title: Transcriptomic response of tall fescue to heat stress and improved thermotolerance by melatonin and 24-epibrassinolide

city: Wuhan

first: Zhulong

last: Chan

state: Hubei Province

address: No. 1, Shizishan St, Nanhu

department: College of Horticulture and Forestry Sciences

institute: Huazhong Agricultural University

summary:
Purpose: We aimed to dissect heat response of tall fescue and possible roles of melatonin and 24-epibrassinolide during thermotolerance.
Methods: A total amount of 3 µg RNA was used for generation of sequencing libraries using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Clean reads were obtained by removing low quality reads, reads containing adapter and ploy-N from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. Index of the Arabidopsis genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of drought stress versus control condition was performed using the DESeq R package (1.18.0).
Results:In total, six samples with two biological replicates per genotype/treatment combination were used for RNA sequencing analysis. At least 2 G clean bases were generated for each sample. Comparative analysis identified genes modulated by short term heat (2h) and long term heat (12h) treatments.

overall_design: In this study, 10-day-old tall fescue seedlings were treated with 38 °C temperature for 2 and 12h. The leaves of control and heat treated tall fescue plants were then collected for RAN isolation.


title: RNAseq of CCRF-CEM, a T-cell acute lymphoblastic leukemia cell line, after knockdown with 2 control hairpins and 6 hairpins targeting the PRC2 complex.

status: Public on Mar 02 2018

city: Boston

first: Ingrid

middle: M

last: Aries

state: MA

address: 1 Blackfan circle

department: Hematology/Oncology

country: USA

institute: Boston Children's Hospital

type: Expression profiling by high throughput sequencing

summary: The data was used to study mechanisms of apoptosis resistance induced by loss of PRC2.

overall_design: CCRF-CEM cells infected with shLuciferase, shGFP, shEZH2.1, shEZH2.4, shEED2, shEED5, shSUZ12.2, shSUZ12.3 were harvested, RNA isolated, and RNAsequencing performed on HiSeq 2000.

first: Alejandro

last: Gutierrez


title: CARM1-expressing ovarian cancer depends on the histone methyltransferase EZH2 activity

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

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: Single-cell RNA-seq reveals a subpopulation of prostate cancer cells with enhanced cell cycle-related transcription and attenuated androgen response

city: San Antonio

first: Tim

middle: Hui-Miing

last: Huang

state: TX

address: 8403 Floyd Curl Dr.

department: Molecular Medicine

institute: University of Texas Health San Antonio

summary: Increasing evidence indicates that minor subpopulations intrinsic to androgen-independence are present in prostate cancer cells, poised to become clonal dominance under prolonged androgen-deprivation selection. To stratify different subpopulations, we conduct transcriptome profiling of 144 single LNCaP prostate cancer cells treated and untreated with androgen after cell cycle synchronization. At least eight subpopulations of LNCaP cells are identified, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displays stem-like features, the advanced growth of which depends more on enhanced expression of 10 cell cycle-related genes and less on androgen-dependent signaling. Concordant upregulation of these genes appears to be linked to recurrent prostate cancers and can be used for early detection of tumors that subsequently develop androgen independence. Moreover, this single-cell approach provides a better understanding of how cancer cells respond heterogeneously to androgen-deprivation therapies and to reveal which subpopulations are resistant to this treatment.

overall_design: For each of 3 treatment groups, forty eight LNCaP single cells and 1 representative bulk cell RNA sample (1ng) were collected for SMART-seq2 amplification and later single-cell RNA-seq (total 144 single cells and 3 bulk cell samples). All of the treatment groups were harvested from after synchronizing the cells at the G1/S phase with a double thymidine block and androgen depriving the cells for ~24 hours. Treatment groups 2 and 3 were cultured in the absence and presence of androgen (1 nM R1881) for 12 hours, respectively. Treatment group 1 was a baseline comparison treatment group and was collected right after cell synchronization and androgen deprivation (considered 0 hour).

first: Aaron

last: Horning

first: Yao

last: Wang


title: CARM1-expressing ovarian cancer depends on the histone methyltransferase EZH2 activity [RNA-Seq]

summary: CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 is often overexpressed in cancers and stimulates growth.  However, clinically applicable therapeutic strategies based on CARM1 expression in cancer remains to be explored.  Here we show that epithelial ovarian cancer is among the cancers with the highest CARM1 amplification rates that predicates a shorter survival. Our unbiased screen show that CARM1-expressing ovarian cancer cells are selectively sensitive to the inhibition of EZH2, another epigenetic regulator that silences its target genes. Inhibition of EZH2 activity using a clinically applicable small molecule inhibitor significantly suppressed the growth of CARM1-expressing ovarian tumors in two xenograft models.  The observed selectivity correlates with upregulation of EZH2 target genes in a CARM1-dependent manner.  CARM1 promotes EZH2 dependent gene silencing by methylating BAF155 to alter the antagonism between EZH2 and BAF155.   Together, these results indicate that pharmacological inhibition of EZH2 is a novel therapeutic strategy for CARM1-expressing cancers.

overall_design: CARM1 wild type and knockout samples assayed by RNA-seq

first: Rugang

last: Zhang

first: Sergey

last: Karakashev


title: Functional analysis of HD2C in Arabidopsis

status: Public on Mar 01 2018

city: Madison

first: Xuehua

last: Zhong

state: WI

address: 330 N. Orchard St.

department: Epigenetics

institute: Wisconsin Institute for Discovery

summary: Ribosome biogenesis is a fundamental process required for all cellular activities. Histone deacetylases play critical roles in many biological processes including transcriptional repression and rDNA silencing. However, their function in pre-rRNA processing remains poorly understood. Here, we discovered a previously uncharacterized role of Arabidopsis histone deacetylase HD2C in pre-rRNA processing via both canonical and non-canonical manners. HD2C interacts with HD2B and forms homo- and/or hetero-oligomers in the nucleolus. Depletion of HD2C and HD2B induces a ribosome biogenesis deficiency morphological phenotype and aberrant accumulation of 18S pre-rRNA intermediates. Our genome-wide analysis revealed that HD2C binds and represses the expression of key genes involved in ribosome biogenesis. Using RNA immunoprecipitation and sequencing, we further uncovered a non-canonical mechanism of HD2C directly associating with pre-rRNA and small nucleolar RNAs to regulate rRNA methylation. Together, this study reveals a multifaceted role of HD2C in ribosome biogenesis and provides mechanistic insights into how histone deacetylases modulate rRNA maturation at both transcriptional and post-transcriptional levels.

overall_design: HD2C binding site were studied with ChIP-seq; Gene expression change in hd2b hd2c double mutant was studied using RNA-seq; H4K16 hyperacetylation site were identtified with H4K16ac ChIP-seq in hd2c mutant; HD2C binding RNAs were identified with HD2C RIP-seq.

first: Li

last: Lu

first: Xiangsong

last: Chen


title: Transcriptome analysis of the litchi fruit borer (Conopomorpha sinensis)

status: Public on Mar 03 2018

city: Guangzhou

first: Haibin

last: Sun

state: Guangdong

address: 7 Jinying Road

department: Plant Protection Research Institute

country: China

institute: Guangdong Academy of Agricultural Sciences

summary:
Purpose: transcriptome sequencing of Conopomorpha sinensis
Methods: high-through Illumina HiSeqTM 2000
Results:66017 transcripts,35383 unigenes
Conclusions:This study provided valuable transcriptome data for the litchi fruit borer, which was the first fundamental genomic basis for exploiting gene resources from the litchi fruit borer

overall_design: Identification of different expression genes in 2 groups of the litchi fruit borer by Illumina HiSeqTM 2000

first: Yanping

last: Liu

first: Siwei

first: Jianshan

last: Bai

first: Pengyan

last: Li


title: CARM1-expressing ovarian cancer depends on the histone methyltransferase EZH2 activity [ChIP-Seq]

overall_design: CARM1 wild type and knockout samples assayed by EZH2 and H3K27me3 CHIP-seq


title: RNA-Sequencing of Running-dependent and Running-independent Environmental Enrichment in the Adult Mouse Dentate Gyrus

city: Montreal

first: Karl

middle: JL

last: Fernandes

state: Quebec

address: 900 Rue St-Denis, R09-474

department: Neurosciences

country: Canada

institute: Université de Montréal

summary: We used RNA sequencing to perform an unbiased interrogation of DG gene expression in CD1 mice exposed to either a voluntary running disc (RUN), a running-independent complex environment (CE, containing a locked disc, social enrichment and tunnels), or a locked disc alone (LD). RNA sequencing revealed that both RUN and CE mice showed significant, and largely non-overlapping, transcriptomic differences versus the LD control. Our findings reveal stimulus-dependent transcriptional signatures of EE on the DG, and provide a resource for generating unbiased, data-driven hypotheses about novel mediators of EE-induced cognitive changes.

overall_design: RNA sequencing to ask whether running-dependent and running-independent EE paradigms differentially affect the transcriptome of the hippocampal dentate gyrus, a brain region critical for learning and memory

first: Catherine-Alexandra

last: Grégoire


title: 5hmC-seq in human kidney DNA

city: Edinburgh

first: John

middle: Paterson

last: Thomson

address: Crewe Road

department: MRC Human Genetics Unit

country: United Kingdom

institute: University of Edinburgh

type: Methylation profiling by high throughput sequencing

summary: DNA IP for 5hmC modified CpGs to determine 5hmC paterns across male human kidney

overall_design: We carry out 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) on covaris sheared genomic DNA (Amsbio), prior to sequencing Ion Proton P1 to report on the genome-wide 5hmC patterns.

middle: P

first: Angie

last: Fawkes


title: CRISPR RNA-dependent binding and cleavage of endogenous RNAs by the Campylobacter jejuni Cas9

city: Würzburg

first: Thorsten

last: Bischler

address: Josef-Schneider-Straße 2 / D15

department: Core Unit SysMed

country: Germany

institute: University of Wuerzburg

summary: This study investigates the RNA targets and cleavage sites of endogenous Cas9 in the food-borne pathogen Campylobacter jejuni. Direct RNA binding targets of Cas9 in C. jejuni strain NCTC11168 were determined using RIP-seq. The Cleavage sites were then predicted in the RNA targets by comparing total transcriptome data from WT and deletion (cas9, crRNA3, tracrRNA, CRISPR-tracrRNA) strains. PAMs for the CjeCas9 were enriched using the PAM-SCANR platform, which operates through a GFP reporter gene. Upon GFP (and thus functional PAM) enrichment, fluorescing cells were isolated using FACS and prepared plasmid DNA was amplified and prepared for sequencing.

