title: Single cell RNA-seq data of human hESCs to evaluate SCnorm: robust normalization of single-cell rna-seq data

city: Gainesville

first: Rhonda

last: Bacher

state: FL

address: 2004 Mowry Rd.

institute: University of Florida

summary: Normalization of RNA-sequencing data is essential for accurate downstream inference, but the assumptions upon which most methods are based do not hold in the single-cell setting. Consequently, applying existing normalization methods to single-cell RNA-seq data introduces artifacts that bias downstream analyses. To address this, we introduce SCnorm for accurate and efficient normalization of scRNA-seq data.

overall_design: Total 183 single cells (92 H1 cells, 91 H9 cells), sequenced twice, were used to evaluate SCnorm in normalizing single cell RNA-seq experiments. Total 48 bulk H1 samples were used to compare bulk and single cell properties. For single-cell RNA-seq, the identical single-cell indexed and fragmented cDNA were pooled at 96 cells per lane or at 24 cells per lane to test the effects of sequencing depth, resulting in approximately 1 million and 4 million mapped reads per cell in the two pooling groups, respectively.

first: Li-Fang

last: Chu

first: Christina

last: Kendziorski

first: Scott

last: Swanson


title: Genome-wide histone modification profiling in endometriosis cells

status: Public on Apr 05 2017

city: Los Angeles

first: Ranjani

last: Lakshminarasimhan

state: California

address: 1441 Eastlake ave NOR7336

department: Urology

country: USA

institute: University of Southern California

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We show that knocking down ARID1A in an immortalized endometriosis cell line leads to phenotypic changes  in vitro.  Additionally, we find strong increases in the active H3K27ac mark in promoter regions but decrease of H3K27ac at potential enhancers.

overall_design: Examination of 4 different histone modifications in endometriosis control and ARID1A knockdown cell lines.

first: Claudia

last: Andreu-Vieyra

first: Kate

last: Lawrenson

first: Chris

middle: E

last: Duymich

first: Simon

middle: A

last: Gayther

first: Gangning

last: Liang

first: Peter

last: Jones


title: Natural variation of piRNA expression affects immunity to transposable elements

city: Moscow

first: Sergei

last: Ryazansky

address: Kurchatov sq., 2

department: Department of Molecular Genetics of Cell

country: Russia

institute: Institute of Molecular Genetics

type: Non-coding RNA profiling by high throughput sequencing

summary: In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins.  Here, we explore the variation in the ability to restrain an alien TE in different Drosophila strains.  The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster.  Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements.  The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which we have observed in one particular transposon-resistant R strain.

overall_design: The fractions of small RNAs (19-29 nt) from ovaries of four R strains of Drosophila melanogaster, and from embryos of two R strains of Drosophila melanogaster, were sequenced using Illumina HiSeq 2000.

first: Elizaveta

last: Radion

first: Anastasia

last: Mironova

first: Natalia

last: Akulenko

first: Yuri

last: Abramov

first: Valeriya

last: Morgunova

first: Maria

last: Kordyukova

first: Ivan

last: Olovnikov

first: Alla

last: Kalmykova


title: Dynamic gene regulatory networks of human myeloid differentiation

status: Public on Apr 06 2017

city: Irvine

first: Ricardo

middle: Noel

last: Ramirez

address: 2300 Biological Sciences III

department: Developmental and Cell Biology

institute: University of California Irvine

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: KAT–independent gene regulation by Tip60 promotes ESC self-renewal but not pluripotency

city: Worcester

first: Thomas

last: Fazzio

state: MA

address: 364 Plantation street

institute: UMMS

summary: Although histone-modifying enzymes are assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here we show the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT–deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT–independent role of Tip60 in ESC maintenance. In contrast, KAT–deficient ESCs exhibited impaired differentiation into mesoderm and endoderm, demonstrating a second, KAT–dependent function in differentiation. Consistent with this phenotype, KAT–deficient mouse embryos exhibited post-implantation developmental defects. These findings establish separable KAT–dependent and KAT–independent functions of Tip60 in ESCs and during embryonic development, raising the possibility of undiscovered catalysis-independent functions of additional KATs.

overall_design:
The data represent several experiments: (1) RNA-seq experiments examining gene expression changes in Tip60 catalytic inactive, Ep400 catalytic inactive, double mutants, or knockdowns (RNAi) in ESCs relative to controls, (2) RNA-seq experiments examining changes in gene expression of wild type controls and Tip60 catalytic inactive mutants during ESC differentiation, and (3) ATAC-seq experiments examining chromatin accessibility in control, Tip60 catalytic inactive, and Tip60 knockdown ESCs.
The data represent ChIPseq experiment examining changes in H4 acetylation and H2AZ occupany in Tip60 catalytic inactive and Ep400 catalytic inactive mutant ESCs respectively, relative to controls.

first: Diwash

last: Acharya

first: Sarah

middle: J

last: Hainer

first: Yeonsoo

last: Yoon

first: Feng

last: Wang

first: Ingolf

last: Bach

first: Jaime

last: Rivera-Pérez

middle: G


title: Genetic Analysis of a Metazoan Pathway using Transcriptomic Phenotypes

status: Public on Apr 04 2017

city: Medford

first: David

last: Angeles-Albores

address: 61 Locust St

institute: Northwestern University

summary: RNA-seq is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Linear models can quantify expression effects of individual mutants and identify epistatic effects in double mutants. However, interpreting these high-dimensional measurements is unintuitive. We developed a single coefficient to quantify transcriptome-wide epistasis which accurately reflects the underlying interactions. To demonstrate the power of our approach, we sequenced four single and two double Caenorhabditis elegans mutants. From these mutants, we successfully reconstructed the known hypoxia pathway. Using this approach, we uncovered a class of 31 genes that have opposing changes in expression in egl-9(lf) and vhl-1(lf) but for which the egl-9(lf);vhl-1(lf) mutant phenocopies egl-9(lf). These changes violate the classical model of HIF-1 regulation, but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.

overall_design: 21 samples; 3 replicates

first: Carmie

last: Puckett Robinson

first: Brian

last: Williams

first: Barbara

last: Wold

first: Paul

middle: W

last: Sternberg


title: A microRNA-129-5p/Rbfox crosstalk coordinates homeostatic downscaling of excitatory synapses.

city: Marburg

first: Gerhard

last: Schratt

address: Karl-von-Frisch-Straße n.1

department: Institute for Physiological Chemistry

country: Germany

institute: Philipps-University Marburg

summary: Using quantitative proteomics we identified group of synaptic genes with decreased protein synthesis during homeostatic plasticity. To obtain further information about their mRNA levels/sequences (3’UTR) we performed polyA RNAseq. At the end, small RNAseq helped us to identify miRNAs that are increased during homeostatic plasticity and might regulate downregulated genes.

overall_design: Picrotoxin was applied for 48h to mature hippocampal cultures. Changes in mRNA/small RNA levels were assessed by different types of RNAseq. Three biological replicates are included in all analysis. Poly RNAseq and small RNA seq were done from the same RNA preparation.

first: Marek

last: Rajman

first: Franziska

last: Metge

first: Sharof

last: Khudayberdiev

first: Christoph

last: Dieterich


title: ENL Links Histone Acetylation to Oncogenic Gene Expression in AML

city: NEW YORK

first: Liling

last: Wan

state: NY

address: 1230 York Avenue, Box 78

institute: The Rockefeller University

summary: Cancer cells are characterized by aberrant epigenetic landscapes and often exploit the chromatin machinery to activate oncogenic gene expression programs1. The recognition of modified histones by “reader” proteins constitutes a key mechanism underlying these processes; therefore targeting such pathways holds clinical promise, as exemplified by the recent development of BET bromodomain inhibitors2,3. We recently identified the YEATS domain as a novel acetyllysine-binding module4, yet its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralog AF9, is required for disease maintenance in a variety of acute myeloid leukaemias (AML). CRISPR-Cas9 mediated depletion of ENL led to anti-leukemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies in vitro and ChIP-seq analyses in leukaemia cells revealed that ENL binds to acetylated histone H3, and colocalizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemias. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced RNA polymerase II recruitment on ENL target genes, thus leading to suppression of oncogenic gene expression programs. Importantly, disruption of ENL’s functionality further sensitized leukaemia cells to BET inhibitors. Together, our study identifies ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in AML and suggests that displacement of ENL from chromatin is a promising epigenetic therapy alone or in combination with BET inhibitors for AML

overall_design: iCas9-MOLM-13 or MV411 cells were transduced with sgRNA or shRNA targeting control or ENL in indicated conditions. RNA-seq was then performed to identify differentially expressed genes.

first: Hong

last: Wen

first: Yuanxin

last: Xi


title: Dehydration stress extends mRNA 3' untranslated regions with noncoding RNA functions in Arabidopsis

first: Nam-Hai

last: Chua

state: New York

address: 1230 York Avenue

summary: The 3' untranslated regions (3' UTRs) of mRNAs play important roles in the regulation of mRNA localization, translation, and stability. Alternative cleavage and polyadenylation (APA) generates mRNAs with different 3' UTRs, but the involvement of this process in stress response has not been clarified. Here, we report that a subset of stress-related genes exhibits 3' UTR extensions of their mRNAs during dehydration stress. These extended 3' UTRs have characteristics of long noncoding RNAs and likely do not interact with miRNAs. Functional studies using T-DNA insertion mutants reveal that they can function as antisense transcripts to repress expression levels of sense genes from the opposite strand, or can activate the transcription of downstream genes from the same strand. Poly(A) signal analysis reveal that mRNAs with 3' UTR extensions have weaker poly(A) signals than mRNAs without 3' UTR extensions. Finally, we show that their biogenesis is partially dependent on a trans-acting factor FPA. Taken together, we report that dehydration stress could induce mRNA 3' UTR extensions, and elucidate a novel function for these stress-induced 3' UTR extensions as long noncoding RNAs in the regulation of their neighboring genes.

overall_design: To study the effects of stress on APA, we performed ssRNA-seq on Arabidopsis seedlings grown in normal, ABA-treated, and dehydration-stressed conditions. We collected samples at two time points (1 h and 3 h) for each treatment and for each time point we had three biological replicates.

first: Hai-Xi

last: Sun

first: Yan

last: Li

first: Qi-Wen

last: Niu


title: Genome-wide DNA methylation profile of developing deciduous tooth germ in miniature pigs

city: Beijing

first: Yingying

last: Su

address: Tian Tan Xi Li No.4

country: China

institute: Capital Medical University School of Stomatology

type: Methylation profiling by high throughput sequencing

summary: Background: DNA methylation is an important epigenetic modification critical to the regulation of gene expression during development. To date, little is known about the role of DNA methylation in tooth development in large animal models. Thus, we carried out a comparative genomic analysis of genome-wide DNA methylation profiles in E50 and E60 tooth germ from miniature pigs using methylated DNA immunoprecipitation-sequencing (MeDIP-seq).Results: We observed different DNA methylation patterns during the different developmental stages of pig tooth germ. A total of 2,469 differentially methylated genes were identified. Functional analysis identified several signaling pathways and 104 genes that may be potential key regulators of pig tooth development from E50 to E60.Conclusions: The present study provided a comprehensive analysis of the global DNA methylation pattern of tooth germ in miniature pigs and identified candidate genes that potentially regulate tooth development from E50 to E60.

