title: Blimp1 controls plasma cell physiology and function

city: Clayton

first: Wei

last: Shi

state: Victoria

address: Wellington Rd

institute: Monash University

summary: Antibody-secreting plasma cells are the terminal stage of the B-cell lineage. Plasma cell differentiation requires a major resetting of gene expression to silence the B cell transcriptional program, whilst establishing secretory function and long-term survival. The transcription factors Blimp1 and Irf4 are essential for the initial differentiation of activated B cells to antibody-secreting cells, however their function in mature plasma cells remains elusive. We have found that while Irf4 was essential for plasma cell survival, Blimp1 was dispensable. Blimp1-deficient cells retained the unique plasma cell transcriptional signature, but lost the ability to secrete antibody or to maintain the characteristic size and ultrastructure of plasma cells. Blimp1 was required for full expression of many components of the unfolded protein response (UPR), including Xbp1 and Atf6, as well as for the appropriate processing of Igh mRNA. The overlap of Blimp1 and Xbp1 function was restricted to the UPR genes, with Blimp1 uniquely regulating activity of the mTOR pathway, plasma cell size and morphology. These studies establish Blimp1 as a major regulator of the UPR pathway that is also required for the unique metabolic requirements of plasma cells enabling the secretion of protective antibody.

overall_design: RNA-seq was performed on wild type, Blimp1-/- and Xbp1-/- mouse plasma cells. Between two to four biological replicates were generated and sequenced for each sample.

first: Julie

last: Tellier

first: Yang

last: Liao

first: Gordon

last: Smyth

first: Stephen

last: Nutt


title: Transcriptome-wide distribution and function of RNA hydroxymethylcytosine

status: Public on Jan 15 2016

city: Bruxelles

first: Matthieu

last: Defrance

address: CP614 route de Lennik 808

country: Belgium

institute: Université Libre de Bruxelles

summary: Our understanding of the posttranscriptional modifications that decorate RNA, a regulatory layer positioned between DNA and proteins, is in its infancy. Hydroxymethylcytosine, well described in DNA, occurs also in RNA, where its distribution, localization and function remain unexplored. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We provide the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We find that RNA hydroxymethylation can favor mRNA translation. Tet-deficient fruitflies show impaired brain development, accompanied by decreased RNA hydroxymethylation. Together, these steps into uncharted territory uncover central roles for RNA hydroxymethylation in Drosophila, highlighting the importance of epitranscriptomics.

overall_design: RNA hydroxymethylation landscape in S2 cell lines.

first: Benjamin

last: Delatte

first: Fei

last: Wang

first: Long

middle: V

last: Ngoc

first: Evelyne

last: Collignon

first: Elise

last: Bonvin

first: Rachel

last: Deplus

first: Emilie

last: Calonne

first: Bouchra

last: Hassabi

first: Pascale

last: Putmans

first: Stephan

last: Awe

first: Collin

last: Wetzel

first: Judith

last: Kreher

first: Romuald

last: Soin

first: Catherine

last: Creppe

first: Patrick

last: Limbach

first: Cyril

last: Gueydan

first: Véronique

last: Kruys

first: Alexander

last: Brehm

first: Svetlana

last: Minakhina

first: Ruth

last: Steward

first: François

last: Fuks


title: Whole transcriptome sequencing of phage φR1-37 and its host Yersinia enterocolitica

status: Public on Jan 22 2016

city: Helsinki

first: Päivi

last: Saavalainen

address: Haartmaninkatu 8

department: TRIMM

country: Finland

institute: University of Helsinki

type: Expression profiling by high throughput sequencing

summary: RNA-sequencing was preformed from RNA isolated from bacteria infected with the bacteriophage. In order to  reveal the phage-host interactions between φR1-37 and Yersinia enterocolitica throughout the phage infection cycle, both the transcriptomes were scrutinized during all the stages of infection.

sample_organism:
Yersinia enterocolitica
Yersinia phage phiR1-37

overall_design: Fresh cultures (logarythmic phase) of Yersinia enterocolitica YeO3-R1 were infected with bacteriophage R1-37. The RNA samples were taken at following time points post-infection: 0 (negative control), 2, 5, 10, 15, 21, 28, 35, 42 1nd 49 min.

first: Katarzyna

last: Leskinen

first: Bob

middle: G

last: Blasdel

first: Rob

last: Lavigne

first: Mikael

last: Skurnik


title: Transcriptomic analysis of germline tumor in fasted C. elegans

city: Frankfurt am Main

first: Devang

middle: Savdas

last: Odedra

state: Hesse

address: Max-von-Laue-Street 15

department: Biochemistry

country: Germany

institute: Buchmann Institute for Molecular Life Sciences

summary: Transciptomic analysis of germline tumor cells to understand the role of autophagy and neuronal differentiation in lifespan extension.

overall_design:
Methods: Worms were grown on control L444 seeded plates or gld-1 RNAi seeded plates and subjected to RNA isolation and sequencing using standard Illumina protocols.
Conclusions: Fasting of animals expressing tumors increases their lifespan two-fold through autophagy and modular changes in transcription as well as metabolism.

first: Ligia

last: Gomes

first: Dikic

last: Ivan

first: Christian

last: Pohl


title: Genome-wide transcriptome analyses of developing seeds from low and normal phytic acid soybean lines

city: Blacksburg

first: M.A. Saghai

last: Maroof

state: VA

address: 220 Ag Quad Ln

department: Crop & Soil Environmental Sciences

country: USA

institute: Virginia Tech

summary: Purpose: Comprehensive comparison of gene-expression profiles between 3mlpa (mutant with low phytic acid) and 3MWT (non-mutant with normal phyic acid) soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. Methods: mRNA sequencing reads (101SE) were generated in triplicate from five seed developmental stages of 3mlpa and 3MWT soybean line using Illumina HiSeq 2000. Results: A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. Conclusion: This study provides a global perspective of transcriptomal changes during soybean seed development in 3mlpa mutant. The results suggest that low phtic acid-causing mutations in 3mlpa play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively.

overall_design: RNA-Seq of five seed developing soybean seeds from mips1/mrp-l/mrp-n triple mutant line (with low phytic acid) and MIPS1/MRP-L/MRP-N non-mutant line (with normal phytic acid). The mRNA libraries from three biological replicates of each sample were sequenced as 101 SE using Illumina HiSeq 2000.

first: M

middle: A

last: Saghai-Maroof


title: A high-throughput functional screening identifies non-canonical cis-regulatory sequences in the POU5F1 Locus

city: La Jolla

first: Bing

last: Ren

state: CA

address: 9500 Gilman Dr., CMM-East, Admin Area/Rm 2071

institute: University of California, San Diego School of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: With less than 2% of the human genome coding for proteins, a major challenge confronting researchers today is to interpret the function of the non-coding DNA. Millions of regulatory sequences have been predicted in the human genome through the analyses of DNA methylation, chromatin modification, hypersensitivity to nucleases, and transcription factor binding, but few have been shown to regulate transcription in their native contexts.  To begin to address this issue, we have developed a high throughput CRISPR/Cas9-based genome-editing strategy and used it to interrogate 174 candidate regulatory sequences within the 1Mbp POU5F1 locus in the human ES cells. We identified two classical regulatory elements - including a promoter and a proximal enhancer - that are essential for POU5F1 transcription in the ES cells.  Unexpectedly, we also discovered a new class of cis elements that contribute to POU5F1 transcription in an unusual way: disruption of such sequences led to a temporary loss of POU5F1 transcription that is fully restored after a few rounds of cell division. These results demonstrate the utility of a high throughput screening for functional characterization of non-coding DNA, and reveal a previously unrecognized layer of gene regulation in human cells.

overall_design: ChIP-Seq for H3K27ac, 4C-Seq for human POU5F1 bait

first: Yarui

last: Diao

first: Bin

last: Li

first: Zhipeng

last: Meng

first: Inkyung

last: Jung

first: Ah

middle: Y

last: Lee

first: Jesse

last: Dixon

first: Lenka

last: Maliskova

first: Kun-liang

last: Guan

first: Yin

last: Shen


title: Expression analysis by RNAseq of roots and shoots of the pho1 mutant expressing the EXS domain of PHO1

city: Lausanne

first: Sylvain

last: Pradervand

address: Genopode

department: CIG

country: Switzerland

institute: UNI Lausanne

summary: Expression of the EXS domain of PHO1 in the Arabidopsis thaliana pho1 mutant leads to improved shoot growth in comparison to the parental pho1 mutant. We have used RNAseq to study the gene expression profile in the roots and shoots of the pho1 mutant, two independent transgenic lines of pho1 expressing the EXS domain of PHO1 (lines EXS32 and EXS34) as well as a pho1 mutant transformed with the full length PHO1 gene (complemented line).

overall_design: Plants were grown for 25 days in pots, roots and shoots were harvested and RNA was extracted

first: Stefanie

last: Wege

first: Yves

last: Poirier


title: Understanding the role of Shoc2 protein in spatio-temporal regulation of extracellular signal-regulated kinase (ERK1/2) cascade

city: Louisville

first: Eric

middle: Christian

last: Rouchka

state: Kentucky

address: 522 East Gray Street

department: Biochemistry and Molecular Genetics

institute: University of Louisville

summary: This study was designed to understanding changes in RNA transcription in cells depleted of Shoc2 scaffold.

overall_design: 2 samples were analyzed (3 replicates for each sample were included). Cos1 is a control sample.

first: Emilia

last: Galperin

first: Myoungkun

last: Jeoung

middle: C

first: Xiaohong


title: mRNA-seq of ros1-3 and ros1-4 with ABA treatment

city: Seoul

first: Jin Hoe

last: Huh

address: Gwanak-gu

department: Dept Plant Science

country: South Korea

institute: Seoul National University

summary:
To survey transcriptome changes by the mutations of a DNA demethylase ROS1 responding to a phytohormone abscisic acid, we performed the Next-gen sequencing (NGS) associated RNA-seq analysis.
Two ROS1 knockout lines (ros1-3, ros1-4; Penterman et al. 2007 [PMID: 17409185]) with the wild-type Col line (wt) were subjected.

