title: The WRN protein accumulates at tRNA and G-rich regions during S phase in HEK293 cells.

city: Philadelphia

first: Sager

middle: Jayesh

last: Gosai

state: Pennsylvania

address: 433 S University Ave.

department: Biology

institute: Universiry of Pennsylvania

summary: Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The mutated gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and DNA repair. In this study, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify regions of the genome bound by the WRN protein in Human Embryonic Kidney (HEK) 293 cells. We found 409 such genomic sites, one third of which corresponded to tRNA genes and another third of which were within RNA polymerase II transcribed genes that tended to contain guanine-rich sequences. We confirmed the specificity of several sites using WRN-depleted HEK293 cells by ChIP followed by quantitative PCR. Expression profiling of the HEK293 cells and RT-PCR analyses indicated that the transcription of the genes (including tRNAs) bound by WRN were not changed. Instead, we found a greater accumulation of WRN protein at these sites during S phase. These results suggest that the WRN protein accumulates at specific genomic sites during S phase of the cell cycle in HEK293 cells.

overall_design: ChIP-seq analysis of WRN genome binding

first: Davinder

last: Sandhu

first: Brian

middle: D

last: Gregory


title: B-cell Differentiation is Regulated by Targeted DNA Hypomethylation [RRBS_1]

status: Public on May 01 2016

city: Atlanta

first: Benjamin

middle: G

last: Barwick

state: GA

address: 1365 Clifton Rd. NE WCI-C 4th Floor Benches 36-37

department: Hematology and Medical Oncology

country: USA

institute: Emory University

type: Methylation profiling by high throughput sequencing

summary: B cells provide humoral immunity by differentiating into antibody secreting plasma cells.  Differentiation is dependent upon division and transcriptional changes, with commitment to B cell lineages associated with epigenetic changes.  Analysis of early transcriptional and epigenetic events in B cell differentiation revealed that plasmablasts and plasma cells undergo dynamic changes in gene expression and a progressive DNA hypomethylation targeted to at least 10% of genes/loci.  Of the differentially methylated loci, more than 99.7% were demethylated during differentiation and these clustered in cis-regulatory features such as enhancers and transcription factor binding sites.  Changes in gene expression and DNA methylation coincided with each other at specific divisions during differentiation and inhibition of DNA methylation resulted in augmented plasma cell commitment in a division-dependent manner.  These data identify a major epigenetic reprogramming event during early B cell differentiation coupled division and provide an approach to modulating humoral immune responses.

overall_design: Splenic B cells (B220+ CD43-) from naïve C57/BL6J mice, and Day 3 LPS-induced Plasmablasts (B220mid CD138+) and Plasma Cells (B220low CD138+) were analyzed by Illumina Gene Expression Microarray and Reduced Representation Bisulfite Sequencing

first: Jeremy

middle: M

last: Boss


title: B-cell Division DNA methylation [RRBS_2]

overall_design: Splenic B cells (B220+ CD43-) from naïve C57/BL6J mice were labeled with CFSE or CTV and transferred into uMT mice and allowed to rest overnight prior to challenge with LPS.  Three days post challenge adoptively transferred B cells representing distinct divisions were sorted out for molecular analysis.  These divisions are labelled Div0, Div1, Div3, Div5, Div8- and Div8+.  Division 8- refers to cells that divided at least 8 times but were CD138-, whereas Division 8+ refers to cells that divided at least 8 times but were CD138+.  Cells were subjected to RNA-seq and Reduced Representation Bisulfite Sequencing.


title: B-cell Division RNA-seq [RNA-Seq]

type: Expression profiling by high throughput sequencing


title: Regulation of Nucleosome Ejection by the SWI/SNF-Family Chromatin Remodeller RSC/Sth1

city: Salt Lake City

first: Timothy

middle: J

last: Parnell

state: UT

address: 2000 Circle of Hope

institute: Huntsman Cancer Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: SWI/SNF-family chromatin remodelling complexes conduct nucleosome sliding and ejection to provide DNA-binding proteins access to their sites in chromatin. RSC is an essential and abundant SWI/SNF-family chromatin remodeller from S. cerevisiae that both slides and ejects nucleosomes. However, how ejection versus sliding is chosen, regulated and implemented by any remodeller remains largely unknown. The RSC catalytic subunit Sth1 conducts ATP-dependent DNA translocation, pumping DNA around the nucleosome, providing a property that might underlie both sliding and ejection. Sth1 shares with other SWI/SNF-family ATPases direct binding to two nuclear actin-related proteins (ARPs), but how ARPs impact DNA translocation, sliding and ejection is unknown. Here, we reveal that nucleosome ejection by RSC requires ARPs, and that ARPs improve ‘coupling’ – the efficiency of DNA translocation by Sth1 relative to ATP hydrolysis – thus, enhancing DNA translocation. We further characterize two domains within Sth1 (termed PTH and P1), showing they separately regulate ATP hydrolysis or ‘coupling’, respectively. Interestingly, gain-of-function PTH or P1 mutations suppress arpΔ lethality, and improve ATPase or coupling activities, enabling Sth1 to efficiently slide and eject nucleosomes without ARPs. Moreover, PTH mutations greatly improved DNA translocation velocity. Overall, our results provide a logic for regulating nucleosome sliding versus ejection: both modes involve DNA translocation, but ejection requires a higher magnitude. Here, low-to-moderate ATPase and coupling activity integrate to confer low-to-moderate sliding, whereas high ATPase and/or coupling combine to provide efficient and rapid DNA translocation, consistent with the simultaneous rupture of multiple histone-DNA contacts, causing histone loss/ejection. Our discovery of an ARP module and regulatory domains that regulate sliding and ejection suggests a mechanistic platform through which activators and histone epitopes may guide the remodelling outcome of SWI/SNF-family chromatin remodellers.

overall_design: Genome-wide nucleosome maps were generated from four different yeast strains representing sth1 null, Sth1 wild type, sth1-deltaDL mutant, and sth1-L392P mutant. Two separate biological samples were grown, isolated, sequenced, and analyzed separately.

first: Cedric

middle: R

last: Clapier

first: Margaret

last: Kasten

first: Ramya

last: Viswanathan

first: Heather

last: Szerlong

first: Bradley

last: Cairns


title: Large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection [RNA-Seq]

city: New Haven

first: Haifan

last: Lin

state: Connecticut

address: 10 Amistad St.

department: Cell Biology

institute: Yale Univeristy

summary: Rice blast is a recurrent fungal disease, and resistance to fungal infection is a complex trait. Therefore, a comprehensive examination of rice transcriptome and its variation during fungal infection is necessary to understand the complex gene regulatory networks. In this study, adopting Next-Generation Sequencing we profiled the transcriptomes and microRNAomes of rice varieties, one susceptible and the other resistant to M. oryzae, at multiple time points during the fungal infection.

overall_design: In order to investigate variations of rice transcriptome and microRNAome in response to blast infection, we sequenced, profiled, compared and analyzed the gene expressions of two rice cultivars, one susceptible (CO39) and the other resistant (C101LAC) to M. oryzae, at different time points after M. oryzae infection.

first: Ze-Yuan

last: Li

first: Cong-Ying

last: Wang

first: Weixiong

last: Zhang

first: Yue-Qin

last: Chen


title: Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation.

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

overall_design: Refer to individual Series


title: Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation

city: Paris Cedex 05

first: Nicolas

last: Servant

address: 26 rue d'ulm

country: France

institute: Institut Curie

summary: How the parental genomes of the very specialized sperm and oocyte cells are remodelled upon fertilization to confer totipotency has remained a tantalizing open questions. Indeed, in the case of mammals, the parental genomes undergo dramatic reprogramming upon fertilization, including differential dynamics of histone post-translational modifications. The roles of histone modifying enzymes in this process, which are maternally provided, are only just starting to emerge. Here, we explore the function of the oocyte inherited pool of Lsd1/Kdm1a, which encodes a histone H3K4 and K9 demethylase, during early mouse development. Maternal deficiency of Lsd1/Kdm1a results in developmental arrest by the two-cell stage, associated with dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns depending on its demethylase activity. At the transcriptional level, two major changes occur. On one hand, switch from maternal-to-zygotic program fails to be induced. On the other hand, LINE-1 retrotransposons are not properly silenced, along with evidences for increased LINE-1 activity. We propose that Lsd1/Kdm1a is involved in the correct establishment of epigenetic information harboured by histones and is involved in the initiation of new pattern of genome expression driving early mouse development and preserving genome integrity

overall_design: RNA-seq of invidual mouse two-cell stage embryos

first: K

last: Ancelin

first: L

last: Syx

first: N

first: C

first: E

last: Heard


title: Native elongating transcript sequencing (NET-seq), nascnet RNA-seq, and total RNA-seq of wild type and RNA polymerase II C-terminal domain mutants and ChIP-nexus of RNA polymerase II C-terminal domains phosphoisoforms and splicing factors in S. cerevisiae

city: Boston

first: Stirling

last: Churchman

state: MA

address: NRB Rm 356, 77 Ave Loius Pasteur

department: Genetics

institute: Harvard Medical School

summary: Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7), we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a novel role for threonine-4 in 3’ end processing through controlling the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3’ splice sites through a direct interaction with spliceosomal subcomplex, U1. Strikingly, threonine-4 phosphorylation also impacts splicing through serving as a mark of spliceosomal release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

overall_design: NET-seq of WT, rai1, rtt103 and Pol II CTD threonine-4 mutants. Nascent RNA-seq of WT and Pol II CTD threonine-4 mutant. RNA-seq of WT and Pol II CTD threonine-4 mutants. ChIP-nexus analysis of phospho-Ser5 and phospho-Thr4 of the Pol II CTD and ChIP-nexus of splicing factors.