first: Zhipeng

last: Fan

first: Xiaoshan

last: Wu

first: Yang

first: Fu

last: Wang

first: Chunmei

last: Zhang

first: Jinsong

first: Jie

last: Du

first: Songlin


title: polyA+ RNA expression profiles of Drosophila melanogaster male and female brains after treatment with Sodium salicylate, Chlorpromazine, or Trifluoperazine2HCl

city: Bethesda

first: Brian

last: Oliver

state: MD

address: 50 South Drive

department: LBG

institute: NIDDK, NIH

summary: To explore some of the sex differential responses to drug treatment, we exposed 3 day post-eclosion virgin Drosophila melanogaster (genotype DGRP-900, FlyBase line ID: FBst0028261) to 3 FDA approved drugs and characterized transcript abundance among male and female brains after 24 hours of drug exposure. The 3 tested drugs are water-soluble group N (Nervous system) compounds according to The Anatomical Therapeutic Chemical (ATC) classification system. Two (Chlorpromazine and Trifluoperazine2HCl) are primarily used to treat psychotic disorders, while the third (Sodium salicylate) is used as an analgesic and antipyretic. Following drug treatment, we dissected fly brains, isolated polyA+ RNA and performed high-throughput RNA-Seq analysis. We conducted quantification and statistical analysis of gene expression difference between tested drugs, sexes and their interaction.

overall_design: We analyzed 48 brain samples in 6 independent biological replicates with 8 samples each. Eight samples of each replicate included 2 sexes (female and male) in 4 different conditions: (1) control, (2) Chlorpromazine, (3) Trifluoperazine2HCl, (4) Sodium Salicylate. Replicates were performed on different days. Each sample was a pool of 5 individual brains. Brains were dissected from DGRP-900  virgin 3 days post-eclosion flies after 24 hours of drug exposure. We isolated total RNA from the samples, purified polyA+ mRNA, and generated multiplexed stranded polyA+ libraries. We added external RNA controls during library preparation to quality check and performed 75bp single end sequencing on Illumina Hiseq 2500.

first: Kseniya

last: Golovnina


title: Transcriptome analysis of planktonic, initial pellicle, and mature pellicle of Pseudoalteromonas sp. SCSIO 11900

city: Guangzhou

first: Pengxia

state: Guangdong

address: 164 Xingang West Rd

department: South China Sea Institute of Oceanology

institute: Chinese Academy of Sciences

summary: One of the most distinct features of Pseudoalteromonas sp. SCSIO 11900 is its ability to form a very robust pellicle than most Pseudoalteromonas strains. Thus we want to identify the genes essential for the pellicle formation of SCSIO 11900. We compared transcriptom profiles of planktonic cells, initial pellicle and mature pellicle of coral Pseudoalteromonas sp. SCSIO 11900 and revealed that some unique genes from horizontal gene transfer is involved in the pellicle formation of SCSIO 11900.

overall_design: mRNA profiles of  planktonic cells, initial pellicle cells and mature pellicle cells Pseudoalteromonas sp. SCSIO 11900 were generated by Illumina Hiseq2000.

first: Zhenshun

last: Zeng

first: Xingsheng

last: Cai

first: Xiaoxue


title: Interaction dynamics determine signaling and output pathway responses

city: Barcelona

first: Christina

last: Kiel

address: Dr Aiguader 88

department: Systems Biology

country: Spain

institute: CRG

summary: Study of the response to the osmotic shock and its effect on gene expression regulation in different strains of Saccharomyces cerevisiae (WT and Sln1/Ypd1 mutants). The cells received a sodium chloride stimulation (NaCl 0.4M), and were sampled at the time of stimulation (0 min) and in 10 and 20 minutes after the stimulation.

overall_design: 24 Samples

first: Klement

last: Stojanovski

first: Tony

last: Ferrar

first: Hannah

last: Benisty

first: Friedemann

last: Uschner

first: Javier

last: Delgado

last: Jimenez

first: Carme

last: Solé

first: Eulalia

last: de Nadal

first: Edda

last: Klipp

first: Francesc

last: Posas

first: Luis

last: Serrano


title: RNA-seq analysis of MCT8-deficient and healthy control brain microvascular endothelial cells (BMECs) and neural cells

first: Gad

middle: David

last: Vatine

address: 8700 Beverly blvd

department: RMI

institute: Cedars-Sinai Medical Center

summary: MCT8-deficient, healthy control and mutated/corrected isogenic iPSCs were differentiated into brain microvascular endothelial cells and neural cells. Neural cells were cultured with or without T3.

overall_design: MCT8-deficient and healthy control iPSCs were differentiated into brain microvascular endothelial cells for 8 days and purified based on metrix preferences then total RNA was extracted. In addition, MCT8-deficient and healthy control mixed neural cells were differentiated in the presence or abscence of T3 for 32 days, then total RNA was extracted.

middle: D

first: Clive

middle: N

last: Svendsen


title: Modeling the mutational and phenotypic landscapes of Pelizaeus-Merzbacher Disease using human iPSC-derived oligodendrocytes

city: Clev

first: Paul

middle: J.

last: Tesar

state: OH

address: 10900 Euclid Ave

department: Genetics and Genome Sciences

institute: Case Western Reserve University School of Medicine

summary: Pelizaeus-Merzbacher disease (PMD) is a pediatric disease of myelin in the central nervous system and manifests with a wide spectrum of clinical severities. Although PMD is a rare monogenic disease, hundreds of mutations in the X-linked myelin gene proteolipid protein 1 (PLP1) have been identified in humans. Attempts to identify a common pathogenic process underlying PMD have been complicated by an incomplete understanding of PLP1 dysfunction and limited access to primary human oligodendrocytes. To address this, we generated panels of human induced pluripotent stem cells (hiPSCs) and hiPSC-derived oligodendrocytes from 12 individuals with mutations spanning the genetic and clinical diversity of PMD—including point mutations and duplication, triplication, and deletion of PLP1—and developed an in vitro platform for molecular and cellular characterization of all 12 mutations simultaneously. We identified individual and shared defects in PLP1 mRNA expression and splicing, oligodendrocyte progenitor development, and oligodendrocyte morphology and capacity for myelination. These observations enabled classification of PMD subgroups by cell-intrinsic phenotypes and identified a subset of mutations for targeted testing of small-molecule modulators of the endoplasmic reticulum stress response, which improved both morphologic and myelination defects. Collectively, these data provide insights into the pathogeneses of a variety of PLP1 mutations and suggest that disparate etiologies of PMD could require specific treatment approaches for subsets of individuals. More broadly, this study demonstrates the versatility of a hiPSC-based panel spanning the mutational heterogeneity within a single disease and establishes a widely applicable platform for genotype-phenotype correlation and drug screening in any human myelin disorder.

overall_design: Examination of iPSC stranded RNA expression in 12 disease samples (in duplicate) and 7 unrelated controls

first: Zachary

middle: S

last: Nevin

first: Daniel

middle: C

last: Factor


title: Activity-dependent gene expression in the mammalian olfactory epithelium

city: Lexington

first: Timothy

last: McClintock

state: Kentucky

address: 508 Medical Science Buildi

department: Physiology

institute: University of kentucky

summary: We access the activity-dependent genes in olfactory neuron cells with unilateral naris occlusion model with mouse.

overall_design: mRNA profile of olfactory epithelia between closed and open sides of mice naris was compared

first: Qiang

last: Wang


title: Transcriptomic Analysis of Endothelial Cells from Fibrovascular Membranes in Proliferative Diabetic Retinopathy

city: Boston

first: Joseph

last: Arboleda-Velasquez

state: Massachusetts

address: 20 Staniford St.

institute: Schepens Eye Research Institute

summary:
Purpose: Identification of RUNX1 via next-generation sequencing (NGS) of fibrovascular membranes in patients with proliferative diabetic retinopathy.
Methods: Transcriptomic analysis with Illumina HiSeq2000 of fibrovascular membrane and control retina CD31+ samples. The sequence reads were analyzed with ANOVA (ANOVA) and targets with significance (fold change > +/-1.5 and p-value < 0.05) were selected for with Cufflinks, DeSeq2, Partek E/M, and EdgeR. qRT–PCR validation was performed using SYBR Green assays along with Western blots, siRNA, and MUSE proliferation assays.
Results: Using an optimized data analysis workflow, we mapped sequence reads per sample to the human genome (hg19) and identified genes that were statistically significant in all four statistical packages. P-values ranged from 8.78E-10 to 0.05. Using this gene list for ontology, highly significant annotation clusters included inflammatory, vascular development, and cell adhesion pathways.
Conclusions: Our study represents the first detailed transcriptomic analysis of CD31+ cells from fibrovascular membrane and CD31+ cells from control retinas with biologic replicates, generated by RNA-seq technology. The preferential selection of inflammatory and angiogenic pathways using this gene list is highly consistent with DR pathogenesis, which involves leaky and aberrant vessel growth.

overall_design: CD31+ retinal mRNA profiles were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

last: Oh

first: Jonatham

last: Lam

first: Patricia

last: D'Amore

first: Leo

last: Kim


title: Comprehensive transcriptomes of mouse pancreatic beta cells that transdifferentiated from alpha cells

city: Davis

first: Mark

middle: O.

last: Huising

state: CA

address: One Shields Avenue

department: Neurobiology, Physiology and Behavior

institute: University of California, Davis

summary: Rodent models are widely used to study diabetes. Yet, significant gaps remain in our understanding of mouse islet physiology, including the maintenance of mature endocrine cell fate. We generated comprehensive transcriptomes beta cells that transdifferentiated from alpha cells using a triple transgenic mouse models generated for this purpose.

overall_design:
FACS purified beta cells with an alpha cell lineage label that reflect beta cells that transdifferentiated from alpha cells, along with alpha cells and beta cells [GSE80673] from the same islets. Islets were isolated from triple transgenic offspring of a cross between Ins1-H2b-mCherry (Jax # 028589) and Gcg-cre (alpha) cells and a floxed YFP allele to lineage-label alpha cells. Islets from replicate groups of 10 to 12 triple transgenic animals for each group were pooled by sex to obtain sufficient material. Pooled islets were dissociated, sorted and collect in Trizol for RNA isolation and library construction.
Islets from replicate groups of 10 to 12 bitransgenic females for each group were pooled to obtain sufficient material. Pooled islets were dissociated, sorted and collect in Trizol for RNA isolation and library construction.