overall_design: Three samples (ros1-3, ros1-4 and wt), biological triplicates, ABA or mock treatment, using Illumina HiSeq 2500 system

first: June-Sik

last: Kim

first: Jin

middle: H


title: mRNA-Seq expression and MS3 proteomics profiling of human post-mortem BA9 brain tissue for Parkinson Disease and neurologically normal individuals

city: Boston

first: Alexandra

last: Dumitriu

address: 72 East Concord Street

department: Neurology

institute: Boston University

summary: Parkinson disease (PD) is a neurodegenerative disease characterized by the accumulation of alpha-synuclein (SNCA) and other proteins in aggregates termed “Lewy Bodies” within neurons. PD has both genetic and environmental risk factors, and while processes leading to aberrant protein aggregation are unknown, past work points to abnormal levels of SNCA and other proteins. Although several genome-wide studies have been performed for PD, these have focused on DNA sequence variants by genome-wide association studies (GWAS) and on RNA levels (microarray transcriptomics), while genome-wide proteomics analysis has been lacking. After appropriate filters, proteomics identified 3,558 unique proteins and 283 of these (7.9%) were significantly different between PD and controls (q-value<0.05). RNA-sequencing identified 17,580 protein-coding genes and 1,095 of these (6.2%) were significantly different (FDR p-value<0.05), but only 166 of the FDR significant protein-coding genes (0.94%) were present among the 3,558 proteins characterized. Of these 166, eight genes (4.8%) were significant in both studies, with the same direction of effect. Functional enrichment analysis of the proteomics results strongly supports mitochondrial-related pathways, while comparable analysis of the RNA-sequencing results implicates protein folding pathways and metallothioneins. Ten of the implicated genes or proteins co-localized to GWAS loci. Evidence implicating SNCA was stronger in proteomics than in RNA-sequencing analyses. Notably, differentially expressed protein-coding genes were more likely to not be characterized in the proteomics analysis, which lessens the ability to compare across platforms. Combining multiple genome-wide platforms offers novel insights into the pathological processes responsible for this disease by identifying pathways implicated across methodologies.

overall_design: The study consists of mRNA-Seq (29 PD, 44 neurologically normal controls) and three-stage Mass Spectrometry Tandem Mass Tag Proteomics (12 PD, 12 neurologically normal controls) performed in post-mortem BA9 brain tissue. The proteomics samples are a subset of the RNA-Seq samples.

first: Javad

last: Golji

first: Adam

middle: T

last: Labadorf

first: Benbo

last: Gao

first: Thomas

last: Beach

first: Richard

last: Myers

first: Kenneth

last: Longo

first: Jeanne

last: Latourelle


title: Paf1C plays novel subunit-specific roles in alternative cleavage and polyadenylation

city: Bridgewater

first: Wencheng

last: Li

state: NJ - New Jersey

institute: PTC Therapeutics

summary: The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites.

overall_design: ChIP-seq, RNA-seq and 3'READS of Paf1C factors in mouse C2C12 myoblast cells

first: Yan

last: Yang

first: Mainul

last: Hoque

first: Liming

last: Hou

first: Steven

last: Shen

first: Bin

last: Tian

first: Brian

middle: D

last: Dynlacht


title: Epigenomic profiling of Non-small-cell Lung Cancer (NSCLC) cells with or without Eed

city: Berlin

first: Gaetano

last: Gargiulo

address: Robert-Rössle-Straße 10

department: Molecular Oncology

institute: Max Delbrüch Center

summary: Polycomb repressive complexes (PRC) are frequently implicated in human cancer acting either as oncogenes or tumor suppressors. Here we show that PRC2 is a critical regulator of Kras-driven non-small-cell lung cancer (NSCLC) progression. Modulation of PRC2 by either Ezh2 overexpression or Eed deletion enhances Kras-driven adenomagenesis and inflammation, respectively. Eed-loss-driven inflammation leads to massive macrophage recruitment and marked decline in tissue function. Additional Trp53 inactivation activates a cell autonomous epithelial-to-mesenchymal transition (EMT) program leading to an invasive mucinous adenocarcinoma. A switch between methylated/acetylated chromatin underlies the tumor phenotypic evolution, prominently involving genes controlled by Hippo/Wnt-signaling. Our observations in the mouse models were conserved in human cells. Importantly, PRC2 inactivation results in context-dependent phenotypic alterations, with implications for its therapeutic application.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We generated ChIP-seq from primary Kras;p53 (KP) cells in culture with and without Eed (KPE) and from KP primary tumors generated by injection of NSCLC into the tail vein. Mice were sacrificed on the onset of shortness of breath. We generated genome-wide expression profiles (RNA-seq) and Nuclease Accessibility (NA)-seq in primary KP and KPE tumor cells. NA-seq was also performed in A549 cells.

first: Michela

last: Serresi

first: Matteo

last: Cesaroni

first: Iris

last: de Rink


title: The equine embryo influences immune related gene expression in the oviduct

city: Gent

first: Dieter

last: De Coninck

address: J. Plateaustraat 22

department: Applied Ecology and Environmental Biology

institute: Ghent University

summary: While the equine oviduct clearly affects early embryo development and while the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, the oviduct epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected. mRNA was extracted and samples were sequenced using the Illumina Hiseq-2000 sequencer. Data analysis was performed with the CLC Genomics software and differentially expressed genes (DEGs) were determined (p-value ≤ 0.05 and absolute fold change ≥ 2). ClueGO was used for functional interpretation. A total of 26,991 genes was identified and 253 genes were found to be upregulated and 108 to be downregulated in the pregnant ipsilateral oviduct, when compared to the cyclic ipsilateral oviduct. Comparison of the ipsilateral and the contralateral oviduct indicated 164 DEGs in pregnant mares and 77 DEGs in cyclic mares. Functional enrichment analysis only detected differences in the comparison of pregnant and cyclic ipsilateral oviducts and showed that the equine embryo affects the expression of immune response related genes in the oviduct, with marked upregulation of interferon associated genes. This research represents the foundation for further assessment of the role of specific genes in the early embryo-maternal dialogue of the horse.

overall_design: mRNA profiles of epithelial cells of equine oviducts ipsilateral and contralateral to the ovulation side of pregnant and cyclic mares four days after ovulation were examined, resulting in four different conditions (pregnant ipsilateral, pregnant contralateral, cyclic ipsilateral and cyclic contralateral, consisting of five biological replicates each.

first: Katrien

last: Smits

middle: M

first: Filip

last: Van Nieuwerburgh

first: Jan

last: Govaere

first: Mario

last: Van Poucke

first: Luc

last: Peelman

last: Deforce

first: Ann

last: Van Soom


title: Improved transcription and translation with L-leucine stimulation of mTORC1

city: Kansas City

first: Madelaine

last: Gogol

state: MO

address: 1000 E. 50th Street

department: Computational Biology Core

institute: Stowers Institute

summary: Roberts syndrome (RBS) is a human developmental disorder caused by mutations in the cohesin acetyltransferase ESCO2. We previously reported that mTORC1 was inhibited and overall translation was reduced in RBS cells. Treatment of RBS cells with L-leucine partially rescued mTOR function and protein synthesis, correlating with increased cell division. In this study, we use RBS as a model for mTOR inhibition and analyze transcription and translation with ribosome profiling to determine genome-wide effects of L-leucine. The translational efficiency of many genes is increased with Lleucine in RBS cells including genes involved in ribosome biogenesis, translation, and mitochondrial function. snoRNAs are strongly upregulated in RBS cells, but decreased with L-leucine. Imprinted genes, including H19 and GTL2, are differentially expressed in RBS cells consistent with contribution to mTORC1 control. This study reveals dramatic effects of L-leucine stimulation of mTORC1 and supports that ESCO2 function is required for normal gene expression and translation.

overall_design: 42 samples of human fibroblast cell lines with various genotypes (wt, corrected, and esco2 mutants) are treated with l-leucine or d-leucine (control) for 3 or 24 hours. Biological replicates are present.

first: Baoshan

last: Xu

first: Karin

last: Zueckert

first: Jennifer

last: Gerton


title: Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1

status: Public on Jan 17 2016

city: Toronto

first: Pamela

last: Ohashi

state: Ontario

address: 610 University Ave

country: Canada

institute: Princess Margaret Cancer Centre

summary: Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GAB2 as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transduced Gab2-/- bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors PI3K or SHP2. GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph+ hematologic neoplasms.

overall_design: RNA-Seq expression profiling of 6 mouse bone marrow samples: 3 GAB2 WT (+/+) and 3 GAB2 NULL (-/-)

first: Azin

last: Sayad

first: Shengqing

last: Gu


title: Medial prefrontal cortex transcriptome changes in germ-free mice

status: Public on Jan 14 2016

city: Cork

first: Roman

last: Stilling

address: Western Road

department: Anatomy and Neuroscience

country: Ireland

institute: University College Cork

summary: We sequenced mRNA from 12 samples extracted from mouse prefrontal cortex tissue to generate the first prefrontal cortex-specific murine transcriptome for germ-free mice (GF), conventionally raised controls (CON) and germ-free mice that have been colonized with normal microbiota from postnatal day  21 (exGF).

overall_design: Equal amounts of RNA from two to three animals were pooled to yield 4 samples per group (CON, GF, and exGF). Pairwise comparisons for CONvsGF, CONvsexGF, GFvsexGF were performed using DESeq2.

first: Alan

middle: E

last: Hoban

first: Feargal

last: Ryan

first: Gerard

last: Clarke

first: Marcus

last: Claesson

first: John

middle: F

last: Cryan

first: Timoth

last: Dinan

first: Fergus

last: Shanahan


title: The Functional Genomic Landscape of Human Breast Cancer Drivers, Vulnerabilities, and Resistance (RNASeq)

summary:
Large-scale genomic studies have identified multiple somatic aberrations in breast cancer, including copy number alterations, translocations, and point mutations. Still, identifying causal variants and emergent vulnerabilities that arise as a consequence of genetic alterations remain major challenges. We performed whole genome shRNA “dropout screens” on 77 breast cancer cell lines. Using a new hierarchical linear regression algorithm to score our screen results and integrate them with accompanying detailed genetic and proteomic information, we identify novel vulnerabilities in breast cancer, including new candidate “drivers,” and reveal general functional genomic properties of cancer cells. Comparisons of gene essentiality with drug sensitivity data suggest potential resistance mechanisms, novel effects of existing anti-cancer drugs, and new opportunities for combination therapy.  Finally, we demonstrate the utility of this large dataset by identifying BRD4 as a potential target in luminal breast cancer, and PIK3CA mutations as a resistance determinant for BET-inhibitors.