first: Kevin

last: Harlen

middle: S


title: Expression data for melanoma cell lines with different resistance to SBI-0640756

city: Rockville

first: Tongwu

state: MD

address: 9609 Medical Center Drive

department: Division of Cancer Epidemiology & Genetics

institute: National Cancer Institute

summary:
The emergence of effective inhibitors for BRAF-mutant melanoma has had major impact on clinical management of melanoma 1,2. The initial success of such treatments, however, has been limited due to the propensity of melanomas to develop resistance 3-5. In most cases, mechanisms underlying BRAFi-resistance include activation of genetic or epigenetic pathways that circumvent targeted BRAF and restore MAPK and related signaling to levels sufficient to encourage tumorigenesis 3-7. This outcome has led to development of combination therapies targeting both BRAF and associated pathways, such as MEK and PI3K 8,9, albeit, with limited success. Furthermore, 50% of melanomas, such as those harboring NRAS and NF1 mutations, lack BRAF mutations and are thus not amenable to BRAFi therapy 10,11. Thus tumor chemoresistance and the lack of therapies for BRAF wild-type (WT) tumors remains a major clinical challenge.
Disrupting the eukaryotic translation initiation factor 4F (eIF4F) complex has been an appealing strategy to potentiate effectiveness of existing cancer therapies. We identify that the small molecule SBI-0640756 (SBI-756) binds to eIF4G1 and dissociates it from eIF4E, leading to disruption of the eIF4F complex. SBI-756 treatment also inhibited AKT, NF-κB and mTOR signaling. We detected the melanoma cell lines have different response to SBI-756.  It important to invertigate genes and pathways in melanoma cells resistant to SBI-756.

overall_design: Dose response analysis of SBI-756 in 21 melanoma lines identified two groups representing respective SBI-756-sensitive and -resistant lines (>2-fold expression difference in IC50 between groups (7 cell lines for each). Total RNA from 14 melanoma lines was profiled per manufacturer’s protocol on Human Genome U133plus2.0 GeneChips (Affytmetrix), containing ~47,000 probe sets for 38,000 characterized human genes. GeneChips were scanned using the Affymetrix GeneChip Operating Software (GCOS) and the .CEL files were generated for downstream data analysis.

last: Brown


title: Dynamic regulation of the histone variant H1.0 contributes to intratumor epigenetic and functional heterogeneity [FAIRE-Seq]

city: London

first: Paola

last: Scaffidi

address: 1 Midland Road

country: United Kingdom

institute: Francis Crick Institute

summary: Identification of nucleosome-free, active regulatory regions in in vitro generated CSCs and their parental hTERT-immortalized cell line.

overall_design: FAIRE-seq analysis of biological replicates from SSEA1+ in vitro-transformed human skin fibroblasts and parental hTERT-immortalized cells.

last: Morales Torres

first: A

last: Biran

first: T

last: Henser-Brownhill

first: H

last: Patel

last: Hashahar

first: Y

last: Nye

first: R

last: Ben Hamo

last: Matthews

last: Misteli

last: Meshorer

first: P


title: Dynamic regulation of the histone variant H1.0 contributes to intratumor epigenetic and functional heterogeneity [RNA-Seq]

summary: Identification of genes regulated by the histone variant H1.0 by RNA-seq analysis of cell lines expressing inducible specific shRNAs.

overall_design: RNA-seq analysis of in vitro-transformed human skin fibroblasts expressing inducible shRNAs targeting H1.0. Two different cell lines expressing distinct shRNAs are analyzed. For ech cell line, uninduced cells (NT), cells induced with doxycycline for 16h  (SHOT), cells induced with doxycycline for 14 days (DOX) and cells washed out of doxycycline for 4 days (washDOX) are compared.

last: Li


title: Altered microRNA expression and pre-mRNA splicing events reveal new mechanisms associated with early stage Mycobacterium avium subspecies paratuberculosis infection

city: Edmonton

first: Guanxiang

last: Liang

state: Alberta

address: 410 Agriculture/Forestry Centre

country: Canada

institute: University of Alberta

type: Non-coding RNA profiling by high throughput sequencing

summary: The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was “proliferation of endothelial cells”, indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as “proliferation of endothelial cells” (bta-miR-196b), “bacteria recognition” (bta-miR-146b), and “regulation of the inflammatory response” (bta-miR-146b).

overall_design: Five male, Holstein calves that were 10-14 days old were inoculated with MAP using surgically isolated intestinal segments.The distal compartments were injected with 1- 3×10^8 CFU of MAP strain K10 in a final volume of 5 ml phosphate-buffered saline (PBS). The proximal compartment of the intestinal segment was injected with 5 ml PBS. The miRNA expression and mRNA expression were compared between two groups.


title: Dynamic regulation of the histone variant H1.0 contributes to intratumor epigenetic and functional heterogeneity


title: Temporal comparison of transcriptomic alterations in human, mouse and rat primary B lymphocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

status: Public on Apr 30 2016

city: East Lansing

first: Norb

last: Kaminski

state: MI

institute: MSU

summary: RNA-Seq was used to analyze and compare temporal TCDD-induced gene expression changes in primary human, mouse and rat B cells. Most of the differentially expressed genes exibited species-specific expression. Cross-species comparison identified 28 orthologs commonly deregulated in all 3 species.  The study provides new insight into mechanisms of TCDD-induced suppression of B cell effector function.

sample_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Primary human, mouse, and rat mRNA profiles of primary B cells treated with either vehicle (0.02% DMSO) or 30nM TCDD for 4, 8, and 24h  were generated by sequencing.  Human B cell treatments was performed in triplicate while primary B cells from 20 mice or 6 rats were pooled.

first: Natalia

last: Kovalova

first: Rance

last: Nault

first: Timothy

last: Zacharewski

first: Norbert

middle: E

first: Robert

last: Crawford


title: Nuclear PKCq binding to DNA in T lymphocytes.

city: Bruce

first: Kristine

last: Hardy

state: ACT

address: University of Canberra

country: Australia

institute: University of Canberra

summary:
Adaptive immune responses to infection result in the formation of memory T and B cells that respond more rapidly and robustly to reinfections, providing the basis of the immunological memory targeted by vaccines. Underlying the enhanced responsiveness of memory cells is their ability to rapidly up-regulate the transcription of key effector genes at a higher level compared to naïve cells (termed transcriptional memory). While transcriptionally permissive histone modifications are known to provide chromatin structures that facilitate transcriptional memory, the molecular mechanisms that underpin this process still remain elusive.
The role of the cytoplasmic signalling kinase, PKC-θ in T cell signalling cascades is well established, however PKC-θ has also recently been described as a chromatin-modifying enzyme. We have previously demonstrated that PKC-θ forms a part of an active transcription complex that docks at the IL2 gene - a gene which displays transcriptional memory. In this study, we perturbed PKC-θ expression using approaches such as siRNA knockdown in primary human naïve and memory CD4+ T cells to show that transcriptional memory is highly dependent on PKC-θ, particularly in memory CD4+ T cells. Chromatin-tethered PKC-θ was immuno-precipitated with high levels of the activating marks, H3K4me3 and H3K9ac and low levels of the repressive mark, H3K27me3 at the IL2 promoter in ex vivo-derived, activated memory CD4+ T cells. Further ChIP-sequencing analysis revealed that PKC-θ was prevalent at transcriptionally permissive domains such as promoters, upstream regions and exons but also at intronic regions containing putative enhancers annotated to a subset of genes preferentially expressed in memory CD4+ T cells. Collectively, these data argue that chromatin-tethered PKC-θ can directly regulate genes to establish and maintain a permissive chromatin state that facilitates transcriptional memory in human CD4+ T cells.

overall_design: 8 different biological samples were analyzed. Immunoprecipitated and total input samples were obtained.

first: Sudha

last: Rao

first: Jasmine


title: Expression data from GFP-labeled c-Kit+ mouse APL cells and normal Lin-Sca-1-c-Kit+ mouse bone marrow cells

city: Shanghai

first: Jiang

last: Zhu

address: 197, rui-jin 2nd Road

country: China

institute: Shanghai institute of hematology

summary: To characterize the differentially expressed genes between leukemic and normal myeloid progenitors

overall_design: Using FACS, we isolated GFP-labeled c-Kit+ mouse APL cells and normal Lin-Sca-1-c-Kit+ mouse bone marrow cells. Each sample had 3 biological replicates.

first: Mengmeng

last: Huang


title: Digital gene expression tag profile of five development stages of pearl sac from pearl oyster Pinctada martensii reveals potential candidates contributing to nacre formation

city: zhanjiang

first: xiaoxia

last: zhao

state: guangdong

address: huguangyaneast

department: Fisheries University

institute: Guangdong Ocean University

summary: A sequencing depth of 5 million raw tags per sample was obtained and a large number of differentially expressed genes were identified. The results of the DGE tag profile were validated by examining gene expression in a subset of genes using qRT-PCR. Gene expression profiles at different pearl sac development stages implicated that a great change in the body of the host pearl oyster was induced by the surgical implantation and achieved the maximum at 15 days. An enrichment of genes relevant to purine metabolism was obtained by KEGG pathway analysis of the genes differentially expressed between 15 and 60 days. Analyzing gene expression trends, most of the immune-related genes, such as HSP70, TLR1 and lectin, were highly expressed before 30 days. While nacre formation-related genes, such as DPT and perlucin-like protein isoformA, and some immune-related genes, such as galectin and HSP90 like protein, were highly expressed at 60 days, indicating some immune-related gene may participate in nacre formation. Furthermore, we found 286 unigenes highly expressed at 60 days. These unigenes may be potential candidates contributing to nacre formation, such as leucine-rich repeat-containing protein, secreted protein with cysteine rich repeats and Cathepsin O.

overall_design: 5 different development stages Digital Gene Expression Profiling(DGE) in pearl sac were generated by deep sequencing, using Illumina hiseq-2000

first: Qingheng

last: wang

first: Yu

last: jiao

first: Xiaoxia

first: Xiaodong

last: du

first: Yuewen

last: deng

first: Ronglian

last: huang


title: mRNA expression profile of dop-1 mutants to wild- type animals during adulthood (L4+48 hours) using RNA-seq

city: Piscataway

first: Christopher

last: Rongo

state: NJ

address: 190 Frelinghuysen Road

department: Department of Genetics

institute: Waksman Institute, Rutgers The State University of New Jersey

summary: Developmentally synchronized animals were obtained by hypochlorite treatment of gravid adults to release embryos. Synchronized embryos were hatched on NGM plates and grown at 20°C until 48 h after the L4 stage of development. Fluorodeoxyuridine was used to prevent the development of second-generation embryos once animals reached fertile adulthood. For each RNA-seq experiment, populations for odIs77[Pcol-19::UbG76V-GFP] and dop-1(vs100); [Pcol- 19::UbG76V-GFP] were grown simultaneously under the same conditions. Total RNA was isolated from animals using trizol (Invitrogen) combined with Bead Beater lysis in 3 biological replicates, and an mRNA library (single-end, 50-bp reads) was prepared for each sample/replicate using Illumina Truseq with PolyA selection.

overall_design: Examination of mRNA levels in adults dop-1 mutants and wild-type animals.