sample_organism:
Campylobacter jejuni
Escherichia coli K-12

overall_design: Identification of Cas9 binding and cleavage sites in C. jejuni strain NCTC11168. Four total files are included for the PAM-SCANR dataset. Raw reads are available for both biological replicates, both pre- and post-FACS. After extracting the variable PAM region from the reads, enrichment was calculated by comparing the pre-FACS PAM counts to the post-FACS counts. These calculations are presented in the included processed data file.

first: Gaurav

last: Dugar

first: Ryan

middle: T

last: Leenay

first: Sara

middle: K

last: Eisenbart

first: Belinda

middle: U

last: Aul

first: Chase

middle: L

last: Beisel

first: Cynthia

last: Sharma


title: MAP kinase-dependent cell-to-cell movement drives plant tissue invasion by the rice blast fungus

city: Exeter

first: Darren

middle: Mark

last: Soanes

address: RILD Building, RD&E Hospital Wonford, Barrack Road

department: College of Medicine and Health

institute: University of Exeter

summary: We created a mutant in the MAP kinase-encoding Pmk1 gene of the rice blast fungus Magnaporthe oryzae (pmk1AS) that renders the gene sensitive to inhibition by 1NA-PP1. Fungal gene expression was compared during infection of rice sheath by the M. oryzae pmk1AS mutant in the presence and absence of 1NA-PP1.

overall_design: Rice leaf sheaths inoculated with pmk1AS mutant of Magnaporthe oryzae with or without 1NA-PP1 (three independent biological replicates for each condition).

first: Wasin

last: Sakulkoo

first: Miriam

last: Osés-Ruiz

first: Ely

middle: O

last: Garcia

first: George

middle: R

last: Littlejohn

first: Christian

last: Hacker

first: Ana

last: Correia

first: Barbara

last: Valent

first: Nicholas

last: Talbot


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and miR-211-/- Whole Eye Transcriptomes

city: Napoli

first: Annamaria

last: Carissimo

address: via Castellino

country: Italy

institute: Tigem

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS transcriptome profiling (RNA-seq) from whole eye, after removal of the lens and cornea from 1-2 month old miR-211-/- mice and compare it with wt mice
Methods: Whole eye (after removal of the lens and cornea) mRNA profiles of 1-2 month old  wild-type (WT) and neural miR-211-/-mice were generated by deep sequencing, in multiple biological replicates, five for WT and six for miR-211-/- animals, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays RNA-Seq libraries were prepared from whole eye, after removal of the lens and cornea from miR-211-/- mice.
Results: Each library was sequenced using 100 bp paired-end sequencing on the Illumina HiSeq 1000 system. Gene abundances from RNA-Seq data were quantified using RSEM45. Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome. This approach yielded read count values for a total of 38253 mouse genes annotated in GenCode. We only considered genes that had at least 1 count per million in at least five out of 11 samples as expressed, yielding a total of 15590 genes. Next we performed differential gene expression analysis to determine the transcriptional effects of miR-211 deletion. This analysis yielded a total of 63 genes that were differentially expressed with a False Discovery Rate (FDR) <0.1 (Fig. 4). Of these, the expression levels of 61 genes were significantly decreased upon miR-211 deletion, while only 2 genes were upregulated.
Conclusions: Our study represents the first detailed analysis of whole eye transcriptomes, with biologic replicates, generated by RNA-seq technology on miR-211-/-.

first: Ivan

last: Conte

first: Sandro

last: Banfi

last: Barbato


title: Circular RNAs are super abundant in cervical tumor and plasma detected by high throughput microarray [RNA-Seq]

city: ANN ARBOR

first: Shasha

last: Li

state: MI

address: 1109 Maiden Ln, ct apt 104

institute: University of Michigan

type: Non-coding RNA profiling by high throughput sequencing

summary: Circular RNAs (circRNAs) are a new class of endogenous and regulatory non-coding RNAs, but their expressions in tumor and plasma are largely unknown. Here, our study firstly suggested that microarray should be more efficient than RNA sequencing for circRNA profiling. Then, we detected ~80,000 circRNAs expressed in cervical tumors and matched normal tissues by microarray, and ~23,000 of them are differently expressed. The numbers of up- and down-regulated circRNAs during tumorigenesis are almost equal. In addition, we discovered that the expression of different circRNA isoforms from the same linear RNA also different. Strikingly, as much as ~18,000 circRNAs could be robustly detected in plasmas, and ~8,000 circRNAs show different expression after surgery of tumor removal. Altogether, taking advantage of the huge number of circRNAs detected in tumor and plasma, we provide strong evidence for circRNA expression, regulation and potential clinical application as non-invasive biomarkers.

overall_design: The circRNA expression in Universal Human Reference RNA (UHRR, 2 replicated samples) was compared to circRNA expression in Ambion’s Human Brain Reference total RNA (HBRR, 2 replicated samples) from the Microarray Quality Control (MAQC) project; The circRNA expression in cervical tumor (10 samples) was compared to circRNA expression in adjacent normal tissue (control, 10 samples) of 10 patients; The mRNA expression in cervical tumor (10 samples) was compared to mRNA expression in adjacent normal tissue (control, 10 samples) of 10 patients; The circRNA expression in preoperative plasma samples (control,7 samples) was compared to circRNA expression in postoperative plasma samples (14 samples) of 8 out of 10 cervical cancer patients.Microarray based circRNA expression profiles were acquired using CapitalBio circRNA Human Gene Expression Microarray V1.0 screening for 87,935 distinct human circRNA candidates.

first: Shuaishuai

last: Teng

first: Dong

last: Wang


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and ibm1-3 trancriptomes in normal light and high light condition

city: ChongQing

first: Di

last: Fan

address: NO.2 Tiansheng Road

institute: Southwest University

summary: n Arabidopsis and other species, anthocyanin accumulation specifically depends on light signaling. The CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex is required for the control of anthocyanin biosynthesis in response to light, but the precise mechanism underlying this process is largely unknown. Here we report that Increase in BONSAI Methylation 1 (IBM1), a JmjC domain-containing histone demethylase, participates in the regulation of light-induced anthocyanin biosynthesis in Arabidopsis. Loss-of-function mutations of IBM1 lead to accelerated anthocyanin accumulation in high light. We further identified that IBM1 is directly associated with SPA1/2/3 chromatin in vivo, whereas knockout of IBM1 resulted in an elevated level of H3K9 methylation and DNA non-CG sites within SPA genes, thereby repressing their expression. Transgenic ibm1 plants overexpressing SPA1demonstrated that SPA1 was capable of complementing the mutant phenotype. Our results reveal a novel mechanism that controls the epigenetic regulation of anthocyanin biosynthesis and offer insight into how plant adapt to high light stress. This study could contribute to a better understanding of the linkage between light signaling and anthocyanin biosynthesis in plants

overall_design: Total RNA was extracted using RNeasy Plant Mini Kit (QIAGEN) from Arabidopsis rosette leaves of 40-d-old ibm1-3 and WT plants grown under low light, or of 30-d-old plants treated by high light for 48 hours. mRNA samples were purified and subjected to Illumina sequencing as described in the Illumina mRNA sequencing sample preparation guide.

first: Keming

last: Luo


title: Differential miRNA profiles in cumulus and mural granulosa cells from human pre-ovulatory follicles

city: Groningen

first: Joost

last: Kluiver

address: Hanzeplein 1

country: Netherlands

institute: UMCG

summary: Small RNA seq analysis identified several hundreds of miRNAs being expressed in MGC and CC. 53 miRNAs were differentially regulated

overall_design: We performed small RNA sequencing analysis using cultured human primary MGCs and CCs collected from  three individual healthy patients to identify the respective miRNA profile.

first: Daniela

last: Andrei

first: Ruxandra

last: Nagy

first: Aafke

last: van Montfoort

first: Uwe

last: Tietge

first: Martijn

last: Terpstra

first: Klaas

last: Kok

first: Anke

last: van der Berg

first: Annemieke

last: Hoek

first: Rogier

last: Donker


title: An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis

city: San Francisco

first: Angela

last: Oliveira Pisco

state: CA

address: 499 Illinois St

department: Data Science

institute: Chan Zuckerberg Biohub

summary: mRNA quality control mechanisms ensure fidelity of protein translation. An evolutionarily conserved component of the quality control machinery, Dom34/Pelota (Pelo), rescues stalled ribosomes. Here we show that Pelo is required for mammalian epidermal homeostasis. Our study reveals a novel role for the ribosome-rescue machinery in mammalian tissue homeostasis and an unanticipated specificity in its impact on different stem cell populations.

overall_design: RNA-seq of mouse epidermal stem cell subpopulations (Lrig1+, Lgr5+ and Lgr6+) in WT and Pelota KO mice. We have triplicates for each experimental condition.

first: Kifayathullah

last: Liakath-Ali

first: Ines

last: Sequeira

first: Fiona

last: Watt


title: The effect of MAC3 and MAC5A on small RNA accumulation

city: Lincoln

first: Chi

last: Zhang

state: NE

address: 1901 Vine Street

institute: University of Nebraska - Lincoln

summary: MAC5A and MAC3 are conserved proteins that are required for development and immunity. They promotes the accumulation of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs), which repress gene expression. In this project, we aim to determine the global effect of MAC3 and MAC5Aon the accumulation of miRNAs and siRNAs.  We compared the small RNA profile in mac3a mac3b and mac5a with that in Col (Wild-type, WT). Small RNA libraries prepared from inflorescences of mac3a mac3b, mac5a and Col was subjected to Illumina deep sequencing analyses. The results show that many miRNAs  and siRNAs were reduced in abundance in mac5a and mac3a mac3b relative to Col in two biological replicates.

overall_design: non coding RNA deep sequencing

first: Li

last: Shengjun

first: Yu

last: Bin

first: Zhang

last: Chi


title: Molecular consequences of Dlx3 deletion by tamoxifen inducible K14 Cre in adult mouse epidermis (P42-P46) as determined by RNASeq analysis

city: Bethesda

first: Maria

middle: Irene

last: Morasso

state: Maryland

address: Bldg 50

institute: NIAMS NIH

summary:
DLX3 is a homeodomain transcription factor involved in epidermal and hair follicle differentiation. Epidermal specific deletion of DLX3 leads to IL17 related inflammation.
However, the downstream signals from DLX3-deficient keratinocytes that initiates IL17 associated inflammation is not well-characterized. Therefore we performed acute Dlx3 deletion to identify the initial molecular changes causing the skin inflammatory reponse and tried to rescue the immune phenotype by STAT3 inhibitor treatment (Cryptotanshinone).
In this study, we compared the epidermal and dermal transcriptome from Dlx3 wild-type (WT) and K14CreERT;DLX3 fl/fl (icKO).