first: Talitha

last: van der Meulen

first: Alex

last: Mawla

middle: O


title: Co-clinical analysis of a genetically-engineered mouse model and human prostate cancer reveals significance of NKX3.1 expression for response to 5α-reductase inhibition (Mouse Finasteride Prostate RNASeq)

city: Newark

first: Antonina

last: Mitrofanova

state: NJ

address: 65 Bergen Street, Rm 923B

institute: Rutgers University

summary:
Analysis of transcriptome from 12 month old finasteride (and vehicle) treated mouse prostatic tissue
Prostate tissue from 12 month old Nkx3.1+/+ and Nkx3.1-/- mice treated with finasteride (or vehicle) was harvested, and snap frozen for subsequent molecular analysis

overall_design: Total RNA obtained from prostate tissues. Prostate tissues were harvested and processed for RNA isolation and transcriptome analysis using the MagMAX RNA isolation kit (Ambion).

first: Aditya

last: Dutta

first: Cory

last: Abate-Shen


title: The neuron-specific transcription factor Myt1l represses many non-neuronal fates

city: Stanford

first: Marius

last: Wernig

address: 265 Campus Drive, Rm. G3145

department: Institute for Stem Cell Biology & Regenerative Medicine

institute: Stanford University

summary:
Normal differentiation and induced reprogramming require
the activation of target cell programs and silencing of donor cell
programs. In reprogramming, the same factors are often used to
reprogram many different donor cell types. As most developmental
repressors, such as RE1-silencing transcription factor (REST) and
Groucho (also known as TLE), are considered lineage-specific
repressors, it remains unclear how identical combinations of
transcription factors can silence so many different donor programs.
Distinct lineage repressors would have to be induced in different
donor cell types. Here we found that the pan neuron-specific
transcription factor Myt1-like (Myt1l) exerts its pro-neuronal
function by direct repression of many different somatic lineage
programs but not the neuronal program during reprogramming
and neurogenesis and in primary mouse neurons. The repressive
function of Myt1l is mediated by recruitment of a complex
containing Sin3b by binding to a previously uncharacterized
N-terminal domain. In agreement with its repressive function, the
genomic binding sites of Myt1l are similar in neurons and fibroblasts
and are preferentially in an open chromatin configuration. The
Notch signalling pathway is repressed by Myt1l through silencing
of several members, including Hes1. Acute knock-down of Myt1l
in the developing mouse brain mimicked a Notch gain-of-function
phenotype, suggesting that Myt1l allows newborn neurons to escape
Notch activation during normal development. Depletion of Myt1l in
primary postmitotic neurons de-repressed non-neuronal programs
and impaired neuronal gene expression and function, indicating
that many somatic lineage programs are actively and persistently
repressed by Myt1l to maintain neuronal identity. It is now tempting
to speculate that similar ‘many-but-one’ lineage repressors exist for
other cell fates; such repressors, in combination with lineage-specific
activators, would be prime candidates for use in reprogramming
additional cell types.

overall_design: We utilized ChIP-seq of Myt1L, Sin3b, or HDAC1 in MEFs and the developing mouse brain to observe Myt1L chromatin localization and binding partner co-localization (31 samples). Additionally RNA-seq was performed to observe the consequence of Myt1L overexpression in MEFs or Myt1L knock-down in hippocampal neurons (10 samples).

first: Moritz

last: Mall

first: Michael

last: Kareta


title: Mouse Pancreatic Islet Maturation Time Series

summary: Beta cells are the sole source of insulin in our body, yet we do not understand how they mature into fully functional, glucose-responsive beta cells. We generated transcriptomes of FACS-purified beta cells using the Ins1-H2b-mCherry reporter line (Jax # 028589) at different peri- and postnatal maturation stages. This enables a systematic comparison across thousands of genes as beta cells mature.

overall_design:
Breeders homozygous for Ins1-H2b-mCherry (Jax #028589) were crossed to wild type C57bl6 mates to ensure offspring were uniformly hemizygous for the mCherry reporter. Islets from single litters (a mix of male and female is expected) were pooled for each sample to obtain sufficient material. Pooled islets were dissociated, sorted and collect in Trizol for RNA isolation and library construction.
Each time point was collected in the morning and FACS-sorted at the conclusion of the islet prep the same day. Each time point was done in duplicate or triplicate.


title: Genome-wide identification and characterization of small RNAs in Rhodobacter capsulatus and identification of small RNAs affected by loss of the response regulator CtrA

city: St John's

first: Marc

middle: Patrick

last: Grüll

state: Newfoundland

address: 230 Elizabeth Ave

department: Biology

country: Canada

institute: Memorial University of Newfoundand

summary: We present the first genome-wide identification and characterizaion of 422 novel sRNAs in R. capsulatus. In addition we report a comparative analysis of conserved sRNAs across 24 bacterial species.

overall_design: Genome-wide identification of sRNAs in R. capsulatus

middle: P

first: Lourdes

last: Peña-Castillo

first: Martin

last: Mulligan

first: Andrew

last: Lang


title: Developmental constraints shape the evolution of the nematode mid-developmental transition

city: Haifa

first: Harel

last: Zalts

state: Israel

address: Technion city

country: Israel

institute: Technion

summary: Evolutionary theory assumes that genetic variation is uniform and gradual in nature, yet morphological and gene expression studies have revealed that different life-stages exhibit distinct levels of cross-species conservation. In particular, a stage in mid-embryogenesis is highly conserved across species of the same phylum, suggesting that this stage is subject to developmental constraints, either by increased purifying selection or by a strong mutational bias. An alternative explanation, however, holds that the same ‘hourglass’ pattern of variation may result from increased positive selection at the earlier and later stages of development. To distinguish between these scenarios, we examined gene expression variation in a population of the nematode C. elegans using an experimental design that eliminated the influence of positive selection. By measuring gene expression for all genes throughout development in twenty strains, we found that variations were dramatically uneven throughout development, with a significant depletion during mid-embryogenesis. In particular, the family of homeodomain transcription factors, whose expression generally coincides with mid-embryogenesis, evolved under high constraint. Our data further shows that genes responsible for the integration of germ layers during morphogenesis are the most constrained class of genes. Together, these results provide the first evidence for developmental constraints as the mechanism underlying the hourglass model of animal evolution. Understanding the pattern and mechanism of developmental constraints provides a framework to understand how evolutionary processes have interacted with embryogenesis and led to the diversity of animal life on earth.

overall_design: Embryonic gene expression time course for 20 Mutation-Accumulation (MA) S. elegans strains in seven stages.

first: Itai

last: Yanai


title: Assessing the clear cell Renal Cell Carcinoma (ccRCC) DNA-methylome by MethylCapSeq

city: Ghent

first: Tim

last: De Meyer

address: Coupure Links 653

department: Dept. of Mathematical Modelling, Statistics and Bioinformatics

country: Belgium

institute: Ghent University

summary: MethylCap-Seq experiments of 4 ccRCC cell lines (SKRC1, SKRC10, SKRC52, SKRC59) and one "normal" kidney cell line (HK2) were performed, and used, in combination with DAC/TSA re-expression data, to mine the ccRCC specific cancer methylome. Identified candidate DNA-methylation markers were evaluated by MSP in two independent series, yielding 4 validated markers (GREM1, NEURL, LAD1, NEFH) of which promoter methylation was highly predictive of survival.

overall_design: After enrichment of methylated DNA by MBD-based affinity purification, captured fragments were paired-end sequenced on the GAIIx.

first: Iris

last: Vlodrop

first: Kim

middle: M

last: Smits

first: Leander

last: Van Neste

first: Marcella

last: Baldewijns

first: Peter

middle: T

last: Moerkerk

last: Schouten

first: Piet

last: van den Brandt

first: Joo

last: Mi Yi

first: Jana

last: Jeschke

first: Marilia

last: de Freitas Calmon

first: Kornel

last: Schuebel

first: Nita

last: Ahuja

first: Stephen

middle: B

last: Baylin

first: James

last: Herman

first: Patricia

last: Soetekouw

first: Fred

last: Bosman

first: Wim

last: Van Criekinge

first: Manon

last: Van Engeland


title: Identification of Factors Required for m6A mRNA Methylation in Arabidopsis Reveals a Role for the Conserved E3 Ubiquitin Ligase HAKAI

status: Public on Apr 03 2017

city: Nottingham

first: Nigel

last: Mongan

address: University Park

department: Biodiscovery Institute

country: United Kingdom

institute: University of Nottingham

summary: Illumina RNA-Seq of Arabidopsis virilizer-1 root tips

overall_design: N6-adenosine methylation (m6A) of mRNA is an essential process in most Eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identify a core set of mRNA m6A writer proteins in Arabidopsis thaliana. The components required for m6A in Arabidopsis include MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins leads to reduced m6A levels and shared pleiotropic phenotypes, which include aberrant vascular formation in the root, indicating that correct m6A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst Eukaryotes and the demonstration of a role in writing m6A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

first: Kamil

last: Ruzicka

first: Rupert

last: Fray


title: Whole transcriptome expression of trigeminal ganglia compared to dorsal root ganglia in Rattus norvegicus

city: Glostrup

first: Lisette

last: Kogelman

address: Nordre Ringvej 67

department: Danish Headache Centre

country: Denmark

institute: Rigshospital Glostrup

summary: RNA-Sequencing of the trigeminal ganglia and dorsal root ganglia in male naive rats (Wistar Han) of 10 weeks old.

overall_design: 6 naive rats were killed after 2 weeks of arrival, both trigeminal ganglia and dorsal root ganglia were dissected of each rat.

first: Rikke

last: Elgaard-Christensen

first: Sara

middle: H

last: Pedersen

first: Thomas

middle: F

last: Hansen

first: Inger

last: Jansen-Olesen

first: Jes

last: Olesen


title: Transcriptomic profiling of HeLa cells infected with Shigella flexneri

city: Würzburg

first: Ana

last: Eulalio

address: Josef Schneider Str. 2, D-15

institute: IMIB, University of Würzburg

summary: We evaluated the transcriptome changes induced by infection of Hela 229 cells with Shigella flexneri. The sample set consists of a control (mock), total population of infected sample and infected sample sorted into Shigella positive and Shigella negative population.

overall_design: Transcriptmic profiles of HeLa cells infected with Shigella were generated by high throughput sequencing using Illumina HiSeq2000.