Additional formatted data can be found at http://neellab.github.io/bfg/.
Code and tutorials for the siMEM algorithm can be found at http://neellab.github.io/simem/.

overall_design: RNA-Seq expression profiling of 82 breast cancer cell lines without replicates or control samples


title: Expression profiling of S2 cells overexpressing wildtype or polymerization-defective Ph

city: Montreal

first: Nicole

last: Francis

state: Quebec

address: 110 Ave des Pins Ouest

institute: IRCM/University of Montreal

summary: Chromatin in eukaryotic nuclei is organized at multiple scales, from individual nucleosomes to specific loops between regulatory sequences, to the folding of large genomic regions into topological domains and segregation of whole chromosomes into territories. Many of the chromatin proteins that regulate this architecture, including the essential Polycomb Group (PcG) proteins, are themselves organized into subnuclear structures. Deciphering mechanistic links between protein organization and genome architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, we characterized the nanoscale organization of PcG proteins in Drosophila cells and find hundreds of small protein clusters, distinct from the large PcG bodies present in just a few copies per cell that have been the focus of previous investigations. We manipulated PcG clusters either by disrupting the polymerization activity of the conserved Sterile Alpha Motif (SAM) of the PcG protein Polyhomeotic (Ph) or increasing Ph levels in Drosophila S2 cells. Disrupting clustering using Ph SAM mutations disrupts chromatin interactions on scales from 50kb to 13Mb while increasing Ph levels increases both cluster number and long range chromatin interactions. RNA-seq and qPCR indicate that both perturbations also alter expression levels of many genes.  Molecular simulations suggest a model in which PcG cluster formation on chromatin is governed by the kinetics of association between Ph SAMs and PcG cluster size is bounded by the affinity and occupancy of chromatin binding sites.  Our results suggest that nanoscale organization of PcG proteins into small, abundant clusters on chromatin through the polymerization activity of Ph SAM shapes genome architecture by mediating numerous long-range chromatin interactions.

overall_design: Two biological replicates of three RNA-seq samples from S2 cells, cells overexpresing wild-type Ph, and cells overexpressing polymerization defective Ph-ML

first: Ajazul

last: Wani


title: Impact of age and hypoxia on proliferation and microRNA expression profile of bone marrow-derived human mesenchymal stem cells

city: Cheras

first: norlaily

last: mohd ali

state: Selangor

address: Universiti Tunku Abdul Rahman, Jln Sg Long

department: Faculty of Medicine and Health Sciences

country: Malaysia

institute: Universiti Tunku Abdul Rahman

type: Non-coding RNA profiling by high throughput sequencing

summary:
Intro: Donor age has been widely demonstrated to affect the characteristic of multipotent mesenchymal stem cells derived from bone marrow (BM-MSCs). The cellular features including decrease in cell proliferation, differentiation capacity and telomere length as well as increase of senescence may reduce the yield and clinical application for cell therapy.
Methods: Expansion of BM-MSCs from young and aged donors under prolonged passage (P15) and hypoxic (5% O2) platforms was performed and the biological, cellular and miRNA profiles were assessed using Next Generation Sequencing.
Results: miRNA sequencing revealed differential expression of hypoxia-inducible miRNAs when young donor MSC was cultured under hypoxia.Here, we demonstrate for the first time the link between age, hypoxia and microRNA expression and conclude that MSC isolated from both young and aged donors have a greater expansion potential when cultured under hypoxic condition.

overall_design: miRNA profiles of bone marrow MSC from young and aged donors expanded under normal and hypoxic culture condition were generated in triplicate by deep sequencing using TruSeq small RNA illumina

first: Norlaily

last: Ali

first: Lily

last: Boo

first: Swee

middle: K

last: Yeap

first: Huynh

last: Ky

first: Dilan

last: Satharasinghe

first: Woan

last: Liew

first: Han

last: Ong

first: Soon

last: Cheong

first: Tunku

last: Kamarul


title: Functional neurons generated from T cell-derived iPSCs for neurological disease modeling [MBD-seq]

city: Shinjuku-ku

first: Takuya

last: Matsumoto

state: Tokyo

address: 35 Shinanomachi

country: Japan

institute: Keio University School of Medicine

type: Methylation profiling by high throughput sequencing

summary: In this study we determine the genomic methylation profile by MBD-seq of T cell derived iPSCs and aHDF derived iPSCs.

overall_design: T cell derived iPSCs and aHDF derived iPSCs subjected to MBD-seq analysis.

first: T

first: K

last: Fujimori

first: W

last: Akamatsu

first: H

last: Okano


title: The Histone Variant MacroH2A1.2 is Necessary for the Activation of Muscle Enhancers and Recruitment of the Transcription Factor Pbx1

city: Bethesda

first: Hossein

last: Zare

address: 5o South Dr #1347

institute: NIH

summary: Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart due to the presence of a ~25kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation.

overall_design: To establish the role of the  histone variant mH2A1.2 in skeletal muscle differentiation we employed the mouse skeletal muscle C2C12 cell line and examined the genome wide distribution mH2A1.2  in myoblast (MB) and myotube (MT) (two replicates). We intersected the distribution of mH2A1.2 with active (H3K4me3 and H3K4me1 from published dataset, and H3K27ac, two replicates) and repressive (H3K27me3, two replicates) epigenetic marks in MB and MT. To gain insite on how  chromatin accessibility is remodelled when muscle cells are induced to differentiate, we performed  ATAC-seq in C2C12 MB and MT (2 replicates). We then evaluated whether mH2A1.2 was involved in conferring H3K27 acetylation during skeletal muscle differentiation by performing H3K27ac ChIP-seq (two replicates) upon mH2A1.2i in MB and MT. We also prefomred the ChIP-Seq for transcription factor Pbx1 in control and mH2A1.2i cells in MT to address the potential role of mH2A in recruitment of Pbx1.   RNA-seq experiments were performed in control and mH2A1.2i C2C12 cells at the stage of MB and MT (three replicates). When mH2A1.2i C2C12 MB were induced to differentiate, a global effect on transcription was observed.

first: Stefania

last: Dell’Orso

first: A

last: Wang

first: Han-Yu

last: Shih

first: Kayoko

last: Saso

first: Libera

last: Berghella

first: Gustavo

last: Gutierrez-Cruz

first: Andreas

last: Ladurner

first: John

middle: J

last: O’Shea

first: Vittorio

last: Sartorelli


title: Functional neurons generated from T cell-derived iPSCs for neurological disease modeling

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

overall_design: Refer to individual Series


title: Transcriptome analysis of salt stress responsiveness in the seedlings of Dongxiang wild rice (Oryza rufipogon Griff.)

status: Public on Jan 13 2016

city: Nanchang

first: Yi

last: Zhou

state: Jiangxi

address: No. 99, Ziyang Rd

country: China

institute: Jiangxi Normal University

summary: Dongxiang wild rice (Oryza rufipogon Griff.) is the progenitor of cultivated rice (Oryza sativa L.) and is well known for its superior level of tolerance against cold, drought and diseases. To date, however, little is known about the salt-tolerant character of Dongxiang wild rice. To elucidate the molecular genetic mechanisms of salt-stress tolerance in Dongxiang wild rice, the Illumina HiSeq 2000 platform was used to analyze the transcriptome profiles of the leaves and roots at the seedling stage under salt stress compared with those under normal conditions. The analysis results for the sequencing data showed that 6,867 transcripts were differentially expressed in the leaves (2,216 up-regulated and 4,651 down-regulated) and 4,988 transcripts in the roots (3,105 up-regulated and 1,883 down-regulated). Among these differentially expressed genes, the detection of many transcription factor genes demonstrated that multiple regulatory pathways were involved in salt stress tolerance. In addition, the differentially expressed genes were compared with the previous RNA-Seq analysis of salt-stress responses in cultivated rice Nipponbare, indicating the possible specific molecular mechanisms of salt-stress responses for Dongxiang wild rice. A large number of the salt-inducible genes identified in this study were co-localized onto fine-mapped salt-tolerance-related quantitative trait loci, providing candidates for gene cloning and elucidation of molecular mechanisms responsible for salt-stress tolerance in rice.

overall_design: Leaf and root mRNA profiles of Dongxiang wild rice at the seedling stage with or without salt stress were generated by deep sequencing, on Illumina Hiseq 2000 platform.