overall_design: Total RNA was extracted epidermis and dermis of WT and icKO mice. cDNA libraries were generated using NEBnext and NuGen kits. Sequencing was performed on the HiSeq 2500 and HiSeq 3000.

first: Shreya

last: Bhattacharya

first: Jinchul

last: Kim


title: Gene expression profiles in lens from KLPH/lctl knockout mice versus wild type controls by RNA-seq

first: Jianguo

last: Fan

state: MD

address: 6 Center Drive Rm 108A

institute: National Eye Institute

summary:
Lenses from 6 week old KLPH/lctl knockout mice versus wild type controls were used to prepare total RNA for RNA-sequencing.
Differential mRNA expression between KLPH/lctl knockout mice and age matched C57bl6/J control mice was quantified by RNA-seq data analysis using the Tuxedo package (i.e. the Tophat-Cufflinks pipeline)

first: Lijin

last: Dong

first: Graeme

last: Wistow


title: Substrate-based regulation in Pycnoporus cinnabarinus PB94

city: Santa Barbara

first: John

last: Henske

address: Engineering II Building

department: Department of Chemical Engineering

institute: University of California, Santa Barbara

summary: Differential expression analysis of overall transcriptome in response to changes in energy substrate.

overall_design: Fungal cultures grown on glucose, maltose, cellobiose, poplar, switchgrass, and no energy substrate (control). RNA isolated from growing cultures and sequenced on an Illumina NextSeq using TruSeq stranded mRNA library prep.

first: Stephen

last: Springer

first: Alison

last: Butler

first: Michelle

last: O'Malley


title: Genome-wide maps of histone modification in mast cells

city: Denver

first: Hua

last: Huang

state: CO

address: 1400 Jackson St

department: Biomedical Research

institute: National Jewish Health

summary: Epigenomic studies demonstrate that monomethylation of lysine residue 4 on histone 3 (H3K4me1) marks genes that are poised to be transcribed, whereas acetylation of lysine residue 27 on histone 3 (H3K27ac) identifies genes that are actively being transcribed. The combined presence of H3K4me1 and H3K27ac modifications predicts enhancer activity. To detect these marks genome-wide, we performed H3K4me1 and H3K27ac ChIP-seq analysis of mast cells to identify the putative mast cell enhancers.

overall_design: H3K4me1 and H3K27ac ChIP-seq to identify the putative enhancers in mast cells.

first: Yapeng

last: Li

first: Laura

last: Harmacek

first: Sonia

last: Leach

first: Brian

last: O'Connor


title: Super-enhancer analysis of human sarcomas reveals YAP1 control of NF-κB-dependent tumorigenesis [RNA-seq]

city: Boston

first: Jun

last: Qi

state: Massachusetts

address: 450 Brookline Avenue

department: Cancer Biology

institute: Dana-Farber

summary: Recent sequencing efforts have failed to identify consistent oncogenic driver mutations in most adult soft tissue sarcomas. Therefore, we investigated alternate genetic/epigenetic mechanisms underlying sarcomagenesis to facilitate development of novel therapeutics. Our previous work showed that deregulation of the Hippo pathway increases proliferation in many types of sarcoma. We have now identified the mechanism of Hippo-mediated sarcomagenesis using autochthonous mouse models, ChIP-seq (H3K27Ac) and super-enhancer (SE) analysis of human undifferentiated pleomorphic sarcoma (UPS), an aggressive muscle-derived tumor. We found that the Hippo effector, Yes-Associated Protein 1 (YAP1) enhances NF-êB signaling and that YAP1 is constitutively active in these tumors due to epigenetic silencing of its inhibitor Angiomotin (AMOT). Treatment with epigenetic modulators (SAHA and JQ1) inhibited YAP1 transcription and restored expression of AMOT. These changes initiated a muscle differentiation program and reduced tumorigenesis.

overall_design: Expression study by high throughput sequencing of a human Undifferentiated Pleomorphic Sarcoma tumor.

first: Karin

last: Eisinger


title: An Arabidopsis Nucleoporin NUP85 modulates plant responses to ABA and salt stress

city: West Lafayette

first: Jian-Kang

last: Zhu

state: IN

address: 625 Agriculture Mall Dr.

department: Department of Horticulture and Landscape Architecture

institute: Purdue University

summary: Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.

overall_design: RNA-Seq of 4 samples, each has 3 replicates. The samples are wild type ( WT ) with mock treatment, nup85 with mock treatment, WT with 50 μM ABA treatment for 3 hours, and nup85 with 50 μM ABA treatment for 3 hours.

first: Yingfang

first: Kai

last: Tang

first: Shaojun

last: Xie


title: Super-enhancer analysis of human sarcomas reveals YAP1 control of NF-κB-dependent tumorigenesis

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

overall_design: Refer to individual Series


title: Analysis of gene expression in the skin of mice under high-fat diet regimen following topical association with Corynebacterium accolens.

city: BETHESDA

first: abdel

middle: G

last: Elkahloun

address: 50, SOUTH DRIVE

institute: NHGRI-NIH

summary:
The resident skin microbiota plays a fundamental role in the control of skin physiology and growing evidence support the idea that, at this barrier site, both immunity and inflammatory processes are controlled by skin resident microbiota. However, how defined skin microbes influence the skin immune system under both steady state conditions and inflammatory settings remains poorly understood. Obesity has been linked to increased prevalence of skin inflammatory disorders.
We used microarray analysis to investigate how Corynebacterium spp., dominant members of the skin microbiota of mice and humans, influence gene expression in the skin in mice under normal diet or a high-fat diet regimen.

overall_design: Three weeks-old mice were placed under a high-fat diet regimen (60% total calories from fat) or a control diet regimen (10% total calories from fat) for a month and then topically associated on their skin with the bacterium Corynebacterium accolens. Some mice were left unassociated. The experiment had four groups of 5 mice: control diet unassociated; control diet associated with C. accolens; high fat diet unassociated; high fat diet associated with C. accolens. Two weeks after topical association, the ear pinnae of each mouse was collected and placed in RNA later.

first: Yasmine

last: Belkaid

first: Nicolas

last: Bouladoux

first: Vanessa

last: Ridaura

first: Allyson

last: Byrd


title: Super-enhancer analysis of human sarcomas reveals YAP1 control of NF-κB-dependent tumorigenesis [ChIP-seq]

overall_design: Study of H3K27Ac localization in three Undifferentiated Pleomorphic Sarcoma patient samples.


title: Genome Scale Analysis of miRNA and mRNA regulation during preterm labor

city: Seattle

first: alison

middle: g

last: paquette

state: WA

address: 401 Terry Avenue N

institute: Institute of Systems Biology


title: ZFP36 RNA binding proteins restrain T-cell activation kinetics and anti-viral immunity

city: New York

first: Robert

last: Darnell

state: New York

address: 1230 York Ave

institute: Rockefeller University


title: ZFP36 RNA binding proteins restrain T-cell activation kinetics and anti-viral immunity [ribo-seq]

summary: Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in T-cells, which confirmed regulation of cytokine expression and revealed unanticipated actions in regulating T-cell activation and proliferation. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, most robustly through a novel class of AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics by attenuating activation marker expression, limiting T-cell expansion, and promoting apoptosis in a cell autonomous manner. Strikingly, loss of ZFP36 in vivo accelerated T-cell responses to acute viral infection, and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T-cell expansion and effector functions, and suggest ZFP36 inhibition as a novel strategy to enhance immune-based therapies.

overall_design: Ribosome profiling by was done for WT and Zfp36 KO CD4+ T-cells activated under Th1 polarizing conditions for 4 hours, to determine differentially translated mRNAs. Naïve CD4+ T-cells were purified with the CD4+CD62L+ isolation kit (Miltenyi) and incubated in co-cultures with formalin-fixed bone-marrow derived dendritic cells (BMDCs), anti-CD3e, 10 U/ml IL-2 and 5 ng/ml IL-12. After 4 hours, cells were harvested in the presence of cycloheximide and snap frozen in liquid nitrogen. Cytoplasmic lysates were prepared and treated with micrococcal nuclease, then monosomes were isolated by sucrose gradient fractionation as described in the accompanying protocol  Ribosome-protected-fragments (RPFs) were released from monosomes, and ligated to a pre-adenylated 3? linker (GTGTCAGTCACTTCCAGCGG), then amplified using the Br-dU incorporation, cDNA circularization, and PCR strategy described in detail in the accompanying publication.  RT primers were designed to incorporate sample indices for multiplexing (trimmed from uploaded sequences) and a 7 nucleotide degenerate sequence (NNNNNNN; present in uploaded reads) at the 5? end of the sequenced read. Amplified libraries were sequenced on the Illumina Miseq, with 75 nucleotide single-end reads. For analysis, adapter sequences were trimmed on both ends, samples were de-multiplexed using indices, and degenerate barcodes were appended to read IDs.  RPF reads were then aligned against the mouse Ensembl transcripts using Bowtie2.

first: Michael

last: Moore

first: Christopher

middle: Y

last: Park

middle: B


title: Successful transplantation of islets into inguinal subcutaneous white adipose tissue

city: Yokohama

first: Takaho

middle: A.

last: Endo

state: Kanagawa

address: 1-7-22 Suehiro, Tsurumi

department: IMS

country: Japan

institute: RIKEN

summary: Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2-3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved. Here we describe a novel site for islet transplantation, the inguinal subcutaneous white adipose tissue. In this site, transplanted islets are engrafted as clusters and function to reverse diabetes in mice. Importantly, transplanted islets can be visualized by CT and are easily retrievable, and allograft rejection is preventable by blockade of co-stimulatory signals. Of much interest, the efficiency of islet transplantation is superior to the liver, with increased mass of transplanted β cells. Furthermore, transplanted human islets function to reverse diabetes in immunodeficient mice. Thus, this adipose tissue site may be ideal for clinical islet transplantation.