first: Malvika

last: Sharan

first: Claire

last: Maudet


title: Conservation and diversity of the IrrE/DdrO-controlled radiation response in radiation-resistant Deinococcus bacteria

city: Saint Paul lez Durance

first: Arjan

last: de Groot

address: CEA Cadarache

department: BIAM-SBVME

country: France

institute: CEA

summary: The extreme radiation resistance of Deinococcus bacteria requires the radiation-stimulated cleavage of protein DdrO by a specific metalloprotease called IrrE. DdrO is the repressor of a predicted radiation/desiccation response (RDR) regulon, composed of radiation-induced genes having a conserved DNA motif (RDRM) in their promoter regions. Here, we showed that addition of zinc ions to purified apo-IrrE, and short exposure of Deinococcus cells to zinc ions, resulted in cleavage of DdrO in vitro and in vivo, respectively. Binding of IrrE to RDRM-containing DNA or interaction of IrrE with DNA-bound DdrO was not observed. The data are in line with IrrE being a zinc peptidase, and indicate that increased zinc availability, caused by oxidative stress, triggers the in vivo cleavage of DdrO unbound to DNA. Transcriptomics and proteomics of Deinococcus deserti confirmed the IrrE-dependent regulation of predicted RDR regulon genes and also revealed additional members of this regulon. Comparative analysis showed that the RDR regulon is largely well conserved in Deinococcus species, but also showed diversity in the regulon composition. Notably, several RDR genes with an important role in radiation resistance in Deinococcus radiodurans, for example pprA, are not conserved in some other radiation-resistant Deinococcus species.

overall_design: Using Illumina HiSeq 2000, RNA-Seq was performed to explore the transcriptome in non-irradiated and irradiated Deinococcus deserti strains lacking gene irrE or ddrOc.

first: Laurence

last: Blanchard


title: Transcriptomic profiling of HeLa cells treated with miR-29b-2-5p and control-miR

summary: To have a global picture of the targets of the mir-29b-2-5p, we assessed transcriptome changes, by deep-sequencing, of HeLa cells transfected with this miRNA or a control miRNA (cel-miR-231).

overall_design: Transcriptmic profiles of HeLa cells treated miR-29b-2-5p and control-miR  were generated by deep sequencing, using Illumina HiSeq2000.


title: Bidirectional terminators in Saccharomyces cerevisiae prevent cryptic transcription from invading neighbouring genes

city: Montreal

first: Francois

last: Robert

state: QC

address: 110 av des Pins Ouest

institute: IRCM

summary: Transcription can be quite disruptive for chromatin so cells have evolved mechanisms to preserve chromatin integrity during transcription, hence preventing the emergence of cryptic transcript from spurious promoter sequences. How these transcripts are regulated and processed by cells remains poorly characterized. Notably, very little is known about the termination of cryptic transcription. Here we used RNA-Seq to identify and characterize cryptic transcripts in Spt6 mutant cells (spt6-1004) in Saccharomyces cerevisiae. We found polyadenylated cryptic transcripts running both sense and anti-sense relative to genes in this mutant. Cryptic promoters were enriched for TATA boxes, suggesting that the underlying DNA sequence defines the location of cryptic promoters. While intragenic sense cryptic transcripts terminate at the terminator of the genes that host them, we found that anti-sense cryptic transcripts preferentially terminate at the 3’-end of upstream genes. These findings led us to demonstrate that most terminators in yeast are bidirectional, leading to termination and polyadenylation of transcripts coming from either direction. We propose that S. cerevisiae has evolved this mechanism in order to prevent spurious transcription from invading neighbouring genes, a feature particularly critical for organisms with small compact genomes.

overall_design: Cells from spt16-1004 and its respective WT strain were grown to an OD600 of 0.5 at 30°C and shifted to 37°C for 80 min before RNA extraction. Total RNA was extracted using the hot phenol method. Prior to library preparation, total RNA was either depleted for ribosomal RNA using the Ribo-zero Gold yeast kit (Epicentre-Illumina) or enriched for polyadenylated RNA using  the NEBnext Poly(A) kit (New England Biolabs). Strand specific RNA-seq libraries were prepared using the KAPA stranded RNA-Seq library preparation kit prior to paired-end sequencing on an Illumina Hi-Seq2000. Reads were mapped to the sacCer3 assembly of the S. cerevisiae genome using Tophat2 (23). Intron length range was set at 50 to 1000 bp and a reference annotation file was provided to guide the assembly.

first: Nicole

last: Uwimana

first: Pierre

last: Collin

first: Célia

last: Jeronimo

first: Benjamin

last: Haibe-Kains

first: François


title: Genome-wide map of H3K4me3 and H3K27Ac in the protozoan parasite Trichomonas vaginalis

city: Seoul

first: Hyoung-Pyo

last: Kim

address: Yonsei-ro 50-1

country: South Korea

institute: Yonsei University

summary: In this study, we investigated genome-wide transcriptional and epigenetic responses of T. vaginalis to histone deacetylase (HDAC) inhibitors.

overall_design: Examination of mRNA expression and 2 different histone modifications in T. vaginalis in the absence or presence of HDAC inhibitors, apicidin or Trichostatin A


title: MicroRNA profiles and potential regulatory pattern during the early stage of spermatogenesis in mice

city: Beijing

first: lili

last: hao

address: Beichen west road

institute: BIG

summary: Spermatogenesis is a complicated and poorly understood process that relies on the precise regulation of the self-renewal and differentiation of spermatogonia. In many organisms, microRNAs (miRNAs) are involved in multiple developmental processes as critical regulators of transcriptional and post-transcriptional gene silencing. This study investigated the expression pattern of miRNAs in type B spermatogonia cells (BSc) and primary spermatocytes (PSc) of mice, using a high-throughput small RNA sequencing system.

overall_design: Using sRNA-seq technology to examine miRNA expression pattern's change between mouse type B spermatogonia and primary spermatocytes cell types.

first: Mengmeng

last: Luo

first: Lili

last: Hao

first: Fen

last: Hu

first: Lixia

last: Gou

first: Wendian

last: Zhang

first: Xin

last: Wang

first: Yuhui

last: Zhao

first: Mengchun

last: Jia

first: Songnian

first: Xiujun


title: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 is essential for p53-null cancer cells

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

summary: The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) controls metabolic flux through allosteric regulation of glycolysis. Here we show that p53 regulates the expression of PFKFB4 and that p53-deficient cancer cells are highly dependent on the function of this enzyme. We found that p53 down-regulates PFKFB4 expression by binding to its promoter and mediating transcriptional repression via histone deacetylases. Depletion of PFKFB4 from p53 deficient cancer cells increased levels of the allosteric regulator fructose 2,6-bisphophate, leading to increased glycolytic activity but decreased routing of metabolites through the oxidative arm of the pentose phosphate pathway. PFKFB4 was also required to support the synthesis and regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) in p53 deficient cancer cells. Moreover, depletion of PFKFB4 attenuated cellular biosynthetic activity and resulted in the accumulation of reactive oxygen species and cell death in the absence of p53. Finally, silencing of PFKFB4 induced apoptosis in p53 deficient cancer cells in vivo and interfered with tumour growth. These results demonstrate that PFKFB4 is essential to support anabolic metabolism in p53-deficient cancer cells and suggest that inhibition of PFKFB4 could be an effective strategy for cancer treatment.

overall_design: Gene expression changes in HCT116 p53+/+ and p53-/- xenograft tumours after PFKFB4 silencing

first: Susana

last: Ros

first: Jessica

last: Flöter

first: Irem

last: Kaymak

first: Clive

last: DaCosta

first: Amina

last: Houddane

first: Sébastien

last: Dubuis

first: Beatrice

last: Griffiths

first: Richard

last: Mitter

first: Susanne

last: Walz

first: Sophia

last: Blake

first: Axel

last: Behrens

first: Kevin

last: Brindle

first: Nicola

last: Zamboni

first: Mark

last: Rider

first: Almut

last: Schulze


title: Structural transitions during yeast large ribosomal subunit maturation analyzed by tethered nuclease structure probing

status: Public on Apr 01 2017

city: Regensburg

first: Philipp

last: Milkereit

address: Universitätsstr. 31

department: Lehrstuhl für Biochemie III

institute: Universität Regensburg

summary: Yeast large ribosomal subunit (LSU) precursors are subject to substantial changes in protein composition during their maturation due to coordinated transient interactions with a large number of ribosome biogenesis factors and due to the assembly of ribosomal proteins. These compositional changes go along with stepwise processing of LSU rRNA precursors and with specific rRNA folding events, as revealed by recent cryo-electron microscopy analyses of late nuclear and cytoplasmic LSU precursors. Here we aimed to analyze changes in the spatial rRNA surrounding of selected ribosomal proteins during yeast LSU maturation. For this we combined a recently developed tethered tertiary structure probing approach with both targeted and high-throughput readout strategies. Several structural features of late LSU precursors were faithfully detected by this procedure. In addition, the obtained data let us suggest that early rRNA precursor processing events are accompanied by a global transition from a flexible to a spatially restricted rRNA conformation. For intermediate LSU precursors, a number of structural hallmarks could be addressed which include the fold of the internal transcribed spacer between 5.8S rRNA and 25S rRNA, the orientation of the central protuberance and the spatial organization of the interface between LSU rRNA domains I and III.

overall_design: Major rRNA cleavages by nuclease fusion proteins incorporated in LSU precursors were identified by random primer extension reactions followed by high-throughput sequencing of the resulting cDNA fragments.

first: Gisela

last: Pöll

first: Christian

last: Müller

first: Malena

last: Bodden

first: Fabian

last: Teubl

first: Norbert

last: Eichner

first: Gerhard

last: Lehmann

first: Joachim

last: Griesenbeck

first: Herbert

last: Tschochner


title: STAT5 antagonism of B cell enhancer networks drives leukemia and poor patient surviva

city: Minneapolis

first: Rendong

last: Yang

state: MN

address: 117 Pleasant St. SE

institute: University of Minnesota

summary: The transcription factor STAT5 plays a critical role in B cell Acute Lymphoblastic Leukemia (B-ALL).  How STAT5 mediates this effect is unclear.  Here we demonstrate that STAT5 activation cooperates with defects in the pre-BCR signaling components Blnk, Btk, Prkcb, Nfkb1, and Ikzf1 to initiate B-ALL.  STAT5 antagonizes NFkB and IKAROS by opposing regulation of shared target genes.  STAT5 binding was enriched at super-enhancers, which were associated with an opposing network of transcription factors, including PAX5, EBF1, PU.1, IRF4, and IKAROS.  Patients with high ratios of active STAT5 to NFκB or IKAROS have more aggressive disease.  Our studies illustrate that an imbalance of two opposing transcriptional programs drive B-ALL, and suggest that restoring the balance of these pathways may inhibit B-ALL.

overall_design: STAT5 ChIP-seq in primary mouse Stat5b-CAxBlnk+/- leukemic cells

first: Casey

last: Katerndahl

first: Michael

middle: A

last: Farrar


title: Identification of potential biomarkers in donor cows for in vitro embryo production by granulosa cells transcriptomics

city: Kongens Lyngby

first: Haja

last: Kadarmideen

address: Richard Petersens Plads, Building 324

department: Department of Applied Mathematics and Computer Science

institute: Technical University of Denmark

summary: In this study we sequenced RNA of granulosa collected from Holstein cows at oocyte aspiration prior to IVP, to identify candidate genes and biological mechanisms for favourable IVP-related traits in donor cows.