first: Jiankun

last: Xie


title: Clarifying the translational pausing landscape in bacteria by ribosome profiling

city: Baltimore

first: Allen

middle: R

last: Buskirk

state: MD

address: 725 N. Wolfe St

department: Molecular Biology and Genetics

institute: Johns Hopkins University School of Medicine

summary: The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Ribosome profiling (three replicates) and RNAseq (two replicates) of E. coli MG1655


title: Mode of action analysis: Distinct Staphylococcus aureus transcriptome changes upon antivirulence hydroxy amide treatment

city: Garching

first: Jan

last: Vomacka

address: Lichtenbergstraße 4

department: Department of Chemistry

institute: Technische Universität München

summary: To elucidate the antivirulent hydroxy amide (R*,R*)-3 mode of action, next generation sequencing was applied to analyze the transcriptome of NCTC 8325 cells treated with either compound or DMSO as control.

overall_design: Two biological replicates for compound treated and also two biological replicates for DMSO treated Staphylococcus aureus cultures were analysed.

first: Bianca

last: Schwanhäußer

first: Katrin

last: Lorenz-Baath


title: Satb1 Overexpression Drives Tumor-Promoting Activities In Cancer-Associated Dendritic Cells

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Special AT-rich sequence-binding protein-1 (Satb1) governs genome-wide transcriptional programs. Using a new conditional knockout mouse, we found that Satb1 is required for normal differentiation of conventional dendritic cells (DCs). Furthermore, Satb1 governs the differentiation of inflammatory DCs by regulating MHC-II expression through Notch1 signaling. Mechanistically, Satb1 binds to the Notch1 promoter, activating Notch expression and driving RBPJ occupancy of the H2-Ab1 promoter, which activates MHC-II transcription. However, tumor-driven, unremitting expression of Satb1 in activated Zbtb46+ inflammatory DCs that universally infiltrate ovarian tumors results in an immunosuppressive phenotype characterized by increased secretion of tumor-promoting Galectin-1 and IL-6. Correspondingly, specific in vivo silencing of Satb1 in tumor-associated DCs reverses their tumorigenic activity and boosts protective immunity. Therefore, dynamic fluctuations in Satb1 expression govern the generation and immunostimulatory activity of steady-state and inflammatory DCs, but relentless Satb1 overexpression in differentiated DCs converts them into tolerogenic/pro-inflammatory cells that contribute to malignant progression.

overall_design: RNA-seq with whild type and knocked-down Satb1

first: Jose

last: Conejo-Garcia

first: Amelia

last: Tesone


title: Polynucleotide phosphorylase can interact with small noncoding RNAs in both stabilizing and degradative modes

city: Houston

first: Nicholas

last: De Lay

state: Texas

address: 6431 Fannin St

department: Microbiology and Molecular Genetics

institute: University of Texas Health Science Center

summary: In all bacterial species examined thusfar, small regulatory RNAs (sRNAs) contribute to intricate patterns of genetic regulation. Many of the actions of these nucleic acids are mediated by chaperones such as the Hfq protein, and genetic screens have identified the exoribonuclease polynucleotide phosphorylase (PNPase) as a stabilizer and facilitator of sRNAs in vivo. We observe that the protective and facilitating effects of PNPase in vivo require the RNA-binding KH and S1 domains and the catalytic site, suggesting a requirement for physical interation of the ribonuclease with either the sRNA itself or other RNAs acting upstream of the process. Although purified PNPase can readily degrade sRNAs in vitro, those same substrates, as well as numerous other sRNAs and transcripts, can be co-purified from cells by immunoprecipitation with neither degradation nor modification to the 3’ end.  Our results indicate that PNPase can bind RNA in two modes in vivo – either destructive or stabilizing, and that there is active flux of RNAs on PNPase so that the stable molecules do not accumulate. In the presence of Hfq, PNPase and sRNA form a ternary complex in which the enzyme plays a non-destructive, structural role, but the complex does not confer protection against the action of RNase E in vitro. Taken together, our results indicate that PNPase, Hfq and additional factors form a protective ribonucleoprotein assembly that stabilizes certain sRNAs and facilitates their activities.

platform_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Using cells from the same original culture, immunoprecipitiations using the anti-FLAG M2 resin were performed in the presence or absence of tungstate.  Total RNA and immunoprecipitated RNA were sequenced from two independent biological replicates.  The number of normalized reads (RPKM) were compared between the total RNA (input) and the immunoprecipitated RNA (output).


title: Regulation of gene expression in SK-N-SH cell line

city: Basel

first: Quan

last: Xu

address: Grenzacherstrasse 124

institute: Institute of Human Biology (IHB)

summary: The closely linked human protocadherin (Pcdh) α, β, and γ gene clusters encode 53 distinct protein isoforms, which are expressed in a combinatorial manner to generate enormous diversity on the surface of individual neurons. This diversity is a consequence of stochastic promoter choice and alternative pre-mRNA processing. Here, we show that Pcdhα promoter choice is achieved by DNA looping between two downstream transcriptional enhancers and individual promoters driving the expression of alternate Pcdhα isoforms. In addition, we show that this DNA looping requires specific binding of the CTCF/cohesin complex to two symmetrically aligned binding sites in both the transcriptionally active promoters and in one of the enhancers. These findings have important implications regarding enhancer/promoter interactions in the generation of complex Pcdh cell surface codes for the establishment of neuronal identity and self-avoidance in individual neurons.

overall_design: Examination of CTCF, Rad21, RNAPII and 2 different histone modifications in SK-N-SH

first: Ya

last: Guo

first: Daniele

last: Canzio

first: Jia

last: Shou

first: Jinhuan

last: Li

first: David

middle: U

last: Gorkin

first: Inkyung

last: Jung

first: Haiyang

last: Wu

first: Yanan

last: Zhai

first: Yuanxiao

last: Tang

first: Yichao

last: Lu

first: Yonghu

first: Zhilian

last: Jia

first: Wei

first: Michael

middle: Q

last: Zhang

first: Bing

last: Ren

first: Adrian

last: Krainer

first: Tom

last: Maniatis

first: Qiang


title: Genome-wide hydroxymethylcytosine pattern changes in response to oxidative stress

city: Bruxelles

first: Matthieu

last: Defrance

address: CP614 route de Lennik 808

institute: Université Libre de Bruxelles

summary: The TET-family enzymes (TETs) convert methylcytosine to hydroxymethylcytosine, a lately discovered epigenetic modification that can modulate transcription. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain. Here we show that Tet1 is sensitive to peroxide and report a global decrease in hydroxymethylcytosine in cells treated with BSO and in the intestinal epithelium of mice lacking the major antioxidant enzymes glutathione peroxidases 1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in genes involved in responses to oxidative stress. Intriguingly, a considerable proportion of these regions lie in genes encoding microRNAs predicted to target transcripts involved in oxidative stress response. This work thus demonstrates a profound effect of oxidative stress on the hydroxymethylome and opens exciting new avenues of research by highlighting a set of microRNAs that may participate in the prevention or etiology of oxidative-stress-related diseases.

overall_design: Examination of DNA hydroxymethylation landscape in SY5Y cell lines and in intestinal epithelium of mice.

first: Benjamin

last: Delatte

first: Jana

last: Jeschke

first: Catherine

last: Creppe

first: Emilie

last: Calonne

first: Martin

last: Bizet

first: Rachel

last: Deplus

first: Laura

last: Marroquí

first: Myriam

last: Libin

first: Françoise

last: Mascart

first: Decio

middle: L

last: Eizirik

first: Albert

last: Jeltsch

first: Tomasz

middle: P

last: Jurkowski

first: François

last: Fuks


title: Identification and characterization of transcript polymorphisms in soybean lines varying in oil composition and content

city: St. Louis

first: Yong-qiang (Charles)

last: An

address: 975 North Warson Road

department: Plant Genetics Research

institute: United States Department of Agriculture, Agricultural Research Service at Donald Danforth Plant Science Center

summary: Genetic/genome diversity underlying variation in seed oil composition and content among soybean varieties is largely attributed to differences in transcript sequences and/or transcript accumulation of oil production related genes in seeds. Discovery and analysis of sequence and expression variations in these genes will accelerate soybean oil quality improvement. In an effort to identify these variations, we sequenced the transcriptomes of soybean seeds from nine lines varying in oil composition and/or total oil content. Our results showed that 69,338 distinct transcripts from 32,885 annotated genes were expressed in seeds. A total of 8,037 transcript expression polymorphisms and 50,485 transcript sequence polymorphisms (48,792 SNPs and 1,693 small Indels) were identified among the lines.  Effects of the transcript polymorphisms on their encoded protein sequences and functions were predicted. The studies also provided independent evidence that the lack of FAD2-1A gene activity and a non-synonymous SNP in the coding sequence of FAB2C caused elevated oleic acid and stearic acid levels in soybean lines M23 and FAM94-41, respectively. As a proof-of-concept, we developed an integrated RNA-seq and bioinformatics approach to identify and functionally annotate transcript polymorphisms, and demonstrated its high effectiveness for discovery of genetic and transcript variations that result in altered oil quality traits. The collection of transcript polymorphisms coupled with their predicted functional effects will be a valuable asset for further discovery of genes, gene variants, and functional markers to improve soybean oil quality.

overall_design: transcriptome comparison of nine different soybean varieties

first: Yong-qiang

first: Wolfgang

last: Goettel

first: Rick

last: Meyer


title: T-TRAP Profiling Reveals Dynamic Changes in the Transcriptome during Circuit Assembly

city: Los Angeles

first: Lawrence

middle: S.