overall_design: Gene expression of was observed before and after islet translplantation

first: Yohichi

last: Yasunami

first: Satoshi

last: Kojo

middle: A


title: Genome Scale Analysis of miRNA and mRNA regulation during preterm labor [whole blood]

summary: The goal of this study was to define relationships between peripheral blood miRNAs and mRNAs of women undergoing idiopathic preterm labor (PTL) and compare network level changes to control women that deliver at term.Using RNA Sequencing we have performed global miRNA and mRNA profiling in both monocytes and whole blood leukocytes of women who underwent PTL (N=15) matched to non-pathological controls (N=30) as a part of the Ontario Birth Study cohort.  We have identified differentially expressed miRNAs, mRNAs and pathways associated with PTL.  Intriguingly, we found perturbations in many cellular signaling pathways, particularly in interleukin signaling. We also predicted mRNA targets for specific miRNAs and used these predictions to build putative miRNA-mRNA networks. We identified 6 miRNAs significantly associated with PTL whose expression is negatively correlated with expression of 14 predicted mRNA targets that are also significantly associated with PTL.

overall_design: miRNA and mRNA were quantified from whole blood and monocytes of women undergoing spontaneous preterm labor compared to nonlabor controls matched on gestational age

first: Stephen

last: Lye

first: Nathan

last: Price


title: Genome Scale Analysis of miRNA and mRNA regulation during preterm labor [monocytes]


title: ZFP36 RNA binding proteins restrain T-cell activation and anti-viral immunity [HITS-CLIP]

overall_design: Determination of ZFP36 binding sites by HITS-CLIP in primary CD4+ T-cells at two time points post-activation (4 hours and 3 days)

middle: J


title: ZFP36 RNA binding proteins restrain T-cell activation kinetics and anti-viral immunity [RNA-seq]

overall_design: Transcriptome profiling by RNAseq was done for WT and Zfp36 KO CD4+ T-cells activated under Th1 polarizing conditions for 4 hours or 72 hours, to determine differentially expressed transcripts. Naïve CD4+ T-cells were FACS-sorted from pooled spleen and lymph nodes of WT or Zfp36 KO mice, and incubated in co-cultures with formalin-fixed bone-marrow derived dendritic cells (BMDCs), anti-CD3e, 10 U/ml IL-2 and 5 ng/ml IL-12. Total RNA was extracted with Trizol (Life Technologies) four hours post-activation, or 72 hours post-activation after 2 hours? restimulation with 20 ng/ml PMA and 1 ?M ionomycin. RNA was further purified on HiPure columns with DNAse treatment (Roche).  500 ng total RNA was depleted of rRNA with Ribo-Zero (Epicentre), then used to generate libraries with the TruSeq kit (Illumina).  Libraries were analyzed on the Hiseq 2000, pooling 4-5 samples per sequencing lane. RNAseq libraries were aligned against the mouse genome (mm10) with STAR, and read counts per feature were determined with HTSeq.


title: Functional involvement of microRNAs in T cell ageing-related decline of CD28 expression

first: Joost

last: Kluiver

summary: MicroRNA expression profiling of T-cell clones with high or low population doublings as a longitudinal model of age-associated gene expression changes.

overall_design: T cell clones were generated from phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) by limiting dilution in the presence of IL-2 and pooled irradiated PBMC feeder cells. Three T cell clones from two healthy old and three T cell clones from one healthy young donor with low and high population doublings were selected for small RNA sequencing (n=12 samples).

first: Nato

last: Teteloshvili

first: Gerjan

last: Dekkema

first: Peter

last: Heeringa

first: Pytrick

last: Jellema

first: Debora

last: de Jong

first: Martijn

last: Terpstra

first: Elisabeth

last: Brouwer

first: Graham

last: Pawelec

first: Annemieke

last: Boots

first: Klaas

last: Kok

first: Anke

last: van den Berg

first: Bart-Jan

last: Kroesen


title: RNA sequencing (RNA-SEQ) of 21 HBV-HCC patients with non-neoplastic liver and tumor tissues

first: Seungyeul

last: Yoo

state: NY

address: 1425 Madison Avenue

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

summary:
Purpose: Chronic Hepatitis B virus (HBV) infection leads to liver fibrosis which is a major risk factor in Hepatocellular carcinoma (HCC) and an independent risk factor of recurrence after HCC tumor resection. HBV genome can be inserted into human genome, and chronic inflammation may trigger somatic mutations. Several studies characterized HBV integration sites in HCC patients with regard to frequently occurring hotspots. However, how HBV integration and other genomic changes contribute to the risk of tumor recurrence with regard to different degree of liver fibrosis is not clearly understood. In this study, we aim to find potential molecular mechanisms underlying tumor recurrence of HBV-associated HCC (HBV-HCC) with different degree of liver fibrosis.
Methods: We performed RNA sequencing of 21 pairs of tumor and non-neoplastic liver tissues of HBV-HCC patients and performed comprehensive genomic analysis of our RNAseq data and public available sequencing data related to HBV-HCC. We developed a robust pipeline for sensitively identifying HBV integration sites based on sequencing data. Simulations with sequencing data showed that our method outperformed existing methods.  We also compared SNPs of each sample with SNPs in cancer census database and inferred patient’s pathogenic SNP loads in tumor and non-neoplastic liver tissues.
Conclusions: The HBV-integration and pathogenic SNP load patterns for HCC recurrence risk vary depending on liver fibrosis stage, suggesting potentially different tumorigenesis mechanisms for low and high liver fibrosis patients.

overall_design: Total RNA (1-3μg/sample) extracted from surgical resection specimens were submitted to the Mount Sinai Genomic Core Facility for quality control analysis. The RNA quality was assessed using the Agilent 2100 Bioanalyzer, and the RNA integrity number for all 21 samples measured 8.2 ± 0.7 (mean ± SD). The poly(A)-RNA was captured using oligo-dT beads and used for cDNA library preparation using the standard TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA). Briefly, total RNA was poly-A selected and then fragmented. The cDNA was synthesized using random hexamers, end-repaired and ligated with appropriate adaptors for sequencing. The library then underwent size selection and purification using AMPure XP beads (Beckman Coulter, CA, USA). The appropriate Illumina recommended 6 bp barcode bases are introduced at one end of the adaptors during PCR amplification step. The size and concentration of the RNAseq libraried was measured by Bioanalyzer and Oubit fluorometry (Life Technologies, NY, USA) before loading onto the sequencer. The mRNA libraries were sequenced on the Illumina HiSeq 2500 System with 100 nucleotide paired-end reads, according to the standard manufacturer’s protocol (Illumina, CA, USA).

first: Qin

last: Wang

first: Jun

last: Zhu

first: Spiros

last: Hiotis


title: Genome-wide expression analysis of Caenorhabditis elegans AXIN mutant

city: boston

first: Ayush

middle: Vasant

last: Ranawade

state: MA

address: 360 Huntington Ave

department: Bioengineering

institute: Northeastern University

summary:
Purpose: The goal of this study is to identify differentially expressed genes in pry-1/Axin mutant compare to N2 wild-type (WT).
Our study represents the first analysis of Axin transcriptome in C. elegans and facilitates investigations of axin mediated processes.

overall_design: Whole animal total RNA was extracted from L1 synchronized worms and mRNA profiles of WT and pry-1(mu38) animals were generated by paired end deep sequencing, using Illumina HISeq 2000. The sequence reads that passed quality filters were analyzed using ce6 with Burrows–Wheeler Aligner (BWA) followed by eXpress to estimate transcript abundances. Differentially-expressed genes were called at a false discovery rate (FDR) of 0.05% using the DESeq package in R.

first: Bhagwati

last: Gupta


title: Expression data from ependymal cell cultures treated with MLN4924.

status: Public on Feb 28 2018

city: Durham

first: Khadar

last: Abdi

state: NC

address: 300 North Duke St.

department: DMPI

institute: Duke University

summary:
We identified that Foxj1 is degraded by the ubiquitin proteasome system. Foxj1 protein is stabilized by the cullin-RING ligase inhibitor MLN4924.
We evaluated global changes to ependymal cell culture gene expression profiles during MLN4924 treatment.

overall_design: Fully differentiated ependymal cell cultures (2 weeks old) were either treated with DMSO as a control or with 0.25 uM MLN4924 for 36 hours.

first: K

first: C

last: Kuo


title: JAZ1 attenuates drought resistance by regulating JA and ABA signaling in rice

city: Wuhan

first: Jie

last: Fu

state: Hubei

address: 1 lion hill street

institute: Huazhong Agricultural University

summary: Jasmonates (JA) and abscisic acid (ABA) are phytohormones known to play important roles in plant response and adaptation to various abiotic stresses including salinity, drought, wounding, and cold. JAZ (JASMONATE ZIM-domain) proteins have been reported to play negative roles in JA signaling. However, direct evidence is still lacking that JAZ proteins regulate drought resistance. In this study, OsJAZ1 was investigated for its role in drought resistance in rice. Expression of OsJAZ1 was strongly responsive to JA treatment, and it was slightly responsive to ABA, salicylic acid, and abiotic stresses including drought, salinity, and cold. The OsJAZ1-overexpression rice plants were more sensitive to drought stress treatment than the wild-type rice Zhonghua 11 (ZH11) at both the seedling and reproductive stages, while the jaz1 T-DNA insertion mutant plants showed increased drought tolerance compared to the wild-type plants. The OsJAZ1-overexpression plants were hyposensitive to MeJA and ABA, whereas the jaz1 mutant plants were hypersensitive to MeJA and ABA. In addition, there were significant differences in shoot and root length between the OsJAZ1 transgenic and wild-type plants under the MeJA and ABA treatments. A subcellular localization assay indicated that OsJAZ1 was localized in both the nucleus and cytoplasm. Transcriptome profiling analysis by RNA-seq revealed that the expression levels of many genes in the ABA and JA signaling pathways exhibited significant differences between the OsJAZ1-overexpression plants and wild-type ZH11 under drought stress treatment. Quantitative real-time PCR confirmed the expression profiles of some of the differentially expressed genes, including OsNCED4, OsLEA3, RAB21, OsbHLH006, OsbHLH148, OsDREB1A, OsDREB1B, SNAC1, and OsCCD1. These results together suggest that OsJAZ1 plays a role in regulating the drought resistance of rice partially via the ABA and JA pathways.

overall_design: Transcriptome analysis of ZH11, OsJAZ1-OE plants under both severe drought stress and normal growth conditions.