overall_design: 24 RNA-Seq profiles of granulosa cells collected from antral follicles of 24 cows. Each animal was scored for In Vitro Production performances (blastocysts rate, morphology and kinetic scores). Half of the oocytes were inseminated with semen from high genetic merit bulls and half with semen from low genetic merit bulls.

first: Gianluca

last: Mazzoni

first: Suraya

last: Salleh

first: Kristine

last: Freude

first: Hanne

last: Skovsgaard Pedersen

first: Lotte

last: Stroebech

first: Henrik

last: Callesen

first: Poul

last: Hyttel


title: Analysis of gene expression patterns of arabidopsis whitening mutant wn1 and wild-type

status: Public on Apr 02 2017

first: Sun Hyun

last: Chang

address: Rm7112 Bd200 Gwanakro1 Gwanak-gu

department: Applied Biology and Chemistry

institute: Seoul National University

summary: This data analyzes gene expression patterns in Arabidopsis whitening mutants, wn1, with defects in chloroplast development. We found that wn1 was a T-DNA insertion line of Arabidopsis AtPTAC10/PAP3 (At3g48500) and exhibited a lethal, albino-like phenotype in the seedling stages.  pTAC10 was known as a component of RNA polymerase complex called plastid-encoded RNA polymerase, PEP, in mature chloroplasts. Because wn1 mutants were lethal, we extracted RNA from cotyledons of 7-day-old seedlings from wn1 mutants and Col-0. RNA seq analysis was commissioned by NICEM in Seoul National University, Korea and transcriptome resequencing was confirmed in Macrogen, Korea. Through analysis, we compared gene expression patterns of wn1 mutant and Col-0 seedlings.

overall_design: Total mRNA was extracted shoot parts of Col-0 and whitening mutant seedlings grown in 1/2 MS solid media for 7 days. We extract RNA by using Quiagen Plant RNA extraction kit. Transcriptome analysis was commissioned by NICEM Genome Analysis Center in Seoul National University, Korea. We commissioned Macrogen to reanalyze the raw data. Raw data we attached were reanalyzed transcriptome.

first: Geupil

last: Jang

first: Sun

first: Tae

middle: Y

last: Um

first: Sangyool

last: Lee

first: Yang

last: Choi


title: Activation of Lung Macrophages in Hypoxic Pulmonary Hypertension

city: denver

first: steve

last: pugliese

state: CO

address: 12700 East 19th Avenue

department: Medicine

institute: University of Colorado

summary: Studies in various animal models suggest an important role for pulmonary macrophages in the pathogenesis of pulmonary hypertension (PH). Yet, the molecular mechanisms characterizing the functional macrophage phenotype relative to time and pulmonary localization/compartmentalization remain largely unknown.  Here, we utilized a hypoxic murine model of PH in combination with flow cytometry assisted cell sorting (FACS) to quantify and isolate lung macrophages from two compartments over time and characterized their programing via RNA sequencing (RNAseq) approaches.  In response to hypoxia, we found an early increase in macrophage number that was restricted to the interstitial/perivascular compartment, without recruitment of macrophages to the alveolar compartment or changes in the number of resident alveolar macrophages.  Principle component analysis demonstrated significant differences in overall gene expression between alveolar (AMs) and interstitial macrophages (IMs) at baseline and after 4 and 14 days hypoxic exposure.  AM’s at both day 4 and 14 and IM’s at day 4 shared a conserved “hypoxia program” characterized by mitochondrial dysfunction, pro-inflammatory gene activation and mTORC1 signaling, while IM’s at day 14 demonstrated a unique anti-inflammatory/ pro-reparative programming state.  We conclude that the pathogenesis of vascular remodeling in hypoxic PH involves an early compartment independent activation of lung macrophages towards a conserved hypoxia program, with the development of compartment specific programs later on in the course of disease.  Thus, harnessing time and compartment specific differences in lung macrophage polarization needs to be considered in the therapeutic targeting of macrophages in hypoxic PH and potentially other inflammatory lung diseases.

overall_design: FACS sorting of lung macrophages from hypoxic mouse lung

first: Steven

last: Pugliese

first: Karim

last: El Kasmi


title: Acetici acid treated A.thaliana (Col-0)

city: Yokohama

first: Takaho

middle: A.

last: Endo

state: Kanagawa

address: 1-7-22 Suehiro, Tsurumi

department: IMS

country: Japan

institute: RIKEN

summary: Changes in histone H4 acetylation of Arabidopsis thaliana (Col-0) by treatment with acetic acid.

overall_design: 2weeks-old wild-type plants of A.thaliana (Col-0) were treated with/without 10mM aetic acid for 9days.

first: Jong-Myong

last: Kim

first: Taiko

first: Junko

last: Ishida

first: Motoaki

last: Seki


title: Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms

city: Heidelberg

first: Benjamin

middle: Andrew

last: Story

address: Meyerhofstrasse 1

department: Genomics

institute: European Molecular Biology Laboratory (EMBL)

summary:
Purpose: Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through piRNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear if it plays any roles in early GSC lineage development.
Results: The study reveals a novel function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors.

overall_design: iCLIP experiments were conducted to obtain RNA binding profiles of Aub in cultured Drosophila bam mutant germline stem cells

first: Xing

last: Ma

first: Xiujuan

last: Zhu

first: Yingying

last: Han

first: Trieu

last: Do

first: Xiaoqing

last: Song

first: Su

last: Wang

first: Ying

last: Zhang

first: Marco

last: Blanchette

first: Madelaine

last: Gogol

first: Kate

last: Malanowski

first: Allison

last: Peak

first: Perera

last: Anoja

first: Ting

last: Xie


title: Virgin Beta Cells Persist throughout Life at a Neogenic Niche within Pancreatic Islets

city: Davis

first: Mark

last: Huising

address: One Shields Avenue

institute: University of California, Davis

summary: Beta cells are the sole source of insulin in our body, yet we do not understand how they mature into fully functional, glucose-responsive beta cells. We discovered a persistent population of immature beta cells that is marked by the absence of Ucn3 and is functionally immature. We generated transcriptomes of these immature beta cells, along with mature beta cells and the mixed pool of non-beta cells from the same islets. This enables a systematic comparison across thousands of genes between two distinct maturation stages of the beta cell.

overall_design:
FACS purified immature or mature beta cells, or double negatives from the same islets. Islets were isolated from bitransgenic offspring of a cross between Ins1-H2b-mCherry (Jax # 028589) and Ucn3-eGFP. Immature beta cells are mCherry single positive, mature beta cells are eGFP and mCherry co-positive. Non beta cells are negative for mCherry and eGFP.
Islets from replicate groups of 10 to 12 bitransgenic females for each group were pooled to obtain sufficient material. Pooled islets were dissociated, sorted and collect in Trizol for RNA isolation and library construction.

first: Talitha

last: van der Meulen

first: Alex

last: Mawla


title: RNA_seq and Ribo_seq analyses of control and CPT-treated MCF7 cells

city: Tel Aviv

first: Ran

last: Elkon

address: Ramat Aviv

department: Human Genetics

institute: Tel Aviv University

summary: We used RNA-seq and Ribo-seq analyses to examine the effect of CPT treatment of translation efficiency (TE)

overall_design: We measured expression levels (RNA.seq) and ribosome densities (ribo-seq) using biological duplicates of control and CPT-treated (5 hrs) MCF7 cells

first: R

last: Agami


title: The Project for High-Confidence Coding and Noncoding Transcriptome Maps

first: Jin-Wu

last: Nam

address: Seongdong-Gu Hangdang-dong

department: Department of Life Science

institute: Hanyang University

summary:
The advent of high-throughput RNA sequencing (RNA-seq) has led to the discovery of unprecedentedly immense transcriptomes encoded by eukaryotic genomes. However, the transcriptome maps are still incomplete partly because they were mostly reconstructed based on RNA-seq reads that lack their orientations (known as unstranded reads) and certain boundary information. Methods to expand the usability of unstranded RNA-seq data by predetermining the orientation of the reads and precisely determining the boundaries of assembled transcripts could significantly benefit the quality of the resulting transcriptome maps. Here, we present a high-performing transcriptome assembly pipeline, called CAFE, that significantly improves the original assemblies, respectively assembled with stranded and/or unstranded RNA-seq data, by orienting unstranded reads using the maximum likelihood estimation and by integrating information about transcription start sites and cleavage and polyadenylation sites. Applying large-scale transcriptomic data comprising 230 billion RNA-seq reads from the ENCODE, Human BodyMap Projects, The Cancer Genome Atlas, and GTEx, CAFE enabled us to predict the directions of about 220 billion unstranded reads, which led to the construction of more accurate transcriptome maps, comparable to the manually curated map, and a comprehensive lncRNA catalogue that includes thousands of novel lncRNAs. Our pipeline should not only help to build comprehensive, precise transcriptome maps from complex genomes but also to expand the universe of non-coding genomes.

This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Homo sapiens
Mus musculus

overall_design:
The direction of unstranded reads (from ENCODE, Human BodyMap Projects, GTEx and TCGA as well as from HeLa and mES cells) were predicted using k-order Markov chain models (kMC) generating a read with a predicted direction (RPD) and were used to assemble transcriptome maps (BIGTranscriptome). Those transcriptome maps were next used for quantification of RPDs.