first: Kishore

middle: K

last: Joshi


title: RNA-seq data from livers of 6 month old Akt1F/F;Akt2-/- versus Akt1F/F;Akt2-/-;Alb-Cre mice

city: Chicago

first: Nissim

last: Hay

state: IL

address: 900 South Ashland Ave

department: BMG

institute: University of Illinois Chicago

summary: Finding diffrential gene expression gene expression in the livers of male mice after the deletion  of Akt1 in th eliver of Akt2-/- mice.

overall_design: mRNA was isolated from 4 indivdual livers of each group of mice of the same genotype. mRNAs from each group was pooled to generate 1 sample per each group. The 2 samples were used to generate cDNA libraries for RNA-seq.


title: YAP mediates tumorigenesis in neurofibromatosis type 2 through a COX2-EGFR signaling axis

city: Jupiter

first: Joseph

last: Kissil

state: Florida

address: 130 Scripps Way

department: Cancer Biology

institute: The Scripps Research Institute

summary: The Hippo-YAP signaling pathway has emerged as a major driver of tumorigenesis in a wide spectrum of human cancers.  YAP functions as a transcriptional activator and while details of YAP regulation are emerging at a fast pace, it remains unknown what downstream targets are critical for YAP oncogenic functions. To establish the mechanisms involved and identify disease-relevant targets we examined the role of YAP in neurofibromatosis type 2 (NF2) using cell and animal models. YAP function is required in NF2-null Schwann cells to promote cell survival and for tumor growth, in vivo. Moreover, YAP promotes transcription of several targets including Prostaglandin-endoperoxide synthase 2 (PTGS2), which controls production of prostaglandin E2 (PGE2) and amphiregulin (AREG), a member of the epidermal growth factor family. Both AREG and PGE2 converge to activate survival signaling through EGFR, in a Src-dependent manner, thus promoting cell survival. Importantly, treatment with the COX2 inhibitor celecoxib significantly repressed the growth of NF2-null Schwann cells in vitro and tumor growth in a mouse model of NF2-associated schwnnoma.

overall_design: YAP-dependent differential gene expression was assessed by RNA-Seq on Nf2-null Schwann cells treated with 1) Control siRNA, or 2) YAP pooled siRNA for 48 hours.

middle: L

first: William

last: Guerrant

first: Mohammad

last: Fallahi


title: U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3’ End Formation

city: Worcester

first: Michael

middle: R.

last: Green

address: 364 Plantation Street

department: Molecular, Cell and Cancer Biology

institute: University of Massachusetts Medical School

summary: We sequenced mRNA from Ba/F3 cells with U2AF35(S34F) expressing or parental Ba/F3

overall_design: Examination of alternative splicing between U2AF35(S34F) expressing and parental Ba/F3

first: Sung Mi

last: Park

first: Jianhong

last: Ou

first: Lihua


title: The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila

city: Berkeley

first: Qingqing

last: Wang

state: CA

address: 161 Koshland Hall, UC Berkeley

department: Molecular and Cell Biology

institute: University of California, Berkeley

summary: Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms.  Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown.  Using genome-wide approaches and quantitative behavioral assays, we show that the P element somatic inhibitor (PSI) and its interaction with U1 snRNP control male courtship behavior.  PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts.  The PSIΔAB mutant results in significant changes in the AS patterns of ~1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior.  PSI directly regulates the AS of at least a third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior.  Importantly, one of these direct targets is fruitless — the master regulator of courtship.  Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system.  This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

overall_design: Two PSIΔAB male fly head replicate samples and one wildtype male fly head sample were prepared for RNA-seq for differential alternative splicing analyses; two Drosophila S2 cell line replicate samples were prepared for the iCLIP experiment and libraries were pooled before sequencing. Barcode information are provided.

first: Matthew

last: Taliaferro

first: Donald

last: Rio


title: Human islets contain four distinct subtypes of β cells

status: Public on Apr 29 2016

city: Philadelphia

first: Jonathan

last: Schug

address: 12-156 Smilow Ctr Trans Research

department: School of Medicine

institute: University of Pennsylvania

summary: The transcriptomes of four subpopulations of beta cells isolated by FACS from five healthy human donors. Populations were defined using cell surface-labeling antibodies, avoiding the need for fixation.

overall_design: There are 5 biological replicates of 4 different cell types.   Each donor yielded all 4 subtypes.

first: Craig

last: Dorrell

first: Pamela

last: Canaday

first: Holger

middle: A

last: Russ

first: Branden

last: Tarlow

first: Maria

middle: T

last: Grompe

first: Tamara

last: Horton

first: Matthias

last: Hebrok

first: Philip

last: Streeter

first: Klaus

middle: H

last: Kaestner

first: Markus


title: Gene expression analysis of mice liver tumors isolated from β2SP+/-; SMAD3+/- mice

city: Houston

first: Jian

last: Chen

state: Texas

address: 1515 Holcombe Blvd.

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

summary: Gene expression profiling was carried out in two liver tumors and one normal liver isolated from β2SP+/-; SMAD3+/- mice, and one normal liver isolated from wild type mouse. Whole-transcriptome sequencing of these 4 liver tissues.

overall_design: Whole-transcriptome RNA sequencing of the 4 different samples

first: Lopa

last: Mishra


title: Recessive mutations in TRMT10C cause defects in mitochondrial RNA processing and multiple respiratory chain deficiencies

status: Public on Apr 28 2016

city: Perth

first: Stefan

last: Siira

state: Western Australia

address: 15 Hospital Ave, Nedlands

department: Precision Health

institute: The Kids Research Institute Australia

summary: We investigated the changes in the abundance of reads across the entire mitochondrial transcriptome, we found an increase in the regions that span gene boundaries, where RNA processing is required to release individual mitochondrial RNAs from the precursor transcripts. These data confirm an impairment of mt-tRNA processing efficiency without severe effects on mature mt-mRNA or mt-tRNA steady-state levels. Strand-specific coverage profiles were generated in bedGraph format and normalised to library size (RPM; reads per million mapped).

overall_design: Mitochondrial RNA from control and patient fibroblast cells, with a wild type and biallelic TRMT10C variants, respectively, were sequenced and the RNA processing analyzed

first: Aleksandra

last: Filipovska


title: Psychoactive pharmaceuticals at environmental concentrations induce in vitro gene expressions associated with neurological disorders

city: Pocatello

first: Michael

last: Thomas

state: ID

address: 921 S. 8th Avenue

department: Biological Sciences Department

institute: Idaho State University

summary: We tested if psychoactive pharmaceuticals (fluoxetine, carbamazapine, venlafaxine) at lower concentrations (in ppb) could induce gene expressions linked with neurological disoders.

overall_design: We used human nueronal cells as a model to study gene expressions induced by psychoactive pharmaceuticals at lower concentrations.


title: Transcriptionally inactive ATF2 variant drives melanomagenesis [Seq]

city: San Diego

first: brian

last: james

address: 10901 North Torrey Pines Road

institute: Sanford Burnham

summary: Characterized by striking metastatic propensity and chemoresistance, melanoma is among the most lethal cutaneous malignancies. The transcription factor ATF2 was shown to elicit oncogenic activities in melanoma, and its inhibition attenuates melanoma development. Here, a mouse model engineered to express a transcriptionally inactive form of Atf2 (Atf2?8,9) was found to be sufficient to induce nevi formation and, when crossed with BrafV600E animals, to promote melanoma development. The cross of Atf2?8,9 with BrafV600E;Pten-/- mice augmented pigmentation, tumorigenicity, and metastasis. Similar to mouse Atf2?8,9, the human ATF2 splice variant 5 enhanced growth and migration capacity of cultured melanoma and immortalized melanocytes. Induced Melan-A, CXCL9, S100A8, CCR7 expression, seen in Atf2?8,9-driven tumors associate with their enhanced pigmentation, immune infiltration and propensity to metastasize. Notably, elevated ATF2SV5 expression in melanoma specimens coincided with poor prognosis. The gain-of-function activity elicited by the truncated ATF2 form offers unexpected insight into mechanisms underlying melanoma development and progression.

overall_design: Compared silencing of ATF2SV5 in H3A cells vs. silencing of ATF2WT via Ampliseq whole transcriptome analysis on the Ion Proton

first: Giuseppina

last: Claps

first: Yann

last: Cheli

first: Tongwu

last: Zhang

first: Marzia

last: Scortegagna

first: Eric

last: Lau

first: Hyungsoo

last: Kim

first: Jianfei

last: Qi

first: Jian-Liang

last: Li

first: Brian

last: James

first: Mitchell

last: Levesque

first: Reinhard

last: Dummer

first: Nicholas

last: Hayward

first: Marcus

last: Bosenberg

first: Kevin

last: Brown

first: Ze’ev

last: Ronai


title: Transcriptome of IL-13 treated small intestinal tissue in Nod2-/- mice

city: Saint Louis

first: Mei San

last: Tang

state: MO

address: 660 S Euclid, Campus Box 8118

department: Pathology & Immunology

institute: Washington University in St Louis

summary: We previously found that mice deficient in the CD susceptibility gene Nod2 develop small intestinal abnormalities including impaired mucus production by goblet cells and susceptibility to injury, which were associated with interferon-gamma producing intraepithelial lymphocytes. These abnormalities were caused by a striking expansion of a common member of the microbiota, Bacteroides vulgatus. Remarkably, infection of Nod2-deficient mice with the helminth Trichuris muris led to a TH2 response that eliminated B. vulgatus colonization and intestinal abnormalities. In addition, treatment with recombinant IL13 (rIL13) or recombinant IL4 reduced B. vulgatus levels and eliminated goblet cell defects, suggesting that type 2 cytokines alone can reverse intestinal abnormalities in the absence of helminth infection. To determine the mechanism by which type 2 cytokines protected Nod2-/- mice from intestinal abnormalities, we performed RNA-seq on small intestinal tissue from WT, Nod2-/- and rIL13 treated Nod2-/- mice. We found that rIL13 treatment induced a wound healing response characterized by M2 macrophage activation genes. Hence, type 2 cytokines can reverse inflammatory imbalances in the composition of the gut microbiota that occurs in a genetically susceptible host.

overall_design: Comparison of small intestinal transcriptome in WT, Nod2-/-, and rIL-13 treated Nod2-/- mice.