first: Lizhong

last: Xiong


title: Biogeographic conservation and CO2-dynamics of the cytosine methylome in the globally important cyanobacterium Trichodesmium [WGBS]

city: Los Angeles

first: Eric

middle: Alva

last: Webb

address: 3616 Trousdale Parkway

institute: University of Southern California

summary: Our paper presents the results of a study in which we used whole genome bisulfite sequencing (WGBS), RNA-Seq (i.e. transcriptomics), high-CO2 physiology experiments, and spatiotemporally separated samples isolated in situ (i.e. directly from the ocean) to examine the metabolic potential of genome-wide cytosine (5mC) methylation (i.e. epigenomics), its potential impacts to transcriptional dynamics under both present-day and future ocean acidification conditions, and its biogeographic conservation in the globally-significant, biogeochemically-critical marine cyanobacterium Trichodesmium.

sample_organism:
Trichodesmium
Trichodesmium erythraeum IMS101
Trichodesmium erythraeum 21-75
Trichodesmium thiebautii VI-1

overall_design: Characterize the 5mC cytosine methylation landscape of Trichodesmium erythraeum IMS101 and examine its relationship to its transcriptome under both 380 and 750 µatm CO2, respectively. WGBS of other T. erythraeum isolates (IMSB and 2175) as well as samples from natural populations isolated in situ in order to biogeographically characterize the Trichodesmium cytosine methylome.

first: Nathan

last: Walworth


title: Circular RNAs are super abundant in cervical tumor and plasma detected by high throughput microarray

city: ANN ARBOR

first: Shasha

last: Li

address: 1109 Maiden Ln, ct apt 104

institute: University of Michigan


title: Biomodal role of H3.3 in safe-guarding and establishing cell identities during reprogramming [RNA-Seq H3.3 KD]

city: Singapore

first: Chadi

last: El Farran

address: 61 Biopolis Drive

country: Singapore

institute: Institute of Molecular and Cell Biology

summary: Determination of transcriptome rewiring during cellular reprogramming in cells in which H3.3 had been depleted.

overall_design: mRNA-Seq of H3.3-depleted cells undergoing cellular reprogramming.

last: EL  Farran

first: Hai-Tong

last: Fang

first: Qiao

last: Xing

first: Li-Feng

last: Zhang

first: Hu

first: Yuin-Han

last: Loh


title: HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes

city: Rome

first: Viviana

last: M

address: Via A. Scarpa 16

department: SAIMLAL

institute: La Sapienza University of Rome


title: Genome-wide maps of chromatin bound by HDAC4 in proliferating muscle cells

summary: NGS technology was used for high-throughput profiling of DNA loci bound by HDAC4 in  muscle cells in growth condition. Chromatin was immunoprecipitated from C2C12 cells and IP with anti-HDAC4 antibody. Libraries were then generated, processed with Illumina cBot for cluster generation on the flowcell, and sequenced on single-end 50 bp mode at the multiplexing level requested on HiSeq2500. ChIP-Seq standard bioinformatics analysis and functional annotation revealed that HDAC4 predominantly binds to the gene body (UTR5, introns and exons) of coding genes, rather than to upstream regulatory regions or non-coding RNAs in proliferating muscle cells.

overall_design: HDAC4 was IP from proliferating C2C12 cells and chromatin was sequenced

first: Nicoletta

last: Marroncelli

first: Daniela

last: Palacios


title: Biogeographic conservation and CO2-dynamics of the cytosine methylome in the globally important cyanobacterium Trichodesmium

platform_organism:
Trichodesmium
Trichodesmium erythraeum IMS101
Trichodesmium erythraeum 21-75
Trichodesmium thiebautii VI-1


title: Analysis of chromatin accessibility in decidualizing human endometrial stromal cells

city: Coventry

first: Jan

last: Brosens

address: University Hospital Coventry and Warwickshire

department: Warwick Medical School

institute: University of Warwick

summary: Spontaneous decidualization of the endometrium in response to progesterone signaling is confined to menstruating species, including humans and other higher primates. During this process, endometrial stromal cells (EnSCs) differentiate into specialized decidual cells that control embryo implantation. We subjected undifferentiated and decidualizing human EnSCs to Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility upon decidualization was strongly associated with differential gene expression. Analysis of 1,533 opening as well as closing chromatin regions revealed overrepresentation of DNA binding motifs for known decidual transcription factors (TFs) and identified putative new regulators. ATAC-seq footprint analysis provided evidence of TF binding at specific motifs. One of the largest footprints involved the most enriched motif, basic leucine zipper (bZIP), as part of a triple motif that also comprised the estrogen receptor- and Pax domain-binding sites. Without exception, triple motifs were located within Alu elements, suggesting a role for this primate-specific transposable element (TE) in the evolution of decidual genes. Although other TEs were generally underrepresented in open chromatin of undifferentiated EnSCs, several classes contributed to the regulatory DNA landscape that underpins decidual gene expression.

first: Raffaella

last: Lucciola

first: Emma

middle: S

last: Lucas

first: Pavle

last: Vrljicak

first: Sascha

last: Ott


title: Transcriptome analysis of satellite cells with a genetic deletion of HDAC4 to identify the gene modulated by HDAC4

summary: NGS technology was used for high-throughput profiling of the transcriptome by comparing satellite cells lacking or not HDAC4.

overall_design: Total RNA was isolated from control and HDAC4 KO satellite cells in growth conditions


title: Isolation and functional examination of the long non-coding RNA Redrum

city: Goyaongsi

first: Charny

last: Park

state: Gyeonggi-do

address: 323 Ilsan-ro,

country: South Korea

institute: National Cancer Center

summary: Sought to isolate multiple long non-coding RNAs (lncRNAs) specifically expressed during murine embryogenesis. Multiple tissue-specifically expressed lncRNAs were identified based on differential expression levels between telencephalon and whole body. Detailed funtional analysis of one of the lncRNAs were carried out, and a report is being prepared.

overall_design: Total RNA was isolated from telencephalon and whole body and was subjected to RNAseq analysis. Differentially expressed lncRNAs were identified.

first: Jaesang

last: Kim

first: Sanghyuk

last: Lee

first: Yerim


title: Transcriptomic analysis of healthy donor and Sickle Cell Disease (SCD) hematopoietic stem/progenitor cells

city: Padova

first: Oriana

last: Romano

address: Via U. Bassi 58/B

department: Molecular Medicine

institute: University of Padova

summary: Human hematopoietic stem/progenitor cells (HSPCs) were either collected from bone marrow or mobilized from healthy donors and SCD patients. We performed RNA-seq analysis on RNA extracted from purified CD34+ HSPCs coming from the different sources to compare their gene expression profiles.

overall_design: RNA-sequencing of CD34+ cells collected from bone marrow or mobilized from healthy donors and SCD patients.

first: Tristan

last: Felix

first: Annarita

last: Miccio


title: Single cell transcriptomic profiling of pluripotent stem cell-derived SCGB3A2+ airway epithelium reveals fate plasticity

first: Ignacio S.

last: Caballero

address: 620 Albany St., NEIDL 401V

department: NEIDL/Microbiology

institute: Boston University School of Medicine

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

overall_design: Refer to individual Series


title: Single cell transcriptomic profiling of pluripotent stem cell-derived SCGB3A2+ airway epithelium reveals fate plasticity  *- 10x Chromium: bronchospheres vs. alveolospheres day 41*

summary: Many lung diseases involve alterations in the cellular identity of the lung epithelium. Improved insight into airway development and the changes to cellular identity that result from abnormal signaling may therefore improve understanding of the etiology of these complex diseases. We have previously described a protocol to generate epithelial airway spheres from human pluripotent stem cells (hPSCs), but still poorly understand the identity, development, and heterogeneity of these early airway cells. Here we use novel murine and human PSC lines to study developing secretory cells derived in vitro from hPSCs. Using an SCGB3A2CherryPicker (SC) reporter system together with population-based or single-cell global transcriptomic profiling we track, purify, and analyze hPSC-derived putative secretory airway progenitors and find that SC+ cells are enriched for expression of airway epithelial markers, including secretory cell markers, SCGB3A2 and SCGB1A1. Unexpectedly, some SC+ human cells also co-express distal type 2 cell genes, including SFTPC, ABCA3, NAPSA, CTSH and functional lamellar bodies, suggesting a significant level of plasticity within the hPSC-derived secretory population relative to concurrently generated proximal airway TP63+ cells or distal alveolar SFTPC+ cells. We establish that this plasticity is minimized by inhibiting low levels of endogenous canonical Wnt signaling post-lung specification, thus depleting the co-expressed type 2 cell program. Taken together, these findings suggest that, similar to in vivo mouse genetic models and diseased human lungs, hPSC-derived airway cells exhibit cellular plasticity in response to signaling cues, providing a human model system in which to study cellular identity in a disease-relevant context.

overall_design: Single-cell RNA-seq; NKX2-1 GFP + lung progenitors in proximal or distal media

first: Katherine

last: McCauley

first: Darrel

middle: N

last: Kotton

first: Ignacio


title: Single cell transcriptomic profiling of pluripotent stem cell-derived SCGB3A2+ airway epithelium reveals fate plasticity* - Fluidigm C1: bronchospheres day 27*


title: Biomodal role of H3.3 in safe-guarding and establishing cell identities during reprogramming [RNA-Seq]

first: Chadi

last: El Farran

summary: Determination of transcriptome rewiring during cellular reprogramming using RNA-Seq

overall_design: mRNA-Seq of cells undergoing cellular reprogramming at different time-points

last: EL  Farran

first: Hai-Tong

last: Fang

first: Qiao

last: Xing

first: Li-Feng

last: Zhang

first: Hu

last: Li

first: Yuin-Han

last: Loh


title: Biogeographic conservation and CO2-dynamics of the cytosine methylome in the globally important cyanobacterium Trichodesmium [RNA-Seq]

first: Eric

last: Webb

overall_design: Characterize the 5mC cytosine methylation landscape of Trichodesmium erythraeum IMS101 and examine its relationship to its transcriptome under both 380 and 750 µatm CO2, respectively. WGBS of other T. erythraeum isolates (IMSB and 2175) as well as samples from natural populations isolated in situ in order to biogeographically characterize the Trichodesmium cytosine methylome.)

first: Nathan

last: Walworth


title: Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples

city: RTP

first: Susan

last: Hester

address: 109 TW Alexander Dr

institute: US EPA

summary: Low RNA yield and quality limit use of formalin-fixed paraffin-embedded (FFPE) tissue samples for genomic analyses. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and target gene responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 hours followed by ethanol (18F); and 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. The latter group received no additional treatment (3F) or the following demodification protocols: short heated incubation with TAE buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst. TruSeq Stranded Total RNA libraries with Ribo-Zero were built and sequenced using the Illumina HiSeq platform. Extended incubation with or without organocatalyst increased RNA yield >3-fold and enhanced quality compared to 3F, as indicated by higher RNA integrity number (>1.5-fold) and fragment analysis values (>3.0-fold). Post-sequencing metrics showed reduced bias in gene coverage and deletion rates for all extended incubation groups. Following PB-induced differential gene expression analysis, all demodification groups showed increased overlap with FR in genes (73-83%) and pathways (91-94%) compared to 3F overlap with FR (60% and 63%, respectively). These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.