Refer to individual Series


title: miR-205 is a critical regulator of lacrimal gland development

city: San Francisco

first: D'juan

middle: T.

last: Farmer

address: 513 Parnassus Ave

institute: UCSF

summary: Tears are essential for the maintenance of the terrestrial animal ocular surface and the lacrimal gland is the source of the aqueous layer of the tear film. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an essential gene for lacrimal gland development. Knockout mice lacking miR-205 fail to develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal bud initiation. RNA-seq analysis uncovered several up-regulated miR-205 targets, including Inppl1, a negative regulator of Akt signaling. Data indicate that Akt signaling is required within lacrimal gland epithelia and is activated by Fgf10. Furthermore, combinatorial epistatic deletion of Fgf10 and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways downstream of Fgf10 to regulate glandular development. These data show that a single microRNA is a key regulator for lacrimal gland initiation in mice and highlights the important role of microRNAs during organogenesis.

overall_design:
Laser capture and RNA isolation. The laser capture staining protocol was adapted from the Laser Capture Molecular Core at Ohio State University. Briefly, E12.5 embryo heads were dissected and flash frozen in OCT for cyrosectioning. Sections were collected at 10 µM onto RNase-Zap treated 1.0 PEN membrane slides from Zeiss and stored at -80°C. Frozen sections were immediately submerged into ice cold RNase free 70% ethanol, stained in Vector® Hematoxylin QS for 30 sec, and then rinsed in RNase water, following by 95% ethanol and 100% ethanol. Slides were then air dried and returned to -80°C before laser capture. The surface ectoderm between the eye and brain was isolated using the Zeiss PALM into 0.5 mL tubes containing Qiagen RLT buffer. Three embryos were pooled together per sample and four wildtype and four knockout samples were collected for future analysis. RNA was isolated using a Qiagen RNeasy Plus Micro kit and assessed on an Agilent 2100 Bioanalyzer using Agilent RNA Pico Chips. All samples had a RIN above 7.0.
cDNA synthesis and library preparation. cDNA was generated using Clontech’s SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing according to manufacturer’s recommendations. Sequencing libraries were prepared with 1 ng of cDNA using the Nextera XT DNA Library Prep Kit according to manufacturer’s recommendations. The final product was then run on a Novex 4-12% TBE gel and DNA between 300-500 nt was selected. Samples were run on an Illumina HiSeq 2500 (UCSF Institute for Human Genetics Genomics Core).
RNA-seq data analysis. Reads were mapped to the mouse GRCm38 genome using Tophat v.2.0.9 (Trapnell et al., 2009). Gene expression was measured from the mapped reads by using HT-seq-count (Anders et al., 2014) in intersection-strict mode, which counts the reads aligning to each annotated gene (gene set, Ensembl.org). Differentially expressed genes were called using the DESeq2 R package, (Anders and Huber, 2010) considering genes differentially expressed with FDR < 0.1. Only genes with normalized count values above 10 in all samples were evaluated. One WT samples (WT #1) was removed due to contamination of non-surface ectoderm tissues, as evident by decreased markers of Pax6, Krt5, and others that constant across all other samples.
Please note that

first: D'Juan

first: Michael

last: McManus


title: An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells

city: Stanford

first: Pascale

last: Guiton

address: Sherman Fairchild Science Building  Stanford School of Medicine 299 campus Drive

department: Microbiology and Immunology

institute: Stanford University

summary: Toxoplasmosis is a zoonotic infection affecting approximately 30% of the world’s human population. After sexual reproduction in the definitive feline host, Toxoplasma oocysts, each containing 8 sporozoites, are shed into the environment where they can go on to infect humans and other warm-blooded intermediate hosts. Here, we use an in vitro model to assess host transcriptomic changes that occur in the earliest stages of such infections. We show that infection of rat intestinal epithelial cells with mature sporozoites primarily results in higher expression of genes associated with Tumor Necrosis Factor alpha (TNFα) signaling via NF-κB. Furthermore, we find that, consistent with their biology, these mature, invaded sporozoites display a transcriptome intermediate between the previously reported day 10 oocysts and that of their tachyzoite counterparts. Thus, this study uncovers novel host and pathogen factors that may be critical for the establishment of a successful intracellular niche following sporozoite-initiated infection.

overall_design: Rat intestinal epithelial cells were infected in duplicate with either Toxoplasma sporozoites or tachyzoites in the presence or absence of frozen sporozoites for 8 hours. Frozen sporozoites and lysates from uninfected cells were used as controls. The experiment was performed twice. Total RNA was extracted from the resulting 24 samples and the transcriptional profiles of both host and parasites were determined using RNA sequencing on the Illumina NextSeq platform

first: Janelle

last: Sagawa

first: Heather

last: Fritz

first: John

last: Boothroyd


title: Clonally stable Vκ allelic choice instructs Igκ repertoire

city: Jerusalem

first: Yehudit

last: Bergman

address: Hebrew University Medical School, Ein Kerem

department: Developmental Biology and Cancer Research

institute: The Hebrew University of Jerusalem

summary: Although much research has been done to understand how rearrangement of the Igκ locus is regulated during B-cell development, very little is known about the way the variable (V) segments themselves are selected. Using B6/Cast hybrid pre-B cell clones, we show that a limited number of V segments on each allele is stochastically activated as characterized by the appearance of ncRNA and histone modifications. The activation states are clonally distinct, stable across cell division and developmentally important in directing the Ig repertoire upon differentiation. By employing a new approach of allelic ATAC-seq we demonstrate that the Igκ V alleles have differential chromatin accessibility, which may serve as the underlying basis of clonal maintenance at this locus, as well as other instances of monoallelic expression throughout the genome. These findings highlight a new level of immune-system regulation that appears to be geared at optimizing gene diversity.

overall_design: ATAC-seq was performed in biological duplicate on mouse pre-B cell clones and pool grown in IL-7 cultures, as well as B220+IgM-CD43-CD25+ bone marrow derived pre-B cells on a C57BL6/Castaneous background. Nuclear enriched total stranded RNA-seq was performed in biological duplicate on the same clones, and B220+IgM-CD43-CD25+ bone marrow derived pre-B cells. Kappa_VJ-seq was performed on  mouse pre-B cell clones and pool where IL7 was removed from culture for 12-48 hours.

first: Rena

last: Levin-Klein


title: ChIPseq and RNAseq of ER mutant breast cancer cell lines

city: New York City

first: MacIntosh

middle: Grant

last: Cornwell

department: School of Medicine

institute: New York University

summary: We performed several extensive phenotypic, genomic, and transcriptomic analyses in an effort to discern the biological differences between normal breast cancer cell lines and breast cancer cells with a mutation in the ESR1 Ligand Binding Domain.

overall_design: ER ChIP-seq and mRNA-seq of Several MCF7 and T47D Cell lines

first: Rinath

last: Jeselsohn

first: Matthew

last: Pun


title: Regulation of KMT2D by PI(3)K/AKT signalling orchestrates oestrogen receptor mediated transcriptional activation in breast cancer

city: New York

first: Bic

last: MSKCC

address: 1275 York Ave.

institute: Memorial SLoan-Kettering Cancer Center

summary: We aim to elucidate the mechanisms of how PI3K inhibition is promoting estrogen receptor (ER) activation and ER dependency in breast cancer.

overall_design: Genome-wide analysis of KMT2D upon treatment with the PI3K inhibitor (BYL719).

first: Eneda

last: Toska

first: Hatice

last: Osmanbeyoglu


title: Quantitative Specificity of STAT1 and Several Variants.

city: St. Louis

first: Basab

last: Roy

state: MO

address: 4515 Mckinley avenue

department: Department of Genetics

institute: Washington University School of Medicine

summary:
The specificity of STAT1 transcription factor was determined quantitatively by measuring the relative binding affinity to hundreds of variants of the gonsensus site. The binding specities of eight mutants of STAT1 were also investigated. The mutants showed either overall reduced binding specificity or altered specificity accross the binding site. The effect of CpG methylaton within the binding site was also investigated, which demonstrated verry little effect on binding.
A previously described method, Spec-Seq, was used to determine the binding specificititys of STAT1 and the mutants. Libreries of DNAs were enzymatically methylated and the same method was used for studying their binding affinities in comparison with the non-methylated counterparts. The proteins and DNAs were incubated for 30 min in in suitable buffer and the bound and unbound bands were resolved in 12% polyacrylamide gels. the DNAs were purified from the bound and unbound bands and sequenced.

overall_design: 18 independent  Spec-Seq experiments were designed for each of the STAT1 and its mutants. four other Spec-Seq experiments were performed for the comperative analysis of the effect of CpG methylation across the binding site. One of these libreries were enzymatically methylated using CpG-methyltransferase (NEB).

first: Gary

middle: D

last: Stormo


title: Analysis of the in planta Transcriptome Expressed by the Corn Pathogen Pantoea stewartii subsp. stewartii via RNA-Seq

city: Blacksburg

first: Holly

last: Packard

state: VA

address: 926 West Campus Drive

department: Biological Sciences

institute: Virginia Tech

summary: Pantoea stewartii subsp. stewartii is a bacterial phytopathogen that causes Stewart’s wilt disease in corn.  It uses quorum sensing to regulate expression of some genes involved in virulence in a cell density-dependent manner as the bacterial population grows from small numbers at the initial infection site in the leaf apoplast to high cell numbers in the xylem where it forms a biofilm. There are also other genes important for pathogenesis not under quorum-sensing control such as a Type III secretion system. The purpose of this study was to compare gene expression during a high-density in planta infection versus a low-density pre-inoculum liquid culture and in a high-density culture grown on agar medium to identify genes specifically expressed in planta that may also be important for colonization and/or virulence.  RNA was purified from each sample type to determine the transcriptome via RNA-Seq using Illumina sequencing of cDNA.  Fold gene expression changes in the high-density in planta data set in comparison to the two in vitro grown samples were determined and a list of the most differentially expressed genes was generated to elucidate genes important for plant association.  Quantitative reverse transcription PCR (qRT-PCR) was used to validate expression patterns for a select subset of genes. Analysis of the transcriptome data via gene ontology revealed that bacterial transporters and systems active under low oxygen tensions appear to play a critical role for P. stewartii as it colonizes and causes wilt disease in corn plants.

sample_organism: Pantoea stewartii subsp. stewartii DC283

overall_design: Duplicate samples of wild-type P. stewartii were obtained for RNA-Seq after a 10 day in planta corn infection, pre-inoculum liquid culture in RM medium, and a high cell density plate culture

first: Alison

middle: K

last: Burke

first: Roderick

middle: V

last: Jensen

first: Ann

last: Stevens


title: Analysis in C. merolae

city: Berlin

first: Pawel

last: Mikulski

address: Koenigin-Luise-Str. 12-16

department: Biology

institute: Free University Berlin

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

overall_design: Refer to individual Series


title: Transcriptomic analysis in C. merolae

summary: RNA-seq experiment in wildtype Cyanidioschyzon merolae cultures. The aim of the experiment was the comparison of the transcriptomic activity with H3K27me3 profile. The results revealed a negative correlation between expression and H3K27me3 occupancy. The experiment contains 3 biological replicates.

overall_design: RNA isolation, reverse-transcription, cDNA library generation and Illumina sequencing.

first: Daniel

last: Schubert


title: Programming of a pathogenic Th2 state by TSLP signaling in CD4+ T cells

city: Cincinnati

first: Yrina

last: Rochman

address: 3333 Burnet Avenue

department: Allergy and Immunology

institute: Cincinnati Children's Hospital Medical Center

summary: Pathogenesis of allergic diseases including asthma is strongly associated with robust responses of allergen-specific Th2 cells, which produce high levels of IL-4, IL-5, IL-9, and IL-13. Despite evidence for pathogenic Th2 cells in the induction and propagation of allergic disorders, signals required for differentiation of such cells remain largely unknown. Thymic stromal lymphopoietin (TSLP) is a cytokine that is expressed upon epithelial injury, dysfunction or infection and is strongly implicated in the pathogenesis of atopic dermatitis (AD) and asthma. Although indirect regulation of Th2 differentiation via TSLP-stimulated dendritic cells well known, direct effects of TSLP on Th2 differentiation in vivo have not been rigorously analyzed. We show that TSLP may initiate Th2 response independent on IL-4 signal and increases sensitivity of CD4 cells to IL-4 stimulation inducing more robust expression of IL-4, IL-5, and IL-13 by human and mouse CD4 cells. This more potent effector state appears to be stably programmed in memory Th2 cells. As part of molecular mechanism, we demonstrate that TSLP and IL-4 signals induce distinctive genome wide alterations in activating histone modification (H3K27Ac, H3K36Me3, and H3K4Me3). We propose that TSLP acts in coordination with IL-4 to generate a more potent Th2 state that could underlie persistence and propagation of allergic disorders.