last: Zipursky

address: Howard Hughes Medical Institu675 Charles E. Young Drive South 5-784 MacDonald Research Laboratories

institute: University of California, Los Angeles

summary:
Purpose: Communication between growth cones and their environment plays a central role in assembling neural circuits. We use Tandemly-Tagged Ribosome Affinity Purification (T-TRAP) of mRNA from R cells followed by RNA-seq for multiple time points during development to follow gene expression during target selection and synapse formation.
Methods: We chose a ribosome trap method by modifying the N-terminus of the Drosophila ribosomal protein RpL10 with two tandemly arranged epitopes, 3X FLAG and GFP, separated by the Tobacco Etch Virus (TEV) protease site and expressed this in specific cell types using the GAL4/UAS system. cDNA libraries were prepared from mRNA associated with the affinity purified ribosomes and sequenced using an Illumina HiSeq 2000. We mapped raw reads to the D. melanogaster reference genome (release FB2013_01) with the gapped aligner Tophat. Only reads uniquely aligned were collected.Transcript expression levels were quantified using RPKM units using customized scripts written in Perl.
Results: In this study, we observed massive changes in expression of cell surface proteins over short time scales (i.e. 5 fold differences in the expression of many hundreds of genes over 5 hr intervals) as R cell growth cones encounter the processes of many different neurons during their conversion from growth cones to synaptic terminals. In addition, to changes in transcripts encoding cell surface proteins, other mRNAs changed significantly as did non-coding RNAs (lincRNAs) associated with ribosomes. Although dramatic changes in transcript levels of presynaptic proteins were not observed preceding the onset of synapse formation, marked changes in the 3'-untranslated regions of these transcripts were seen.
Conclusions: These studies provide a step towards merging traditional genetic and global genomic approaches to understanding cellular recognition underlying the assembly of neural circuits.

overall_design: We chose 7 time points for RNA-seq analysis of R cells during pupal development corresponding to 24, 35, 40, 45, 53, 65 and 96 hrs after pupal formation (APF).

first: S

first: Kelvin

last: Zhang

first: Jason

last: McEwen


title: Quantitative reduced representation bisulfite sequencing with unique molecular identifiers of single cell

city: Chong Qing

first: Xianfeng

last: Li

address: Da ping

department: Department of Gastroenterology, Daping Hospital

institute: Army Medical University (Third Military Medical University)

summary: Reduced representation bisulfite sequencing (RRBS) has been proven a powerful method in DNA methylome profiling. Since the initial development of this method, the RRBS protocol has been modified in order to optimize it for genomic coverage, starting material, and library-construction throughput, which has resulted in new methods such as enhanced RRBS (ERRBS), double-enzyme RRBS (dRRBS), gel-free and multiplexed RRBS (mRRBS), and single-cell RRBS (scRRBS). However, each of these methods has failed to address PCR-derived duplication artifacts, which can bias the results of DNA methylation analyses. To overcome the aforementioned complication, we developed quantitative RRBS (Q-RRBS), a method in which unique molecular identifiers (UMIs) are used to eliminate PCR-induced duplication. By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.

overall_design: Quantitative Reduced Representation Bisulfite Sequencing (Q-RRBS) was performed on genomic DNA, single-cell (SC) samples to quantify the effects of duplication-induced artifacts on methylome profiling when using ultra-trace amounts of starting material.

first: Kangli

last: Wang

first: Zhongsheng

last: Sun


title: The Histone Methyltransferases MLL1 and DOT1L Cooperate with Meningioma-1 to Induce AML [Mouse Dot1l ko RNA-seq]

status: Public on Jan 12 2016

city: Aurora

first: Tobias

last: Neff

state: CO

department: Pediatric Oncology

institute: University of Colorado Denver AMC

summary:
Purpose: To characterize transcriptional changes associated with homozygous inactivation of Dot1l or Mll1 in MN1 driven AML
Methods: We sequenced mRNA from murine LSK-cells transformed using forced expression of MN1 (MSCV-MN1-IRES-GFP), and transduced with Cre-vector to inactivate either Dot1l or Mll1. Cells were sorted for Cre-expression (pTomato fluorescent marker) or expression of an inert control vector.
Results: Inactivation of either Dot1l or Mll1 in this model leads to a substantial delay or complete abrogation of leukemia development. Loss of Dot1l or Mll1 are associated with gene expression changes that have substantial overlap. In addition, genes that are downregulated follwing inactivation of Dot1l or Mll1 have substantial overlap with the gene set upregulated in MN1 transduced CMPs.
Conclusions: MN1 mediated leukemogenesis is associated with a gene expression program that dependes on Mll1 and Dot1l

overall_design: Examination of mRNA levels between Dot1l f/f and Dot1l ko, and Mll1 f/f  and Mll1 ko.

first: Simone

last: Riedel

first: Kathrin

last: Bernt


title: The Histone Methyltransferases MLL1 and DOT1L Cooperate with Meningioma-1 to Induce AML [Human ChIP-seq]

summary:
Purpose: To characterize the genome-wide distribution of H3K79me2 in human leukemia cell lines treated with the Dot1l inhibitor EPZ004777 or control
Methods: We performed Chip-seq for the H3K79me2 on the leukemia cell lines Mutz3, Loucy and Molm14 after 6 days in culture in the presence of 3uM EPZ004777 or DMSO control
Results: H3K79me2 is completely erased from key target genes such as the HOXA cluster.
Conclusions: Exposure of Mutz3, Loucy and Molm14 to 3uM EPZ004777 erases H3K79 methylation globally as well as on key loci

overall_design: ChIP-Seq for H3K79me2 on human leukemia cell lines exposed for 6 days to the Dot1l inhibitor EPZ004777


title: Effect of mitochondria deficiency on senescence-associated gene expression

city: Glasgow

first: Peter

last: Adams

address: Switchback Rd, Bearsden

country: United Kingdom

institute: University of Glasgow, Beatson Institute for Cancer Research

summary: We used parkin –overexpressing MRC5 fibroblasts to investigate the role of mitochondria deficiency on senescence-associated gene expression.

overall_design: RNA-seq analysis on proliferating and senescent Parkin-expressing MRC5 fibroblasts treated with CCCP (treated) or DMSO (Untreated).

first: Clara

last: Melo

first: Joao

last: Passos

first: John

last: Cole


city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

country: France

institute: IPMC/CNRS

summary: Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells.

overall_design: Ribosome profiling or stranded RNAseq (ribominus) with murine embryonic stem cells treated with either DMD-pateamineA, Puromycin, Harringtonine or vehicle (no drug control). Two replicates per condition.

first: R

last: Waldmann

first: A

last: Popa

first: K

first: P

last: Barbry


title: The Histone Methyltransferases MLL1 and DOT1L Cooperate with Meningioma-1 to Induce AML


title: Effects on the transcriptome upon deletion of distal elements are not correlated with the size of H3K27Ac peaks in human cells

city: LA

first: Peggy

last: Farnham

address: 1450 Biggy St.

department: GMCB

institute: University of Southern California

summary: Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for colorectal cancer (CRC). A molecular understanding of the functional consequences of this genetic variation is complicated because most GWAS SNPs are located in non-coding regions. After identifying H3K27Ac peaks in HCT116 colon cancer cells that harbor SNPs associated with an increased risk for CRC, we used CRISPR/Cas9 nuclease to delete 2 CRC risk-associated H3K27Ac peaks (E7 and E24), a peak that is not associated with CRC risk (18qE) and a region that doesn?t have H3K27Ac peak (18qNE) from HCT116 cells and analyzed effects on the transcriptome and epigenome. We also deleted E7 region from HEK 293 cells and analyzed effects on the transcriptome of HEK 293. We also confirmed the physical interaction between enhancers of our interest and their putative target genes.

overall_design: Analysis of RNA-seq data and ChIP-seq between control clones and enhancer deleted clones in HCT116 cell. For Control, gRNA empty vectorplasmid was transfected with Cas9-GFP. For Deletion, gRNAs that have enhancer target sequences were transfected along with Cas9-GFP. Cells with high GFP expression were identified using fluorescence-activated cell sorting. Sorted cells were plated into individual wells of a 24 well plate and then re-plated as single cells in 10cm dishes and subsequently expanded for further analyses. Samples used in this study were clonal populations. Interaction profiling of enhancers of interest with putative target genes using 4C-seq

first: Yu

middle: G

last: Tak


title: Epigenomic annotation of gene regulatory alterations during evolution of the primate brain

city: Rotterdam

first: Menno

last: Creyghton

address: Doctor Molewaterplein 40

country: Netherlands

institute: Erasmus MC

summary: While genome sequencing has identified numerous non-coding alterations between primate species, which of these are regulatory and potentially relevant to the evolution of the human brain is unclear. Here, we annotate cis-regulatory elements (CREs) in the human, rhesus macaque and chimpanzee genome using ChIP-sequencing in different anatomical parts of the adult brain. We find high similarity in the genomic positioning of CREs between rhesus macaque and humans, suggesting that the majority of these elements were already present in a common ancestor 25 million years ago. Most of the observed regulatory changes between humans and rhesus macaque occurred prior to the ancestral separation of humans and chimpanzee, leaving a modest set of regulatory elements with predicted human-specificity. Our data refine previous predictions and hypotheses on the consequences of genomic changes between primate species, and allow the identification of regulatory alterations relevant to the evolution of the brain.

sample_organism:
Macaca mulatta
Pan troglodytes
Homo sapiens
Mus musculus

overall_design: ChIP-Sequencing for H3K27ac on 8 distinct brain regions from human (three biological replicates per brain region), chimpanzee (two biological replicates per brain region) and rhesus macaque (three biological replicates per brain region).