overall_design: Frozen mouse liver samples from control and phenobarbital exposed groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR, high quality RNA control); 70% ethanol (OH, fixative control); 10% buffered formalin for 18 hours followed by ethanol (18F); and 10% buffered formalin (3F, demodification control). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. The latter group received no additional treatment (3F) or the following demodification protocols: short heated incubation with TAE buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst (catalyst control). TruSeq Stranded Total RNA libraries with Ribo-Zero were built and sequenced using the Illumina HiSeq platform.

first: Leah

middle: C

last: Wehmas

first: Charles

middle: E

last: Wood

first: Remi

last: Gagne

first: Andrew

last: Williams

first: Carole

last: Yauk

first: Mark

last: Gosink

first: Deidre

last: Dalmas

first: Ruixin

last: Hao

first: Raegan

last: O'Lone


title: RNASeq analysis to detect any molecular changes in skin of Dlx4 ablated mice compared to Wild type littermates.

city: Bethesda

first: Maria

last: Morasso

address: Bldg 50

institute: NIAMS NIH

summary:
DLX4 is a member of DLX family of mammalian homeobox genes and cis linked sister gene of Dlx3. Misexpression of Dlx4 is associated with tumorigenicity.
However,Dlx4 role in epidermal development and homeostasis is not characterized.
In this study, we compared Wild type (DLX4-WT) and DLX4 deficient (DLX4-KO) Skin transcriptomes.

overall_design: Total RNA was extracted from dorsal skin of Dlx4-WT and Dlx4-KO mice at P1 and P60. cDNA libraries were generated using NEBnext and NuGen kits. Sequencing was performed on the HiSeq3000.

first: Shreya

last: Bhattacharya


title: Small RNA Next Generation Sequencing of healthy and myeloma plasma cells

city: Ferrara

first: Stefano

last: Volinia

address: Via Fossato di Mortara 64

institute: University of Ferrara

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the small RNA profile of healthy and myeloma plasma cells derived from non cancer donors and newly diagnosed myeloma patients
Methods: Plasma Cell small RNA profiles of 3 healthy plasma cells and 3 newly diagnosed myeloma patients were generated by deep sequencing, using Illumina GAIIx.
Conclusions: Our study represents the first detailed analysis of small RNA sequencing in plasma cells. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive quantitative and qualitative evaluation of small RNA content within the healthy and myeloma plasma cells.

overall_design: The RNA was extracted from the plasma cells obtained from non-cancer donors and newly diagnosed myeloma patients without high risk features

first: Flavia

last: Pichiorri


title: Designer epigenome modifiers enable robust and sustained gene silencing in clinically relevant human cells [RNA-seq]

status: Public on Feb 27 2018

city: Freiburg

first: Claudio

last: Mussolino

address: Breisacherstr. 115

institute: Institute for Transfusion Medicine and Gene Therapy

summary: Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkable high specificity, paving the way for the establishment of a potential new class of therapeutics.

overall_design:
We established a novel platform for epigenome editing. With this RNA-seq experiment we intend to profile specificity of our platform
We analyzed the transcriptome of primary human T lymphocytes in three independent experiment comparing cells receiving an inactive designer epigenome modifier (DEM) with cells receiving its active counterpart.

first: Tafadzwa

last: Mlambo

first: Sandra

last: Nitsch

first: Markus

last: Hildenbeutel

first: Marianna

last: Romito

first: Claudia

last: Bossen

first: Sven

last: Diederichs

first: Tatjana

middle: I

last: Cornu

first: Toni

last: Cathomen


title: Designer epigenome modifiers enable robust and sustained gene silencing in clinically relevant human cells [ATAC-seq]

overall_design:
We established a novel platform for epigenome editing. With this ATAC-seq experiment we intend to profile specificity of our platform
Control T cells and T cells with DEM.


title: Selective modulation of inflammatory Natural Killer (NK) cell phenotypes following histone H3K27 demethylase inhibition

city: Oxford

first: Adam

last: Cribbs

address: Windmill Road

department: NDORMS

institute: University of Oxford


title: Selective modulation of inflammatory Natural Killer (NK) cell phenotypes following histone H3K27 demethylase inhibition [RNA-Seq]

summary: Natural Killer cells are innate lymphocytes, participate in immune surveillance and elimination of stressed or transformed cells and critically shape the inflammatory cytokine environment to interact with cells of the innate and adaptive immune system, including macrophages, dendritic and Tcells. By performing a focused compound library screening, further validated by knockdown approaches, we here identify Jumonji-type histone 3 lysine 27 (H3K27) demethylases as key regulators of cytokine production in various human NK cell subsets. The prototypic H3K27 demethylase inhibitor GSK-J4 increases global levels of the repressive H3K27me3 mark around transcription start sites including NK cell effector cytokines, and thereby reduces IFN-γ, TNFα, GM-CSF and IL-10 levels in IL-15 stimulated NK cells whilst sparing the cytotoxic killing capacity against cancer cells. The anti-inflammatory effect of GSK-J4 in highly inflammatory NK cell subsets, isolated from peripheral blood or tissue from rheumatoid arthritis patients, coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggests a wider role and utility of histone demethylase inhibition in immunity and inflammation.

overall_design: We constructed 6 RNA-seq libraries; two conditions in triplicate. NK cells were cultured in media in the presence of IL-15 stimulation then cultured in the presence of DMSO and GSK-J4 treated NK cells. RNA was collected at 24 hours.

middle: P

first: Edward

last: Hookway

first: Udo

last: Oppermann


title: Selective modulation of inflammatory Natural Killer (NK) cell phenotypes following histone H3K27 demethylase inhibition [ATAC-Seq and ChIP-Seq]

summary: Natural Killer cells are innate lymphocytes, participate in immune surveillance and elimination of stressed or transformed cells and critically shape the inflammatory cytokine environment to interact with cells of the innate and adaptive immune system, including macrophages, dendritic and Tcells. By performing a focused compound library screening, further validated by knockdown approaches, we here identify Jumonji-type histone 3 lysine 27 (H3K27) demethylases as key regulators of cytokine production in various human NK cell subsets. The prototypic H3K27 demethylase inhibitor GSK-J4 increases global levels of the repressive H3K27me3 mark around transcription start sites including NK cell effector cytokines, and thereby reduces IFN-γ, TNF, GM-CSF and IL-10 levels in IL-15 stimulated NK cells whilst sparing the cytotoxic killing capacity against cancer cells. The anti-inflammatory effect of GSK-J4 in highly inflammatory NK cell subsets, isolated from peripheral blood or tissue from rheumatoid arthritis patients, coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggests a wider role and utility of histone demethylase inhibition in immunity and inflammation.

overall_design: ATAC-seq and ChIP-Seq of histone modifications H3K4me3 and H3K27me3 in human NK cells treated with GSK-J4 with/without reference SP1 spike-in.


title: Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naïve embryonic stem cells

city: Lausanne

first: Julien

last: Duc

state: VAUD

address: Station 19 CH-1015 Lausanne

department: School of Life Science

country: Switzerland

institute: EPFL

summary: The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in the silencing of transposable elements (TEs) and the maintenance of parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of active demethylation (operated by TET proteins) and lack of de novo methylation; in these cells, KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation. Here, upon removing either KAP1 or the cognate KZFP, we observed rapid TET-dependent accumulation of 5hmC at both ICRs and TEs. In absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild type, Dnmt and Tet triple knockout mESCs. We found that KAP1 represents the main effector of TEs repression in all three settings, yet we could additionally identify specific groups of TEs also controlled by DNA methylation. Furthermore, activation upon Kap1 removal in the absence of TET proteins could be either blunted or increased depending on TE subsets, indicating a complex interplay between heterochomatin and active demethylation in regulating the expression of the endovirome.

overall_design:
Profile of H3K4me3 and H3K27me3 in naive murine emrbyonic stem cells
Profile of 5-hydroxymethylcytosine enrichment in mure embryonic stem cells upon Kap1 knockdown
Analysis of transcriptional profiles of wild type, Dnmt triple knockout and Tet triple knockout murine embryonic stem cells upon Kap1 knockdown

first: Andrea

last: Coluccio


title: Direct reprogramming of spiral ganglion non-neuronal cells into neurons: Towards ameliorating sensorineural hearing loss by gene therapy

city: Toronto

first: Yutaka

last: Amemiya

state: ON

address: 2075 Bayview Ave.

institute: Sunnybrook Research Institute

summary: The loss of primary auditory neurons (PANs) leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs), comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs). The overexpression of both Ascl1 and NeuroD1 in vitro generated iNs at high efficiency and these iNs expressed several key markers of neuron identity. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, demonstrating that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs.

overall_design: P1 Tau-EGFP cochleae were dissected and isolated SGs were sorted by EGFP fluorescence with flow cytometer. EGFP positive fraction contained endogenous PANs, and EGFP negative fraction containing SGNNCs.  SGNNCs were cultured and transfected with transcription factors or control vector - induced neurons (iN) or vector control (VC) respectively. PANs, iNs, and VC cells were collected and RNA extracted for transcriptome analysis.

first: Teppei

last: Noda

first: Jumpei

last: Nogami

first: Yasuyuki

last: Ohkawa

first: Alain

last: Dabdoub


title: Age-dependent increase of oxidative stress regulates microRNA-29 family preserving cardiac health

city: Bad Nauheim

first: Carsten

last: Kuenne

address: Ludwigstrasse 43

department: Bioinformatics

institute: Max Planck Institute for Heart and Lung Research

summary: The short-lived turquoise killifish Nothobranchius furzeri (Nfu) is a valid model for aging studies. Here, we investigated its age-associated cardiac function. We observed oxidative stress accumulation and an engagement of microRNAs (miRNAs) in the aging heart. MiRNA-sequencing of 5 week (young), 12-21 week (adult) and 28-40 week (old) Nfu hearts revealed 23 up-regulated and 18 down-regulated miRNAs with age. MiR-29 family turned out as one of the most up-regulated miRNAs during aging. MiR-29 family increase induces a decrease of known targets like collagens and DNA methyl transferases (DNMTs) paralleled by 5´methyl-cytosine (5mC) level decrease. To further investigate miR-29 family role in the fish heart we generated a transgenic zebrafish model where miR-29 was knocked-down. In this model we found significant morphological and functional cardiac alterations and an impairment of oxygen dependent pathways by transcriptome analysis leading to hypoxic marker up-regulation. To get insights the possible hypoxic regulation of miR-29 family, we exposed human cardiac fibroblasts to 1% O2 levels. In hypoxic condition we found miR-29 down-modulation responsible for the accumulation of collagens and 5mC. Overall, our data suggest that miR-29 family up-regulation might represent an endogenous mechanism aimed at ameliorating the age-dependent cardiac damage leading to hypertrophy and fibrosis.