overall_design: Mouse CD4 cells activated with TCR signal in the presence or absence  of TSLP or IL-4 for different time points in vitro.

first: Andrey

last: Kartashov

first: Artem

last: Barski

first: Harinder

last: Singh


title: Specific small-RNA signatures in the amygdala at premotor and motor stages of Parkinson’s disease revealed by deep sequencing analysis

city: Boston

first: Lorena

last: Pantano

state: MA

address: 655 Huntington Ave

department: Biostatistics

institute: Harvard Chan School of Public Health

summary: Motivation: Most computational tools for small non-coding RNAs (sRNA) sequencing data analysis focus in microRNAs (miRNAs), overlooking other types of sRNAs that show multi-mapping hits. Here, we have developed a pipeline to non-redundantly quantify all types of sRNAs, and extract patterns of expression in biologically defined groups. We have used our tool to characterize and profile sRNAs in post-mortem brain samples of control individuals and Parkinson’s disease (PD) cases at early-premotor and late-symptomatic stages.Results: Clusters of co-expressed sRNAs mapping onto tRNAs significantly separated premotor and motor cases from controls. A similar result was obtained using a matrix of miRNAs slightly varying in sequence (isomiRs). The present framework revealed sRNA alterations at premotor stages of PD, which might reflect initial pathogenic perturbations. This tool may be useful to discover sRNA expression patterns linked to different biological conditions.

overall_design:
We subjected 21 human brain samples to small RNA sequencing (sRNA-seq). We specifically focused in the amygdala, a brain area presenting Lewy bodies (LB) at pre-motor stages, the characteristic neuropathological hallmark of PD

Availability and Implementation: The full code is available at http://github.com/lpantano/seqcluster

first: Eulalia

last: Marti


title: Grainyhead-like 2 is essential for androgen receptor expression and activity in prostate cancer (RNA-seq)

city: Bedford Park

first: Luke

last: Selth

state: SA

address: Flinders Drive

department: College of Medicine and Public Health

country: Australia

institute: Flinders University

summary: The mainstay treatment for men with metastatic prostate cancer is androgen deprivation therapy (ADT), which inhibits androgen biosynthesis and/or binding of ligand to the androgen receptor (AR). Most men respond to ADT, but all subsequently develop resistance and progression to incurable and lethal castration-resistant prostate cancer (CRPC). CRPC generally remains driven by the AR: therefore, the development of novel AR-targeted therapies, as well as elucidating therapy-driven changes to the AR signalling axis that mediate resistance, remain priorities for the field. Using a novel and powerful proteomic technique, rapid immunoprecipitation of endogenous proteins (RIME), we have identified a new AR binding protein, the transcription factor Grainyhead-like 2 (GRHL2), and shown that it plays an essential, multifaceted role in signalling by the canonical AR and by constitutively active truncated AR variants (ARVs). GRHL2 is necessary for the maintenance of AR/ARV expression in multiple prostate cancer model systems, co-localises with AR at specific sites on chromatin to enhance the androgen-regulated transcriptional program, and is required for optimal prostate cancer growth. Interestingly, we found that GRHL2 is itself an AR/ARV-regulated gene, creating a positive feed-forward loop between the two factors. The GRHL2 gene is frequently amplified and upregulated in CRPC, thereby potentially enabling further amplification AR signalling in the drug-resistant setting. In summary, this study has identified a critical new AR coregulator and potential therapeutic target in prostate cancer.

overall_design: RNA-seq following GRHL2 knockdown in LNCaP cells

first: L


title: Grainyhead-like 2 is essential for androgen receptor expression and activity in prostate cancer


title: Grainyhead-like 2 is essential for androgen receptor expression and activity in prostate cancer (ChIP-seq)

overall_design: ChIP-seq for GRHL2 and AR in LNCaP cells


title: Gene expression profile in mouse esophageal squamous cell carcinoma

city: Bethesda

first: Sean

last: Davis

state: MD

address: 37 Convent Drive, Room 6138

institute: National Cancer Institute

summary: Human esophageal cancer is the sixth leading cause of cancer death worldwide. More than 90% of esophageal cancer is esophageal squamous cell carcinoma (ESCC). However, the etiological cause of ESCC remains unclear. By using gene expression microarray analysis, we aimed to find whether fungal infection is involved in ESCC development. We identified a wide spectrum of molecular signatures in a fungal infection and ESCC mouse model, including alterations involved in epigenetic regulation, cell cycle control, cell proliferation and survival signaling, and inflammation, which share many similarities with human ESCC.

overall_design: Esophagus from Amphotericin B treated KA mouse, ESCC from untreated KA mouse, Esophagus from WT mice

first: Feng

last: Zhu

first: Sun

last: David


title: Characterization of human Merkel cells and their response to Merkel cell polyomavirus

first: Christian

middle: J

last: Berrios

address: 450 Brookline Ave; Mayer 443

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: Merkel cells are epidermal mechanoreceptor cells responsible for the perception of gentle touch. Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer. Although MCC histologically resembles Merkel cells, the cell of origin for MCC is unknown. MCC frequently contains integrated Merkel cell polyomavirus (MCPyV), a small DNA tumor virus with widespread prevalence. Whether MCPyV can transform Merkel cells is unknown. Here, we describe the isolation and long-term expansion of human Merkel cells from neonatal foreskin. We validated the expression of several Merkel cell-related factors by RNASeq, and assessed the ultrastructure by electron microscopy. Culture of Merkel cell preparations on an artificial basement membrane promoted the formation of structures containing both Merkel and non-Merkel cell populations. To determine whether Merkel cells were susceptible to transformation, we expressed tumor-derived MCPyV T antigens and additional oncogenes. We were unable to demonstrate tumorigenesis in immunodeficient mice, but were able to detect T antigen expression from excised cells weeks after implantation. These results highlight that foreskin-isolated Merkel cells can be propagated extensively, sustain expression of MCPyV T antigens, but are not susceptible to transformation by MCPyV, suggesting that Merkel cells from non-glabrous skin may not be a cell of origin for MCC.

overall_design: Comparison of MKL-1 Merkel cell carcinoma cell line to primary CD56 Merkel cells expressing either GFP or Merkel cell poylomavirus (MCCL21) early-region.

first: Victor

last: Huang

first: Kellie

last: Jurado

first: Rosa

last: Yoon

first: Alan

middle: N

last: Engelman

first: Rachael

last: Clark

first: James

last: DeCaprio


title: Identification of mRNA bound by TRIM25 binding in triple negative breast cancer

first: Logan

last: Walsh

state: NY

address: 1275 York Ave

department: Human Oncology and Pathogenesis Program

institute: Memorial Sloan Kettering Cancer Center

summary: We report high-throughput profiling of TRIM25 bound mRNA in triple negative breast cancer cells.

overall_design: Examination of TRIM25 bound mRNA in 2 triple negative breast cancer cell lines.

first: Timothy

last: Chan


title: Genome-wide maps of TRIM25 binding sites in triple negative breast cancer cell

summary: We report high-throughput profiling of TRIM25 binding sites in triple negative breast cancer cells.

overall_design: ChIP-seq was performed using a TRIM25 antibody on 2 triple negative breast cancer cell lines


title: Expression data from thymic epithelial cells of 5-6 weeks old C57BL/6J mice.

summary:
Defects in thymic epithelial cell development causes autoimmune diseases due to breakdown of central tolerance, including skin inflammation. IKKa may be essential to link central tolerance to skin inflammation.
We used microarrays to examine the global gene expression with or without kinase inactive IKKa, and identified a distinct set of down-regulated genes in the presence of kinase-dead IKKa in thymic epithelial cells.

overall_design: Thymic epithelial cells were isolated from 5-6 weeks old mice. Total RNAs were extracted, and then subjected to Affymetrix mouse genome 430 2.0 array.

first: Yinling

last: Hu


title: Next generation sequencing facilitates quantitative analysis of changes in mRNA after knock-down of putative master regulators of the breast cancer metastasis transcriptome.

summary:
Purpose:  To identify regulatory proteins that are potential drivers of a coordinated breast cancer metastasis gene expression signatures.
Methods: Knockdown of target genes in breast cancer cell lines was achieved using scramble and/or gene-specific siRNA (ON-TARGET SMARTpool, Thermo Scientific) and Lipofectamine RNAiMAX.  48h post transfection, total RNA was isolated from cell lines using the RNeasy Plus mini prep kit (Qiagen). Nucleic acid quality was determined with the Agilent 2100 Bioanalyzer. RNA Sequencing was also performed at the New York Genome Center (Manhattan, NY, USA) using a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA, USA).
Results: Raw reads in the fastq format were aligned to Human Genome HG19 using the RNA-seq STAR aligner version 2.4.0d (http://www.ncbi.nlm.nih.gov/pubmed/23104886, http://www.ncbi.nlm.nih.gov/pubmed/26334920) as recommended by user manual downloaded along with the software. STAR aligner was chosen for mapping accuracy and speed (http://www.nature.com/nmeth/journal/v10/n12/full/nmeth.2722.html).  Mapped reads for each sample were counted for each gene in annotation files in GTF format (gencode.v19.annotation.gtf available for download from GENECODE website (http://www.gencodegenes.org/releases/19.html)) using the FeatureCounts read summarization program (http://www.ncbi.nlm.nih.gov/pubmed/?term=24227677) following the user guide (http://bioinf.wehi.edu.au/subread-package/SubreadUsersGuide.pdf). Individual count files were merged to generate the raw-counts matrix by an in-house R script, normalized to account for differences in library size and the variance was stabilized by fitting the dispersion to a negative-binomial distribution as implemented in the DESeq R package (http://bioconductor.org/packages/release/bioc/html/DESeq.html)(Anders and Huber, 2010).
Conclusions:  Our data suggest that targeting keystone proteins in the breast cancer metastasis transcriptome can effectively collapse transcriptional hierarchies necessary for metastasis formation, thus representing a formidable cancer intervention strategy.