first: Deepshika

last: Ramanan

first: Rowann

last: Bowcutt

first: Mei

first: P'ng

last: Loke

first: Ken

last: Cadwell


title: E3 ubiquitin ligase Rfwd2 inactivation effect on embryonic lung development

city: Madison

first: Yan

last: Zhang

state: WI

address: 425 Henry Mall Room 5240

institute: UW-Madison

summary: Transcriptome analysis by RNA-seq of lungs from control and Rfwd2 epithelial-specific conditional knockout mice at embryonic 13.5 day age. RFWD2, is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. Rfwd2 deficiency led to a striking halt in branching morphogenesis shortly after secondary branch formation. In the mutant lung, two ETS transcript factors essential for normal lung branching, ETV4 and ETV5, were upregulated at the protein, but not transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions at least in part through degrading ETV proteins. As a number of E3 ligases are known to target factors important for lung development, our findings provides a preview of a protein-level regulatory network essential for lung branching morphogenesis.

overall_design: Total mRNA obtained from three samples per group (control and Rfwd2 conditional knockout)

first: Shigetoshi

last: Yokoyama

first: John

middle: C

last: Herriges

first: Zhen

first: Jamie

last: Verheyden

first: Randee

last: Young

first: Xin

last: Sun


title: Proteasome machinery is instrumental in a common gain-of-function program of the p53 missense mutants in cancer

city: Milan

first: Marco

middle: J.

last: Morelli

address: via Adamello 16

department: Center for Genomic Science of IIT@SEMM

country: Italy

institute: Istituto Italiano di Tecnologia

summary: Mutant p53 proteins, resulting from the missense mutations of the TP53 tumor suppressor gene, possess gain-of-function activities and are among the most robust oncoproteins in human tumors. They are potentially important therapeutic targets. To complement our mutant p53 transcriptomic studies we have performed ChIP-seq analysis in MDA-MB-231 (R280K) using the mouse anti-p53 DO1 antibody or control mouse IgG versus the input material.

overall_design: ChIP-seq analysis was performed in MDA-MB-231 (R280K) using the mouse anti-p53 DO1 antibody (Santa Cruz Biotech.) and  MDA-MB-231 (R280K) with mouse unspecific IgG antibody (Santa Cruz Biotech.) as negative control.

first: Dawid

last: Walerych


title: Gene expression analysis of human cell lines established from normal patient's fibroblast and BWS patients with known mutations in the CDKN1C and KCNQ1OT1

first: Jian

last: Chen

summary: Gene expression profiling was carried out in one normal human fibroblast cell line established from normal people and three different cell lines established from BWS patients to characterize the molecular mechanisms relevant to the etiology of BWS and tumor development. Whole-transcriptome sequencing of three BWS fibroblastic cell lines was established from patients with mutation in the CDKN1C mutation (CDKN1C+ cell line), and loss of methylation in the KCNQ1OT1 region (KvDMR+ cell line: with KvDMR molecular defect, and KvDMR- cell line: absence of KvDMR molecular defect but it had some clinical signs of BWS)

overall_design: Whole-transcriptome RNA sequencing of the 4 different cell lines

first: Lopa

last: Mishra


title: The RNAi machinery in Colletotrichum higginsianum has a role in antiviral defense

first: James

last: Carrington

address: 975 North Warson Road

institute: Donald Danforth Plant Science Center

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

overall_design: Refer to individual Series

first: S

last: Campo

first: K

middle: B

last: Gilbert

first: J


title: The RNAi machinery in Colletotrichum higginsianum has a role in antiviral defense [RNAi_mutants_RNAseq]

summary: Here we describe the identification and regulation of a novel dsRNA virus in Colletotrichum higginsianum. High throughput sequencing of small RNAs and strand-specific RNA-seq was performed on single gene knock-out mutants created for each RNAi component gene: rdr1, rdr2, rdr3, dcl1, dcl2, ago1, and ago2, and the double mutant: ∆dcl1∆dcl2. De novo assembly of the ∆dcl1 RNA-seq data identified two contigs that represented the forward and reverse strands of an uncharacterized dsRNA virus, designated here as Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1). We found increased presence of the viral RNA in the RNA-seq datasets of the ∆dcl1, ∆dcl1dcl2, and ∆ago1 strains, suggesting that these genes are required for control of the virus. We show that viral small RNAs co-immunoprecipitate with a 6xFLAG-3xHIS-tagged AGO1 protein by sequencing the small RNAs from immunoprecipitated fractions. Additionally, analyses of the small RNA datasets from the RNAi mutants revealed control of the virus through small RNA-mediated silencing required both AGO1 and DCL1.

overall_design: Strand-specific RNA-seq libraries were created from total RNA treated with RiboZero Magnetic Gold Kit (Yeast) to deplete ribosomal RNAs. Four independent transformed replicates were sequenced for all RNAi mutant strains, except for rdr1 where three independent transformed replicates were sequenced. Three empty-vector and two untransformed strains were sequenced as controls


title: A TGF- -CTCF regulated pathway suppresses stem cell associated tumor progression


title: Sp7/Osterix is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification [ChIP-Seq 2]

city: Bunkyo-ku

first: Hironori

last: Hojo

state: Tokyo

address: 7-3-1 Hongo

department: Center for Disease Biology and Integrative Medicine

country: Japan

institute: The University of Tokyo

summary: To identify Sp7’s molecular interactions in vivo, we used gene-targeting strategies to generate an Sp7-Biotin-3xFLAG knock-in mouse (Sp7-BioFL mouse). This approach appends a biotin (Bio) recognition motif and three copies of the FLAG (FL) epitope at the C-terminus of the Sp7 protein. To validate this knockin system, we compared ChIP-seq results in the MC3T3E1 osteoblast cell line, introducing Sp7 forms that were epitope-tagged at either the N- (N-terminal FLAG tag; MC3_FLAG-Sp7) or C-terminus (C-terminal Biotin-FLAG tag, as in the in vivo targeted allele; MC3_Sp7-BioFLAG). These data show an extensive overlap, suggesting that FLAG tagging at different positions gives comparable outcomes. Second, we examined ChIP on wild-type calvarial primary osteoblasts (POB) using an anti-Sp7 antibody (POB_Sp7Ab-ChIP). The majority of Sp7-Ab ChIP-seq peaks (95%) overlapped with the larger set identified by Sp7-BioFL ChIP-seq.

overall_design: ChIP-seq experiments with primary osteoblasts and a osteoblast cell line were performed

first: Shinsuke

last: Ohba

first: Xinjun

last: He

first: Lick

middle: P

last: Lai

first: Andrew

last: McMahon


title: Gene expression profiling of six MEF cell genotypes (wild type, β2SP+/-, β2SP-/-, SMAD3+/-, SMAD3-/-, and β2SP+/-; SMAD3+/-)

summary: Gene expression profiling was carried out in six (wild type, β2SP+/-, β2SP-/-, SMAD3+/-, SMAD3-/- and β2SP+/-/ SMAD3+/-) different mouse knockout embryonic fibroblast (MEF) cells.  Beta-2-spectrin (β2SP) is a dynamic intracellular non-pleckstrin homology (PH)-domain protein that belongs to a family of polypeptides that have been implicated in conferring cell polarity. Spectrins have been linked to multiple signaling pathways, including cell cycle regulation, DNA repair and TGFβ signaling. In this study, we report a major role of the TGFβ/Smad3 adaptor β2-Spectrin in conserving genomic integrity from alcohol-induced DNA damage and describe a novel pathway that protects genomes from genotoxic stresses. To determine the mechanism for the oncogenic switch, and whether it is related to the role of β2SP in TGF-β signaling transduction or secondary to its cytoskeletal functions, we analyzed disruption of two elements of the TGF-β pathway by generating double heterozygous Sptbn1+/−/Smad3+/− mice.

overall_design: Whole-transcriptome RNA sequencing MEF cells of the following genotypes was carried out on an Illumina HiSeq 2000 sequencer: wildtype, heterozygous Beta-2-spectrin knockout (β2SP+/-), homozygous Beta-2-spectrin knockout (β2SP-/-), heterozygous SMAD3 (Mothers against decapentaplegic, Drosophila, Homolog of 3, SMAD3+/-), homozygous knockout SMAD3-/-, and double heterozygous mutation of Beta-2-spectrin and SMAD3 (β2SP+/-/ SMAD3+/-).


title: The RNAi machinery in Colletotrichum higginsianum has a role in antiviral defense [AGO_IP_smallRNA]

overall_design: 6xFLAG-3xHIS-AGO1 (6F3H-AGO1) and 6xFLAG-3xHIS-AGO2 (6F3H-AGO2) proteins were immunoprecipitated from C. higginsianum mycelia tissue, along with a non-tagged protein control. RNAs isolated in the IP fraction were used as the starting material for the small RNA library protocol. The input and IP fractions of three independent transformants containing 6F3H-AGO1, two independent transformants containing 6F3H-AGO2, and one replicate of wild-type (mock-IP) were sequenced.


title: RNASeq Analysis of the Shoot Apex of Flax (Linum usitatissimum) to Detect Phloem Fiber Specification Genes

status: Public on Apr 27 2016

city: Kelowna

first: Michael

last: Deyholos

state: British Columbia

address: 1177 Research Road

institute: University of British Columbia, Okanagan

summary: We used RNAseq to compare transcript expression patterns in two segments of the vegetative stem of 14d flax plants, from which all visible leaves had been removed.  The segments were: (i) the apical region (AR) of the shoot apex, which contained the apical-most 0.5 mm of the stem, including the SAM and its immediate derivatives; and (ii) the basal region (BR), which contained the entire stem except for the apical-most 1 cm, and therefore represented all stem and vascular tissues at later stages of differentiation as compared to the AR.  These data will help identify genes that contribute to specification of phloem fiber identity.

overall_design: Two tissue systems compared using RNASeq, with at least two biological replicates each

first: Ningyu

last: Zhang


title: Identification of microRNA targets in Arabidopsis by transcriptome profiling of wild-type and ago1-36 mutant plants

city: Beijing

first: xiaofeng

last: cao

address: West Lincui Road, Chaoyang District

department: State Key Laboratory of Plant Genomics

institute: Institute of Genetics and Developmental Biology

summary: The goal of this study is to establish the global gene expression profiles of Arabidopsis wild type and ago1-36 mutant plants, and identify novel potential miRNA targets.

overall_design: RNA-seq was performed using Arabidopsis wild type and ago1-36 mutant seedlings.

first: Chunyan

last: Liu

first: Xuan

last: Ma

first: Xiaofeng

last: Cao


title: Comparison of gender-biased liver gene expression from transgenic mice with chronic IFN gamma expression

city: Bethesda

first: Anand

last: Merchant

address: 41 Center Dr

institute: NIH

summary: We analyzed liver gene expression from male and female ARE-Del mice, which have prolonged and chronic expression of IFN gamma through deletion of the IFN gamma 3’ UTR AU-rich element.