overall_design: RNA was isolated from zebrafish heart samples (3 wt and 3 miR-29-sponge) and sequenced.

first: Johanna

last: Heid

first: Chiara

last: Cencioni

first: Roberto

last: Ripa

first: Mario

last: Baumgart

first: Sandra

last: Atlante

first: Giuseppina

last: Milano

first: Alessandro

last: Scopece

first: Stefan

last: Guenther

first: Valerio

last: Azzimato

first: Antonella

last: Farsetti

first: Giacomo

last: Rossi

first: Thomas

last: Braun

first: Giulio

last: Pompilio

first: Fabio

last: Martelli

first: Andreas

middle: M

last: Zeiher

last: Cellerino

first: Carlo

last: Gaetano

first: Francesco

last: Spallotta


title: A versatile approach for assessing human stem cell-derived retinal progeny using single cell transcriptomics

city: Cleveland

first: Peng

last: Jiang

state: OH

address: 2121 Euclid Ave

institute: Cleveland State University

summary: To study photoreceptor (PR) production in human pluripotent stem cells (hPSCs), we created a reporter line that robustly labels PRs throughout differentiation. We then performed scRNAseq to define the molecular signature of hPSC-PRs

overall_design: We employed both a genetically engineered reporter line and scRNAseq to examine PRs and other NR cell types in serum-free cultures of differentiating hPSC-Ovs (D070* and  D218* samples). Adult* samples are from adult retinal tissue.

first: M

last: Phillips

first: Sara

last: Howden


city: Jena

first: Marco

last: Groth

address: Beutenbergstraße 11

department: Core Facility - Next Generation Sequencing

institute: Leibniz Institute on Aging - FLI

summary: Comparison of miRNA expression profiles from male Nothobranchius furzeri (strain MZM-0410) hearts in different age groups. The small-RNA-seq data comprises 3 groups

overall_design: 12 samples in 3 groups: 5 wph (4 samples), 12 wph (4 samples), 27-29 wph (4 samples)

first: Mario

last: Baumgart

first: Johanna

last: Heid

first: Chiara

last: Cencioni

first: Roberto

last: Ripa


title: Drosophila transcription factors, Séance, Ouija board and Molting defective, cooperatively control ecdysone biosynthesis

city: Edmonton

first: Kirst

last: King-Jones

state: Alberta

address: 11455 saskatchewan driver

department: Biological Sciences

institute: University of Alberta

summary: RNA sequencing was performed to examine differential gene expression profiles in the ring gland of PG-specific Séance RNAi animals versus control.

overall_design: Drosophila larvae with PG-specific knockdown of Séance and control animals were carefully staged at the larval L2/L3 molt. Ring glands were dissected at 44 hours L3. RNA isolated from ring glands were subject to RNA sequencing. Differential gene expression profiles were compared between control and RNAi animals.

first: Qiuxiang

last: Ou


title: RNA-Seq of in vitro Beta-Catenin-Stabilized Mouse Dermal Fibroblasts

first: Radhika

middle: Paresh

last: Atit

state: Ohio

address: 2074 Adelbert Road, Millis 316

department: Biology

institute: Case Western Reserve University

summary: Gene expression changes in mouse ventral dermal fibroblasts in response to Beta-Catenin stabilization in vitro

overall_design: Goal of study was to examine gene expression changes upon stabilization of beta-catenin in P4 ventral mouse dermal fibroblasts in vitro


title: Synergistic activity of BET inhibitor BI 894999 with PLK inhibitor volasertib in AML in vitro and in vivo

city: Vienna

first: Daniel

last: Gerlach

address: Dr.-Boehringer-Gasse 5-11

department: Global Computational Biology and Digital Sciences

country: Austria

institute: Boehringer Ingelheim RCV GmbH & Co KG

summary: Interactions between a new potent Bromodomain and extraterminal domain (BET) inhibitor BI 894999 and the polo-like kinase (PLK) inhibitor volasertib were studied in acute myeloid leukemia cell lines in vitro and in vivo. We provide data for the distinct mechanisms of action of these two compounds with a potential utility in AML based on gene expression, cell cycle profile and modulation of PD biomarkers such as MYC and HEXIM1. In contrast to BI 894999, volasertib treatment neither affects MYC nor HEXIM1 expression, but augments and prolongs the decrease of MYC expression caused by BI 894999 treatment. In vitro combination of both compounds leads to a decrease in S-Phase and to increased apoptosis. In vitro scheduling experiments guided in vivo experiments in disseminated AML mouse models. Co-administration of BI 894999 and volasertib dramatically reduces tumor burden accompanied by long-term survival of tumor-bearing mice and eradication of AML cells in mouse bone marrow. Together, these preclinical findings provide evidence for the strong synergistic effect of BI 894999 and volasertib, warranting future clinical studies in patients with AML to investigate this paradigm.

overall_design: MV-4-11B cells were treated with either BI 894999 (1 nM or 35 nM) or volasertib (20 nM) or the combination of both for 4h or 24h. Subsequently cells were harvested for total RNA isolation using TriZol (Ambion) and further purified using miRNAeasy columns (Qiagen). Approximately 500ng of total RNA were subjected to library preparation using the TruSeq RNA Library Prep Kit v2 with polyA selection as recommended by the manufacturer (Illumina) without generating stranded information. Library were multiplexed and sequenced on a HiSeq1500 using paired-end sequencing with 50 cycles each.

first: Ulrike

last: Tontsch-Grunt

last: Savarese

first: Norbert

last: Schweifer

first: Onur

last: Kaya


title: Gene regulation by CnpB in Mycobacterium tuberculosis

city: Albany

first: Guangchun

last: Bai

address: New Scotland Ave

department: DIMD

institute: Albany Medical College

summary: We have previously reported that Mycobacterium tuberculosis Rv2837c (cnpB) encodes a phosphodiesterase that specifically cleaves cyclic di-AMP (c-di-AMP) into AMP. Deletion of cnpB results in significant virulence attenuation in a mouse pulmonary infection model, which is very likely due to the significantly elevated c-di-AMP levels as overexpression of Mtb diadenylate cyclase, disA, also leads to a similar outcome. An earlier study also demonstrated that CnpB functions similarly to E. coli oligoribonuclease (Orn) that hydrolyzes 2-5-mer nanoRNAs (short oligonucleotides of five residues or shorter in length) except that CnpB prefers 2-mer nanoRNA as a substrate. Additionally, a recent report showed that CnpB also degrades cyclic di-GMP (c-di-GMP), although we demonstrated that CnpB prefers c-di-AMP to c-di-GMP according to an in vitro enzymatic kinetics analysis  In this study, we initially attempted to determine c-di-AMP-mediated gene regulation in Mtb by comparing the expression profiles between WT and ∆cnpB using RNA-Seq. We found that the CRISPR-Cas system of M. tuberculosis was highly upregulated by deletion of cnpB.

overall_design:
This experiment is designed to study gene regulation by cnpB in Mycobacterium tuberculosis. We prepared RNA samples of M. tuberculosis WT and ∆cnpB with 3 sets of cultures. RNA samples were treated with DNase to remove contaminated DNA. rRNA was removed using Ribo-Zero magnetic kit (Epicentre). cDNA library was then generated using ScriptSeq Complete Kit for Bacteria (Epicentre). Samples 1, 3, 5, 7, 9, 11 were amplified with index PCR primers 1, 3, 5, 7, 9, 11, respectively, from Script Seq Index PCR primers (Epicentre). RNA-Seq was performed at the Next Generation Sequencing and Expression Analysis Core of University of Buffalo.

>>> Grant <<
Funder: American Heart Association (AHA)

Grant number: 12SDG12080067

Grantee: Guangchun Bai

Title: Interaction between TB bacilli and host via cyclic nucleotides


title: In planta bacterial RNA-Seq without bacterial isolation

city: Cologne

first: Tatsuya

last: Nobori

address: Carl-von-Linne Weg 10, Max-Planck Institute for Plant Breeding Research

summary: Plant pathogens can cause serious diseases that impact global agriculture1. Understanding how the plant immune system naturally restricts pathogen infection holds a key to sustainable disease control in modern agricultural practices. However, despite extensive studies into the molecular and genetic basis of plant defense against pathogens since the 1950s2,3, one of the most fundamental questions in plant pathology remains unanswered: how resistant plants halt pathogen growth during immune activation. In the case of bacterial infections, a major bottleneck is an inability to determine the global bacterial transcriptome and metabolic responses in planta. Here, we developed an innovative pipeline that allows for in planta high-resolution bacterial transcriptome analysis with RNA-Seq, using the model plant Arabidopsis thaliana and the foliar bacterial pathogen Pseudomonas syringae. We examined a total of 27 combinations of plant immunity and bacterial virulence mutants to gain an unprecedented insight into the bacterial transcriptomic responses during plant immunity. We were able to identify specific bacterial transcriptomic signatures that are linked to bacterial inhibition during two major forms of plant immunity: pattern-triggered immunity and effector-triggered immunity. Among them, regulation of a P. syringae sigma factor gene, involved in iron regulation and an unknown process(es), was found to play a causative role in bacterial restriction during plant immunity. This study unlocked the enigmatic mechanisms of bacterial growth inhibition during plant immunity; results have broad basic and practical implications for future study of plant diseases.

sample_organism: Pseudomonas syringae pv. tomato str. DC3000

overall_design: Two leaves from three four-and-one-half week-old A. thaliana plants were infiltrated with either 0.005% dimethyl sulfoxide (DMSO, mock) or 500 nM flg22 using a needleless syringe. Twenty hours post-infiltration, plants were inoculated with a bacterial suspension at an OD600 of 0.75 (10^9 CFU ml-1) of Pto DC3000. Seven hours after Pto DC3000 inoculation, leaves were collected for RNA extraction. Three independent replicates (including 3 technical replicates for flg22 treated samples) were taken.