first: Marit

middle: W

last: Vermunt

first: Sander

last: Tan

first: Bas

last: Castelijns

first: Geert

last: Geeven

last: Reinink

first: Ewart

last: de Bruijn

first: Ivanela

last: Kondova

first: Stephan

last: Persengiev

first: Ronald

last: Bontrop

first: Edwin

last: Cuppen

first: Wouter

last: de Laat

last: Creygthon


title: Stromal cells secrete exosomes carrying specific molecular signature involved in the support of Hematopoietic Stem and Progenitor Cells

city: Barcelona

first: Gregoire

last: Stik

state: select a state

address: 88 Dr Aiguader

country: Spain

institute: CRG

summary:
Purpose: The study focused on the RNA content of supportive (AFT024) and non supportive (BFC012) stromal lines and their respective exosomes to analyze the molecular complexity of the Hematopoietic Stem and Progenitor Cell niche
Methods: All the samples from small RNA (SR) and polyA-RNA libraries were sequenced using 1×50 bp single reads high-throughput sequencing (RNA-Seq) in single lane on Illumina HiSeq 2500.  The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks and by Fisher's test for polyA-RNA, or with bowtie and parse tools for smallRNA (galaxy server https://lbcd41.snv.jussieu.fr/).
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10). We focused on differentially expressed transcripts between i) exosomes from supportive and non supportive stromal cells and ii) exosomes and their respective cells.Using a subtractive strategy, we identified 324 mRNAs and 23 miRNAs specific to the exosomes of the supportive stromal line AFT024. We used gene ontology annotation to study the biological functions associated to these specific gene sets. Transcripts involved in negative regulation of apoptosis were found enriched in AFT exosomes.
Conclusions: Our RNA seq analysis combined with biological assays reveal that exosomes serve as an important and novel mediator for the HSPC supporting capacity of stromal cells. This unprecedented effort to resolve the molecular complexity of the HSPC-targeted exosomes, provide new candidat genes of the HSPC support and may help designing innovative stromal-free culture conditions to deliver specific molecules to HSPCs.

overall_design: Fetal liver derived stromal lines and their corresponding secreted exosomes were sequenced using Illumina HiSeq2500 technology (Fasteris, CH). Due to low amount of exosomal RNAs, several RNA extraction were pooled and split to perform 2 technical sequencing replicates that were merged during bionformatic analyses.

first: G

first: C

last: Durand


title: Differentiation of Mammary Tumors and Reduction in Metastasis Upon Malat1 LncRNA Loss

city: Dunedin

first: Sarah

last: Diermeier

address: 710 Cumberland Street

department: Biochemistry

country: New Zealand

institute: University of Otago

summary: Genome-wide analyses have identified thousands of long non-coding RNAs (lncRNAs). Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genomic loss, as well as systemic knockdown of Malat1 using antisense oligonucleotides, in the MMTV-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by differentiation into highly cystic tumors and a significant reduction in lung metastasis. Further, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT and Her2/neu amplified tumor organoids consistent with the in vivo reduction in lung metastasis. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and pro-tumorigenic signaling pathways. Together, these data indicate that the lncRNA Malat1 regulates critical processes in mammary cancer pathogenesis and represents a promising therapeutic target for inhibiting breast cancer metastasis.

overall_design: Transcriptome profiles of tumors and organoids after Malat1 knockdown using antisense olgonucleotides (ASOs).

first: Gayatri

last: Arun

first: Kung-Chi

last: Chang

first: David

last: Spector


title: RNA-seq of Mesp1-induced cardiopharyngeal mesoderm derivatives from ES cells

city: Minneapolis

first: Sunny

middle: Sun-Kin

last: Chan

state: MN

department: Pediatrics

institute: University of Minnesota

summary: We have developed a protocol to generate cardiopharyngeal mesoderm (CPM) in vitro by Mesp1 induction in ES cells.  The goal of this study is to compare the transcriptome of CPM-derived cardiac and skeletal myogenic progenitors to identify novel lineage-specific markers.

overall_design: mRNA profiles of CPM-derived D6 (early) and D12 (late), cardiac (BMP) and skeletal myogenic (control) progenitors were generated

middle: S

first: Michael

last: Kyba

first: Scott

middle: A

last: Swanson

first: Ron

last: Stewart

first: James

last: Thomson


title: Transcriptome profiling in knock-in mouse models of Huntington's disease. [gonadal adipose mRNA]

status: Public on Jan 11 2016

city: Princeton

first: Jeff

last: Aaronson

state: New Jersey

address: 155 Village Boulevard, Suite 200

department: Discovery & Systems Biology

institute: CHDI

summary: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that is characterized by motor, cognitive, and psychiatric alterations. The mutation responsible for this disease is an abnormally expanded and unstable CAG repeat within the coding region of the gene encoding huntingtin (Htt). Knock-in mouse models of HD with human exon 1 containing expanded CAG repeats inserted in the murine huntingtin gene (Hdh) provide a genetic reconstruction of the human causative mutation within the mouse model. The goal of this study is RNA expression profiling by RNA sequencing (RNA-seq) in 6 month old knock-in mice with CAG lengths of 20, 80, 92, 111, 140, 175 along with littermate control wild-type animals

overall_design: mRNA expression profiles were obtained via RNA-seq analysis performed on samples from the White Adipose tissue around Gonad of 6 month old knock-in mice with CAG lengths of 20, 80, 92, 111, 140, 175 along with littermate control wild-type animals.

first: Jim

last: Rosinski


title: The Histone Methyltransferases MLL1 and DOT1L Cooperate with Meningioma-1 to Induce AML [Mouse Mll1 ko RNA-seq]

first: Tobias

last: Neff

summary:
Purpose: To characterize transcriptional changes associated with homozygous inactivation of Dot1l or Mll1 in MN1 driven AML
Methods: We sequenced mRNA from murine LSK-cells transformed using forced expression of MN1 (MSCV-MN1-IRES-GFP), and transduced with Cre-vector to inactivate either Dot1l or Mll1. Cells were sorted for Cre-expression (pTomato fluorescent marker) or expression of an inert control vector.
Results: Inactivation of either Dot1l or Mll1 in this model leads to a substantial delay or complete abrogation of leukemia development.Loss of Dot1l or Mll1 are associated with gene expression changes that have substantial overlap. In addition, genes that are downregulated follwing inactivation of Dot1l or Mll1 have substantial overlap with the gene set upregulated in MN1 transduced CMPs.
Conclusions: MN1 mediated leukemogenesis is associated with a gene expression program that dependes on Mll1 and Dot1l

first: Simone

last: Riedel

first: Kathrin

last: Bernt


title: Identification and characterization of Wor4, a new transcriptional regulator of white-opaque switching

city: San Francisco

first: Matthew

last: Lohse

address: 44 Vicksburg Street

institute: BioSynesis, Inc.

summary: The human fungal pathogen Candida albicans can switch between two cell types, “white” and “opaque,” each of which is heritable through many cell divisions. Switching between these two cell types is regulated by six transcriptional regulators which form a highly interconnected circuit with multiple feedback loops. Here, we identify a seventh regulator of white-opaque switching, which we have named Wor4. We show that ectopic expression of Wor4 is sufficient to drive switching from the white to the opaque cell type and that deletion of Wor4 blocks switching from the white to the opaque cell type. A combination of ectopic expression and deletion experiments indicates that Wor4 is positioned upstream of Wor1 and that it is formally an activator of the opaque cell type. The combination of ectopic expression and deletion phenotypes for Wor4 is unique; none of the other six white-opaque regulators show this pattern. We determined the pattern of Wor4 binding across the genome by ChIP-seq and found it is highly correlated with that of Wor1 and Wor2, indicating that Wor4 is tightly integrated into the existing white-opaque regulatory circuit. We previously proposed that white-to-opaque switching relies on the activation of a complex circuit of feedback loops that remains excited through many cell divisions. The identification of a new, central regulator of white-opaque switching supports this idea by indicating that the white-opaque switching mechanism is considerably more complex than those controlling conventional, non-heritable patterns of gene expression.

overall_design: C. albicans Wor4-GFP versus untagged control in both white and opaque cell types. Cells grown in SD+aa+Uri at 25°C with three technical replicates of each strain/cell type (12 total).

middle: B


title: Integrative analysis identifies targetable CREB1/FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence

city: Columbus

first: Qianben

last: Wang

state: Ohio

address: 460 W 12th Avenue

department: Molecular and Cellular Biochemistry and the Comprehensive Cancer Center

institute: Ohio State University

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this work is comprehensive analysis of target genes co-regulated by CREB1 and FoxA1 in prostate cancer cell models, tissues, and circulating tumor cells (CTCs). Expression of CREB1/FoxA1 target genes corresponds with disease recurrence and aggressive clinical features.
Methods: LNCaP cells between passage number 32-34 and abl between 62-64 were used for assay. For RNA-seq, cells are transfected with FoxA1,CREB1 specific or nonspecific siRNA and total RNA is extracted with Qiangen RNeasy Mini kit(cat 74104) and library is generated with  TruSeq RNA Sample Preparation Kit v2 from Illumina(cat  RS-122-2001). For Chip-seq,  LNCaP cells between passage number 32-34 and abl between 62-64 were used for assay. After crosslinking with 1% formaldehyde, ChIP was performed. the ChIP producted was further used to generate library with illumina ChIP-seq kit. Hi-seq 2500 was used for sequencing and the data was analyzed by MACs for peaks.

overall_design: LNCaP cells and LNCaP cells were used as cell model. Library was sequenced using Illumina HISeq 2500.

first: Dayong

last: Wu

first: Xiangtal

last: Liu

first: Zhenqing

last: Ye

first: Benjamin

middle: D

last: Sunkel


title: Transcriptome profiling in knock-in mouse models of Huntington's disease. [gonadal adipose miRNA]

overall_design: miRNA expression profiles were obtained via RNA-seq analysis performed on samples from the White Adipose tissue around Gonad of 6 month old knock-in mice with CAG lengths of 20, 80, 92, 111, 140, 175 along with littermate control wild-type animals.


title: Transcriptome profiling in knock-in mouse models of Huntington's disease [gonadal adipose mRNA and miRNA]

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

overall_design: Refer to individual Series


title: The Histone Methyltransferases MLL1 and DOT1L Cooperate with Meningioma-1 to Induce AML [Human RNA-seq]

summary:
Purpose: To characterize transcriptional changes associated with inhibition of Dot1l in 2 inv(16) patient AML samples
Methods: We sequenced mRNA from patient samples that were exposed to 5 uM EPZ004777 or DMSO control for 7  days.
Results: Inhibition of Dot1l leads to gene expression changes in genes related to cell growth and cell cycle.