overall_design: Compare liver gene expression from male and female ARE-Del compared to control littermates (n=3)

first: Heekyong

last: Bae

first: Howard

last: Young


title: Transcription co-regulator Clim2 plays a role in the maintenance of limbal stem cells

city: Irvine

first: Bogi

last: Andersen

address: 839 Health Sciences Dr.

department: Medicine

institute: University of California, Irvine

summary: Clim1 and Clim2 are transcriptional cofactors that mediate protein-protein interactions within DNA-binding complexes in many cell types. Epithelial specific expression of a dominant negative Clim construct causes epithelial hyperplasia, neovascularization, inflammation, and blistering, followed by epithelial thinning and subsequent epidermal-like differentiation of the cornea epithelium. This phenotype resembles some aspects of limbal stem cell deficiency, suggesting that Clim proteins may be involved in regulating stem cell mainenance for the cornea. K14 DN-Clim cornea epithelium has a decrease in progenitor cells and altered proliferation during epithelial development. Gene expression profiling on DN-Clim corneas identified disrupted genes and pathways, including pathways crucial for stem cell mainenance, as well as regulation of proliferation. In vivo ChIP-Seq experiments confirm that many genes that are highly expressed in the limbus, as well as genes with known roles in stem cell maintenance are directly regulated by Clim factors. Additionally, factors that regulate corneal epithelial differentiation are direct targets of Clim. Together our results identify a role for Clim proteins in regulating stem cell maintenance and the proliferation dynamics of cornea epithelial proliferation.

overall_design: Whole corneas were dissected from P7 K14 DN-Clim mouse eye globes, taking care to remove all non-corneal tissues.  Corneal epithelium was isolated by digestion in 50% EMEM/dispase II with 50 mM sorbitol and 10 ml/ml bME for two hours at 37°C. ChIP was performed with an anit-Myc antibody to the Myc tag of the DN-Clim construct.

first: Denise

last: Stephens


title: Sp7/Osterix is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification [ChIP-Seq]

summary: Sp7/Osterix is a master regulator of osteoblast specification. To identify the Sp7-mediated gene regulatory network in osteoblasts, we performed Sp7 ChIP-seq on primary mouse calvarial osteoblasts comparing the DNA binding profile with the transcriptional profile of Sp7-positive osteoblasts. Analysis of these identified a network of Sp7 regulated osteoblast targets and provides a new insight into the mode of Sp7 action in osteoblast. To further study for the Sp7 mode, we performed ChIP-seq for Sp1, Sp7, Dlx5, as a potential Sp7 partner identified in this study and mutated Sp7 which has mutations in the zinc finger domain, by using in vitro system with a pre-osteoblast mouse cell line, MC3T3E1.

overall_design: ChIP-seq experiments with primary osteoblasts and an osteoblast cell line were performed.


title: Musashi-2 attenuates AHR signalling to expand human haematopoietic stem cells

city: Montreal

first: Brian

last: Wilhelm

address: 2950 Chemin Polytechnique

institute: University of Montreal

summary: A greater understanding of the molecular pathways that underpin the unique human hematopoietic stem and progenitor cell (HSPC) self-renewal program will improve strategies to expand these critical cell types for regenerative therapies. The post-transcriptional mechanisms guiding HSPC fate during ex vivo expansion have not been closely investigated. Using shRNA-mediated knockdown, we show that the RNA-binding protein (RBP) Musashi-2 (MSI2) is required for human HSPC self-renewal. Conversely, when overexpressed, MSI2 induces multiple pro-self-renewal phenotypes, including significant ex vivo expansion of short- and long-term repopulating cells through direct attenuation of aryl hydrocarbon receptor (AHR) signaling. Using a global analysis of MSI2-RNA interactions, we determined that MSI2 post-transcriptionally downregulates canonical AHR pathway components in cord blood HSPCs. Our study provides new mechanistic insight into RBP-controlled RNA networks that underlie the self-renewal process and provides evidence that manipulating such networks can provide a novel means to enhance the regenerative potential of human HSPCs expanded ex vivo.

overall_design: 4 samples were used for RNA-seq (4 biological duplicate) including 2 sets of control samples (irrelvant shRNA kncok-downs)

first: Kristin

last: Hope

first: Stefan

last: Rentas

first: Nicholas

last: Holzapfel

first: Muluken

last: Belew

first: Gabriel

last: Pratt

first: Gary

last: Bader

first: Veronique

last: Voisin

first: Gene

last: Yeo


title: Musashi-2 Post-transcriptionally Attenuates Aryl Hydrocarbon Receptor Signaling to Expand Human Hematopoietic Stem Cells

city: La Jolla

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

overall_design: CLIP-seq against MSI2 in NB4 Cells

middle: D

middle: W


title: The functional impact of RNA Polymerase II pausing across multiple mamamlian cell types

city: Boston

first: Peter

last: Park

state: MA

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

summary: We analyzed the breadth and functional relationships of RNA Polymerase II pausing across many human and mouse cell types to understand what roles RNA Polymerase II pausing plays in gene regulation. We identified a novel association of H2A.Z at the TSS of increasingly paused genes. We knocked down H2A.Z to test whether H2A.Z positively or negatively affects RNA Polymerase II pausing to find that pausing globally increased upon knockdown.

overall_design: We tested the effect of H2A.Z knockdown on RNA Polymerase II pausing by setting up a case vs. control experiment in which MCF7 breast cancer cells were treated with a siRNA against H2A.Z. We tested for changes in RNA Polymerase II pausing as a result.

first: William

last: Pu

first: Bing

last: Zhang


title: Whole genome nucleosome positioning by MNase-Seq on the mouse tumor line, RMA

city: Ottawa

first: Andrew

last: Makrigiannis

state: Ontario

address: 451 Smyth Rd

department: Microbiology & Immunology

institute: University of Ottawa

summary: We have employed MNase-Seq technology to determine the nucleosome positioning across the Ly49-expressing RMA cell line. This information was compared to the default nucleosome landscape of these cells as predicted by NuPop computations to identify transcription factor binding site regions that significantly deviate from our predictions, potentially indicating an interfering role for nucleosome binding at these sites. We report that expressed Ly49 genes significantly deviate in their nucleosome coverage at AML-1a sites when compared to other, non-expressed Ly49 genes within the same sample. This information has implications for our understanding of NK cell biology, and also presents the Ly49 family as a convenient model system for discovering how various genetic and epigenetic elements impact expression state, since Ly49 genes are stochastically expressed within a given population, but have similar transcription factor requirements. It is our hope that other work on the epigenetic control of gene expression can benefit from this model system.

overall_design: Examination of ten genetic regions in one population

last: Wight

first: Doo

last: Yang

first: Ilya

last: Ioshikhes


title: A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species

city: Göttingen

first: Nico

last: Posnien

address: Justus-von-Liebig-Weg 11

department: Department of Developmental Biology

country: Germany

institute: University of Göttingen

summary:
Background: RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. Since the more reliable statistical methods for differential gene expression inference are based on the use of raw read count data, the reference genomes of the species to be compared need to be highly comparable.
Results: We show that the published genomes and annotations of the three closely related Drosophila species, D. melanogaster, D. simulans and D. mauritiana, have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant length differences in the different species. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. For that we present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. We carried out a RNA-seq experiment in combination with quantitative real-time PCR to confirm that the newly generated gene sets do not result in a high number of false positives as observed with references that still show a clear length difference of gene models between species.
Conclusions: We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses.

sample_organism:
Drosophila mauritiana
Drosophila melanogaster
Drosophila simulans

overall_design: mRNA profiles of larval eye-antennal imaginal discs (late L3) of three species of Drosophila (D. melanogaster OreR, D. simulans YVF and D. mauritiana TAM16) were generated by deep sequencing using Illumina HiSeq 2000. 3 biological replicates were generated for D. melanogaster sample and sequenced in 50 bp single-end reads; 3 biological replicates were generated for D. simulans sample and sequenced in 100 bp paired-end reads; 6 biological replicates were generated for D. mauritiana sample, 3 of these were sequenced in 50 bp single-end reads and the other 3 in 100 bp paired-end reads.

first: Montserrat

last: Torres-Oliva

first: Isabel

last: Almudi

first: Alistair

last: McGregor


title: A Lysosomal In Vitro Exposure (LivE) Model to Identify Pathways Critical for Mycobacterium tuberculosis Intracellular Persistence

city: Singapore

first: Ahmad Nazri

last: Mohamed Naim

address: 60 Biopolis Street, Genome, #02-01

department: Infectious disease

country: Singapore

institute: Genome institute of Singapore

summary: Increasing experimental evidence supports that Mycobacterium tuberculosis (Mtb) has evolved strategies to survive within the lysosomes from activated macrophages, which may represent a reservoir for persistent mycobacteria. To further our knowledge in Mtb response to the lysosomal environment, we profiled the global transcriptional activity of Mtb in a lysosomal in vitro exposure (LivE) model. At inhibitory conditions of lysosomal SF (iLivE), which did not kill but arrested mycobacterial replication thereby mimicking persistence, Mtb expresses a unique transcriptome, where genes involved in general stress response, metabolic reprogramming, cell wall remodeling, respiration, oxidative stress and dormancy response were found to be significantly modulated. Genes encoding for protein families involved in Mtb virulence including ESAT-6, PE-PPE and Mce proteins were also distinctly regulated. Extensive meta-analysis of the Mtb transcriptomes from iLivE and previously reported ex vivo, in vivo and in vitro stress models revealed most significant overlap between iLivE and primary murine macrophage infection model. Our study also highlighted the α-glucan synthesis pathway and a distinct set of toxin-antitoxin systems as toxicity mechanisms that could be exploited as novel antimicrobial approaches for intra-macrophage persistent bacilli. The specificity in responses generated in the LivE model was supported by the significant number of iLivE genes that did not overlap with any of the previously reported in vitro stress models. Finally, to validate the relevance of the LivE model, rv1258c encoding for the efflux pump protein Tap, was selected from the iLivE Mtb transcriptome for further characterization. An Mtb Δrv1258c mutant was constructed and displayed increased susceptibility to killing by lysosomal SF and by the antimicrobial peptide LL-37 found in lysosomes. Furthermore, Δrv1258c Mtb was attenuated in its ability to survive in primary murine macrophages.