first: André

last: Velásquez

first: Brian

middle: H

last: Kvitko

first: James

last: Kremer

first: Sheng

last: He

first: Kenichi

last: Tsuda


title: In planta bacterial RNA-Seq with bacterial isolation.

institute: Max-Planck Institute for Plant Breeding Research

platform_organism: Pseudomonas syringae pv. tomato str. DC3000

overall_design: In planta bacterial RNA-Seq was profiled in 27 combinations (100 samples) of four P. syringae pv. tomato DC3000 and nine Arabidopsis thaliana genotypes. Bacterial suspension was syringe infiltrated to plant leaves at OD600=0.5. At 6 h after infection, the infected leaves were harvested and bacterial cells were isolated, followed by RNA extraction. Ribosomal RNA of bacteria and plants were removed with a commercial kit to enrich bacterial mRNA. Then, cDNA libraries were generated and RNA-Seq was performed. Bacteria grown in liquid media (Minimal medium and/or King’s B medium) were also analyzed. For most of the samples, three independent replicates were taken.


title: DNA regions bound by Myxococcus xanthus ParB and FrzCD

city: Marselle

first: Emanuele

middle: G

last: Biondi

address: 31 chemin Joseph Aiguier

department: LCB

country: France

institute: CNRS

summary: Our ChipSeq analysis show that while FrzCD does not  bind DNA specific regions, ParB binds parS consistent with what as been previously shown. The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. FrzCD directly binds to the nucleoid and the FrzCD binding to the DNA leads to the formation of chemosensory complexes. This supra-molecular organization is required for cooperative interactions between clustered receptors, in turn important for the modulation of bacterial social behaviors.

overall_design: ChIp-seq was performed as previously described (Fioravanti et al., 2013). Cells were grown and then fixed with formaldehyde as previously described (Fioravanti et al., 2013).

first: Emilia

last: Mauriello

first: Tam

last: Mignot

first: Hanna

last: Bismuth

first: Matteo

last: Brilli


title: Single-cell RNAseq Reveals Seven Classes of Visceral Sensory Neuron

city: Cambridge

last: Hockley

address: Tennis Court Road

institute: University of Cambridge

summary: Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. The principal neural unit in the bowel encoding these stimuli is the visceral sensory neuron with endings at the mucosa, intramurally and along mesenteric blood vessels. Sensory neurons activate reflex pathways and give rise to conscious sensation, however, the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by the current paucity of molecular markers. Here we overcome these limitations by comprehensive transcriptomic profiling and unsupervised clustering of single colonic sensory neurons revealing 7 classes characterised from both lumbar splanchnic (LSN) and pelvic nerves (PN). We identify and classify neurons based on novel marker genes, confirm translation of patterning to protein expression and show subtype-selective differential agonist activation, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity.

overall_design: Sensory neurons innervating the mouse colorectum were labelled by retrograde tracer injection. Single-cell RNAseq was performed on 399 dissociated colonic sensory neurons isolated from thoracolumbar (T10-L1) and lumbosacral (L5-S2) dorsal root ganglia distributed over six 96-well plates. 13 additional negative controls were collected.

middle: R

first: Toni

last: Taylor

first: Gerard

last: Callejo

first: Anna

middle: L

last: Wilbrey

first: Alex

last: Gutteridge

first: Karsten

last: Bach

first: Wendy

last: Winchester

first: David

last: Bulmer

first: Gordon

last: McMurray

first: Ewan

last: St. John Smith


title: Designer epigenome modifiers enable robust and sustained gene silencing in clinically relevant human cells

first: Claudio

last: Mussolino

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

overall_design: Refer to individual Series


title: Genome-wide binding reveals intrinsic specificity and regulatory function of lineage-specifying bHLH transcription factors (RNA-Seq).

city: Dallas

first: Jane

last: Johnson

state: TX

address: 5323 Harry Hines Blvd

department: Neuroscience

institute: UT Southwestern Medical Center

summary: Developmental mechanisms of lineage specification are crucial to tissue-specific gene regulation, and crucial lineage-specification factors are associated with developmental and oncogenic diseases. The mechanisms by which these factors regulate their distinct transcriptional profiles remains unclear. Comparison of their binding in a common context reveals that such specificity is intrinsic to these factors, and not simply a consequence of distinction in preference for, or availability of accessible chromatin at their respective binding sites.

overall_design: ChIP-seq performed on murine ESCs as part of bHLH transcription factor binding screen. The antibodies used were directed against bHLH factors ASCL1, ASCL2-FLAG, and MYOD-FLAG to observe TF binding. H3K27ac enrichment was assayed using an antibody recognizing this mark for comparison to TF binding, gene expression, and chromatin accessibility from the same cell lines.

first: Bradford

last: Casey

first: Rahul

middle: K

last: Kollipara


title: A quantitative chemotherapy genetic interaction map identifies new factors associated with PARP inhibitor resistance

city: San Francisco

first: Sourav

last: Bandyopadhyay

state: California

address: 1450 3rd Street

department: Helen Diller Family Comprehensive Cancer Center

institute: University of California, San Francisco

summary: The goal of this study was to understand the gene expression changes in MCF10A cells with GPBP1 knockdown in presence and absence of BMN673.

overall_design: Examination of RNA expression changes in drug treated and untreated celllines with GPBP1 knockdown

first: Hsien-Ming

last: Hu

first: Xin

last: Zhao

first: Swati

last: Kaushik


title: Genome-wide binding reveals intrinsic specificity and regulatory function of lineage-specifying bHLH transcription factors (ChIP-Seq).


title: Genome-wide binding reveals intrinsic specificity and regulatory function of lineage-specifying bHLH transcription factors (ATAC-Seq).


title: Argonaute2 and LaminB modulate gene expression by controlling chromatin topology (ChIP-seq)

city: Bethesda

first: Ryan

last: Dale

state: MD

address: Rm 10D39, 10 Center Drive

department: National Institute of Child Health and Human Development

institute: National Institutes of Health

summary: Drosophila Argonaute2 (AGO2) is known to regulate expression of certain loci in an RNA interference-independent manner, but its genome-wide function on chromatin remains unknown.  Here, we identified RNA Polymerase II (Pol II) and LaminB as AGO2 nuclear interactors. AGO2 and LaminB attenuate transcription at borders between both LaminB-associated domains (LADs) and topologically-associated domains (TADs). We identified a somatic target of AGO2 transcriptional repression, nht, which is immersed in a LAD located within a repressive TAD. Null mutation of AGO2 leads to ectopic expression of spermatogenesis genes dependent on nht. Finally, depletion of either AGO2 or LaminB results in reduced looping interactions within the TAD as well as ectopic inter-TAD interactions. Overall, our findings reveal coordination of AGO2 and LaminB to dictate overall genome architecture and thereby regulate gene expression.

overall_design: ChIP-seq of AGO2 in mock and LaminB knockdown, 2 replicates per experiment.

first: Ezequiel

last: Nazer

first: Elissa

last: Lei


title: High-efficiency RNA-based reprogramming of human primary fibroblasts

city: Aurora

last: Astling

state: CO

address: 12801 East 17th Ave.

department: Department of Biochemistry and Molecular Genetics

institute: University of Colorado Denver

summary: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the reprogramming of human patient’s fibroblasts into iPSCs can be enhanced to an unprecedented efficiency via the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNAs-367/302s delivered as mature miRNA mimics. This synergism is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to primary human fibroblasts. As a result, we have developed a novel, integration- and feeder-free reprogramming approach that generates up to 4,019 TRA-1-60 positive colonies from only 500 starting human primary neonatal fibroblasts and reprograms up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant iPSCs from a variety of human patient’s fibroblasts and opens up new horizons for studying the biology of reprogramming.

overall_design: Gene expression profiles of induced pluripotent stem cells (iPSCs), derived from neonatal, adult and senescent human fibroblasts, were compared to the primary cell lines


title: Argonaute2 and LaminB repress transcription by controlling RNA Pol II recruitment and chromatin topology in Drosophila melanogaster (4C-Seq)

summary: Although best known as core components of RNA silencing pathways in the cytoplasm, Argonautes have been shown to regulate gene expression by functioning in the nucleus. Previous work showed that Drosophila AGO2 is preferentially associated with active promoters and can activate gene expression by promoting looping to an enhancer. In order to elucidate the genome-wide role of AGO2 in regulating transcription, we performed mass spec analysis of factors that interact with AGO2 in nuclear extracts and found RNA Pol II and LaminB among the top-associated proteins. Using nascent RNA sequencing, we showed that AGO2 and LaminB co-repress transcription at borders between both LaminB-associated domains (LAD) and topologically-associated domains (TADs). Furthermore, LaminB and AGO2 specifically prevent RNA Pol II recruitment at repressed genes, and AGO2 additionally impairs the ability of RNA Pol II to extend into active elongation. Using mRNA-seq profiling, we identified a somatic target of AGO2 repression, nht, which leads to ectopic expression of spermatogenesis genes in AGO2 null mutants. Interestingly, nht is immersed in a LAD located within a repressive TAD flanked by AGO2 binding sites. 4C-seq analysis showed that depletion of either AGO2 or LaminB results in a significant decrease in frequency of interactions within the TAD as well as ectopic inter-TAD interactions. Overall, our findings reveal a coordinated function for AGO2 and LaminB in dictating the overall architecture of the genome to control the recruitment of RNA Pol II and thereby regulate gene expression.

overall_design: 4C-seq analysis using as a bait the nht promoter in mock, AGO2 and LaminB knockdowns. The experiment has 2 biological replicates


title: Zebrafish Rfx4 controls dorsal and ventral midline formation in the neural tube

city: Madison

first: Oleg

last: Moskvin

state: WI

address: 1220 Capitol Court

institute: University of Wisconsin-Madison

summary: RNA-seq transcriptome analysis identified a functional requirement for zebrafish Rfx4  in the developing neural floor plate and roof plate.

overall_design: Embryos derived from an rfx4uw8013/+  incross were sorted by phenotype into mutant and sibling groups.  RNA was prepared from each individual embryo at ~ 25 hpf

first: Irina

last: Sedykh

first: Abigail

last: Keller

first: Baul

last: Yoon

first: Laura

last: Robertson

first: Yevgenya

last: Grinblat


title: Genome-wide binding reveals intrinsic specificity and regulatory function of lineage-specifying bHLH transcription factors

last: Johnson