overall_design: Examination of mRNA levels between cells treated with  5 uM EPZ004777 or DMSO control


title: Alternative cleavage and polyadenylation in spermatogenesis coordinates chromatin regulation and post-transcriptional control

city: Bridgewater

first: Wencheng

last: Li

institute: PTC Therapeutics

summary: Most mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3’UTRs in testes. Here we show widespread shortening of 3’UTR by APA during the first wave of spermatogenesis in mouse, with 3’UTRs being the shortest in spermatids. Shortening of 3’UTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear to be highly potent for transcript elimination during spermatogenesis. We additionally found widespread regulation of APA in introns and global activation of upstream antisense transcripts during spermatogenesis. Interestingly, genes that display 3’UTR shortening tend to have higher levels of H3K4me3, consistent with the open chromatin feature previously observed in spermatids. Since genes with 3’UTR shortening tend to have functions important for further sperm development after spermatids, when transcription is halted, this result indicates that expression of short, stable mRNAs may serve the purpose of mRNA storage for later translation. Thus, APA in spermatogenesis connects regulation of chromatin status with post-transcriptional control, and impacts sperm maturation.

overall_design: 3'READS of 1 week to 6 week of testis development

first: JiYeon

last: Park

first: Dinghai

last: Zheng

first: Mainul

last: Hoque

first: Ghassan

last: Yehia

first: Bin

last: Tian


title: NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers  [ChIP-seq]

city: St Louis

first: Sabine

last: Dietmann

address: 4565 McKinley Avenue

institute: Washington University School of Medicine

summary: Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.

overall_design: Nanog ChIP-seq

first: Kazuhiro

last: Murakami

first: Ufuk

last: Günesdogan

first: Walfred

last: Tang

first: Jan

last: Zylicz

first: Roopsha

last: Sengupta

first: Toshihiro

last: Kobayashi

first: Shinseog

last: Kim

first: Azim

last: Surani


title: NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers

summary: Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naïve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naïve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.


title: The Histone Methyltransferases MLL1 and DOT1L Cooperate with Meningioma-1 to Induce AML [Mouse ChIP-seq]

first: Tobias

last: Neff

summary:
Purpose: To characterize the genome-wide distribution of H3K79me2 in murine MN1 driven myeloid leukemia
Methods: We performed Chip-seq for the H3K79me2 in leukemias isolated from moribund mice that had been injected with common myeloid progenitors (CMPs) transduced with MSCV-MN1-GFP
Results: H3K79me2 is enriched at key loci that 1. are bound by MN1 in the data set of Heuser et al, (Cancer Cell. 2011 Jul 12;20(1):39-52.), 2. upregulated upon transduction with MN1, and lose expression upon deletion of the H3K79 methyltransferase Dot1l.
Conclusions: A leukemogenic program in MN1 leukemias is marked by H3K79me2 and dependent on this mark

overall_design: ChIP-Seq for H3K79me2 using MN1 driven leukemias isolated from the bone marrow of moribund mice.

first: Kathrin

last: Bernt


title: Dynamic control of enhancer repertoires drives lineage and developmental stage-specific transcription during human erythropoiesis

city: Memphis

first: Jian

last: Xu

state: Tennessee

address: 262 Danny Thomas Place, MS 345

department: Pathology

institute: St. Jude Children's Research Hospital

summary: Transcriptional enhancers are the primary determinants of tissue-specific gene expression and influence a variety of cellular phenotypes. The regulatory components controlling enhancer assembly and turnover during stem cell development remain largely unknown. Here we compared the similarities and differences in enhancer landscape, transcriptional factor (TF) occupancy and transcriptomic changes in human primary fetal and adult hematopoietic stem/progenitor cells (HSPCs) and committed erythroid progenitors. We find that enhancers are modulated dynamically and extensively, and direct lineage and developmental stage-specific transcriptional programs. GATA2-to-GATA1 switch is prevalent within transcriptionally dynamic enhancers and drives enhancer commissioning. Further examination of lineage-specific enhancers identified TFs and their combinatorial patterns with known and unknown roles as putative drivers of enhancer turnover during differentiation. Importantly, by site-directed loss-of-function analysis of individual lineage-selective enhancers within the SLC25A37 super-enhancer using CRISPR/Cas9-mediated genomic editing, we uncover unexpected functional hierarchy of constituent enhancers within the super-enhancer cluster. Despite the indistinguishable chromatin features between the GATA switch enhancers at the GATA2 gene, we reveal through genomic editing the functional diversity of GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate gene expression during development. Thus, genome-wide enhancer profiling coupled with in-depth enhancer editing in situ provide critical insights into the functional hierarchy and complexity of enhancers during stem cell development.

overall_design: Analysis of chromatin occupancy of hematopoietic transcription factors (GATA1, GATA2, TAL1, NFE2, IRF2, PU.1, and RUNX1,) and various histone marks (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in primary human fetal liver and adult bone marrow CD34+ HSPCs and differentiated proerythroblasts (ProEs) by ChIP-seq

first: Jialiang

last: Huang

first: Zhen

last: Shao

first: Stuart

middle: H

last: Orkin


title: Increased DNA Methylation of Dnmt3b-Targets Impairs Leukemogenesis

city: Heidelberg

first: Christian

last: Rohde

address: Im Neuenheimer Feld 410

institute: Heidelberg University


title: Pioneer factor FOXA1 defines GATA2- and AR-mediated transcriptional program in prostate cancer [ChIP-Seq]

city: Atlanta

first: Jindan

last: Yu

state: GA

address: E330, 1760 Haygood Dr NE

department: Urology

institute: Emory University

summary: AR is tightly regulated by many transcriptional cofactors, including key pioneer factor such as FOXA1 and GATA2.  While FOXA1 was recently shown to be able to redistribute AR across the genome, how GATA2 regulates AR cistrome has not been carefully investigated.  Here, we report that, unlike FOXA1, GATA2 is unable to reprogram AR, but instead it enhances AR program by inducing AR expression and augmenting AR co-occupancy, thereby acting as a pure AR coactivator, rather than a pioneer factor.  On the other hand, AR co-occupancy enhances both GATA2 and FOXA1 binding on the chromatin, forming a positive feedback loop.  Importantly, we found that FOXA1 is also capable of reprogramming GATA2 by recruiting GATA2 from GATA motif to FKHD-containing regions, being analogous to its pioneering effect on AR signaling.  By contrast, GATA2 simply acts as a co-activator of FOXA1 with a lack of pioneering ability.

overall_design: ChIP_Seq examination of AR, FoxA1 and GATA2 binding sites in LNCaP and DU145 cells

first: Jonathan

last: Zhao


title: Tunable protein synthesis by transcript isoforms in human cells (Transcript Isoforms in Polysomes sequencing: TrIP-seq)

first: Stephen

middle: N.

last: Floor

state: CA

address: 513 Parnassus Ave, HSW 740

department: Cell and Tissue Biology

institute: University of California, San Francisco

summary: Eukaryotic genes generate multiple mRNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell-type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.

overall_design: Total cytoplasmic and eight polysomal fractions of RNA were purified from HEK 293T cells in biological duplicate. Ribosomal RNA was depleted using Ribo-Zero (Human/Mouse/Rat; Epicenter) and libraries were prepared using the TruSeq RNA v2 kit (RS-122-2001; Illumina) skipping the polyA selection step. Reads are paired-end 75bp and sequencing adapters are GATCGGAAGAGCACACGTCTGAACTCCAGTCAC (read1) and AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT (read2).

middle: N

first: Jennifer

last: Doudna


title: Genome–wide transcriptional profiling with spatial resolution identifies Bone Morphogenetic Protein signaling as essential regulator of zebrafish cardiomyocyte regeneration.

city: Utrecht

first: Fabian

middle: Klaus

last: Kruse

address: Uppsalalaan 8

institute: Hubrecht Institute

summary: In contrast to mammals, zebrafish regenerate heart injuries via proliferation of cardiomyocytes located at the wound border. Here, we show that tomo-seq can be used to identify whole-genome transcriptional profiles of the injury zone, the border zone and the healthy myocardium. Interestingly, the border zone is characterized by the re-expression of embryonic cardiac genes that are also activated after myocardial infarction in mouse and human, including targets of Bone Morphogenetic Protein (BMP) signaling. Endogenous BMP signaling has been reported to be detrimental to mammalian cardiac repair. In contrast, we find that genetic or chemical inhibition of BMP signaling in zebrafish reduces cardiomyocyte dedifferentiation and proliferation, ultimately compromising myocardial regeneration, while bmp2b overexpression is sufficient to enhance it. Our results provide a resource for further studies on the molecular regulation of cardiac regeneration and reveal intriguing differential cellular responses of cardiomyocytes to a conserved signaling pathway in regenerative versus non-regenerative hearts.

overall_design: To generate spatially-resolved RNA-seq data for injured zebrafish hearts (3 and 7 days-post-injury), we cryosectioned samples, extracted RNA from the individual sections, and amplified and barcoded mRNA using the CEL-seq protocol (Hashimshony et al., Cell Reports, 2012) with a few modifications. Libraries were sequenced on Illumina NextSeq  using 75bp paired end sequencing.

middle: K

first: Chi-Chung

last: Wu

first: Mohankrishna

last: Vasudevarao

last: Junker

first: David

middle: C

last: Zebrowski

first: Kristin

last: Fischer

first: Emily

last: Noël

first: Dominic

last: Grün

first: Berezikov

last: Eugene

first: Felix

last: Engel

first: Alexander

last: van Oudenaarden

first: Gilbert

last: Weidinger

first: Jeroen

last: Bakkers


title: Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphoma

city: New York

first: Tharu

middle: M

last: Fernando

state: NY

address: 413 East 69th St BB1462

department: Medicine, Heme-Onc

institute: Weill Cornell Medical College

summary: Aggressive double and triple hit (DH/TH) DLBCL feature activation of Hsp90 stress pathways.  Herein, we show that Hsp90 controls post-transcriptional dynamics of key mRNA species including those encoding BCL6, MYC and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E (eukaryotic translation initiation factor 4E). EIF4E drives nuclear export and translation of BCL6, MYC and BCL2 mRNA. eIF4E RIP-sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes BCR signaling, cellular metabolism and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counter-regulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative anti-lymphoma activity in DH/TH DLBCL in vitro and in vivo.

overall_design:
We found that eIF4E activity regulates the nuclear export of BCL6, MYC, and BCL2 in DH/TH DLBCLs. To determine the extent of nuclear eIF4E activity in DH/TH DLBCLs and how these programs can support the oncogenic activity of BCL6, MYC and/or BCL2 transcripts, we conducted eIF4E-RIP of nuclear RNA followed by RNA-sequencing in OCI-Ly1 cells in triplicates.