overall_design: Illumina sequencing was performed on mRNA extracted from in vitro mid-log phased cultures of Mtb  co-incubated with lysosomal soluble fraction (SF) at non-inhibitory, sub-inhibitory and inhibitory conditions, in triplicates.

first: Lin

last: Wenwei

first: Paola

middle: F

last: De Sessions

first: Michelle

last: Ang

first: Ahmad

middle: N

last: Naim

first: Garrett

last: Keong

first: Vanessa

last: Qi

first: Martin

last: Hibberd

last: Dedon

first: Sylvie

last: Alonso


title: Montelukast counteracts the influenza virus-induced block in unfolded protein stress response and reduces virus multiplication

city: Cantoblanco

first: Juan

middle: Carlos

last: Oliveros

state: Madrid

address: Darwin 3

country: Spain

institute: CNB, CSIC

summary: Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and economy. Therefore, a large effort has been devoted to the development of new anti-influenza drugs directed to viral targets, as well as to the identification of cellular targets amenable for anti-influenza therapy. Here we describe a new approach to identify such potential cellular targets by screening collections of drugs approved for human use. We reasoned that this would most probably ensure addressing a cellular target and, if successful, the compound would have a well known pharmacological profile.  In addition, we reasoned that a screening using a GFP-based recombinant replicon system would address virus trancription/replication and/or gene expression, and hence address a stage in virus infection more useful for inhibition. By using such strategy we identified Montelukast as an inhibitor of virus gene expression, which reduced virus multiplication in virus-infected cells but did not alter virus RNA synthesis in vitro or viral RNA accumulation in vivo. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated or not with Montelukast, we identified the PERK-mediated unfolded protein response as the pathway responsible for Montelukast action. Accordingly, PERK phosphorylation was inhibited in infected cells but stimulated in Montelukast-treated cells. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection.

overall_design: Comparison of gene expression measured by deep sequencing (single-ends, 50nt, RNA-seq) of "Infected", "Not infected", "Infected+Montelukast" and "Not infect+Montelukast" in human A549 cells. Infected means "Infected with influenza virus".

first: Sara

last: Landeras-Bueno

first: Yolanda

last: Fernández

first: Ana

last: Falcón

last: Ortín


title: RNA-Seq results of six Chinese Sutai piglets from ear tissue based on Illumina HiSeq 2000 platform

status: Public on Apr 26 2016

city: Nanchang

first: chenlong

last: liu

state: Jiangxi province

address: Lushanzhong street NO.888, Nanchang, China

department: Animal science and technology department

institute: Jiangxi Agricultural University

summary:
Purpose: The paired-end sequencing strategy of RNA-Seq improves sequencing efficiency and extends short read lengths for better understanding pig transcriptome. The goals of this study are to identify whether alternative splicing sites or alternative 3' gene boundary caused by a CNV located in 3' region of MSRB3 gene and find differential expression genes in ear tissue between QQ and qq genotype piglets for QTL of ear size
Methods: Ear tissue mRNA profiles of 6 piglets including 3 QQ and 3 qq genotypes responsible for QTL of ear size were generated by deep sequencing, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks.
Results: Using an optimized data analysis workflow, we mapped about 93.6-95.3 million 2 x 90 bp paired-end clean reads per sample to the pig genome (Sscrofa 10.2) and identify 19,819-20,136 annotated genes in each sample. Our ranking analysis identified a list of 281 differentially expressed genes with a >1 fold change at a false discovery rate lower than 0.001. But we found no alternative transcripts and differential expression of MSRB3 gene.
Conclusions: In our study, the expression levels of MSRB3 gene had no significant difference in the ear tissue between QQ and qq individuals, and no alternative splicing was found for MSRB3 gene between QQ and qq individuals.

overall_design: Ear tissue mRNA profiles of 3 QQ and 3 qq genotype piglets were generated by deep sequencing, using Illumina HiSeq 2000.

first: Congying

last: Chen

first: Chenlong

last: Liu

first: Lusheng

last: Huang


title: Transcriptome analysis of E. coli REL4536 grown aerobically and anaerobically

city: Palmerston North

first: Christina

last: Moon

address: Tennent Drive

country: New Zealand

institute: AgResearch Ltd

summary: Transcriptome analysis using RNA-Seq of E. coli REL4536 grown aerobically and anaerobically was undertaken to investigate the activities of various DNA replication and repair pathways involved in maintaining genome integrity, and to assess if their expression is consistent with observed patterns of mutation in the aerobic and anaerobic environments. Consistent with mutation rate and spectra observations, genes for recombination repair, associated with SVs, were generally up-regulated during anaerobic growth.

overall_design: Transcriptional profiling of 3 aerobically grown cultures of  E. coli REL4536 and 2 anaerobically grown cultures E. coli REL4536.

first: Sonal

last: Shewaramani

first: Sinead

last: Leahy


title: Proteasome machinery is instrumental in a common gain-of-function program of the p53 missense mutants in cancer.

city: Trieste

first: Silvano

last: Piazza

address: localita' Padriciano 99

institute: ICGEB

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

overall_design: Refer to individual Series


title: The RNAi machinery in Colletotrichum higginsianum has a role in antiviral defense [RNAi_mutants_smallRNA]

city: Saint Louis

first: James

last: Carrington

summary: Here we describe the identification and regulation of a novel dsRNA virus in Colletotrichum higginsianum. High throughput sequencing of small RNAs and strand-specific RNA-seq was performed on single gene knock-out mutants created for each RNAi component gene: rdr1, rdr2, rdr3, dcl1, dcl2, ago1, and ago2, and the double mutant: ∆dcl1∆dcl2. De novo assembly of the ∆dcl1 RNA-seq data identified two contigs that represented the forward and reverse strands of an uncharacterized dsRNA virus, designated here as Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1). We found increased presence of the viral RNA in the RNA-seq datasets of the ∆dcl1, ∆dcl1dcl2, and ∆ago1 strains, suggesting that these genes are required for control of the virus. We show that viral small RNAs co-immunoprecipitate with a 6xFLAG-3xHIS-tagged AGO1 protein by sequencing the small RNAs from immunoprecipitated fractions. Additionally, analyses of the small RNA datasets from the RNAi mutants revealed control of the virus through small RNA-mediated silencing required both AGO1 and DCL1.

overall_design: Small RNAs were extracted from total RNA from Colletotrichum higginsianum mycelia tissue using 17% polyacrylamide, 7M urea gel electrophoresis for size fractionation. Four independent transformation replicates of each mutant strain were sequenced. Small RNAs were also extracted from 6 different control strains: three empty vector strains and three untransformed, wild-type strains

first: S

last: Campo

first: K

last: Gilbert

first: J


summary: Mutant p53 proteins, resulting from the missense mutations of the TP53 tumor suppressor gene, possess gain-of-function activities and are among the most robust oncoproteins in human tumors. They are potentially important therapeutic targets. No studies to date have distinguished common, therapeutically relevant mutant p53 gain-of-function effects from effects specific to different mutant variants and cell backgrounds. here we performed RNA-seq analysisin MDA-MB-231 (R280K) upon silencing TP53 or the control siRNA.

overall_design: MDA-MB-231 (R280K) cell line was transfected with control or p53 siRNA.So The study comprises one experimental cell line,in triplicate.

first: Dawid

last: Walerych

first: Kamil

last: Lisek

first: Giannino

last: Del Sal


title: TBX18 regulates the differentiation of periductal smooth muscle stroma and the maintenance of epithelial integrity in the prostate

city: Urbana

first: Lisa

middle: J

last: Stubbs

address: 1206 West Gregory Drive | MC-195

department: IGB

institute: University of Illinois Urbana-Champaign

summary: The T-box transcription factor TBX18 is essential to mesenchymal cell differentiation in several tissues and Tbx18 loss-of-function results in dramatic organ malformations and perinatal lethality. Here we demonstrate for the first time that Tbx18 is required for the normal development of periductal smooth muscle stromal cells in prostate, particularly in the anterior lobe, with a clear impact on prostate health in adult mice. Prostate abnormalities are only subtly apparent in Tbx18 mutants at birth; to examine postnatal prostate development we utilized a relatively long-lived hypomorphic mutant and a novel conditional Tbx18 allele. Similar to the ureter, cells that fail to express Tbx18 do not condense normally into smooth muscle cells of the periductal prostatic stroma. However, in contrast to ureter, the periductal stromal cells in mutant prostate assume a hypertrophic, myofibroblastic state and the adjacent epithelium becomes grossly disorganized. To identify molecular events preceding the onset of this pathology, we compared gene expression in the urogenital sinus (UGS), from which the prostate develops, in Tbx18-null and wild type littermates at two embryonic stages. Genes that regulate cell proliferation, smooth muscle differentiation, prostate epithelium development, and inflammatory response were significantly dysregulated in the mutant urogenital sinus around the time that Tbx18 is first expressed in the wild type UGS, suggesting a direct role in regulating those genes. Together, these results argue that Tbx18 is essential to the differentiation and maintenance of the prostate periurethral mesenchyme and that it indirectly regulates epithelial differentiation through control of stromal-epithelial signaling.

overall_design: Embryos were collected from timed matings of Tbx18Gfp/+ knock-in mutants at E16.5 and E18.5, and genotyped to identify Tbx18Gfp/Gfp null mutants and wild-type (WT) littermates.  The urogenital sinus (UGS) was dissected and used to extract RNA from each of three animals of each genotype.  The RNA samples were pooled to generate libraries for sequencing.

first: C

last: Bolt


title: Mice carrying a human GLUD2 gene recapitulate aspects of human transcriptome and metabolome development

city: Okazaki

first: Yasuhiro

last: Go

address: 38 Nishigonaka Myodaiji

department: Department of Brain Sciences, Center for Novel Science Initiatives

institute: National Institutes of Natural Sciences

summary: Whereas all mammals have one glutamate dehydrogenase gene (GLUD1), humans and apes carry an additional gene (GLUD2), which encodes an enzyme with distinct biochemical properties. We inserted human genomic region containing the GLUD2 gene into mice and analyzed the resulting changes in the transcriptome and metabolome during postnatal brain development. Effects were most pronounced early postnatally and affected predominantly genes involved in neuronal development. Remarkably, the effects in the transgenic mice partially parallel the transcriptome and metabolome differences seen between humans and macaques analyzed. Notably, the introduction of GLUD2 did not affect glutamate levels in mice, consistent with observations in the primates. Instead, the metabolic effects of GLUD2 center on the tricarboxylic acid cycle, suggesting that GLUD2 affects carbon flux during early brain development, possibly stimulating lipid biosynthesis.