overall_design: Examination of mRNA profiling of breast cancer cell lines after knock-down of putative master regulators of the breast cancer metastasis transcriptome


title: Gene expression analysis of human hepatocellular cancers

city: Houston

first: Jian

last: Chen

state: TX

address: 1515 Holcombe Boulevard - Unit 1466

institute: The University of Texas M. D. Anderson Cancer Center

summary: Gene expression profiling was carried out in 4 matched human hepatocellular cancer samples/normal liver smapls, and 4 human hepatocellular cancer samples to characterize the molecular mechanisms relevant to the etiology of human hepatocellular cancer development.

overall_design: Whole-transcriptome RNA sequencing of the 4 matched human hepatocellular cancer samples/normal liver samples, and 4  human hepatocellular cancer samples.

first: Lopa

last: Mishra


title: TRIM25


title: Transcription elongation factors are in vivo-specific cancer dependencies in glioma

city: Cleveland

first: Tyler

middle: E

last: Miller

state: Ohio

department: Stem Cell Biology and Regenerative Medicine

institute: Cleveland Clinic Lerner Research Institute


title: RNA-seq Profiles in Transcription elongation factors are in vivo-specific cancer dependencies in glioma

summary: Glioblastoma ranks as one of the most lethal human cancers, with no effective therapies.  To discover novel  therapeutic targets, here we performed parallel in vivo and in vitro RNA interference screens of epigenetic regulators and show that transcription elongation factors are essential for human glioblastoma cell survival in vivo, but not in vitro.  Context-specific dependency in vivo is driven by microenvironment-induced global changes in the cancer epigenome. JMJD6, a top in vivo-specific hit, binds at enhancers and correlates with increased transcription of known pause-controlled genes.  JMJD6 knockdown in patient-derived glioblastoma cells enhances survival of mice bearing orthotopic tumors.  Moreover, elevated levels of JMJD6 alone, as well as transcription elongation factors collectively, informs tumor grade and predicts poor prognosis for patients.  Our work provides a rationale for targeting transcription elongation as a therapeutic strategy in glioblastoma and, more broadly, the power of in vivo phenotypic screening to identify therapeutically relevant targets in cancer.

overall_design: RNA-seq of primary patient-derived GBM cells grown in in vivo tumor microenvironment or in vitro in serum free cell culture

first: Daniel

middle: C

last: Factor

first: Paul

last: Tesar

first: Jeremy

last: Rich


title: Identification of long noncoding RNAs regulated by p53

first: Javed

last: Khan

address: 37 Convent Dr.

department: Genetics Branch

institute: NCI, NIH

summary: Analyize the transcriptpme regulated by p53 in 3 pairs of isogenic p53WT and p53KO colorectal cancer cell lines untreated or treated with Doxorubicin. The hypothsis of this study is that p53 can regulate the expression of subset of lncRNAs to mediate its biological functions.

overall_design: Total RNA was isolated from 3 pairs of isogenic colon cancer cell lines; HCT116-p53_WT and p53_KO; RKO-p53_WT and p53_KO: and SW48-p53_WT and p53_KO. Cells were untreated or treated with Doxorubicin (DOXO) at a final concentration of 300 nM for 16 hours.

first: Ashish

last: Lal

first: Xinyu

last: Wen


title: Epigenetic Profile in Transcription elongation factors are in vivo-specific cancer dependencies in glioma

overall_design: H3K27Ac histone modifications and JMJD6 binding were mapped by ChIP-seq in glioblastoma cells grown either in an in vivo intrancranial tumor or in serum free cell culture

first: Brian

last: Liau

first: Bradley

last: Bernstein


title: Genome-wide profiling of H3K27me3 occupancy in C. merolae

first: Pawel

last: Mikulski

summary: Chromatin immunopreciptation coupled with Illumina sequencing to H3K27me3 occupancy in model red alga, C. merolae. The results revealed preferential abundance over repetitive elements, instead of the genes. This histone mark enriches also over subtelomeric and telomeric regions of the chromosomes. C. merolae H3K27me3 has a canonical repressive function as in the higher eukaryotes and forms differential clusters over the gene bodies with different levels of repression. Overall, our study provides important insights toward the function of chromatin-mediated repression in the evolutionary aspect.

overall_design: ChIP-seq experiment in triplicate in C. merolae cell cultures. The samples include: H3K27me3, H3 and input. The H3K27me3 domain occupancy was calculated with normalization, either to input or H3 samples.

last: Schubert


title: Porcine oocytes maturation in vitro

status: Public on Mar 31 2017

city: Poznań

first: Marcin

last: Ruciński

state: Wielkopolska

address: Święcickiego 6

department: Histology and Embryology

country: Poland

institute: Poznań University of Medical Sciences

summary:
The proper mammalian oocytes maturation is recognized as reaching MII stage and accumulation of mRNA and proteins in cell cytoplasm following fertilization. The proper course of folliculogenesis and oogenesis is orchestrated with morphogenesis significantly influencing further zygote formation and embryos growth. This study was aimed to determinate new transcriptomic markers of porcine oocytes morphogenesis associated with cell maturation capacity.
We used microarrays to detail the global programme of gene expression underlying changes before and after in vitro maturation of oocytes in sus scrofa

overall_design: Pig oocytes were aspired from porcine ovaries, selected to two groups by BCB test, subjected to in vitro maturation, RNA extraction, reverse transcription, and hybridization on Affymetrix microarrays. Only oocytes of grade I with homogeneous ooplasm were considered for the following steps of the experiment, resulting 300 grade I oocytes (3 x n=50 before in vitro maturation, 3 x n=50 after In vitro maturation).

first: Bartosz

last: Kempisty

first: Joanna

last: Budna

first: Piotr

last: Celichowski


title: Pyroptosis by Caspase11/4-Gasdermin-D Pathway in Alcoholic Hepatitis

city: Los Angeles

first: Hidekazu

last: Tsukamoto

state: CA

address: 1333 San Pablo Street MMR414

department: Pathology

institute: University of Southern California

summary: Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment.  Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH, is largely unknown. To address this question, unbiased RNA-seq and proteomic analyses were performed on livers of the recently developed AH mouse model which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared with gene expression profiling data from AH patients.  This cross-analysis has identified Casp11 (CASP4 in man) as a commonly upregulated gene known to be involved in non-canonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice but not in chronic ASH. Gasdermin-D (GSDMD) which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and the severity of AH. Conversely, the deficiency of IL-18, the key anti-microbial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Further, hepatocyte-specific expression of constitutively active GSDMD worsens hepatocellular lytic death and PMN inflammation.  These results implicate pyroptosis induced by CASP11/4-GSDMD pathway in the pathogenesis of AH.

overall_design: 9 total samples = 3 AH liver, 3 ASH liver, 3 control

first: Yibu

last: Chen

first: Charles

last: Ansong

first: Richard

last: Smith

first: Raymond

last: Wu


title: The de novo Autism Spectrum Disorder RELN R2290C Mutation Reduces Reelin Secretion and Increases Protein Disulfide Isomerase Expression

city: Syracuse

first: Frank

last: Middleton

address: 505 Irving Avenue

department: Neuroscience and Physiology

institute: SUNY Upstate Medical Univ

summary: To determine if changes in Protein Disulfide Isomerase (PDIA1) expression in mice with a null Reelin allele were caused by accumulation of intracellular Reelin or were an effect of reduced Reelin protein, we examined expression of PDIA1 and other stress markers in heterozygous RELN +/- null allele mice. The levels of PDIA1 as well as PERK, BIP, phospho-eIF2alpha and total eIF2alpha were unchanged between wild-type and RELN +/- null allele mice. This suggested that there are phenotypic differences in the cerebella between mice that carry a RELN allele that fails to produce a protein (null allele) and those that make a protein that fails to be secreted (Orl allele). Each of the three major ER stress pathways ultimately leads to changes in gene transcription. Thus, we compared wild-type and heterozygous RELN Orl +/- mice cerebellum by RNAseq. Analysis was performed on 3 heterozygous (HET) and 3 wild-type (WT) cerebella, obtained from 6-week old male mice.

overall_design: Comaprison of gene expression in the cerebellum of Reelin heterozygous null mice and wildtype mice using RNA-seq

last: Howell

first: Dawn

last: Lammert


title: Conserved roles for murine mDUX and human DUX4 in activating cleavage stage genes and MERVL/HERVL retrotransposons [ChIP-Seq Human]

city: Salt Lake City

first: Peter

middle: G

last: Hendrickson

state: UT

address: 2000 Circle of Hope Dr.

department: HHMI/Oncological Sciences

institute: University of Utah

summary: To better understand transcriptional regulation during human oogenesis and pre-implantation embryonic development, we defined stage-specific transcription, which revealed cleavage stage as highly distinctive.  We present multiple lines of evidence that two cleavage-specific homologs, mouse mDUX and human DUX4, each activate hundreds of cleavage-specific endogenous genes (e.g. ZSCAN4, ZFP352, KDM4E) and retroviral elements (MERVL/HERVL-family).  Remarkably, mDux expression converts mouse ESCs into two-cell embryo-like (2C-like) cells by binding to MERVL promoters/enhancers and restoring the chromatin landscape (via ATACseq) to the pattern of mouse two-cell embryos

overall_design: We ectopically overexpressed human DUX4  (or luciferase control) in human pluripotent stem cells (iPSCs) by dox -induction for 18hrs. Cells were then lysed and chromatin was immunoprecipitated using a rabbit monoclonal anti-DUX4 antibody [E5-5] (ab124699) . 125bp libraries were prepped (2 technical replicates/condition) and sequenced in paired-end format. All sequencing was done on the Illumina HiSeq 2500 platform.

first: Jessie

last: Dorais

first: Edward

last: Grow

first: Jennifer

middle: L

last: Whiddon

first: Jong-Won

last: Lim

first: Candice

last: Wike

last: Weaver

first: Christian

last: Pfluegar

first: Benjamin

middle: R

last: Emery

first: Aaron

last: Wilcox

first: David

last: Nix

first: Matthew

last: Peterson

first: Stephen

last: Tapscott

first: Douglas

last: Carrell

last: Cairns


title: Mouse Liver tumor SRC-2

city: Dallas

first: Shruthy

last: Suresh

state: Texas

address: 6000 Harry Hines Blvd NA6.300

department: Molecular Biology

institute: UT Southwestern Medical Center

summary: We studied the effects of loss of SRC-2 in MYC driven mouse liver tumorigenesis

overall_design: Src-2+/-; LAPtTA females were bred with Src-2+/-; tet-o-MYC males to obtain tet-o-MYC; LAPtTA mice with SRC-2 WT or homozygous null alleles. Doxycycline was withdrawn at 6 weeks of age to induce MYC and animals were dissected at 15 weeks of age. RNA was isolated from liver tumors of 5 SRC-2 WT and 5 SRC-2 -/- animals and processed for RNA sequencing to identify differentially expressed genes between the two groups.

first: Kathryn

last: O'Donnell