools of animals.


title: Role of intratumoral CD11b+ cells in immune checkpoint resistance in GBM

city: Heidelberg

first: Katrin

last: Deumelandt

address: INF 280

department: DKTK Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology

institute: German Cancer Research Center (DKFZ)

summary: Glioblastomas show low response rates to immune checkpoint blockade with few hypermutated glioblastomas showing responses. Moreover, a growing body of evidence suggests that macrophages in the glioblastoma microenvironment impair anti-tumor immunity and immunotherapy approaches. Here, we investigate macrophage-mediated mechanisms of response and resistance to combinatory immune checkpoint blockade targeting PD-1 and CTLA-4 in the murine syngeneic Gl261 glioblastoma model. Nanostring analysis from CD45highCD11b+ cells isolated from ICB responder and non-responder Gl261 tumors was performed. Here, we provide evidence for a differential expression of pro- and anti-inflammatory genes in CD45highCD11b+ cells from ICB responder and non-responder tumors pointing towards a suppressive phenotype of ICB non-responder CD45highCD11b+ cells. Targeting these suppressive CD45highCD11b+ cells in the glioblastoma microvenvironment might thus potentiate ICB efficacy.

overall_design: Gl261-infiltrating CD11b+ cells (CD45highCD11b+) were purified for anti-CTLA-4 + anti-PD-1 responder and non-responder mice by fluorescence-activated cell sorting and gene expression analysis was performed by NanoString analysis.

first: Michael

last: Platten

last: Aslan