overall_design: RNA-sequencing of frontal cortex of GLUD2+/- transgenic mice (two lines: line a and line b) and control littermates

first: Qian

last: Li

first: Song

last: Guo

first: Xi

last: Jiang

first: Jaroslaw

last: Bryk

first: Ronald

last: Naumann

first: Wolfgang

last: Enard

first: Masaru

last: Tomita

first: Masahiro

last: Sugimoto

first: Philipp

last: Khaitovich

first: Svante

last: Pääbo


title: Drought stress tolerance strategies revealed by RNA-Seq in two sorghum genotypes contrasting for WUE

city: Piacenza

first: Alessandra

last: Fracasso

state: Piacenza

address: via Emilia Parmense 84

department: DiProVeS

institute: Università Cattolica del Sacro Cuore

summary:
Background: Drought stress is the major environmental stress that affects plant growth and productivity. It triggers in plants a wide range of responses detectable at different scales: molecular, biochemical and physiological levels. At the molecular level the response to drought stress results in the differential expression of several metabolic pathways. For this reason, explore the subtle differences existing in gene expression of drought sensitive and drought tolerant genotypes allows to identify drought-related genes that could be used for selection of drought tolerance traits. Genome-wide RNA-Seq technology was used to compare the drought response of two sorghum genotypes characterized by contrasting water use efficiency.
Results: the physiological measurements carried out confirmed the drought sensitivity of IS20351 and the drought tolerance of IS22330 previously studied. The expression of drought-related genes was more abundant in the sensitive genotype IS20351 compared to the tolerant IS22330. The Gene Ontology enrichment highlighted a massive increase in transcript abundance in “response to stress” and “abiotic stimulus”, “oxidation-reduction reaction” in the sensitive genotype IS20351 under drought stress. “Antioxidant” and “secondary metabolism”, “photosynthesis and carbon fixation process”, “lipids” and “carbon metabolism” were the pathways most affected by drought in the sensitive genotype IS20351. The sensitive genotype IS20351 showed under well-watered conditions a lower constitutive expression level of “secondary metabolic process” (GO:0019748) and “glutathione transferase activity” (GO:000004364).
Conclusions: RNA-Seq analysis revealed to be a very useful tool to explore differences between sensitive and tolerant sorghum genotypes. The transcriptomic results supported all the physiological measurements and were crucial to clarify the tolerance of the two genotypes studied. The connection between the differential gene expression and the physiological response to drought states unequivocally the drought tolerance of the genotype IS22330 and the strategy adopted to cope with drought stress.

overall_design: mRNA profiles of 42-day old leaves of well-watered and drought-stressed Sorghum bicolor plants were generated by deep sequencing, in triplicate, using Illumina GAIIx.

first: Luisa

middle: M

last: Trindade

first: Stefano

last: Amaducci


title: The second subunit of DNA-polymerase delta is required for genomic stability and epigenetic regulation

city: Beijing

first: Jinkui

last: Cheng

address: No.2 Yuanmingyuan West Road

institute: China Agricultural University

summary: DNA polymerase δ plays crucial roles in DNA repair and replication, and maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis. During a genetic screen for release of TGS, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with ATR and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination (HR). It also exhibits hypersensitivity to DNA-damaging reagents, and short telomere length. Whole-genome ChIP-seq and RNA-seq analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggest that POLD2 is required for maintaining genome integrity, and properly establishing the epigenetic markers during DNA replication to modulate gene expression.

overall_design: Examine DNA methylation, histone modification and RNA expression changes by whole-genome bisulfite sequencing; RNA sequencing; H3K27me3-, H3K4me3-, H3K9me2-, H3-ChIP-seq in A. thaliana wild type and pold2 mutant

first: Jixiang

last: Zhang

first: Shaojun

last: Xie

first: Jinsheng

last: Lai

first: Jian-Kang

last: Zhu

first: Zhizhong

last: Gong


title: The mRNA-bound proteome of the early fly embryo

city: New York

first: Hans-Hermann

last: Wessels

address: 101 Avenue of the Americas

institute: New York Genome Center

summary: Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first two hours of embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 were previously characterized as DNA-binding transcription factors (TFs), signaling proteins, etc., and were not known to bind RNA. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first two hours of embryonic development. In situ hybridization data from existing databases showed that RNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation.

overall_design: Protein on ecdysone puffs (PEP) and CG3800 CLIPseq small RNA sequencing data in Drosophila S2 cells stably transfected with FLAG/HA-tagged PEP or CG3800. CLIP experiments were conducted in biological duplicates, including rRNA-depleted, size selected, matched total RNA inputs

first: Hans

first: Koshi

last: Imami

first: Alexander

last: Baltz

first: Marcin

last: Kolinski

first: Anastasia

last: Beldovskaya

first: Matthias

last: Selbach

first: Stephen

last: Small

first: Uwe

last: Ohler

first: Markus

last: Landthaler


title: Effect of pubertal BPA exposure on mammary stem cells

city: San Antonio

first: Yidong

last: Chen

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: Perinatal exposure to bisphenol A (BPA) has been shown to cause aberrant mammary gland morphogenesis and mammary neoplastic transformation. Yet, the underlying mechanism is poorly understood. We tested the hypothesis that mammary glands exposed to BPA during a susceptible window may lead to its susceptibility to tumorigenesis through a stem-cell mediated mechanism. We exposed 21-day-old Balb/c mice to BPA by gavage (25 µg/kg/day) during puberty for 3 weeks, and a subset of animals were further challenged with one oral dose (30 mg/kg) of 7,12-dimethylbenz[a]anthracene (DMBA) at 2 months of age. Primary mammary cells were isolated at 6 weeks, and 2 and 4 months of age for mammary stem cell (MaSC) quantification and function analysis. Pubertal exposure to the low-dose BPA increased lateral branches and hyperplasia in adult mammary glands and caused an acute increase of MaSC in 6-week-old glands and a delayed increase of luminal progenitors in 4-month-old adult gland. Most importantly, pubertal BPA exposure altered the function of MaSC from different age groups, causing pre-neoplastic lesions in their regenerated glands similar to those induced by DMBA exposure, which indicates that MaSCs are susceptible to BPA-induced transformation. Deep sequencing analysis on MaSC-enriched mammospheres identified a set of aberrantly expressed genes associated with pre-neoplastic lesions in human breast cancer patients. Thus, our study for the first time shows that pubertal BPA exposure altered MaSC gene expression and function such that they induced early neoplastic transformation.

overall_design: Four samples were analyzed (control, BPA, DMBA, BPA+DMBA), and for each treatment group, mammospheres from 5 animals were pooled for RNA extraction.

first: Qiaoxiang

last: Dong

first: Lu

middle: Z

last: Sun


title: Comparison of the transcriptomes of a Burkholeria pseudomallei bprR [bpss0688] mutant, a bprS [bpss0687] mutant and a bprRS double mutant with the wild-type B. pseudomallei K96243 parent strain.

city: Clayton

first: David

middle: Richard

last: Powell

state: VIC

address: Wellington Road

department: Bioinformatics Platform

institute: Monash University

summary: Objectives: The Burkholderia pseudomallei bprR and bprS genes are predicted to encode the response regulator and sensor kinase components respectively of a two component signal transduction system. Inactivation of either single component of this two component signal transduction system led to attenuation of B. pseudomallei virulence and also reduced motility. However, inactivation of both components resulted in wild-tpe virulence and motility. Therefore, we used RNA-seq to compare the transcritptomes of the three mutant strains (bprR single, bprS single, bprRS double mutant) with the wild-type parent strain in order to identify the genes differenty regulated in the different mutants.

overall_design: Methods: RNA sequencing was employed to determine the transcriptomes of three mutant strains (bprR single, bprS single, bprRS double mutant) with the wild-type parent strain B. pseudomallei K96243. B. pseudomallei parent strain K96243 and the directed bprS, bprR and bprRS  mutants were grown overnight at 37 °C in LB with appropriate antibiotics (K96243 in gentamicin 64 µg/ml; mutants in gentamicin 64 µg/ml and tetracycline 25 µg/ml). Strains were subcultured 1/50 into fresh antibiotic-free medium and grown at 37 °C with shaking (200 rpm) to an OD600 of 0.5. The cells were harvested by centrifugation, and RNA purified using Trizol reagent (Gibco/BRL) as specified by the manufacturer. RNA was DNase treated using the TURBO DNA-free kit (Ambion), followed by processing with a QIAGEN RNeasy kit clean-up with on-column DNase digestion as per the manufacturer’s instruction. Double-stranded cDNA synthesis and high-throughput RNA-seq were performed as described previously (Henry R, Crane B, Powell D, Deveson Lucas D, et al: The transcriptomic response of Acinetobacter baumannii to colistin and doripenem alone and in combination in an in vitro pharmacokinetics/pharmacodynamics model. J Antimicrob Chemother. 2015; 70:1303-1313). For each strain three biological replicates were sequenced.

first: John

last: Boyce


title: RNA-Seq: assessment of transcript level analysis tools [seq]

city: Rehovot

first: Gilgi

last: Friedlander

address: Hertzel st

country: Israel

institute: Weizmann Institute of Science

summary: This study uses spiked-in transcript in order to compares various bioinformatics approaches and tools to assemble, quantify abundance and detect differentially expressed transcripts using RNA-Seq data. Mouse total RNA seq was extracted from embryonic stem cells (ES) before (designated as day 0) and four days after the addition of retinoic acid. 48 spikes were made in vitro from plasmid constructs and added to the total RNA in different concentrations (each mix has a set of different spike concentrations, see paper's method). We found that detection of differential expression at the gene level is acceptable, yet on the transcript-isofom level all tools tested were lacking accuracy and precision.

overall_design: Mouse total RNA was extracted from embryonic stem cells (ES) before (designated as day 0) and four days after the addition of retinoic acid (RA) (designated as day 4). Mouse spike-ins consisting of 48 different mouse RNA transcripts were generated in vitro from plasmid constructs and added to the total RNA. 23 of the spike-ins originate from 10 different locus regions, so that each locus is represented by at least two different transcripts. The remaining 25 spike-ins represent different loci.