To understand the changes in gene expression after ribavarin in a clinically relevant sample, we generated a patient-derived xenograft (PDX) in NSG mice from a de-identified specimen isolated from a patient prior to treatment harboring a triple-hit ABC-type DLBCL. PDX cells from passage four (PDX-4) were implanted into NSG mice. When tumors were palpable, mice were randomized to receive vehicle or 80 mg/kg/b.i.d. ribavarin intraperitoneally for 10 days.  We isolated RNA from tumors treated with vehicle (n=2) or ribavarin (n=2) and performed mRNA-seq.

first: Leandro

last: Cerchietti


title: Genome-wide studies reveal novel biological pathways regulated by SIN3 isoforms

status: Public on Jan 09 2016

city: Detroit

first: Lori

middle: Ann

last: Pile

state: Michigan

address: 5047 Gullen Mall

department: Biological Sciences

institute: Wayne State University


title: mRNA- and RISC-sequencing of mouse hearts overexpressing miR-133a

city: Indianapolis

first: Scot

middle: J

last: Matkovich

state: IN

address: Lilly Corporate Center

institute: Eli Lilly and Company

summary: miR-133a-3p is a highly abundant cardiomyocyte-enriched microRNA whose expression is persistently decreased in response to pressure overload (or transverse aortic constriction, TAC) in mice. Overexpression of miR-133a in cardiomyocytes of mouse hearts in vivo (under the control of the Myh6 promoter) decreases pressure overload-induced apoptosis and fibrosis. In previous studies using microarray platforms, we detected numerous mRNAs whose transcript levels were altered by either or both of miR-133a overexpression and pressure overload.  The data set presented here builds upon our previous study in these mice by examining mRNA-RISC associations (using Ago2-immunoprecipitated RNA) and global mRNA abundances via RNA-sequencing procedures, and tests the hypothesis that mRNAs targeted by overexpressed miR-133a are dissimilar between sham and TAC contexts.

overall_design: Cardiac polyadenylated RNA (mRNA) profiles were generated from nontransgenic and transgenic mouse hearts of FVB/N background, on Illumina HiSeq 2000 instruments.  Male mice 8-12 weeks of age were used in these studies, and subjected to sham surgery or 1 week of pressure-overload via transverse aortic constriction (TAC). 3 nontransgenic sham, 7 transgenic sham, 5 nontransgenic TAC, 4 transgenic TAC, each with mRNA-seq and RISC-seq (mRNA-seq of Ago2 immunoprecipitate) data.


title: The human cellular nucleic acid binding protien binds G-rich elements close to translation initiation sires and promotes translation.

status: Public on Jan 08 2016

city: Wuerzburg

first: Sanjay

middle: Kumar

last: Gupta

state: Bavaria

address: Biozentrum, Am Hubland

department: Dept of Biochemistry

institute: University of Wuerzburg


title: Transcriptome analysis upon overexpression of SIN3 187HA  in Drosophila cultured cells

summary: SIN3 is a master transcriptional scaffold protein. SIN3 interacts with RPD3 and other accessory proteins to form a histone modifying complex. A single Sin3A gene encodes multiple isoforms of SIN3, of which SIN3 187 and SIN3 220 are the predominant isoforms. Previous studies demonstrated that SIN3 isoforms play non-redundant roles during fly development. In the current study, we sought to investigate the genes regulated by SIN3 187.

overall_design: S2 cells and cells carrying a stable transgene of SIN3 187HA (SIN3 187HA cells) were treated with 0.07 µM CuSO4. CuSO4 treatment led to ectopic expression of SIN3 187HA. S2 cells were used as a control. Following induction, total mRNA was extracted. mRNA profiling of these samples were performed by deep sequencing using Illumina Hiseq2500. Three biological replicates were performed.

first: N

last: Saha

first: A

last: Gajan

first: M

last: Liu

first: L


title: sec66∆ RNAseq

city: Kansas City

first: Madelaine

last: Gogol

state: MO

address: 1000 E. 50th Street

department: Computational Biology Core

institute: Stowers Institute

summary: Identification of the difference in transcriptome between wild-type and SEC66 deletion mutant cells

overall_design: poly-A stranded RNAseq of three isolates of wild-type and three isolates of sec66∆ cells

first: Sreenivasulu

last: Santharam Katta

first: Kate

last: Hall


title: Pet-1 Switches Transcriptional Targets Postnatally to  Regulate Maturation of Serotonin Neuron Excitability.

city: Cleveland

first: Evan

last: Deneris

state: OH

address: 2109 Adelbert Rd.

department: Neurosciences

institute: Case Western Reserve University

summary: Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known aboutthe regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1 / neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. 5-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturationthrough secondary postmitotic regulatoryfactors. The early postnatal switch in Pet-1targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability.

overall_design: 5-HT neuron mRNA profiles of E11.5, E15.5, and postnatal (P1-P3) wild type (WT) and Pet-1-/- mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Myc-tagged Pet-1 ChIP-seq was performed on E12.5 to E14.5 hindbrains and sequencing using NextSeq 500.

first: Steven

last: Wyler

first: William

last: Spencer

first: Noah

last: Green

first: Benjamin

last: Rood

first: LaTasha

last: Crawford

first: Caryne

last: Craige

first: Paul

last: Gresch

first: Douglas

middle: G

last: McMahon

first: Sheryl

last: Beck


title: Increased DNA Methylation of Dnmt3b-Targets Impairs Leukemogenesis (RRBS)

first: Christian

last: Rohde

summary: The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells, and Dnmt3b has recently been shown to play a role in genic methylation. Forced Dnmt3b expression induced widespread DNA hypermethylation in myc-bcl2 induced leukemias, especially at promoters and gene bodies of stem cell-related genes. MLL-AF9 induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Our findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.

overall_design: To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knockin mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function.

first: Isabell

last: Schulze

first: Carsten

last: Müller-Tidow


title: Transcriptome profile of liver at different physiological stages in laying hens

city: Zheng

first: HONG

last: LI

address: 95 Wenhua Road, Jinshui District

institute: Henan Agriculture University

summary: In this study, RNA-Seq technology was adopted to investigate the differences in expression profiles of the hepatic lipid metabolism-related genes and the associated pathways between juvenile and laying hens. RNA-Seq analysis was carried out to estimate total RNA harvested from the liver of juvenile hens (n = 3) and laying hens (n = 3). Compared with juvenile hens, 2574 differentially expressed (DE) genes (1487 down and 1087 up) with P ≤ 0.05 were obtained, and 955 of these genes were significantly DE (SDE) at a false discovery rate (FDR) of 0.05 and fold-change ≥ 2 in laying hens. There were 198 SDE novel genes (107 down-regulated and 91 up-regulated) (FDR ≤ 0.05) that were obtained from the transcriptome, and most of them were highly expressed. Moreover, 332 SDE isoforms were identified. Gene Ontology (GO) enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that SDE genes were significantly associated with steroid biosynthesis, PPAR signaling pathway, biosynthesis of unsaturated fatty acids, glycerophospholipid metabolism, three amino acid pathways, and pyruvate metabolism (P ≤ 0.05). The top significantly enriched GO terms included lipid biosynthesis, cholesterol and sterol metabolic, and oxidation reduction suggesting the principal lipogenesis in the liver of laying hens. This study suggests that the major changes at the level of transcriptome in laying hen liver are closely related to fat metabolism. Some highly differentially expressed uncharacterized novel genes and alternative splicing isoforms detected might also take part in lipid metabolism, though it needs investigation. Therefore, this study provides valuable information of mRNA of chicken liver, and deeper functional investigations on the mRNAs could help explore or provide new insights into molecular networks of lipid metabolism in chicken liver.

overall_design: The liver expression profile of juvenile hens and laying hens were generated by RNA-seq.

first: Hong

last: Li

first: Xiaojun

last: Liu

first: Xiangtao

last: Kang


title: Genome-wide DNase-seq profiling of Saccharomyces cerevisiae (W303 strain)

city: Durham

first: Jason

middle: Alan

last: Belsky

state: NC

address: LSRC Room C333

department: Pharmacology and Cancer Biology

institute: Duke University

summary: Intact nuclei from an asynchronous population of W303 Saccharomyces cerevisiae in log-phase growth were subjected to a 16-minute DNase I digestion (0.1 U/μL) at 37 °C.  DNA was then recovered, and single-end Illumina sequencing libraries were prepared using the Crawford DNase-seq method (Song and Crawford, 2010).

overall_design: Two biological replicates of DNase-seq were sequenced in single-end mode on an Illumina HiSeq 2000.

first: Jianling

last: Zhong

first: Peter

last: Winter

first: Gregory

middle: E

first: Alexander

last: Hartemink