first: Dena

last: Leshkowitz

first: Ester

last: Feldmesser

first: Ghil

last: Jona

first: Elena

last: Ainbinder

first: Yisrael

last: Parmet

first: Shirley

last: Horn-Saban


title: Identification of transcriptome of mouse non-neural ectoderm during nueral tube closure and gene enrichment compared to remaining neural tube tissue

city: Aurora

first: Lee

last: Niswander

state: Colorado

address: 12801 E. 17th Ave Bld RC1 South 12400

department: Pediatrics

institute: UC Denver Anschutz Medical Campus

summary: The process of neural tube closure is a highly complex morphogenetic event that results in the generation of the primordial central nervous system. During formation of the neural tube, the non-neural ectoderm separates from the neighboring neural ectoderm and forms a single layer epithelial sheet that overlies the closed neural tube. Previous work has shown that the non-neural ectoderm is necessary for proper cranial neural tube closure, however little is known about this cell population at the molecular level or how the non-neural ectoderm contributes to neural tube closure. In this study, we used a mouse genetic system to fluorescently label the non-neural ectoderm cells and FACS sorted these cells away from the other cell populations in the neural tube. We performed high throughput RNA-sequencing to identify the transcriptome of the non-neural ectoderm and compared the gene expression profile of non-neural ectoderm cells to the remaining population of cells within the neural tube in order to identify which genes are enriched within the non-neural ectoderm. This analysis provides a clue as to which underlying molecular processes may be important for non-neural ectoderm function during neural tube closure.

overall_design: mTomato/mGFP dual fluorescent reporter mice were mated to Grhl3-cre mice to generate embryos that expressed a membrane-bound RFP throughout the embryo with specific expression of membrane-bound GFP in the Grhl3+ non-neural ectoderm. Embryos were dissected at 9.5 days post-fertilization and scored for correct genetic recombination. Non-neural tube tissue was dissected away and single cell suspensions were made followed by FACS to sort the GFP+ and RFP+ populations. RNA was isolated immediately and pooled samples of 1ug total RNA were used to generate libraries for sequencing with the Illumina TruSeq RNA sample preparation kit. Pooled RNA was generated from 25 individual embryos all aged betweed 21 and 25 somites. RNA-sequencing was performed on an Illumina HiSeq 2000.

first: Heather

last: Ray


title: RNA-Seq: assessment of transcript level analysis tools


title: Hobit and Blimp1 instruct a universal transcriptional program of tissue-residency in lymphocytes

status: Public on Apr 25 2016

city: Vienna

first: Moritz

last: Gaidt

address: Campus-Vienna-Biocenter 1

country: Austria

institute: IMP

summary: Tissue-resident memory T cells (Trm) permanently localize to portals of pathogen entry, where they provide immediate protection against re-infection. To enforce tissue retention, Trm upregulate CD69 and downregulate molecules associated with tissue egress including CCR7 and S1PR1. Although suppression of the transcription factor KLF2 in Trm prevents S1PR1-driven migration, a Trm-specific transcriptional regulator has not been identified. Here, we show that the transcription factor Hobit is specifically upregulated in Trm and together with related Blimp1, mediates the development of Trm in skin, gut, liver and kidney. Importantly, the Hobit/Blimp1 transcriptional module is also required for other populations of tissue-resident lymphocytes including NKT cells and liver-resident NK cells, all of which share a common transcriptional program that includes repression of Ccr7, S1pr1, and Klf2. Our results identify Hobit and Blimp1 as central regulators of this universal program that instructs tissue retention in diverse tissue-resident lymphocyte populations.

overall_design: Examination of Blimp1 and Hobit binding-sites in CD8 T cells

first: Martina

last: Minnich

last: Jaritz

first: Meinrad

last: Busslinger


title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells (RNA-Seq)

first: Yang

last: Wang

state: Beijing

address: Xueyuan Road 38#

department: Molecular Biology and Biochemistry

institute: Peking University Health Science Center

summary: ISL1 is expressed in cardiac progenitor cells and plays critical roles in cardiac lineage differentiation and heart development. Cardiac progenitor cells hold great potential for clinical and translational applications. However the mechanisms underlying ISL1 function in cardiac progenitor cells have not been fully elucidated. Here we uncover a hierarchical role of ISL1 in cardiac progenitor cells, showing that ISL1 directly regulates hundreds of potential downstream targets that are implicated in cardiac differentiation, through an epigenetic mechanism. Specifically, ISL1 promotes the demethylation of tri-methylation of histone H3K27 (H3K27me3) at the enhancers of key downstream target genes, including Myocd and Mef2c, which are core cardiac transcription factors. ISL1 physically interacts with JMJD3, a H3K27me3 demethylase, and conditional depletion of JMJD3 leads to impaired cardiac progenitor cell differentiation, phenocopying that of ISL1 depletion. Interestingly, ISL1 is not only responsible for the recruitment of JMJD3 to specific target loci during cardiac progenitor differentiation, but also modulates its demethylase activity. In conclusion, ISL1 and JMJD3 partners to alter the cardiac epigenome, instructing gene expression changes that drive cardiac differentiation.

overall_design: To investigate the transcriptional effects of ISL1 binding, we performed RNA-seq on day 7 Isl1 knock-down EBs versus control EBs.

first: Chunyan

last: Zhou


title: Small RNAs from sweet potato upon wounding

city: Taipei

first: Jeng-Shane

last: Lin

address: No. 1, Sec. 4, Roosevelt Road

department: Plant Biology

country: Taiwan

institute: National Taiwan University

summary: Small RNAs (sRNAs) play important roles in plants encountering stress environments. However, limited research has been conducted on the sRNAs involved in plant wound responses. To identify potential roles for the wounding-related sRNAs, sRNA deep sequencing was used. After leaves were wounded for 0.5 hour, total RNAs from unwounded and wounded leaves were isolated for sRNA library construction. The Illumina platform was used to sequence sRNA libraries. About 12 million sequence reads were obtained for each sample.

overall_design: Examination of sweet potato leaves with either unwound or wound treatment

first: Jeng

last: Shih-Tong

first: Lin

last: Jeng-Shane


title: Parental epigenetic asymmetry of PRC2-mediated histone modifications in the Arabidopsis endosperm

city: Uppsala

first: Juan

last: Santos-González

state: Uppsala

address: Almas Allé 5

department: Department of Plant Biology and Forest Genetics

country: Sweden

institute: SLU

summary:
Parental genomes in the endosperm are marked by differential
DNA methylation and are therefore epigenetically distinct. This
epigenetic asymmetry is established in the gametes and maintained
after fertilization by unknown mechanisms. In this manuscript,
we have addressed the key question whether parentally
inherited differential DNA methylation affects de novo targeting of
chromatin modifiers in the early endosperm. Our data reveal that
polycomb-mediated H3 lysine 27 trimethylation (H3K27me3) is
preferentially localized to regions that are targeted by the DNA
glycosylase DEMETER (DME), mechanistically linking DNA
hypomethylation to imprinted gene expression. Our data furthermore
suggest an absence of de novo DNA methylation in the early
endosperm, providing an explanation how DME-mediated
hypomethylation of the maternal genome is maintained after
fertilization. Lastly, we show that paternal-specific H3K27me3-
marked regions are located at pericentromeric regions, suggesting
that H3K27me3 and DNA methylation are not necessarily exclusive
marks at pericentromeric regions in the endosperm.

overall_design: Analysis of H3K27m3, H3k9m2 and H3K27m1 modifications and DNA methylation in endosperm and leaves in Arabidopsis. The ChIP-seq experiment consists of 37 samples. These include two replicates for each combination of cross/tissue/histone modification analyzed, except for the sample with H3k27m1 in leaf which has only one replicate. Most inputs and H3 samples are common for two or more H3-methylation samples. The BS-seq experiment consists of two samples that are replicates.

first: Jordi

last: Moreno-Romero

first: Hua

last: Jiang

first: Claudia

last: Köhler


title: Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells

city: La Jolla

first: Xiang-Dong

last: Fu

state: California

address: 9500 Gilman Drive

department: Department of Cellular and Molecular Medicine

institute: University of California, San Diego


title: Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells [RNA-seq]

summary: Direct conversion of somatic cells into neurons holds great promise for regenerative medicine. However, neuronal conversion is relatively inefficient in human cells compared to mouse cells. It has been unclear what might be the key barriers to reprogramming in human cells. We recently elucidated an RNA program mediated by the polypyrimidine tract binding protein PTB to convert mouse embryonic fibroblasts (MEFs) into functional neurons. In human adult fibroblasts (HAFs), however, we unexpectedly found that invoking the documented PTB–REST–miR-124 loop generates only immature neurons. We now report that the functionality requires sequential inactivation of PTB and the PTB paralog nPTB in HAFs. Inactivation of nPTB triggers another self-enforcing loop essential for neuronal maturation, which comprises nPTB, the transcription factor BRN2, and miR-9. These findings suggest that two separate gatekeepers control neuronal conversion and maturation and consecutively overcoming these gatekeepers enables deterministic reprogramming of HAFs into functional neurons.

overall_design:
Six RNA-seq libraries are generated by MAPS approach. Total RNA is extracted from induced neuronal cells derived from control shRNA or PTB shRNA treated human adult fibroblasts.

Please note that the 'RNA-seq HAF hygro' sample was on 6 days after switching to N3 media *without* nPTB depletion. The other two 'shPTB 6d' and 'shPTB 3w' were on 6 days and 3 weeks respectively after  switching to N3 media *with* nPTB depletion.

first: Yuanchao

last: Xue

first: Xihao

last: Hu

first: Yu


title: Ultraefficient irCLIP platform for characterization of protein-RNA interactions

city: Stanford

first: Ryan

middle: Alexander

last: Flynn

state: CA

address: 380 Roth Way, Room 265

institute: Stanford University

summary: The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce an advanced UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, nonisotopic method for the detection of protein-RNA interactions using 100-fold less material than standard protocols.

overall_design: Nonisotopic interrogation of in vivo RNA-protein interactions

first: Brian

last: Zarnegar

first: Ying

last: Shen

last: Do

first: Howard

middle: Y

last: Chang

first: Paul

last: Khavari


title: Comparative Analysis of Cas9 Activators Across Multiple Species

city: Boston

first: Emma

middle: Joanna Katharina

last: Kowal

address: 3 Blackfan Circle

department: Genetics

institute: Harvard Medical School

summary: RNA-Seq after Cas9-gRNA transfection with different activators targeting HBG1

overall_design: we performed PolyA Selection and RNA-Seq on cells transfected with a dCas9-based transcriptional activator (VP64, VPR, SAM or Suntag) and a gRNA targeting HBG1

first: Alejandro

last: Chavez

first: Dmitry

last: Ter-Ovanesyan